Subtilis Chromosome Research Articles

Correlation between Bacillus subtilis scoC phenotype and gene expression determined using microarrays for transcriptome analysis.

The availability of the complete sequence of the Bacillus subtilis chromosome (F. Kunst et al., Nature 390:249-256, 1997) makes possible the construction of genome-wide DNA arrays and the study of this organism on a global scale. Because we have a long-standing interest in the effects of scoC on late-stage developmental phenomena as they relate to aprE expression, we studied the genome-wide effects of a scoC null mutant with the goal of furthering the understanding of the role of scoC in growth and developmental processes. In the present work we compared the expression patterns of isogenic B. subtilis strains, one of which carries a null mutation in the scoC locus (scoC4). The results obtained indicate that scoC regulates, either directly or indirectly, the expression of at least 560 genes in the B. subtilis genome. ScoC appeared to repress as well as activate gene expression. Changes in expression were observed in genes encoding transport and binding proteins, those involved in amino acid, carbohydrate, and nucleotide and/or nucleoside metabolism, and those associated with motility, sporulation, and adaptation to atypical conditions. Changes in gene expression were also observed for transcriptional regulators, along with sigma factors, regulatory phosphatases and kinases, and members of sensor regulator systems. In this report, we discuss some of the phenotypes associated with the scoC mutant in light of the transcriptome changes observed.

YveB, Encoding endolevanase LevB, is part of the sacB-yveB-yveA levansucrase tricistronic operon in Bacillus subtilis.

Transcription of sacB, yveB and yveA, three clustered genes on the Bacillus subtilis chromosome, is simultaneously induced by sucrose. Northern blotting analyses with specific probes showed three distinct mRNAs: a monocistronic 1.7 kb sacB mRNA, a bicistronic 3.3 kb sacB-yveB mRNA and a tricistronic 4.9 kb sacB-yveB-yveA mRNA. These results indicate that sacB, encoding levansucrase, is the proximal gene of a sucrose-inducible operon that includes the two other genes. The yield of the full-length transcript is lower than that of the bicistronic transcript, whose yield is itself lower than that of the monocistronic transcript. This suggested that the 3' terminal parts of sacB and yveB genes worked as internal terminator structures. The protein encoded by yveB, which remains anchored to the membrane, displays an endolevanase activity, which, coupled with exolevanase activity of SacB, leads to a complete degradation of levan, a branched fructosyl polymer. It is proposed to rename yveB as levB.

Analysis of promoter recognition in vivo directed by sigma(F) of Bacillus subtilis by using random-sequence oligonucleotides.

Formation of spores from vegetative bacteria by Bacillus subtilis is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase sigma factors. Of these, sigma(F) is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing sigma(F) (E-sigma(F)), and only two genes of known function are exclusively under the control of E-sigma(F), spoIIR and spoIIQ. In order to investigate the features of promoters that are recognized by E-sigma(F), we studied the effects of randomizing sequences for the -10 and -35 regions of the promoter for spoIIQ. The randomized promoter regions were cloned in front of a promoterless copy of lacZ in a vector designed for insertion by double crossover of single copies of the promoter-lacZ fusions into the amyE region of the B. subtilis chromosome. This system made it possible to test for transcription of lacZ by E-sigma(F) in vivo. The results indicate a weak sigma(F)-specific -10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated sigma factors, as well as to sigma(A). There was a rather stronger -35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated sigma factors. The looseness of the sigma(F) promoter requirement contrasts with the strict requirement for sigma(A)-directed promoters of B. subtilis. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-sigma(F) in vivo.

The Bacillus subtilis yabQ gene is essential for formation of the spore cortex.

An extensive screening for transcripts with probes specific for the genes in a 108 kb region from rrnO to spo0H of the Bacillus subtilis chromosome led to identification of an operon, yabP--yabQ--divIC--yabR, the expression of which was initiated at the second hour of sporulation and in a sigma(E)-dependent manner. Among three y genes in the operon, deletion of the yabQ gene, which is predicted to encode a protein product of 468 residues with five membrane-spanning domains, resulted in a large decrease in numbers of chloroform-, lysozyme- and heat-resistant spores, compared to findings with the wild-type strain. Electron microscopy revealed that development of the spore cortex was blocked in the yabQ mutant. In addition, although the spore coat was visible, the inner coat layer of the mutant seemed partially detached from the outer coat. In sporangia of the strains harbouring an in-frame fusion of the green fluorescent protein gene to yabQ, fluorescence was detected around the forespore. This localization did not depend on SpoIVA or on CotE functions, both of which determine proper localization of coat proteins and cortex formation. The yabQ deletion did not affect expression of genes involved in cortex synthesis. These results suggest that the YabQ protein localizes in the membrane of the forespore and plays an important role in cortex formation.

Crystallization of the Bacillus subtilis RTP-DNA complex prepared using NMR spectroscopy.

The replication terminator protein (RTP)-DNA complex of Bacillus subtilis is responsible for the arrest of DNA replication at terminator sites in the B. subtilis chromosome. The crystallization and preliminary diffraction data analysis for the complex of an (15)N-labelled mutant form of RTP and a symmetrical form of its DNA-binding site is reported. NMR spectroscopy was used to assess the stoichiometry of complex formation, with the sample containing the most homogeneous solution of complex giving rise to diffracting crystals. Synchrotron-radiation data to 2.5 A were collected from a crystal of space group P3(2)21, unit-cell parameters a = b = 44.780, c = 395.582 A, containing an RTP dimer within the asymmetric unit.

DNA amplification occurs between the core region of replicationori + of the ts plasmid integrated in the chromosome and its analog in the bacterial genome

Integrated in theBacillus subtilis chromosome, hybrid plasmid pGG10 is capable of thermosensitive amplification. One amplification site corresponds to the core region of replicationori + of pE194, a component of pGG10; the other is a homologous region of theB. subtilis chromosome. A model of illegitimate amplification mediated by pE194 RepF is proposed.

The CitST two-component system regulates the expression of the Mg-citrate transporter in Bacillus subtilis.

citS and citT genes encoding a new two-component system were identified in the 71 degrees region between the pel and citM loci on the Bacillus subtilis chromosome. citS- and citT-deficient strains were unable to grow on minimal plates including citrate as a sole carbon source. In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg-citrate complex, exhibited the same phenotype on this medium. Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM-yflN operon. Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant. Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose. We found a sequence exhibiting homology to a catabolite-responsive element (cre) in the citM promoter region. Glucose repression was lost in ccpA and citM-cre mutants. From the result of a citM-promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from -62 to -74 and from -149 to -189, relative to the citM start point. Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from -36 to -84 and from -168 to -194. From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA.

Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view.

Bacillus species and other microbes with pH optima for growth higher than pH 9 are defined as alkaliphiles. A large number of alkaliphilic Bacillus strains producing useful enzymes, have been isolated from various environments. Some of these enzymes, such as proteases and cellulases from alkaliphilic Bacillus strains, have been commercialized and have brought great advantages to industry and domestic life. To support further development of the enzyme industry, we initiated analysis of the genome of Bacillus halodurans C-125, which is 4.25Mb in size, and constructed a physical and genetic map for comparison with the Bacillus subtilis chromosome. Systematic sequencing of the whole genome of Bacillus halodurans C-125 has been automated since the beginning of May 1998, and sequencing of 98% of the whole genome has been done so far. Through genome analysis, it became apparent that the genome organization of alkaliphilic Bacillus halodurans C-125 is totally different from that of B. subtilis orthologues.

The Bacillus subtilis cysP gene encodes a novel sulphate permease related to the inorganic phosphate transporter (Pit) family.

Sulphate permeases in the plasma membrane are responsible for uptake of environmental sulphate used in the sulphate assimilation pathway in bacteria and plants. Here it is reported that the ORF designated cysP, located on the Bacillus subtilis chromosome between cysH and five putative genes involved in sulphate assimilation, encodes a sulphate permease. cysP is able to complement Escherichia coli cysteine auxotrophs with mutations affecting either the membrane or periplasmic components of the sulphate-thiosulphate permease. Transport studies with cell suspensions of a cysA97 E. coli strain transformed with a plasmid expressing the B. subtilis cysP gene indicated that CysP catalyses sulphate uptake. Analysis of the primary sequence showed that CysP (354 amino acids, estimated molecular mass 24 kDa) is a highly hydrophobic protein which has 11 putative transmembrane helices. Sequence comparisons revealed that CysP, together with the phosphate permease of Neurospora crassa, Pho-4, and E. coli PitA, belongs to the family of related transporters, the inorganic phosphate transporter (Pit) family. Among the putative phosphate permeases, CysP shows a similar size and the same domain organization as the archaeal transporters. This is the first report of a sulphate permease in a Gram-positive organism.

Interspecific transformation of Bacillus subtilis competent cells by chromosomal DNA in lysates of protoplasts of Bacillus amyloliquefaciens.

Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B. subtilis or B. amyloliquefaciens. The interspecific transformation frequency of B. subtilis by cysA in a conserved region was 3.1 x 10(4) transformants per microg DNA, 60 times higher than that for conventional transformation using purified DNA. Increased interspecific transformation frequencies of B. subtilis were also observed for arg-1, lys-1, leuB, aroG, thr-5, hisH, or metC markers outside the conserved region (3.1 x 10 approximately 5.2 x 10(2) transformants per microg DNA). An interspecific cotransformation ratio (33-50%) as high as an intraspecific one (46%) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B. subtilis chromosome. Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation. The involvement of mutS in the interspecific transformation was not significant.

Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis.

Biosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-step oxidation process of choline, with glycine betaine aldehyde as the intermediate. We have exploited the sensitivity of B. subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC) were required for these phenotypes. Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein-dependent ABC transport system that consists of four components. The presumed binding proteins of both ABC transporters were shown to be lipoproteins. Kinetic analysis of [14C]-choline uptake via OpuB (K(m) = 1 microM; Vmax = 21 nmol min-1 mg-1 protein) and OpuC (K(m) = 38 microM; Vmax = 75 nmol min-1 mg-1 protein) revealed that each of these ABC transporters exhibits high affinity and substantial transport capacity. Western blotting experiments with a polyclonal antiserum cross-reacting with the presumed substrate-binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium. Primer extension analysis confirmed the osmotic control of opuB and allowed the identification of the promoter of this operon. The opuB and opuC operons are located close to each other on the B. subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved from a duplication event of a primordial gene cluster. Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate specificity for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.

GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis.

We report the development of a series of plasmid vectors for the construction of fusions to mutants of the intrinsically fluorescent green fluorescent protein, GFPmut1 (Cormack et al., 1996. Gene 173, 33-38) and GFPuv (Crameri et al., 1996. Nature Biotechnology 14, 315-319). Both N- and C-terminal fusions can be produced, and their expression can be finely controlled from the inducible Pxyl promoter following double crossover integration into the amyE locus of the Bacillus subtilis chromosome. Other vectors designed for single crossover insertion into the chromosome allow downstream genes to be placed under inducible control. We also show that fusions to GFPmut1 and GFPuv can be co-localized within the cell by virtue of their different excitation spectra.

Genetic engineering of Bacillus subtilis for the commercial production of riboflavin

Recombinant DNA engineering was combined with mutant selection and fermentation improvement to develop a strain of Bacillus subtilis that produces commercially attractive levels of riboflavin. The B. subtilis riboflavin production strain contains multiple copies of a modified B. subtilis riboflavin biosynthetic operon (rib operon) integrated at two different sites in the B. subtilis chromosome. The modified rib operons are expressed constitutively from strong phage promoters located at the 5′ end and in an internal region of the operon. The engineered strain also contains purine analog-resistant mutations designed to deregulate the purine pathway (GTP is the precursor for riboflavin), and a riboflavin analog-resistant mutation in ribC that deregulates the riboflavin biosynthetic pathway.

Cyclophilin and Trigger Factor from Bacillus subtilis Catalyze in Vitro Protein Folding and Are Necessary for Viability under Starvation Conditions

Cyclophilin (the product of the ppiB gene) and the trigger factor (the product of the tig gene) are the only cytosolic peptidyl-prolyl cis-trans isomerases that are known in Bacillus subtilis. Both enzymes catalyze the in vitro refolding of ribonuclease T1, a reaction that is limited in rate by a prolyl cis/trans isomerization. The efficiency of cyclophilin as a folding catalyst is only modest with a kcat/KM value of 3.8 x 10(4) M-1 s-1, but the trigger factor shows an almost 40-fold higher specific activity with a kcat/KM value of 1.4 x 10(6) M-1 s-1. This high catalytic activity originates from the tight binding to the protein substrate as reflected in both the low KM value of 0.5 microM and in the strong inhibition of the trigger factor by unfolded proteins. By use of a protein-folding assay, the concentrations of cyclophilin and the trigger factor in the cytosol of B. subtilis could be determined as 26 and 35 microM, respectively. Together they account for the entire folding activity that is detectable in crude extracts of wild-type B. subtilis cells. The genes encoding cyclophilin and the trigger factor in the B. subtilis chromosome were disrupted individually and simultaneously. Even in combination, these disruptions had no effect on cell viability in rich medium or under several stress conditions, such as heat, osmotic, or oxidative stress. However, in poor medium and, in particular, in the absence of amino acids, the growth of the double mutant strain was strongly decelerated, indicating that the prolyl isomerases become essential for growth under starvation conditions. It is not yet known whether this function relates to the catalysis of the proline-limited folding of essential proteins.

Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates.

A 5 kb region upstream of katA at 82 degrees on the Bacillus subtilis chromosome contains five ORFs organized in an operon-like structure. Based on sequence similarity, three of the ORFs are likely to encode an ABC transport system (ssuBAC) and another to encode a monooxygenase (ssuD). The deduced amino acid sequence of the last ORF (ygaN) shows no similarity to any known protein. B. subtilis can utilize a range of aliphatic sulfonates such as alkanesulfonates, taurine, isethionate and sulfoacetate as a source of sulfur, but not when ssuA and ssuC are disrupted by insertion of a neomycin-resistance gene. Utilization of aliphatic sulfonates was not affected in a strain lacking 3'-phosphoadenosine 5'-phosphosulfate (PAPS) sulfotransferase, indicating that sulfate is not an intermediate in the assimilation of sulfonate-sulfur. Sulfate or cysteine prevented expression of beta-galactosidase from a transcriptional ssuD::lacZ fusion. It is proposed that ssuBACD encode a system for ATP-dependent transport of alkanesulfonates and an oxygenase required for their desulfonation.

DNA polymerase template switching at specific sites on the phi29 genome causes the in vivo accumulation of subgenomic phi29 DNA molecules.

The accumulation of subgenomic phage phi29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb. Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage phi29 genome. The nature of the 4, 2.3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single-stranded self-complementary strands forming hairpin structures. The other molecules consisted of palindromic linear double-stranded DNA molecules. Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo. Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5'-TGTTtCAC-GTGg-AACA-3' being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the phi29 genome. In addition, the almost identical sequence 5'-TgGTTTCAC-GTGGAAtCA-3' was found once. These five DSEs are all located in the right half of the phi29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene of the B. subtilis chromosome. Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE. As the same subgenomic phi29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.

The yvsA-yvqA (293 degrees-289 degrees) region of the Bacillus subtilis chromosome containing genes involved in metal ion uptake and a putative sigma factor.

The region between yvsA (293 degrees) and yvqA (289 degrees) of the Bacillus subtilis chromosome has been sequenced within the framework of the B. subtilis 168 international sequencing programme. A primary analysis of the 42 ORFs identified in this 43 kb region is presented. The region included a high proportion of genes that did not show homology with genes in other bacteria. The identified ORFs showed homology to proteins involved in the transport of metal ions, two-component signal transducers, ATP-binding-cassette-type transporters and a sigma factor.

Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis.

We describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 microm in an 11 min period of abrupt movement, representing an average velocity of 0.17 microm min(-1), and reaching a maximum velocity of greater than 0.27 microm min(-1). The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.

A 35.7 kb DNA fragment from the Bacillus subtilis chromosome containing a putative 12.3 kb operon involved in hexuronate catabolism and a perfectly symmetrical hypothetical catabolite-responsive element.

The Bacillus subtilis strain 168 chromosomal region extending from 109 degrees to 112 degrees has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and beta-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase alpha-subunit (FdhA), protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.

The 172 kb prkA-addAB region from 83 degrees to 97 degrees of the Bacillus subtilis chromosome contains several dysfunctional genes, the glyB marker, many genes encoding transporter proteins, and the ubiquitous hit gene.

A 171812 bp nucleotide sequence between prkA and addAB (83 degrees to 97 degrees) on the genetic map of the Bacillus subtilis 168 chromosome was determined and analysed. An accurate physical/genetic map of this previously poorly described chromosomal region was constructed. One hundred and seventy open reading frames (ORFs) were identified on the DNA fragment. These include the previously described genes cspB, glpPFKD, spoVR, phoAIV, papQ, citRA, sspB, prsA, hpr, pbpF, hemEHY, aprE, comK and addAB. ORF yhaF in this region corresponds to the glyB marker. Among the striking features of this region are: an abundance of genes encoding (putative) transporter proteins, several dysfunctional genes, the ubiquitous hit gene, and five multidrug-resistance-like genes. These analyses have also revealed the existence of numerous paralogues of ORFs in this region: about two-thirds of the putative genes seem to have at least one paralogue in the B. subtilis genome.