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Bacteriophages are able to hijack host essential machineries to benefit their fitness and assemble their own progeny. Phage proteins targeting major bacterial pathways can be powerful tools to understand cell functions and have possible applications in human health and industry. Bacterial genomes also harbor cryptic prophages carrying genes that may contribute to their host fitness and properties. The cryptic prophages are mostly transcriptionally silent and most of the functions they encode are not annotated. In B. subtilis , the 48 kb-long skin element is a prophage carrying the yqaF-yqaN operon, which is tightly regulated by the Xre-like repressor sknR . The small yqaH gene potentially encodes the protein YqaH in absence of SknR. It was previously reported that YqaH interacts with the replication initiator DnaA in yeast two-hybrid assay and its expression in B. subtilis causes defects in the chromosomal cycle. In this study, we report that, in addition to DnaA, YqaH interacts with Spo0A, a master regulator of sporulation. To decipher yqaH mode of action, we used the yeast two-hybrid to isolate single mutations in yqaH that separate interactions with DnaA and Spo0A. We isolated mutations that caused loss-of-interaction (LOI) with DnaA but not Spo0A. However, all mutations disrupting the interaction with Spo0A were also DnaA-LOI functions, suggesting that these functions could not be separated. We found that expression YqaH carrying DnaA-LOI mutations affects both chromosome integrity and DnaA-mediated transcription, leading to growth inhibition as well as preventing bacterial development such as sporulation and biofilm formation. These results show that YqaH acts as an antimicrobial peptide in B. subtilis and pave the way for the structural design of mutants with improved antibacterial action.

In most firmicutes expression of the mannitol operon is regulated by MtlR. This transcription activator is controlled via phosphorylation of its regulatory domains by components of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). We found that activation of Bacillus subtilis MtlR also requires an interaction with the EIIBMtl domain of the mannitol permease MtlA (EIICBMtl). The constitutive expression of the mtlAFD operon in an mtlF mutant was prevented when entire mtlA or only its 3 part (EIIBMtl) were deleted. Yeast two-hybrid experiments revealed a direct interaction of the EIIBMtl domain with the two C-terminal domains of MtlR. Complementation of the 3-mtlA mtlF or mtlAFD mutants with mtlA restored constitutive MtlR activity, whereas complementation with only 3-mtlA had no effect. Moreover, synthesis of EIIBMtl in strains producing constitutively active MtlR caused MtlR inactivation. Interestingly, EIIBMtl fused to the trans-membrane protein YwqC restored constitutive MtlR activity in the above mutants. Replacing the phosphorylatable Cys with Asp in MtlA or soluble EIIBMtl lowered MtlR activation, indicating that MtlR does not interact with phosphorylatyed EIIBMtl. Induction of the B.subtilis mtl operon therefore follows a novel regulation mechanism where the transcription activator needs to be sequestered to the membrane by unphosphorylated EIICBMtl in order to be functional.

Since 1996, induction of competence for genetic transformation of Streptococcus pneumoniae is known to be controlled by the ComD/ComE two-component regulatory system. The mechanism of induction is generally described as involving ComD autophosphorylation, transphosphorylation of ComE and transcriptional activation by ComE similar to P of the early competence (com) genes, including comX which encodes the competence-specific sigma(X). However, none of these features has been experimentally established. Here we document the autokinase activity of ComD proteins in vitro, and provide an estimate of the stoichiometry of ComD and ComE in vivo. We report that a phosphorylmimetic mutant, ComE(D58E), constructed because of the failure to detect transphosphorylation of purified ComE in vitro, displays full spontaneous competence in Delta comD cells, an that in vitro ComE(D58E) exhibits significantly improved binding affinity for P-comCDE. We also provide evidence for a differential transcriptional activation and repression of P-comCDE and P-comX. Altogether, these data support the model of ComE similar to P-dependent activation of transcription. Finally, we establish that ComE antagonizes expression of the early com genes and propose that the rapid deceleration of transcription from P-comCDE observed even in cells lacking sigma(X) is due to the progressive accumulation of ComE, which outcompetes ComE similar to P.

International audience; Eubacteria have evolved multicomponent protein machines, termed replisomes, which duplicate their chromosomes rapidly and accurately. Extensive studies in the model bacteria Escherichia coli and Bacillus subtilis have revealed that in addition to the replication core machinery, other proteins are necessary to form a functional replication fork. Specific subsets of proteins mediate: a) the assembly of the replisome at the chromosomal origin of replication [initiation]; b) the progression of the replication forks along the chromosome [elongation] and their maintenance by providing solutions for replication restart, which are adapted to overcome possible 'roadblocks' encountered on the DNA template; and c) the physiological arrest of replication when chromosome duplication is completed [termination]. Within the cell, DNA replication takes place within a factory positioned at the cell centre. This review summarises recent knowledge about chromosomal replication in Bacillus subtilis and related Gram-positive bacteria. It is focused on the events governing the assembly and the fate of the replication fork, describes protein networks connected with the replisome, and emphasises several novel aspects of DNA replication in this group of bacteria.

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The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases that includes the human Bloom's syndrome and Werner's syndrome proteins. In this work, we report studies on the interaction between Sgs1 and DNA topoisomerase III in vitro and in vivo. Affinity chromatography experiments with various fragments of Sgs1, a 1447-amino acid polypeptide, suggested that its N-terminal one-fifth was sufficient for interaction with DNA topoisomerase III. Gel electrophoretic mobility shift assays also indicated that a fragment Sgs1(1-283), containing residues 1-283, inhibited the binding of DNA topoisomerase III to single-stranded DNA. A shorter protein fragment containing residues 1-107 also showed partial inhibition in these assays. Studies of a sgs1 top1 double mutant lacking both Sgs1 and DNA topoisomerase I showed that the slow growth phenotype of this double mutant is suppressed by expressing full-length Sgs1, but not Sgs1 without the N-terminal 107 amino acid residues. In sgs1 top3 cells devoid of DNA topoisomerase III, however, expression of full-length Sgs1 or Sgs1 lacking the N-terminal 107 amino acid residues has the same effect of reducing the growth rate of the double mutant. These in vitro and in vivo data indicate that Sgs1 and DNA topoisomerase III physically interact and that this interaction is physiologically significant.

International audience; In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes.

The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pIJ101, pSB24, and pJV1.