JOURNAL OF BACTERIOLOGY, Aug. 2011, p. 4199–4213
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 193, No. 16
Genome Sequences of the Biotechnologically Important
Bacillus megaterium Strains QM B1551 and DSM319?†
Mark Eppinger,1‡ Boyke Bunk,2‡ Mitrick A. Johns,3Janaka N. Edirisinghe,3Kirthi K. Kutumbaka,3
Sara S. K. Koenig,1Heather Huot Creasy,1M. J. Rosovitz,4§ David R. Riley,1Sean Daugherty,1
Madeleine Martin,5Liam D. H. Elbourne,6Ian Paulsen,6Rebekka Biedendieck,5Christopher Braun,3
Scott Grayburn,3Sourabh Dhingra,3Vitaliy Lukyanchuk,3Barbara Ball,3Riaz Ul-Qamar,5
Ju ¨rgen Seibel,7Erhard Bremer,8Dieter Jahn,5Jacques Ravel,1* and Patricia S. Vary3*
Institute for Genome Sciences and Department of Microbiology and Immunology, University of Maryland, School of Medicine,
Baltimore, Maryland 212011; German Collection for Microorganisms and Cell Cultures, Braunschweig 38124, Germany2;
Northern Illinois University, Department of Biological Sciences, DeKalb, Illinois 601153; J. Craig Venter Institute,
Rockville, Maryland 208504; Technische Universita ¨t Braunschweig, Institute of Microbiology, Braunschweig 38106,
Germany5; Macquarie University, Department of Chemistry and Biomolecular Sciences, Sydney 2109,
Australia6; Julius-Maximilians-Universita ¨t Wu ¨rzburg, Institute of Organic Chemistry,
Wu ¨rzburg 97074, Germany7; and Philipps-Universita ¨t Marburg, Laboratory for
Molecular Microbiology, Marburg 35043, Germany8
Received 2 April 2011/Accepted 10 June 2011
Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of
particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megate-
rium is a commercially available, nonpathogenic host for the biotechnological production of several substances,
including vitamin B12, penicillin acylase, and amylases. Here, we report the analysis of the first complete
genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551,
which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while
QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced
in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the
chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences
in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B12through an
oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic path-
ways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible
for the large cell size of members of this species, as well as genes for gas vesicles, a second ?-galactosidase gene,
and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus
gene pool showed that only an asymmetric region around the origin of replication was syntenic across the
genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to
their sporulating lifestyle.
Bacillus megaterium was first described by Anton De Bary
more than 1 century ago in 1884 (14). Named for its large size,
a “megat(h)erium” (Greek for big animal) of 1.5 by 4 ?m, this
microorganism is the largest of all bacilli. Long before Bacillus
subtilis was introduced as a Gram-positive model organism, B.
megaterium was used for studies on biochemistry as well as
bacteriophages (13). The French microbiologist Maurice Lem-
oigne in 1925 discovered the polyester polyhydroxybutyrate in
B. megaterium as an important energy storage molecule in
bacteria (32), and Andre Lwoff discovered UV induction of
bacteriophage in a lysogenic B. megaterium strain (35). Due to
its large cell size, B. megaterium is well-suited for research on
cell morphology, such as cell wall and cytoplasmic membrane
biosynthesis, sporulation, spore structure and cellular organi-
zation, DNA partitioning, and protein localization (10, 61). In
the 1960s, B. megaterium was used to study sporulation, since it
sporulates and germinates efficiently (18). Because of its bio-
technological use in the production of several substances, the
nonpathogenic B. megaterium is of general interest to industry
(7). In contrast to Gram-negative organisms like Escherichia
coli, B. megaterium does not produce endotoxins associated
with the outer membrane, which, combined with its growth on
a variety of carbon sources and simple media, has made it a
workhorse in food and pharmaceutical production processes
for decades (i.e., ?- and ?-amylases used for starch modifica-
tion in the baking industry and penicillin acylases essential for
the synthesis of novel ?-lactam antibiotics, among others ).
Further, it is one of the most efficient producers of vitamin B12
* Corresponding author. Mailing address for Jacques Ravel: Insti-
tute for Genomic Sciences and Department of Microbiology and Im-
munology, University of Maryland, School of Medicine, Baltimore,
MD 21201. Phone: (410) 706-5674. Fax: (410) 706-1482. E-mail: jravel
@som.umaryland.edu. Mailing address for Patricia S. Vary: Northern
Illinois University, Department of Biological Sciences, DeKalb, IL
60115. Phone: (815) 787-2502. Fax: (815) 753-0461. E-mail: pvary@niu
† Supplemental material for this article may be found at http://jb
‡ Mark Eppinger and Boyke Brunk made equal contributions to this
§ Present address: National Biodefense Analysis and Countermea-
sures Center, Frederick, MD 21702.
?Published ahead of print on 24 June 2011.
FIG. 1. Circular representation of the B. megaterium chromosome. Circles (numbered 1 to 13, from outer to inner circle): circles 1 and 2,
predicted open reading frames encoded on the QM B1551 plus (circle 1, blue) and minus (circle 2, red) strands; circle 3, predominant genes in
conserved Bacillus region, showing tRNAs (brown), rRNA clusters (black), and sporulation and germination (dark green); circle 4, GC skew; circle
5, chromosomal regions of interest, including gas vesicle operons I and II (gvp; magenta), vitamin B12operons I and II (B12; brown), flagellar
operons (fla; aqua), RecA genes (rec; black), arsenate resistance operons (ars; green), ?-galactosidase genes (lac; orange), ethanolamine
utiliazation (eut; brown), histidine biosynthesis (his; gray), and competence operons CEFG (com; purple); circles 6 and 7, comparative analysis of
the QM B1551 chromosome identities (circle 6) and proteome (circle 7) to DSM319; circles 8 and 9, nonrandom distribution of genomic islands
and islets in QM B1551 (circle 8) and DSM319 (circle 9); circles 10 and 11, chromosomal genes with QM B1551 plasmid-borne orthologs in QM
B1551 (circle 10) and DSM319 (circle 11); circle 12, chi-square values to show GC deviations; circle 13, region of conserved synteny (red)
neighboring the ori identified by the comparative study of genome architectures in related Bacilli (see Fig. S4 in the supplemental material), with
the remainder of the genome colored green.
4200 EPPINGER ET AL.J. BACTERIOL.
(63, 64). B. megaterium has been extensively studied genetically
and is amenable to genetic manipulation (63, 66). Hundreds of
auxotrophs, division mutants, antibiotic-resistant, and UV-sen-
sitive mutants have been characterized and have been previ-
ously mapped in QM B1551 (17, 20, 31, 55, 57, 62, 65). Strain
QM B1551 is second only to B. subtilis in the number of
multiply marked, characterized strains that are available from
the Bacillus Genetic Stock Center (Vary collection, BGSC,
Ohio State University; http://www.bgsc.org/). Several biotech-
nological mutants have been constructed in strain DSM319
and are commercially available (38, 40, 72). Recent analysis
using phylogenetic analysis based on 16S rRNA genes led to
the division of the genus Bacillus into 4 different families and
37 genera (34). Even after the taxonomic reorganization, Ba-
cillus is a diverse genus with G?C content ranging from 34 to
35% (Bacillus cereus and related pathogens) to 44% (B. subti-
lis) and species that differ radically in lifestyles and metabolic
properties. Most of the sequenced Bacillus genomes are closely
related to B. cereus or B. subtilis. Recently, Porwal et al.
showed that B. megaterium (with a G?C content of 38 to 39%)
is only distantly related to the B. cereus and B. subtilis groups
and that it is more deeply rooted in the phylogenetic tree than
previously thought (44, 51). To gain insights into the genome
evolution and the metabolic versatility that facilitate biotech-
nological applications, we have sequenced the complete ge-
nomes of B. megaterium strains QM B1551 and DSM319. We
have used this information to examine the genetic diversity,
genome dynamics, and phylogenetic relationships within B.
megaterium and among members of the genus in great detail.
Our genomic analysis reveals new genetic traits not previously
seen in Bacillus and has resulted in a refined model for genome
evolution and adaptation of B. megaterium.
MATERIALS AND METHODS
Bacterial strains. Freeze-dried spores of B. megaterium QM B1551 prepared
in 1967 by James C. Vary were provided by Patricia S. Vary (Northern Illinois
University). These spores are closest in time to the original QM B1551 (Quarter
Master Bacterium 1551; ATCC 12872) isolation by Hillel Levinson at the Pio-
neering Research Division, Quartermaster and Engineering Center, Natick, MA.
B. megaterium DSM319 is a naturally plasmidless strain isolated by Stahl and
Esser (53) and obtained from DSMZ (http://www.dsmz.de/). The plasmidless
QM B1551 derivative, B. megaterium strain PV361 (56), was used for high-
throughput phenotype arrays. PV361 was grown on rich and minimal media and
showed no differences in sporulation compared to QM B1551. Germination of
PV361 was performed on rich medium because it lacks pBM700, which carries
key germination genes (55). Strain PV586, a Lac?derivative of PV361 (29), was
used as transformation recipient in the replicon analysis of pBM600.
Detailed materials and methods describing genome sequencing, assembly,
annotation, comparative genome analysis, pangenome computation, and the
different experimental assays can be found in the supplemental material.
Nucleotide sequence accession numbers. The B. megaterium complete genome
sequences (QMB1551 project ID 30165; DSM319 project ID 33377) have been
deposited in the NCBI GenBank under accession numbers CP001983 (chromo-
some, QM B1551), CP001984 to CP001990 (pBM100 to pBM700, respectively),
and CP001982 (chromosome, DSM319).
RESULTS AND DISCUSSION
Genome architecture. The chromosomes of B. megaterium
strains QM B1551 and DSM319 are circular molecules of
5,097,129 bp and 5,097,447 bp, respectively, with an average
G?C content of 38.2%. A well-defined G?C bias was ob-
served, with the right-hand replichore significantly enriched in
G?C relative to the left-hand replichore (Fig. 1 and Table 1).
In addition, strain QM B1551 contains seven indigenous plas-
mids, pBM100 to pBM700, with sizes from 5.4 kb to over 164
kb, while strain DSM319 is a naturally plasmidless isolate (53)
(Fig. 2 and Table 2; see also Fig. S1 in the supplemental
material). The plasmids have significantly lower G?C contents
than the chromosomes (33.0 to 36.5 versus 38.2%). The chro-
mosome of strain QM B1551 contains 5,284 genes, and that of
strain DSM319 contains 5,272 genes. The two chromosomes
display a high level of genome conservation, having a nucleo-
tide sequence identity of more than 95% over 83.3% of the
length of the two chromosomes. The genomes are mostly co-
linear (Fig. 3), with only a single rearrangement larger than
three genes: a 17-gene block in QM B1551 (Fig. 3, red arrows)
is inverted and displaced by 1.7 Mbp relative to DSM319
(genes BMQ_1765 to BMQ_1781 are homologous with genes
BMD_3632 to BMD_3616). The rearranged region does not
appear to be a single functional unit: genes are on both strands
and are involved in several different biological processes. Most
of the genetic differences between the two genomes are due to
insertions or deletions (indels) of single genes or small groups
of genes at scattered and independent genomic locations
throughout both chromosomes. We cataloged a total of 300
and 254 isolate-specific genes that are organized in 96 and 106
TABLE 1. Genomic properties of B. megaterium compared to related species
No. of protein-
No. of tRNAs
No. of rRNA
B. megateriumQM B1551 5.1 38.25,130, plus 499 on
120, plus 19 on
11, plus 1 on
VOL. 193, 2011GENOME SEQUENCES OF B. MEGATERIUM QM B1551 AND DSM3194201
independent clusters for strains QM B1551 and DSM319, re-
spectively (see Table S1 in the supplemental material). The
distribution of these indels is not random (Fig. 1, circles 8 and
9): we found that the gene insertions in both chromosomes are
rare in a 2-Mb region around the origin of replication (ori)
(Fig. 1 and Fig. 3; see also Table S1). Compared to the function
of genes common to both strains, strain-specific genes are
increased in functions affecting interactions with the environ-
ment: cell envelope, transport, signal transduction, and gene
regulation (Fig. 4; see also Fig. 9, below). In contrast, relatively
fewer strain-specific genes are associated with basic cellular
processes, such as amino acid, nucleotide, or cofactor biosyn-
thesis, central intermediary metabolism, fatty acid metabolism,
protein synthesis or degradation, or transcription. The isolate-
specific genes are also more frequently annotated as conserved
hypothetical genes or genes coding for enzymes of unknown
Plasmid analyses. Plasmids make up 11% of the QM B1551
genome (24). This strain harbors seven indigenous plasmids
with plasmid copy numbers ranging between 1 and 18 copies
(Table 2). Since many other strains of B. megaterium carry
multiple plasmids (63), this is a critical part of the genome
analysis for this species. The three largest plasmids are shown
in Fig. 2. Plasmids pBM100 and pBM200 of QM B1551 were
previously sequenced and deposited in GenBank. Plasmids
pBM300 and pBM400 have been analyzed (30, 52, 63) and
have been resequenced to higher coverage and reannotated for
this study (see Fig. S1 in the supplemental material). The
plasmids carry a variety of genes, including genes for sporula-
tion, germination, regulation, transport, and lantibiotic synthe-
sis, as well as erythromycin and rifampin resistance. There are
also genes for fatty acid metabolism, cell wall hydrolysis, sigma
factors, and cell division, as well as integrons, insertion se-
quence (IS) elements, and transposons. Because B. megaterium
is frequently found with Pseudomonas species in contaminated
environments, it has long been suspected of having the capa-
bility of metabolizing unusual substrates of possible bioreme-
dial use (30, 52, 61). To that extent, B. megaterium plasmids
carry genes for heavy metal resistance, including Cu and Cd
export, and genes such as styrene monooxygenase are present.
Several metabolic genes on the larger plasmids are organized
in what appear to be functional operon structures and may
enable this strain to survive in unusual habitats (Fig. 2). The
possible substrates for associated transporters cannot be de-
duced from genomic annotation, but the identification of these
regions provides the basis for further experimental character-
Of special note is that all seven indigenous plasmids ap-
pear to be unique to B. megaterium compared to a broad
panel of diverse Bacillus spp. plasmids. Besides similarities
in insertion sequence genes among the plasmids, we found
three genes similar to the pXO1 virulence plasmid of B.
anthracis, including a reverse transcriptase (52) (see Fig. S1
in the supplemental material). This finding may indicate
either a common phylogenetic origin or genetic exchange
among these plasmid-borne genes. Plasmid pBM500 (Fig. 2)
is intriguing because of the presence of three possible
sigma factors (BMQ_pBM50015, BMQ_pBM50023, and
BMQ_pBM50090), a collagen-like gene (BMQ_pBM50081),
and an erm(B) resistance gene (BMQ_pBM50045). In addi-
tion, a cytochrome P450 gene (BMQ_pBM50008) is plasmid-
borne. The P450 enzymes of B. megaterium have long been
used as a model system (15, 20, 21). There is a gene cluster that
appears to be responsible for the biosynthesis of a fatty acid
compound (BMQ_pBM50048 to BMQ_pBM50053). Plasmid
pBM600 seems to be a depository for mobile genetic elements,
FIG. 2. Circular representation of the B. megaterium plasmids pBM500, pBM600, and pBM700. Circles 1 and 2, predicted open reading frames
encoded on the QM B1551 plus (circle 1, blue) and minus (circle 2, red) strands with the replication gene dnaA (green); circle 3 (circle 4 in pBM700
representation), GC skew; circle 4 (circle 5 in pBM700 representation), plasmid genes with chromosomal orthologs (green) in strains QM B1551
and DSM319. The two innermost circles are the chi square deviations from the average trinucleotide distribution and the results of a comparative
analysis of each plasmid with the other seven indigenous plasmids of QM B1551, showing nucleotide identities (Fig. 1). (A) Regions and genes
of interest in pBM700. Circle 3, tRNA-Cys and mobile genetic elements (black); circle 6, regulatory functions, including transcription regulators
(red), two-component sensor histidine kinase systems (TCS; brown), adaptive response (light blue), and sporulation (orange); circle 7, metabolic
features, including carbohydrate metabolism (brown) and two peptidases (gold); circle 8, several enzymes of unknown specificity (blue) and for
antibiotic resistance (yellow). (B) Regions and genes of interest in pBM600. The origin amplicon is shown in black, with the identified replication
gene in green. Circle 5, mobile genetic elements (red). The IS3 transposase is adjacent to an aggrecan core (proteoglycan) protein that is further
distinguished by a deviating GC content, which suggests horizontal acquisition; circle 6, carbohydrate metabolism (brown) and sporulation genes
(orange). (C) Regions and genes of interest in pBM500. Circle 5, regulatory functions, including transcription regulators (red) and three sigma
factors (light blue); circle 6, metabolism features, including carbohydrate metabolism (brown) and fatty acid biosynthesis (magenta); circle 7,
sporulation (orange) and erm(B) macrolide resistance (yellow); circle 8, regions of deviating GC content, membrane protein (blue), collagen-like
protein (gold), and cytochrome P450 (green).
TABLE 2. Plasmid content of B. megaterium strain QM B1551
Plasmid Size (bp)Read countRead coverageCopy no. CDS rRNA tRNA % GCReplicon
Theta (this study)
VOL. 193, 2011GENOME SEQUENCES OF B. MEGATERIUM QM B1551 AND DSM3194203
since it carries four transposases, three integrase genes, and a
group II intron. It also has eight glycosyltransferases and other
glucose carrier proteins within a 25-kb region (kb 13.9 to 39.6).
Plasmid pBM700 encodes several transferases and transport-
ers, transcriptional regulators, and bacteriocin and antibiotic
synthesis operons. In addition, two two-component histidine
kinase regulatory systems are present (BMQ_pBM70140/141
and BMQ_pBM70155/156), strengthening the role of this plas-
mid in metabolic activities and strain-specific niche adapta-
tions. Previous studies on the pBM700 germination (ger) oper-
ons have helped identify specific amino acid residues that
define receptor specificity to germinant compounds in Bacillus
B. megaterium QM B1551
B. megaterium DSM319
B. megaterium DSM319
B. megaterium QMB1551
less similarity to more similarity
FIG. 3. Genome synteny of B. megaterium. The genomes of strains QM B1551 and DSM319 are mostly colinear, but the distribution of
isolate-specific genes is not random: gene insertions are rare in a 2-Mbp region around the origin of replication (ori). Each protein of the reference
genome plotted on the x axis was used as the query sequence with BLASTP against the y axis subject genome. The N-terminal coordinates of the
best hit proteins were plotted. The colors represent the level of similarity of the match, expressed by the BLAST score ratio (48).
FIG. 4. Functional classification of the isolate-specific gene inventory of B. megaterium strains QM B1551 and DSM319 compared to the
DSM319 chromosome. Unique genes in strains QM B1551 (A) and DSM319 (B) are indicated. (C) Functional classes of total chromosomal genes
4204 EPPINGER ET AL. J. BACTERIOL.
(11, 12). Strains of QM B1551 cured of pBM700 can no longer
germinate on single germinant compounds (55), and the com-
plete germination operon, with gerUA, gerUC, and gerUB
and including the downstream monocistronic gene gerVB
(BMQ_pBM70070 to BMQ_pBM70073), has been identified
Plasmid replicon analyses. With the exception of pBM600
(Fig. 2), all replicons have been previously identified and func-
tionally analyzed (30, 52, 64). A region containing a putative
replicon gene, BMQ_pBM60001, was identified and cloned in
pJM103, a plasmid that cannot replicate in B. megaterium. The
clone was successfully maintained in B. megaterium PV586,
indicating that it is the pBM600 replicon. The presence of a
characteristic plasmid replication motif, including six copies of
a 21-bp iteron upstream from BMQ_PBM60001, indicates it is
most likely a theta replicon. However, the identified replicon
shows no homology with the other four theta plasmid replicons
in QM B1551. We note that orthologs of QM B1551 plasmid
genes are found not only on the genome of QM B1551 but also
on DSM319 (Fig. 1; see also Table S2 in the supplemental
material). This observation indicates that strain DSM319 is
potentially capable of acquisition and maintenance of plas-
Evidence for the exchange of genes between plasmids and
chromosomes. We have observed extensive gene exchange be-
tween QM B1551 plasmids and the main chromosomes of both
strains (Fig. 1, circles 10 and 11). Of the QM B1551 499
protein-coding plasmid genes, 104 have homologs (BSR, ?0.4)
on either the QM B1551 or DSM319 chromosome (see Table
S2). Some genes share more than 90% sequence identity over
the full peptide length with their chromosomal homologs. Plas-
mid genes with homologs on the chromosomes are found on all
plasmids except pBM100 (5.4 kb). The majority of genes are
encoded on pBM400, pBM500, and pBM700, while only a few
genes are found on pBM200, pBM300, and pBM600 (see Ta-
ble S2). These genes can be classified into three groups. There
are 19 genes present on QM B1551 plasmids that have ho-
mologs on the QM B1551 chromosome but not on the
DSM319 chromosome. Conversely, 32 plasmid genes have ho-
mologs on the DSM319 chromosome but not the QM B1551
chromosome. The remaining 53 genes have homologs on both
the QM B1551 and DSM319 chromosomes (see Table S2).
These findings demonstrate that some of the plasmid-borne
capabilities of QM B1551 are also found in the plasmidless
DSM319 strain. The fact that the number of genes shared
between the plasmids and QM B1551 chromosome is smaller
than the number shared with the DSM319 chromosome sug-
gests that the plasmidless state of DSM319 might be the result
of a recent event. The homologous genes are found scattered
in different locations, both on the plasmids and on the
DSM319 and QM B1551 chromosomes. Several of the genes
are found in clusters of 2 to 11 genes and appear to have been
cotransferred, as they have the same relative positions and
orientations on both the plasmid and chromosomes with con-
served intergenic nucleotide homology. This set of genes en-
codes a variety of physiological functions, such as metabolism,
transport, regulation, sporulation, and germination, and in-
clude 45 conserved hypothetical CDS with no assigned func-
Spore coat biosynthesis. We detected major differences in
the genes involved in spore coat structure and its biosynthesis
between B. megaterium and B. subtilis strain 168 (see Table S3
in the supplemental material). More than half of the B. subtilis
genes involved in spore coat formation are missing and could
not be identified in either B. megaterium strain, which suggests
that B. megaterium possesses a different spore coat structure
than B. subtilis (23).
Natural competence genes of B. megaterium. Transformation
competence has been well-studied in the Gram-positive model
organism B. subtilis 168 (16). To date, no conditions have been
found that induce natural competence in B. megaterium. B.
megaterium genomes contain 33 competence orthologs to
genes known to mediate competence in B. subtilis (see Table
S4 in the supplemental material) (28) and that may allow B.
megaterium to incorporate foreign genetic material. The ge-
nome data revealed that B. megaterium lacks the comQXPA
gene cluster and two other competence genes, comFB and
comS, that are present in B. subtilis. Having detected almost all
known genes for competence in other bacilli (see Table S4), we
speculate that competence would be easy to engineer in the
well-studied B. megaterium strains. Furthermore, it may occur
naturally in some strains of the species.
Flagellar genes of B. megaterium. B. megaterium QM B1551
is motile but requires an oxygen-permeable coverslip for sus-
tained motility (P. S. Vary, unpublished data). The flagellar
genes of both QM B1551 and DSM319 form two clusters that
are almost identical to those found in B. subtilis (Fig. 1 and
5D). Cluster one corresponds to the fla/che 26-kb operon in B.
subtilis (42). In this cluster, only CheC, a signal-terminating
phosphatase important in chemotaxis protein methylation, is
missing from the operon and not found elsewhere in B. mega-
terium. The second and larger cluster is more complex (Fig.
5D). The fliT, fliS, and fliD genes match those of B. subtilis, but
flaG is not found in B. megaterium. Moreover, the B. subtilis
gene hag, which codes for flagellin, is not part of the cluster but
is found almost 2 Mb away (BMQ_1093), not near any other
flagellar genes except an adjacent short flagellin domain gene
(BMQ_1092), which is presumably a gene fragment that may
have been created during the transposition events that moved
the hag gene to its new location. In its place in the cluster are
six genes that apparently mediate unrelated metabolic func-
tions (BMQ_5103 to BMQ_5108). These genes code for a
GABA permease and associated symporter, a betaine alde-
hyde dehydrogenase, an alcohol dehydrogenase, and a GbsR
transcriptional regulator, as well as a hypothetical protein. The
syntenic genes consist of flagellar genes (fliWLKS), two com-
petence-associated genes (comFA and comFC), and the degS-
degU genes, which code for a two-component system that reg-
ulates the transition from exponential to stationary growth. We
note here that DegU has also been shown to regulate motility
via ?Din B. subtilis (36).
Metabolic and physiologic capabilities. The genomic se-
quences were used to investigate the underlying molecular
nature of some of the unique physiological and metabolic fea-
tures of B. megaterium. The transporter inventory of both
B. megaterium strains and potential substrates (http://www
.membranetransport.org/index.html) has been analyzed using
B. megaterium carries genes for the glyoxylate pathway. In
VOL. 193, 2011GENOME SEQUENCES OF B. MEGATERIUM QM B1551 AND DSM3194205
keeping with its known phenotype, B. megaterium uses the
Embden-Meyerhof pathway for glycolysis, followed by a clas-
sical Krebs cycle. In contrast to other bacilli, B. megaterium
contains an intact glyoxylate pathway (see Fig. S2A in the
supplemental material). From this starting point, the B. mega-
terium genome contains genes for the synthesis of all 20 pro-
teinogenic amino acids, all nucleotides and cofactors used by
other enzymes, and all necessary fatty acids. Genes for the
transporters and special enzymes needed to utilize a wide va-
riety of carbon sources have been found. Aerobic respiration
involves three membrane-bound dehydrogenases to transfer
electrons from NADH, glycerol-3-phosphate, and succinate to
menaquinone (see Fig. S2B). Menaquinone is oxidized by two
menaquinone oxidases of the cytochrome bd type, by a cyto-
Vitamin B12 biosynthesis I
Vitamin B12 biosynthesis II
Gas vesicle operon I
Gas vesicle operon II
Flagellar biosynthesis cluster
Vitamin B12 dependent Ethanolamine locus
FIG. 5. Genome features of B. megaterium QM B1551. (A) Gas vesicle-forming proteins. The B. megaterium QM B1551 chromosome features
two unique gas vesicle protein-forming loci (gvp) not found in any of the related species or genera. DSM319 has only operon II. The scale (in bp)
indicates the genomic location of the gas vesicle loci, which are composed of 8 and 14 genes (gvpBRFGLSKJ and gvpAPQBRNFGLSKJTU). A
possible araC regulator is also present. The loci are syntenic and share 8 homologous genes, with the notable exception of gvpN. The absence of
colocalizing mobile genetic loci and average GC content suggest a duplication event in strain QM B1551 rather than acquisition via horizontal gene
transfer. Homologous genes have been given identical colors and shaded in gray. (B) Vitamin B12biosynthesis. Both B. megaterium strains encode
three phylogenetically unrelated loci that are associated with vitamin B12biosynthesis; the two multigene loci are shown here. The scale (in bp)
indicates the genomic location of these loci in strain QM B1551. Regulatory genes are colored magenta. (C) Vitamin B12-dependent loci. Several
enzymes with dependencies on vitamin B12have been identified in the B. megaterium genome, such as ethanolamine ammonia-lyase (EutBC) and
methylmalonyl-CoA mutase (MutAB). The genes eutCBA are also found at Mb 2.52 to 2.53 in both strains. (D) Flagellar biosynthesis. The two
flagellar biosynthesis clusters, fla/che and fla/com/deg in QM B1551, are shown, with the upstream regulatory genes colored magenta. The
fla/com/deg cluster contains flagellar, competence, and the extracellular enzyme regulatory genes degU and degS.
4206 EPPINGER ET AL.J. BACTERIOL.
chrome aa3-type oxidase, which is not found in other bacilli, or
by the cytochrome bc1complex, followed by cytochrome c and
cytochrome c oxidase. B. megaterium also contains a fourth
oxidase with high sequence similarity to E. coli cytochrome o
ubiquinol oxidase, which is not found in other bacilli. Under
anaerobic conditions, B. megaterium has all the genes required
to perform a mixed acid fermentation that produces lactate,
2,3-butanediol, and acetate, as previously described for B. sub-
tilis (see Fig. S2C) (41, 47). Unlike most other bacilli, B. mega-
terium is not capable of using nitrate as an electron acceptor,
since it lacks a membrane-bound nitrate reductase of the Nar
type. Further details about B. megaterium energy metabolism
are found in Fig. S2 of the supplemental material.
Strain-specific carbon source utilization. Predictions for the
metabolic capabilities of each strain were deduced from in
silico comparative genome analysis and validated by growth
experiments in vivo (Fig. 6). For strain QM B1551, several
unique glucarate-degrading enzymes were found in an operon-
like structure, including glucarate permease (BMQ_1892), glu-
carate dehydratase (BMQ_1893), and the degradative enzymes
galactarate dehydratase (BMQ_1888) and 5-dehydro-4-deoxy-
glucarate dehydratase (BMQ_1890). The final product of this
glucarate degradation process is 2,5-dioxopentanoate, which is
discharged into the citrate cycle via 2-oxoglutarate. Further-
more, a complete galactitol-specific phosphotransferase system
(PTS) is found to be unique in B. megaterium QM B1551
(BMQ_3201 to BMQ_3204). The genes for the corresponding en-
zymes galactitol-1-phosphate 5-dehydrogenase (BMQ_3200) and an
alcohol dehydrogenase (BMQ_3199) are also part of this genomic
island. We demonstrated experimentally that B. megaterium QM
B1551 (in contrast to strain DSM319) can utilize both glucarate and
galactitol as carbon sources (Fig. 6A and B). Additionally, growth
experiments on pullulan showed that B. megaterium QM B1551
grows more effectively on pullulan than does DSM319 (Fig. 6C),
although both strains are equipped with the same chromosomal
pullulanases, AmyX (BMQ_4837) and PulA (BMQ_2037). Strain
DSM319 encodes a cellulase-like glycosyl hydrolase (BMD_1113),
of a sucrose-specific PTS (BMD_3721), sorbitol dehydrogenase
GutB (BMD_3588) with its transcriptional activator GutR
(BMD_3589), and a probable sorbitol transporter (BMD_3587).
The sorbitol uptake system proved to be very effective in DSM319
(Fig. 6D), with optical growth densities of 6, while that for QM
B1551 was only 1.2.
Osmotic adjustment and compatible solute accumulation.
In its varied habitats, B. megaterium is exposed to increased
FIG. 6. Growth of B. megaterium strains DSM319 (blue) and QM B1551 (red) on different nutrients. Strain-specific gene differences deduced
from the analysis of the isolate-specific genes were validated by growth on different nutrients: (A) glucarate, (B) galactitol, (C) pullulan,
(D) sorbitol. The optical density was recorded over a time course of up to 13 h. The experimental data were fitted to logistic or exponential growth
VOL. 193, 2011GENOME SEQUENCES OF B. MEGATERIUM QM B1551 AND DSM3194207
osmolarity that triggers water efflux from the cell. It therefore
has to adjust the osmotic potential of its cytoplasm to prevent
dehydration. The physiology and genetics of osmotic adjust-
ment to high-osmolarity surroundings has been studied inten-
sively in B. subtilis (6). In the initial phase of adjustment to high
osmolarity, B. subtilis accumulates large amounts of potassium
via the KtrAB and KtrCD systems to curb the efflux of water
(22). Both Ktr potassium uptake systems are also present in B.
megaterium (BMQ_3903/3904 and BMQ_1339/1233). In the
second phase of osmotic adjustment, B. subtilis synthesizes
large quantities of the compatible solute proline and acquires
various types of organic osmoprotectants (e.g., glycine betaine)
from environmental sources via high-affinity transport systems.
Recent studies have shown that B. megaterium DSM32 synthe-
sizes proline when it is challenged by high salinity (9). The in
silico analysis of the genome sequences of QM B1551 and
DSM319 revealed a complete proline biosynthetic gene cluster
(proHJA; BMQ_2287 to BMQ_2289) whose counterparts are
osmotically induced in B. subtilis, B. licheniformis, and Halo-
bacillus halophilis (26, 71). B. megaterium may also use an
exogenous supply of proline for osmoprotective purposes,
since it possesses a homolog (BMQ_1420) of the osmotically
induced proline uptake system OpuE from B. subtilis (6). Gly-
cine betaine is a very effective osmoprotectant, and B. subtilis
can acquire it either from environmental sources or synthesize
it from an exogenous supply of the precursor choline via a
two-step oxidation process. Based on homology to B. subtilis,
several glycine betaine import systems were identified in B.
megaterium. Two ABC transporters of the OpuA type,
opuACAB (BMQ_0858 to BMQ_0860) and opuAABC
(BMQ_1542 to BMQ_1544), an OpuC-like glycine betaine/
choline importer (BMQ_3925/3926), and an OpuD-like
BCCT-type transport system (BMQ_1351) are present. In ad-
dition, a gene cluster encoding the glycine betaine biosynthetic
enzymes GbsA and GbsB (BMQ_5106/5107) are present.
Adaptations to an aquatic lifestyle. Besides its most com-
mon soil habitat, B. megaterium is also found in diverse envi-
ronments, including rice paddies, dried food, seawater, sedi-
ments, fish, and even in bee honey (63). Analysis of B.
megaterium genome sequences strongly indicates that it is well-
adapted to an aquatic lifestyle, since it possesses genes for the
formation of gas vesicles. These intracellular structures are
often found in marine microorganisms and function as flota-
tion devices, allowing the bacterium to float up and down by
adjusting the gas pressure inside the gas vesicle (60). The
genome of B. megaterium QM B1551 has two gas vesicle-
forming operons (Fig. 5A). The two gvp loci share eight ho-
mologous genes (gvpBRFGLSKJ) and are syntenic except for
gvpN, which is found between gvpR and gvpF in the larger
operon. The smaller locus (BMQ_3224 to BMQ_3231) is not
present in DSM319. The larger operon encompasses 14 genes,
gvpAPQBRNFGLSKJTU (BMQ_3290 to BMQ_3303), and an
araC-like regulator. This gene cluster was previously discov-
ered in B. megaterium strain VT1660, and its functional expres-
sion in E. coli renders this bacterium buoyant (33). It should be
noted that genes for gas vesicles have been found in genomes
of many microorganisms that do not typically live in aquatic
habitats, fostering discussions on the physiological function of
these floating devices (60). The proteins involved in gas vesicle
formation in B. megaterium show a phylogenetic relationship
far outside the Bacillus group and are related to proteins pres-
ent in aquatic Cyanobacteria but also in members of Archaea.
One explanation for the gas vesicles could be related to the
FIG. 7. Oxygen-independent biosynthesis of adenosylcobalamin. B. megaterium is able to synthesize vitamin B12in the absence of oxygen due
to the presence of the signature gene cbiX, which results in insertion of cobalt at an early stage, in contrast to the oxygen-dependent pathway.
4208EPPINGER ET AL.J. BACTERIOL.
ability of the bacterium to leave aquatic regions of low oxygen
Biotechnological use of B. megaterium for protein produc-
tion. One major advantage of B. megaterium for industrial
protein production is its ability to secrete proteins directly into
the growth medium (2, 4, 37, 45). B. megaterium employs Sec
and Tat systems for this purpose; the relevant genes were all
detected in the genomes. The Sec-dependent secretion system
is especially widely used and has been investigated for indus-
trial recombinant protein production. A look at both genome
sequences of B. megaterium revealed potential genes for ap-
proximately 30 secreted proteases (see Table S5 in the supple-
mental material). Only a few proteases have experimentally
been found to be secreted, including metalloprotease InhA,
extracellular protease Vpr, aminopeptidase YwaD, and neutral
protease NprM (67). The gene for the major extracellular
protease NprM was deleted from production strain MS941
(69), which allowed up to 1.2 g/liter functional proteins to be
recovered from the medium (4, 20, 25, 54, 66). These data
clearly demonstrate the advantage of B. megaterium strains for
recombinant protein production.
Vitamin B12production. B. megaterium was one of the first
biotechnological vitamin B12producers described for bacteria
(61, 64, 70). In agreement with the well-studied biosynthetic
pathway in Salmonella enterica, which is known for its ability to
synthesize vitamin B12in the presence and absence of oxygen
(5, 8, 49), the genes for oxygen-independent vitamin B12bio-
synthesis genes were found in the genomes of B. megaterium.
They are organized in two distinct, independent operons (Fig.
5B). The larger operon, cbiWHXJCDETLFGA-cysG-cbiY-
btuR, has been thoroughly described (49); the smaller operon,
cbiB-cobDUSC, is associated with an uncharacterized ATP:
cob(I)alamin adenosyltransferase and codes for the enzymes
involved in the final steps of the biosynthetic pathway (Fig. 7).
The gene cbiP coding for adenosylcobyrinic acid synthase (EC
22.214.171.124) is isolated in the genome at Mb 2.9 as a single gene
with no further B12biosynthesis context. The genome of both
B. megaterium strains now allows the full reconstruction of the
vitamin B12biosynthetic pathway (Fig. 7).
Vitamin B12-dependent enzymes. Several enzymes with de-
pendencies on vitamin B12have been identified in silico in the
B. megaterium genome. Ethanolamine ammonia-lyase EutBC
(EC 126.96.36.199) and methylmalonyl coenzyme A (CoA) mutase
MutAB (EC 188.8.131.52) are two that have been shown to con-
tribute to the ability of B. megaterium to degrade lipids. Other
than B. megaterium, only Geobacillus kaustophilus and B. ha-
FIG. 8. Distinct genomic locations for different functional classes of genes in B. megaterium QM B1551. The number of genes classified by
function that were encoded within the conserved syntenic region flanking the ori (red) was compared to the number present in the remainder of
the chromosome (green), according to Fig. 1, and to the total chromosomal number (gray). Those functions concentrated in the conserved region
that were compared to the remainder of the functions are lettered in red.
VOL. 193, 2011 GENOME SEQUENCES OF B. MEGATERIUM QM B1551 AND DSM319 4209
4210EPPINGER ET AL.J. BACTERIOL.
lodurans have been found to carry the MutAB genes. EutBC
is part of a large B. megaterium species-specific ethanol-
amine utilization operon, eutHSPABCLEM (BMQ_3678 to
BMQ_3686) (Fig. 5C), together with an upstream ethanol-
amine two-component response regulator system (BMQ_3687/
3688) and further uncharacterized genes contributing to etha-
nolamine utilization (BMQ_3675 to -3677). A second EutBC
can also be found as part of a small eutABC-eat cluster
(BMQ_2531 to -2534), where Eat is an ethanolamine per-
mease. It is noteworthy that a phage integrase (BMQ_2528) is
found three genes downstream, which may indicate a lateral
acquisition of this functional unit. Furthermore, the vitamin
B12-dependent methionine synthase MetH (BMQ_1293) and a
vitamin B12-dependent ribonucleoside-diphosphate reductase
(BMQ_4885/4886) were identified in both strains.
Relationship of B. megaterium to other bacilli. (i) Phyloge-
netic position of B. megaterium. B. megaterium belongs to a
deeply rooted lineage within the genus Bacillus, based on its
phenotypic characteristics, genome size, intermediate G?C
content, and 16S rRNA phylogeny (Table 1) (1, 46). Whether
B. megaterium is more closely related to B. cereus and its
relatives or to B. subtilis and its relatives has been difficult to
resolve based on 16S rRNA gene phylogeny alone. Trees show-
ing all three possible relationships between these groups have
been published recently (27, 44, 68). To address this issue, we
performed a phylogenetic analysis on 385 orthologous genes
identified using BSR analysis (see Fig. S3 in the supplemental
material) (48). A neighbor-joining tree was generated, using
Listeria monocytogenes as the outgroup. In this tree B. mega-
terium, B. cereus, and B. subtilis each anchor clades that are
supported in all bootstrap samples. However, the order with
which these clades join varies between bootstrap samples, with
each of the three possible orders supported by 21 to 51% of the
bootstrap samples. Thus, the ambiguous relationship between
B. megaterium, B. cereus, and B. subtilis that is seen with 16S
rRNA phylogenies is shown to also exist when a large set of
orthologous genes is examined, possibly indicating a large
amount of lateral gene transfer between these species.
(ii) Genome dynamics in the Bacillus group of organisms.
When orthologous genes were compared between B. megate-
rium and other Bacillus species, a region of striking synteny was
observed neighboring the ori (Fig. 1), from about the 10
o’clock to the 2 o’clock position (Fig. 8; see also Fig. S4 in the
supplemental material). The Gram-positive spore former
Oceanobacillus iheyensis and Listeria monocytogenes, the caus-
ative agent of listeriosis, show similar regions of synteny. In-
creased phylogenetic distance generally correlated with an in-
creased number of insertions and inversions in the syntenic
regions. The region of synteny is dominated by genes involved
in sporulation, cell envelope biosynthesis, and the transcription
and translation machinery. This phenomenon might be ex-
plained by a need for these genes to be readily accessible
during germination and sporulation (73).
The core and pan-genome of the Bacillus group of organ-
isms. The availability of two complete genomes of B. megate-
rium (Fig. 1; see also Table S1 in the supplemental material)
provides the opportunity to investigate the genomic plasticity
and global gene reservoir of the Bacillus group of organisms,
the Bacillus pan-genome (39) (Fig. 9). The core genome, genes
expected to be present in all genomes, is estimated at 2,009
protein-coding genes (Fig. 9A). We estimated the Bacillus pan-
genome to contain 10,534 unique protein-coding genes (Fig.
9C). The pan-genome defines the total number of genes found
at least once among all Bacillus genomes. Our estimates of the
gene discovery rate indicate that Bacillus has an open pan-
genome: on average, 96 new genes would be expected from
each new Bacillus genome sequenced (Fig. 9B). These results
are comparable to data obtained for E. coli (59) or for Staph-
ylococcus agalacticae and S. pneumoniae (58). As evidenced
by the genomic analyses of B. megaterium QM B1551 and
DSM319, there is a high degree of genetic diversity even
among closely related strains. The gene discovery rate in Ba-
cillus was significantly increased by the fact that each strain had
about 300 genes not found in the other strain, with an addi-
tional 499 genes found on the QM B1551 plasmids. In a species
featuring an open pan-genome, such as E. coli, the predicted
pan-genome size is almost 75% larger than the average ge-
nome size of the species (59), a finding comparable to our
results within the Bacillus group of organisms.
Conclusions and future directions. The analysis of the ge-
nome sequences of the historically and biotechnologically im-
portant strains QM B1551 and DSM319 has advanced our
knowledge of the metabolic versatility and the evolution of the
Gram-positive aerobic spore-forming bacteria. We have iden-
tified numerous unique genetic traits not seen in any Bacillus
species previously studied, including the presence of gas vesicle
proteins, a glyoxylate pathway, and vitamin B12biosynthesis,
and also a lack of homology with the spore coats of B. subtilis.
Our data suggest there is considerable DNA exchange between
Bacillus and other Gram-positive bacteria (including patho-
gens), as well as on the intraspecies level between the seven
indigenous plasmids and chromosomes. The observed phe-
nomenon of large conserved syntenic regions neighboring the
chromosomal ori is key in understanding the lifestyle of B.
FIG. 9. Pan-genome analysis and genomic plasticity in the Bacillus group of organisms. (A) Core genes. For each reported number of genomes
(n), the circles represent the number of genes in common in different randomly chosen combinations of Bacillus species, with a sampling size of
1,000. Diamonds show the median values for each distribution. The curve represents the exponential regression of the least squares fit of the
function Fcore(n) ? ?cexp[?n/?c] ? tgc(?), based on the medians of the distribution. The extrapolated core genome size is shown as a horizontal
dashed red line. (B) Gene discovery. By using the same sampling method as for panel A, the number of new genes found was plotted for increasing
values of n. A power law regression for new genes discovered was fitted to the means of new gene counts (diamonds) for each value of n. The curve
is the least squares fit of the exponential decay equation Fnew(n) ? ?nexp[?n/?n] ? tgn(?), based on the means of the distribution. The value of
tgn(?) shown in this figure represents the number of new genes asymptotically predicted for further genome sequencing. (C) The Bacillus
pan-genome. The total numbers of genes found according to the pan-genome analyses are shown for increasing values of the number (n) of Bacillus
genomes sequenced, using medians and an exponential fit. Red diamonds indicate the means of the distributions. The dashed line represents the
asymptotic prediction of the total number of genes expected to be found in the Bacillus pan-genome.
VOL. 193, 2011GENOME SEQUENCES OF B. MEGATERIUM QM B1551 AND DSM3194211
megaterium and other related spore formers. The availability of
the genome sequences of these two B. megaterium strains, the
reconstruction of key metabolic and energetic pathways, and
the finding that most of the known Bacillus species competence
genes are present, together with well-established protocols for
genetic modifications, offer a promising future for the further
development of B. megaterium as a model organism for systems
biotechnology (3, 19, 64).
The sequencing of B. megaterium strains QM B1551 and DSM319
was supported with federal funds from the National Science Founda-
tion under NSF contract no. 0802327 and the German Research Foun-
dation under contract no. SFB578.
We thank Vanessa Hering for the growth experiments, Anni Moore
and Melvin Duvall for phylogeny discussions, and Stefan Mu ¨nnich and
students of the Bioinformatics classes at NIU for help with the anno-
We declare that we have no competing financial interests.
1. Ash, C., J. A. E. Farrow, S. Wallbanks, and M. D. Collins. 1991. Phylogenetic
heterogeneity of the genus Bacillus revealed by comparative analysis of small
subunit ribosomal RNA sequences. Lett. Appl. Microbiol. 13:202–206.
2. Biedendieck, R., et al. 2007. Export, purification, and activities of affinity
tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium.
Appl. Microbiol. Biotechnol. 74:1062–1073.
3. Biedendieck, R., et al. 2009. Systems biology of recombinant protein pro-
duction in Bacillus megaterium, p. 133–161. In T. Scheper (ed.), Biosystems
engineering, vol. 113, Springer, Berlin, Germany.
4. Biedendieck, R., et al. 2007. A sucrose-inducible promoter system for the
intra- and extracellular protein production in Bacillus megaterium. J. Bio-
5. Biedendieck, R., et al. 2010. Metabolic engineering of cobalamin (vitamin
B12) production in Bacillus megaterium. Microb. Biotechnol. 3:24–37.
6. Bremer, E. 2002. Adaptation to changing osmolarity, p. 385–391. In A. L.
Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest
relatives: from genes to cells. ASM Press, Washington, DC.
7. Bunk, B., R. Biedendieck, D. Jahn, and P. S. Vary. 2010. Bacillus megaterium
and other bacilli: industrial applications, p. 1–15. Encyclopedia of industrial
biotechnology: bioprocess, bioseparation, and cell technology. John Wiley &
Sons, Hoboken, NJ.
8. Bunk, B., et al. 2010. A short story about a big magic bug. Bioeng. Bugs
9. Bursy, J., A. J. Pierik, N. Pica, and E. Bremer. 2007. Osmotically induced
synthesis of the compatible solute hydroxyectoine is mediated by an evolu-
tionarily conserved ectoine hydroxylase. J. Biol. Chem. 282:31147–31155.
10. Christie, G., H. Gotzke, and C. R. Lowe. 2010. Identification of a receptor
subunit and putative ligand-binding residues involved in the Bacillus mega-
terium QM B1551 spore germination response to glucose. J. Bacteriol. 17:
11. Christie, G., M. Lazarevska, and C. R. Lowe. 2008. Functional consequences
of amino acid substitutions to GerVB, a component of the Bacillus megate-
rium spore germinant receptor. J. Bacteriol. 190:2014–2022.
12. Christie, G., and C. R. Lowe. 2007. Role of chromosomal and plasmid-borne
receptor homologues in the response of Bacillus megaterium QM B1551
spores to germinants. J. Bacteriol. 189:4375–4383.
13. Clarke, N. A., and P. B. Cowles. 1952. Studies on the host-virus relationship
in a lysogenic strain of Bacillus megaterium. II. The relationship between
growth and bacteriophage production in cultures of Bacillus megaterium. J.
14. De Bary, A. 1884. Vergleichende Morphologie und Biologie der Pilze, My-
cetozoen und Bacterien. Wilhelm Engelmann, Leipzig, Germany.
15. Dietrich, J. A., et al. 2009. A novel semi-biosynthetic route for artemisinin
production using engineered substrate-promiscuous P450(BM3). ACS Chem.
16. Dubnau, D. 1993. Genetic exchange and homologous recombination, p.
555–584. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus
subtilis and other gram-positive bacteria. American Society for Microbiology,
17. English, J. D., and P. S. Vary. 1986. Isolation of recombination-defective and
UV-sensitive mutants of Bacillus megaterium. J. Bacteriol. 165:155–160.
18. Foerster, H. F., and J. W. Foster. 1966. Response of Bacillus spores to
combinations of germinative compounds. J. Bacteriol. 91:1168–1177.
19. Fu ¨rch, T., et al. 2007. Effect of different carbon sources on central metabolic
fluxes and the recombinant production of a hydrolase from Thermobifida
fusca in Bacillus megaterium. J. Biotechnol. 132:385–394.
20. Gamer, M., D. Frode, R. Biedendieck, S. Stammen, and D. Jahn. 2009. A T7
RNA polymerase-dependent gene expression system for Bacillus megate-
rium. Appl. Microbiol. Biotechnol. 82:1195–1203.
21. He, J.-S., and A. J. Fulco. 1991. A barbiturate-regulated protein binding to
a common sequence in the cytochrome P450 genes of rodents and bacteria.
J. Biol. Chem. 266:7864–7869.
22. Holtmann, G., E. P. Bakker, N. Uozumi, and E. Bremer. 2003. KtrAB and
KtrCD: two K?uptake systems in Bacillus subtilis and their role in adapta-
tion to hypertonicity. J. Bacteriol. 185:1289–1298.
23. Imamura, D., R. Kuwana, H. Takamatsu, and K. Watabe. 2010. Localization
of proteins to different layers and regions of Bacillus subtilis spore coats. J.
24. Kieselburg, M. K., M. Weickert, and P. S. Vary. 1984. Analysis of resident
and transformant plasmids in Bacillus megaterium. Biotechnology 2:254–259.
25. Kim, J. Y. 2003. Overproduction and secretion of Bacillus circulans endo-
beta-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium. Bio-
technol. Lett. 25:1445–1449.
26. Kocher, S., B. Averhoff, and V. Muller. 1 March 2011. Development of a
genetic system for the moderately halophilic bacterium Halobacillus halo-
philus: generation and characterization of mutants defect in the production
of the compatible solute proline. Environ. Microbiol. doi:10.1111/j.1462-
27. Kolsto, A. B., N. J. Tourasse, and O. A. Okstad. 2009. What sets Bacillus
anthracis apart from other Bacillus species? Annu. Rev. Microbiol. 63:451–
28. Kramer, N., J. Hahn, and D. Dubnau. 2007. Multiple interactions among the
competence proteins of Bacillus subtilis. Mol. Microbiol. 65:454–464.
29. Kunnimalaiyaan, M., D. M. Stevenson, Y. Zhou, and P. S. Vary. 2001.
Analysis of the replicon region and identification of an rRNA operon on
pBM400 of Bacillus megaterium QM B1551. Mol. Microbiol. 39:1010–1021.
30. Kunnimalaiyaan, M., and P. S. Vary. 2005. Molecular characterization of
plasmid pBM300 from Bacillus megaterium QM B1551. Appl. Environ. Mi-
31. Lach, D. A., V. K. Sharma, and P. S. Vary. 1990. Isolation and character-
ization of a unique division mutant of Bacillus megaterium. J. Gen. Microbiol.
32. Lemoigne, M., C. P. Lenoel, and M. Croson. 1950. Assimilation of acetyla-
cetic acid and beta-hydroxybutyric acid by B. megatherium. Ann. Inst. Pasteur
(Paris) 78:705–710. (In French.)
33. Li, N., and M. C. Cannon. 1998. Gas vesicle genes identified in Bacillus
megaterium and functional expression in Escherichia coli. J. Bacteriol. 180:
34. Ludwig, W., K.-H. Schleifer, and W. B. Whitman. 2009. Revised road map to
the phylum Firmicutes, p. 1–14. In P. D. Vos et al. (ed.), Bergey’s manual of
systematic bacteriology, vol. 3, 2nd ed. Springer, New York, NY.
35. Lwoff, A., L. Siminovitch, and N. Kjeldgaard. 1950. Induction of the pro-
duction of bacteriophages in lysogenic bacteria. Ann. Inst. Pasteur (Paris)
79:815–859. (In French.)
36. Mader, U., et al. 2002. Bacillus subtilis functional genomics: genome-wide
analysis of the DegS-DegU regulon by transcriptomics and proteomics. Mol.
Genet. Genomics 268:455–467.
37. Malten, M., et al. 2006. A Bacillus megaterium plasmid system for the pro-
duction, export, and one-step purification of affinity-tagged heterologous
levansucrase from growth medium. Appl. Environ. Microbiol. 72:1677–1679.
38. Malten, M., R. Hollmann, W. D. Deckwer, and D. Jahn. 2005. Production
and secretion of recombinant Leuconostoc mesenteroides dextransucrase
DsrS in Bacillus megaterium. Biotechnol. Bioeng. 89:206–218.
39. Medini, D., C. Donati, H. Tettelin, V. Masignani, and R. Rappuoli. 2005. The
microbial pan-genome. Curr. Opin. Genet. Dev. 15:589–594.
40. Nahrstedt, H., and F. Meinhardt. 2004. Structural and functional character-
ization of the Bacillus megaterium uvrBA locus and generation of UV-sensi-
tive mutants. Appl. Microbiol. Biotechnol. 65:193–199.
41. Nicholson, W. L. 2008. The Bacillus subtilis ydjL (bdhA) gene encodes
acetoin reductase/2,3-butanediol dehydrogenase. Appl. Environ. Microbiol.
42. Ordal, G. W., L. Marquez-Magana, and M. J. Chamberlin. 1993. Motility
and chemotaxis, p. 765–784. In A. L. Sonenshein, J. A. Hoch, and R. Losick
(ed.), Bacillus subtilis and other gram-positive bacteria. American Society for
Microbiology, Washington, DC.
43. Panbangred, W., K. Weeradechapon, S. Udomvaraphant, K. Fujiyama, and
V. Meevootisom. 2000. High expression of the penicillin G acylase gene (pac)
from Bacillus megaterium UN1 in its own pac minus mutant. J. Appl. Micro-
44. Porwal, S., S. Lal, S. Cheema, and V. C. Kalia. 2009. Phylogeny in aid of the
present and novel microbial lineages: diversity in Bacillus. PLoS One
45. Priest, F. G. 1977. Extracellular enzyme synthesis in the genus Bacillus.
Bacteriol. Rev. 41:711–753.
46. Priest, F. G., M. Goodfellow, and C. Todd. 1988. A numerical classification
of the genus Bacillus. J. Gen. Microbiol. 134:1847–1882.
47. Ramos, H. C., et al. 2000. Fermentative metabolism of Bacillus subtilis:
physiology and regulation of gene expression. J. Bacteriol. 182:3072–3080.
4212EPPINGER ET AL.J. BACTERIOL.
48. Rasko, D. A., G. S. Myers, and J. Ravel. 2005. Visualization of comparative Download full-text
genomic analyses by BLAST score ratio. BMC Bioinformatics 6:2.
49. Raux, E., A. Lanois, A. Rambach, M. J. Warren, and C. Thermes. 1998.
Cobalamin (vitamin B12) biosynthesis: functional characterization of the
Bacillus megaterium cbi genes required to convert uroporphyrinogen III into
cobyrinic acid a,c-diamide. Biochem. J. 335:167–173.
50. Ren, Q., K. Chen, and I. T. Paulsen. 2007. TransportDB: a comprehensive
database resource for cytoplasmic membrane transport systems and outer
membrane channels. Nucleic Acids Res. 35:D274–D279.
51. Rossler, D., et al. 1991. Phylogenetic diversity in the genus Bacillus as seen
by 16S rRNA sequencing studies. Syst. Appl. Microbiol. 14:266–269.
52. Scholle, M. D., C. A. White, M. Kunnimalaiyaan, and P. S. Vary. 2003.
Sequencing and characterization of pBM400 from Bacillus megaterium QM
B1551. Appl. Environ. Microbiol. 69:6888–6898.
53. Stahl, U., and K. Esser. 1983. Plasmid heterogeneity in various strains of
Bacillus megaterium. Eur. J. Appl. Microbiol. Biotechnol. 17:248–251.
54. Stammen, S., et al. 2010. High-yield intra- and extracellular protein produc-
tion using Bacillus megaterium. Appl. Environ. Microbiol. 76:4037–4046.
55. Stevenson, D. M., D. Lach, and P. S. Vary. 1993. A gene required for
germination in Bacillus megaterium is plasmid-borne, p. 197–207. In E. Balla
and G. Berencsie (ed.), DNA transfer and gene expression in microorgan-
isms. Intercept, Budapest, Hungary.
56. Sussman, M. D., P. S. Vary, C. Hartman, and P. Setlow. 1988. Integration
and mapping of Bacillus megaterium genes which code for small, acid-soluble
spore proteins and their protease. J. Bacteriol. 170:4942–4945.
57. Tao, Y.-P., and P. S. Vary. 1991. Isolation and characterization of sporulation
lacZ fusion mutants of Bacillus megaterium. J. Gen. Microbiol. 137:797–806.
58. Tettelin, H., et al. 2005. Genome analysis of multiple pathogenic isolates of
Streptococcus agalactiae: implications for the microbial “pan-genome.” Proc.
Natl. Acad. Sci. U. S. A. 102:13950–13955.
59. Touchon, M., et al. 2009. Organised genome dynamics in the Escherichia coli
species results in highly diverse adaptive paths. PLoS Genet. 5:e1000344.
60. van Keulen, G., D. A. Hopwood, L. Dijkhuizen, and R. G. Sawers. 2005. Gas
vesicles in actinomycetes: old buoys in novel habitats? Trends Microbiol.
61. Vary, P. S. 1992. Development of genetic engineering in Bacillus megaterium,
p. 251–310. In R. Doi and M. McGloughlin (ed.), Biology of bacilli: appli-
cation to industry. Butterworths-Heinemann, Boston, MA.
62. Vary, P. S. 1993. The genetic map of Bacillus megaterium, p. 475–487. In
A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and
other gram-positive bacteria. American Society for Microbiology, Wash-
63. Vary, P. S. 1994. Prime time for Bacillus megaterium. Microbiology 140:
64. Vary, P. S., et al. 2007. Bacillus megaterium: from simple soil bacterium to
industrial protein production host. Appl. Microbiol. Biotechnol. 76:957–967.
65. Vary, P. S., J. C. Garbe, M. Franzen, and E. W. Frampton. 1982. Genetics of
leucine biosynthesis in Bacillus megaterium. J. Bacteriol. 149:1112–1119.
66. von Tersch, M. A., and H. L. Robbins. 1990. Efficient cloning in Bacillus
megaterium: comparison to Bacillus subtilis and Escherichia coli cloning hosts.
FEMS Microbiol. Lett. 70:305–310.
67. Wang, W., J. Sun, R. Hollmann, A.-P. Zeng, and W.-D. Deckwer. 2006.
Proteomic characterization of transient expression and secretion of a stress-
related metalloprotease in high cell density culture of Bacillus megaterium.
J. Biotechnol. 126:313–324.
68. Wang, W., and M. Sun. 2009. Phylogenetic relationships between Bacillus
species and related genera inferred from 16S rDNA sequences. Braz. J.
69. Wittchen, K. D., and F. Meinhardt. 1995. Inactivation of the major extra-
cellular protease from Bacillus megaterium DSM319 by gene replacement.
Appl. Microbiol. Biotechnol. 42:871–877.
70. Wolf, J. B., and R. N. Brey. 1986. Isolation and genetic characterizations of
Bacillus megaterium cobalamin biosynthesis-deficient mutants. J. Bacteriol.
71. Wood, J. M., et al. 2001. Osmosensing and osmoregulatory compatible solute
accumulation by bacteria. Comp. Biochem. Physiol. A Mol. Integr. Physiol.
72. Yang, Y., et al. 2006. High yield recombinant penicillin G amidase produc-
tion and export into the growth medium using Bacillus megaterium. Microb.
Cell Fact. 5:36.
73. Zupancic, M. L., H. Tran, and A. E. Hofmeister. 2001. Chromosomal orga-
nization governs the timing of cell type-specific gene expression required for
spore formation in Bacillus subtilis. Mol. Microbiol. 39:1471–1481.
VOL. 193, 2011GENOME SEQUENCES OF B. MEGATERIUM QM B1551 AND DSM3194213