Life Sciences and Medicine Research, Volume 2010: LSMR-18
Isolation Of A Potential New Member Of The Bacillus cereus Group From Snow
Covered Soil
*Sarmad Saman, Sudad Saman, Patty Slattery
Life Sciences Department, Massachusetts Bay Community College, 50 Oakland Street, Wellesley, MA 02481,
USA
*Correspondence to: Sarmad Saman, ssaman@massbay.edu, sarmadsh@yahoo.com
Published online: September 12, 2010
Abstract
C1 is an endospore forming gram-positive rod isolated from snow-covered soil in Massachusetts. Colonies were white and feathery in appearance
and grew after 22 hours. The isolate was motile with ellipsoidal spores that are subterminal. The strain grew best at temperatures between 5-25˚ C
with a pH range between 5–7 and NaCl concentration 1% (w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated that C1
belonged to the genus Bacillus, and the Bacillus cereus group. C1 was closest to Bacillus cereus with 40% alignments and 0.69% to Bacillus
thuringiensis. The cellular fatty acid profiles were closest to Bacillus mycoides with the iso-C15 making 21.25% of the total cellular membrane fatty
acids. On the basis of phenotypic and molecular data, the strain C1 represents a novel species within the Bacillus cereus group. A culture deposit
was made to the Biodefense and Emerging Infections Research Resources Repository (BEI Resources) with the BEI code (NR-4056).
Keywords: Bacillus cereus; new species; isolation; soil.
1.
Introduction
Bacillus sp was discovered by Cohn, and Koch in the 19th century, in which this group contains gram positive endospore forming
rods. The spore forming ability enabled these bacteria to survive in extreme environments. The Bacillus cereus group is composed of
B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B. weihenstephanensis [10, 12, 13]. Members of this
group share many of their biochemical, morphological and very closely related gene sequence based on their 16S rRNA [4,18]. The
genomic and proteomics analysis between members of this group yield important findings to understand their evolution and
interrelationship. B. thuringiensis can be differentiated from B. cereus by the presence of the CRY crystal protein and plasmidencoded CRY genes thus loss of this gene would mean that B. thuringiensis could not be distinguished from B. cereus [1,2,7,8]. The
sequence of the 16S rRNA gene has been widely used as a molecular timepiece to estimate relationships among bacteria phylogeny
and recently it has also become important as a means to identify unknown bacterium not only by its genus but by its species too.
The 16S rRNA gene sequences of the B. cereus group have high levels of sequence similarity (>99%). Thus using 16S rRNA gene
sequencing would show this minimal diversity within the group [1,2,3,10]. The objective of this work is to use the sequence from the
16S rRNA of the newly isolated Bacillus samanii sp nov and compare it to other members of this group along with that of Bacillus
subtilis to see the degree of matching and their phylogenic relationship.
2.
Methods
2.1 Isolation of C1
Snow covered soil samples were collected during the month of February of 2006 in the woodland surrounding Massachusetts Bay
Community College in Wellesley, Massachusetts USA. The collected snow covered soil samples were placed in 15ml sterile falcon
tubes and placed in the incubator at 250C to thaw. Dilution series of 3 test tubes containing sterile 0.9% peptone water in 9 ml tubes
rd
were carried out. From the 3 dilution 1ml and .1 ml were removed and placed then spread on Nutrient Agar, Tryptic Soya Agar,
EMB, McConkey Agar, Mannitol Salt Agar and Muller Hinton. These plates were incubated at 5 0C, 25 0C, 370C and 48 0C. Feathery
rhizoid colonies coded C1 were selected and sub-cultured on TSA agar plates to obtain pure colonies of this isolate for identification.
2.2 Identification of C1
The isolate C1 was identified through Morphology, physiology, 16SrRNA and Fatty acids methylation Ester FAME.
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2.3 Morphology
Morphological characterization includes Gram staining using a kit supplied by BD for the C1 strain according to the standard method
of Gram staining and Spore staining using 5% Malachite green supplied by Carolina and using the method described in [6]. Cell
morphology and that of spores were observed using a Motic® DMBA 200-C Digital Laboratory Microscope at 1000 magnification.
2.4 Effect of Environmental Factors
The isolate was grown in TSB at 5˚, 25˚, 30˚, 35˚, 60˚ and 70˚ C to determine its optimum growth temperature. The isolate was also
grown at various pH levels such as pH 5, 7 and 9. The isolate was grown in a TSB solution with 1%, 3%, 5% and 7.5%, Sodium
Chloride.
2.5 Physiology
Biochemical activity included fermentation of sugars, enzymatic activity and motility. Catalase activity was determined by the
formation of bubbles when 3% hydrogen peroxide was added to a TSA slant that had growth of the C1 isolate on it. Oxidase ability
was determined by oxidation of 1% p-aminodimethylanine oxalate. Acidic fermentation of sugars was determined using a 1% sugar
solution with either phenol red added as an indicator. Durham tube was including in the test-tube to determine gas evolution from
the fermentation of sugars. Other biochemical tests included the utilization of citrate using Simmons citrate slant, urease production
using urea agar, protein degradation and production of hydrogen sulfide using Kliger iron agar slant, SIM and triple sugar iron.
Orthinine production from MIO, motility using motility agar slants. Gelatin hydrolysis was determined using nutrient gelatin slant.
Litmus milk was used to determine peptonization and Phenylalanine Agar is used to determine the isolates ability to produce
phenylpyruvic acid by oxidative deamination. Nitrate Broth was used to determine the nitrate reduction ability of the isolate. Blood
agar and CDC blood agar were used to determine the isolate hemolysis ability while starch/casein agar plate was used to determine
its ability to carry out hydrolysis of casein and starch. Basal media OF using Mannose, Dextrose, Xylose, Rhamnose, and Arabinose
were used to determine the oxidative and fermentative metabolism of carbohydrates. All the materials for the biochemical testing
were from Northeast labs.
2.6 16S rRNA
The GenBank accession numbers for the 16S rRNA gene sequences of the isolate C1 is EF036537. The GC ratio and percentage was
also determined for the isolate. The 16S rRNA was sequenced by MIDI Systems Inc. through PCR amplification of the 16S rRNA gene
will be carried out using universal bacterial primers. The primers to be used are universal 16S primers that correspond to positions
0005F and 0531R for a 500bp sequence, and 0005F and 1513R for the 1500bp sequence. Amplification products are purified from
excess primers and dNTPs and checked for quality and quantity by running a portion of the products on an agarose gel. Cycle
sequencing of the 16S rRNA amplification products was done using DNA polymerase and dye terminator chemistry. Data analysis
was carried out using MicroSeq Analysis Software and Sequence Database [11].
2.7 FAME
Fatty Acids Methyl Esters (GC-FAME) for the C1 isolate is provided. Cellular fatty acids were analyzed by fine capillary column gasliquid chromatography of fatty acid methyl esters (FAME) using a dedicated GC analyzer (MIDI Systems Inc., Newark, Del.) and the
corresponding proprietary interpretive software (Sherlock, version 2.01; MIDI Systems Inc.).
3.
Results and Discussion
3.1 Morphology
The isolate C1 was found to be a gram-positive endospore forming motile rod that occurred either as a single rod or in chains. The
spores were sub-terminal and ellipsoidal in shape. Colonies grow well and formed within 22 hours on various types of agar media
such as Nutrient agar, TSA, Muller-Hinton, TSA plus 5% sheep’s blood and CDC anaerobic blood agar. Colonies were white and
feathery in appearance with the growth seemed to be bearing to the outside of the main branch growth then to the inside.
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Life Sciences and Medicine Research, Volume 2010: LSMR-18
3.2 Effect of Environmental Factors
Table-1 shows the effect of temperature, pH and Sodium Chloride concentration of the growth of the isolate C1. The table shows
that the isolate grow well between 5˚C–25˚C and the growth slowed down above 35˚C and there was no growth at 60˚C. Table-1
does show that C1 grow optimum in neutral pH with a moderate growth at low acidic pH however its growth was inhibited at basic
pH. Salt solution that contains 1% (W/V) NaCl showed the best growth for C1 as shown in table-1. Salt concentration of 3% (w/v)
showed weak growth however growth was inhibited at above of that.
Table–1 Effect of pH, Temperature and Salt Concentration on the rate of growth of C1.
pH
5
7
9
+++ = Heavy Growth.
++= Moderate growth.
+= Light Growth.
- = No growth.
Growth
++
+++
-
0
Temp C
5
25
30
35
60
70
Growth
++
++
+++
+
-
NaCl % w/v
1
3
5
7
Growth
+++
+
-
3.3 Physiology
Table-2 showed C1 biochemical ability produced acidic fermentation from glucose, lactose, maltose, sucrose and trehalose. However
there was no gas production and the isolate did not ferment mannitol, Sorbitol, Arabinose, mannose and cellbiose. There were also
negative results for the utilization of citrate, urease production, gelatin hydrolysis, oxidase production and the break down of litmus
milk. The isolate did not reduce nitrate or produce hydrogen sulfide, Ornithine, lysine and Phenylalanine. The isolate was however
positive for production of catalase. The isolate produced β-hemolysin, reflecting its ability to carry out β-hemolysis as it was
detected on TSA with 5 % sheep’s blood (blood agar).
Table–2 Physiological characteristics for C1.
Test
Dextrose
Sucrose
Lactose
Maltose
Mannitol
Sorbitol
Trehalose
Fructose
Cellbiose
Galactose
Glycerol
Simmons Citrate
Litmus Milk
Nitrate Reduction
Gelatin Hydrolysis
Urea
Oxidase
Catalase
Result
+A
+A
+A
+A
+
+
+
+
+
A = Acid production
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3.4 16S rRNA Sequencing
Figure-1 shows the sequence comparison of 1513 bp for the 16S rRNA gene sequence (sequence GenBank accession EF036537)
Bacillus samanii sp nov. With 2034 bp of the 16S rRNA gene sequence Bacillus subtilis showed no significant similarity between the
two. The phylogenic tree showed that Bacillus samanii sp nov falls closer to Bacillus cereus than other members of the group. There
was 0.40% alignment between the two compared to 0.69% with B. thuringiensis and 0.93% with B. mycoides for the full-length
sequencing. However, for the 500 bp the alignment results were 0.56%, 1.40% and 1.49% with B. cereus, B. thuringiensis and
B. mycoides respectively. Areas of mismatch were seen in bp number 185 to 244 in 3 bases with B. cereus, B. thuringiensis and bp
1025-1084 in 2 bases. There was also a marked difference in the protein regions as B. cereus has a region of low complexity between
bp400-500, which Bacillus samanii sp nov does not have. The DNA G+C mol% content of C1 was 53.67 mol%. The 196bp showed C in
B. samanii compared to T in the others and 204 bp showed C in B. samanii compared to G in the others. Remarkable was the first 60
bp
that
were
composed
of
the
following
sequence:
(TGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCG) and shared by all tested members with the
exception in base pair 59 in which B. samanii has C compared to T in Bacillus cereus E33L. The other observation is that B. samanii
shared the sequence from 1500-1512 (TCGTAACAAGGT) with the other group members.
Figure-1 Full Length Alignment Report for C1 16S rRNA.
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Life Sciences and Medicine Research, Volume 2010: LSMR-18
3.5 FAME
Table-3 shows the cell membrane fatty acids analysis of the isolate that were mostly made up from C12 to C17 and included
saturated, monoenoic and iso-branched components. Iso-C17: 0 made up the highest percentage of 14.65% then iso- C13: 0, which
made up 9.97%. The lowest was iso-C18: 0 which only made up 0.23% of the total cellular fatty acids.
Table-3 FAME Analysis of C1.
These results indicate that the isolated bacterium is very closely matched on genetic levels with other members of this group with
distinctive differences in its production of proteins. The use of the 16S rRNA gene sequence is of prime importance to distinguish bacteria
and enable to identify those that have not been isolated or described before. The 16S rRNA gene sequence alignment similarity between C1
to Bacillus cereus was 40%, which was the closest match for the full length of 1513BP, and 56% for the 500BP with the same
bacterium. There are widely accepted criteria for delineating species in current bacteriology: these state that strains with DNA
relatedness values of less than 70% or with more than 3% difference in their 16S rRNA gene sequences are considered to represent
different species [5,19].
Physiological comparison of this isolate with other members of the Bacillus cereus groups clearly shows that there is very little
difference. This would support the fact that it’s a member of the group however the difference the gelatin hydrolysis ability, motility,
indole production and lactose fermentation does reflect enough difference in its biochemical; ability which would reflect difference
in its genetic makeup [14,10]. Short chain fatty acid analysis, which are also known as volatile fatty acids (VFA), could be used for the
identification of bacteria as they could seve as a second finger print for bacteria. These fatty acids which are found as part of the cell
membrane bilipdi layer have a length chain of 9 - 20 carbons. This method was used for genus and species identification that shared
most of their fermentative abilities. Gram-positive bacteria had more branched fatty acid chains compared to gram negatives that
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had short chains of fatty acids as this is related to their lipopolysaccharides that make up most of their cell wall. Cellular fatty acids
analysis uses gas chromatography of fatty acid methyl esters (FAME) interpret the results. Isolates should be inoculated onto
Trypticase soy agar for 24 hours at their optimum growth temperatures [15,16,17].
Table–4 Physiological characteristics comparison between C1 and other Bacillus sp.
Character
Motility
Casein hydrolysis
Starch hydrolysis
Gelatin hydrolysis
Nitrate reduction
Indole production
Fructose
Galactose
Mannitol
Glucose
Lactose
Maltose
Sucorse
Xylose
Catalase
C1
+
+
+
+
+
+
+
+
+
+
+
B. cereus
+
+
+
+
+
+
+
+
+
+
+
B. mycodies
+
+
+
+
+
+
+
+
+
+
+
B. megaterium
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
B. subtilis
+
+
+
+
+
+
+
+
+
+
+
+
+
+
[14]
C1 is a Gram-positive endospore forming, rod-shaped facultative anaerobic bacterium that is 0.75–1.2 mm wide and 2.0–2.5 mm
long, motile and occurs singularly or in chains. They produce ellipsoidal endospores that are subterminal positions with very little
sporangia swelling. Colonies formed after 22 hours at 30˚C on blood agar, TSA and Muller-Hinton were white and feathery in
appearance and grows best at pH 7. Growth slowed down at 37˚C and it was inhibited at 65˚C and growth was also inhibited at pH 9.
NaCl concentration of 1% (w/v) resulted in optimal growth but growth was inhibited by 5% (w/v) NaCl. Phylogenetic analysis based
on 16S rRNA gene sequences indicated that it is in the genus Bacillus, and the Bacillus cereus group. The G+C content of C1 was
53.67%. The cellular fatty acid profiles showed that were iso-C15 made up 21.25% of the total cellular membrane fatty acids.
Deposits have been made to the Biodefense and Emerging Infections Research Resources Repository (BEI Resources) catalogue
number NR-4056 and designated at Bio-level Safety 2. On the basis of the previously mentioned morphology, biochemistry, fatty
acid analysis, phenotypic and genetic characteristics of the isolate C1, this isolate is distinct from other Bacillus species and we
propose that it be considered as a new member of the Bacillus cereus group.
Competing Interests
The authors declare that they have no competing interests.
Authors' Contributions
SS and PS carried out the initial isolation and purification of C1. They also analyzed the morphology, physiology and effect of
environmental factors on the growth of C1. SS drafted the manuscript. Sudad Saman was responsible for the genetic variation and
other bioinformatics work that was required for this project.
References
1. Ash C, Collins MD, 1992. Comparative analysis of 23S rRNA gene sequences of Bacillus anthracis and emetic Bacillus cereus
determined by PCR-direct sequencing. FEMS Microbiology Letters, 94: 75-80.
E-ISSN: 19487886
Life Sciences and Medicine Research, Volume 2010: LSMR-18
2. Ash C, Farrow JAE, Wallbanks S, Collins MD, 1991. Phylogenetic heterogeneity of the genus Bacillus revealed by comparative
analysis of small-subunit-rRNA sequences. Letters in Applied Microbiology, 13: 202-206.
3. Ash C, Farrow JAE, Dorschy M, Stackebrandty R, Collins MD, 1991. Comparative Analysis of Bacillus anthracis, Bacillus cereus, and
Related Species on the Basis of Reverse Transcriptase Sequencing of 16s rRNA. International Journal of Systematic Bacteriology,
41(3): 343-346.
4. Bavykin SG, Lysov YP, Zakhariev V, Kelly JJ, Jackman J, Stahl DA, Cherni A, 2004. Use of 16S rRNA, 23S rRNA, and gyrB gene
sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. Journal of Clinical Microbiology,
42: 3711-3730.
5. Bavykin S, Vladimir M, Zakharyev K, Lysov P, Kelly J, Oleg A, Igor M, 2008. Discrimination of Bacillus anthracis and closely related
microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray. Chemico-Biological Interactions, 171(2): 212-235.
6. Brown A, 2006. Benson's Microbiological Applications: Laboratory Manual in General Microbiology. 10 th Ed. McGraw-Hill
Science/Engineering /Math.
7. Cherif A, Ettoumi B, Raddadi N, Daffonchio D, Boudabous A, 2007. Genomic diversity and relationship of Bacillus thuringiensis
and Bacillus cereus by multi-REP-PCR fingerprinting. Canadian Journal of Microbiology, 53(3): 343–350.
8. Daffonchio D, Borin S, Frova G, Manachini P, Sorlini C, 1998. PCR fingerprinting of whole genomes, the spacers between the 16S
and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and
Bacillus licheniformis. International Journal of Systematic Bacteriology, 48: 1081.
9. Daffonchio D, Raddadi N, Merabishvili M, Cherif A, Carmagnola L, Brusetti L, Rizzi A, Chanishvili N, Visca P, Sharp R, Borin S, 2006.
Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis. Applied and
Environmental Microbiology, 72(2): 1295-1301.
10. Lechner S, Mayr R, Francis KP, Pruss BM, Kaplan T, Wiessner-Gunkel E, Stewart GS, Scherer S, 2006. Bacillus weihenstephanensis
sp. nov. is a new psychrotolerant species of the Bacillus cereus group. International Journal of Systematic Bacteriology, 48(4): 13731382.
11. MIDI Inc, 2001. Sherlock Microbial Identification System References.
12. Nakamura L, 1998. Bacillus pseudomycoides sp. nov. International Journal of Systematic Bacteriology, 48: 1031 – 1035.
13. Nakamura L, Jackson M, 1995. Clarification of the Taxonomy of Bacillus mycoides. International Journal of Systematic
Bacteriology, 45: 46 - 49.
14. Ogunshe A, Omotosho M, Ayansina A, 2007. Microbial Studies and Biochemical Characteristics of Controlled Fermented Afiyo- a
Nigerian Fermented Food Condiment from Prosopis africana (Guill and Perr.) Taub. Pakistan Journal of Nutrition, 6 (6): 620-627.
15. Saman S, Slattery P, Saman S, 2009. Isolation, Description and Sequencing of a number of Novel microorganisms isolated from
the Hockomock Swamp in Massachusetts. 3rd ASM Conference on Prokaryotic Development.
16. Saman S, Saman S, Slattery P, 2008. Isolation and Characterization of protein toxins produce by A Related Bacillus anthracis and
cereus Novel Species Bacillus samanii sp nov. ASM Biodefense and Emerging Diseases Research Meeting, Washington DC.
17. Sasser M, 2006. Identification of bacteria by gas chromatography of cellular fatty Acids. MIDI Labs Incorporated Technical Note
#101, p. 1-6.
18. Wang L, Lee F, Tai C, Kasai H, 2007. Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA–DNA hybridization
in the Bacillus subtilis group. International Journal of Systematic and Evolutionary Microbiology, 57: 1846 - 1850.
http://astonjournals.com/lsmr
7
8
Research Article
19. Wayne LG, Brenner DJ, Colwell RR, 1987. International Committee on Systematic Bacteriology. Report of the ad hoc committee
on reconciliation of approaches to bacterial systematics. International Journal of Systematic Bacteriology, 37: 463–464.
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