Biomedical subjects
Publications and source records attributed to S Autret.
The MinCDE system regulates the position of the division plane in rod-shaped bacteria. New results from Escherichia coli provide insight into how this operates by showing that MinE stimulates the ATPase activity of MinD.
Spo0J and Soj belong to the ParB/ParA family of proteins involved in chromosome and plasmid segregation in bacteria. In Bacillus subtilis, Spo0J protein binds to several specific sites, parS, located on both sides of the origin of DNA replication, oriC, and apparently self-associates to form large discrete foci visible by fluorescence microscopy. Soj protein forms large 'patches' probably associated with the nucleoid, which can undergo dynamic, co-operative jumping from nucleoid to nucleoid in the presence of Spo0J. Patches of Soj protein somehow help to bring about the condensation of Spo0J foci. Soj is also a negative regulator of transcription. In the absence of Spo0J, Soj is statically distributed on each of the nucleoids in the cell and blocks the transcription of several sporulation genes. To analyse the functional interaction between Spo0J and Soj further, we have constructed and studied a collection of spo0J mutants. Most of the mutants completely prevent Spo0J from interacting with DNA. One mutation impairs the formation of compact Spo0J foci and simultaneously results in loss of Soj movement. We also isolated one spo0J mutant, in which the frequency of Soj internucleoid oscillation is highly increased. Both mutations affecting the interaction with Soj lie in the N-terminal coding part of spo0J, whereas the substitutions affecting DNA binding lie in the mid- to C-terminal coding region.
We have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively. This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus. In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP. DNA-DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (-128 kb from oriC). A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome. This LSTer2 site was further mapped to a 3.65 kb fragment overlapping the next40 probe. Remarkably, this fragment contains a 17 bp sequence (B'-1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This B'-1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting. Importantly, precise deletion of this sequence abolished the replication arrest. We propose that this modified B site is an essential constituent of the LSTer2 site. The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.
When DNA replication is interrupted in bacteria, a specific inhibitor (SfiA), a component of the SOS system, is synthesised which transiently blocks cell division. This is the prototype, dispensable, cell cycle checkpoint, essential for maximal survival under a particular stress. In contrast, no process specifically signalling the termination of chromosomal replication to activate the subsequent division event, which might be termed an essential checkpoint, has yet been demonstrated. In E coli, a specific mechanism is apparently required to reactivate replication forks blocked by damage, but its molecular basis is unclear. Induction of the stringent response, mediated by RelA via the level of ppGpp, presumably to optimise macromolecular synthesis according to the availability of nutrients, activates a control system which inhibits DNA replication in both E coli and B subtilis. In E coli, this blocks new rounds of initiation at oriC, although the mechanism is not clear. Conversely, initiation is not blocked in B subtilis, but replication is blocked apparently at a number of distinct sites 100-200 kb downstream and either side of oriC. This nutrient-dependent replicating checkpoint specifically requires RTP, the chromosomal terminator protein, and new evidence indicates that specific RTP binding sites may be involved in this post-initiation control mechanism. A similar post-initiation control mechanism appears to block replication reversibly after premature initiation in B subtilis, indicating that this system may have a dual function, limiting replication in starvation conditions and as a mechanism to compensate for premature initiations.
The approximately 10 kbp region encompassing nprB and argJ at 102 degrees on the Bacillus subtilis chromosome was sequenced, revealing 12 ORFs, four known genes (argJ, argC, ipi and nprB) and two genes, yitY and yitS, whose products respectively display significant homology with L-gulono-gamma-lactone oxidase of rat and dihydrofolate reductase of Staphylococcus aureus. The data also indicated that nprB mapped to a different position than previously published.
The nucleotide sequence of 17.3 kbp downstream of addA (98 degrees) on the Bacillus subtilis chromosome was determined. Twenty putative ORFs were identified. Three of them coincided with known B. subtilis genes, addA, sbcD and wprA. The product of four other ORFs showed similarity to SbcC of Clostridium perfringens, CotH of B. subtilis, 2-hydroxyhepta-2,4-diene-1,7-diodate isomerase of Methanococcus jannaschi and a putative ORF of Pseudomonas syringae. In addition, a sequence of 7.6 kbp downstream of citG (189 degrees) was analysed. Among 10 putative ORFs identified, two coincided with known genes, citG and mrgA, whilst three showed homology with X86780, a sensory protein kinase of Streptomyces hygroscopicus, an alkaline phosphatase regulatory protein and a hypothetical protease, YyxA, of B. subtilis.
The 25.9 kbp region upstream of nprB at 100 degrees-102 degrees on the Bacillus subtilis chromosome was sequenced. This revealed a known gene, degA, which was previously mislocated on the genetic map. A total of 29 putative ORFs were identified including a cluster of three ORFs whose products show clear homology with sulphate adenylyl pathway enzymes and, in addition, 11 ORFs whose products have one or more membrane domains, as indicated by their hydropathy profiles.
Regulation of DNA replication in Bacillus subtilis involves a post-initiation mechanism which is subject to control by the Stringent System, an essential regulatory network, mediated by the alarmone, ppGpp. In detailed studies using DNA-DNA hybridization procedures, we have now shown that, following the induction of the Stringent Response, replication is blocked downstream of the origin, on the left, close to the hut marker (-175 kb) and on the right, beyond the soft10 marker (+199 kb). In addition, we provide evidence that inhibition of replication under these conditions requires the replication terminator protein (RTP). In a mutant lacking RTP, a protein normally involved in termination of chromosomal replication through recognition of specific terminator sequences, replication continues past the sites normally blocked by the Stringent Response. These data strengthen the argument that this second level of control of DNA replication occurs at specific sites, the Strigent Terminus (STer) sites, either side of orlC. Such sites are presumably related to the sequence involved in RTP recognition at the terminus, terC. We propose that the binding of RTP must be modulated, perhaps through the action of ppGpp, to recognize post-initiation control sequences during the Stringent Response, in order to block replisome movement. This, therefore, acts as a checkpoint in chromosome elongation.