Table of Contents
Abbreviations ............................................................................................................... 3
Chapter 1 ........................................................................................................................... 5
Introduction .................................................................................................................. 5
Archaea ........................................................................................................................ 6
Archaea: phylogeny ..................................................................................................... 7
History .......................................................................................................................... 8
Halobacteria ................................................................................................................. 9
Hypersaline Lakes ...................................................................................................... 10
Adaptation mechanisms ............................................................................................. 12
The Square Haloarchaea of Walsby (SHOW) ........................................................... 13
Cultivation of “uncultivables”.................................................................................... 13
Isolation ...................................................................................................................... 15
Chapter 2 ......................................................................................................................... 18
Materials and Methods ............................................................................................... 18
Sample Collection ...................................................................................................... 19
Microscopic Observation ........................................................................................... 19
Medium Formulations ................................................................................................ 19
DBCM1 ................................................................................................................. 19
DBCM2 ................................................................................................................. 19
MGM ..................................................................................................................... 20
Incubation Conditions ................................................................................................ 20
Liquid Medium ...................................................................................................... 20
Plate Incubation Conditions: ................................................................................ 20
Extinction cultures:................................................................................................ 21
Standard PCR Protocols ........................................................................................ 21
Polymerase Chain Reaction (PCR) Amplification of Isolate 16S rDNA .............. 22
PCR Protocol DB194 ........................................................................................... 22
DB195 ................................................................................................................... 22
Multiplex PCR Protocol DB239............................................................................ 23
PCR Cleanup ......................................................................................................... 23
Sequencing PCR .................................................................................................... 23
DNA visualisation: ................................................................................................ 24
Clone library generation ........................................................................................ 24
16S rRNA gene identification and phylogenetic tree reconstructions. ...................... 24
Phenotypic tests ......................................................................................................... 25
Media and growth conditions .............................................................................. 25
Anaerobic respiration ........................................................................................... 25
Antibiotic sensitivity ............................................................................................. 26
Casein hydrolysis ................................................................................................. 26
Catalase Test.......................................................................................................... 26
GC content and DNA-DNA hybridisation ........................................................... 27
Indole test .............................................................................................................. 27
Lipids ..................................................................................................................... 27
Magnesium Optima ............................................................................................... 27
Oxidase Test .......................................................................................................... 27
Salinity Optima...................................................................................................... 28
Substrate utilisation and acid production .............................................................. 28
Temperature Optima.............................................................................................. 28
Chapter 3 ......................................................................................................................... 29
Characterisation of Natronomonas-like, and Halogeometricum-like isolates, and
isolates related to the Deep-10 phylotype (Antarctic Deep Lake) ............................. 29
Introduction ..................................................................................................................... 30
Part 1: Isolates 1.15.5, 2.27.5, 5.24.4 and 6.14.5 ....................................................... 31
Introduction ................................................................................................................ 31
Results and Discussion............................................................................................... 32
Part 2: Isolates 4.03.5 and 8.8.11 ............................................................................... 40
Natronomonas-like group........................................................................................... 40
Results and Discussion............................................................................................... 40
Part 3: Isolate 2.24.4................................................................................................... 46
Results and Discussion............................................................................................... 46
Chapter 4 ......................................................................................................................... 50
Isolation of the Square Haloarchaea of Walsby ......................................................... 50
Overview ......................................................................................................................... 51
Solid medium cultivation ...................................................................................... 51
Liquid isolation ..................................................................................................... 51
Low-substrate liquid isolation ............................................................................... 52
Screening techniques ............................................................................................. 52
SHOW cultivation using natural water extinction culturing ................................. 53
Passaging of mixed SHOW culture............................................................................ 54
Improving SHOW isolation medium ......................................................................... 54
Low substrate medium supplementation – amino acids and acetate ..................... 54
Low substrate medium supplementation – pyruvate ............................................. 55
SHOW isolation ......................................................................................................... 56
SHOW medium variation and optimisation .......................................................... 59
Isolate Purity .............................................................................................................. 60
Microscopic examination ...................................................................................... 61
RFLP ..................................................................................................................... 61
Sequencing ............................................................................................................ 62
Isolate Selection ......................................................................................................... 62
Chapter 5 ......................................................................................................................... 64
Characterisation of Haloquadratum walsbyi .............................................................. 64
Microscopy................................................................................................................. 65
Macroscopic appearance ............................................................................................ 67
Further characteristics ................................................................................................ 67
Bibliography: .................................................................................................................. 75
Abbreviations
aa
amino acid/s
ATCC
American Type Culture Collection
BLAST
Basic Local Alignment Search Tool
bp
base pair(s)
BSA
Bovine Serum Albumin
C
Celsius
DNA
deoxyribonucleic acid
dNTP
deoxyNucleotide TriPhosphate
DSM
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
(German Collection of Microorganisms and Cell Cultures)
EDTA
ethylene diamine tetra-acetic acid
EM
electron microscope
EtBr
ethidium bromide
G+C
guanosine plus cytosine
JCM
Japan Collection of Microorganisms
hr
hour
kb
kilobase pairs
MGM
Modified Growth Medium
min
minute
NCBI
National Center for Biotechnology Information
nt/s
nucleotide/s
OD
optical density
PCR
polymerase chain reaction
PHA – Polyhydroxyalkanoate
PHB – Polyhydroxybutyrate
RNA
ribonucleic acid
rpm
revolutions per minute
rRNA
ribosomal RNA
sec
second
spp.
species
SW
salt water
TE
Tris-EDTA
vol
volume
wt
weight
X-gal
5-bromo-4-chloro-3-indolyl--galactopyroanoside
Chapter 1
Introduction
Archaea
The advent of molecular techniques applied to natural ecologies heralded a dramatic
expansion in our understanding of the diversity of microbial populations. Early
molecular studies began with protein sequencing, and later moved to nucleotide
sequencing and then specifically the 5S universally-conserved ribosomal RNA gene,
and later again to the larger and more complex (and current benchmark) 16S rRNA
gene. This last gene codes for the 16S small subunit rRNA ribosome component, an
essential part of all living cells. The genetic sequence for the 16S rRNA gene is highly
conserved, but with sufficient slow changes that its many variants may be compared to
determine phylogenetic relationships between organisms.
Molecular techniques have provided a radically different picture of ecological diversity
compared to traditional, cultivation-based techniques. Based on rRNA gene
phylogenetic trees, most of Earth’s biodiversity is microbial (Hugenholtz et al., 1998).
These molecular studies have largely been based on the 16S rRNA gene, although other
genes have also been used. It was through molecular studies that the Archaea were first
realised to be distinct from the other prokaryotic Domain, the Bacteria; Woese and Fox
in 1977 found that the universal phylogenetic tree did not correspond to the expected
bifurcated Bacteria/Eukaryote pattern but instead consisted of the three Domains
recognised today. (Woese et al.,1990). Archaea are now fully accepted as the third
Domain of life alongside Bacteria and Eucarya, and exhibit features of both of the other
Domains, as well as having their own unique characteristics. Current phylogenetic
studies indicate that the Bacteria seceded from the Eucarya and Archaea, before the
Archaea and Eucarya themselves diverged into the present tri-Domain arrangement.
On account of their unique evolutionary history, Archaea represent a remarkable
opportunity to study evolutionary relationships and the development of life. Archaea are
also, in their own right, a diverse and significant part of the biosphere, particularly in
“extreme” conditions such as high-temperature, pressure, or salinity locales, although
their distribution is otherwise as broad as Bacteria.
The separation of Domains is supported by other characteristics than just 16S rRNA
gene sequence variation, including unique glycerol isopranyl ether-linked lipids specific
to Archaeal cell envelopes (and absence of acyl ester lipids) (Boone and Castenholz,
2001, Seghal et al., 1962), as well as the nature of transcription systems, being
dissimilar to both Bacteria and Eukarya, but with features of both (Soppa, 1999).
Ribosomal RNA gene sequencing revealed many of the shortcomings of purely
morphological and biochemical based classifications, and as such, this form of
phylogenetic classification has become the preferred method of assessing diversity,
identity and evolutionary relationships in the majority of microbial communities and
also for cultivated organisms. However, formal classification still requires a range of
taxonomic features to be considered, including membrane lipids, cell morphology, ionic
requirement (both H+ and salts) and growth characteristics, as 16S rRNA gene sequence
alone is not necessarily indicative of phenotype.
While there have been some counterarguments against the monophyly or branch point
of the Archaea (Gupta, 1998; Rivera and Lake, 1992), the current Domain divisions
appear robust and are strongly supported by the currently available data.
Archaea: phylogeny
The Archaeal Domain consists of four phlya. The two major phyla are the
Crenarchaeota and the Euryarchaeota. In general, they are separated by phenotype as
well as by molecular means; the Crenarchaeota consist of the extremely thermophilic,
sulphur metabolising groups such as Thermoproteales, Desulfurococcales and
Sulfolobales. In contrast, the Euryarchaeota is a much more diverse phylum, including
the methanogens, extreme halophiles and hyperthemophiles, represented by classes such
as the Halobacteria, Archaeoglobi, Methanobacteria and Thermoplasmata.
The remaining two phyla contain very few known representatives; the Korachaeota and
Nanoarchaeaota. These phyla are both recent additions to the Archaea; Korarchaeaota
was recognised in 1996 (Barns et al., 1996) and does not contain any isolated
organisms, and is only represented by 16SrRNA gene sequences recovered from the
environment. Its distribution is very limited, appearing thus far only in high temperature
springs and deep sea vents (Auchtung et al., 2006). The Nanoarchaeaota includes only
one organism, which has been isolated in binary culture with Ignicoccus spp. (Huber et
al, 2003) However, the longevity of this phylum appears in doubt, with recent studies
arguing the sole organism in the clade (Nanoarchaeum equitans) is in fact a fastevolving member of the phylum Euryarchaeaota (Brochier et al., 2005).
History
Haloarchaea, or halobacteria as they have been called, have been the subject of human
interest for centuries. The colouration of brines was recorded by the ancient Chinese
(Baas-Becking, 1931) and also the Romans (Jones, 1963). More recently, halobacteria
were studied for their role in food spoilage; with salting of food a common method of
preservation, these organisms were unusual in their ability to grow in these harsh
conditions. The strain Halobacterium cutirubrum (“red skin”) was isolated in 1934
(Loackhead, 1934, Boone and Castenholz, 2001) and, along with other strains such as
Pseudomonas salinaria (Harrison and Kennedy, 1922; Boone and Castenholz, 2001)
and Halobacterium halobium (Petter, 1931; Boone and Castenholz, 2001), were in fact
Halobacterium salinarum strains, a haloarchaeon that has a particular prevalence in
proteinaceous salted products.
Later researchers realised haloarchaea could be isolated from almost any hypersaline
environment, including salt mines and deposits (McGenity et al., 2000), on beach sand
particles (Onishi et al., 1985), and from crude solar salt, as well as the aforementioned
salted products (hides and other food products) (Boone and Castenholz, 2001).
Haloarchaea, and particularly, the family Halobacteriaceae are members of the Domain
Archaea, and comprise the majority of the prokaryotic population in hypersaline
environments (Oren 2002). There are currently 15 recognised genera in the family
(Gutierrez et al., 2002). The domain Bacteria can comprise up to 25% of the prokaryotic
community, but is more commonly a much lower percentage of the overall population
(Anton et al., 2000). At times, the alga Dunaliella salina can also proliferate in this
environment (Casamayor et al., 2002).
A comparatively wide range of taxa have been isolated from saltern crystalliser ponds,
including members of the following genera: Haloferax, Halogeometricum, Halococcus,
Haloterrigena, Halorubrum, Haloarcula and Halobacterium families (Oren 2002).
However, the viable counts in these studies have been small when compared to total
counts, and the numerical significance of these isolates has been unclear. Recently it has
become possible to determine the identities and relative abundances of organisms in
natural populations, typically using PCR-based strategies that target 16S rRNA genes.
While comparatively few studies of this type have been performed, results from the
majority of these suggest that some of the most readily isolated and studied genera may
not in fact be significant in the in-situ community. This is seen in cases such as the
genus Haloarcula, which is estimated to make up less than 0.1% of the in situ
community (Anton et al., 1999) but commonly appears in isolation studies. An
exception to this was seen in an Australian saltern, in which it was found to be possible
to isolate and cultivate the majority of the diversity observed by 16S rRNA gene
libraries (Burns et al., 2004). Even this study, however, failed to isolate one of the more
abundant organisms in this lake, the Square Haloarchea of Walsby.
Halobacteria
The Halobacteriaceae are by no means the only prokaryotic representatives at
saturating salinities. The halophilic bacterium Salinibacter ruber can make up from 527% of the prokaryotic population (Anton et al., 2000). While at saturation, particularly
for thassohaline lakes, this is generally the only or dominant member of the Bacterial
community, in some hypersaline environments Salicola marasensis can make up to
10% of the prokaryotic community (Maturrano et al., 2006). At lower salinities, the
Domain Bacteria are more abundant than Archaea, although both halophilic Archaea
and Bacteria are represented across the entire salinity range from seawater to saturation.
Hypersaline Lakes
The oceans are about 3.5% (w/v) salt, mainly sodium chloride (NaCl), and contain the
majority of halophilic microorganisms, including a wide variety of Bacteria and
Eukarya (eg. protists and algae), and some Archaea. Moderate halophiles (both Bacteria
and Archaea) exist in the range from above seawater to approximately 15% salts, while
the extremely halophilic microrganisms grow from this point up to saturation (around
37%). Previous studies have demonstrated that the dominant microorganisms in lakes
with
salinities
approaching
saturation
are
haloarchaea
from
the
Family
Halobacteriaceae (Benlloch et al., 2002; Oren 2002).
While the diversity is low in these environments, the productivity and cell densities can
be quite high. The distinctive pink colouration of these lakes is reflective of the fact that
rather than being hostile to life, hypersaline lakes harbour large and active populations
of microorganisms. The colour in these lakes is brought about from pigment contained
in the haloarchaea, and to a lesser extent, halophilic algae and halobacteria. Even in
lakes with saturated salt concentrations (when not dried out to salt pans, or after recent
rains, salt lakes are usually near saturation, with a thick layer of crystalline salt as the
lake bed) microbial cell densities can reach up to 107 to 108 cells per millilitre. These
populations reflect a lack of predation and often quite high nutrient levels (Oren, 2002).
Saline lakes are widely recognized as highly productive aquatic habitats, harbouring
specialised assemblages of species and often supporting large populations of both
migrating and breeding birds (Rodriguez-Valera, 1988).
Natural hypersaline environments are a significant part of Australia’s landscape and
ecology. Of Australia’s largest five lakes, four are salt lakes - Lake Eyre is 9500 km2
when full, and the next two largest salt lakes in Australia, Lake Torrens and Lake
Gairdner are 5745 and 4351 km2 respectively. The Northern Territory’s (salt) Lake
Amadeus is the fifth largest lake on the continent at 1032 km2 (Geoscience, 2002). In
addition, artificial hypersaline lakes are abundant, being created to alleviate the
country’s salinity problems and also for economic reasons, such as salt harvesting. It is
important to have an understanding of the microbial ecology of these environments,
given the increasing occurrence, and importance of these environments in Australia, and
also internationally; we note that salt lakes make up about the same volume of the
world’s bodies of water as freshwater systems (excluding oceans).
Salt lakes usually form when a body of water becomes landlocked, in which water
cannot exit the lake except by evaporation, leading to an increase in the salinity of the
remaining water. The ionic composition of the lake is therefore dependent on the
composition of the source water, itself often dependent on local and regional geology.
There are considered to be two main types of salt lake. The first is thalassohaline, in
which the ionic ratios in the lake are close to that of seawater, and therefore containing
sodium as the dominant cation. The other is athalassohaline, in which the composition
and/or origin is unlike that of seawater. Athassohaline lakes are mainly soda lakes,
containing high levels of divalent cations and bi-carbonate. They also include some of
unusual composition such as the Dead Sea in Israel, which has a high magnesium
content. Thassohaline lakes can occur as natural inland salt lakes, whose source can be
salt mine drainage water, brine springs from underground salt deposits, coastal splash
zones and tide pools, as well as solar salterns (Litchfield and Gillevet, 2002), while
athalassohaline lakes are usually reflective of the local geochemistry.
As might be expected, organisms populating these lakes have physiologies optimised to
take advantage of the local conditions; by way of example, the genera Natronomonas,
Natronococcus and Natronobacterium, typically found in hypersaline soda (alkaline)
lakes require high pH and low Mg2+, while Haloferax volcanii, isolated from the
magnesium-rich Dead Sea has a relatively high Mg2+ requirement of 0.5-1.5 M. (Tindall
& Truper, 1986).
The low total diversity in the lakes makes hypersaline lakes an ideal candidate system
for ecological studies. Investigation into subsystems in this environment such as
nutrient cycling, predator/prey, and population dynamics, is a more manageable task
than in a high diversity environment such as soil, which can contain as many as 12-
18,000 species per gram (Torsvik et al., 1996). In particular, multi-pond solar salterns
represent ideal candidate model systems due to their managed nature, in which salt
concentrations are kept relatively constant over time, in contrast to natural systems
which are more susceptible to external variables such as climatic variation.
Additionally, salterns exist around the world, albeit under somewhat different
conditions. This provides a greater degree of international comparability than most
natural systems.
Adaptation mechanisms
Few organisms have been able to successfully adapt to and occupy the extreme
environment of high salinity. Most halophilic and all halotolerant organisms expend
energy to exclude salt from their cytoplasm to avoid protein aggregation (‘salting out’).
In order to survive the osmotic stress caused by the high salinities, halophiles employ
two differing strategies to prevent dessication (through osmotic movement of water out
of their cytoplasm). Both strategies work by increasing the internal osmolarity of the
cell to closer match that of the surrounding water. In the first (that employed by the
majority of Bacteria, some Archaea, yeasts, algae and fungi) specific low molecular
weight organic compounds are accumulated in the cytoplasm. These compounds are
known as compatible solutes and can be synthesised de novo or accumulated from the
environment (Santos and da Costa, 2002). The most common compatible solutes are
neutral or zwitterionic and include amino acids, sugars, polyols, betaines and ectoines,
as well as deriavatives of some of these compounds.
The second, more radical, adaptation involves the selective influx of potassium ions into
the cytoplasm. This adaptation is restricted to the moderately halophilic bacterial Order
Halanerobiales, the extremely halophilic archaeal Family Halobacteriaceae and the
extremely halophilic bacterium Salinibacter ruber. The presence of this adaptation in
three distinct evolutionary lineages suggests convergent evolution of this strategy, it
being unlikely to be an ancient characteristic retained in only scattered groups or
through massive lateral gene transfer (Santos and da Costa, 2002). The primary reason
for this is that the entire intracellular machinery (enzymes, structural proteins etc) must
be adapted to high salt levels, becoming highly acidic (Grant et al., 2001; Danson and
Hough, 1998), whereas in the compatible solute adaptation little or no adjustment is
required to intracellular macromolecules. In fact, the compatible solutes often act as
more general stress protectants as well as just osmoprotectants (Santos and da Costa,
2002).
The Square Haloarchaea of Walsby (SHOW)
This organism was first observed in samples taken from a salt flat, or sabkha, by A.
Walsby in 1980 (Walsby, 1980). It is a thin, flat, gas-vacuolated cell with square
morphology (Walsby, 1980, Kessel and Cohen, 1982). Obviously of interest on account
of its unusual shape – square, and also so thin its membranes are almost touching, with
a total thickness in the order of 0.1 µm (Stoeckhenius, 1981). It is also one of the most
dominant phylotypes seen in salterns and salt lakes, making up to 80% of the
haloarchaeal community. Owing to these factors, many attempts have been made to
cultivate it (Walsby, 1980, Benlloch et al. 2001; Rosello-mora, 2003), but despite nearly
a quarter-century passing since its discovery, it has remained uncultivated. This has not
prevented significant research being performed on it; some of which has indicated that it
is relatively easily to gather enriched samples from saltern water using low-speed
centrifugation, while more recently, studies involving FISH (Anton et al., 1999) and
autoradiography (Rossello-Mora et al., 2003) have aided in elucidating probable
substrates for the organism.
While there are other haloarchaea that are sometimes seen in a square shape, these
organisms do not exhibit the true square shape of the SHOW organism, having greater
pleomorphism typical to the haloarchaea (Boone and Castenholz, 2001). Further, nor
are they, in general, dominant in the haloarchaeal community in the way the SHOW
organism is.
Square cell shapes have not been observed in environments other than hypersaline
water; perhaps the increased freedom afforded by non-rigid cell walls, coupled with the
ability to tolerate a wide range of internal osmolarities permits the unusual shapes often
seen in haloarchaea.
Cultivation of “uncultivables”.
It has been a longstanding quest for microbiologists to bring into culture representative
isolates of the microbial world. It has been recognised since the early history of
microbiology itself that this is no mean feat; the “great plate count anomaly” was first
described by Razumov in 1932 (Razumov, 1932; Staley & Konopka, 1985), in which
the number of colonies on a plate is orders of magnitude lower than the cell count as
observed by direct microscopy.
There are some clear reasons for the recalcitrance of most microorganisms to be brought
into culture. The first is simply that scientists are often unable (whether through
ignorance or technical limitation) to provide an adequate growth medium that closely
recreates their natural environment. This may be due to a lack of knowledge about the
requirements of the target organism(s), leading to the omission of important substrates,
nutrients or other growth factors. Even the choice of very basic components such as the
gelling agent for solid media can play a role in improved cultivability (Sait et al., 2002).
A second reason may be that insufficient time is provided for organisms to establish
colonies or initiate growth after transition from their natural environment to laboratory
setting. This phenomenon is particularly often observed in the plate cultivation
technique, where it is observed that greatly increased viable counts are obtained when
the incubation time is increased appreciably over the commonly-used incubation times
of days to a week or two (Ochsenreiter et al., 2002). This effect was also observed when
cultivating haloarchaea, with 2-3 times higher viable counts obtained after twelve weeks
incubation compared to those obtained after the (still longer than normal) incubation
time of three weeks (Burns et al, 2004).
Finally, researchers may not properly identify that they have succeeded in isolating a
novel organism. This can occur due to reasons such as the colony being smaller than
expected, or a liquid culture not reaching visible turbidity (Leadbetter, 2003). However,
new or careful screening techniques can be used to overcome these oversights, such as
the use of microscopes to detect microcolony formation or proliferation at low cell
densities (Ferrari et al., 2005; Connon and Giovannoni, 2002),
Isolation
Isolation of representative populations of the target environment offers significant
potential both for the understanding of the ecological system and also for research into
one of the most easily accessed Archaea, the Haloarchaea, but is limited by the lack of
cultured representatives of the dominant species present in the environment as identified
by Fluorescent In-Situ Hybridisation (Anton et al., 1999), clone library and microscopic
analysis.
However, by combining molecular analytical methods with newer cultivation
techniques, it may be possible to gain insight into the community structure and culture a
greater percentage of the population such that most major groups are represented. At
present many currently isolated representatives are thought to be only minor
components of the total community (Anton et al., 1999), although a recent study
indicated that the majority of organism observed could be cultivated (Burns et al.,
2004).
It has been shown in soil that reduced sample inoculum increases the cultivation success
rate, presumably due to reduced competition (Olsen and Bakken, 1987). Further, the
same study found that lower nutrient levels lead to greater cultivation success. Both of
these approaches have been applied to various extents to saltern crystallizer ponds with
varying but limited to reasonable degrees of success (Benlloch et al., 2001; Burns et al.,
2004).
However, the currently popular alternative to cultivation-based techniques for surveying
communities, molecularly-based studies, are subject to their own biases and limitations.
In particular, the majority of studies provide no other information about the organism
than its 16S rRNA gene sequence, relative relatedness to other organisms by 16S
comparison, and perhaps relative abundance in a community. The majority of studies
also obtain clones after a possibly bias-inducing PCR-amplification step, although some
clone directly from the environment (Hugenholtz et al., 1998).
In either case, biases can include the fact that many organisms contain more than one,
often dissimilar, 16S rRNA genes (including the Haloarchaeal genus Haloarcula
(Boone and Castenholz, 2001; Klappenbach et al., 2000), leading to an overestimate of
true abundance and/or relative diversity present (Farrelly et al., 1995). There can also be
DNA extraction biases, in which some organisms release their DNA more readily than
others, leading to some groups being under or overrepresented (Frostegard et al., 1999).
Despite these drawbacks, molecular techniques generally allow for a more complete
analysis of microbial communities than cultivation-based approaches alone, as standard
cultivation techniques are estimated to recover less than 1% of the diversity of
microorganisms in nature (Hugenholtz et al., 1998).
The metagenomic studies recently undertaken address some of the deficiences of using
only 16S rRNA genes to survey communities by also gleaning genetic information that
may indicate possible metabolic functions, lysogenic viruses and other functional
information. However, scaffolding the metagenomic information, or properly assigning
sequence information to its correct host may still be fraught with pitfalls. Additionally,
in relatively understudied environments much of the sequence information may not
correlate to any existing data, rendering it essentially meaningless.
Isolation of pure strains is an essential requirement to properly study organisms as it
allows characterisation of their metabolic capabilities and hence an insight into their
role in the natural environment. Functional genes and promoters can be mapped and
understood, adding value to future metagenomic studies. In addition, pure cultures
allow investigations into the interaction of specific strains and/or other organisms in the
home ecology.
For example, pure strains are required for viral isolation to be undertaken. Viral
populations are believed responsible for the majority of nutrient cycling and population
turnover in hypersaline lakes through the lysis of infected cells. Bacterivory is absent in
ponds above about 20% salts, while viruses begin to be detectable at salinities above
15% (Pedros-Alio et al., 2000). Viral lysis is estimated to affect 7% of the total
population per day, significant given the low doubling time of about 2 days for the insitu prokaryotic population.
Viruses present a possible problem with isolation in liquid media due to the fact that
viral loads are estimated at up to 10 times that of the prokaryotic population. Hence, an
extinction culture containing only one cell may contain 10 times as many phage, some
of which may have the potential to lyse the cell. This possibility is increased when the
fact that the dominant organisms (and hence most likely to be at the end-point of the
dilution series) are most likely to be the hosts for the largest phage populations.
However, it is also possible that the dominant organisms are abundant because they are
resistant to the most common phages; this theory is yet to be tested, on account of the
lack of pure isoaltes of the dominant organisms, and consequently also their viruses.
The dominant uncultured organism, the Square Haloarchaea of Walsby, is presumably
responsible for a significant part of the nutrient cycling in the environment.
Improvements to standard cultivation techniques need to be made to ensure that this
dominant microbial species present be isolated and characterised if any meaningful
analysis of the natural ecology is to be made. In the case of the Halobacteriaceae, and
hypersaline environments in general, it is alarming that after several decades that one of
the most important organisms remains uncultivated.
It is the aim of this investigation to isolate and characterise the Square Haloarchaea of
Walsby, and additionally to characterise novel isolates of previously unknown, but
numerically significant organisms present.
Chapter 2
Materials and Methods
Sample Collection
Sample collection involved collecting water by submerging a sterile Schott bottle 5cm
beneath the surface of a froth and debris free edge of one of the Cheetham Salt Works
(Geelong, Victoria; 38°09.841' S, 144°25.274' E) crystallisers. All samples were
collected during the full phase of saltern harvesting (as opposed to after salt harvesting
had taken place).
Microscopic Observation
Cultures and samples were observed with a Leitz Diaplan microscope at 1000x
magnification using both phase contrast, and UV illuminescence in conjunction with
acridine orange (3,6-bis(dimethylamino)-acridinium chloride) (AO) staining.
When viewing cells with UV illuminescence, a BP 450-490 nm exciter filter, 510 nm
beam splitter and an LP 520 nm barrier filter were used. Cells were stained with AO by
adding AO to the sample to a final concentration of 10 µg / mL, as well as a
fluorescence anti-quenching agent, para-phenylene diamine (Sigma) to a final
concentration of 0.5 %. Visibly turbid cultures were observed without concentration,
while those with little or no visible turbidity were concentrated by means of
centrifugation and re-suspension in a smaller volume. Five µl of sample was spotted
onto normal glass slides when using a full cover slip; alternatively, multiple samples
were observed on the same slide using 2 µl of sample and 22 mm x 22 mm cover slips.
Medium Formulations
DBCM1
Identical to DBCM2 (see next), with the exception that Yeast Extract and Peptone are at
1/10th the level of that in DBCM2, and without any added substrate (i.e, pyruvate). This
is essentially a minimal medium, used primarily for characterization, and is ill-suited for
general cultivation and passaging of strains.
DBCM2
This medium is described in detail in The Halohandbook, chapter 2.8.2.
Salts (NaCl; 180 g, MgCl2.6H2O; 25 g, MgSO4.7H2O; 29 g, KCl; 5.8 g) were dissolved
in 950ml glass distilled water, and 4.15 ml 1 M CaCl2.2H2O slowly added from a stock
solution. Oxoid Peptone (0.25 g) and Oxoid Yeast Extract (0.05 g) were added then the
pH adjusted to 7.5 with 1 M Tris.Cl buffer (pH 7.5) and the volume topped up to ~995
ml. For solid medium, agar was added at this at 1.5% w/v; agar solutions were preboiled to dissolve the agar prior to autoclaving. The solution (with or without agar) was
then autoclaved (20 min / 101kPa).
Once cooled (<55 oC), 5 ml of a 1 M NH4Cl stock, 2ml of K2HPO4 buffer, 1 ml SL10
Trace Elements Solution (Widdell et al., 1983), 3 ml of Vitamin 10 solution (Vit10 is a
combination of 0.25 ml of Vitamin Solution 1 and 0.75 ml of Vitamin Solution 2 per 1
ml as described in Janssen et al., 1997) and 4.4 ml of filter-sterilised, 25% w/v Napyruvate refrigerated stock solution were added, and the completed medium dispensed
aseptically for refrigerated storage or immediate use. It is not possible to autoclave the
medium once the vitamins and pyruvate have been added.
MGM
The medium MGM was prepared as described in the Halohandbook (Item 2.3) always
using 23 % salts. Solid MGM medium used unwashed 1.5 % agar (Difco-Bacto) as the
gelling agent.
Incubation Conditions
Liquid Medium
Liquid medium was incubated shaken at 120 rpm at 37 oC in the dark, or low light
conditions. Most cultures were grown as 10 ml volumes in MacCartney 30 ml tubes,
while larger volumes were grown in Schott bottles of varying volume.
Plate Incubation Conditions:
Aerobically incubated plates were placed in plastic containers, and additionally sealed
with Parafilm ‘M’ (American National Can) to prevent moisture loss if extended
incubation periods were expected. All plates were incubated at 37 oC in the dark or low
light conditions.
Extinction cultures:
A dilution series was prepared using 0.8 µm filtered, autoclaved water taken from the
Cheetham Salt Works crystalliser overwinter storage pond in September 2002 as the
diluent and unmodified water from the same source as the inoculum. Ten-fold serial
dilutions were performed with a final volume of 5 ml in sterile 30 ml MacCartney tubes.
At the 1 x 10-6 dilution step the series was expanded to 25 tubes per dilution level and
this continued to the 1 x 10-10 dilution level. Additional dilutions of 2 x 10-9 and 2 x 1010
were also made. The tubes were incubated in the dark at 37 oC for 4 weeks. These
tubes were screened using the multiplex PCR protocol DB239 with crude lysates
prepared as earlier described.
AMINO ACIDS ACETATE 5UM EACH FIRST TIME AROUND. Addded to
stetile slatern water
(a) MGM cultures. Crystallizer water was diluted in a ten-fold series using MGM as
the diluent. Between the 10À6 and 10À10 dilutions the series was expanded to 20
tubes per dilution, and these tubes were incubated in the dark at 37 °C for 3 weeks.
Most probable number(MPN) calculations were performed using the software program
MPN3 [18]. (b) Natural water cultures. Ten- fold serial dilutions were made in
crystallizer pond water that had been previously filtered through a 0.2 lm mem-brane
and then autoclaved (15 min, 121 °C, 101 kPa). The dilution series was expanded to 25
tubes per dilution between 10À6 and 10À10 dilution steps and addi- tional dilutions of 2 · 10À9 and
2 · 10À10 were also made. Tubes were incubated, stationary, in the dark at 37 °C for 4
weeks, then screened for growth using the multiplex PCR protocol DB239 (see below).
Four formulations using natural saltern water supplemented with selected nutrients were also
used. They contained sterile saltern water with: 5 lM amino acids and 5 lM pyruvate
(Medium A); 5 lM amino acids, 5 lM pyru- vate and 5 lM acetate (Medium B); 50 lM
amino acids and 0.5% (wt/vol) pyruvate (Medium C); and 50 lM amino acids, 50 lM
pyruvate and 50 lM acetate (Med- ium D).
Standard Polymerase Chain Reaction (PCR) Protocols
All PCRs were performed in either a MJ Research PTC-100 or Thermo Electron
Corporation PxE 0.2 thermal cycler in a volume of 50 µl, with the exception of
sequencing PCRs, described separately.
PCR Amplification of Isolate 16S rDNA
Due to the susceptibility of the Family Halobacteriaceae (with only a few exceptions)
to lysis in distilled water, crude DNA preparations were made using a modified version
of the Alternative Chromosomal DNA Isolation method originally described by
Matthias Pfeiffer in the Halohandbook (Dyall-Smith 2001). Briefly, 0.5 - 1 ml of each
broth culture was centrifuged for 3 minutes (for visibly turbid cultures), or 10 minutes
for non-turbid cultures, in a benchtop centrifuge at 16,000 g. The supernatant was
aspirated with a separate pipette tip for each sample and the sample centrifuged for an
additional 2 minutes. For tubes containing visible pellets, 200 µl of sterile distilled
water was added and the cell pellet lysed by vigorous mixing with the pipette. For tubes
without a visible pellet, 100 µl of distilled water was used, and the predicted location of
the cell pellet subjected to vigorous mixing. In each case, 1 µl of the lysate was added to
a 50 µl PCR. Primers used are listed in Table X.
PCR Protocol DB194
Each reaction comprised 1.75 mM MgCl2
pmol forward primer (F1 or 344mod) and 50 pmol reverse primer (1391R or 1492Ra)
µl was made up
with distilled water. 1 µl of DNA preparation (lysate or DNA extract) was added and
the following thermal cycling performed: 15 min at 95 oC; 30 cycles of: 1 min
denaturation at 95 oC, 30 sec annealing at 46 oC and 2 min extension at 72 oC; followed
by 10 min of final extension at 72 oC. A Haloferax volcanii lysate was used as a
positive control in this reaction and distilled water as a negative.
DB195
An abbreviated version of DB194, sometimes required to improve stringency; identical
composition but cycling 15 min at 95 oC; 25-30 cycles of: 1 min denaturation at 95 oC,
30 sec annealing at 52 oC and 1 min extension at 72 oC; followed by 10 min of final
extension at 72oC.
Multiplex PCR Protocol DB239
Each reaction comprised 1.75 mM MgCl2, PCR buffer (Qiagen), 200 µM dNTPs, 37.5
pmol F1 forward primer, 37.5 pmol SHOWprb forward primer and 60 pmol reverse
primer (1492Ra) and 2 U HotStarTaq DNA polymerase(Qiagen). The balance to 49 µl
was made up with distilled water. 1 µl of DNA preparation (lysate or DNA extract) was
added and the following thermal cycling performed: 15 min at 95 oC; 30 cycles of: 1
min denaturation at 95 oC, 30 sec annealing at 46 oC and 90 sec extension at 72 oC;
followed by 10 min of final extension at 72 oC. A Haloferax volcanii lysate was used as
a positive control in this reaction for the large (~1500bp) fragment and the clone
CSWFA61 used as a positive for both the large and SHOW-specific (~800bp)
fragments. The negative control in this reaction was distilled water.
PCR Cleanup
PCR cleanup was performed using the UltraClean PCR Clean-Up DNA purification kit
(MoBio Laboratories) according to the manufacturer’s instructions, eluting into a final
volume of 50 µl of distilled water.
Sequencing PCR
Between 30-100 ng of PCR product was used in sequencing reactions. Reactions also
contained 3.2 pmol of primer and 4 µl of ABI PRISM Big Dye Terminator Mix v2 or v3
(PE Applied Biosystems) and dd.H2O to 20 µl. Reactions that failed were repeated
using up to 200 ng PCR product and either 9.6 or 32 pmol of primer. Thermal cycling
was performed in a Gene Amp PCR System 9600 (Perkin-Elmer). Thermal cycling
consisted of 25 cycles of a denaturing step at 96oC for 10 sec, an annealing step at 50oC
for 5 sec and an extension step at 60oC for 4 min. Sequence PCR products was
precipitated in 65% (v/v) isopropanol and pelleted by centrifugation. Dry samples were
submitted to the Australian Genome Research Facility DNA Sequencing Laboratory
(Walter and Eliza Hall Institute, Parkville, Melbourne) for gel separation and
chromatograph generation.
Alternatively, sequencing was performed by the Applied Genetic Diagnostics
Sequencing Service, Department Pathology, University of Melbourne, Parkville,
Melbourne.
DNA visualisation:
DNA was visualised by loading 5 µl of PCR product alongside either 2-log or 100 bp
NEB DNA ladders into 1-1.5 % TAE (40 mM Tris to pH 8.0 with acetic acid, 1 mM
EDTA to pH 8.0 with NaOH) or TBE (9 mM Tris pH 8.0, 0.2 mM EDTA, 89 mM boric
acid and 25 mM NaOH) agarose gels. Gels were run at 120 V for 20 mins for routine
PCR and 90 V for 75 mins for PCR reactions requiring higher resolution. DNA was
added to 2 µl of Orange G Loading dye (0.1mM EDTA, 40 % (v/v) glycerol, 0.15g
Orange G powder [ICN]) prior to loading into wells. Gels were stained in an ethidium
bromide bath for 10 minutes and de-stained in distilled water for 30 mins, or run with
EtBr in the gel. Gels were viewed using a UV transilluminating table and digital camera
using the Kodak 1D 2.0.2 software program.
Clone library generation
All clone libraries were generated using the TOPO TA Cloning (Invitrogen) system as
specified in the manufacturer's instructions. Briefly, cleaned PCR product was ligated
and cloned into One-Shot TOP10 chemically competent E. coli, with the exception that
the ligation was left overnight at 4 oC. Transformed cells were plated onto X-Gal
Kanamycin Luria plates, a substrate that permits blue/white colony differentiation, and
white colonies (containing an insert) patched after overnight incubation at 37 oC for
further analysis. Two blue (without insert) colonies were also patched as controls.
RFLP
Restriction digests were performed using 2 units of restriction enzyme, BSA (if
required), enzyme buffer and PCR product to 25 µl in a PCR tube. Digests were
incubated from 2.5 to 3.5 hours and the entire reaction run on a TBE high-resolution
agarose gel at 60 V for approximately 120 mins.
16S rRNA gene identification and phylogenetic tree reconstructions.
16S rRNA genes were amplified by PCR and sequenced as described above. Initial
identification of sequences was made using the NCBI BLAST web-based nucleotidenucleotide search program. Sequences were aligned and phylogenetic trees constructed
or inferred using the DNA parsimony algorithm that is part of the ARB phylogeny
software package (Ludwig et al., 2004).
Phenotypic tests
The minimum standards for the description of new taxa within the Order
Halobacteriales were followed (Oren et al., 1977). The methods have been described
previously (Torreblanca et al., 1986; Gutierrez et al., 2002) and are based on standard
microbiological protocols (Gerhardt et al., 1981) but adapted for use with extremely
halophilic microorganisms. Where there were variations used, the methods are
described below. The tests performed are included in the species description. Hfx.
volcanii NCIMB 2012T was used as a control in most tests.
Media and growth conditions
Two types of characterization media were used; DBCM1 and DBCM2. All culture tests
were incubated unshaken at 37 °C in glass test tubes, unless stated otherwise. Growth
was measured spectrophotometrically at 550 nm using a Bausch and Lomb Spectronic
20 spectrophotometer (capable of measuring directly from the test tube).
Strains. Isolation and preliminary descriptions of the two SHOW isolates C23 (JCM
12705 = DSM 16854) and HBSQ001 (JCM 12895 = DSM 16790) have been reported
previously (Burns et al., 2004; Burns et al., 2004a; Bolhuis et al., 2004). Other,
reference strains included in this study were Hfx. mediterranei ATCC 33500 T, Hfx.
volcanii NCIMB 2012T, Hgm. borinquense JCM 10706 T, Hbt. salinarum NRC-1 JCM
11081, and Htg. turkmenica JCM 9101 T.
Anaerobic respiration
Anaerobic respiration was tested using DBCM1, but without MGM, and with only 0.1 
the concentration of NH4Cl. Dimethyl sulfoxide (DMSO) or NaNO3 were added to a
final concentration of 10 mM. Media was dispensed aseptically into 30 ml plastic tubes,
each containing a Durham tube. Cultures were incubated under either anaerobic
conditions using Anaerogen sachets (Oxoid, Basingstoke, UK), or aerobically, for 3
weeks at 37 °C without shaking, after which the presence of nitrogen gas bubbles and
nitrite were determined.
Antibiotic sensitivity
Cells were inoculated into DBCM2 media with the addition of 50 µg ml-1 of antibiotic.
Cells were incubated for 3 weeks before growth was determined.
Casein hydrolysis
Cells were cultured for 1 week in 10 ml of medium DBCM1 with the addition of a low
level (5 mM) of pyruvate and with 0.5 % filter-sterilised casein solution. Then 0.1%
azocasein was added and the cultures incubated a further 9 days. Cells and saltprecipitated casein (and azocasein) were removed by centrifugation and azocasein
hydrolysis measured spectrophotometrically (at 405 nm).
Catalase Test
As described in the Microbiological Techniques Manual, Dept of Microbiology and
Immunology, The University of Melbourne.
Briefly, a drop of 3% solution of H2O2 is placed on a microscope slide, and a cell pellet
/ colony smeared onto a cover slip. The cover slip is placed inverted (cell side down)
onto the H2O2 and a positive reaction recorded if gas bubbles occur immediately. A
negative result is recorded when there is no gas or delayed gassing (>10 min required).
Also performed as described in 25.1.15.2 Methods for General and Molecular
Bacteriology, edited: Gerhardt P, Murray R.G.E., Wood, W.A., Krieg N.R., American
Society for Microbiology, Washington D.C. 1994.
The following exceptions were used in both cases: a positive result was recorded if
gassing occurred within 10 minutes, owing to the slow growth of isolates being tested;
and a cell pellet was used instead of a picked colony on the cover slip (again associated
with small colonies; a larger liquid culture could produce a greater amount of biomass).
Cryo-electronmicroscopy
Light microscopy was performed as described previously, and was used to check cell
morphology, Gram stain, and motility. For cryo-electronmicroscopy (performed by
Zhuo Li), a 4 µl drop of cell culture was applied to glow-discharged Quantifoil grids
(Quantifoil Micro Tools, Jena, Germany) and then plunge-frozen in liquid ethane using
a Vitrobot (FEI). Images were recorded in an FEG G2 Polara FEI transmission electron
microscope operating at 300 keV. Liquid nitrogen was used as the cryogen, the energy
slit-width was 20 eV and the defocus was ~30 µm.
GC content and DNA-DNA hybridisation
The G+C content of whole cell DNA was determined by HPLC (Tamaoka, 1994).
Relatedness by DNA-DNA hybridization was performed by a fluorometric method
(Ezaki et al., 1989).
Indole test
Cells were grown in DBCM1 with the addition of 1 % w/v peptone for 1 week at 37 °C
before testing for indole using Kovac's reagent.
Alternatively, cells were grown in DBCM1 with the addition of 5 mM tryptophan, and
in both the presence, or absence, of 10 mM pyruvate. This variation was used to test
whether the growth-promoting effects of pyruvate would also enhance tryptophan
utilization, while the tryptophan alone variant was designed to force utilization of this
substrate.
Lipids
Lipids were extracted with chloroform/methanol and separated by thin-layer
chromatography as previously described (Kamekura et al., 1993; Gutierrez et al., 2002).
Magnesium Optima
The magnesium requirement was assessed by increasing Mg against a constant NaCl
concentration. Solutions contained 2.37 M NaCl and 0.08 M KCl, and either MgCl2 or
MgSO4 ranging from 0 to 1.5 M in steps of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.25, 1.5 M. MGM
was added to 0.1  final concentration (0.5 g peptone, 0.1g YE per l) was added before
autoclaving. Cultures were incubated for 3 weeks.
Motility
Motility was tested by both light microscopy (as above) and also using Craigie tubes.
Briefly, a 200 µl plastic pipette tip was placed point-down in a 30 ml MacCartney tube
containing DBCM1 with the addition of 10 mM pyruvate. The medium was semisolidified with the addition of 0.3 % (w/v) agar. Cells were inoculated using a straight
wire into the pipette tip and a positive recorded if growth was observed in the medium
outside the pipette tip. Hbt salinarum was used as a positive control in place of
Haloferax volcanii, which is non-motile.
Oxidase Test
As described in the Microbiological Techniques Manual, Dept of Microbiology and
Immunology, The University of Melbourne, except that a cell pellet instead of a picked
colony is dabbed onto the impregnated filter paper strip. (Briefly, a freshly grown cell
pellet is smeared on filter paper impregnated with a 1% solution of tetramethyl-pphenyldiamine dihydrochloride; a postive results is indicated by purple colouration of
the paper within 10 seconds.)
Salinity Optima
For growth at different salinities, DBCM2 was prepared using salt water solutions of
increasing salinity (2% increments), keeping the same ratios of specific salts as
normally used (as for 30% SW (2.2, The Halohandbook)). After inoculation, cultures
were incubated for ~3 weeks.
Substrate utilisation and acid production
Cells were inoculated into DBCM1 containing 10 mM of substrate (see species
description for full list) except for cellulose, chitin and starch, which were added at 0.1
% w/v. After inoculation, cultures were incubated for ~3 weeks. In cultures that showed
growth, the final pH was measured using a calibrated pH meter, and compared to the
uninoculated control of the same substrate to test for acid production.
Temperature Optima
Growth at different temperatures was performed in DBCM2 media. Cultures were made
in duplicate and incubated at 4, 25, 30, 37, 40, 42, 45 and 55 °C for two weeks. Growth
was followed spectrophotometrically at 550 nm.
Chapter 3
Characterisation of Natronomonas-like, and Halogeometricum-like
isolates, and isolates related to the Deep-10 phylotype (Antarctic Deep
Lake)
Introduction
Previous studies led to the isolation of many novel isolates from an Australian solar
saltern (Burns, 2004). Isolates included representatives that fell within well studied
genera, such as Haloferax and Halorubrum, as well as isolates that were not closely
related to established species or genera of the family Halobacteriaceae. In the latter
category
were
isolates
Halogeometricum-like.
initially
ascribed
as
being
Natronomonas-like
and
A third group had 16S rRNA sequences that grouped most closely to a clade known
only by cloned 16S rRNA gene sequences recovered from Deep Lake in Antarctica
(Bowman, 1999), and were thus tentatively assigned as ADL (Antarctic Deep Lake)
isolates.
Seven isolates from this study, representing the Natronomonas, Halogeometricum and
ADL groups, were selected for full taxonomic charactersisation with the results
presented here. Note that the putative taxonomic assignment given to these isolates
during the course of my Honours in 2002, and following isolation paper (Burns, 2004)
are not necessarily reflective of their final taxonomic status provided here, as studies
undertaken during my PhD have clarified the taxonomic status of these isolates.
Part 1: Isolates 1.15.5, 2.27.5, 5.24.4 and 6.14.5
Putative ADL Group
Introduction
In 1999, Bowman et al. described clone sequences recovered from Deep Lake, a
perenially ice-free, thassohaline, monomictic 36m deep lake in the Vestfold Hills of
Eastern Antarctica. Similar sequences were also obtained from Organic lake, a
meromictic, shallow lake with a high concentration of dimethylsulphide in its bottom
waters. There were two main clades of sequences; one comprising the Deep2, 5 and 10
sequences, and the other including sequences from both the Deep and Organic lakes
(such as Organic13, Deep6). Attempts at the time to cultivate these organisms from
which these sequences were recovered were unsuccessful.
Five years later we reported (Burns et al., 2004) the recovery of sequences similar to the
Deep10 clade from a southern Australian solar saltern, a physical environment similar
only in salinity. The crystallizer pond these sequences were detected in is very shallow
and experiences much higher temperatures than the Antarctic lakes. Cultivation
attempts yielded, after extended incubation, five isolates that closely matched the
Deep10 clade sequences, and both the clone sequences recovered from this saltern, and
isolate sequences, initially appeared to belong to the same phylotype as the Bowman
Deep10 clade. We referred to the Deep10-like clade we had recovered the Antarctic
Deep Lake group (Burns et al., 2004).
However, more detailed phylogenetic analysis of four of our isolates indicated that the
they did not cluster, as originally thought, with the Bowman et al. Deep10 sequences,
but in fact form a novel grouping within the Halobacteriaceae. These results, combined
with the physiological characteristics of these isolates, indicated that they belong to a
new species and a new genus within the Halobacteriaceae.
Results and Discussion
Five related strains were isolated from a southern Australian solar saltern, however, one
(6.21.5, Burns et al., 2004) was lost upon passaging, and the remaining four constitute
the subject of this section. Isolation was made using a variety of media types; however
in all cases, isolation required extended incubation times (> 8 weeks) rather than any
specific media formulation to successfully isolate members of this phylogenetic group.
Once cultivated, colonies took 4 – 8 weeks to grow on solid media; optimally on
DBCM2, but possible on MGM, with agar or agarose as the gelling agent. After 8
weeks, colonies were small (0.5 – 1.0 mm diameter), convex, round, with an entire
edge, and intense red colour. Liquid cultures were also red or pink in colour, depending
upon the cell density. Under optimal growth conditions, cells exhibit a flat, rod shaped
morphology. Additionally, many cells appear to have a “twisted rod” shape, distinctive
to these isolates.
All strains are motile and are Gram stain negative. Flagella are observed by TEM (Fig.
3.1.2). These strains were initially thought to be non-motile, but in light of the TEM
images, the lack of apparent motility by either microscopy or culture tests suggested
that further experimentation was be required to determine under which circumstances
motility was induced (if at all). Certainly some other Halobacteriaceae, such as Har.
hispanica do not exhibit motility under microscopic examination, but in this example
culture tests do indicate motility (K. Porter, Dyall-Smith Haloarchaeal Laboratory, Dept
of
Microbiology
&
Immunology,
The
University
of
Melbourne;
personal
communication). When the strains were re-examined following the TEM imaging,
motility was observed by phase microscopy with between 1-5 % of cells exhibiting
motility. The discrepancy is most likely explained that initial microscopic motility tests
were performed during the initial phases of characterization, prior to the development of
the optimized media DBCM1 and DBCM2; thus the organisms may not have been
under conditions conducive to expression of motility in their phenotype at this time. The
negative culture tests may have been influenced by several factors. “Halonotius”
cells usually require a substantial inoculum to initiate growth in new media. The stab /
straight wire inoculation may have been insufficient to reliably initiate growth inside the
pipette tip. This is not generally an issue with the control organism, Halobacterium
salinarum. Additionally, no other experiments were performed in which the organisms
were encased in agar (albeit at low concentrations) and any possible inhibitory effects
associated with this have not been quantified. Further, the organisms may have been
insufficiently motile to swim through the agar, even at the reduced concentration used.
Despite a wide range of substrates being tested, under the characterisation conditions,
cells were able to grow only on glucose, glycerol and pyruvate. Cells are oxidase and
catalase negative using conventional testing. They do not hydrolyse starch or casein,
and do not show ß-galactosidase activity. Indole is not produced in tryptophancontaining media. All strains were unable to use nitrate (with or without the presence of
L-arginine) or DMSO as alternative electron acceptors under anaerobic conditions.
Figure 3.3.1: Representative morphology of “Halonotius”.
.Composite phase microscopy images of strain 1.15.5, 1000x
magnification. Cells display the some of the pleomorphism typical of
Haloarchaea, with rods, and some triangular cell shapes visible.
Although difficult to visualize by 2D micrographs, many cells exhibit
a “twisted rod” morphology, with the twist occurring usually in the
cell midsection, although occasionally toward the end. All other
“Halonotius” (2.27.5, 5.24.4, 6.14.5) isolates exhibit similar
morphology.
Figure 3.1.2: “Halonotius” cells possess flagella. Transmission
Electron Micrograph of “Halonotius torquis" strain 2.27.5.
Temperature Optimum
The optimum growth temperature for strains 1.15.5 and 5.24.4 was 40 °C, while 2.27.5
and 6.14.5 shared an optimum of 37 °C. The minimum growth temperature in the tested
range was 25 °C for all strains; no growth was observed at 4 °C or 55 °C. The
mesophilic optimum growth temperature initially lead us to believe that this further
separated the likelihood of these isolates being “Antarctic” related, but recent studies
(D. Oh, Dyall-Smith Haloarchaeal Laboratory, Dept of Microbiology and Immunology,
The University of Melbourne; personal communication) have indicated that few true
psychrophiles exist in Antarctic hypersaline lakes; most organisms seeming to simply
grow at far below their optimum growth temperature, rather than being truly adapted to
the Antarctic conditions.
Salinity and Magnesium Optimum
Growth occurred over a wide range of salinity, from a minimum of 16% up to
saturation. The optimum salinity was 20 - 24% for 1.15.5 and 2.27.5, and 22% for
5.24.4 and 6.14.5. All strains had no minimal magnesium requirement but poor growth
was observed in the absence of Mg2+ ions, and low to no growth above 1.25 M Mg2+.
Optimum growth varied in respect of both Mg-associated anion and isolate. 1.15.5 had
an equal optimum for both MgCl2 and MgSO4 of 0.4 M, while 2.27.5 and 5.24 shared
the MgSO4 optimum of 0.4 M but had MgCl2 optima of 0.6 and 0.2-0.4 M, respectively.
Isolate 6.14.5 had the broadest optima of all strains, demonstrating a MgSO4 optimum
of 0.2-0.6 M and MgCl2 of 0.4-0.8 M.
pH Optimum and Antibiotic Sensitivity
All isolates had similar responses to pH, exhibiting growth over the range 5.5 - 8.5.
Optimum growth was at pH 7.0 - 7.5 for 1.15.5 and 2.27.5, and 7.0 for 5.24.4 and
6.14.5, with strains 1.15.5 and 2.27.5 and 6.14.5 exhibiting a slightly greater tolerance
for higher pH than 5.24.4. All strains were sensitive to the antibiotics anisomycin,
novobiocin, rifampicin, and simvastatin. They were resistant to ampicillin, bacitracin,
chloramphenicol, cycloheximide, erythromycin, kanamycin, mycostatin, neomycin,
streptomycin, and tetracycline.
The polar lipid profiles of the all isolates were identical. The isolates possessed only the
glycolipid S-DGA-1 (data not shown), the same lipid that occurs in Haloferax and
Halococcus species (Boone and Castenholz, 2001).
16S rRNA gene phylogeny
All of the group's 16S rRNA gene sequences were tightly clustered, with strains 2.27.5
and 5.24.4 being identical and the greatest divergence occurring between isolates 1.15.5
and 6.14.5, with 98.4% similarity. These results indicate they are members of the same
species. Phylogenetic tree reconstructions placed the Halonotius sequences as a separate
clade within the Halobacteriaceae (Fig. 3.2). Their relationship to the Antarctic lake
organisms (Bowman et al, 1999) is relatively close (91.6%). However, owing to the
short lengths and imperfect quality of Antarctic lake sequence available from the
Bowman study, the “Halonotius” organisms' initial assignment to the “ADL”/Deep10
clade (Burns et al, 2004) was premature and more comprehensive analysis has lead to
their reassignment as a separate clade.
Among the recognised members of the Halobacteriaceae, the closest relative was
Halogeometricum borinquense (85.5 - 87%), isolated from a solar saltern (MontalvoRodriguez et al., 1998). The 16S rRNA gene sequence similarity to Hfx. volcanii (86.3 86.8%) and Hrr. saccharovorum (86.6 - 87.6%) was much lower.
Figure 3.1.3. Phylogenetic tree showing relative placement of
strains 4.03.5 and 8.8.11. Phylogenetic tree reconstruction based on
complete or nearly complete (>1300nt) 16S rRNA sequences. The
tree shown was derived by maximum likelihood (using the ARB
package). Scale bar represents 0.10 expected nucleotide
substitutions per site. Bootstrap values (using distance matrix
methods) were derived from 1000 replicates and significant nodes
(>75%) are indicated by filled circles at branch points. The outgroup
sequences consisted of sequences representing most of the other
genera of the Halobacteriaceae as well as that of Methanosaeta
concilii ATCC 35969T (accession X16932.1) (not shown). OPEN
NODES?????????????????????????????????
The phenotypic characterisation and phylogenetic data support the placement of these
isolates in a new species and a new genus within the Halobacteriaceae, for which we
propose the name Halonotius torquis gen. nov., sp. nov.
GENUS and SPECIES description
Description of Halonotius gen. nov.
-----halonotius meaning? (the salty southern
one)
Halonotius tor' quis. L. masc. n. torquis the twisted one
Gram negative, flat rods, often with a twist located along the length. Aerobic
heterotrophs. Oxidase and catalase tests are negative. Phylogenetically belonging to the
family Halobacteriaceae. Habitat: salt lakes and saltern crystallizer ponds. The type
species is Halonotius torquis. Following the published guidelines (Oren and Ventosa,
2000) we propose the three-letter genus abbreviation Hnt.
Species description
Gram-negative. Cells are flat rods, often with a twist located along its length. Colonies
on agar medium are red with entire edge. Strictly aerobic; only oxygen is used as the
final electron acceptor. Cannot utilise NO3- or DMSO as alternative electron acceptors.
Growth occurs at pH 5.5 – 8.5, 25 – 45°C and 16 – 36% (w/v) NaCl. Halophilic; cells
lyse immediately in distilled water, and a minimum ~16% salts required for growth.
Optimal growth occurs at neutrophilic conditions, above 18% salinity. Capable of
growing in defined media, but is very restricted in the substrates utilised. Grows best on
pyruvate, but also capable of utilising glucose and glycerol, as sole carbon source.
Incapable of utilising acetate, alanine, arabinose, arginine, aspartate, benzoate, betaine,
butanol, butyrate, cellobiose, citrate, ethanol, formate, fructose, fumarate, galactose,
galacturonate, gluconuronate, glycine, glycolate, lactate, lactose, leucine, lysine, malate,
malonate, mannitol, mannose, methanol, propanol, propionate, ribose, serine, succinate,
sucrose, tartrate, threonine, urea, valine, xylose, at 10mM; and; cellulose, chitin and
starch at 0.1% (w/v) as the sole carbon and energy source. Acid is not produced from
carbohydrate utilisation. Produces poly-hydroxybutyrate granules. Negative for ßgalactosidase, oxidase, catalase activity and indole production. Sensitive to anisomycin,
novobiocin, rifampicin and simvastatin, and resistant to ampicillin, bacitracin,
chloramphenicol, cycloheximide, erythromycin, kanamycin, mycostatin, neomycin,
streptomycin and tetracycline at 50 µg/ml. All strains isolated from solar saltern
crystallizer ponds. Phylogenetically affiliated to the Halobacteriaceae. The type species
of the genus is Halonotius torquis, isolated from Cheetham Salt Works, Geelong,
Australia. The type strain is 1.15.5T (= JCM 14355 T = DSM 18729T), with additional
reference strains 2.27.5 (= JCM 14356 = DSM 18671), 5.24.4 (= JCM 14357 = DSM
18673) and 6.14.5 (= JCM 14358 = DSM 18692).
Part 2: Isolates 4.03.5 and 8.8.11
Natronomonas-like group
Results and Discussion
Strains 4.03.5 and 8.8.11 were isolated by independent methods from a saltern
crystallizer pond at Moolap, Geelong, Australia. Isolation methods and origin of strain
4.03.5 has been reported previously (Burns et al., 2004; see also Materials and Methods,
Chapter Two). The isolation of 8.8.11 was achieved using extinction culturing as
previously described (Burns et al., 2004a) with growth being detected by non-specific
binding of the SHOWprb primer. Colonies took 2 – 4 weeks to grow on solid media.
After 4 weeks, colonies were small (0.5 – 1.0 mm diameter), convex, round, with an
entire edge, and pink or red in colour. Liquid cultures were also red or pink in colour,
depending upon the cell density, but faded to an off-white when left undisturbed for
extended periods ( > ~ 4 weeks). Under optimal growth conditions, cells exhibit a
stumpy rod shaped or flat tetragonal morphology.
Figure 3.2.1: Representative morphology of “Placeholder”.
Composite phase microscopy images of strains 4.03.5 (left, lighter
side of image) and 8.8.11 (darker, right), 1000x magnification. Cells
display the pleomorphism typical of haloarchaea, with rods (less
common), rounded, tetragonal and triangular cell shapes all visible.
Most cells exhibit a flat, rather than rounded morphology.
Both strains are motile and are Gram stain negative. Under the characterisation
conditions, cells were able to grow only on acetate, butanol, butyrate, ethanol,
glycerol,lactate, propanol, propionate and pyruvate, with pyruvate providing the best
growth. Cells are oxidase and catalase negative using conventional testing. Cells were
left for up to 10 minutes without displaying activity, while Haloferax volcanii was
positive within this time. They do not hydrolyse starch or casein, and do not show βgalactosidase activity. Indole is not produced in tryptophan-containing media. 8.8.11
was unable to use nitrate (with or without the presence of L-arginine) or DMSO as
alternative electron acceptors under anaerobic conditions, but 4.03.5 reduced NO3- to
NO2-, to a final nitrite concentration of 10 mg / ml.
Temperature Optimum
The optimum growth temperature for strain 4.03.5 was 37-40 °C, compared with 45 °C
for 8.8.11. The minimum growth temperature in the tested range was 25 °C; no growth
was observed at 4 °C or 55 °C.
Salinity and Magnesium Optimum
Growth occurred over a wide range of salinity, from a minimum of 12-14% (8.8.11) and
14% (4.03.5) up to saturation (both). The optimum salinity was 18-20% for both strains.
Neither strain had a minimal magnesium requirement but poor growth was observed in
the absence of Mg2+ ions, and low to no growth above 1.25 M Mg2+. Isolate 4.03.5 had
an MgCl2 optimum of 0.2 M, and MgSO4 optimum of 0.4 M, while 8.8.11 had a
comparatively elevated preference for 0.4 M MgCl2 and 0.4-0.6 M MgSO4.
pH Optimum and Antibiotic Sensitivity
The isolates had somewhat different responses to pH. Isolate 4.03.5 reached high
turbidity from relatively acidic conditions (pH 6.0) while 8.8.11 reached this growth
level 0.5 pH units later. Both strains exhibited a very flat yield response from these pH
levels until pH 8.5, at which yield reduced marginally. By way of comparison, the
isolating saltern typically has a pH of about 7.0 – 7.5, comfortably within these
organisms range. Both strains were sensitive to the antibiotics mycostatin, novobiocin,
rifampicin, and simvastatin. They were resistant to ampicillin, anisomycin, bacitracin,
chloramphenicol,
cycloheximide,
erythromycin,
kanamycin,
neomycin
and
streptomycin, and tetracycline at 50 µg / ml.
16S rRNA gene phylogeny
The 16S rRNA gene sequences of these isolates were 99.8% similar, and they clustered
tightly together in phylogenetic tree reconstructions, supporting placement of these
isolates as members of the same species. Phylogenetic tree reconstructions placed these
sequences as a separate clade within the Halobacteriaceae (Fig. 3.2.2). Among the
recognised members of the Halobacteriaceae, the closest relative was Halogeometricum
borinquense (85.5-87%), isolated from a solar saltern (Montalvo-Rodriguez et al.,
1998). The 16S rRNA gene sequence similarity to Hfx. volcanii (86.3-86.8%) and Hrr.
saccharovorum (86.6-87.6%) was much lower.
Figure 3.2.2. Phylogenetic tree showing relative placement of
strains 4.03.5 and 8.8.11. Phylogenetic tree reconstruction based on
complete or nearly complete (>1300nt) 16S rRNA sequences. The
tree shown was derived by maximum likelihood (using the ARB
package). Scale bar represents 0.1 expected nucleotide substitutions
per site. Bootstrap values (using distance matrix methods) were
derived from 1000 replicates and significant nodes (>75%) are
indicated by filled circles at branch points. The outgroup sequences
consisted of sequences representing most of the other genera of the
Halobacteriaceae as well as that of Methanosaeta concilii ATCC
35969T (accession X16932.1) (not shown).
The phenotypic characterisation and phylogenetic data support the placement of isolates
4.03.5 and 8.8.11 in a new species and a new genus within the Halobacteriaceae, for
which we propose the name Halonotius torquis gen. nov., sp. nov.
GENUS and SPECIES description
Description of Halonotius gen. nov.
-----halonotius meaning? (the salty southern
one)
Halonotius tor' quis. L. masc. n. torquis the twisted one
Gram negative rods; aerobic heterotrophs. Oxidase and catalase tests are negative.
Phylogenetically belonging to the family Halobacteriaceae. Habitat: salt lakes and
saltern crystallizer ponds. The G+C content of the type species is 46.9 mol%. The type
species is Halonotius torquis. Following the published guidelines (Oren and Ventosa,
2000) we propose the three-letter genus abbreviation Hnt.
Species description
Gram-negative. Cells are rod shaped. Colonies on agar medium are red with entire edge.
Strictly aerobic; only oxygen is used as the final electron acceptor. Strain 4.03.5 can
reduce NO3- to NO2-, but cannot utilize DMSO as an alternative electron acceptor, while
strain 8.8.11 cannot utilize either NO3- or DMSO as alternative electron acceptors.
Growth occurs at pH 5.5–8.5, 25–45 °C and 12/14–36% (w/v) NaCl. Halophilic; cells
lyse immediately in distilled water, and a minimum ~ 14% salts required for growth.
Optimal growth occurs at neutrophilic conditions, above 18% salinity. Capable of
growing in defined media, but is very restricted in the substrates utilised. Grows best on
pyruvate, but also capable of utilising acetate, butanol, butyrate, ethanol, glycerol,
lactate, propanol and propionate as sole carbon source. Incapable of utilising alanine,
arabinose, arginine, aspartate, benzoate, betaine, cellobiose, citrate, formate, fructose,
fumarate, galactose, galacturonate, gluconuronate, glycine, glycolate, lactose, leucine,
lysine, malate, malonate, mannitol, mannose, methanol, ribose, serine, succinate,
sucrose, tartrate, threonine, urea, valine, xylose, at 10mM, and cellulose, chitin and
starch at 0.1% (w/v) as the sole carbon and energy source. Acid is not produced from
carbohydrate utilisation. Produces poly-hydroxybutyrate granules. Negative for βgalactosidase, oxidase, catalase activity and indole production. Sensitive to anisomycin,
novobiocin, rifampicin and simvastatin, and resistant to ampicillin, bacitracin,
chloramphenicol, cycloheximide, erythromycin, kanamycin, mycostatin, neomycin,
streptomycin and tetracycline at 50 µg/ml. All strains isolated from solar saltern
crystallizer ponds. Phylogenetically affiliated to the Halobacteriaceae. The type species
of the genus is Halonotius torquis, isolated from Cheetham Salt Works, Geelong,
Australia. The type strain is 4.03.5T (= JCM 14360T = DSM 18672T), with the
additional reference strain 8.8.11 (= JCM 14361 = DSM 18674).
Part 3: Isolate 2.24.4
Halogeometricum-like group
Results and Discussion
Once isolated in pure culture, colonies of isolate 2.24.4 took 2 – 4 weeks to grow on
solid media (including MGM, but optimally on DBCM2). After 4 weeks, colonies were
small (0.5 – 1.0 mm diameter), convex, round, with an entire edge, and red in colour.
Liquid cultures were also white to pink in colour, fading to an off-white when left
undisturbed for extended periods. Under optimal growth conditions, cells exhibit a rod
shaped morphology.
Figure 3.3.1: morphology of “Placeholder”. Composite phase
microscopy images of strain 2.24.4, Phase contrast microscopy of
isolate 2.24.4 at 1000x magnification using oil immersion. Cells
display the pleomorphism typical of Haloarchaea, with rods,
rounded and triangular cell shapes all visible. Most cells exhibit a
flat, rather than rounded morphology.
Isolate 2.24.4 is motile and Gram stain negative. Under the characterisation conditions,
cells were able to grow on betaine, fructose, fumarate, galactose, glucose, glycerol,
lactose, malate, and pyruvate, with the last providing optimal growth. Cells are oxidase
and catalase negative using conventional testing. They do not hydrolyse starch or
casein, and do not show β-galactosidase activity. Indole is not produced in tryptophancontaining media. Isolate 2.24.4 was unable to use nitrate (with or without the presence
of L-arginine) or DMSO as alternative electron acceptors under anaerobic conditions.
Temperature Optimum
The optimum growth temperature for strain 2.24.4 was 45 °C, and the minimum growth
temperature in the tested range was 4 °C; no growth was observed at 55 °C.
Salinity and Magnesium Optimum
Growth occurred over a wide range of salinity, from a minimum of 16% up to
saturation. The optimum salinity was 22%. The strain had no minimal magnesium
requirement but poor growth was observed in the absence of Mg2+ ions, and low to no
growth above 1.25 M Mg2+. Isolate 2.24.4 had a MgCl2 optimum of 0.4 M, and MgSO4
optimum of 0.2 M.
pH Optimum and Antibiotic Sensitivity
Isolate 2.24.4 was able to grow from pH 5.5 to 8.5, with optimal growth occurring at pH
7.0-7.5. Isolate 2.24.4 was sensitive to the antibiotics novobiocin, rifampicin, and
simvastatin, and resistant to ampicillin, anisomycin, bacitracin, chloramphenicol,
cycloheximide, erythromycin, kanamycin, mycostatin, neomycin and streptomycin, and
tetracycline at 50 µg / ml.
16S rRNA gene phylogeny
The 2.24.4 16S rRNA gene sequence was dissimilar to most recognised members of the
Halobacteriaceae, with the closest recognised species being Halogeometricum
borinquense with 92.7 % similarity. It does, however, share 99% similarity with an
environmental sequence recovered from a hypersaline endoevaporitic microbial mat at
the bottom of a solar saltern, ArcH05 (DQ103672; Sørenson et al., 2005). ArchH05 was
encountered as one of 53 clones, a similar population frequency to that of 2.24.4,
although with the caveat that the Sørenson library included a much wider variety of
phylotypes, only a fifth of which are readily identifiable as haloarchaea typically found
in the water column of salterns (thus inflating the expected abundance of this phylotype
in free water). Regardless, this suggests that the 2.24.4 phylotype is global in
distribution, albeit at a relatively low proportion of saltern populations.
Phylogenetic tree reconstructions placed isolate 2.24.4 and ArcH05 together as a group
(Fig. 3.3.2), with its closest relatives belonging to the Haloquadratum and
Halogeometricum genera (91.0 % and 92.7 % to the type strains, respectively) along
with Haloferax volcanii (91.3 %).
The phenotypic characterisation and phylogenetic data support the placement of these
isolates in a new species and a new genus within the Halobacteriaceae, for which we
propose the name Halonotius torquis gen. nov., sp. nov.
GENUS and SPECIES description
Description of Halonotius gen. nov.
-----halonotius meaning? (the salty southern
one)
Halonotius tor' quis. L. masc. n. torquis the twisted one
Gram negative rods; aerobic heterotrophs. Oxidase and catalase tests are negative.
Phylogenetically belonging to the family Halobacteriaceae. Habitat: Hypersaline
environs such as salt lakes and saltern crystallizer ponds. The G+C content of the type
species is 46.9 mol%. The type species is Halonotius torquis. Following the published
guidelines (Oren and Ventosa, 2000) we propose the three-letter genus abbreviation
Hnt.
Species description
Gram-negative. Cells are rod shaped. Colonies on agar medium are red with entire edge.
Strictly aerobic; only oxygen is used as the final electron acceptor. Cannot utilise NO3or DMSO as alternative electron acceptors. Growth occurs at pH 5.5–8.5, 4–45 °C and
16 - 36 % (w/v) NaCl. Halophilic; cells lyse immediately in distilled water, and a
minimum ~16% salts required for growth. Optimal growth occurs at neutrophilic
conditions, above 18% salinity. Capable of growing in defined media, but is restricted
in the substrates utilised. Grows optimally on pyruvate, but also capable of utilising
betaine, fructose, fumarate, galactose, glucose, glycerol, lactose and malate as sole
carbon sources. Incapable of utilising acetate, alanine, arabinose, arginine, aspartate,
benzoate, butanol, butyrate, cellobiose, citrate, ethanol, formate, galacturonate,
gluconuronate, glycine, glycolate, lactate, leucine, lysine, malonate, mannitol, mannose,
methanol, propanol, propionate, ribose, serine, succinate, sucrose, tartrate, threonine,
urea, valine, xylose, at 10mM, and cellulose, chitin and starch at 0.1% (w/v) as the sole
carbon and energy source. Acid is not produced from carbohydrate utilisation. Negative
for β-galactosidase, oxidase, catalase activity and indole production. Sensitive to
novobiocin, rifampicin and simvastatin, and resistant to ampicillin, anisomycin,
bacitracin, chloramphenicol, cycloheximide, erythromycin, kanamycin, mycostatin,
neomycin, streptomycin and tetracycline at 50 µg / ml. Phylogenetically affiliated to the
Halobacteriaceae. The type species of the genus is Halonotius torquis, isolated from
Cheetham Salt Works, Geelong, Australia. The Genbank accession number for the 16S
rRNA gene sequence of isolate 2.24.4 is AY498650. The type strain is 2.24.4T (JCM
14359T = DSM 18730T).
Figure 3.3.2: Phylogenetic tree showing relative placement of strain 2.24.4. Phylogenetic tree
reconstruction based on complete or nearly complete (>1300nt) 16S rRNA sequences. The tree shown
was derived by maximum likelihood (using the ARB package). Scale bar represents 0.1 expected
nucleotide substitutions per site. Bootstrap values (using distance matrix methods) were derived from
1000 replicates and significant nodes (>75%) are indicated by filled circles at branch points. The
outgroup sequences consisted of sequences representing most of the other genera of the Halobacteriaceae
as well as that of Methanosaeta concilii ATCC 35969T (accession X16932.1) (not shown).
Chapter 4
Isolation of the Square Haloarchaea of Walsby
(Haloquadratum walsbyi)
Overview
The isolation of the Square Haloarchaea of Walsby began in 2002, during the final
portion of my Honours year. As such, a brief overview of the work undertaken in that
period is provided here followed by the continuation of this work through into
postgraduate study.
Solid medium cultivation
Cultivation experiments performed earlier (Burns et al., 2004), utilising solid media
failed to permit growth of SHOW organisms (although other novel species were
cultivated). At the time, we postulated that SHOW growth may be inhibited by agar,
after it was found that higher viable counts could be obtained in soil using different
gelling agents (Janssen et al., 2002). A further consideration (later shown to be
incorrect) was that the unusual square shape may prevent stacking of cells into visible
colonies.
Liquid isolation
To avoid these issues, I began conducting isolation experiments in liquid media by the
use of extinction cultures; more specifically, isolates were taken from the terminal and
sub-terminal positive (those containing growth) tubes on the basis that their growth
originates from only one or a few cells, thus increasing the chances that a tube will
contain an axenic culture.
Contrary to the modern opinion that low substrate levels improve cultivation success
(Janssen et al., Bussmann et al., 2001), earlier haloarchaeal isolation attempts using
solid media had indicated that the complex medium MGM (0.5 % peptone, 0.1 % yeast
extract (w/v)) was suitable for supporting isolation of both novel and previously-
cultivated organisms, and so this medium was used for initial extinction culture
experiments.
Results from the first culturing attempt indicated that MGM in liquid format was not
suitable for an isolation strategy aimed solely at the SHOW; all cultures were of the
genus Halorubrum. Further analysis (D. Burns, Honours Thesis [2002]), indicated
pursuing further with MGM liquid cultureswould have been fruitless. The spatial
separation of cells on solid media may have afforded the possibility of slower-growing
cells being able to initiate growth and form colonies, whereas in a high-nutrient liquid
medium, such cells become overwhelmed by faster-growing species able to capitalise
on the high substrate levels and disseminate throughout the entire medium volume.
Low-substrate liquid isolation
Subsequently, the decision was made to pursue a natural-water based medium approach,
as demonstrated in the successful isolation of other “uncultivable” organisms (Rappe et
al., 2002), (Connon and Giovannoni, 2002). Possible benefits of this medium change
included both the lower substrate concentrations present in the sterilised saltern water
compared to the medium MGM (reducing possibility of overgrowth by fast-growing
species) , and the fact that the natural water has an almost saturating salinity (about
33%) while the MGM formulation used previously had a total salinity of 23 %. While
SHOW group organisms are known to grow at the lower salinity, the additional
selective pressure of higher salinity was thought to increase potential for SHOW
organism isolation, as it is only at saturating salinities that SHOW organisms dominate
the haloarchaeal community (Benlloch et al, 2002).
Screening techniques
Owing to the low substrate concentrations present in sterilised saltern water, screening
was performed using PCR-based techniques instead of by the presence of visible
turbidity in the culture medium. This technique was chosen after similar cultivation
techniques used in the isolation of the SAR11 bacterioplankton revealed that molecular
detection methods were advisable as cultures grown under these low substrate
conditions do not always reach sufficient cell densities to create visible turbidity (Rappe
et al., 2002). This screening decision proved fortuitous given the overwhelming
majority of tubes detected as having growth detectable by PCR had no or barely
detectable visible turbidity after 4 weeks of incubation. (The PCR was not sensitive
enough to amplify DNA from a single haloarchaeon; the cells must have multiplied for
the PCR positive).
The (multiplex) PCR screening tested for both the presence of archaeal growth using
universal archaeal 16S rRNA gene primers, and also for the presence of the SHOW
group using a specific primer pair. The primer was designed using the ARB software
package (Ludwig et al., 1998) using both SHOW clone sequences from other studies,
and also SHOW clone sequences from the clone library derived in the earlier part of my
Honours year (2002), to ensure specificity.
SHOW cultivation using natural water extinction culturing
The screening of the natural-water extinction cultures yielded three possible SHOWspecific bands from 17 candidate positives. Further investigation found that one of these
was unable to be successfully sequenced using the SHOW primer while another
returned a non-specific product that did not return any matches in the NCBI database.
Upon detailed examination both were close but not exactly the same size as the SHOWspecific control ~800 bp band and therefore both were presumed to be non-specific
product. The SHOW-specific ~ 800 bp PCR product from the 2 x 10-9 dilution was
successfully sequenced and when placed in a phylogenetic tree, grouped within the
SHOW clade of the Haloarchaea. However, the universal product sequence returned an
identity matching Halorubrum Group 2 demonstrating that the culture was mixed.
These results, in conjunction with a microscopic analysis of the culture, indicated the
SHOW organism to be about 15% of the total culture with the balance made up by
small, round cells.
Table 4.1: Natural water extinction cultures multiplex PCR screen results
Number of growth positives as detected by PCR
SHOW-positive
Dilution level
screen (out of 25)
(culture)
1 x 10-7
25
-
1 x 10-8
11
2
band
2 x 10-9
5
1
1 x 10-9
1
0
2 x 10-10
not screened
-
1 x 10-10
not screened
-
In the studies that follow I outline and discuss isolation and cultivation of
Haloquadratum walsbyi.
Passaging of mixed SHOW culture
Multiple passaging of the mixed SHOW culture was unsuccessful. It was not over-run
by the co-cultured organism (Halorubrum spp.), but simply died out. Growth was
monitored by both PCR, and microscopy techniques during this phase, and the
proportion of the two organisms in the co-culture did not appear to change, rather, both
simply reduced in number until a non-viable state was reached (data not shown).The
demise of the culture was postulated to most likely be a result of the extremely low
nutrient concentrations present – only what existed in the saltern water following
filtration and autoclaving.
Improving SHOW isolation medium
Given that artificial media formulations had not had any success in cultivating the
SHOW organisms, and with limited success in using neat sterilised saltern water, the
obvious step was supplementation of the sterilised saltern water. This had been
performed in other aquatic enviroments with some success, particularly cultivation of
the SAR11 marine bacterium (Candidatus Pelagibacter ubique; Rappe 2002) but the
technique was not novel, having been reported earlier (Schut et al., 1993; Button et al.,
1993).
The choice of supplement was considered important; the earlier experiments utilising
MGM (high levels of yeast extract and peptone) had suggested that supplements used in
traditional media formulations might discriminate against the SHOW organism in
favour of microorganisms of a type more easily cultured.
An attempt had previously been made (Rossello-Mora et al., 2003) to identify substrate
uptake by members of the hypersaline lake microbial community, including the SHOW
organism. Their results indicated that the SHOW organism incorporated both aminoacids and acetate.
Low substrate medium supplementation – amino acids and acetate
In order to test whether the addition of amino-acids (a.a.) and acetate would aid in the
isolation of SHOW organisms, an extinction culturing experiment was established,
adding 5 µm of these supplements (each) to sterile saltern water, as described
previously (Materials and Methods). Owing to the number of samples being screened,
culturing from this point onward utilized tissue culture trays (6 x 4 3ml wells; total 24
wells per tray).
Screening results from these samples (Table 4.2) indicated that the addition of amino
acids and acetate was beneficial for the isolation of the SHOW organism. Of the 280
samples supplemented with amino-acids and acetate, there was only one positive for
growth – and it was a SHOW organism. Of the 284 samples that did not contain the
supplements, there were 19 positive for haloarchaeal growth – without any being the
SHOW organism.
These results suggested that the amino acids and acetate were generally inhibitory to
haloarchaeal growth under these conditions, but were permissive to the growth of
SHOW cells.
Low substrate medium supplementation – pyruvate
Concurrent research with the “Halonotius” group had indicated pyruvate was an ideal
growth substrate for those previously uncultivated organisms. Pyruvate has also been
previously reported as being a useful substrate for other Haloarchaea, such as Haloferax
volcanii and others. As such, pyruvate was added to SHOW isolation media in an
attempt to improve the growth of the SHOW organism. Variations of the previous
supplements were retained, so as to keep the possibility of the perceived inhibitory
effect on other strains.
All dilutions were screened for the first two trays (6 samples per dilution; 4 dilutions per
tray) of every medium to gain an indication of the appropriate dilutions to target for
screening. Low numbers of growth-positive cultures were observed from Medium A (5
µm amino-acids, 5 µm pyruvate) and B (as for A, plus 5 µm acetate), so the majority of
screening focussed on Medium C (0.5 % pyruvate, 5 µm amino acids), and to a lesser
extent, Medium D (Medium B supplements at 10 x concentration), these having greater
numbers of growth-positive wells (as detected by PCR). Medium C had 60 samples of
the intermediary 2 x 10-8 and 1 x 10-8 dilutions, and Medium D had 24 samples of the
same dilutions screened.
Results indicated that the addition of pyruvate improved SHOW cultivation efficiency,
but only when added in high concentrations (0.5 % w/v). All SHOW isolates were
recovered from this media containing pyruvate at this concentration; none were
identified in media containing no pyruvate, or at the lesser concentrations of 5 or 50 µm.
The lack of SHOW isolates in all media other than that containing 0.5% (w/v) pyruvate
was not unexpected; the recovery rate in earlier experiments was very low, and fewer
samples were screened in this subsequent iteration. The supplements of acetate and
amino acids seem to have retained their suppresive effect on haloarchaeal species other
than the SHOW, while the pyruvate delivered the desired growth-promoting effect
observed on the “Halonotius” and other groups.
SHOW isolation
From the extinction cultures described above, Medium C yielded three cultures positive
for SHOW growth, a recovery of 2.5 %. This compares unfavourably with the
population abundance detected in 2002 of 33 % (Burns et al., 2004), but in the context
of being the first isolation of this organism after a quarter-century of attempts seems
reasonable. In addition, success rates of approximately 10% of the target population
abundance of an organism may be considered reasonable; these sorts of recovery rates
would previously have been considered excellent, as historically cultivation efficiencies
of even 1% of total population would have been considered acceptable, although
recently, efficiencies as high as 20-50% have been reported (Chin et al., 1999, Joseph et
al., 2003).
Fig 4.1: Gel photograph of SHOW screening PCR showing
SHOW-positive cultures. Lanes 1-6: Tray C13-18, 2 x 10-8 dilution.
Lanes 7-12: Tray C13-18, 1 x 10-8 dilution. Lanes 13-18: Tray C1924, 2 x 10-8 dilution. 19-24: Tray C19-24, 1 x 10-8 dilution. Lane 25:
SHOW clone control. Lane 26: Negative control. Lane 27: Saltern
water lysate. Lanes 12 and 23 show amplification of SHOW-specific
product, while lane 14 contains a non-SHOW haloarcheaon. This is
the screening gel that detected the type strain, Haloquadratum
walsbyi, strain C23T. Growth levels are unusually low in this set of
samples.
NB: The gel has had some post-capture contrast modification to
enable easier viewing of the relevant bands.
One of the SHOW isolates recovered was recovered from the 2 x 10-8 and two from the
1 x 10-8 dilution.
Growth of “other” haloarchaeal species in Medium C was in the range of 33-66% of
samples at the 2 x 10-8 dilution level and 0-33% of samples at the 1 x 10-8 dilution. It
would be expected that all of these other organisms were most likely previouslycultivated species, in line with results gleaned from earlier cultivation experiments
performed in this laboratory, and the field of cultivation generally (Burns et al., 2004).
These species were thus not identified any further, as cultivation studies performed in
my Honours year had recovered multiple isolates of all the major organisms in the
target saltern, as detected by 16S rRNA gene clone libraries, except the SHOW
organism, and it was considered unnecessary to devote resources to isolation of
organisms other than the SHOW given this isolate collection.
Medium
No. of SHOW positives
Total Samples
Recovery
0
25
0%
1 (not pure)
42
2.3 %
SSW + a.a. + acetate
1
280
0.4 %
Medium C (SSW, a.a.,
3
60 (Medium C only)
5%
MGM
Sterile Saltern Water
(SSW)
acetate and pyruvate)
Table 4.2: Summary of extinction culture medium and SHOW
recovery by medium type.
Passaging of SHOW cultures
The three SHOW cultures were passaged 7 days after detection by PCR into Medium C,
the isolating medium formulation. The dilution was approximately double (2 ml). A low
dilution level was used to reduce the chance of a sudden nutrient burst inhibiting or
killing the cultures (Jensen et al., 1996); the remaining isolating medium volume
containing the SHOW isolates was only ~ 1.5 ml after removal of 0.5 ml for the PCR
screening. Additionally, an often observed effect in “difficult” microorganisms (such as
methanogens and other extremophiles) is that adding large volumes of fresh media to a
culture is toxic or lethal. Suggestions into the cause of this effect have included, for
instance, that there may be enough oxygen radicals in a large addition of new medium
to completely overwhelm the microorganisms' defences against these toxic molecules –
adding a lesser amount of medium reduces the number of radicals being added and thus
the culture is able to survive the medium addition.
This parallels the idea of 'conditioned media', in which spent, or partially-used media is
recycled (usually by sterilisation or centrifugation to remove existing organisms) with
the addition of fresh nutrients and added to the existing culture. When used in this
manner the specific function or substances in the spent media are usually unknown but
have a proven beneficial effect. In the instance of the SHOW cultures, this 'conditioned
media' effect was achieved by using smaller culture dilution levels when adding new
growth medium.
SHOW medium variation and optimization
After three doubling dilutions (from ~ 2 to 4 ml, 4 to 8, and 8 to 16 ml) of the cultures
(~ 5 weeks), and presence of growth confirmed by microscopic examination, sufficient
culture was available to begin experimentation with the actual medium formulation to
improve yield and growth rate. Three variations were trialled (see Table 4.3, below)
while cells also continued to be passaged in Medium C.
Volume of Medium C /
Trial Medium
Compared to Medium
Trial medium
2 ml + 2 ml
C
0.05% pyruvate + 50 µm amino
1
/10 pyruvate
acids
concentration
2 ml + 2 ml
0.5 % pyruvate
(-) amino acids
2 ml + 2 ml
50 µm amino acids
(-) pyruvate
2 ml + 2 ml
Medium C
identical
Table 4.3: Initial SHOW isolation medium variations.
Cultures were passaged several times for 6 weeks using doubling dilutions; repeat
passaging was necessary to permit the diluting-out of the original medium formulation
given the low dilution levels being used.
After three passagings, a further variation was added; each existing variation (as
described in Table 4.3) was split and 50 µm (NH4)HPO4 added to one of the culture
splits.
The (NH4)HPO4 was added to provide nitrogen and phosphate, in the event that the
cultures were limited for either or both of these substances. As with pyruvate, these
substances were added (albeit separately as NH4Cl and KH2PO4) to “Halonotius”
cultures and, at the minimum, had no ill-effect and were believed to be growth
promoting.
It was found that, as expected, pyruvate was the critical medium addition; cultures with
reduced pyruvate (0.05 %) still grew to acceptable levels, while those without grew very
poorly. The addition or removal of amino acids had no noticeable effect on growth.
Isolate Purity
Purity tests were constantly performed to ensure culture purity. These included
microscopic examination, sequencing and restriction fragment length polymorphism
analysis.
Microscopic examination
Microscopic examination was considered relatively reliable, given the unusual
morphology of the SHOW organism. However, low-level contamination of the cultures
may be overlooked with this technique, and other complicating factors included the
pleomorphic nature of other Haloarchaea. Examples of other Haloarchea with a square
morphology include Haloarcula quadrata (Oren et al. , 1999), occasional cells in
Haloarcula japonica culture and some Halorubrum spp. (personal observations).
Crucially, the SHOW organism is the only organism of those observed to form square
cells that possesses many and obvious gas vesicles. Additionally, when under nutrientlimiting conditions, the SHOW cells may also possess large numbers of
polyhydroxyalkanoate, (PHA), most likely polyhydroxybutryrate (PHB) granules.
Restriction Fragment Length Polymorphism (RFLP)
Purity by RFLP was also regularly performed. In-silico digestion identified a restriction
enzyme that would differentiate SHOW organisms from other Haloarchaea; this
enzyme was also used to differentiate the other major isolates from the Cheetham Salt
Works, including those described in Chaper Three.
RFLP digests, using amplified DNA, would be expected to display additional or
incorrectly sized bands if there were any contaminating cells in the culture. None were
observed in the SHOW isolates, indicating the cultures were almost certainly axenic.
Figure 4.2: RFLP purity gel photograph showing differences in
fragment pattern between various members of the Family
Halobacteriaceae. Lanes 1 and 12: NEB 100 bp DNA ladder. Lanes
2-5: 1.15.5, 2.27.5, 5.24.4, 6.14.5 (“Halonotius” group). Lanes 6-7:
4.03.5, 8.8.11 (Natronomonas-like group). Lane 8: Isolate 2.24.4 Lane
9-10: Haloquadratum walsbyi, strains C23 and HBSQ001. Lane 11:
Halobacterium salinarum.
Sequencing
Consistent with the RFLP data, sequencing of the 16S rRNA gene indicated no traces of
other organisms, although higher levels of contamination would be expected to be
required to observe base changes in a sequence chromatogram. The 16S rRNA gene
sequence matched with >99% similarity existing SHOW 16S rRNA gene clone
sequences (from environmental clone libraries) such as X84084 (Benlloch et al., 1995)
and CSWF61 (Burns et al., 2004).
Isolate Selection
The best growing isolate, C23, was selected for continued passaging This culture was
then underwent comprehensive characterisation in a bid to improve yield and growth
rate, and also to more fully understand the organism's physiology and role in the
environment it was isolated from.
Chapter 5
Characterisation of Haloquadratum walsbyi
Preface
After publication of the isolation of C23 (Burns et al., 2004a) another SHOW isolate
was reported by Bolhuis and colleagues (Bolhuis et al., 2004). Different isolation
methods were used: isolate C23 (Australia) was recovered using a rapid extinctiondilution culturing technique, and isolate HBSQ001 (Spain) was obtained by serial
enrichment over a 2-year period. Despite the time difference in isolation method, in
both cases low-nutrient media containing pyruvate was used, which appears to be a
critical factor in isolation of this organism.
Following these publications, a formal arrangement for collaboration with this group
was proposed and agreed upon to expedite characterisation of both of these novel
SHOW isolates. However, owing to the European laboratories having difficulties
cultivating these organisms, all characterisation was eventually completed in this
laboratory, with the exception of lipid analyses (Kamekura, Japan), mol G+C % and
DNA/DNA hybridization (Tamekura, Japan) and cryo-electron microscopy (Jensen and
Li, USA).
Microscopy
Light microscopy of all SHOW cultures was consistent with C23; as C23 exhibited the
best growth, this isolate was selected for more extensive characterisation and its results
are those described here.
Isolate C23 cultures under optimal growth conditions (Figure 5.1A, 5.1B) showed very
thin (0.2 µm), square (average of 2.6 µm) or slightly rectangular (average 2.3 x 3 µm)
cells with well-defined, sharp corners, prominent, phase-bright, gas vesicles and dark
granules. Apart from rare triangular cells, the culture appeared homogeneous. Division
borders between cells were not always easy to see, making estimates of cell sizes
difficult. At times, cells appeared to have divided but maintained edge proximity,
resulting in 'sheets' of (usually) four or more cells (akin to sheets of postage stamps).
Much larger cell aggregates were also observed; HBSQ001 displays cell sheets of up to
40  40 µm (Bolhuis et al., 2004), and C23 can form sheets of at least 12 µm a side
(data not shown). At salinities below ~23%, the cell morphology deteriorates to a
ragged square or other flat pleomorphs, presumably due to increased cell turgor or
perhaps changes to protein behaviour under the reduced ionic concentrations of lower
salinity.
The gas vesicles were easily collapsed by pressure (such as dropping, rather than
placing, a cover slip onto the sample). The dark granules were shown by Nile Blue A
staining to be PHA (probably PHB) (Figure 5.1C and 5.1D). By negative stain electronmicroscopy (figure 5.1E) the gas vesicles are seen as thin cylinders of variable length
with cone-shaped ends (~ 0.1 µm x 0.2 - 0.6 µm). The shape of these vesicles contrasts
with the spindle-shaped vesicles common in another haloarchaeon, Halobacterium
salinarum (Walsby, 1994; Pfeifer et al., 1997), but is similar to that found in the
majority of haloarchaea and other organisms. The gas vesicles are most commonly
found near the periphery of the cell. Three, electron-dense PHB granules (~ 0.3 µm
diameter) can also be seen at the right hand corner of the cell in Figure 5.1E.
Figure 5.1: Cryo-electron microscopy.
Electron cryomicroscopy (conducted by Zhuo Li and Grant Jensen, California Institute
of Technology, Pasadena, California, USA) resolved internal cell structures very clearly
(Fig. 5.1). The two isolates showed similar types of gas vesicles and
polyhydroxyalkanoate (PHA) granules but differed significantly in cell wall structure.
Isolate C23T possessed a typical two-layer cell wall consisting of a simple S-layer above
the cell membrane (Fig. 5.1A, D, E). The cell wall of isolate HBSQ001 displayed a
more complex, apparently three-layered structure, unlike other haloarchaea (Fig. 5.1B,
F, G, H). Fig. 5.1H shows the density profiles across both cell walls. In HBSQ001, two
peaks were resolved outside the cytoplasmic membrane, corresponding to two layers of
surface structure. Each layer appeared ~14 nm away from the next (peak-to-peak). In
C23T, there was only one surface
layer covering the cytoplasmic membrane, also ~14 nm above the membrane.
All of the physical characteristics shown by isolate C23 and HBSQ001 are very similar
to those of naturally occurring SHOW organisms, as reported originally by Walsby
(Walsby, 1980) and in later studies.
Macroscopic appearance
Colonies take 4 – 8 weeks to grow on solid media (DBCM2 with agar as the gelling
agent) under aerobic conditions. We were unable to produce growth on solid media for
some time. Optimisation of the liquid media was first required before using that
formulation, in conjunction with agarose (not agar) as the gelling agent eventually
proved successful. The colonies now grow on unwashed agar as well; presumably the
slowly-improved media formulation, combined with some 'laboratory adaptation' of the
strains themselves have contributed to this result.
After 8 weeks, colonies are small (0.5 – 1.0 mm diameter), convex, round, with an
entire edge, and intense red to pink in colour. Liquid cultures are also a light pink to
intense, almost iridescent pink/red colouration, depending upon the cell density.
Further characteristics
Both strains are non-motile and stain Gram negative. A large number of potential
growth substrates were tested (see species description) but pyruvate was the only carbon
source to reproducibly permit measurable growth of both SHOW isolates. Cells are
oxidase and catalase negative using conventional testing. They do not hydrolyse starch
or casein, and do not show -galactosidase activity. Indole is not produced in
tryptophan-containing media. Neither strain was able to use nitrate (either by itself, or in
the presence of L-arginine) or DMSO as alternative electron acceptors under anaerobic
conditions.
The optimum growth temperature for both strains was 45 °C. The minimum growth
temperature was 25 °C for C23 and 30 °C for HBSQ001. No growth was observed at 56
°C. Growth occurred over a wide range of salinity, from a minimum of 12% (w/v) for
C23 or 14% (w/v) for HBSQ001, up to saturation. From 18–36% salinity, the growth
profiles of both isolates were relatively flat, and did not show a steep rise and fall
around an optimum concentration. The optimum salinity was 18% (w/v) for both
HBSQ001 and C23. Neither strain required magnesium ions, but growth was poor in
their absence. Optimum growth varied both by strain and Mg-associated anion. C23 had
no specific optimum for MgCl2, but concentrations above 1 M MgCl2 yielded higher
cell densities than lower Mg2+ concentrations, while growth peaked at 0.4–0.6 M
MgSO4. HBSQ001 grew optimally at 0.2 M MgCl2, but required 0.6 M MgSO4 to reach
the same density. In both strains, growth declined markedly at high MgCl2
concentrations compared to MgSO4, suggesting the high chloride ion concentration (>5
M) may have been inhibitory.
The two isolates had similar responses to pH, exhibiting growth over the range 5.5-8.5.
Optimum growth rate was at pH 6.5 for C23 and pH 7.0 for HBSQ001. Both SHOW
strains were sensitive to the antibiotics anisomycin, chloramphenicol, erythromycin,
novobiocin, rifampicin, simvastatin and tetracycline. They were resistant to ampicillin,
bacitracin, cycloheximide, kanamycin, mycostatin, neomycin and streptomycin.
These results are unusual in that chloramphenicol, erythromycin and tetracycline are
normally considered to affect only Bacteria. Under the same conditions, all other
Halobacteriaceae tested were resistant to these antibiotics. This implies that these
antibiotics are targeting in some manner a feature specific to the SHOW group.
Fig 5.2: TLC showing unique polar lipid profile of SHOW isolates.
Hfx. mediterranei ATCC 33500T (lane 1), Hfx. volcanii NCIMB
2012T (2), Hgm. borinquense JCM 10706T (3), strain HBSQ001 (4),
strain C23 (5) and Hbt. salinarum NRC-1 (6). The origin is at the
bottom.
PG,
Phosphatidylglycerol;
PGP-Me,
phosphatidylglycerophosphate methyl ester; S-DGD-1, sulfated
diglycosyl diether lipid. Glycolipids were detected as purple spots and
circled in pencil. Lane 4 showed a visible but faint PG spot that did
not reproduce in the photograph. Cells were grown as described in
Materials and Methods.
As shown in Figure 5.2, the mobilities of the polar lipids detected by TLC (performed
by M. Kamekura, Noda Institute for Scientific Research, Japan) were identical in the
two isolates, although the proportion of phosphatidyl glycerol (PG) observed in the
HBSQ001 sample was lower than that of C23T. The PG spot for HBSQ001 sample was
faint in the original TLC and did not reproduce in the photograph shown in Fig. 5.2. In
addition to PG and phosphatidyl glycerate-methyl ester (PGP-Me), the isolates
possessed a glycolipid that co-chromatographed with S-DGD-1 (Fig. 5.2, lanes 4 & 5), a
lipid that occurs in the Haloferax species (lanes 1 & 2). This is consistent with previous
data derived from the extraction of lipids from natural samples containing almost pure
populations of SHOW cells (Oren et al., 1996).
Figure 5.3: 16S rRNA gene sequence alignments showing relative
position of SHOW clade. Phylogenetic tree reconstruction based on
complete or nearly complete (>1300nt) 16S rRNA sequences. The tree
shown was derived by maximum likelihood (using the ARB package).
Scale bar represents 0.1 expected nucleotide substitutions per site.
Bootstrap values (using distance matrix methods) were derived from
1000 replicates and significant nodes (>75%) are indicated by filled
circles at branch points. The outgroup sequences consisted of
sequences representing most of the other genera of the
Halobacteriaceae as well as that of Methanosaeta concilii ATCC
35969T (accession X16932.1) (not shown)..
The G+C content of both isolates was 46.9 mol %, and whole genome DNA-DNA
hybridisation gave a relatedness of ~ 80 %. Their 16S rRNA gene sequences were
almost identical, with only 2 base differences. These results indicate they are members
of the same species. Phylogenetic tree reconstructions placed both SHOW sequences in
a separate clade within the Halobacteriaceae (Fig. 5.3). This clade also contained
environmental clone sequences from SHOW organisms, including X84084 (Antón et
al., 1999). The closest cultivated isolate to this clade was T1.3 (93.2 %), isolated from
a salt mine (McGenity et al., 2000) (not shown). Among the recognised members of the
Halobacteriaceae, the closest relative was Halogeometricum borinquense (91.2 %
sequence similarity). The 16S rRNA gene sequence similarity to Hfx. volcanii (90.3 %),
Hrr. sodomense (85.6 %), and Htg. turkmenica (87 %), the control organism used in the
DNA-DNA hybridisation, was much lower.
The phenotypic characterisation and phylogenetic data support the placement of the
isolates C23 and HBSQ001 in a new species and a new genus within the
Halobacteriaceae, for which we propose the name Haloquadratum walsbyi gen. nov.,
sp. nov.
GENUS and SPECIES description
Description of Haloquadratum gen. nov.
Haloquadratum (Ha.lo.qua.dra' tum. Gr. masc. n. hals, halos salt; L. neut. n.
quadratum square; N.L. neut. n. Haloquadratum, salt square)
Flat, square cells that usually contain gas vesicles and polyhydroxyalkanoate storage
granules. Aerobic heterotrophs. Oxidase and catalase tests are negative. Cells stain
Gram negative. Phylogenetically belonging to the family Halobacteriaceae. Habitat:
salt lakes and saltern crystallizer ponds. The G+C content of the type species is 46.9
mol%. The type species is Haloquadratum walsbyi. Following the published guidelines
(Oren and Ventosa, 2000) we propose the three-letter genus abbreviation Hqr.
Description of Haloquadratum walsbyi sp. nov.
Haloquadratum walsbyi (wals' by.i . N.L. gen. masc. n. walsbyi, of Walsby, named after
A.E. Walsby, who first published observations on this organism.)
Gram-negative. Cells are square (~ 2  2 µm) and flat (0.2 µm thick), and can form
large sheets. Under suboptimal conditions, such as reduced salinity, they show flat,
pleomorphic forms. Colonies on agar medium are iridescent pink with entire edge.
Strictly aerobic; only oxygen is used as the final electron acceptor. Cannot utilise NO3or DMSO as alternative electron acceptors. Does not grow anaerobically on L-arginine.
Growth occurs at pH 6.0 – 8.5, 25–45 °C and 14–36 % (w/v) NaCl. Halophilic; cells
lyse immediately in distilled water, and a minimum ~14 % (w/v) salts required for
growth. Optimal growth occurs at neutrophilic to alkaliphilic conditions, above 18 %
salinity. Capable of growing in defined media, but is severely restricted in the substrates
utilised. Grows best on pyruvate as sole carbon source. No growth enhancement
occurred with acetate, alanine, arabinose, arginine, aspartate, benzoate, betaine, butanol,
butyrate, cellobiose, citrate, ethanol, formate, fructose, fumarate, galactose,
galacturonate, gluconuronate, glucose, glycerol, glycine, glycolate, lactate, lactose,
leucine, lysine, malate, malonate, mannitol, mannose, methanol, propanol, propionate,
ribose, serine, succinate, sucrose, tartrate, threonine, urea, valine, xylose, at 10 mM;
and; cellulose, chitin and starch at 0.1% (w/v) as the sole carbon and energy source.
Acid was not produced from carbohydrate utilisation. Produces polyhydroxyalkanoate
granules. Negative for -galactosidase, oxidase, catalase activity and indole production.
Sensitive to anisomycin, chloramphenicol, erythromycin, novobiocin, rifampicin,
simvastatin and tetracycline, and resistant to ampicillin, bacitracin, cycloheximide,
kanamycin, mycostatin, neomycin and streptomycin at 50 µg ml-1. The polar lipids are
C20C20 derivatives of PG, PGP-Me and S-DGD-1. The DNA G+C content of the two
strains is 46.9 mol%. Both strains isolated from solar saltern crystallizer ponds.
Phylogenetically affiliated to the Halobacteriaceae. The type species of the genus is
Haloquadratum walsbyi, isolated from Cheetham Salt Works, Geelong, Australia. The
type strain is C23T ( =JCM 12705 = DSM 16854). Strain HBSQ001 has been deposited
as JCM 12895 ( = DSM 16790).
Chapter 6
Final Discussion
This study set out to isolate and characterise the organism now recognised as
Haloquadratum walsbyi, while also characterising previously isolated novel organisms
from an Australian solar saltern.
The isolation of Haloquadratum was achieved using relatively simple extinction
culturing methods; a contrast to some of the more exotic attempts at isolating
recalcitrant microbes, such as encapsulating cells in gel microdroplets (Zengler et al.,
2002), complex simulations of the natural environment (Kaeberlein et al., 2002) and
micromanipulation and separation of cells (Fröhlich and H. König, 2000). This suggests
that variations on more traditional cultivation techniques such as plating (Joseph et al.,
2003, Burns et al., 2004) and extinction culturing (this study) still have much to offer in
their ability to isolate novel microbial diversity, with significantly reduced costs (in both
financial and temporal terms) and complexity compared to 'advanced' cultivation
techniques. The extinction culturing technique combined with PCR screening
techniques could be easily adapted to target other genera of interest. Using this
technique, 16S rRNA sequence is the only prerequisite information required to make an
attempt on any other genus (provided an appropriately specific primer can be designed,
of course). Alternatively, a functional gene could be targeted, yielding organisms
hosting these genes.
Having now characterised Haloquadratum walsbyi, and demonstrated it it can grow on
solid media (despite initial attempts failing to isolate the organism on solid media), I
now believe that directly isolating this organism from hypersaline waters using plating
techniques, with a medium optimised for its growth (such as DBCM2), presents as an
achievable target.
While this was study was successful in characterising a range of novel haloarchaeal
isolates, as many questions as answers have been raised about these organisms. In vitro,
Haloquadratum walsbyi grows only on pyruvate as its primary substrate, but it is highly
unlikely that free pyruvate exists in the natural environment. The Haloquadratum
genome contains catabolic genes that should permit a wider variety of substrates to be
utilised (Bolhuis et al., 2006), but these seem not to be expressed in the laboratory
setting, despite a wide variety of media and conditions being trialled. It is possible that
further work may elucidate a trigger compound or suitable conditions for expression of
these genes and activation of other metabolic pathways.
This example of a schism existing between the phenotype and genotype highlights both
the advantages and drawbacks of utilising either molecular or cultivation techniques in
isolation. In this case, if the genome sequence had been performed first (acquired
through a metagenomic study, for instance), then the pyruvate dependency that was
critical in its isolation may not have been considered as an avenue worthy of pursuit.
Alternatively, the characterisation work taken in isolation would suggest a limited
genome, when in fact, this is not the case.
Indeed, it seems likely that all of the isolates characterised in this study most likely have
a greater metabolic potential than determined in the laboratory, given their generally
restricted substrate utilization. However, it would seem unlikely that they would
encounter the characterization conditions in the natural environment; such taxonomic
tests being a relatively crude and artificial method of grouping organisms, and not
accounting for the likely presence of multiple complex substrates and secondary
metabolites in the natural environment, and interaction of these in determining cell
metabolism. Nevertheless, some conclusions can be drawn about the lifestyle and role
performed by these organisms. All are aerobic mesophiles, obligate halophiles and
neutrophilic. Perhaps surprisingly, optimum growth for all of the isolates was in the
range of 20-24 % salinity, well below that of the isolating environment (saturation,
>36%). This suggests that crystallizer ponds are a sub-optimal environment for these
organisms, but they nevertheless can outcompete all other species in these adverse
conditions. Strangely though, they are not always dominant at their own salinity
optimum (Benlloch et al., 2002) suggesting that their growth may not be as fast as their
competitors at lower salinity, but is faster than them at higher salinities, despite the
higher salinities leading to an overall reduction in their growth rate.
For the first time, a complete library of isolates comprising all of the numerically
significant Haloarchaea in a thalassohaline solar saltern is now available to researchers.
This represents a remarkable opportunity to explore community relationships both
between the Haloarchaea themselves, and also investigate their interaction with the
broader environment (in particular, haloviruses). Viral isolates are easily made with, for
example, Halorubrum spp. (Kate Porter, personal communication), which often
comprise a large fraction of the total haloarchaeal population (Burns et al., 2004),
indicating that at least in some cases, high population does appear to correlate with an
abundance of genus-specific viruses.
Unfortunately, owing to the inability to isolate any haloviruses on the novel species
described in this thesis, this study does not investigate whether the abundance of
Haloquadratrum or Halonotius (or any of the other isolates) is reflective of either a
increased resistance to the ever-present halophages in hypersaline waters, or a lack of
phage that specifically target these genera. Either scenario may account for the lack of
viral isolates, but with the improved media now available for these new species, and
consequent improved culture density and growth rate, novel viruses targeting these
organisms may be able to be isolated in future work, permitting ecological experiments
to be undertaken.
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