The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication

The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA. We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins. This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B. subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.     

The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion

A weak Ca²⁺-binding site (K_a= 0.8× 10³ M^?1, at pH7) was identified in the mature part of levansucrase. An amino acid substitution (Thr-236 →lle) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca²⁺ (>2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium-binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process.     

Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene

We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241-248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.     

Cosegregation of cell wall and DNA in Bacillus subtilis.

Cosegregation of cell wall and DNA of a lysis-negative mutant of Bacillus subtilis was examined by continuously labeling (i) cell wall, (ii) DNA, and (iii) both cell wall and DNA. After four to five generations of chase in liquid media it was found by light microscope autoradiography that the numbers of wall segregation units per cell are 29 and 9 in rich and minimal medium, respectively. Under the same conditions the numbers of segregation units of DNA were almost 50% lower: 15 and 5, respectively. Simultaneous labeling of cell wall and DNA (iii) provided figures almost identical to those obtained for cell wall alone, (i), implying cosegregation of the two components. Statistical analysis ruled out their random distribution into daughter cells. Measurements of the positions of grain clusters at the end of the chase period along chains of cells, each derived from a single cell at the beginning of chase, show that cell wall units are localized according to a symmetrical pattern, whereas those of DNA are distributed in an asymmetrical but highly regular way. It appears that of two cell wall units of the same age one only has a strand of DNA attached to it. We present a simple diagrammatic model of cell wall organization and DNA-cell wall association which is compatible with our observations. Finally, we discuss previous experiments pertinent to cosegregation of cell wall and DNA obtained with cells grown on solid media as well as with germinating spores; an explanation for the independent segregation of cell wall and DNA observed in the latter case is advanced.     

The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis

Adaptation of bacteria to the prevailing environmental and nutritional conditions is often mediated by two-component signal transduction systems (TCS). The Bacillus subtilis YycFG TCS has attracted special attention as it is essential for viability and its regulon is poorly defined. Here we show that YycFG is a regulator of cell wall metabolism. We have identified five new members of the YycFG regulon: YycF activates expression of yvcE, lytE and ydjM and represses expression of yoeB and yjeA. YvcE(CwlO) and LytE encode endopeptidase-type autolysins that participate in peptidoglycan synthesis and turnover respectively. We show that a yvcE lytE double mutant strain is not viable and that cells lacking LytE and depleted for YvcE exhibit defects in lateral cell wall synthesis and cell elongation. YjeA encodes a peptidoglycan deacetylase that modifies peptidoglycan thereby altering its susceptibility to lysozyme digestion and YdjM is also predicted to have a role in cell wall metabolism. A genetic analysis shows that YycFG essentiality is polygenic in nature, being a manifestation of disrupted cell wall metabolism caused by aberrant expression of a number of YycFG regulon genes.     

MinJ (YvjD) is a topological determinant of cell division in Bacillus subtilis

In Bacillus subtilis, FtsZ ring formation and cell division is favoured at the midcell because the inhibitor proteins MinC and MinD are indirectly restricted to the cell poles by the protein DivIVA. Here we identify MinJ, a topological determinant of medial FtsZ positioning that acts as an intermediary between DivIVA and MinD. Due to unrestricted MinD activity, cells mutated for minJ exhibited pleiotropic defects in homologous recombination, swarming motility and cell division. MinJ restricted MinD activity by localizing MinD to the cell poles through direct protein-protein interaction. MinJ itself localized to cell poles in a manner that was dependent on DivIVA. MinJ is conserved in other low G+C Gram-positive bacteria and may be an important component of cell division site selection in these organisms.     

Characterization of a combined DNA initiation and cell division mutant of Bacillus subtilis.

Summary The temperature-sensitive mutation in Bacillus subtilis 168-134ts, a conditional lethal DNA initiation mutant, was transferred to the minicell producing strain, CU 403 div IV-B1, to study he relationship of DNA synthesis to cell division. Markers in the combined mutant were verified by transduction. DNA replication kinetics, genome location by autoradiography, and clonal analysis of cell division patterns during spore outgrowths were investigated. Growth of the double mutant at the restrictive temperature results in an impressive reduction of the percentage cell length covered by DNA grain clusters (60.2% at 30° C compared to 8.6% after 2 h at 45° C). The probability of a minicell producing division in double mutant clones is essentially the same at 30° C and during the initial 2–3 h growth at 45° C at which time lysis begins. Residual division at 45° C is attributable to processes initiated at 30° C. The CU 403 div IV-B1, 134ts, double mutant divides about 25% as frequently relative to growth as do wild type CU 403 clones when incubated at permissive temperature. This is approximately 15% greater division suppression than previously found in the CU 403 div IV-B1 mutant strain, and is presumably due to interactions of the mutant gene products both of which affect DNA.     

Purification and characterization of a cell wall hydrolase encoded by the cw1A gene of Bacillus subtilis

A cell wall hydrolase of Bacillus subtilis was prepared from Escherichia coli cells harboring a plasmid containing the B. subtilis cwlA gene and purified by hydroxyapatite column chromatography and HPLC through TSK-gel G3000SWXL. In contrast to the molecular mass of 29,919 Da deduced from its nucleotide sequence, the purified CWLA is a 23 kDa protein. Characterization of the specific substrate bond cleaved by CWLA indicated the enzyme is an N-acetylmuramyl-L-alanine amidase. A 32-kDa precursor protein was detected on zymography of a crude cell homogenate. Some of the enzymatic properties of CWLA are also described.     

Characterization of the essential cell division gene ftsL (yllD ) of Bacillus subtilis and its role in the assembly of the division apparatus

We have identified the Bacillus subtilis homologue of the essential cell division gene, ftsL , of Escherichia coli . Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of cell division. The filaments show no sign of invagination, indicating that division is blocked at an early stage. FtsL is also shown to be required for septation during sporulation, and depletion of FtsL blocks the activation but not the synthesis of the prespore-specific sigma factor, σ^F. Immunofluorescence microscopy shows that depletion of FtsL has little or no effect on FtsZ ring formation, but the assembly of other division proteins, DivIB and DivIC, at the site of division is prevented. Repression of FtsL also results in a rapid loss of DivIC protein, indicating that DivIC stability is dependent on the presence of FtsL, in turn suggesting that FtsL is intrinsically unstable. The instability of one or more components of the division apparatus may be important for the cyclic assembly/disassembly of the division apparatus.     

Peptidoglycan hydrolase LytF plays a role in cell separation with CwlF during vegetative growth of Bacillus subtilis.

Peptidoglycan hydrolase, LytF (CwlE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the yhdD gene. YhdD exhibits high sequence similarity with CwlF (PapQ, LytE) and p60 of Listeria monocytogenes. The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues. The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity. The histidine-tagged LytF protein produced in Escherichia coli cells hydrolyzed the linkage of D-gamma-glutamyl-meso-diaminopimelic acid in murein peptides, indicating that it is a D,L-endopeptidase. Northern hybridization and primer extension analyses indicated that the lytF gene was transcribed by EsigmaD RNA polymerase. Disruption of lytF led to slightly filamentous cells, and a lytF cwlF double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the cwlF sigD mutant.     

Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division

Bacillus subtilis sporulation is a last-resort phenotypical adaptation in response to starvation. The regulatory network underlying this developmental pathway has been studied extensively. However, how sporulation initiation is concerted in relation to the environmental nutrient availability is poorly understood. In a fed-batch fermentation set-up, in which sporulation of ultraviolet (UV)-mutagenized B. subtilis is repeatedly triggered by periods of starvation, fitter strains with mutated tagE evolved. These mutants display altered timing of phenotypical differentiation. The substrate for the wall teichoic acid (WTA)-modifying enzyme TagE, UDP-glucose, has recently been shown to be an intracellular proxy for nutrient availability, and influences the timing of cell division. Here we suggest that UDP-glucose also influences timing of cellular differentiation.     

Influence of the nucleoid and the early stages of DNA replication on positioning the division site in Bacillus subtilis

Although division site positioning in rod‐shaped bacteria is generally believed to occur through the combined effect of nucleoid occlusion and the Min system, several lines of evidence suggest the existence of additional mechanisms. Studies using outgrown spores of Bacillus subtilis have shown that inhibiting the early stages of DNA replication, leading up to assembly of the replisome at oriC, influences Z ring positioning. Here we examine whether Z ring formation at midcell under various conditions of DNA replication inhibition is solely the result of relief of nucleoid occlusion. We show that midcell Z rings form preferentially over unreplicated nucleoids that have a bilobed morphology (lowering DNA concentration at midcell), whereas acentral Z rings form beside a single‐lobed nucleoid. Remarkably however, when the DnaB replication initiation protein is inactivated midcell Z rings never form over bilobed nucleoids. Relieving nucleoid occlusion by deleting noc increased midcell Z ring frequency for all situations of DNA replication inhibition, however not to the same extent, with the DnaB‐inactivated strain having the lowest frequency of midcell Z rings. We propose an additional mechanism for Z ring positioning in which the division site becomes increasingly potentiated for Z ring formation as initiation of replication is progressively completed.     

The membrane-bound cell division protein DivIB is localized to the division site in Bacillus subtilis

The cell division gene divIB of Bacillus subtilis is essential for the normal rate of growth and division. The gene product, DivIB, is a membrane-bound protein in which the bulk of the protein (at the C-terminal end) is on the exterior surface of the cell membrane. DivIB is involved in the early stages of septum formation, but its exact role in cell division is unknown. To gain more information about the mode of action of DivIB in septum formation, we determined the location of DivIB within the cell membrane using immunofluorescence. This immunolocalization approach established that DivIB becomes localized to the division site before visible septation and remains localized to this site throughout the division process. Various DivIB immunostaining patterns were observed in immunofluorescence experiments and, together with cell length and nucleoid distance measurements, have allowed us to propose two models to describe DivIB localization during the cell cycle.     

PBP1 is a component of the Bacillus subtilis cell division machinery

Bacillus subtilis penicillin-binding protein PBP1 has been implicated in cell division. We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum. Localization of PBP1 to the vegetative septum is dependent on various cell division proteins. This study proves that PBP1 forms part of the B. subtilis cell division machinery.     

Identification and characterization of new DNA replication terminators in Bacillus subtilis

A functional DNA replication terminator of Bacillus subtilis contains two overlapping binding sites, A and B, for the replication terminator protein (RTP). A degenerate 17-mer oligonucleotide corresponding to the consensus B site has been used to detect four new terminators in the B. subtilis chromosome, in addition to the previously identified and closely spaced IRI and IRII. All the new terminators lie in the terminus region of the chromosome, on both sides of IRI and IRII, with their positions spanning <1O% of its length. Their DNA sequences are characterized by clearly identifiable A- and B-binding sites. They bind RTP in a manner indistinguishable from IRI, although precise affinities have not been compared. Each new terminator is functional in causing fork arrest when present in a plasmid replicating in B. subtilis . Three of the four were tested for polarity in fork-arrest activity and exhibited the polarity expected. The total of six terminators now identified in B. subtilis have been named TerI-TerVI . TerI and TerII correspond to the previously identified IRI and IRII, respectively. The chromosomal orientations of all but one of the terminators ( TerIV ) have been established and they conform to an arrangement similar to that in Escherichia coli in which two opposed groups of polar terminators provide a replication-fork trap ensuring that the approaching forks meet within a restricted region of the chromosome. The development of a strikingly similar arrangement of terminators in the two organisms, despite the lack of any detectable similarity in their respective DNA terminators and terminator proteins, emphasizes the importance of the replication-fork trap in each case.