Whole genome sequencing of Bacillus subtilis a gram positive organism

Genus to Genome
“ Whole genome sequence of the Gram- positive bacterium
Bacillus subtilis ”
Indian Agricultural Research Institute
Division of Plant Pathology
Speaker - M. Ashajyothi, 10863
Ph.D First year

Ferdinand Julius Cohn 1828-1898
Vibrio subtilis (Ehrenberg 1835)
Bacillus subtilis Cohn 1872
It is used as an “indicator organism” during gas sterilization procedures, to ensure
a sterilization cycle has completed successfully.

Taxonomy
 Cellular organisms
› Bacteria
› Terrabacteria group
› Firmicutes
› Bacilli
› Bacillales
› Bacillaceae
› Bacillus
› Bacillus subtilis group
› Bacillus subtilis
› Bacillus subtilis subsp. subtilis 168
Characteristics: Rod shaped, gram positive, flagellate, soil bacterium
Synonyms - Bacillus uniflagellatus, Bacillus globigii, and Bacillus natto

Nature - Vol: 390
20 November, 1997
 Till 1997 - 40 prokaryotic genomes.
 Gram-positive bacteria, which are
important for the environment,
medicine and industry.
 Bacillus subtilis has been chosen to
fill this gap.

But why Bacillus subtilis??
 Good model for cellular development and differentiation (Entrez Genome
Project).
 Its biochemistry, physiology and genetics have been studied intensely for more
than 40 years.
 An important source of industrial enzymes (such as amylases and proteases),
and capacity to secrete these enzymes at gram per litre concentrations.
 Used for the study of protein secretion and for development as a host for the
production of heterologous proteins.
 Under nutritional starvation, B. subtilis stops growing and initiates responses
to restore growth by increasing metabolic diversity.
 These responses include 1. Induction of motility and chemotaxis
2. Production of macromolecular hydrolases
(proteases and carbohydrases) and antibiotics.

 B. subtilis (natto) is also used in the production of Natto, a traditional Japanese
dish of fermented soya beans.
 Its surface-binding properties play a role in safe radionuclide waste [e.g. thorium
and plutonium] disposal.
 B. subtilis is used as a soil inoculant in horticulture and agriculture.
 It provide benefit to saffron growers by speeding corm growth and increasing
stigma biomass yield.
 Ingestion of significant quantities of Bacillus subtilis is thought to restore the
normal microbial flora following extensive antibiotic use or illness.

• In September 1989 a consortium of five European laboratories started a joint project
aimed at :
Developing the physical map
Constructing appropriate DNA libraries
Launching on a pilot scale the systematic sequencing of the B.
subtilis genome
Bacillus subtilis strain168
International consortium
Labs: European(25) Japanese(7) Korean(1) Biotechnology
companies(2)
2.68Mb 1.36Mb
 Sequences from strain 168 published previously were not resequenced when long
overlaps did not indicate differences.

METHODS
Bacterial DNA Extraction
Restriction digestion
Cloning in to Vectors (E.Coli, Cosmids)
Sanger Sequencing
Sequence annotation and verification
Organisation and accesibility of data
PCR In vitro Amplification
 Plasmid rescue chromosome walking method
 Marker rescue genome walking method
6 ORF’s - each 100 codon in length
Start codon - ATG,TTG,GTG
TIS - 5’ -AAGGAGGTG-3’
SubtiList
Gene Mark coding-sequence prediction method

General features
 Analysis at the replicon level
 Chromosome has 4,214,810 base pairs (bp)
 Origin of replication coinciding with the base numbering start point, and the
terminus at about 2,017 kilobases (kb).
 The average G : C ratio - 43.5%
 This average is different in the nucleotide content of coding sequences.
 Asymmetry of the nucleotide composition between the replication leading
strand and the lagging strand.
 Several A : T-rich islands are likely to reveal the signature of bacteriophage
lysogens or other inserted elements.

Distribution of A þ T-rich islands along the chromosome of B. subtilis

Abundance of oligonucleotides in the genome:
 Dinucleotide bias - Dinucleotides most overrepresented are AA, TT and GC,
whereas those less represented are TA, AC and GT.
 The frequencies of AG, GA, CT and TC show dramatic decreases or increases
around the origin and terminus of replication.
 The genome of B. subtilis contains a plethora of duplications that contains
oligonucleotides longer than 24 nucleotides
 Significant duplications in the regions involved in the transcriptional control of
several genes (such as 118 bp repeated three times between yxbB and yxbC).
 several repetitions were found at the borders of regions that might be involved
in bacteriophage integration.

• The most prominent duplication was a 190-bp element that was repeated 10
times in the chromosome.
• Multiple alignment of the ten repeats showed that they could be classified
into two subfamilies with six and three copies each.
• Similar sequences have also been described in the closely related species
Bacillus licheniformis.
• A striking feature of these repeats is that they are only found in half of the
chromosome, at either side of the origin of replication, with five repeats on
each side.

Analysis at the transcription and translation level
• Putative protein coding sequences (CDSs) - 4,000
• With an average size - 890 bp
• Coverage of the genome sequence - 87%
• 78% of the genes started with - ATG - 85%
• 13% with - TTG - 3%
• 9% with - GTG -14%
• Fifteen genes (eight in the predicted CDSs in bacteriophage SPβ) exhibiting
unusual start codons (namely ATT and CTG).
• The gene coding for translation initiation factor 3, the similarity with its E.
coli counter part strongly suggests that the initiation codon is ATT, as is the
case in E. coli.
• The estimated number of B. subtilis CDSs will fluctuate around the present
figure of 4,100.
E.coli

Coding sequences classes
• Class 1 : The majority of the genes (3,375 CDSs), including most of the
genes involved in sporulation.
• Class 2 : (188 CDSs) genes that are highly expressed under exponential growth
1. Genes encoding the transcription and translation machineries
2. Core intermediary metabolism
3. Stress proteins
4. 1/3 of genes of unknown function.
• Class 3 : (537 CDSs) high proportion of genes of unidentified function (84%), and
codons enriched in A þ T residues.
The codon usage of B. subtilis CDSs was analysed using factorial
correspondence analysis

• These genes are usually clustered into groups between 15 and 160 genes (for
example, bacteriophage SPb).
• They usually correspond to functions associated with, bacteriophages or
transposons, as well as functions related to the cell envelope.
• This includes the region ydc/ydd/yde where gene products showing similarities
to bacteriophage and transposon proteins are intertwined.
• Many of these genes are associated with virulence genes identified in
pathogenic Gram-positive bacteria.
• Annotation of the corresponding regions often reveals the presence of genes
that are similar to bacteriophage lytic enzymes.

• The ribosomal RNA genes organized into 10 rRNA operons, mainly clustered around the
origin of replication of the chromosome.
• 84 tRNA genes identified and four more proposed putative new tRNA loci specific for
lysine, proline and arginine.
• There is a strong transcription orientation bias with respect to the movement of the
replication fork: 75% of the predicted genes are transcribed in the direction of
replication.
• 14 sigma factors, recognizing different promoter sequences, have been identified .
• The consensus of the main vegetative sigma factor (jA) appears to be identical to its
counterpart in E. coli (j70): 5ᶦ- TTGACA-n17-TATAAT-3ᶦ.

Classification of gene products
• Using the BLAST2P software running against a composite protein databank
compound of SWISS-PROT , TrEMBL .
• They have assigned at least one significant counterpart with a known function
to 58% of the B. subtilis 168 proteins.
• 42% of the gene products the function cannot be predicted by similarity to
proteins of known function:
– 4% of the proteins are similar only to other unknown proteins of B. Subtilis
– 12% are similar to unknown proteins from some other organism
– 26% of the proteins are not significantly similar to any other proteins in databanks

Regulatory systems
• Transcription regulatory proteins
• Using BLAST searches consensus matrices for helix–turn–helix proteins
were constructed to analyse the B. subtilis protein library.
• 18 sigma or sigma-like factors, of which nine (including a new one) are of
the SigA type.
• Identified 20 regulators : GntR family
19 regulators : LysR family
12 regulators : LacI family
• Other transcription regulatory proteins were : AraC family
Lrp family
DeoR family
MarR, ArsR or TetR families

Two-component signal-transduction pathways
• 34 genes encoding response regulators adjacent genes encoding histidine kinases
• Response regulators possess a well-conserved N-terminal phospho-acceptor
domain, C-terminal DNA-binding domains
• Share similarities with response regulators in E. coli, Rhizobium meliloti, Klebsiella
pneumoniae
• Response regulators are representatives of the four subfamilies identified in E.
coli31 (OmpR, FixJ, CitB and LytR).

Quorum sensing
• It contains 11 aspartate phosphatase genes, whose products are involved in
dephosphorylation of response regulators.
• Downstream from the corresponding genes are some small genes, called phr,
encoding regulatory peptides that may serve as quorum sensors.
• Seven phr genes have been identified so far, including three new genes (phrG,
phrI and phrK).

Protein secretion
• It is known that B. subtilis and related Bacillus species have a high capacity to
secrete proteins into the culture medium.
• Several genes encoding proteins of the major secretion pathway have been
identified: secA, secD, secE, secF, secY, ffh and ftsY.
• There is no gene for the SecB chaperone.
• Ffh and FtsY, may take over the SecB function.
• Five type I signal peptidase genes (sipS, sipT, sipU, sipV and sipW) have been
found .
• The lsp gene, encoding a type II signal peptidase required for processing of
lipo-modified precursors.
• PrsA, located at the outer side of the membrane, is important for the
refolding of several mature proteins after their translocation through the
membrane.

Other families of proteins
• ABC transporters were the most frequent class of proteins found in B. subtilis.
• ABC transporters allow bacteria to escape the toxic action of many compounds.
• 77 transporters are encoded in the genome.
• In general they involve the interaction of at least three gene products, specified
by genes organized into an operon.
• General stress proteins are important for the survival of bacteria under a variety
of environmental conditions.
• 43 temperature-shock and general stress proteins displaying strong similarity to
E. coli counterparts.

Metabolism of small molecules
• The type and range of metabolism used provide important clues to an
organism’s natural environment and its biological activity.
• Intermediary metabolism
• B. subtilis can use a variety of carbohydrates.
• It encodes an Embden–Meyerhof–Parnas glycolytic pathway, coupled to a
functional Tricarboxylic acid cycle.
• B. subtilis is also able to grow anaerobically in the presence of nitrate as an
electron acceptor.
• This metabolism is regulated by the FNR protein, binding to sites upstream of
at least eight genes
• A noteworthy feature of B. subtilis metabolism is requirement of branched
short-chain carboxylic acids for lipid biosynthesis.
• Branched chain 2-keto acid decarboxylase activity exists suggesting it can
synthesize and utilize linear branched short-chain carboxylic acids and alcohols.

Amino-acid and nucleotide metabolism
• Pyrimidine metabolism of B. Subtilis different from that of E. Coli.
• It has two carbamylphosphate synthetases (one specific for arginine synthesis,
the other for pyrimidine).
• Pyrimidine deoxyribonucleotides are synthesized from ribonucleoside
diphosphates, not triphosphates.
• Genes involved in amino-acid degradation (such as the roc operon, which
degrades arginine and related amino acids).
• A large number of genes involved in the degradation of molecules such as
opines and related molecules, derived from plants.
• B. subtilis also degrades polygalacturonate, and suggests that, in its biotope, it
forms specific relations with plants.

Secondary metabolism
• 4% of the B. subtilis genome codes for large multifunctional enzymes (srf, pps
and pks loci).
• Natural isolates of B. subtilis produce compounds with antibiotic activity,
such as surfactin, fengycin and difficidin.
• This bacterium provides a simple and genetically amenable model to study
the synthesis of antibiotics and its regulation.
• These pathways are often organized in very long operons (for example, the
pks region spans 78.5 kb, about 2% of the genome).
• The corresponding sequences are mostly located near the terminus of
replication, together with prophages and prophage-like sequences.

Structurally unrelated genes of similar function
1. The helicase loader genes, E. coli dnaC and B. subtilis dnaI
2. The genes coding for the replication termination protein, E. coli tus and
B.subtilis rtp
3. The division topology specifier genes, E. coli minE and B. subtilis divIVA.
• E. coli DNA polymerase II is structurally related to DNA polymerase α of
eukaryotes, whereas B. subtilis YshC is related to DNA polymerase β.

Paralogues
• Many of the paralogues constitute large families of functionally related
proteins, involved in the transport of compounds into and out of the cell.
• Several approximate DNA repetitions, associated within regions putatively
identified as prophages (PBSX and the skin element).
• This suggests that these prophage-like elements share a common ancestor and
have diverged relatively recently.
 The study of paralogues showed that, as in other genomes, a few classes of
genes have been highly expanded.
 This argues against the idea of the genome evolving through a series of
duplications of ancestral genomes.

Orthologues
 Among the 450 genes encoded by M. genitalium, the products of 300 are similar
to proteins of B. subtilis.
 Only 90 genes that would be specific to M. genitalium and might be involved in
the interaction of this organism with its host.
 The B. subtilis genome is similar in size to that of E. coli.
 About 1,000 B. subtilis genes have clear orthologous counterparts in E. coli (one-
quarter of the genome).
 100 putative operons or parts of operons were conserved between E. coli and B.
subtilis {ATP synthesis (atp operon) and electron transfer (cta and qox operons)}.

EVOLUTION
Non pathogenic Vs Pathogenic

Quorum sensing operon
Regulatory sequences
Transcriptional networks
PlcR-Pap R Operon
More rapidly evolving and evolutionarily flexible
Differential regulation modulates virulence!!
Pap R - Secrete a peptide PlcR- Transcription activator

• The sigma factor gene family encoded by the Bc species-group is result of a dynamic
gene-duplication.
• Expansion of the sigma factor gene family appears to have preferentially occurred
within the extra cytoplasmic function (ECF) sigma factor genes.
• Primary alternative (PA) sigma factor genes are highly conserved with those found
in B. subtilis.
• Divergence of the sigma-controlled transcriptional regulons among various
members of the Bc species-group likely has a major role in explaining the diversity
of phenotypic characteristics seen in members of the Bc species-group.
Schmidt et al. 2011 (BMC Genomics)
Bacillus cereus Vs Bacillus subtilis

SUMMERY
• Bacillus subtilis is the best-characterized member of the Gram-positive bacteria.
• Its genome of 4,214,810 bp comprises 4,100 protein-coding genes.
• 53% are represented once, while a quarter of the genome corresponds to several gene
largest family containing 77 putative ATP-binding transport proteins.
• A large proportion of the genetic capacity is devoted to the utilization of a variety of
carbon sources.
• Five signal peptidase genes, secretion apparatus are identified to secrete large
amounts of industrially important enzymes.
• Genes involved in the synthesis of secondary metabolites, including antibiotics, that
are more typically associated with Streptomyces species.
• The genome contains at least ten prophages , indicating that bacteriophage infection
has played an important evolutionary role in horizontal gene transfer.

Conclusion
• B. subtilis can be used to study post exponential phase phenomena such as
sporulation and competence for DNA uptake.
• Provide a means of studying Eubacteria evolutionary divergence.
• The availability of powerful genetic tools will allow the B. subtilis genome sequence
data to be exploited fully within the framework of a systematic functional analysis
program.
current data release of GenoList/SubtiList:
4,215,606 bp - Size
4,244 - Protein genes
86 - tRNA genes
30 - rRNA genes

Importantwebsites:
›onlinelibrary.wiley.com
›www.magma.ca
›www.pasteur.fr
›en.wikipedia.org
›genolist.pasteur.fr
›NCBI

Whole genome sequencing of Bacillus subtilis a gram positive organism

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