Genome engineering reveals large dispensable regions in Bacillus subtilis

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.     

Characterization of signal-sequence-coding regions selected from the Bacillus subtilis chromosome

Signal-sequence-coding regions for protein export were selected from chromosomal Bacillus subtilis DNA. The number of different signals obtained was higher than expected on the basis of known exported proteins in B. subtilis.

Most of the selected regions showed the characteristics of typical signal sequences, including a basic N-terminal region followed by a hydrophobic core and a potential signal-peptidase cleavage site.

The signal-coding regions were functionally interchangeable between the β. licheniformis -amylase and Escherichia coli TEM β-lactamase genes. In addition to the signal-sequence-coding regions, the nature of the host cells, and the mature parts of the reporter proteins contributed to the amounts of protein secreted.     

Stable gene amplification in the chromosome of Bacillus subtilis

We constructed five different structures, consisting of a genetic marker flanked by directly repeated sequences 2-4 kb long, in the Bacillus subtilis chromosome. When a selective pressure was applied amplification of the marker and one of the repeats was observed in all cases. Amplification was not detected with two markers which were not flanked by the repeated sequences. The maximum amplification level observed with the different structures varied between 5 and 50. The size of the most amplified structure corresponded to 7.5% of the chromosome. Amplification was stable upon growth of cells under non-selective conditions. Each copy of an amplified gene was expressed with equal efficiency. These results indicate that chromosomal gene amplification may be useful for constructing genetically engineered B. subtilis strains.     

Transposon-mediated restriction mapping of the Bacillus subtilis chromosome

Analysis of chromosomal DNA depends upon a knowledge of the locations of restriction sites over several thousand kilobases (kb). However determination of even a subset of these sites can be time-consuming, and it can be difficult to link genetic and physical maps. We describe here a significant improvement which can be used in concert with genetically mapped chromosomal insertions. The circular chromosome of Bacillus subtilis 168 was physically examined on contour-clamped homogeneous electric field (CHEF) gels using the restriction enzyme NotI. Restriction mapping of the 4.7-megabase (Mb) DNA was accomplished using a novel technique involving the transposon Tn917, which linked the genetic and physical maps and also significantly increased the rate at which this was performed. The DNA of 54 strains which contained Tn917 at genetically determined locations was cleaved with NotI and used to determine the approximate positions of 31 restriction fragments with sizes between 45 kb and 290 kb, totalling 3589 kb. This information should greatly assist in the construction of a more detailed map using standard methodology.     

Specific labeling of the Bacillus subtilis chromosome terminus

The deoxyribonucleic acid labeled by a procedure described previously for labeling the chromosomal terminus of B. subtilis 168 was substantially enriched for sequences homologous to bacteriophages SP beta and phi 3T, which integrate in the terminal region.     

Replication terminus of the Bacillus subtilis chromosome.

Bidirectional replication of the Bacillus subtilis chromosome terminates at a point on the circular chromosome which is symmetrically opposite to the replication origin. Since replication rates are similar in both "halves" of the chromosome, termination presumably occurs at the meeting point of the two replication forks. To investigate whether the DNA sequence of this region of the chromosome contributes to the termination event, we have determined the latest replicating region of a chromosome in which this DNA sequence is no longer symmetrically opposite to the origin. The merodiploid strain GSY1127 has a very large nontandem duplication (approximately 25% of the total chromosome length) in the left-hand half of the chromosome, so that size and symmetry of this chromosome are grossly different from those of normal strains. We have examined the replication order of genetic markers in this strain by measuring subtilis terminal marker for replication remains a terminal marker in the merodiploid, i.e., replicates later than a marker situated symmetrically opposite to the replication origin. These results were supported by replication orders determined by pulse-density transfer experiments during synchronous replication. The data obtained indicate that there is a preferred site for the termination of replication in the B. subtilis chromosome.     

Control of key metabolic intersections in Bacillus subtilis

The remarkable ability of bacteria to adapt efficiently to a wide range of nutritional environments reflects their use of overlapping regulatory systems that link gene expression to intracellular pools of a small number of key metabolites. By integrating the activities of global regulators, such as CcpA, CodY and TnrA, Bacillus subtilis manages traffic through two metabolic intersections that determine the flow of carbon and nitrogen to and from crucial metabolites, such as pyruvate, 2-oxoglutarate and glutamate. Here, the latest knowledge on the control of these key intersections in B. subtilis is reviewed.     

Stability of reiterated sequences in the Bacillus subtilis chromosome.

The instability of reiterated sequences in the Bacillus subtilis chromosome that was previously reported (M. Young, J. Gen. Microbiol. 130:1613-1621, 1984) results from the presence of a truncated pC194 replication origin together with an intact replication protein A gene in the amplified DNA. Removal of the truncated pC194 replication origin or inactivation of replication protein A stabilizes reiterated sequences, whereas provision of replication protein A in trans destabilizes them. We suggest that residual activity of protein A at the truncated replication origin generates single-stranded DNA, which stimulates recombination between repeated sequences and thus destabilizes amplified structures.     

Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.     

Integration of various plasmids into the Bacillus subtilis chromosome

Some features of integration of temperature-sensitive pE194, pGG10 and pGG20 plasmids into the Bacillus subtilis chromosome were studied. Several auxotrophic mutations were obtained using insertion of these plasmids into the chromosome. The sites of plasmids for illegitimate recombination were determined. It was shown that the integration into the Bac. subtilis chromosome is characteristic not only for the plasmid pE194 but is the property of Staphylococcus aureus plasmid pC194 and Escherichia coli pBR322 plasmid. The influence of different Bac. subtilis rec mutations on the frequency of integration was studied.     

Highly specific labeling of the Bacillus subtilis chromosome terminus

By making use of the sporulation process, the terminus region of the Bacillus subtilis chromosome has been labeled with [3H]thymine in a highly specific manner. The result achieved supports the view that B. subtilis spores contain only completed chromosomes.     

Genetic and physical maps of the Bacillus subtilis chromosome

Sequencing of the complete Bacillus subtilis chromosome revealed the presence of approximately 4100 genes, 1000 of which were previously identified and mapped by classical genetic crosses. Comparison of these experimentally determined positions to those derived from the nucleotide sequence showed discrepancies reaching up to 24 degrees (approximately 280 kb). The size of these discrepancies as a function of their position along the chromosome is not random but, apparently, reveals some periodicity. Our analyses demonstrate that the discrepancies can be accounted for by inaccurate positioning of the early reference markers with respect to which all subsequently identified loci were mapped by transduction and transformation. We conclude (i) that specific DNA sequences, such as recombination hotspots or presence of heterologous DNA, had no detectable effect on the results obtained by classical mapping, and (ii) that PBS1 transduction appears to be an accurate and unbiased mapping method in B. subtilis.     

Macromolecular synthesis in chromosome initiation mutants of Bacillus subtilis

Molecular Genetics and Genomics - Inactivation of the dna B or dna D gene product in Bacillus subtilis stimulates RNA and protein synthesis. Strains containing ts dna B and D mutations have been...     

Novel small RNA-encoding genes in the intergenic regions of Bacillus subtilis

Small, non-coding RNAs (ncRNAs) perform diverse functions in a variety of organisms, but few ncRNAs have been identified in Bacillus subtilis. To search the B. subtilis genome for genes encoding ncRNAs, we focused on 123 intergenic regions (IGRs) over 500 bp in length and analyzed expression from these regions. Seven IGRs termed bsrC, bsrD, bsrE, bsrF, bsrG, bsrH and bsrI expressed RNAs smaller than 380 nt. All small RNAs except BsrD RNA were expressed in transformed Escherichia coli cells harboring a plasmid with PCR-amplified IGRs of B. subtilis, indicating that their own promoters independently express small RNAs. Under the non-stressed condition, depletion of the genes for the small RNAs did not affect growth. Although their functions are unknown, gene expression profiles at several time points showed that most of the genes except for bsrD were expressed during the vegetative phase (4-6 h), but undetectable during the stationary phase (8 h). Mapping the 5' ends of the 6 small RNAs revealed that the genes for BsrE, BsrF, BsrG, BsrH, and BsrI RNAs are preceded by a recognition site for RNA polymerase sigma factor sigma(A). These small RNAs might lack an SD sequence and exert their actions as ncRNAs.     

Amplification of plasmid DNA inserted into the Bacillus subtilis chromosome

Amplification of plasmid pGG10 inserted into the Bacillus subtilis chromosome is described. The possibility of the 3.2 kb fragment of eucaryotic (wheat) DNA to be amplified within the bacterial genome is shown. The models explaining this phenomenon are discussed.     

Utilization of promoter regions of coliphage lambda in Bacillus subtilis

Summary In vivo studies with galactokinase monitoring system demonstrated that the coliphage lambda P_RP_L promoter regions could be utilized in B. subtilis. These promoter regions were preferentially utilized during the stationary growth phase of B. subtilis. However, these promoter regions were not controlled by the cI₈₅₇ gene at permissive or non-permissive temperature in B. subtilis, although the P _RM promoter was utilized in B. subtilis. S1-nuclease mapping suggests that B. subtilis RNA polymerase recognizes specific sequences in P _R promoter region that is not utilized in E. coli.     

Characterization of the major citrate synthase of Bacillus subtilis.

The major citrate synthase of Bacillus subtilis (CS-II) was purified to near homogeneity and shown to correspond to the product of the citZ gene. Accumulation of CS-II during exponential growth and stationary phases paralleled expression of the citZ gene. The physical and kinetic properties of CS-II were similar to those of citrate synthase enzymes from Bacillus megaterium and from eukaryotic cells but differed from those of citrate synthases from many gram-negative bacteria.     

Division septation in the absence of chromosome termination in Bacillus subtilis.

Germinating spores of the temperature-sensitive DNA initiation mutants of Bacillus subtilis, TsB134 and dna-1(Ts), were allowed to undergo a single round of replication by shifting to the restrictive temperature shortly after its initiation. To monitor the progress of the round 5-bromouracil was added at various times and DNA extracted after a further time, sufficient to allow completion of the chromosome. Average replication was measured from the relative amounts of LL and LH material in Cs₂SO₄ gradients. The replication state of origin (purA), intermediate (leuA) and terminus (metB) markers at the times of 5-bromouracil addition were obtained from genetic analysis of the density species fractionated in gradients of CsCl.The DNA replication inhibitor, 6-(p-hydroxyphenylazo)-uracil (HPUra), was added at various stages of the single round and the outgrown cells examined at later times for the frequency and type of septation. Under the conditions of the experiment, central division septation was blocked if HPUra (20 μm) was added before 70% (approximately) of the chromosome was replicated. Using higher concentrations of HPUra, 40 and 100 μm, it was shown that central division septation would occur at about its normal time if replication was blocked after this 70% stage but before termination. In these circumstances there was a distinct tendency for the DNA to remain close to the septum on both sides of it. The B. subtilis spore contains a single chromosome, which means that the central septum that forms in the absence of termination must pass through a partially completed chromosome. Electron microscopic evidence for such a situation has already been described (Van Iterson &; Aten, 1976). It is concluded that, at least under the restrictive conditions of the present experiments, termination of chromosome replication is not obligatory for the formation of the division septum with which it is normally coupled.