Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Introns and intein coding sequence in the ribonucleotide reductase genes of Bacillus subtilis temperate bacteriophage SPbeta.

The two putative ribonucleotide reductase subunits of the Bacillus subtilis bacteriophage SPbeta are encoded by the bnrdE and bnrdF genes that are highly similar to corresponding host paralogs, located on the opposite replication arm. In contrast to their bacterial counterparts, bnrdE and bnrdF each are interrupted by a group I intron, efficiently removed in vivo by mRNA processing. The bnrdF intron contains an ORF encoding a polypeptide similar to homing endonucleases responsible for intron mobility, whereas the bnrdE intron has no obvious trace of coding sequence. The downstream bnrdE exon harbors an intervening sequence not excised at the level of the primary transcript, which encodes an in-frame polypeptide displaying all the features of an intein. Presently, this is the only intein identified in bacteriophages. In addition, bnrdE provides an example of a group I intron and an intein coding sequence within the same gene.

[A case of vaccination-induced polio]. / Ein Fall von Impfpolio.

CASE REPORT: We describe a 69 year-old female who became ill with polio after having changed the nappies of her grandchild who, six weeks earlier, had been vaccinated with oral live-vaccine against polio. Anamnesis and neurological status with limp paralysis in both legs oblige, with their implications, that the patient be isolated initially. Proof of poliomyelitis was to be found, in addition to typical medical findings, in the virus analysis in the stool, determined by polymerase chain reaction and in the discovery of antibodies from the serum by means of a neutralisation test. The neurological disorders could not be treated medically. The main priority was intensive physiotherapy. With walking aids the patient had only limited walking ability three months later. CONCLUSION: Discussed are: the meaning of anamnesis and specific diagnosis of illness and immune deficiency, the morbidity and necessity for certain protection against children recently vaccinated with live-vaccine, also the provision for an individually structured plan for vaccination after satisfactory information.

Sequence of the 305 degrees-307 degrees region of the Bacillus subtilis chromosome.

The nucleotide sequence of the Bacillus subtilis 168 chromosomal segment located between yvhJ (307 degrees) and secA (305 degrees) was determined. This 20.3 kb region encompasses 23 ORFs, 17 of which have been sequenced previously. Comparison of sequences obtained here with the previously obtained ones revealed seven discrepancies. The products of the sequenced genes are involved in the regulation of degradative enzymes, competence, flagellar motility and protein secretion. Putative functions of newly identified genes are based on sequence homologies.

In Bacillus subtilis 168, teichoic acid of the cross-wall may be different from that of the cylinder: a hypothesis based on transcription analysis of tag genes.

Five of the genes known to encode the enzymes for the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons, tagAB and tagDEF. lacZ and gus transcriptional fusions to the first genes of these operons revealed that: (i) in media of different richness, higher growth rates were paralleled by lower transcription levels; (ii) upon transition to stationary phase, the transcription per unit mass of both operons increased abruptly by a factor of about two; and (iii) a rise in temperature was accompanied by decreased transcription of tagA and increased transcription of tagD. Mapping of transcription start points revealed two divergent sigma A-controlled promoters. Although tagD and the neighbouring downstream gene tagE are transcribed from the same promoter, the latter was expressed at a much lower level than the former. Moreover, expression of tagE, and of the translationally coupled tagF, did not increase at the onset of the stationary phase, indicating that additional regulatory signals may act in the intergenic tagD-tagE region. Optimal transcription of these operons appears to require the entire regulatory region, suggesting that tag gene expression may, among other factors, be regulated by the three-dimensional configuration of this segment. The biological implications of these results are discussed.

Sequence analysis of the 308 degrees to 311 degrees segment of the Bacillus subtilis 168 chromosome, a region devoted to cell wall metabolism, containing non-coding grey holes which reveal chromosomal rearrangements.

The 29.71 kb chromosomal region of Bacillus subtilis 168 extending from 308 degrees to 311 degrees contains 18 ORFs. Functions of most of these ORFs were identified and associated with cell wall metabolism. Sequences of two non-coding regions of 0.7 and 2.2 kb flanking the ggaAB operon involved in the synthesis of poly(3-O-beta-D-glucopyranosyl N-acetylgalactosamine 1-phosphate), a minor teichoic acid, correspond to five degenerate segments of neighbouring protein-coding regions. We discuss the possibility that such grey holes are indicative of a chromosomal rearrangement which could have arisen from horizontal gene transfer.

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions.

Five of the genes known to encode the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons (a divergon), denoted tagAB and tagDEF. To monitor their expression, the 399 bp intergenic region separating the first structural genes of these operons was fused, in both orientations, to a lacZ reporter gene, allowing measurement of promoter activity under specific physiological conditions. Under all experimental conditions, tagA and tagD appeared coordinately expressed, the level of tagD being always higher than that of tagA. No influence of the chromosomal context was observed. Phosphate limitation was accompanied by reduced tag gene expression. Following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II. During germination, the activity of tag genes was detectable before the rise in culture turbidity associated with spore outgrowth. In contrast to tagC (dinC), the expression of which is DNA-damage-inducible, the induction of SOS functions had no effect on tagA and tagD gene expression. The biological significance of these results is discussed.

Lytic enzymes associated with defective prophages of Bacillus subtilis: sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX.

Prophage induction in Bacillus subtilis strains 168, S31 and W23 is accompanied by synthesis of two endolysins. The synthesis of those of strain 168, with molecular masses of 32 and 34 kDa, was shown to be controlled by the repressor of the defective phage PBSX. The 32 kDa protein corresponds to an N-acetylmuramoyl-L-alanine amidase, and plays the major role in PBSX-mediated lysis. Its structural gene, xlyA, is the last in the PBSX late operon, whose four most distal open reading frames have been cloned and sequenced. Analysis of the nucleotide sequence suggests that the two open reading frames preceding xlyA, designated xhlA and xhlB, encode polypeptides whose combined action could play the role of a holin. The open reading frame upstream of xhlA, designated xepA, encodes an exoprotein. The phage amidase, although endowed with a signal peptide, is apparently, like Xep, exported by a holin-like mechanism which does not involve the cleavage of the signal peptide. The presence on the B. subtilis chromosome of other, similar, genes, and their possible widespread occurrence, is discussed.

The gene of the N-acetylglucosaminidase, a Bacillus subtilis 168 cell wall hydrolase not involved in vegetative cell autolysis.

lytD, the structural gene of the Bacillus subtilis 168 N-acetylglucosaminidase was localized at 310 degrees, next to the tagABC operon. Sequence analysis revealed a monocistronic operon encoding a 95.6 kDa protein endowed with an export signal, the cleavage of which yields the monomer polypeptide (92.8 kDa) of the dimeric active form of the enzyme. Transcription is initiated at a sigma-D (sigma D)-dependent promoter and ends at a terminator common to lytD and the divergently transcribed tagABC operon. In addition, we report the sequence of the adjacent upstream ORF, transcribed in the same direction as lytD, which shows significant homology to phosphomannose isomerase-encoding genes. Cell separation, motility, autolysis, cell wall turnover and growth were not affected in strains devoid of the N-acetylglucosaminidase. A mutant deficient in the two most abundant autolysins, i.e. the LytC amidase and the glucosaminidase, exhibited the phenotype of the amidase-deficient strains, revealing their non-requirement for growth. This conclusion raises two fundamental questions: how does the cell undo the highly cross-linked peptidoglycan so as to be able to grow, and what is the role of the considerable amount of autolysin normally present? Possible answers to these questions are discussed.

The gerB region of the Bacillus subtilis 168 chromosome encodes a homologue of the gerA spore germination operon.

Spores of gerB spore germination mutants of Bacillus subtilis 168 are defective in response to the germinative mixture of L-asparagine, glucose, fructose and potassium ions (AGFK), but are normal in the L-alanine (ALA) triggered germination response. A lambda clone of 15 kbp carrying the gerB region has been identified. Sequencing of the gerB region of the clone revealed a cluster of three ORFs encoding putative proteins of 53.3, 41.3 and 42.4 kDa (GerBA, GerBB and GerBC, respectively). The first two of these proteins have substantial hydrophobic regions and the third is a possible lipoprotein. At least two, and probably all three products are required for normal germination in AGFK. The three proteins form a set of homologues of the products of the gerA operon, mutations in which cause a defect in the ALA germination pathway, but cause no defect in AGFK. The GerB proteins show 42%, 31% and 35% identity at the amino-acid level to the corresponding GerA proteins, and the homologues occur in the same order in both operons.

Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units.

Insertional mutagenesis has revealed that a 22 kbp segment from the hisA region of the Bacillus subtilis 168 chromosome (310 degrees on the genetic map) contains at least six independent transcription units, all apparently devoted to production of cell envelope components. Genes concerned with synthesis of poly(glycerol phosphate), poly(groP), an essential cell wall polymer in B. subtilis 168, are organized in two divergently transcribed operons denoted tagABC and tagDEF. Nucleotide sequence analysis indicates that three of these six genes encode extremely basic polypeptides. The deduced products of the tagABC operon may be involved in poly(groP) assembly and export, whereas those of the tagDEF operon, which are very hydrophilic, are more likely to be implicated in poly(groP) precursor biosynthesis. The first gene of the tagDEF operon encodes glycerol-3-phosphate cytidylyltransferase (Pooley et al., 1991, Journal of General Microbiology 137, 921-928) and its deduced product has significant homology with cholinephosphate cytidylyltransferase from yeast. There is also substantial homology between the deduced products of tagB in the tagABC operon and tagF in the tagDEF operon.

Pseudo-allelic relationship between non-homologous genes concerned with biosynthesis of polyglycerol phosphate and polyribitol phosphate teichoic acids in Bacillus subtilis strains 168 and W23.

A 60 kbp region of the Bacillus subtilis chromosome encompassing the genes concerned with teichoic acid biosynthesis has been subjected to physical analysis. No homology was detected by Southern hybridization between DNA segments encoding the tag genes of strain 168, concerned with polyglycerol phosphate (poly(groP)) biosynthesis, and the tar genes of strain W23, concerned with polyribitol phosphate (poly-(rboP)) biosynthesis. Analysis of 168/W23 interstrain hybrids that incorporate poly(rboP) instead of poly-(groP) into their cell walls revealed that, in every case, integral substitution of the W23 tar genes for the 168 tag genes had occurred. Interstrain hybrids of the 'W23-like' type have inherited larger segments of W23 DNA than interstrain hybrids of the 'mixed' type. The tag and tar genes are located at equivalent positions on the chromosomes of strains 168 and W23, behaving, in genetic crosses, like an allelic pair. They provide the first example of a pseudo-allelic relationship between non-homologous genes in B. subtilis.

The essential nature of teichoic acids in Bacillus subtilis as revealed by insertional mutagenesis.

A 30 kb DNA segment from the region of the Bacillus subtilis strain 168 chromosome which contains most, if not all, loci specifically involved in teichoic acid biosynthesis, has been cloned. A restriction map was established to which genetic markers were assigned. Four loci, tagA, tagB, gtaA and gtaD, are located on a DNA segment of about 7 kb, whereas the gtaB locus lies some 10 kb distant. The tagA and tagB loci are apparently transcribed independently. Insertional mutagenesis, using integrational plasmids carrying relevant fragments from the tag region, provides strong evidence that biosynthesis of polyglycerol phosphate [poly(groP)], so far largely considered as a dispensable polymer, is in fact essential for growth.

Deduced polypeptides encoded by the Bacillus subtilis sacU locus share homology with two-component sensor-regulator systems.

The sacU locus has been cloned by using two independent strategies, and the presence of two open reading frames was deduced from the nucleotide sequence. Open reading frame 1 encodes a 45,000-dalton polypeptide that is similar to the products of the Salmonella typhimurium cheA and Escherichia coli cpxA genes, which act as sensory transducers. Open reading frame 2 encodes a 26,000-dalton polypeptide that is similar to a family of transcriptional activators, including the products of the Bacillus subtilis spoOA and spoOF and the E. coli ompR and dye genes. These similarities suggest that the products of the B. subtilis sacU locus form a sensor-transducer couple, which functions to relay information about specific environmental changes to the transcription apparatus.

Characterization of proteins induced by mitomycin C treatment of Bacillus subtilis.

A total of 26 polypeptides have been resolved by gel electrophoresis of purified phage PBSX, 3 of which belong to the head and the remainder to the tail. After mitomycin C treatment, synthesis of 11 additional proteins which are not found in the assembled phage particle was demonstrated, all but 4 being under the control of the phage repressor. Existence of a prehead and of a precursor of the main capsid protein (molecular weight, 35,000) suggested phage head maturation which is accompanied by cleavage of the precursor (molecular weight, 36,500). The role of induced proteins related and unrelated to PBSX is discussed. Finally, the estimated phage genome mass of 4 X 10(7) daltons exceeded by more than four times its head capacity, which could explain the defectiveness of the phage.

Prophage induction in thermosensitive DNA mutants of Bacillus subtilis.

Incubation of thermosensitive dna mutants of Bacillus subtilis at the non-permissive temperature leads in some instances to induction of defective prophage PBSX and cell lysis. A clear distinction can be made between mutants affected in DNA replication at the growing point (extension mutants) and those unable to initiate new rounds of replication (initiation mutants). The former promote PBSX induction to a variable and mutation-specific extent, whereas the latter do not exhibit any signs of induction. Analysis of mutants carrying two dna mutations suggests that products of some dna genes involved in initiation and in extension are not essential for induction but can substantially amplify its extent. However, mitomycin C treatment of dna mutants which have completed their residual DNA synthesis leads to a PBSX induction essentially identical to that obtained by mitomycin C treatment of the wild-type strain, which precludes an essential role for any of the mutated proteins in this induction process. On the basis of our observations we propose that the induction signal is related to the number of blocked replication forks: the larger that number, the higher the proportion of induced cells within the population.