Manganese Oxidation by Pseudomonas putida
A thesis presented
by
Steven Robert DePalma
to
The Committee on Higher Degrees in Biophysics
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
in the subject of
Biophysics
Harvard University
Cambridge, Massachusetts
October 1993
1993 by Steven Robert DePalma
All rights reserved
ABSTRACT
This thesis examines the taxonomy of, and manganese (II) oxidation by, a
supposed species of bacteria known as "Pseudomonas manganoxidans", originally
described in the 1970's by Schweisfurth and colleagues. It is shown here that "P. manganoxidans" is more properly considered a member of the common soil and water species
Pseudomonas putida. Another manganese-oxidizing species of doubtful taxonomic
validity, "Arthrobacter siderocapsulatus", is also shown to be P. putida. Furthermore, it
appears that manganese oxidation is a common property of other members of P. putida,
not before known to be metal-oxidizers. Some related strains of Pseudomonas were also
shown to have the ability to oxidize manganese. Contrary to previous reports, cell-free
manganese oxidation in "P. manganoxidans" requires the participation of oxygen, and
can be considered enzymatic. The activity is induced by carbon starvation much more
than by starvation for ammonium or for phosphate. Heat shock and peroxide shock were
not seen to induce manganese oxidation activity, as they do for some other starvationstress activities. The detailed mechanism of and the purpose for manganese oxidation in
this species remain unknown. Pseudomonas putida, as a species well-studied
biochemically and genetically, should be useful as a model system for studying a
mechanism of bacterial manganese oxidation.
To my parents,
for their unending love and support.
iv
TABLE OF CONTENTS
Abstract
iii
Table of Contents ..................................................................................................... v
List of Tables ix
List of Figures x
Acknowlegements .................................................................................................. xii
Chapter One: Overview of Manganese and Bacterial Manganese Oxidation
1.1 Manganese Chemistry ......................................................................... 2
1.2 Manganese in Nature ........................................................................ 8
1.3 Manganese Biochemistry ................................................................. 10 .................
1.4 Practical Implications of Manganese Oxidation .............................. 11
1.5 Biological Manganese Oxidation ..................................................... 13
1.6 Abundance of Manganese-Oxidizing Bacteria ................................. 18
1.7 Bacterial Manganese Oxidation: Mechanisms and Functions ......... 20
Chapter Two: Taxonomy of Manganese-Oxidizing Pseudomonas Species
2.1 "Pseudomonas manganoxidans" ...................................................... 26
2.1.1 Materials and methods ....................................................... 29
2.1.2 Results ................................................................................ 30
v
TABLE OF CONTENTS (continued)
2.1.3 Discussion .......................................................................... 36
2.1.3.1 History of Pseudomonas taxonomy .............................. 37
2.2 "Arthrobacter siderocapsulatus" ...................................................... 41
2.2.1 Materials and methods ....................................................... 43
2.2.2 Results and discussion ....................................................... 44
2.3 New Isolates of Manganese-Oxidizing Bacteria ............................... 48
2.3.1 Materials and methods ....................................................... 48
2.3.2 Results and discussion ....................................................... 50
2.4 Literature Review of Manganese-Oxidizing Pseudomonads ............ 52
Chapter Three: Manganese oxdiation by culture-collection strains
of Pseudomonas putida
3.1 Introduction ....................................................................................... 56
3.1.1 Materials and methods ...................................................... 57
3.1.1.1 P. putida strain histories .............................................. 57
3.1.1.2 Media Formulations ................................................. 62
3.1.1.3 Detection of Mn-oxidation on solid media ............... 65
3.1.1.4 Rates of Mn Oxidation by starved cultures
vi
in liquid medium ............................................................. 65
TABLE OF CONTENTS (continued)
3.1.2 Results
3.1.2.1 Mn oxidation on solid media .................................................... 67
3.1.2.1 Mn oxidation rates for starved P. putida strains
in suspension .................................................................................. 77
3.2 Manganese oxidation by the type strain of
Pseudomonas putida .............................................................................. 85
3.2.1 Materials and methods ................................................................... 86
3.2.1.1 Preparation of crude extracts ............................................... 86
3.2.1.2 In vitro assay of manganese oxidation by
cell-free extracts ............................................................................. 86
3.2.1.3 Effects of heat and protease on manganese oxidation
activity......................................................................................... 87
3.2.2 Results ........................................................................................... 88
3.2.3 Discussion ...................................................................................... 93Chapter Four: In
vii
Phenomenon
4.1 Introduction ................................................................................................. 98
4.2 Materials and methods ............................................................................... 101
4.2.1 Starvation for C, N, and/or P ..................................................... 101
4.2.2 The leucoberbelin blue assay ..................................................... 102
4.2.3 Heat shock/peroxide shock ........................................................ 103
TABLE OF CONTENTS (continued)
4.3 Results and discussion ............................................................................... 105
4.3.1 Nutrient starvation ..................................................................... 105
4.3.2 Heat shock/peroxide shock ..................................................................... 111
Chapter Five: Mechanism of Manganese Oxidation in Pseudomonas putida:
The Role of Oxygen
5.1 Introduction ............................................................................................... 118
5.2 Materials and methods .............................................................................. 121
5.3 Results and discussion .............................................................................. 122
5.4 Discussion: Possible mechanisms of manganese
oxidation in Pseudomonas putida ........................................................ 133
Chapter Six: Summary and Conclusions ................................................................... 139
viii
APPENDIX ONE: Results and Identifications from Commercial
Test Systems ........................................................................................ 143
APPENDIX TWO: Comparison of Jessen Biotypes with P. putida/
P. fluorescens Biovars ......................................................................... 151
REFERENCES .......... ................................................................................................... 159
ix
LIST OF TABLES
Table
Page
I-1
Standard reduction potentials at pH 7 .................................................................... 6
I-2
Free-energy changes for oxidation of manganese .................................................. 7
I-3
Bacteria reported to deposit manganese oxides .................................................. 16
II-1
"Pseudomonas manganoxidans" strains classified under the
scheme of Jessen .................................................................................................. 27
II-2
Characteristics distinguishing fluorescent pseudomonads ................................... 31
II-3
Results of characterization tests:
"Pseudomonas manganoxidans" ............................................................... 32
II-4
Comparison of Jessen's groups with presently-accepted species .............. 35
II-5
Results of characterization tests:
"Arthrobacter siderocapsulatus" ..........................................................................45
II-6
Results of characterization tests:
New isolates of manganese-oxidizing bacteria .....................................................51
III-1 Manganese oxidation by and background of culture-collection
strains of Pseudomonas putida .............................................................................68
III-2 Manganese oxidation by ATCC Pseudomonas putida on
various media 70
III-3 Manganese oxidation by "Pseudomonas manganoxidans" and
"Arthrobacter siderocapsulatus" on various media ..............................................71
III-4 Manganese oxidation by new isolates on various media .........................................72
x
III-5 Manganese oxidation by other species on various media ........................................73
xi
LIST OF FIGURES
Figure
Page
I-1
Stability diagram of manganese in aqueous solution ......................................................... 4
III-1
Manganese oxidation by starved P. putida strains, 0 to 45 hours ........................79
III-2
Manganese oxidation by starved P. putida strains, 0 to 210 hour ........................ 81
III-3
Survival of starved P. putida strains during manganese oxidation assay ............. 83
III-4
Inhibition by protease of manganese oxidation activity
by cell-free extracts of P. putida TS-1 .................................................................. 90
III-5
Inhibition by heat of manganese oxidation activity .............................................. 92
IV-1
Manganese oxidation by P. putida MnB1-A2 under various starvation
regimes, 0 to 10 hours ......................................................................................... 107
IV-2
Manganese oxidation by P. putida MnB1-A2 under various starvation
regimes 0 to 50 hours .......................................................................................... 109
IV-3
Manganese oxidation by P. putida MnB1-A2 after heat and peroxide shock,
Group 1 ............................................................................................................... 114
IV-4
Manganese oxidation by P. putida MnB1-A2 after heat shock,
Group 2 ............................................................................................................... 116
V-1
In vitro manganese oxidation by P. putida A2, 10 μM Mn2+,
0 to 3 hours.......................................................................................................... 126
xii
LIST OF FIGURES (continued)
Figure
V-2
Page
In vitro manganese oxidation by P. putida MnB1-A2, 100 μM Mn2+,
0 to 3 hours.......................................................................................................... 128
V-3
In vitro manganese oxidation by P. putida MnB1-A2, 10 μM Mn2+,
12 to 15 hours...................................................................................................... 130
V-4
In vitro manganese oxidation by P. putida A2, 100 μM Mn2+,
12 to 21 hours...................................................................................................... 132
xiii
ACKNOWLEDGEMENTS
Thanks are offered to innumerable people who helped me out and kept me going
through the years:
To Prof. Ralph Mitchell, for his patience and for giving me the freedom to pursue
this project;
To Profs. Don Wiley and Jim Hogle, who kept me on track when I strayed;
To the late Prof. Richard Schweisfurth, whom I never met but whom I feel I know
intimately after poring so carefully over (and over and over) his articles;
To Jim Maki and Bruce Demple, who were always willing to help me out with the
scientific and the nonscientific aspects of my work;
To Betsy Henry and David Fung, beloved comrades-in-arms in the graduate
school trenches;
To Ned Black, fellow grad student, who turned me on to manganese and to
"Pseudomonas manganoxidans";
To Gayatri Patel, whose zebra mussel isolates I borrowed;
To all the members of the Mitchell lab, past and present, who put a human face on
science for me;
To my roommates and to the Harvard Scottish Country Dancers, who kept me
from working too hard; and especially
To my mother, for coming to the rescue twice in the final days of putting this
thesis together.
xiv
To all, I am eternally grateful.
xv
CHAPTER ONE
OVERVIEW OF MANGANESE AND
BACTERIAL MANGANESE OXIDATION
Chapter One
OVERVIEW OF MANGANESE AND BACTERIAL MANGANESE OXIDATION
Bacterial oxidation of Mn(II) to higher oxides of manganese is a widespread but
poorly understood phenomenon. Bacteria that can oxidize Mn(II) have been known since
the turn of the century (Jackson, 1901; Beijerinck, 1913). However, the identities of
these organisms, the mechanisms of the reactions, and the benefits, if any, to the cells, are
not well known. This thesis presents a reexamination of a known manganese-oxidizing
bacterium, "Pseudomonas manganoxidans," reclassifies it as Pseudomonas putida, and
investigates the mechanism and the regulation of manganese oxidation by this species.
1.1. Manganese chemistry
Manganese is a fairly common element. It is the fifth most abundant metal in the
Earth's crust, and the second most common trace metal after iron, found at about 1/50 the
abundance of iron. Manganese is distinctive for being able to exist in a great number of
oxidation states, from 0 to +7. In nature, however, it is primarily found in the Mn(II),
Mn(III), and Mn(IV) states.
The Mn2+ (manganous) cation is the most important soluble form of manganese in
nature, though certain important Mn(II) salts such as manganous carbonate
(rhodochrosite) have only low or negligible solubility. The Mn3+ (manganic) ion is
2
3
unstable in neutral solution unless strongly complexed; it rapidly disproportionates to
Mn2+ and MnO2. Mn(III) and Mn(IV) are generally found as insoluble oxides or hydrous
oxides, Mn(IV) most notably as MnO2. These oxides are brown- or black-colored.
Mn(II) oxidation can lead to a variety of oxides, depending on the exact
conditions of oxidation; some possibilities are ß-, γ-, and δ-MnO2, α-, ß-, and γMnOOH, Mn2O3, Mn3O4, and Mn(OH)3 (Stumm and Morgan, 1970). Any oxidation not
rigidly controlled is likely to result in a nonstoichiometric mixture of these oxides. Such
mixed oxides are often referred to as "MnOx" for convenience, with x ranging from 1.0 to
2.0. Furthermore, some of the oxides are metastable and convert to higher oxides upon
aging (Hem and Lind, 1983). An important complication of studying Mn(II) oxidation is
that manganese oxides provide binding sites for trace metals, including Mn2+, and that
MnOx catalyzes oxidation of bound Mn(II).
The oxidation of aqueous Mn2+ to MnO2 is energetically favorable under neutral
aerobic conditions, as can be seen from Figure I - 1. However, the kinetics of the reaction
are such that spontaneous oxidation does not occur measurably until the pH increases
above about 8 or 9.
4
FIGURE I - 1:
STABILITY DIAGRAM OF MANGANESE IN AQUEOUS SOLUTION.
Dissolved Mn 0.01 - 100 mg  l─1; HCO3─ 10 mg  l─1; SO42─ 1.0 g  l─1;
c = crystalline. (from Hem, 1964)
5
Manganese oxides are fairly strong oxidizers, as can be seen from Table
I - 1. The Mn3+/Mn2+ and MnO2/Mn2+ redox couples have high reduction potentials,
implying that few molecules present inside a cell can oxidize Mn2+ other than O2 or H2O2.
Oxidation of manganese is suspected by many of being a means by which a
bacterial cell obtains its energy, just as Thiobacillus ferrooxidans does by oxidizing Fe2+,
Nitrobacter by oxidizing ammonium, or E. coli by oxidizing glucose. Nealson et al.
(1988) have calculated ΔG values for oxidation of manganese under various conditions of
pH, [Mn2+], and [O2]. Some of their results are given in Table I - 2.
Table I - 2 shows that the free energy yield from oxidation of Mn2+, while
negative, is not large. For comparison, ΔG' for the reaction
glucose + 6 O2 = 6 CO2 + 6 H2O
is ─686 kcal  mol─1 (Lehninger, 1982), or 57.2 kcal per mole of electron-pairs. Thus the
oxidation of Mn2+ to MnO2 releases only about one-third the free energy of glucose (on
an electron-pair basis), at standard state. Moreover, the free energy yield shows
considerable dependence not only on pH and [Mn2+], but also on the nature of the oxide
produced and the stoichiometry of the reaction.
6
TABLE I - 1:
Standard Reduction Potentials at pH 7 (1M concs., 25 C.)
E0 (volts)
Mn3+ + e─ = Mn2+ ..............................................................................
H2O2 + 2 H+ +2 e─ = 2 H2O ........................................................ 1.357
O2 + 4 H+ + 4 e─ = 2 H2O ................................................................... 0.816
Fe3+ + e─ = Fe2+ ................................................................................. 0.771
MnO2 + 4 H+ + 2 e─ = Mn2+ + 2 H2O ............................................... 0.404
O2 + 2 H+ + 2 e─ = H2O2 .................................................................... 0.295
cytochrome a Fe3+/Fe2+ ...................................................................... . 0.29
cytochrome c Fe3+/Fe2+ ....................................................................... . 0.25
cytochrome b Fe3+/Fe2+ ....................................................................... . 0.08
FAD + 2 H+ + 2 e─ = FADH2 ........................................................... . ─0.22
NAD+ + 2 H+ + 2 e─ = NADH + H+ .................................................. . ─0.32
H+ + e─ =
H2O ................................................................................ . ─0.42
6 CO2 + 4 H+ + 4 e─ = 1/6 glucose + H2O ........................................ . ─0.43
NAD = nicotinamide adenine dinucleotide
FAD = flavin adenine dinucleotide
(From Loach, 1972, and Thauer et al., 1977).
7
TABLE I - 2:
Free Energy Changes for Oxidation of Manganese
Reaction
A.
Mn2++
pH
[O2]
ΔG
kcal mol-1
of Mn oxidized
O2+H2O ───> MnO2 + 2 H+
7
6
7
8
8
B.
[Mn2+]
3 Mn2+ +
1M
1 μM
1 μM
1 μM
100 μM
1 atm. ─ 16.2
225 μM
225 μM
225 μM
225 μM
─ 4.8
─ 7.5
─ 10.2
─ 12.9
O2 + 3 H2O ───> Mn3O4 + 6 H+
7
6
7
8
8
1M
1 μM
1 μM
1 μM
100 μM
1 atm. ─ 10.0
225 μM
+ 1.1
225 μM
─ 1.6
225 μM
─ 4.3
225 μM
─ 7.1
Values of ΔG were calculated from the equation ΔG = ΔG + RT ln K.
1 μM Mn2+ is a generous value for a natural Mn2+ concentration; 225 μM O2 is about the
value for oxygen-saturated seawater.
(from Nealson et al., 1988)
8
A great number of stoichiometries for biological manganese oxidation can be
hypothesized. Nealson et al. (1989), for example, list eleven possible reactions for
biological oxidation of Mn(II) to manganese oxides, but none of these pathways has yet
been confirmed in a real case. A few possibilities are listed below:
Mn2+ +
O2 + H2O ──> MnO2 + 2 H+
Mn2+ + O2 ──> MnO2
Mn2+ + MnO2 ──> [Mn(II)MnO2];
[Mn(II)MnO2] + O2 ──> 2 MnO2
Mn2+ + 2 H2O + 2 A ──> MnO2 + 4 H+ + 2 A─
(where A is some electron acceptor)
As can be seen in the last example, it is conceivable that O2 need not take part in
the oxidation; this possibility is important in Chapter Five. Similar series of reactions to
the above can be hypothesized with MnOOH, Mn2O3, or Mn3O4 as oxidation products,
further complicating the task of determining mechanisms and possible energy yields in
bacterial manganese oxidation.
1.2. Manganese in Nature
An idea of the distribution and abundance of manganese in nature is shown in the
table below (from Nealson, 1983b). Concentrations are given in parts per million (1.00
ppm Mn = 18.2 μM Mn).
9
Distribution and abundance of manganese (ppm)
soil (soluble Mn) ........................... 0.8 - 130
soil and rocks (total Mn) ............... 200 - 3000
lake waters .................................... 0.004 - 0.2
lake sediments ............................... 103 - 105
ocean surface waters ..................... 10─4 - 10─3
river water ..................................... 102 - 103
groundwater .................................. 1 - 10
terrestrial plants ............................. 20 - 500
Manganese is very often found along with iron, and is a common contaminant of
iron deposits. Ferromanganese nodules, valuable for their concentration of trace
minerals, are found on ocean floors and lake floors; they are suspected of having a
biological origin (see, for example, Ehrlich, 1990). Ferromanganese oxides make up the
black "desert varnish" that covers rocks in arid regions; this may also be caused by
manganese-oxidizing bacteria and fungi (Dorn and Oberlander, 1981; Krumbein and
Jens, 1981).
The cation Mn2+ is particularly abundant in environments where reducing
conditions contribute to the stability and abundance of that cation, such as anoxic regions
of lakes and other bodies of water, deep-sea hydrothermal vents, waterlogged soils, and
10
anaerobic sediments. The oxic/anoxic interface of stratified waters is a place where one
would find both an abundance of Mn2+ and the O2 that is presumed to be required to
oxidize it (Tebo et al., 1984).
There is a global biogeochemical manganese cycle, in which manganese is
oxidized and reduced, moving between soluble and insoluble phases. Manganesereducing microorganisms exist as well as manganese-oxidizing ones. Some can
apparently use manganese oxides as terminal electron acceptors in anaerobic respiration
(Nealson et al., 1989).
Biogeochemists have long debated whether manganese oxidation in nature is
primarily a chemical or biological phenomenon. It is generally agreed now to be a
biological one. For example, microbes have been observed in association with
manganese oxide deposits in nature, and microorganisms have been isolated from these
deposits which are capable of oxidizing Mn2+ at pH 7 in the laboratory (Tyler and
Marshall, 1967a,b; Ghiorse and Chapnick, 1983). Manganese oxidation rates in nature
are faster than can be accounted for chemically (Diem and Stumm, 1984). Furthermore,
oxidation (or, at least, deposition) of manganese in soil columns and in natural waters has
been shown to be sensitive to poisons (Mann and Quastel, 1946; Emerson et al. 1982;
Rosson et al., 1984) and to show temperature optima (Tipping, 1983). These examples
strongly suggest a biological role in the oxidation of manganese in natural environments.
1.3. Manganese Biochemistry
Manganese is an essential trace element for all organisms. It is a component of
some metalloenzymes, such as superoxide dismutase, arginase, and some phosphate-
11
transferring enzymes (Lehninger, 1982). In plants, manganese atoms are involved in the
light-mediated oxidation of H2O to O2 in photosystem II. High intracellular
concentrations of Mn2+ have been reported to function as a protectant against superoxide
toxicity in a microaerophilic bacterium, Lactobacillus plantarum (Archibald and
Fridovich, 1981).
Manganese toxicity is known, but manganese is not normally considered a toxic
element. Mn2+ can be toxic and mutagenic for bacteria (Demerec and Hanson, 1951).
Mn2+ can be toxic to plants, especially in low-pH soils (Stevenson, 1986). In humans,
airborne particulate manganese and well water high in soluble manganese have been
linked to illness (National Academy of Sciences, 1973). The U. S. water quality standard
for manganese in drinking water is 0.05 ppm, but this low level is for esthetic purposes
rather than health reasons (Bull and Craun, 1977), because manganese in water supplies
can stain plumbing fixtures and laundry, and may give the water a foul odor.
1.4. Practical Implications of Manganese Oxidation.
Manganese oxidation is of practical concern in agriculture, in industry, and in
drinking water treatment. Oxidation of Mn2+ by soil and rhizosphere bacteria (such as
Pseudomonas putida), it should be noted, has implications for plant nutrition: manganese
is an essential element for plant growth, and only soluble Mn (that is, Mn2+) is available
for uptake by plants. "Gray-speck" in oats and "marsh-spot" in peas are symptoms of
manganese deficiency, remedied by additions of manganous sulfate, but the remedy is
ineffective if the added Mn2+ is oxidized too quickly (Bromfield and Skerman, 1950).
Microbiological treatment methods are sometimes used to precipitate soluble
12
manganese from water supplies, especially in Europe (Griffin, 1960; Mouchet, 1992).
Naturally-occuring manganese-oxidizing bacteria colonize sand filters through which
Mn2+-containing water is passed, resulting in near-complete removal of soluble
manganese (Peitchev and Semov, 1988). Schweisfurth (1973a) isolated some of his
"Pseudomonas manganoxidans" strains from such facilities, and "Siderocapsa",
Ochrobium and Leptothrix species have been found in abundance in the filters (Vuorinen
et al., 1988). Nealson (1992) suggests a novel application for manganese-oxidizing
microorganisms: he notes that manganese oxides, with their strong complexing
properties, can be used to remove radium from water, and speculates that natural
populations of subsurface bacteria could be stimulated to produce manganese oxides and
thus provide an in situ method for removing radium from groundwater supplies.
In industrial pipelines, metal oxide crusts associated with iron- and manganeseoxidizing bacteria can build up to such an extent that water flow is seriously impeded
(Tyler and Marshall, 1967a,b; Tuovinen et al., 1980). Manganese oxidation is also
suspected to be involved in microbiologically-induced metal corrosion (Ford and
Mitchell, 1990).
13
1.5. Biological Manganese Oxidation
For a phenomenon described as "ubiquitous" by workers in the field (Ghiorse,
1984; Nealson, 1992), it is surprising how little is known about the organisms that
oxidize manganese, the mechanisms by which it is done, and the benefits (if any) that are
gained by the organisms involved.
Oxidation of manganese (II) has been observed in cultures of bacteria, fungi,
algae, and occasional protozoa. Algal manganese oxidation appears to be caused by a
localized rise in pH due to photosynthesis, or perhaps by a high concentration of O2 in
algal clumps (Lubbers et al., 1990). A wide variety of fungi are capable of oxidizing
manganese in the laboratory (Schweisfurth, 1971), but the tendency has been to dismiss
this fungal manganese oxidation as a laboratory phenomenon only (Ehrlich, 1990), a
nonenzymatic reaction due to a localized pH rise. The traditional focus of study has been
bacteria. One reason for this is that morphologically-distinct bacteria have been observed
microscopically to be associated with deposits of manganese and iron oxides in natural
samples. Also, from the earliest discovery of iron-oxidizing and manganese-oxidizing
bacteria in the late 19th century and early 20th century (Winogradsky, 1888; Jackson,
1901; Beijerinck, 1913), microbiologists have speculated that the bacteria might be living
autotrophically with reduced iron or manganese as their energy source.
The history of the study of manganese-oxidizing bacteria cannot be separated
from the history of the study of iron-oxidizing bacteria. Since the chemistry of
manganese is similar to that of iron, and since oxides of the two metals are often found
together in association with certain bacteria, there has been a tendency to equate ironoxidizing and manganese-oxidizing bacteria. (Strictly, they should be referred to as
14
"Fe/Mn-oxide-depositing bacteria", since direct bacterial involvement in oxidation of the
metals has not in all cases been proved.)
Bacteria with unusual morphologies have been seen to be associated with deposits
of manganese and iron oxides in neutral pH environments: sheaths (Leptothrix,
Crenothrix), stars ("Metallogenium"), stalks and buds (Pedomicrobium, Planctomyces),
and gelatinous capsules ("Siderocapsa"). These unusually-shaped bacteria have been
termed "iron bacteria", and tend to be considered the primary agents of microbiological
iron oxide deposition in neutral fresh waters (Ehrlich, 1981; Nealson, 1983a; Ghiorse,
1984; Jones, 1986; Mouchet, 1992). They are commonly thought to be specialized for
metal oxidation. Speculation continues as to whether any are autotrophic or mixotrophic
or actively involved in oxidation of the metals. (Autotrophic oxidation of Fe(II) has been
well-established for Thiobacillus ferrooxidans, but this acidophilic bacterium is usually
considered separately from the neutral-pH iron oxidizers).
The iron bacteria are notoriously difficult to grow in the laboratory; only
Leptothrix, Gallionella, and Pedomicrobium have been studied to any great extent in pure
culture. In situ microscopic examination of these bacteria gives strong evidence for their
involvement in the deposition of iron and manganese oxides (see, for instance, Perfil'ev
and Gabe, 1969; Tyler and Marshall, 1967a,b; Ghiorse and Chapnick, 1983). However,
morphologically undistiguished bacteria (small rods and cocci) may be overlooked under
the microscope, and their contribution to the oxide accumulations underestimated.
A variety of these ordinary-looking manganese-oxidizing bacteria have been
isolated by plating natural samples or enrichments on Mn2+-containing solid media and
examining the brown colonies that appear (see Table I - 2, part B). Few of these strains
15
have been identified to the species level. A list of bacteria that have been found to
deposit manganese oxides is presented in Table I - 2, along with information on whether
they have been isolated in pure culture and whether they are available from a culture
collection.
Notice from Table I - 2 that of the few manganese-oxidizing strains that have been
identified to the species level, only eight of the species (Citrobacter freundii,
Pseudomonas eisenbergii, P. putida, P. alcaligenes, Alcaligenes eutrophus and
Arthrobacter globiformis, A. simplex and A. citreus) are not usually thought of as iron- or
manganese-oxidizing organisms, and only Citrobacter freundii has been investigated in
any real detail as to the biochemical mechanism of oxidation. (Indeed, even the
organism's identification as C. freundii is in some doubt, for the authors gave no details as
to how the identification was made, except to give a misleading reference; Douka and
Vaziourakis, 1981.) Certainly examples of manganese-oxidizers exist within many
common genera, as the list shows, but an unidentified bacterium in a common genus is
still an "unusual organism" of sorts, if its relationship to known bacteria is not
determined. (Note also that Pseudomonas and Bacillus are quite diverse genera.) In
some cases, new species names were
16
TABLE I - 3
Bacteria Reported to Deposit Manganese Oxides
A. Morphologically distinctive bacteria
Sheathed
===============================
Leptothrix discophora
Y
Y
Y
Y
(Johnson and Stokes, 1966)
(Sphaerotilus discophorus)
Leptothrix pseudoochraceae
Y
Y
Y
?
(Dubinina, 1978b)
Leptothrix cholodnii LVMW 99
Y
Y
Y
?
(Mulder, 1989)
Leptothrix lopholea LVMW 124 Y
Y
Y
?
(Mulder, 1989)
Leptothrix ochracea
?
Y
N
N
(Mulder, 1989)
Crenothrix polyspora
?
?
N
N
(Hirsch, 1989 b)
"Clonothrix fusca"
?
?
N
N
(Hirsch, 1989 a)
Stalked
Hyphomicrobium
Y
Pedomicrobium manganicum
Y
Blastobacter-Planctomyces group Y
?
N
N
Y
Y
Y
N
Y
?
Y
?
?
N
N
N
N
N
N
?
Y
Y
?
?
?
?
Y
Y
Y
Y
Y
Y
Y
?
?
?
N
N
Y
?
?
N
N
N
(Tyler and Marshall, 1967a)
(Aristovskaia, 1961)
(Schmidt et al., 1982)
Stellate
"Metallogenium symbioticum"
Caulococcus manganifer ?
Kusnezovia polymorpha ?
(Zavarzin, 1989)
"
(Schmidt and Zavarzin, 1981)
Capsulated
"Arthrobacter siderocapsulatus"
"Siderocapsa"
Naumanniella
Ferribacterium
Siderocystis
Siderococcus
KEY: Y = yes, N = no, ? = not known
(Dubinina and Zhdanov, 1975)
(Hanert, 1981)
"
"
"
"
17
TABLE I - 3, continued
Bacteria Reported to Deposit Manganese Oxides
B. Morphologically undistinguished bacteria
Soil and Freshwater Isolates:
===============================
Citrobacter freundii E1
Y*
?
Y
N
(Douka, 1977)
Pseudomonas sp. E4
Y*
?
Y
N
(Douka, 1977)
Pseudomonas eisenbergii Y
?
Y
N
(Zavarzin, 1962)
"Pseudomonas manganoxidans" Y
Y
Y
Y
(Schweisfurth, 1973a)
"strain FM1"
Y
?
Y
N
(Zapkin and Ehrlich, 1983)
Alcaligenes eutrophus 280
Y
Y
Y
N
(Abdrashitova et al., 1990).
Pseudomonas putida 18
Y
Y
Y
N
(Abdrashitova et al., 1990)
Pseudomonas putida
Y
?
Y
N
(Jung and Schweisfurth, 1976)
Pseudomonas alcaligenes Y
?
Y
N
(Jung and Schweisfurth, 1976)
Corynebacterium/Arthrobacter sp Y
?
Y
N
(Bromfield and Skerman, 1950)
Streptomyces sp.
Y
?
Y
N
(Bromfield, 1978)
Nocardia sp.
Y
?
Y
N
(Schweisfurth, 1968)
Arthrobacter simplex BKM 667
Y
Y
Y
Y
(Dubinina and Zhdanov, 1975)
Arthrobacter citreus BKM 654
Y
Y
Y
Y
"
A. globiformis BKM 661 Y
Y
Y
Y
"
Bacillus sp.
Y*
?
Y
N
(Gregory and Staley, 1982)
Caulobacter spp. (4 strains)
Y*
?
Y
N
"
Chromobacterium spp. (2 strains) Y*
?
Y
N
"
Cytophaga sp.
Y*
?
Y
N
"
Pseudomonas spp. (2 strains)
Y*
?
Y
N
"
Marine isolates:
Oceanospirillum BIII45
"strain S13"
"strain SSW22"
"strain BIII82"
Pseudomonas sp. S-36
Arthrobacter sp. 37
Bacillus SG-1
Pseudomonas (many strains)
Y
Y
Y
Y
Y
Y
Y
Y
?
?
?
?
?
?
?
?
Aeromonas (many strains) Y
Flavobacterium (many strains)
Cytophaga spp.
?
Y*
Y*
Y
?
?
Y
Y
Y
Y
Y
Y
Y
Y
N
(Ehrlich and Salerno, 1990)
N
(Ehrlich, 1983)
N
"
N
"
N
(Kepkay and Nealson, 1987)
N
(Ehrlich, 1975)
N
(Rosson and Nealson, 1982a)
N
(Schutt and Ottow, 1978;
Nealson, 1978)
N
"
Y
N
(Nealson, 1978)
Y
N
"
* = Isolated on unbuffered high-peptone, high-Mn2+ medium subject to false positives.
KEY: Y = yes, N = no, ? = not reported
18
created ("Arthrobacter siderocapsulatus", "Pseudomonas manganoxidans"), implying
that those strains are distinct from others of the genus, and that metal oxidation is the
species' primary function.
1.6. Abundance of Manganese-Oxidizing Bacteria
Several censuses have shown manganese-oxidizing bacteria to be fairly abundant
in marine, freshwater, and soil environments. However, "manganese-oxidizing bacteria"
are defined by the methods one uses to detect them.
Some investigators grew colonies on fairly rich, high-peptone, unbuffered media
high in manganese (200 mg/l), then stained the plates with benzidine (a leuco dye which
turns blue upon oxidation by MnOx) and counted blue colonies. Others used low-nutrient
plates with lower manganese concentrations, and scored brown colonies (those visibly
precipitating manganese oxides). Sohngen (1914) and Brantner (1970) have pointed out
that the former approach may lead to false positive results, from bacteria which simply
alkalinize the medium due to deamination of the peptone, leading to chemical oxidation
of the Mn2+ surrounding the colonies. The same result can occur with media containing
high levels of citrate or other carboxylic acids (Van Veen et al., 1978). In my work, I
found that formation of visibly brown, MnOx-containing colonies on a low-nutrient, lowmanganese medium was most likely to reflect directly-bacterially-mediated manganese
oxidation (see Chapter 3).
Gottfreund and Schweisfurth (1983) used four different media in their
investigation of abundance of manganese-oxidizing bacteria in soils of varying
manganese concentration. Their results were not very uniform, but it was apparent that
19
different media gave quite different results. One high-nutrient and one low-nutrient
medium led to results that manganese-oxidizers made up 0.1% to nearly 100% of total
viable heterotrophs, whereas another low-nutrient medium suggested 0.01% to 10%, and
a fourth medium of moderate nutrient level revealed, at best, 0.01% as manganese-oxidizers. They also concluded that manganese-oxidizing bacteria were not enriched in soils
with high total manganese levels.
Schütt and Ottow (1978) examined bacteria isolated from deep-sea manganese
nodules, among other habitats. Rough proportions of manganeseoxidizing bacteria were reported as follows:
Manganese nodules: .................................... 0.1% to 50%
Marine sediments: ....................................... 0.001% to 10%
Sea water: ..................................................... 1% to 10%
Garden soil: ................................................. 0.1%
They used a low-nutrient medium and counted brown colonies as well as benzidinereacting colonies. Sixty-seven nodule strains were characterized by a small array of
diagnostic tests, but positive identifications could not made beyond a very general genus
level. Most (94%) could be classified as Pseudomonas, while the others were classified
as Aeromonas. None of their Pseudomonas strains produced a fluorescent pigment;
however, fluorescent pseudomonads would not be expected to be found in marine
habitats.
Gregory and Staley (1982), sampling in Lake Washington (Washington state) and
in Lake Virginia (Florida), found that benzidine-reacting colonies appeared at a low of a
few tenths of a percent to a high of about seventy percent of total viable heterotrophs,
20
varying with depth and with season. However, they used a high-peptone, unbuffered
medium, and so the results must be interpreted with some caution. Benzidine-reacting
colonies identified to the genus level included Bacillus, Caulobacter, Cytophaga, and
Pseudomonas strains, but from their data, it appears that only one of their isolates, a
Hyphomicrobium, actually forms brown colonies.
Maki et al. (1987), also at Lake Washington, found that manganese-oxidizing
colony-forming units (CFU) occured at about 1/3 to 1/10 the abundance of total CFU,
sampling at depths of 40 to 60 meters. They used a weakly-buffered medium of moderate
peptone content, and scored benzidine-reactive colonies as manganese oxidizers.
1.7. Bacterial Manganese Oxidation: Mechanisms and Functions
The questions of how and why these bacteria oxidize manganese have never been
answered satisfactorily. It appears that different species use different mechanisms, and
probably have different reasons for oxidizing manganese. Excellent reviews on potential
mechanisms and purposes for bacterial manganese oxidation have been published by
Ghiorse (1984) and Nealson et al. (1989).
One mechanism of "biological" manganese oxidation that is not likely to be
important is the simple raising of local pH by bacteria growing on media with high
peptone or carboxylic acid levels, as discussed above, leading to chemical oxidation of
Mn2+ when present in high levels. A brown plate, rather than a brown colony, is usually
the result. This manner of manganese oxidation is likely to be only a laboratory
phenomenon, because nutrient levels in natural environments are too low for
alkalinization to take place to any great extent.
21
A small but growing amount of work has been done to investigate biochemical
mechanisms of manganese oxidation. Cell-free oxidation has been studied in a few
different bacteria. In Leptothrix discophora, an extracellular protein has been purified
which oxidizes Mn2+ to MnOx, utilizes oxygen, and is produced in exponential phase
(Adams and Ghiorse, 1987; Boogerd and de Vrind, 1987). Bacillus sp. SG-1 produces a
spore coat protein which oxidizes Mn2+ in the presence of oxygen (Rosson and Nealson,
1982). The unidentified freshwater strain FMn-1 produces a Mn2+-inducible, proteasesensitive factor in stationary phase, which may be loosely associated with a membrane,
and which requires a small-molecular-weight heat-stable cofactor for oxidation of Mn2+
(Zindulis and Ehrlich, 1983). An intracellular protein from the marine Arthrobacter
strain 37 requires O2 and MnO2 for Mn2+-oxidation to take place (Ehrlich, 1968).
Intracellular manganese-oxidizing proteins in a Pseudomonas sp. and in a strain identified
as Citrobacter freundii were reported by Douka (1977, 1980; Douka and Vaziourakis,
1982) to be constitutively produced, independent of the presence of Mn2+. Jung and
Schweisfurth (1979) reported cell-free manganese oxidation by a protein of "Pseudomonas manganoxidans" MnB-1, which they claimed was expressed only in stationary
phase, was consistently produced, was non-catalytic (hence not a true enzyme), and which
did not require O2 in its reaction.
Since the time of Winogradsky, microbiologists have speculated that manganese,
like iron, could serve as a source of energy for autotrophic bacteria, generating ATP from
the oxidation of Mn(II) to Mn(III) or Mn(IV) via an electron-transport system, and fixing
carbon from CO2 via the Calvin cycle. Or, an organism might be mixotrophic rather than
22
autotrophic, generating ATP from the inorganic substance but requiring reduced organic
compounds to satisfy its demand for carbon. The fact that all manganese-oxidizers so far
isolated are capable of growth in the absence of Mn2+ (unlike, for example, the obligate
demand for reduced nitrogen by denitrifying bacteria) has made the issue problematical,
for manganese mixotrophy is difficult to prove conclusively.
A few reports of energy utilization from manganese oxidation are found in the
literature:
1) Ali and Stokes (1971) claimed autotrophic growth of Sphaerotilus discophorus
(Leptothrix discophora) on Mn2+ in a carbon-free salts medium, but this observation
could not be repeated by others studying this species (Hajj and Makemson, 1976; Adams
and Ghiorse, 1985).
2) Hyphomicrobium manganoxidans (Eleftheriadis, 1976; Schweisfurth et al.,
1978) was claimed to be a chemoautotrophic obligate manganese-oxidizer, growing only
while Mn2+ is oxidized, and incorporating 14C from radiolabeled bicarbonate. However,
this report has not been published in a refereed journal.
3) Growth of Pseudomonas strain S-36 (Kepkay and Nealson, 1987) was shown
to be stimulated by Mn2+ in continuous culture, and cell yield was proportional to the
amount of Mn2+ oxidized.
4) Ehrlich and colleagues, in a number of publications (summarized in Ehrlich,
1981 and 1990) and in a number of different unidentified, heterotrophic marine bacteria,
have shown reduction of cytochromes in the presence of Mn2+. This suggests that at least
mixotrophic energy generation is possible in these strains. Stimulation of growth by
Mn2+, however, has not been reported for these strains.
23
There are other manganese-oxidizing bacteria which show no evidence of using
Mn2+ as an energy source. Manganese oxidation has been suggested to function as a
defense against Mn2+ toxicity (Nealson et al., 1988), an aid to survival in stationary-phase
cultures (Adams and Ghiorse, 1985), as a defense against hydrogen peroxide toxicity (by
oxidizing Mn2+ with H2O2 via the peroxidase function of catalase; Dubinina, 1978a,b;
Bromfield, 1956). Cells coated with manganese oxides tend to adhere more strongly to
surfaces, so that manganese-oxidizing cells might benefit from the "biofilm effect",
concentrating nutrients from a nutrient-poor liquid flowing past (Jung and Schweisfurth,
1976). Or, cells coated with manganese oxides may be less of a target for grazing by
protozoa. It is also conceivable that, for some species, manganese oxidation may provide
no benefit to the cell at all, but is merely an adventitious phenomenon, a by-product of
some other function.
CHAPTER TWO
TAXONOMY OF SOME MANGANESE-OXIDIZING Pseudomonas SPECIES
Chapter Two
TAXONOMY OF SOME MANGANESE-OXIDIZING Pseudomonas SPECIES
Bacterial identification is difficult because the distinguishing characteristics of
bacteria can only be determined by time-consuming biochemical, nutritional, and
molecular tests. Bacterial classification is tenuous, in comparison to that of most higher
organisms, because the concept of a bacterial species is to a large extent subjective. It is
no wonder that few microbiologists spend great effort on extensive characterization and
classification of their isolates. This is unfortunate, because identifying a newly-isolated
bacterium properly is tremendously useful. If the strain can be classified with an existing
species, one can infer that it shares many properties with other members of that species,
and vice versa. If the isolate is proposed to define a new species, then that implies that it
is different in fundamental ways from members of other species.
"Pseudomonas manganoxidans" is the name given to a supposed species of
bacteria which was distinguished by its ability to oxidize reduced manganese. "Arthrobacter siderocapsulatus" is the species defined to include two manganese-oxidizing
isolates which were claimed to be members of the previously uncultured Siderocapsa
group. In this chapter, I show that "Pseudomonas manganoxidans" and "Arthrobacter
siderocapsulatus" strains are more properly classified as belonging to the common soil
and water species, Pseudomonas putida. In addition, I show that many of my own manganese-oxidizing isolates, obtained from various environments, are P. putida.
25
26
2.1 "Pseudomonas manganoxidans"
Schweisfurth (1973a) examined numerous soil, aquatic and industrial sites
containing manganese oxide deposits, and from them isolated about 200 strains of rodshaped bacteria that formed brown colonies on low-nutrient Mn2+-containing agar. He
characterized thirty strains that retained their manganese-oxidizing phenotype after
repeated laboratory subculture. Twenty-three were pseudomonads that secreted a yellowgreen fluorescent pigment, and so Schweisfurth chose to use the classification scheme of
Jessen (1965) for identifying fluorescent pseudomonads. Jessen's monograph was one of
a number of reexaminations of the taxonomy of Pseudomonas that was published in the
1960's. His scheme divided fluorescent pseudomonads into six "Groups" and 82
"Biotypes", rather than into distinct species. (P. aeruginosa was the only grouping among
the fluorescent pseudomonads that Jessen accepted as being homogeneous enough to
deserve a true species rank.)
Within Jessen's scheme, Schweisfurth classified his strains as detailed in Table II 1.
27
Table II - 1:
"Pseudomonas manganoxidans" strains as classified under the scheme of Jessen.
Genus
Group
Biotype
Strain (MnB number)
Pseudomonas I
?
6
Pseudomonas II
11
5, 9/2, 16/1, 41
Pseudomonas II
13
1, 3
Pseudomonas II
?1
8/1, 11, 12/1, 13/2, 14
Pseudomonas II
?2
8/2, 9/1, 13/1, 16/2, 17, 18, 23, 33, 49
Pseudomonas III
46
15
Pseudomonas III
?
48
?
none
none2
10/1, 21/1, 29, 31, 32, 38, 36
bold = strains deposited in the American Type Culture Collection (ATCC)
1 = identical strains
2 = non-identical strains
(from Schweisfurth, 1973a)
28
Schweisfurth, it seems, did not share Jessen's reluctance to assign species names
to these clusters: he named strain MnB-1 Pseudomonas manganoxidans, and referred to
those Pseudomonas strains in other biotypes as the P. manganoxidans-group. He did,
however, consider his classification to be temporary. Six of the strains were deposited
with the American Type Culture Collection in 1967, designated simply as Pseudomonas
spp.
The name "P. manganoxidans" was apparently not accepted as a valid species by
the International Committee on Systematic Bacteriology and was not included on the
Approved Lists of Bacterial Names (Skerman et al., 1980). (Hence the species name is
properly enclosed in quotation marks.)
Schweisfurth himself referred to these strains as "P. manganoxidans" in his
publications of 1973, 1976, and 1978, but as Pseudomonas sp. in 1979 (Jung and
Schweisfurth, 1979). Since then, these strains have been referred to in reviews of the
literature as "P. manganoxidans" or Pseudomonas sp., joining a long list of other poorlyidentified and seemingly unrelated manganese-oxidizing bacteria.
It must be noted that "P. manganoxidans" strain MnB-8/1 (ATCC #23486) was
identified as P. putida biovar A by McManus, et al. (1992), in a study comparing 72
features among 432 miscellaneous fluorescent pseudomonads. The ATCC catalogue
(American Type Culture Collection, 1992) now lists that strain under P. putida. I have
found only one other example in the literature of new work utilizing any of these
organisms (Black, 1991), and no published reports of new isolates of "P. manganoxidans". Results of my classification tests for the five available "P. manganoxidans" strains
29
are examined below.
2.1.1 Materials and Methods
Identification tests were performed by the methods of Stanier et al., 1966, and
Stolp and Gadkari, 1981, with modifications as detailed below.
The test for gelatinase was modified by substituting 30% trichloroacetic acid for 15%
acidic HgCl2 as a protein-precipitating agent (Pitt and Dey, 1970), and testing after three
days of growth rather than two. The oxidase reagent used was N,N,N',N'-tetramethyl-pphenylenediamine in amyl alcohol (Analytab Products, Plainview, N. Y.). Lecithinase
was detected on the egg-yolk medium of Esselmann and Liu (1961). Bacto-Gram stains
and SpotTest-Flagella stains were manufactured by Difco Laboratories, Detroit,
Michigan. Cultures were incubated at 26 C. (30 for denitrification). Positive and
negative controls were included for all tests.
The API Rapid NFT test system (Analytab Products, Plainview, N. Y.) and the
BIOLOG test system were used according to manufacturers' directions. (More
information on these systems is included in Appendix One.)
Gelatin hydrolysis and denitrification, two very important tests in distinguishing
P. putida from P. fluorescens, were done by two different methods each: by the methods
as recommended by Stanier et al., and as part of the API Rapid NFT tests. The only
discrepancy observed was the gelatinase reaction for P. fluorescens ATCC 13525, which
was gelatinase-positive by the method of Stanier et al., but gelatinase-negative according
to the API strip.
30
2.1.2 Results
A pseudomonad is defined as a Gram-negative rod, motile by one or more polar
flagella, aerobic and non-fermentative, and lacking any other special properties (such as
nitrogen fixation) which would lead to another classification (Palleroni, 1992a). An
important group of Pseudomonas species secrete a yellow-green fluorescent pigment on
certain media. Pseudomonas putida, a fluorescent pseudomonad, is distinguished from
other fluorescent pseudomonads by characteristics detailed in Table II - 2. In addition, P.
putida biovars grow or do not grow on certain carbon sources that distinguish this species
from others (Palleroni, 1984).
I chose an array of tests designed especially to distinguish P. putida from P.
fluorescens (the species that appears to be most closely related to P. putida; Stolp and
Gadkari, 1981; Palleroni, 1992a,b; Champion et al., 1980; Barrett et al., 1986). The API
and BIOLOG commercial test systems were used to obtain carbon-source-utilization data,
supplementing standard microbiological tests. I examined characteristics of "P. manganoxidans" strains MnB-1, 5, 6, 8/1, 15, and the unclassified strain MnB-29. The results are
given in Table II - 3 below. Detailed results from the API and BIOLOG tests are given in
Appendix One. These tests do not exactly reproduce the conventional methods of
characterization, and thus are not strictly comparable to data in, for example, Bergey's
Manual, but the information is useful, and the identifications that the systems provide are
consistent with their own data bases.
31
32
33
It can be seen from Table II - 3 that all five "Pseudomonas manganoxidans"
strains clearly match the description of P. putida. Strain MnB1-A2, a variant of strain
MnB-1 which I isolated as a more rapidly-oxiding colony, exhibited characteristics
virtually identical to the parent strain.
Strain MnB-29, not classified by Schweisfurth, was not only not fluorescent, but
non-motile, which suggests this bacterium is not a pseudomonad. The API system
identified this strain only as Pseudomonas species; BIOLOG returned no definite
identification. No further identification tests were performed on it. Schweisfurth (1973a)
reported the %G+C value for this strain to be 62.1%; this value is consistent with a
Pseudomonas identification, leaving open the possibility that the strain is simply a
Pseudomonas with a mutation that makes it non-motile.
Schweisfurth (1973a) determined DNA base compositions for strains MnB-1 and
MnB-6 to be 61.7 to 62.1 mol% G+C, respectively, comparing well with reported values
for P. putida biovar A (62.5%) and P. putida biovar B (60.7%) (Mandel, 1966). Values
for the unidentified strains MnB-29, 32, and 38 were also between 61 and 64 mol% G+C.
Only five of Schweisfurth's twenty-three original "P. manganoxidans" strains
were available for testing. However, it can be seen from Tables II - 1 and II - 3 that the
result for MnB-5 implies that identical strains MnB-9/2, -16/1, and -41 are P. putida; for
MnB-1, that MnB-3 is P. putida, and for MnB-8/1, that MnB-11, MnB-12/1, -13/2, and 14 are P. putida.
The identities of those "P. manganoxidans" strains in unassigned Biotypes of
Groups II and III, not represented among the ATCC strains, can be surmised by
34
comparing the scheme of Jessen (1965) with the conventional scheme for Pseudomonas
classification.
Jessen published his rather obscure monograph at a time in which Pseudomonas
taxonomy was being extensively reexamined and revised. His Group/Biotype scheme
and his reluctance to define species among the fluorescent pseudomonads found little
acceptance among taxonomists. The currently-accepted classification of pseudomonads
(Stanier et al., 1966; Palleroni, 1984, 1992b) now recognizes six species of saprophytic
fluorescent pseudomonads: P. aeruginosa, P. fluorescens, P. putida, P. chlororaphis,
and P. lundensis.
Jessen used many of the same characterization tests that were used by Stanier et
al. (1966) and subsequent workers. By comparing Jessen's results with a minimal,
presumptive definition of P. putida, (namely, a pseudomonad that is positive for
fluorescent pigment production and the oxidase test, but which tests negative for
gelatinase, nitrate reduction, lecithinase, levan formation, and lipase; Palleroni, 1984), the
following Jessen Biotypes are presumed to be P. putida: 5, 6, 11, 12, 13, 14, 16, 28, 30,
33, 34, 43, 44, 45, 46, and 77.
In addition, two hundred of Jessen's strains (recognized by their "PJ" strain
designations) have been reexamined and reclassified in three more-recent studies (Barrett,
et al., 1986; Champion, et al., 1980; McManus, et al., 1992). I have correlated Jessen's
biotypes with species assignments from these three papers in Appendix Two, and I have
summarized Jessen Group correlations with current taxonomy in Table II - 4.
35
TABLE II - 4:
Comparison of Jessen's Groups with Presently-Accepted Species
Jessen Group Jessen Biotype
Pseudomonas species
I
1
P. aeruginosa
II
10 - 29
P. putida biovars A, B, and C,
P. lundensis, and
P. fluorescens bv. VI
III
43 - 47
P. putida bv. A and C, and
P. lundensis.
IV
48 - 58
P. fluorescens bv. III
and unassigned strains
V
61 - 64
P. fluorescens bv. I and II
VI
66 - 73
Phytopathogens, e.g. P. syringae
References: Stanier et al., 1966; Barrett et al., 1986; Champion et al., 1980; McManus et
al., 1992.
P. fluorescens bv. VI of McManus et al. = P. fluorescens bv. V-1 and V-2 of Barrett et al.
(See Appendix Two for details.)
36
The results of this comparison support the results of the classical
biochemical/nutritional characterizations reported above, that "Pseudomonas manganoxidans" is equivalent to Pseudomonas putida. Seven of Schweisfurth's strains fall in P.
putida-equivalent Biotypes 11, 13, and 46. Also, Groups II and III are seen to correspond
broadly to the three P. putida biovars, to P. lundensis, and to P. fluorescens biovar VI;
hence the some of the ten II/? and III/? MnB strains (now presumed lost) listed in Table
II-1 could have been members of P. putida, or were at least closely related.
In all, at least 14 of Schweisfurth's 23 "P. manganoxidans" strains can be
considered P. putida. The other nine are not readily classifiable, but may have been P.
putida, P. fluorescens bv. VI, or P. lundensis. As for the strains that were reported by
Schweisfurth not to be Pseudomonas, no conclusions can be drawn.
2.1.3 Discussion
It is interesting to note not only what Schweisfurth found, but what he did not
find. None of his thirty strains fell in Jessen Groups IV, V, or VI, which cover the bulk of
P. fluorescens biovars I, II, and IV, and the plant-pathogenic pseudomonads. It also
appears that none of his Mn-oxidizing strains were P. aeruginosa. This is true even
though P. aeruginosa and especially P. fluorescens are known to populate soil and water
environments such as the ones Schweisfurth sampled. However, it is also true that
Schweisfurth found other manganese-oxidizing Pseudomonas (sensu strictu) species,
such as P. alcaligenes (Jung and Schweisfurth, 1976). I have also isolated manganese
oxidizers that are not P. putida but appear to be related pseudomonads (see section 2.3
37
below).
One may wonder why Schweisfurth did not recognize that "P. manganoxidans" is
essentially P. putida. Certainly one reason for doing what he did was simply the
common, understandable, very human bias of a microbiologist, in thinking that whatever
property one is studying in an organism is that organism's raison d'etre, its most
important characteristic. It also reflects the misconception that manganese oxidation is an
unusual property, carried out by unusual organisms. However, in Schweisfurth's defense,
it must be pointed out that Pseudomonas taxonomy was truly confused and the confusion
was only beginning to be cleared up around the time he was characterizing and classifying
his strains.
2.1.3.1 History of Pseudomonas Taxonomy
Pseudomonas taxonomy has been complex and chaotic for much of this century.
Since the genus was defined by Migula in 1894, an enormous number of strains and
species have been proposed for membership.
Part of the problem was that the definition of Pseudomonas, until recently, was
not very restrictive: Bacteria fitting this description are ubiquitous in soil, water, plants
(as pathogens and non-pathogens), and in animals as opportunistic pathogens and wound
colonizers.
Another aspect of the problem was that new species were being described
inadequately, using too few biochemical and nutritional characters. This, in turn, made it
difficult for others to relate new isolates to existing ones, leading to new species being
named. If the new species were defined inadequately as well, the problem was
38
compounded. The genus Pseudomonas became a dumping ground for bacteria whose
relatedness to each other, and differences from each other, was often suspect.
By the time that Schweisfurth began isolating and characterizing his rod-shaped
manganese-oxidizers in the 1960's, the contemporary (seventh) edition of Bergey's
Manual of Determinative Bacteriology (Haynes and Burkholder, 1957)
the standard reference for bacterial identification, listed 149 species of Pseudomonas.
Ninety of the species secreted a soluble yellow-green fluorescent pigment, and twentynine of those were found predominantly in soil and water habitats, rather than on diseased
plants. According to this manual, Schweisfurth's fluorescent, gelatinase-negative
pseudomonads could have been classified as P. putida, P. eisenbergii, P. convexa, P.
incognita, P. ovalis, P. rugosa, P. striata, P. arvilla, or P. mildenbergii. It is
understandable that Schweisfurth would have been willing to add one more name to this
long list.
Major attempts to put Pseudomonas taxonomy on a firm foundation were
undertaken by Rhodes (1959), by Jessen (1965), and by Stanier et al. (1966). Rhodes
looked at 134 strains of fluorescent pseudomonads with 69 tests. In her scheme, the
strains clustered so poorly that she concluded there were no valid species within the
fluorescent pseudomonads, and that all should be considered members of a broadlydefined P. fluorescens. Jessen surveyed 859 strains of fluorescent pseudomonads. After
performing 69 biochemical and nutritional tests on them, he concluded that, other than P.
aeruginosa, these bacteria were too heterogeneous for him to confidently establish
species boundaries. He divided his strains into 82 biotypes. Forty-eight of those biotypes,
39
containing the majority of the strains, were clustered into six groups (see Table II - 4).
In 1966, Stanier et al. examined 267 strains in great depth, exploiting the
nutritional versatility of pseudomonads by testing for the ability to grow on 146 different
carbon sources. Twenty-six other biochemical and morphological tests were also
included. These authors concluded that it was indeed possible to reduce twenty-nine
poorly-defined species of saprophytic fluorescent pseudomonads down to three (P.
aeruginosa, seven biovars of P. fluorescens, and two biovars of P. putida) with a
practical number of well-chosen tests.
It was this classification that, with modifications, became the generally-accepted
one, supported by evolutionary data from DNA, ribosomal RNA, and protein sequences.
Modifications to the scheme continue to be made (Palleroni, 1984; Champion et al.,
1980; Barrett et al., 1986; Palleroni, 1992a). Ribosomal RNA sequence analysis has
recently revealed that many species and groups of species within the genus Pseudomonas
are not truly related (Woese et al., 1985). New genera are now being defined for some of
the species. The "true" Pseudomonas species are the fluorescent pseudomonads (see
Table II - 2) and related non-fluorescent species of rRNA group I such as P. stutzeri, P.
fragi, and P. alcaligenes. Ribosomal RNA group II (e.g., "P. solanacearum") is now
called Burkholderia; rRNA group III (e.g., "P. acidovorans") is now Comamonas; some
members of group IV (e.g., "P. paucimobilis") are now called Sphingomonas; and "P.
maltophilia" of group V is now a member of Xanthomonas (Palleroni, 1992). The genus
Pseudomonas is no longer the catch-all taxon it once was.
Although the landmark article by Stanier et al. was published in 1966, it was not
40
until 1974, with the publication of the next (eighth) edition of Bergey's Manual of
Determinative Bacteriology, that the Stanier classification scheme was given an "official"
imprimatur. Schweisfurth's classification of "P. manganoxidans" strains, of course, was
published a year earlier, in 1973, and the strains must have been characterized before
November 1967 (the date on the ATCC "P. manganoxidans" [Pseudomonas sp.] vials I
purchased). Interestingly, in their 1976 paper, Jung and Schweisfurth mention, almost in
passing, that they had isolated a new Mn-oxidizing pseudomonad that they identified,
using the eighth edition of Bergey's Manual, as P. putida. For some reason they did not
equate this P. putida strain with "P. manganoxidans".
41
2.2 "Arthrobacter siderocapsulatus".
In 1975, Dubinina and Zhdanov announced the isolation of the iron- and
manganese-oxide-depositing bacterium "Siderocapsa eusphaera". "Siderocapsa" species
had been described since the turn of the century, but had never been isolated in pure
culture. "Siderocapsa" and similar organisms, commonly found in fresh water
environments, are distinguished by characteristic iron- and/or manganese-oxide deposits
surrounding capsules enclosing one or more rod- or coccus-shaped bacteria (Hanert,
1981). Dubinina and Zhdanov claimed that their two isolates were members of the
Gram-positive genus Arthrobacter, and they placed the strains in a new species,
"Arthrobacter siderocapsulatus". The claim that "A. siderocapsulatus" is a true example
of "Siderocapsa", however, has not been accepted by all authorities (Hirsch et al., 1989;
Ghiorse, 1984).
The classification of the strains as arthrobacters was tenuous as well, dependent
solely on the rod-to-coccus morphological changes shared by species of Arthrobacter.
Bergey's Manual of Systematic Bacteriology (Keddie et al., 1986) considered "A. siderocapsulatus" a species incertae sedis (species of uncertain standing), due to lack of
chemotaxonomic data necessary for inclusion in the species Arthrobacter. Collins (1986)
and Amadi and Alderson (1992) collected some of that chemotaxonomic data, examining
the fatty acids, polar lipids, and isoprenoid quinones of "A. siderocapsulatus" membranes.
All concluded that "A. siderocapsulatus" was not a member of the genus Arthrobacter;
nor, indeed, was it even a Gram-positive organism. They did not speculate further on its
42
proper classification.
Reexamination of the original description of "A. siderocapsulatus" (Zhdanov and
Dubinina, 1975; Dubinina and Zhdanov, 1975), combined with the above-cited lipid data,
strongly suggests that the two described strains of "A. siderocapsulatus" are actually
fluorescent pseudomonads. These two strains were assigned to the genus Arthrobacter
solely on the basis of the observation that in young cultures, cells appeared as short rods
or long filaments, while in older cultures, a mixture of short rods and cocci were present.
Morphological variation is a classical characteristic of Arthrobacter. However, this rodcoccus morphological change is not limited to Arthrobacter (Keddie et al., 1986); other
bacteria, including Pseudomonas, may exhibit a similar transition (Palleroni, 1984;
Kjelleberg and Hermansson, 1987; personal observations).
The original description of "A. siderocapsulatus" also states that the cells are
Gram-negative (a true Arthrobacter has a Gram-positive cell wall, but often does not
retain the Gram stain), obligately aerobic, and motile by multiple polar flagella. This
description fits the general definition of Pseudomonas. Furthermore, Zhdanov and
Dubinina report that their strains secrete a soluble "intense yellow-green pigment" in
meat-peptone broth and agar, just as fluorescent pseudomonads do. This pigment was not
examined by them under UV light for fluorescence.
Collins (1986) and Amadi and Alderton (1992) showed that the dominant
isoprenoid quinone in "A. siderocapsulatus" membranes is a nine-unit ubiquinone (Q-9).
A Q-9 dominant quinone is a rather unusual characteristic in Gram-negative bacteria; of
the five genera reported by Collins and Jones (1981) to have dominant Q-9 quinones,
43
only Pseudomonas rRNA group I (the true Pseudomonas group, which includes the
fluorescent pseudomonads) share with "A. siderocapsulatus" the fundamental
characteristics of having polar flagella and a DNA base composition in the region of 60.8
mol% G+C. Nutritional and biochemical characteristics reported by Zhdanov and
Dubinina match quite well with those
expected for a fluorescent pseudomonad, particularly P. putida.
2.2.1 Materials and Methods
For the above reasons, I decided to reexamine "Arthrobacter siderocapsulatus"
strains A and G using the classification methods described above for "Pseudomonas manganoxidans". Strains A and G were obtained from the National Collections of Industrial
and Marine Bacteria, Aberdeen, Scotland, as strains NCIMB 11286 and NCIMB 11287,
respectively. For comparison, I used Arthrobacter globiformis DK, a variant I isolated
from A. globiformis ATCC #8010, the type strain. Strain DK was selected for its ability
to form brown MnOx-containing colonies on media with Mn2+. Results of the tests are
shown in Table II - 5.
44
2.2.2 Results and Discussion
Table II - 5 shows that both strains of "A. siderocapsulatus" match the expected
results of Pseudomonas putida for these tests, and that the API and BIOLOG systems
identify both strains as P. putida to high probability (see Appendix Two). Weak lipase
activity was observed in both strains only after the six days of observation recommended
by Stanier et al. (1966). These results were labeled "-/+". Stanier et al. and Palleroni
(1984) note that a number of P. putida strains exhibit lipase activity; indeed, in my hands,
ATCC P. putida strains 12633 (the type strain) and 17484 showed this weak and delayed
lipase activity. It is reasonable to conclude that "A. siderocapsulatus" strains A and G are
actually Pseudomonas putida. (These two strains are the only members of "A. siderocapsulatus" reported in the literature, to the best of my knowledge.)
Zhdanov and Dubinina (1975) reported that strains A and G hydrolyzed starch.
Starch hydrolysis is never found in fluorescent pseudomonads (Palleroni, 1984). I
retested the strains for starch hydrolysis by the method given in Stanier et al. (1966) and
saw no evidence of starch hydrolysis in either strain A or strain G, neither after the
recommended two days of incubation nor after one week of incubation. Zhdanov and
Dubinina did not report the method they used for their test.
Other reported characteristics of the "A. siderocapsulatus" strains are consistent
with a identification as P. putida. The fatty acid profiles reported by Collins and by
Amadi and Alderson are consistent with those reported by others for P. putida
(Wilkinson, 1988; Stead, 1992). The oval rods reported in "A. sidero
45
46
capsulatus" cultures by Dubinina and Zhdanov are reminiscent of the oval rods peculiar
to some strains of P. putida, (a synonym of which is P. ovalis; Palleroni, 1984). Like P.
putida/"P. manganoxidans", "A. siderocapsulatus" was shown to oxidize manganese only
in stationary phase (Dubinina and Zhdanov, 1975). Also, like "A. siderocapsulatus",
"Pseudomonas manganoxidans" was reported to deposit iron oxides when growing on a
medium containing high levels of Fe(II) citrate (Kullmann and Schweisfurth, 1978).
Furthermore, P. putida strain was shown to deposit oxides and hydrous oxides of Fe(III)
when grown in a medium containing Fe(III) citrate or FeCl3 (Verhovtseva et al., 1992).
The reclassification of "Arthrobacter siderocapsulatus" as Pseudomonas putida
immediately raises the question of whether "Siderocapsa eusphaera" and/or other species
of "Siderocapsa" are P. putida rather than an Arthrobacter. Granted, the arguments
Dubinina and Zhdanov used in claiming that "A. siderocapsulatus" is "Siderocapsa" are
debatable. Dubinina and Zhdanov include photomicrographs of putative "Siderocapsa"like forms made by pure cultures of "A. siderocapsulatus", but the photos are difficult to
interpret clearly. The "A. siderocapsulatus" strains were isolated based on their
manganese-oxidation phenotype from two lakes in which "Siderocapsa" forms were seen,
but P. putida would be expected to be present as well in a typical lake (as it certainly was
in this case). I show in Chapter Three of this thesis that manganese-oxidation appears to
be a common property of P. putida, and so strain A and strain G could simply have been
"contaminants" of the "Siderocapsa" samples. However, the micrographs that Dubinina
and Zhdanov present are evidence that these two strains may possibly be true
"Siderocapsa", and so future experiments should be done with that hypothesis in mind.
47
Known P. putida strains could be incubated in filtered lake water from which
"Siderocapsa" has been found, and examined for the appearence of the large iron- or
manganese-oxide-impregnated capsules that are characteristic of "Siderocapsa". More
importantly, natural samples of "Siderocapsa" could be stained with a labeled antibody or
ribosomal RNA probe specific for detection of P. putida. "Siderocapsa" has escaped
(unambiguous) pure culture for nearly a century probably not because the organisms will
not grow on laboratory media, but more likely because their characteristic metal-oxide
deposits disappear when a sample is streaked on an agar plate, and the "Siderocapsa"
colonies are no longer distinguishable from neighboring colonies (Hanert, 1981). Perhaps
one could test for a possible enrichment of P. putida in platings from lakewater samples
in which "Siderocapsa" is seen.
Another set of reports of "Siderocapsa" isolation are important to note here.
Schmidt, in a series of papers, investigated the morphology and ultrastructure of
"Siderocapsa geminata" in Lake Pluss, Germany. Transmission electron micrographs of
metal-encrusted "S. geminata" forms showed that these bacteria were Gram-negative
(Schmidt, 1984). Isolates were polarly-flagellated, but characterization tests could not
distinguish between a Vibrio or a Pseudomonas identification (Schmidt, 1979),
suggesting that he was working with impure cultures (Ghiorse, 1984). As with "P. manganoxidans" and "A. siderocapsulatus", manganese oxides were stated to accumulate
only in stationary phase in the isolates (Schmidt and Overbeck, 1984).
Reports continue to appear, mostly from the former Soviet Union, of studies
involving "A. siderocapsulatus". "A. siderocapsulatus" strain A and P. putida were
48
studied side-by-side by Sorokin and Dubinina (1986), and were shown to share the
property of hydroxylamine oxidation in exponential phase. Strain G, however, oxidized
hydroxylamine only in stationary phase.
2.3. New Isolates of Manganese-oxidizing P. putida and Related Bacteria.
I sampled a variety of habitats and tested for the presence of manganese-oxidizing
bacteria. It was quite easy to detect colonies which deposited Mn oxides on non-selective
low-nutrient media, and when isolated and characterized, many were P. putida, and
others appeared to be related Pseudomonas species.
2.3.1. Materials and Methods
All but the three ZM strains were selected for their ability to form brown colonies on
Mn2+-containing media. Recipes for media are given in Chapter Three.
MF-1: Isolated on PYG-Mn agar from Gallionella-associated iron oxide deposits in a
stream, Middlesex Fells, Medford, Massachusetts.
MF-2x: A spontaneous variant of strain MF-1 that formed colorless colonies on PYGMn agar. Has a peculiar, extremely firm colony consistency on low-nutrient agar.
Wh-2: Isolated on PYG-Mn agar from stream sediment, Wheaton, Maryland.
Rk-2: Isolated on PYG-Mn agar from a eutrophic pond, Rockville, Maryland.
49
NEQ-3: Isolated on PYG-Mn agar from a freshwater fish tank, New England Aquarium,
Boston, Mass.
ESL-2a: Isolated on PYG-Mn agar from soil, Harvard University, Cambridge, Mass.
ZM-1, -7, and -12: Isolated on Plate Count Agar (Difco, Detroit, Mich.) from
homogenates of zebra mussels gathered in Lake Michigan and starved in sterile 10%
artificial sea water (Instant Ocean, Aquarium Systems Inc., Eastlake, Ohio). Manganeseoxidation ability was tested only after isolation and identification by the BIOLOG system
as Pseudomonas.
Identification tests were carried out as described above for "Pseudomonas manganoxidans".
50
2.3.2. Results and Discussion
Results of the characterizations are given in Table II - 6. As the table shows,
strains MF-1, NEQ-3, RK-2, and ESL2a match the description of Pseudomonas putida,
and strain WH-2 and the three ZM strains appear to be other Pseudomonas species.
The three zebra mussel isolates (ZM) were identified as Pseudomonas species
before being tested for manganese oxidation. Fourteen other Pseudomonas isolates from
zebra mussels were tested for their ability to form brown colonies on PYGly-Mn agar.
Nine of these strains were fluorescent; none could be identified as P. putida. Only these
three of the seventeen isolates were seen (within eight months) to deposit Mn oxides.
The results here illustrate that P. putida is a common manganese-oxidizing
bacterium in soil and water, yet it is clearly not the only manganese-oxidizing
pseudomonad. Some relatives of P. putida possess the activity as well.
It is also interesting to find manganese-oxidizing bacteria in environments not
normally thought of as being associated with reduced Mn, such as a freshwater aquarium,
or zebra mussels. If manganese oxidation is not associated with manganese in the
environment, perhaps the activity serves some other purpose in the cell, leaving
manganese oxides as an adventitious byproduct.
51
{{{TABLE II-
52
2.4. Literature Review of Other Manganese-oxidizing Pseudomonads
Other manganese-oxidizing pseudomonads have been reported in the literature.
Most notable are the reports of Zavarzin (1961, 1968) of manganese oxidation by
Pseudomonas eisenbergii, because P. eisenbergii is now considered to be a synonym of
P. putida (Palleroni, 1984).
Zavarzin claimed "symbiotic" Mn oxidation between Pseudomonas eisenbergii
strains and Pseudomonas rathonis strains isolated from soil. However, a variant of a P.
eisenbergii strain was obtained which was able to oxidize Mn without the "symbiont".
As was seen by Schweisfurth in "P. manganoxidans", high concentrations of yeast extract
inhibited Mn oxidation in P. eisenbergii, and no oxidation occured when no carbon
source was present.
It is not accurate to refer to this association as "symbiosis", for there is no
evidence that either P. rathonis or P. eisenbergii/P. putida benefits from the association.
It is likely that the non-oxidizing organism alters the local environment in some manner,
perhaps by adding or removing a particular nutrient, in such a way as to enhance or
enable oxidation by P. eisenbergii/P. putida. (Pseudomonas rathonis is considered as a
species incertae sedis in Bergey's Manual (Doudoroff and Palleroni, 1974); there is no
known surviving example of the species, and its original description does not clearly link
it to any presently-accepted species.)
A P. putida strain was mentioned to oxidize manganese by Jung and Schweisfurth
(1976), as mentioned above; the authors also refer to a manganese-oxidizer identified as
P. alcaligenes, a related nonfluorescent pseudomonad. Abdrashitova, et al. (1990)
53
reported oxidation of manganese and iron by an arsenic-oxidizing strain of P. putida
(strain 18); this organism was isolated from a gold-arsenic deposit in the former Soviet
Union.
Advances in molecular phylogeny have shown that many species once considered
to be members of the genus Pseudomonas are not truly related to each other. The
problem arises that manganese-oxidizing bacteria identified in years past as Pseudomonas
species may not be related to P. putida. For instance, the manganese-oxidizing marine
Pseudomonas strain S-36, reported by Kepkay and Nealson (1987) to be autotrophic, was
classified by rRNA oligonucleotide cataloging as a member of the alpha subclass of the
purple eubacteria, whereas P. putida, like all true Pseudomonas species, belongs to the
gamma subclass (Woese, et al., 1985).
Douka (1977) isolated two manganese-oxidizing bacteria with which she was able
to show, indirectly, Mn(II) oxidation by cell-free extracts. One strain, E1, was identified
by her as "Pseudomonas Group III", an archaic classification from the scheme of Shewan
et al. (1960), which means very little with respect to the current convention of
Pseudomonas taxonomy, except that Group III Pseudomonas species are not fluorescent.
Schütt and Ottow (1978) and Nealson (1978) isolated numerous Gram-negative
aerobic heterotrophic bacteria from marine manganese nodules and marine sediments.
Preliminary classifications were done; Pseudomonas-like bacteria were prominently
represented, although species-level classifications were not possible. Schütt and Ottow
suggested species assignments, including P. fluorescens, but they performed too few
characterization tests for those assignments to be reliable, especially considering that they
54
tried to assign marine isolates to species normally associated with soil and freshwater
bacteria. None of their Pseudomonas-like strains secreted a soluble fluorescent pigment.
Finally, it is interesting to speculate on a report of a rod-shaped iron-oxidedepositing bacterium, "Ferribacterium duplex", described by Brussoff in 1916. This
organism, which no longer exists in culture, was claimed to produce a soluble fluorescent
pigment in beef-extract broth (Sauer, 1934; Schweisfurth, 1973b).
CHAPTER THREE:
MANGANESE OXIDATION BY CULTURE-COLLECTION STRAINS OF
Pseudomonas putida
56
CHAPTER THREE
Manganese Oxidation by Culture-collection Strains of Pseudomonas putida
3.1 Introduction
In Chapter Two of this thesis it was shown that two species once thought to be
distinctive for their manganese-oxidizing abilities, "Pseudomonas manganoxidans" and
"Arthrobacter siderocapsulatus", were in fact simply members of the common soil and
water species Pseudomonas putida. It was also noted that Pseudomonas eisenbergii,
some strains of which were known to oxidize manganese, is now recognized as
synonymous with P. putida. Therefore, investigations were undertaken to see if
manganese oxidation is a specialized property of some subspecies within P. putida, or
whether manganese oxidation is a general property of the species. Cultures of P. putida,
purchased from the American Type Culture Collection, were tested for their ability to
oxidize Mn2+ on several media. These cultures originated from a variety of habitats, and
none were initially isolated on the basis of any metal-oxidizing property. P. putida
biovars A, B, and C were represented in the study.
In addition, many different media were evaluated with respect to the speed and
intensity of manganese oxidation on them by a variety of bacteria. The type strain of P.
putida, strain 12633, was investigated in more detail to see if its manganese-oxidation
activity has characteristics similar to that described for "P. manganoxidans" MnB-1,
especially in regard to manganese oxidation by a cell-free extract. Finally, the
57
implications of the findings of Chapters Two and Three are discussed, along with an
assessment of the potential importance of Pseudomonas putida as a contributor to
manganese oxidation in nature.
3.1.1. Materials and Methods
3.1.1.1 P. putida Strain Histories
Given below for each strain of P. putida I tested is its origin, date of first appearence in
literature (when available), synonyms, and some uses. Strains 12633, 17453, 17484, and
33105 are particularly well-known in the literature of P. putida. Biovar assignments are
from Stanier et al. (1966), unless otherwise specified. In some cases, no dates of
isolation or first reference are available; ATCC number provides a relative date of entry
into the culture collection (e.g., 23483 indicates late 1967; 40000 signifies late 1980's).
Some of this information can be found in Stanier et al. (1966) and the ATCC catalog
(American Type Culture Collection, 1992).
Pp 795: Isolated from blue milk (Hammer, 1914). Jessen's Biotype 11 (strain PJ 96).
Originally known as "P. cyanogena."
Pp 950: Biovar A (McManus et al., 1992), although Barrett et al. reported it unclustered
between biovars A and C. Isolated from creamery waste. Originally called "P. ovalis".
Jessen's Biotype 11 (strain PJ 90).
58
Pp 8209: Biovar C (Barrett et al. 1986; McManus et al., 1992). Originally known as "P.
ovalis". Used for production of ketogluconic acid. Jessen's Biotype 11 (strain PJ 91).
Pp 12633: Biovar A. Isolated from soil by enrichment on lactate. Used by Stanier in
studies of enzyme induction and oxidation of aromatic acids, e.g. mandelate. Original
references: Stanier, 1947; Also known as P. fluorescens A.3.12, P. putida PpG3, P.
putida PPN1 (Morgan and Dean, 1985), P. putida PRS1, P. putida RYS 90.
Pp 17390: Biovar A. From soil, amylamine enrichment. Den Dooren de Jong, 1926.
Biotype A. A.k.a. RYS 5
Pp 17391: Biovar A. From soil, propionate enrichment. Den Dooren de Jong, 1926.
Biotype A. A.k.a. RYS 6.
Pp 17453: Biovar A. Isolated by Gunsalus from soil (Urbana, Ill.) by enrichment on D(+)-camphor. Also known as strain P, C1B or C1B, PUG1, PpG1, and RYS 77. Original
reference: Bradshaw, et al., 1959.
Pp 17484: Biotype B. Isolated by naphthalene enrichment. Has NAH plasmid; degrades
naphthalene. Also known as RYS 110, and PpG63.
Pp 33015: Biovar A. Originally "P. arvilla" mt-2. Origin of TOL plasmid; degrades
59
toluene. Parent of ATCC 39213. Isolated from soil by meta-toluate enrichment in early
1950's (Assinder and Williams, 1990).
Pp 39213: Derived from Pp 33015 (above) by genetic manipulation.
Pp 39270: Isolated from sugar beet root; a plant-growth-promoting rhizobacterium.
Pp 43142: Isolated from the inguinal lymph gland of a glandered pony.
Other strains used in this study:
"Pseudomonas manganoxidans" MnB-1, MnB-5, MnB-6, MnB-8/1, MnB-15, and
unidentified strain MnB-29: isolated by Schweisfurth (1973a) from manganese-oxidecontaining deposits from various pipelines, wells, and demanganization filters, West
Germany. Deposited with ATCC as #23483-23488 in 1967. (See Chapter Two.)
"Arthrobacter siderocapsulatus" strains A and G (NCIMB 11286 and 11287), isolated
from two lakes in the former Soviet Union by Dubinina and Zhdanov (1975). (See
Chapter Two.)
New isolates of manganese-oxidizing bacteria, described in Chapter Two, section 2.3.1.
The following strains were isolated as part of this study:
60
"P. manganoxidans"/P. putida MnB1-A2: a spontaneous variant of "P. manganoxidans"
MnB-1, selected as a particularly dark colony on PYG-Mn agar.
P. putida TS-1: a spontaneous variant of P. putida 12633, selected as a particularly dark
colony on PYG-Mn agar.
P. putida TSC: a spontaneous variant of P. putida 12633, selected as a colorless colony
on PYG-Mn agar.
P. putida TSMC: a spontaneous variant of P. putida TS-1, selected as a colorless colony
on PYG-Mn agar.
Other species examined for manganese oxidation:
Pseudomonas aeruginosa ATCC 10145 (type strain)
Pseudomonas fluorescens ATCC 13525 (type strain, biovar I)
Arthrobacter globiformis ATCC 8010 (type strain)
Arthrobacter globiformis DK-1 (from this laboratory; see Chapter Two)
Rhizobium meliloti SU47
Escherichia coli K12
Escherichia coli V517
Salmonella typhimurium ATCC 29631
Klebsiella pneumoniae ATCC 12657
61
Serratia marcesans M2 (colorless; from this laboratory)
Citrobacter freundii M1 (from this laboratory)
Citrobacter freundii ATCC 8090 (type strain)
Cultures were maintained on Trypticase Soy Agar (TSA) or King's B (Flo) agar
(BBL Labs, Cockeysville, Maryland), refrigerated for up to one month, and periodically
restored from cultures frozen in 50% glycerol, kept at -20 C.
62
3.1.1.2. Media Formulations
In general: Glucose was autoclaved separately. MnSO4 was 0.22 μm-filter-sterilized to
avoid oxidation that could occur during autoclaving. For solid media, Bacto-Agar was
added to 1.5% and boiled before autoclaving. Autoclaving was carried out for 20 minutes
at 121C. and 15 psi. The pH of media was normally adjusted to 7.0 - 7.4.
PYG and PYG-Mn
dH2O
Bacto-Peptone
Bacto-Yeast Extract
D-glucose
CaCl2  2H2O
MgSO4  7H2O
1000 ml
250 mg
250 mg
250 mg
70 mg
600 mg
Note: this is equivalent to the "PYG(─H─V)" medium of Ghiorse and Hirsch, 1978.
For PYG-Mn medium, MnSO4H2O was added to 10 mg  l─1 (59 μM).
10% PYG-Mn
As for PYG-Mn, but with 25 mg  l─1 peptone, yeast extract, and glucose. MnSO4H2O
was added to 10 mg  l─1.
10x PYG-Mn
As for PYG-Mn, but with peptone, yeast extract, and glucose added to 2.5 g  l─1.
PYGH-Mn
dH2O
980 ml
Bacto-Peptone
250 mg
Bacto-Yeast Extract
250 mg
D-glucose
250 mg
Hutner's solution w/o Mn
20 ml
─
Note: This is equivalent to the "PYG V" medium of Ghiorse and Hirsch, 1978.
MnSO4H2O was added to 10 mg  l─1.
PYGH-hiMn
As for PYGH-Mn, but with MnSO4H2O added to 100 mg  l─1 rather than 10 mg  l─1.
PYGH-MnCO3
As for PYGH-Mn, but with 1.0 g MnCO3 substituted for the 10 mg  ─1 MnSO4H2O.
63
4xPYGH-Mn
As for PYGH-Mn, but with 1.0 g each of peptone, yeast extract, and glucose.
PYGlyH-Mn
As for PYGH, but with 500 mg glycerol substituted for the 250 mg glucose.
PY-hiGly-Mn
As for PYGH, but with 1.0 g glycerol substituted for the 250 mg glucose.
K1 medium (Gregory and Staley, 1982)
Bacto-peptone
2.0 g
Bacto-yeast extract
0.5 g
MnSO4H2O
0.2 g
dH2O
to 1000 ml
Beef Extract Broth (Jung and Schweisfurth, 1979)
Beef extract
1.5 g
Bacto-peptone
5.0 g
NaCl
2.5 g
Mineral Salts Solution
to 1000 ml
Medium 14a (Schweisfurth, 1973a)
Bacto-Yeast extract
trisodium citrate  2H2O
Na4P2O7  10H2O
Fe(NH4)2(SO4)2  6H2O
MnCO3
dH2O
Bacto-Agar
75 mg
150 mg
50 mg
150 mg
1.0 g
1000 ml
15 g
(pH 7.2)
64
MSS (Mineral Salts Solution; Jung and Schweisfurth, 1979)
Ca(NO3)2  4H2O
CaCl2  2H2O
MgSO4  7H2O
KH2PO4
Na2SiO3  9H2O
NaHCO3
Al2(SO4)3  18H2O
dH2O
50 mg
10 mg
50 mg
10 mg
5 mg
10 mg
1 mg
1000 ml (pH 7.2)
Hutner's Modified Salts Solution w/o Mn (Staley, 1981)
Nitriloacetic acid
10.0 g
CaCl2  2H2O
3.34 g
MgSO4  7H2O
29.7 g
FeSO4  7H2O
0.099 g
NaMoO4  2H2O
12.7 mg
"Metals 44" w/o Mn
50 ml
dH2O
to 1000 ml
(Normally used at 1/50 dilution.)
"Metals 44" trace elements solution w/o manganese (Staley, 1981)
Sodium EDTA
2.5 g
ZnSO4  7H2O
10.95 g
CoCl2  6H2O
0.203 g
FeSO4  7H2O
5.0 g
CuSO4  5H2O
0.392 g
Na2B4O7  H2O
0.177 g
dH2O
to 1000 ml
(This solution normally contains 154 mg  l─1 MnSO4H2O. Normally used at 1/1000
dilution.)
65
3.1.1.3. Detection of Manganese Oxidation on Solid Media
Cultures were streaked on media as formulated above. Plates were typically
streaked from a Trypticase Soy Agar or King's B agar (Flo agar) stock culture (both BBL
Labs, Cockeysville, Md.). Since brown MnOx deposits usually appeared in growth below
the agar surface before that on the agar surface, each plate was stabbed repeatedly with
the inoculation loop. Plates were incubated at 26 C. Plates were kept in plastic bags or
wrapped with Parafilm to prevent dessication after a few days. If incubations longer than
approximately one month were required, plates were moved to room temperature and
stored protected from light. A strain was scored as "manganese-oxidizing" only if a
visible brown coloration appeared in colonies. Brown pigment was confirmed as
manganese oxide by smearing a loopful of cells on filter paper, adding a drop of 0.04%
leucoberbelin blue (LBB) in 45mM acetic acid, and looking for the appearence of a blue
color (Krumbein and Altmann, 1973). The platinum and nichrome wire loops used were
determined not to react with leucoberbelin blue.
3.1.1.4. Rates of Manganese-oxidation by Starved Cultures in Liquid Medium
Cultures were shaken at 180 r.p.m. at room temperature (22-24C) in Trypticase
Soy Broth (BBL), pH 7.0, for 24 hours, well into stationary phase (Absorbance approx.
1.4 in a Spectronic 20 spectrophotometer, 2.0 cm path length). Cells were collected by
centrifugation 20 min. at 6000 x g at 4 C., rinsed thoroughly with sterile Mineral Salts
Solution (MSS), recentrifuged, and resuspended to an absorbance of 0.60 in 125 ml
sterile MSS. MnSO4  H2O was added to a final concentration of 2.0mg  l-1. Flasks
66
were shaken at room temperature. Subsamples of 1.3ml were taken periodically and
amended with 68μl 3M Na acetate, pH 4.8, and 54.7μl leucoberbelin blue (LBB), 1.25mg
 ml-1. These samples were incubated at least 15 min. at room temperature before
centrifugation 5 min. in an Eppendorf microcentrifuge. Supernatants were removed and
optical density was measured at 618nm on Perkin-Elmer model 552 spectrophotometer.
A standard curve of reducing-equivalents for LBB was prepared with a standardized
KMnO4 solution. Cell densities were determined by counting colony-forming units with
the drop-plate method (Hoben and Somassegaran, 1982) immediately upon suspension in
MSS, and periodically during the assay thereafter.
67
3.1.2. Results
3.1.2.1. Manganese Oxidation on Solid Media
The ability to oxidize manganese was tested for eleven different culture-collection
strains of Pseudomonas putida on a variety of different Mn2+-containing solid media.
Results for these strains and for all the strains examined in this study are presented in
Tables III - 1 through III - 5. Nine of the eleven strains of Pseudomonas putida were
found to form visibly brown, MnOx-containing colonies on at least some of the media, as
shown in Tables III - 1 and III - 2. Strain Pp 8209 did not form brown colonies on any
medium tested; strain Pp 950 formed brown colonies only on K1 agar, which I believe
was caused by a pH effect only (discussed below). The other nine strains formed brown
colonies at various rates: some fairly quickly, compared to "P. manganoxidans" MnB-1,
and some very slowly. There was some dependence of a strain's speed and intensity of
manganese-oxide deposition on the makeup of the medium used, but for the most part, a
strain would be relatively weakly-oxidizing on all media or relatively strongly-oxidizing
on all media.
In general, the ATCC P. putida strains did not oxidize as strongly as the "P. manganoxidans" strains (Table III - 3). Pp 12633, the type strain, was nearly as strong an
oxidizer as strain MnB-1. Pp 17390 was a weak oxidizer, and the other P. putida strains
lay between those extremes.
68
TABLE III - 1:
Manganese Oxidiation by and Background of Culture-Collection Strains of
Pseudomonas putida.
ATCC No. Biovar
Isolated from:
or first reference
Date of isol
Mn oxidatio
795
?
Blue milk
1914
moderate
950
?
Creamery waste
?
none detected
8209
C
?
?
none detected
12633
A
soil
1947
mod. strong
17390
A
soil
1926
weak
17391
A
soil
1926
moderate
17453
A
soil
1959
moderate
17484
B
soil
1950's?
moderate
33015
A
soil
1950's
moderate
39213
A
derivative of
ATCC 33105
39270
?
sugar beet root
?
moderate
43142
?
infected pony
?
moderate
moderate
TABLES III-2 -- III-5
Manganese Oxidation by Different Strains on Various Media
Bacteria were streaked on media listed in the tables and incubated at 26 C. Plates were observed and the appearence of brown
pigment was scored in terms of speed of first appearence of pigment, and intensity of coloration one week after first appearence
of brown color (approximate percentage determined by eye).
KEY: Speed of manganese oxidation
++
= Brown coloration appears within 1-3 days
+
= Brown coloration appears within 4-14 days
+/─ = Brown coloration appears between 15-30 days
─/+ = Brown coloration appears only after 30 days
─
= No brown coloration appears
Intensity of manganese oxidation
+++ = (on high-manganese agar only) Colonies become particularly dark brown or black-colored.
++
= >75% of the growth on the plate is dark
+
= 26% to 75% of the growth is dark
+/─ = 5% to 25% of the growth is dark
─/+ = <5% of the growth is dark.
A number of other strains, most not expected to be able to oxidize manganese,
were tested for their behavior on these media. The type strains of Pseudomonas
aeruginosa and Pseudomonas fluorescens, closely related to P. putida, exhibited no
manganese oxide deposition on any medium tested (exception: K1 agar, discussed
below). Of the other species that were not previously known to oxidize manganese, none
formed brown colonies, even over an incubation period as long as 18 months in the case
of PYG-Mn plates. Citrobacter freundii, a strain of which was claimed by Douka (1977)
to deposit manganese oxides, did not do so in my hands. Arthrobacter globiformis,
mentioned by Dubinina and Zhdanov (1975) to oxidize manganese, was the only one of
these other strains to form brown colonies on the media I tested, and the type strain
(ATCC 8010) did so only at very low frequency. Interestingly, some of these colorless
strains exhibited weak LBB reactions on some media (such as P. aeruginosa or
Rhizobium meliloti on PYG agar with or without added manganese). Leuco dyes similar
in structure to leucoberbelin blue are known to be oxidized by H2O2 in the presence of
heme (which has a slight peroxidase activity; Ahlquist and Schwartz, 1975), though in my
hands, a drop of 3% H2O2 had no noticeable effect on LBB/cell-smear spot tests.
It is highly unlikely that the brown colonies seen on the P. putida plates are
caused by, for example, "P. manganoxidans" contamination. For one thing, different P.
putida strains required different times to produce a visible manganese oxide deposit, and
these relative rates of manganese oxidation were consistent through repeated transfer.
For another, different strains exhibited different patterns of manganese oxide deposition
within the colonies: some colonies were uniformly dark (e.g., Pp A2, Pp TS1), some had
concentric light-and-dark patterns (Pp MnB1), some were darkest at the very margins of
the colonies (Pp 795, strain "WH-2"), some had dark centers with light or colorless
margins, some were dark with light-colored centers. These patterns were also
reproducible through many transfers, although colonies of all strains on higher-nutrient
media (4xPYGH-Mn, PYHiGlyH-Mn, and 10xPYG-Mn) were notably less complete in
their pigmentation, similar to the effect seen by Bromfield (1974) for a manganeseoxidizing Arthrobacter strain on media of higher nutrients.
No one medium was found to give optimal results for all strains. Addition of
trace metals (Hutner's salts) to PYG-Mn agar appeared to lead to more reproducible
results; occasional batches of PYG-Mn had inexplicably prevented manganese oxidation
by all but the strongest oxidizers. 4xPYGH-Mn was perhaps the medium most favorable
to the ATCC P. putida strains. Few strains turned brown on 10xPYG-Mn agar, but this
medium lacked added trace minerals; it is therefore not clear whether manganese
oxidation was inhibited by the high nutrient content of the medium or by lack of essential
trace minerals.
Strains plated on K1 medium gave unusual results. Colonies turned brown only
for strains Pp 950, Pp 17390, and Rhizobium meliloti SU47, even after one month.
Instead, after two weeks, all plates showed the following effect: the agar surrounding
colonies, but no closer than about 0.5 cm from a colony, turned brown and reacted
strongly with leucoberbelin blue, indicating that manganese oxidation had occurred away
from the cells. This medium has been used extensively by others (Nealson, 1978;
Gregory and Staley, 1982) to isolate manganese-oxidizers. It contains, relative to PYG-
Mn, high concentrations of peptone and MnSO4, which are conditions noted by others
(e.g., Brantner, 1970) to lead to chemical manganese oxidation due to alkalinization of
the medium, caused by NH4+ released from digestion of the peptone. It seems likely that
alkalinization occurred on this medium, leading to chemical oxidation of the Mn(II), even
though the pH of a brown agar sample from one plate was measured to be 7.3. (The
reaction Mn2+ +
O2 + H2O ──> MnO2 + 2 H+ releases protons; perhaps the pH dropped
after initially rising.) The only strains that formed brown colonies on this medium were
weak- or non-oxidizers on other media, further implying that what was seen on K1 plates
does not reflect biological manganese oxidation in any other than a trivial and artificial
sense.
A few interesting observations were true of the strains in general. A mixture of
darker- and lighter-pigmented colonies was present in many of the strains, most notably
Pp 12633, in which 4%-8% of colonies on PYG-Mn agar darkened faster, and remained
darker, than the rest of the colonies. When such a darker colony was isolated and
restreaked, it retained its more-strongly-oxidizing phenotype (e.g., Pp TS-1, derived from
Pp 12633). Occasional colorless colonies would appear spontaneously, at low frequency
(approx. 10─4 or less). These colonies could be isolated and transferred as well, but no
completely non-oxidizing variant was ever selected this way. A strain that was colorless
on one medium (e.g., Pp TSC or TSMC on PYG-Mn agar) would deposit manganese
oxides weakly, but definitely, on another medium (PYGlyH-Mn agar). Also, it was true
for all strains listed in Tables III - 2 through III - 5, including Arthrobacter globiformis,
that manganese oxides began accumulating first in a stab beneath the agar before
appearing on the agar surface, suggesting that the microaerophilic conditions present
below the surface are advantageous to manganese oxidation by the bacteria. Schweisfurth (1973) and Dubinina and Zhdanov (1975) observed that "P. manganoxidans" and
"Arthrobacter siderocapsulatus", respectively, both appeared to prefer to deposit MnOx
microaerophilically. This effect was also seen in a variety of marine bacteria by Nealson
(1978), and in soil columns by Uren and Leeper (1978). Such an effect is contrary to
what one would expect chemically; by decreasing the O2 tension, the reduced Mn state
should be favored.
3.1.2.2. Manganese Oxidation Rates for Starved P. putida Strains in Suspension
Manganese oxidation rates for five of the P. putida strains (12633, 17390, 17453,
17484, and 39213), as well as "P. manganoxidans" MnB1-A2 and P. fluorescens 13525,
were measured during starvation in Mineral Salts Solution (MSS). The results are shown
in Figures III - 1 and III - 2. All five P. putida strains oxidized manganese within nine
days in suspension, although Pp 17390 was noticeably slower than the others (as it was
on solid media as well). P. fluorescens did not oxidize a detectable quantity within this
time. Cell death was not significant over the nine days of measurements (Figure III - 3).
Measurements of MnOx were not very accurate in old cultures at high MnOx concentrations probably because the oxide-coated cells clumped considerably and adhered to the
sides of the flasks.
FIGURE III - 1:
MANGANESE OXIDATION BY STARVED P. putida STRAINS,
0 TO 45 HOURS
P. putida strains 12633, 17453, 17484, and 39213, along with "P. manganoxidans"/P. putida strain MnB1-A2, are represented in this figure, due to their relatively
rapid accumulation of oxidized manganese. P. putida 17453 is referred to here as "17453
w/Mn" to distinguish it from a parallel flask of P. putida 17453 starved in the absence of
Mn2+. That sample and two others, part of the same experiment, are included in Figure III
- 2.
FIGURE III - 2:
MANGANESE OXIDATION BY STARVED P. PUTIDA STRAINS,
0 - 210 HOURS
P. putida strains 17390 and P. fluorescens 13525, incubated with Mn2+, and P.
putida 17453, incubated without Mn2+, are represented in this figure. Note that this graph
has a longer time scale than Figure III - 1, which was part of the same experiment.
FIGURE III - 3:
SURVIVAL OF STARVED P. putida STRAINS DURING MANGANESE
OXIDATION ASSAY
Cell densities of P. putida 12633 and "P. manganoxidans"/P. putida MnB1-A2
were not measured over time due to the fact that they oxidized the Mn2+ available to them
in a span of only a few hours, and were presumed not to have died off significantly during
that period.
In summary, some things about the culture-collection P. putida strains are
important to note:
1. None of these strains were originally isolated on the basis of any metaloxidizing ability.
2. Most cultures came from soil but plant, animal, and food-spoilage habitats are
represented.
3. Some of these strains have been in laboratory culture for many decades. It
would be assumed that, at least before the advent of lyophilization as a maintenance
technique, the strains had to be transferred repeatedly on media non-selective for
manganese oxidation. It is common in microbiology for a character to be lost if it is not
selected for in transfers; of Schweisfurth's 200 original manganese-oxidizing isolates,
only thirty retained their Mnx+ phenotype after repeated laboratory transfer (Schweisfurth,
1973a). Also, there seems to be no correlation between year of isolation and the time it
takes the strain to form brown colonies.
3.2. Manganese Oxidation by the Type Strain of Pseudomonas putida
The type strain of P. putida, ATCC 12633, was investigated in greater detail to
see if its mechanism of manganese oxidation was similar to that of "P. manganoxidans"
MnB-1. A fraction (4% - 8%) of Pp 12633 colonies were darker than the rest on PYGMn agar; one of these variants was isolated (Pp TS-1) and used in further experiments.
Like "P. manganoxidans", a cell-free extract from starved P. putida TS-1 cells
was able to oxidize Mn2+ in vitro. This activity was completely inhibited by boiling for
10 minutes and was reduced when reactions were incubated in the presence of protease
(See Figure III - 4).
3.2.1. Materials and Methods
3.2.1.1 Preparation of crude extracts
Cells were grown in Beef Extract Broth (or other specified medium) to stationary
phase (approx. 24 hours) at 22-24 C. while shaking at 120 rpm. Cells were
centrifuged at 6,000 x g for 20 minutes at 4 C. The pellet was rinsed twice with Mineral
Salts Solution (MSS) (with recentrifugation) and resuspended to the original volume in
MSS. Cells were starved four hours. Cells were then harvested by centrifugation at
6,000 x g (20 min., 4), rinsed in cold 2.0 mM phosphate buffer (pH 7.0), and
resuspended in 2.0 mM phosphate buffer to approximately 15 ml. This suspension was
sonicated on ice 4 to 8 times for 30 seconds each time, with 2 min. cooling on ice in
between, using a Branson sonifier (Branson Instruments, Inc., Danbury, Conn.) at a power
setting of 500 watts. The sonicate was centrifuged at 48,000 x g for 25 minutes. The
supernatant was saved as the crude extract. Protein content was measured with a
Coomassie Blue assay (Bio-Rad Laboratories, Richmond, Cal.; Bradford, 1976), with
bovine serum albumin as a standard (concentration determined spectrophotometrically,
A280 = 0.660 at 1.00 mg  ml─1).
3.2.1.2 In vitro assay of manganese oxidation by cell-free extracts
These assays followed the procedure of Jung and Schweisfurth, (1979). Reactions
contained 2.0 mM phosphate buffer, pH 7.0, 11.8 μM MnSO4, and crude extract at
various concentrations. Reactions were carried out in 4.0 ml volumes using a Spectronic
20 spectrophotometer (Bausch & Lomb, Rochester, NY) or in 1.0 ml volumes using a
Perkin-Elmer Model 552 spectrophotometer (Perkin-Elmer, Inc., Norwalk, CT). The
extent of reaction was measured by absorbance of MnOx at 366 nm. A molar absorption
coefficient of 1.0 x 10-4 M-1 cm-1 was used for the manganese oxide product (Jung and
Schweisfurth, 1979; Adams and Ghiorse, 1987). The reactions were begun by adding the
MnSO4.
3.2.1.3 Effects of heat and protease on manganese oxidation activity
In vitro assay reactions were made up as above, containing 50 μg  ml-1 of crude
protein from P. putida TS-1. This extract was prepared from cells grown to stationary
phase in King's B broth supplemented with 1.0 mg  l─1 Fe(III) citrate. Two samples
were boiled 10 min. and 20 min., respectively. Other samples were incubated with 50 μg
Pronase E (Sigma) and allowed to incubate 0, 15, 30, 45, and 60 minutes before Mn2+
was added and absorbance at 366nm was followed. Pronase boiled for 20 minutes was
included as a control.
3.2.2. Results
It can be seen from figures III - 4 and III - 5 that there is a manganese-oxidizing
activity in cell-free extracts of starved P. putida TS-1, and that this activity is destroyed
by as little as ten minutes of boiling, and is severely degraded by exposure to the protease
Pronase E in proportion to the length of time of exposure. Boiled Pronase, however, had
no significant effect on manganese oxidation. These results match conclusions of Jung
and Schweisfurth (1979) for a cell-free extract of "P. manganoxidans" MnB-1, and
strongly suggest that the manganese-oxidation activity in the extract is mediated by a
protein. Also like strain MnB-1, no manganese oxidation activity was detected in crude
extracts prepared from P. putida TS-1 cells grown in high-nutrient media but not starved
before sonicating (data not shown).
The activity of strain TS-1 is similar to that of strain MnB-1 in the fact that a lag
time is required before manganese oxides are seen to accumulate. The TS-1 extract,
however, required at least 20 minutes of lag time, whereas the MnB-1 extracts reported
by Jung and Schweisfurth needed only about five minutes before manganese oxides
appeared, and the reaction went to completion within a few more minutes, even though
the amount of crude protein used in their reactions was comparable to the amount used in
the above experiments. The fact that a different medium was used to grow the cells in
before harvesting may have been significant. Perhaps P. putida TS-1 simply has less
intrinsic manganese-oxidation activity than strain MnB-1. In any case, it is likely that P.
putida TS-1 and "P. manganoxidans" MnB-1 share a common mechanism of manganese
oxidation.
FIGURE III - 4:
INHIBITION BY PROTEASE OF MANGANESE-OXIDATION ACTIVITY BY
CELL-FREE EXTRACTS OF P. putida TS-1.
Reactions were carried out at room temperature (22 - 24 C.) in the presence of Pronase
E (12.5 μg  ml-1). Samples were preincubated for 0, 15, or 60 minutes before MnSO4
was added (at t = 0 min.). Note that Pronase would be acting after time t = 0 as well.
FIGURE III - 5:
INHIBITION BY HEAT OF MANGANESE-OXIDATION ACTIVITY BY CELLFREE EXTRACTS OF P. putida TS-1.
Reactions were carried out at room temperature (22 - 24C.) with 200 μg crude protein
in a 4.0 ml volume. In one sample, crude extract was boiled for 10 min. before being
added; in another, crude extract was boiled for 20 min.; in a third, crude extract was not
boiled.
3.3. DISCUSSION: Importance of P. putida in manganese oxidation in nature
The investigations reported above suggest that Pseudomonas putida is a species
whose members, in general, are able to oxidize manganese. What does this imply about
the role of P. putida in the oxidation of manganese in nature? What is P. putida, and
where is it found? What can we learn about manganese oxidation from what we know
about P. putida?
Pseudomonas putida strains are best known to microbiologists for their ability to
degrade aromatic hydrocarbons such as toluene, camphor, and naphthalene. Degradative
pathways, often associated with plasmids, have been examined extensively by
biochemical and genetic means (Assinder and Williams, 1990). In nature, P. putida
would be expected to play a significant role in carbon mineralization along with other
pseudomonads. The species has a characteristic ability to utilize a wide variety of carbon
sources, and it does not require special growth factors (Palleroni, 1992a).
Pseudomonas putida has also been associated with other metal oxidation and
reduction functions. P. putida strains have been isolated which are capable of chromate
reduction (Ishibashi et al., 1990) and mercuric ion reduction (Baldi et al., 1993). Three
strains of P. putida, originally called "Pseudomonas arsenoxydans" (Turner, 1954;
National Collections of Industrial and Marine Bacteria, 1990), were isolated from
arsenical cattle-dipping fluids on the basis of their ability to oxidize arsenite (AsO2─) to
arsenate (AsO43─). Another strain of arsenite-oxidizing P. putida, isolated from goldarsenic deposits, was claimed to be able to oxidize Mn2+ and Fe2+ by Abdrashitova et al.
(1990). An arsenite-oxidizing Alcaligenes eutrophus strain also oxidized iron and
manganese in their hands. Abdrashitova et al. speculated that there was a common
mechanism for oxidation of all three metals. However, Schweisfurth (1976) reported that
the one "P. arsenoxydans" strain he tested did not oxidize manganese.
Pseudomonas putida has been recognized for many years as a common inhabitant
of soils and fresh waters, and is described as "ubiquitous" by Palleroni (1992a). Kremer
et al. (1990) found that P. putida comprised an average of six percent (ranging up to
14%) of all culturable isolates in roots of seven species of common weeds. Read and
Costerton (1987) found that P. putida was the numerically dominant species in lotic
biofilms from rock surfaces in a eutrophic Canadian stream; P. fluorescens was the
dominant species in a similar biofilm from a pristine stream. However, Gennari and
Dragotto (1992) found that fluorescent pseudomonads made up less than 5% of the total
microflora of most of the soil and water samples they examined. P. putida biotypes A
and B together made up 42% of the fluorescent pseudomonads found in soil samples
(city, cultivated, and mountain soils), and 15% of those found in water samples (rivers,
lakes, and canals). They also found P. putida at a lesser abundance in spoiled and fresh
meat and milk products. Sands and Rovira (1971) determined that fluorescent pseudomonads made up only 0.06% to 0.27% of culturable bacteria in 15 different soils and
wheat rhizospheres grown in those soils, and 11% to 17% of the fluorescent pseudomonads were P. putida. P. putida must also be considered a potential pathogen in
humans, as strains have been isolated from a number of medical samples (Bergan, 1981).
P. putida is a wound colonizer rather than the etiologic agent of a disease; the species
poor growth at 37 limits its infective potential, and its obligate oxygen requirement
prevents its growth in the gut.
It has been demonstrated in this chapter that a good number of P. putida strains
can oxidize manganese in the laboratory. They at least have the potential to oxidize
manganese in nature. But is P. putida an important contributor to bacterial manganese
oxidation in nature? I believe it is reasonable to assume so for a number of reasons.
For one thing, strongly-oxidizing members of P. putida have been isolated
repeatedly from iron- and manganese-oxide-bearing environments in many parts of the
world, though usually unrecognized as P. putida at the time ("P. manganoxidans"; "A.
siderocapsulatus"; P. eisenbergii). The fact that they can be isolated from such habitats
is no guarantee that they are active there, of course, but this is no more radical an
assumption than is made by any other microbiologist isolating a morphologicallyundistinguished bacterium from, say, a marine manganese nodule. Indeed, if Dubinina
and Zhdanov are correct that "Siderocapsa" is P. putida, then there will be physical
evidence that P. putida is an important manganese-oxidizing species in nature.
Also, the sheer ubiquity of P. putida in soil and fresh water suggests that when the
conditions are suitable, competent members of this species (and perhaps related
fluorescent pseudomonads) will oxidize available Mn2+. Certainly some P. putida strains
will be more active oxidizers than others, as seen with the strains examined above.
To get a better idea of the importance of this species to manganese oxidation in
nature, a census would have to be done, comparing the fraction of manganese-oxidizing
P. putida isolates among all manganese-oxidizing colonies growing on a variety of
media. It would be interesting to see if strongly-oxidizing P. putida strains were more
abundant in regions of high Mn2+ concentration.
One thing this chapter has demonstrated is that bacterial manganese oxidation
need not be thought of as the domain of specialized or unusual organisms. It is
unnecessary to postulate that the capability of oxidizing manganese sets an organism
apart from others; certainly not that the activity is sufficient to define a new species. The
names "Pseudomonas manganoxidans" and "Arthrobacter siderocapsulatus" imply
something unique about those organisms, whereas the reality is that the bacteria belong to
the common species Pseudomonas putida, whose members, apparently, are generally
capable of oxidizing manganese.
CHAPTER FOUR
INVESTIGATIONS INTO
MANGANESE OXIDATION AS A
STRESS PHENOMENON
98
CHAPTER FOUR
INVESTIGATIONS INTO MANGANESE OXIDATION AS A STRESS
PHENOMENON
4.1. Introduction
Schweisfurth showed that manganese oxidation in "Pseudomonas manganoxidans" took place only in stationary phase or under starvation. In vitro manganese-oxidation activity could be demonstrated only in cells that had been starved for at least two
hours before disruption (Jung and Schweisfurth, 1979). Strains oxidized Mn2+ only on
low-nutrient media; cells suspended in starvation salts stopped oxidizing manganese
when NH4+ or peptone was added (Schweisfurth, 1976; Jung and Schweisfurth, 1976).
Manganese oxidation in some other bacteria has been linked to starvation as well.
Beijerinck (1914) observed that yeast extract prevented manganese oxidation in his
"Bacillus manganicus". Bromfield (1956) noticed that a Corynebacterium sp. (later
reclassified as Arthrobacter) oxidized manganese only in stationary phase. Dubinina and
Zhdanov (1976) showed that "Arthrobacter siderocapsulatus" (which appears to be
Pseudomonas putida; see Chapter 2) oxidized only in stationary phase. Leptothrix
discophora, however, oxidizes in exponential phase (Adams and Ghiorse, 1987).
An obvious link between starvation and manganese oxidation is to presume that
once an organism has exhausted its reduced carbon supply it turns to Mn2+ to use as a
source of electrons. "P. manganoxidans", though, was shown by Jung and Schweisfurth
(1976) not to be an autotrophic manganese-oxidizer. There was no significant increase in
protein or DNA content in cultures starved in the presence of Mn2+, no radioactivity was
99
incorporated into cell polymers during manganese oxidation in the presence of 14CO2, and
no Ru-DP-carboxylase activity was detected in cells. Also, the observation that P. putida
strains from habitats not normally associated with reduced manganese were nevertheless
able to oxidize manganese (Chapter Three) suggests the activity may have some other
function, perhaps not even associated with manganese. It is useful to note that starvation
has other implications beyond nutrition, and that the manganese-oxidation activity might
be involved in a starvation-stress-associated function.
Starvation brings about a variety of responses that tend to make a cell more able to
withstand various stresses; this is true not only for endospore-forming bacteria like
Bacillus, but also for bacteria like Escherichia coli and Vibrio which do not form
protective structures. Upon starvation, E. coli and Vibrio cells do not simply stop
metabolizing; rather, they undergo distinct physical and physiological changes. In some
species, cells fragment into ultramicrocells, and cell surfaces and membranes undergo
changes (Nyström, Albertson et al., 1990; Siegele and Kolter, 1992; Kjelleberg et al.,
1987). Particular proteins are induced by starvation which contribute to cell survival, to
protection against environmental stresses, and to maintenance of the cell in a state such
that it can recover and begin to grow again once nutrients become available. Among
starvation-induced or-enhanced proteins in E. coli are the catalase HPIII (Loewen et al.,
1985) and the DNA repair enzyme exonuclease II (Sak et al., 1989).
One particularly interesting fact about starvation-induced proteins is that some of
them are also induced by other stresses, such as heat shock, peroxide shock, and exposure
to ethanol and CdCl2 (Nyström, Albertson et al., 1990). Starved E. coli cells exhibited
increased resistance to heat and H2O2 challenge which was dependent on new protein
100
synthesis; furthermore, some of the same proteins synthesized upon glucose starvation
were also induced by exposure to high temperature or peroxide (Jenkins et al., 1988).
Starvation-specific proteins are known to fall into certain classes, depending on
what type of starvation brings about expression of that protein (Matin et al., 1989;
Nyström et al., 1992). Some proteins are expressed by carbon starvation, some by
nitrogen, phosphorous, or amino acid starvation, some by any of several starvation
regimes and some only by multiple, simultaneous starvations. Carbon starvation induced
heat-shock proteins in Salmonella typhimurium (Spector et al., 1986) and Vibrio sp. S14
(Nyström, Flärdh and Kjelleberg, 1990). Carbon- and multiple-nutrient-starved cells of
Vibrio sp. S14 showed increased resistance to heat, UV, and CdCl2 stresses, while
nitrogen- and phosphorus-starved cells did not exhibit such resistances (Nyström, Flärdh
and Kjelleberg, 1990).
I tested "P. manganoxidans"/P. putida strain MnB1-A2 (derived from ATCC
28483) to see if its in vivo manganese-oxidation activity was preferentially induced by
starvation for carbon, nitrogen, or phosphorus, and to see if heat shock or H2O2 shock
would induce manganese oxidation activity in growing cells.
101
4.2. Materials and Methods
4.2.1. Starvation for C, N, and/or P
The recipe for defined medium MM4 is as follows, per liter: D-glucose, 1.0 g;
ammonium sulfate, 0.5 g; potassium phosphate, 2.0 mM; PIPES buffer, pH 7.2, 40 mM;
Hutner's salts, 20 ml (see Chapter 2); and when indicated, MnSO4  H2O, 2.0 mg. In
some cases, 2x MM4 was used, containing twice the glucose, ammonium, and phosphate
concentrations as listed above.
"P. manganoxidans" MnB1-A2 cells were grown in 2x MM4 (100ml liquid in a
500-ml Erlenmeyer flask), shaken at 80 rpm in a 26 water bath, and were harvested in
log phase (13 hours) at an absorbance of A660 = 0.56 (18mm path length). Cells were
centrifuged (20 min. at 6,000 x g, 4 C.) and resuspended in 50 ml Hutner's salts. A
volume of 10.0 ml of cells (to an A660 of 0.10; 6.2  0.47 x 107 cells  ml-1) were added
to each of five 500 ml side-arm flasks containing 100 ml of medium, as follows:
1) +CNP
= Full MM4 medium
2) ─C
= MM4 without glucose
3) ─N
= MM4 without ammonium
4) ─P
= MM4 without phosphate
5) ─CNP
= MM4 without glucose, ammonium, or phosphate.
All of these were amended with 2.0 mg  l─1 (= 11.8 μM) MnSO4  H2O. Flasks were
shaken at 26. Absorbances were followed spectrophotometrically at 660nm (Bausch
and Lomb Spectronic 20), and samples were taken periodically to measure manganese
102
oxide levels with the leucoberbelin blue assay (below).
Cell concentrations were determined by the drop-plate method of Hoben and
Somassegaran (1982). Rates of manganese oxidation were calculated from experimental
data by linear regression. The rate for the batch introduced into full MM4 was adjusted to
account for growth of cells (to 9.4  1.2 x 109 cells  ml-1).
The glucose assay was obtained in kit form from Sigma Chemical Co. (St. Louis,
Mo.), utilizing glucose oxidase/horseradish peroxidase/o-anisidine, and was performed
according to the manufacturer's directions.
4.2.2. The Leucoberbelin Blue Assay for Oxidized Manganese
A 900 μl sample, typically containing 0 to 10 nmole of MnO2 (or equivalent
MnOx), was added to 110 μl of 3 M sodium acetate, pH 4.0, and 110 μl of 0.1%
leucoberbelin blue in dH2O (Altmann, 1972; Krumbein and Altmann, 1973) in a 1.5 ml
microcentrifuge tube. Samples were vortexed thoroughly and incubated at room
temperature for 15 minutes before freezing at -20 C. for later spectrophotometric
analysis at a more convenient time. Cells were removed from the sample, if necessary, by
centrifuging in an Eppendorf tabletop microcentrifuge for 5 minutes. The absorbance of
the blue supernatant was measured at 625 nm in a Perkin-Elmer Model 552
spectrophotometer. Standard curves were prepared with standardized KMnO4 solutions;
one nmol of MnO4─ was considered to be equivalent to 5/2 nmol of MnO2. The
oxidation product of the bacteria, for purposes of calculation, was assumed to be MnO2.
Samples were taken in triplicate at each time point.
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4.2.3. Heat Shock/Peroxide Shock
P. putida A2 was grown in 100 ml PYG broth, pH 7.4 (as formulated in Chapter
Three) with MnSO4 added to 2.0 mg  l-1 (= 36 μM). Cultures were grown in 500 ml
sidearm flasks and aerated on a rotary shaker at 100 rpm. Absorbance at 660 nm was
followed in a Bausch & Lomb Spectronic 20 spectrophotometer. MnO2 was measured
with the leucoberbelin blue assay frequently during the experiments, approximately as
often as absorbance measurements were made.
Most batches were grown initially at 23 C. In late-log phase (5 hours growth,
OD660 = 0.07 - 0.09), specified flasks were moved to a 35 shaking water bath for 30
minutes, then moved immediately to a 30 shaking water bath.
Certain flasks were
maintained at 35 as controls. One flask was challenged with hydrogen peroxide to a
final concentration of 0.2 mM at the same time other flasks were heat-shocked. This
concentration was chosen because an H2O2 concentration of 0.3 mM was seen to be toxic
to logarithmic-phase cells of a different strain of P. putida (Katsuwon and Anderson,
1989).
The experiments were run in two groups. Bacteria were subjected to the
following temperature regimens:
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Group 1:
1) 23 only
2) 23  35  30
3) 23  35
4) 35 only
5) 23 only, H2O2 shock
Group 2:
1) 23 only
2) 30 only
3) 23  35  30
This is a modification of a heat-shock regime (26  42  37) often used for E.
coli (Neidhardt and VanBogelen, 1987). "P. manganoxidans" MnB1-A2 grew at 35, but
only rare variants could grow at 37, as determined on solid media (data not shown).
105
4.3. Results and Discussion
4.3.1. Nutrient Starvation
Results of the nutrient starvation experiment are given in Figures IV - 1 and IV 2. In terms of both lag time and rate of MnOx appearence, the preferred starvation regime
for inducing manganese oxidation activity is starvation for all three of glucose,
ammonium, and phosphate, starvation for glucose alone is nearly as effective as
starvation for C, N, and P. The cultures starved for ammonium and phosphate only
exhibited only weak manganese oxidation after a long period of time.
RATES
Batch
Rate of Mn Oxidation
Lag Time
(nmol MnO2  hr-1  108 cells)
+CNP
1.2  12%
2 hr
-C
8.7  8.8%
3 hr
-N
0.23  56% (minimum value)
10 - 22 hr
-P
0.10  61% (minimum value)
10 - 22 hr
-CNP
18
 7.8%
2 hr
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FIGURE IV - 1:
MANGANESE OXIDATION BY STRAIN MnB1-A2 UNDER VARIOUS
STARVATION REGIMES, part A.
Graphs showing cell density (absorbance at 660nm) along with manganese oxide
accumulation for cells starved for C, N, and P; starved for C alone; and for cells
resuspended in full MM4 medium (no starvation). Time scale: 10 hours.
107
108
FIGURE IV - 2:
MANGANESE OXIDATION BY STRAIN MnB1-A2 UNDER VARIOUS
STARVATION REGIMES, part B.
Graphs showing cell density along with manganese oxide accumulation for cells
starved for glucose (as a comparison), starved for ammonium, and starved for phosphate.
Time scale: 48 hours.
109
110
At t = 6 hours, batches 1, 3, and 4 (Full MM4, no N, and no P) were sampled to
determine glucose concentration. All three batches had undetectable glucose levels (less
than 20 mg glucose  ml-1). It may be presumed that in the -N and -P batches, glucose
was converted to, for instance, pyruvate and triose phosphate via the Entner-Doudoroff
pathway (Vicente and Cánovas, 1973) during the six hours of starvation. From previous
experiments (not shown), it is likely that medium MM4, as formulated above, would be
glucose-limited, so that the +CNP batch should be starving for carbon once it reaches
stationary phase. The pH of each batch was measured after manganese oxides began to
appear; values ranged from 7.0 to 7.2.
It is interesting to note that Jung and Schweisfurth (1976) reported that
ammonium ion was the primary inhibitor of manganese oxidation in "P. manganoxidans"
MnB-1. However, their inhibition studies were done with peptone in the "starvation"
salts; peptone (primarily peptides and amino acids) would have served as a carbon source.
The graphs indicate that the rate of manganese oxidation, in most of the batches,
levels off beginning at around 50% of Mn2+ oxidized to MnO2-equivalent. The
concentration of manganese oxides is difficult to determine at these higher levels,
however, because the oxide-covered cells tend to aggregate in liquid and tend to stick to
the sides of the flask. The latter phenomenon leads to an underestimation of manganese
oxide levels, while the former contributes to an increasing imprecision in oxide
measurements.
In the graphs above, manganese oxide levels are shown in terms of "% Mn
oxidized". This assumes, of course, that the oxidation product is MnO2, whereas the true
111
product could be Mn(III) or mixed Mn(III)/Mn(IV) oxides. The fact that more than 50%
of the Mn2+ is oxidized to "MnO2-equivalent" in some of the batches above indicates that
the oxidation product is not solely Mn(III).
4.3.2
Heat Shock/Peroxide Shock
Figure IV - 3 shows that, although heat or peroxide shocks took place about two
hours before the control culture (23 only) reached stationary phase, no manganese
oxides were detected until well after the shocked cultures had entered stationary phase.
(The transition between logarithmic and stationary phases was not sharply defined in
these cultures, probably because of the disturbance necessary in taking frequent samples
in order to detect manganese oxides.) MnOx was first detected in the peroxide-shocked
culture and in the 23 control at approximately the same time. MnOx began appearing in
the heat-shocked culture about ninety minutes earlier than the 23 control, but this
discrepancy is most likely indicates that the strain grows faster at 30 than at 23. This
interpretation is confirmed in Figure IV - 4, where it can be seen that "P. manganoxidans"
A2 does indeed grow faster, reach stationary phase faster, and begin to oxidize Mn2+
faster at 30 than at 23. Again, it is seen that a heat-shocked culture does not begin to
oxidize Mn2+ significantly before the 23 control culture, well into stationary phase. The
pH values of cultures were measured after the experiment and found to be approximately
7.2.
The above experiments suggest that heat shock and peroxide shock do not seem to
112
be involved in induction of the manganese-oxidation (Mnx) activity in "P. manganoxidans" MnB1-A2. Lack of induction of the actual protein (or proteins) that carry out the
activity has not been proved in these experiments, however. It is conceivable that the
Mnx protein was induced by the shock, yet activity is simply masked in log and earlystationary phases. In vitro assays using crude extracts might reveal an activity not present
in the above in vivo experiments; however, a Mnx protein may be present yet masked
even in a crude extract. Indeed, it has not been proved that the Mnx protein is not
expressed in unstarved cells, only that the activity is not present in crude extracts of
unstarved cells (Jung and Schweisfurth, 1979, and personal observations).
The results above, both the nutrient-starvation and the heat-shock experiments, are
not definitive, but it appears that manganese-oxidation is not a generalized stress
phenomenon in this organism, nor does it have a particular relation to nitrogen or
phosphorous metabolism. The activity is preferentially induced by multiple-nutrient or
glucose starvation; if one assumes lack of a carbon source is the key factor in induction of
the activity, the results are consistent with the hypothesis that Mn2+ is being used as an
energy source. There may yet be other explanations, however, for the link between these
starvations and manganese-oxidation activity in Pseudomonas putida.
FIGURE IV - 3:
HEAT AND PEROXIDE SHOCK, GROUP I
At t = 5.0 hr., the temperature was raised from 23 C. to 35 C., or hydrogen
peroxide was added to 0.2 mM in the appropriate flasks. Error bars indicate  one
standard deviation in the measurement of Mn oxidation.
114
115
FIGURE IV - 4:
HEAT SHOCK, GROUP II
At t = 5.0 hr., the temperature was raised or agitation was stopped in the
appropriate flasks.
116
117
CHAPTER FIVE
MECHANISM OF MANGANESE OXIDATION IN
PSEUDOMONAS PUTIDA:
THE ROLE OF OXYGEN
118
CHAPTER FIVE
Mechanism of Manganese Oxidation in Pseudomonas putida:
The Role of Oxygen
5.1. Introduction
Jung and Schweisfurth (1979) reported that oxygen did not take part in manganese
oxidation by the crude extract from "Pseudomonas manganoxidans" MnB-1. In their
experiments, "the same reaction rates were obtained under anaerobic conditions as those
for aerobic controls." No oxygen uptake was detected by an oxygen electrode. They
went on to show that repeated oxidation of small quantities of Mn2+ by the same sample
of crude extract led first to an increase in rate of manganese oxidation, then to a gradual
decrease in rate, proportional to the amount of Mn2+ added/ oxidized. They interpreted
this result to mean that the manganese oxidizing protein is non-catalytic, being "used up"
in the process of oxidizing Mn2+. Since their crude extracts had been dialyzed, small
molecules such as NAD(P)+, FAD, or H2O2 that could potentially act as electron
acceptors were presumably not present in the reaction. Jung and Schweisfurth suggested
that the manganese oxidizing protein may act as a so-called "suicide enzyme", taking part
in the oxidation reaction and thus inactivating itself (perhaps by accepting electrons
without transferring them to another oxidant).
Accordingly, and understandably, the mechanism of manganese oxidation in
"Pseudomonas manganoxidans" has been reported in reviews (Ehrlich, 1981, 1990;
Nealson et al., 1988; Ghiorse, 1984) as being non-enzymatic and not involving oxygen.
119
That interpretation of the mechanism has set this organism apart from other manganeseoxidizing bacteria that have been examined, which, as would be expected, oxidize Mn2+
enzymatically through a reaction in which O2 takes part. In that sense, "Pseudomonas
manganoxidans" is an unusual organism, even among manganese-oxidizers. The
"nonenzymatic" description also implies that the oxidation may be caused simply by a
localized rise in pH.
The question of what is accepting electrons, if not O2, is perplexing. Molecules
such as NAD(P)+ or FAD, even if they were not dialyzed away, would make unlikely
electron acceptors for the reaction because of their low reduction potentials (─0.32 and
─0.22 volts, respectively, versus +0.40 volts for the MnO2/Mn2+ couple). One might
assume that bound cofactors or residues of the protein molecule itself could be accepting
the electrons. Perhaps one or a few bound cofactors each accept one pair of electrons
from one Mn2+ ion.
A simple calculation, however, casts doubt on this view. From Jung and
Schweisfurth's own data (1979; fig. 6), 0.06 mg of crude protein can oxidize at least 0.5
μmole of Mn2+. To estimate how many moles of the manganese-oxidizing (Mnx) protein
there might be in 0.06 mg crude protein, assume the Mnx protein size is of average size,
about 40 kDa (Neidhardt et al., 1990), and that 1% of the total protein is the Mnx protein:
(0.06 mg)(40,000 g/mole)-1(1/100) = 15 pmole Mnx protein.
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If 15 pmole Mnx protein can oxidize 0.5 μmole Mn2+, that means 500/0.015 = 33,000
Mn2+ ions oxidized per molecule of protein. Even if one assumes that the average protein
were very small, say, 10 kDa, and 100% of the crude protein was Mnx protein (two very
unlikely assumptions), then
(0.06 mg)(10,000 g/mole)-1 = 6 nmole Mnx protein,
and (500 nmole Mn2+)(6 nmole Mnx protein)-1 = 83 Mn2+ ions oxidized per molecule of
Mnx protein. This does not sound like the behavior of a suicide enzyme; it does not seem
likely that the protein gets "used up" only after having accepted 83 (or 33,000) pairs of
electrons.
A more reasonable conclusion is that the Mnx protein is a true catalytic enzyme,
and the decrease in activity the authors saw is due to something other than the protein's
taking a necessary part in the reaction. Perhaps the active site of the Mnx protein gets
coated with manganese oxides, for example. Perhaps the MnOx is oxidizing the protein.
Or, the decrease in activity with time might be caused by protease degradation within the
crude extract. The electron acceptor for the reaction, then, is probably not a small
molecule, not the Mnx protein itself, and (by a similar argument) not a different
macromolecule retained by the dialysis membrane. The most reasonable candidate for
electron acceptor in this reaction would seem to be O2, despite the claim of Jung and
Schweisfurth. Therefore, the question of whether O2 was taking part in the "P. manganoxidans" manganese-oxidation reaction was reexamined.
121
5.2. Materials and Methods
Reactions took place in 25 ml rubber-stoppered serum tubes containing 8.0 ml of
2.0 mM phosphate buffer, pH 7.0, amended with either 10 μM or 100 μM MnSO4. The
tubes were sparged with N2 (99.999%) before and after autoclaving 20 minutes at 121
C., 15 psi, to remove as much atmospheric O2 as possible. (The pH dropped 0.1 unit after
autoclaving; no detectable MnOx formed.) Reactions were run in duplicate.
Thirty μl (145 μg) of crude extract from P. putida A2 was added to each tube,
using a 1.0 ml syringe, after diluting the crude extract five-fold with phosphate buffer and
degassing under vacuum. "Aerobic" tubes (2A&B, 4A&B) were opened to the
atmosphere and vortexed thoroughly before crude extract was added. "Anaerobic" tubes
(1A-C, 3A&B) were opened to the air at t = 13.3 hr. One control tube (#5) contained
100μM MnSO4 (aerobic), but no crude extract was added.
The extent of reaction was measured by absorbance of MnOx at 366 nm using a
Bausch and Lomb Spectronic 20 spectrophotometer. A molar absorption coefficient of
1.0 x 104 M-1 cm-1 was used for the manganese oxide product (Jung and Schweisfurth,
1979; Adams and Ghiorse, 1987). Manganese oxides were also measured by the
leucoberbelin blue assay at certain points in the reaction.
Reactions were carried out at room temperature (25 C.); between t = 2.3 and t =
11.5 hours (i.e., overnight), the reaction tubes were kept at 4 C.
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5.3. Results and Discussion
Crude extract of "P. manganoxidans" strain MnB1-A2 was tested for its ability to
oxidize Mn2+ under anaerobic conditions. Strain MnB1-A2 was used, rather than its
parent MnB1, because crude extract from MnB1-A2 is able to oxidize 100 μM Mn2+ in
vitro, whereas that from MnB1 is reported to be completely inhibited above 30 μM (Jung
and Schweisfurth, 1979).
Figures V - 1 and V - 2 show that for the first three hours, with one exception,
only the tubes open to the air exhibited significant manganese oxidation. The manganese
oxidation observed in the anaerobic tubes may be attributable to small quantities of air
that entered the tube when crude extract was injected with a syringe. Some manganese
oxidation did occur, in both the aerobic and anaerobic tubes, overnight at 4 C. This had
leveled off by t = 12.5 hours. At 13.3 hours, when the anaerobic tubes were unstoppered
and vortexed, manganese oxidation took place rapidly in most of the tubes (figures V - 3
and
V - 4).
MnOx assays with leucoberbelin blue at t = 0, t = 13.3 hr, and after maximum
absorbance had been reached confirmed that absorbance at 366 nm was proportional to
amount of MnOx in each tube (data not shown).
Values for the duplicate tubes are not presented as averages because the variation
within some pairs was too great. Part of the problem was that the amount of crude extract
added to each tube was not rigidly controlled, due to the difficulty of handling small
volumes of liquid with a syringe while trying to maintain strict anaerobic conditions. I
123
speculate also that the Mnx assay seems to be sensitive to minute amounts of
contaminants; if the glassware is not scrupulously clean, perhaps, that might explain my
erratic results. I do not know how to explain the discrepencies among the maximum
absorbances reached in the 100 μM manganese tubes.
A separate trial of this experiment gave the same qualitative result, that Mn(II)
oxidation in anaerobic reactions did not proceed until air was introduced into the reaction
tube.
The data are somewhat rough, but the conclusion seems clear that in vitro
manganese oxidation by P. putida A2 cell-free extracts does require oxygen, contrary to
the report of Jung and Schweisfurth. It appears that Jung and Schweisfurth's experiments
with anaerobic manganese oxidation were not truly anaerobic (no details of the method
were given in their 1979 paper). It can be concluded that the manganese oxidation
activity is an oxidase (perhaps requiring more than one protein). In that sense, the
mechanism of manganese oxidation in P. putida is similar to that of other bacteria
purported to possess enzymatic manganese oxidase activities (Ehrlich, 1990).
Oxygen-limitation may explain the decrease in rate of manganese oxidation that
Jung and Schweisfurth attributed to non-catalytic oxidation. At 25, the solubility of
oxygen in water is 8.4 mg  l-1, or 260 μM (American Public Health Association, 1971).
Air-saturated phosphate buffer, as used in the above reactions, should therefore contain
more than enough oxygen to allow the complete conversion of 10 μM or 100 μM MnSO4
to MnO2 (assuming the reaction Mn2+ +
O2 + H2O --> MnO2 + 2H+).
It is possible that Jung and Schweisfurth were seeing the effects of oxygen-limitation
124
when they noticed a decrease in the rate of oxidation when 0.5 μmole Mn2+ was oxidized
in a 5-ml reaction volume. (5 ml x 260 μM = 1.3 μmole O2.)
125
FIGURE V - 1:
In vitro Mn2+ oxidation by "P. manganoxidans" MnB1-A2 crude extract: 10 μM
Mn2+, aerobic and anaerobic tubes, t = 0 - 3 hr.
Extract was added at t = 0 to begin the reaction.
126
127
FIGURE V - 2:
In vitro Mn2+ oxidation by "P. manganoxidans" A2 crude extract: 100 μM Mn2+,
aerobic and anaerobic tubes, t = 0 - 3 hr.
Extract was added at t = 0 to begin the reaction.
128
129
FIGURE V - 3:
In vitro Mn2+ oxidation by "P. manganoxidans" MnB1-A2 crude extract: 10 μM
Mn2+, anaerobic tubes exposed to air, t = 12 - 15 hr.
"Anaerobic" tubes were opened to the atmosphere at t = 13.3 hr.
130
131
FIGURE V - 4:
In vitro Mn2+ oxidation by "P. manganoxidans" MnB1-A2 crude extract: 100 μM
Mn2+, aerobic tubes and anaerobic tubes exposed to air, t = 12 - 21 hr.
"Anaerobic" tubes were opened to the atmosphere at t = 13.3 hr.
132
133
5.4. Possible Mechanisms of Manganese Oxidation in P. putida
A number of mechanisms have been proposed for bacterial manganese oxidation.
It is likely that several different mechanisms will ultimately be found (Ghiorse, 1984;
Nealson et al., 1989). Among the mechanisms proposed are oxidation by H2O2 or O2─,
lithotrophic respiration, and binding of Mn2+ to a polymer or other site on a cell, thereby
promoting autooxidation similar to the way MnOx-bound Mn2+ is autooxidized. The
relevance of these hypotheses to the case of manganese oxidation in P. putida is
discussed below.
Dubinina (1978b) proposed that bacterial manganese oxidation in Leptothrix
pseudoochracea (and, without presenting evidence, in "Arthrobacter siderocapsulatus")
came about by hydrogen peroxide oxidizing Mn2+, with catalase acting as a manganese
peroxidase. This is unlikely to be the mechanism in P. putida for the following reasons:
1) H2O2 does not need to be added to the in vitro manganese oxidation reaction in
order for MnOx to appear. Background levels of H2O2 were not measured in P. putida
crude extracts, but the catalase present would be expected to have reduced the
background peroxide concentration greatly.
2) I detected catalase (by observing bubbling after addition of a drop of 3% H2O2)
in both manganese-oxidizing and non-manganese-oxidizing crude extracts from both
starved and unstarved "P. manganoxidans" MnB1-A2 cells, and from starved P. putida
TSMC cells (a Mnx─ variant of P. putida 12633).
3) Manganese oxidation in P. putida occurs only during starvation, when reduced
carbon compounds would not be available for generation of H2O2 by their incomplete
134
reduction of O2. In Leptothrix, Mn oxidation occurs during logarithmic phase (Adams
and Ghiorse, 1985).
4) In Leptothrix, growth in rich media is weak apparently because of a buildup of
inhibitory quantities of H2O2 (Dubinina, 1978b; Ghiorse, 1984). P. putida grows very
well in rich media.
The work of Fridovich and colleagues (Archibald and Fridovich, 1981) showed
that Lactobacillus plantarum, a lactic acid bacterium, used millimolar levels of
intracellular Mn2+ as a substitute for superoxide dismutase. The Mn2+ apparently reduced
the O2─ to H2O2, while the resulting H2O2 (or intracellular NADH) then reduced the Mn3+
thus formed back to Mn2+ (MnO2 oxidizes H2O2 at neutral pH.). No manganese oxides
were observed to accumulate.
Perhaps P. putida might similarly generate Mn3+ or MnOx in order to use it to
destroy toxic H2O2 or O2─. This hypothesis seems unlikely, for the following reasons:
1) Manganese oxides do accumulate in P. putida cultures, unlike Lactobacillus.
2) Unlike Lactobacillus, P. putida has no requirement for unusually high levels
of Mn(II) in its growth media. P. putida grows well even in minimal media with no
added Mn(II).
3) Lactobacillus plantarum characteristically has no catalase or SOD. P. putida
strains characteristically contain catalase, as determined by evolution of bubbles when a
drop of H2O2 is applied to a colony or smear of cells (Palleroni, 1984). Catalase and
superoxide dismutase isozymes have been studied in two strains of P. putida (Katsuwon
135
and Anderson, 1989; other ref.), and as an aerobic species, it would be expected that all
P. putida strains possess SOD.
4) In my experiments, strongly-manganese-oxidizing P. putida strain TS-1 and its
weakly-manganese-oxidizing derivative, strain TSMC, were both tolerant of at least 10
mM H2O2 in stationary phase (data not shown).
Oxidation of manganese and iron was reported by strains of P. putida and
Alcaligenes eutrophus which are capable of oxidizing arsenite (AsO2─) to arsenate
(AsO43─) (Abdrashitova et al., 1990). Arsenite oxidation was hypothesized to be
involved with lipid peroxidation (Abdrashitova et al., 1986). The authors proposed that
Mn(II) and Fe(II) were oxidized by the same mechanism that causes arsenite oxidation.
However, they showed that arsenite oxidation in these strains took place in logarithmic
phase, rather than stationary phase (the growth phase in which their strains oxidized
manganese was not reported; Mynbaeva et al., 1990). Also, Schweisfurth (1976) detected
no manganese oxidation in a strain of P. arsenoxydans, an arsenite-oxidizing strain now
considered to be P. putida (Turner, 1954; National Collections of Industrial and Marine
Bacteria, 1990). Hence, it seems that arsenite oxidation and manganese oxidation are not
linked in P. putida.
Binding of Mn2+ to extracellular polymers of Pedomicrobium manganicum and to
a spore coat protein of Bacillus SG-1 has been proposed to be important in oxidation of
manganese in these organisms. Manganese-binding was thought to accelerate
136
autooxidation of Mn2+ by lowering the activation energy for the reaction (Ghiorse and
Hirsch, 1979; Rosson and Nealson, 1982). However, Black (1991) determined that "P.
manganoxidans" MnB-1 and MnB-5 exopolymers did not appreciably bind Mn2+.
The most profitable investigations into a mechanism of manganese oxidation are
the studies of Ehrlich and colleagues (summarized in Ehrlich, 1981 and 1990) implicating
lithotrophic Mn(II) respiration in several marine heterotrophic bacteria. Oxidation was
shown to be blocked by electron-transport inhibitors such as cyanide, azide, antimycin A,
and NOQNO. Spectrophotometric scans in the presence of azide indicated that
cytochromes were reduced in conjunction with oxidation of Mn(II) (Arcuri and Ehrlich,
1979). Manganese oxidation coupled to ATP synthesis was demonstrated in membrane
vesicles from an unidentified marine bacterium (Ehrlich and Salerno, 1990). Interaction
between a periplasmic component and membrane particles appeared to be required.
Ehrlich's results seem to be consistent with the hypothesis of Hooper and DiSpirito
(1985) that inorganic reductants are oxidized on the extracytoplasmic (periplasmic) side
of the membrane, in order to establish a proton gradient for generation of ATP.
The relationship of P. putida to the above mechanism has not been tested. In
some ways, it seems consistent: the observed extracellular deposition of MnOx (Schweisfurth, 1973a) suggests the "soluble" Mnx protein is periplasmic rather than cytosolic; the
requirement that cells be starved of other reducing agents (reduced carbon compounds)
before manganese oxidation occurs is consistent with respiratory chain involvement in
oxidation; the observed participation of oxygen in the reaction (see Chapter Four); the
higher rate of manganese oxidation activity I inconsistently observed in uncentrifuged
137
sonicates and unwashed pellets, versus centrifuged crude extracts, implies that a
membrane-associated element is important in P. putida manganese oxidation. This
hypothesis deserves to be pursued further in this organism.
138
CHAPTER SIX
SUMMARY AND CONCLUSIONS
139
CHAPTER SIX
SUMMARY AND CONCLUSIONS
In this thesis, I have elaborated on many implications of two seemingly simple
observations: one, that the previously-described manganese-oxidizing bacteria known as
"Pseudomonas manganoxidans" and "Arthrobacter siderocapsulatus" are actually
members of the common soil and water species Pseudomonas putida; and two, that the
ability to oxidize manganese seems to be common trait within the species P. putida.
Even though that ability has been reported for members of a wide variety of bacterial
genera, this is one of the few examples of manganese oxidation carried out by a member
of a common bacterial species which is not normally associated with metal oxidation.
This helps dispel the misconception that manganese-oxidizing bacteria are "unusual
organisms", specialized for the purpose. Manganese oxidation was observed in culturecollection P. putida strains isolated from a variety of habitats, selected originally for a
variety of traits. Some of the culture collection strains deposited manganese oxides only
after extended incubation, but media containing trace metals helped speed up deposition,
and many of the strains exhibited a low frequency of more-strongly-oxidizing colonies
that might be selected under the proper conditions in nature.
Whether P. putida is an important contributor to manganese oxidation in nature is
not known for certain, but the fact that such strains have been isolated from manganeseoxide-containing habitats by others in the past (by Schweisfurth as "P. manganoxidans"
and P. putida, by Zavarzin as P. eisenbergii, and by Dubinina and Zhdanov as "Arthro-
140
bacter siderocapsulatus") suggests that it is reasonable to assume that these bacteria are
important. The sheer ubiquity of P. putida and related pseudomonads in natural habitats,
combined with their demonstrated potential to oxidize manganese, implies that this
species is likely to play a significant role in manganese cycling.
This thesis has focused on strains of P. putida, but it was also noted in Chapter
Two that isolates apparently related to P. putida can also oxidize manganese. It would be
interesting to see just how narrow or how broad the capacity to oxidize manganese is
among pseudomonads.
Manganese oxidation as a stress phenomenon was investigated in Chapter Four.
The activity in "P. manganoxidans" and in many other manganese-oxidizing bacteria had
been shown earlier to be present only in stationary phase or during starvation, which
suggested that manganese oxidation might be related to other starvation-stress activities.
Starvation for carbon was shown to be much more effective at bringing about manganeseoxidation activity in P. putida than starving for nitrogen or phosphate only. Heat shock
and peroxide shock did not appear to induce the activity. The phenomenon, then, is not
likely to be a generalized stress phenomenon.
The mechanism of manganese oxidation by P. putida was shown in Chapter Four
to be similar to that of other bacteria by demonstrating that the in vitro oxidation reaction
requires oxygen, and by arguing that the putative Mnx protein is indeed an enzyme.
These results contradict two unusual claims by Schweisfurth, claims which seemed to put
this organism in a separate class from other known manganese-oxidizers.
The benefit, if any, that manganese oxidation brings to the organism is not clear.
Hypotheses of contributions to starvation-survival or resistance to oxidative stress seem
141
unlikely. Mixotrophic, as opposed to autotrophic, energy generation cannot be ruled out.
It is certainly possible that manganese oxidation in this species is an adventitious
phenomenon carried out by proteins synthesized for some other purpose, with no benefit
to the cell.
Future experiments could be pursued on many fronts, but a biochemical approach
may be the most fruitful. It should be possible to purify the protein (or proteins) that
brings about in vitro manganese oxidation. Where is the protein located? What does it
interact with? Is an electron-transport system involved that might allow ATP generation?
Detailed investigations into the biochemical mechanism of manganese oxidation in any
strain is sorely needed.
Pseudomonas putida should be a useful species in which to investigate one
mechanism of manganese oxidation in greater detail. The extensive characterization of
the manganese-oxidation activity of "Pseudomonas manganoxidans" carried out by
Schweisfurth, combined with the contributions detailed in this thesis and all the
information in the scientific literature concerning P. putida biochemistry, physiology, and
genetics, should contribute significantly to advancing our knowledge of the poorlyunderstood phenomenon of bacterial manganese oxidation.
142
APPENDIX ONE
RESULTS AND IDENTIFICATIONS FROM COMMERCIAL TEST SYSTEMS
APPENDIX ONE
RESULTS AND IDENTIFICATIONS FROM COMMERCIAL TEST SYSTEMS
The following pages list test results and identifications from two commercial
bacterial identification systems, BIOLOG and API Rapid NFT, for the strains examined
in this thesis.
The BIOLOG system (Biolog, Inc., Hayward, Calif.) consists of a microwell plate
with 95 individual carbon sources to be inoculated with a cell suspension; utilization of a
carbon source results in a change of color in a redox dye present in each well. The API
Rapid NFT system (Analytab Products, Plainview, N.Y.) is specifically designed to
identify non-fermenting Gram-negative bacteria with its 21 biochemical and nutritional
tests. Both systems use statistical methods to achieve a probabilistic identification for a
tested organism, based on strains in each system's data base. BIOLOG calculates a
"similarity" and a "distance" value for each identification; an ideal identification would
have a similarity of 1.0 and a distance of 0.0 from a reference strain.
143
144
145
146
147
148
149
150
151
APPENDIX TWO
COMPARISON OF JESSEN'S BIOTYPES WITH
P. PUTIDA/P. FLUORESCENS BIOVARS
152
APPENDIX TWO
Comparison of Jessen's Biotypes with P. putida/P. fluorescens Biovars
This appendix presents a table comparing the identifications given to the 200
strains of fluorescent pseudomonads originally collected by Jessen and reclassified by
McManus et al. (1992), Barrett et al. (1986), Champion et al. (1980), and Stanier et al.
(1966). The strains are arranged here by Jessen biotype.
Cluster assignments of McManus et al. are also included. Not all of McManus's
clusters were assigned a species name. P. fluorescens biovar VI of McManus seems to
be equivalent to P. fluorescens biovars V-1 and V-2 of Barrett.
Note that three of Jessen's strains listed in this table were included in the
manganese oxidation tests in Chapter Three (PJ 096 = ATCC 795, PJ 091 = ATCC 8209,
and PJ 090 = ATCC 950).
ABBREVIATIONS AND REFERENCES
Pp = P. putida (biovars A, B, or C)
Pf = P. fluorescens (biovars I - VI or A - L)
P.lund = P. lundensis
NONE = no identification
-- = no information
Jessen = Jessen, 1965
McManus = McManus et al., 1992
Barrett = Barrett et al., 1986
Champion = Champion et al., 1980
153
Stanier = Stanier et al., 1966
154
Jessen
Strain Biotype
McManus
Cluster ID
Barrett
ID
Champion
ID
Stanier
ID
PJ839
PJ987
PJ842
PJ499
09
09
09
09
08
08
08
08
Pp A
Pp A
Pp A
Pp A
-----
Pf L
Pf L
Pf L
Pf L
-----
PJ783
PJ873
PJ949
PJ878
10
10
10
10
08
08
08
08
Pp A
Pp A
Pp A
Pp A
Pp C
Pp C
-Pp C
-----
-----
PJ229
PJ399
PJ242
PJ530
PJ276
PJ312
PJ280
PJ305
PJ279
PJ392
PJ999
PJ891
PJ726
PJ892
PJ780
PJ006
PJ897
PJ992
PJ750
PJ096
PJ093
PJ498
PJ098
PJ997
PJ534
PJ542
PJ677
PJ089
PJ067
PJ325
PJ732
PJ091
PJ896
PJ483
PJ698
PJ983
PJ080
PJ088
PJ082
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
08
Pp A
NONE
12
Pp C
12
Pp C
12
Pp C
08
Pp A
09
Pp A
08
Pp A
08
Pp A
08
Pp A
Pp A
Pp A
Pp A
Pp A
Pp C
Pp A
Pp A
Pp A
Pp A
Pp A
Pp A
Pp A
Pp A
Pp C
Pp A
Pp A
Pp C
Pp A
Pp A
Pp A
Pp A
Pp A
Pp C
Pp A
Pp A
Pp A
Pp A
Pp A
Pp A
Pp A
-Pp C
--Pp A
NONE
Pp A
Pp A
Pp A
----------------------------------------
-------------------(ATCC 795)
-----------(ATCC 8209)
--------
155
PJ303
11 (cont'd)
Jessen
Strain Biotype
PJ324
11 (cont'd)
PJ090
11
PJ275
11
08
Pp A
McManus
Cluster ID
08
Pp A
08
Pp A
08
Pp A
Pp A
Barrett
ID
Pp A
NONE
Pp C
-Champion
ID
----
-Stanier
ID
-(ATCC 950)
--
PJ092
PJ777
PJ795
12
12
12
09
09
09
Pp A
Pp A
Pp A
Pp A
Pp A
--
----
----
PJ790
PJ235
13
13
09
09
Pp A
Pp A
Pp A
Pp A
---
---
PJ994
PJ879
PJ993
14
14
14
07
10
09
Pp B
Pf VI
Pp A
Pp B
-Pp B
----
----
PJ948
PJ991d
PJ966
PJ233
15
15
15
15
07
07
07
07
Pp B
Pp B
Pp B
Pp B
Pp B
Pp B
---
-----
-----
PJ764
16
10
Pf VI
--
--
--
PJ707
PJ874
PJ989
17
17
17
07
07
10
Pp B
Pp B
Pf VI
Pp B
Pp B
--
----
----
PJ855
PJ995
PJ977
PJ684
PJ982b
PJ701
PJ867
PJ835
PJ704
18
18
18
18
18
18
18
18
18
10
10
10
12
11
10
10
10
--
Pf VI
Pf VI
Pf VI
Pp C
P.lund
Pf VI
Pf VI
Pf VI
--
Pf V-1
NONE
Pf V-1
-NONE
Pf V-1
Pf V-1
Pf V-1
Pf V-1
----------
----------
PJ760
PJ877
PJ946
PJ770
PJ755
19
19
19
19
19
10
10
10
07
10
Pf VI
Pf VI
Pf VI
Pp B
Pf VI
Pf V-1
Pf V-1
NONE
Pp B
--
------
------
PJ724
20
11
P.lund
NONE
--
--
PJ985
PJ953
PJ856
PJ980
21
21
21
21
10
10
10
10
Pf VI
Pf VI
Pf VI
Pf VI
Pf V-1
Pf V-1
Pf V-1
Pf V-1
-----
-----
156
PJ984
21
10
Pf VI
Pf V-1
--
--
PJ310
PJ057g
23
23
10
01
Pf VI
Pf V-1
--
---
---
McManus
Cluster ID
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
Barrett
ID
Pf V-1
Pf V-2
-Pf V-2
Pf V-2
Pf V-1
Pf V-1
Pf V-2
Pf V-2
Jessen
Strain Biotype
PJ952
24
PJ389
24
PJ391
24
PJ277
24
PJ958
24
PJ715
24
PJ870
24
PJ974
24
PJ942
24
Champion
ID
----------
Stanier
ID
----------
PJ950
25
10
Pf VI
Pf V-1
--
--
PJ981
PJ254
PJ956
PJ965
PJ861
PJ295
PJ700
PJ814
PJ955
PJ387
PJ954
26
26
26
26
26
26
26
26
26
26
26
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
10
Pf VI
NONE
10
Pf VI
10
Pf VI
10
Pf VI
Pf V-1
-Pf V-1
Pf V-1
Pf V-1
---Pf V-1
-Pf V-1
------------
------------
PJ690
27
10
Pf VI
Pf V-1
--
--
PJ767
PJ771
PJ259
28
28
28
12
10
10
Pp C
Pf VI
Pf VI
-Pf V-1
Pf V-1
----
----
PJ943
PJ976
29
29
07
07
Pp B
Pp B
---
-Pf F-II
---
PJ978
PJ962
30
30
05
05
Pf V
Pf V
NONE
NONE
---
---
PJ961
31
07
Pp B
--
--
--
PJ868
32
02
Pf III
--
Pf C
--
PJ197
34
NONE
--
--
--
PJ894
PJ890
PJ967
36
36
36
NONE
06
03
Pf IV
----
-Pf F-II
--
----
157
PJ951
PJ973
37
37
07
07
PJ816
38
PJ990
39
Jessen
Strain Biotype
PJ887
40
Pp B
Pp B
---
---
---
NONE
--
--
--
06
--
Pf, unassgn.
--
McManus
Cluster ID
06
Barrett
ID
--
Champion
ID
--
Stanier
ID
--
PJ944
PJ979
41
41
06
06
---
Pf K
Pf K
---
PJ754
42
NONE
--
--
--
PJ094
PJ095
43
43
09
09
Pp A
Pp A
NONE
NONE
---
---
PJ797
PJ738
44
44
11
11
P.lund
P.lund
NONE
Pf V-3
---
---
PJ758
PJ727
45
45
11
11
P.lund
P.lund
NONE
Pf V-3
---
---
PJ940
PJ805
PJ768
PJ808
46
46
46
46
12
12
-12
Pp C
Pp C
-Pp C
Pp C
Pp C
Pp A
Pp C
-----
-----
PJ728
PJ066
PJ720
PJ893
PJ063
PJ899
PJ922
47
47
47
47
47
47
47
11
11
11
11
11
11
11
P.lund
P.lund
P.lund
P.lund
P.lund
P.lund
P.lund
Pf V-3
Pf V-3
Pf V-3
NONE
Pf V-3
Pf V-3
--
--------
--------
PJ815
48
02
Pf III
--
--
--
PJ864
PJ827b
PJ819
PJ832
PJ905
PJ716
PJ683
PJ132
PJ673c
PJ823a
PJ933
49
49
49
49
49
49
49
49
49
49
49
02
Pf III
NONE
02
Pf III
--02
Pf III
02
Pf III
02
Pf III
02
Pf III
02
Pf III
NONE
02
Pf III
------------
---Pf C-IV
-Pf C-III
-Pf C-V
----
---Pf C
Pf C
-------
158
PJ236
PJ245
PJ253
PJ709
PJ916
PJ837
PJ682
PJ686
PJ693
PJ824
49
49
49
49
49
49
49
49
49
49 (cont'd)
Jessen
Strain Biotype
PJ834
49 (cont'd)
PJ848
49
PJ969
49
02
02
-02
02
02
-----
Pf III
Pf III
-Pf III
Pf III
Pf III
-----
McManus
Cluster ID
-------
----------Barrett
ID
----
Pf C-V
Pf C
-Pf C-III
-------
Pf C
-Pf C
---Pf C
Pf C
Pf C
Pf C
Champion
ID
----
Stanier
ID
Pf C
Pf C
Pf C
PJ880
PJ072
PJ706
PJ238
50
50
50
50
-02
-02
-Pf III
-Pf III
-----
Pf C-II
Pf C-V
Pf C-II
Pf C-V
-----
PJ699
PJ761
51
51
02
02
Pf III
Pf III
---
Pf C-III
--
---
PJ687
PJ931b
PJ691
PJ681
PJ70
PJ929
PJ274
PJ692
52
52
52
52
52
52
52
52
02
02
02
02
-----
Pf III
Pf III
Pf III
Pf III
-----
--
Pf C-III
-------
-------
----Pf C
Pf C
Pf G
Pf G
PJ068c
PJ836
PJ270
53
53
53
01
01
01
Pf V-6
Pf V-6
Pf V-6
----
----
PJ751
PJ282
PJ843
PJ694
54
54
54
54
01
01
NONE
01
Pf V-6
Pf V-6
-Pf V-6
-----
-----
PJ829
55
NONE
--
--
PJ730
56
02
Pf III
Pf C-V
--
--
PJ077g
PJ725
57
57
01
NONE(11-12) --
-NONE
---
--
PJ828
59
03
--
--
--
Pf IV
159
PJ854
60
NONE
--
--
--
PJ185
PJ187
PJ188
PJ251
PJ283
PJ362
PJ365
PJ379
PJ380
PJ381
PJ384
61
61
61
61
61
61
61
61
61
61
61 (cont'd)
------------
------------
------------
Pf B
Pf B
Pf B
Pf B
Pf B
Pf B
Pf B
Pf B
Pf B
Pf B
Pf B
Jessen
Strain Biotype
PJ672
61 (cont'd)
PJ833
61
PJ851
61
PJ883
61
------------
McManus
Cluster ID
---------
Barrett
ID
-----
Champion
ID
-----
Stanier
ID
Pf B
Pf B
Pf B
Pf B
PJ383
62
--
--
--
--
Pf B
PJ073
PJ079
PJ139
PJ160
PJ227
PJ239
PJ288
PJ290
PJ302
PJ311
PJ367
PJ368
PJ372
PJ376
PJ722
PJ776
PJ826
PJ849
PJ885
63
63
63
63
63
63
63
63
63
63
63
63
63
63
63
63
63
63
63
--------------------
--------------------
--------------------
--------------------
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
Pf A
160
REFERENCES
161
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Adams, L. F. and W. C. Ghiorse. 1988. Oxidation state of manganese in the manganese
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