SepF supports the recruitment of the DNA translocase SftA to the Z-ring

In many bacteria, cell division begins before the sister chromosomes are fully segregated. Specific DNA translocases ensure that the chromosome is removed from the closing septum, such as the transmembrane protein FtsK in Escherichia coli. Bacillus subtilis contains two FtsK homologues, SpoIIIE and SftA. SftA is active during vegetative growth whereas SpoIIIE is primarily active during sporulation and pumps the chromosome into the spore compartment. FtsK and SpoIIIE contain several transmembrane helices, however, SftA is assumed to be a cytoplasmic protein. It is unknown how SftA is recruited to the cell division site. Here we show that SftA is a peripheral membrane protein, containing an N-terminal amphipathic helix that reversibly anchors the protein to the cell membrane. Using a yeast two-hybrid screen we found that SftA interacts with the conserved cell division protein SepF. Based on extensive genetic analyses and previous data we propose that the septal localization of SftA depends on either SepF or the cell division protein FtsA. Since SftA seems to interfere with the activity of SepF, and since inactivation of SepF mitigates the sensitivity of a ∆sftA mutant for ciprofloxacin, we speculate that SftA might delay septum synthesis when chromosomal DNA is in the vicinity

Metabolic and chromosomal changes in a <i>Bacillus subtilis whiA</i> mutant

The conserved protein WhiA is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motif and is a transcription regulator of the key cell division gene ftsZ in actinomycetes. In Bacillus subtilis, the absence of WhiA influences both cell division and chromosome segregation; however, the protein does not regulate any gene involved in these processes. In this study, we addressed three alternative mechanisms by which WhiA might exert its activity in B. subtilis and examined whether WhiA influences either (i) central carbon metabolism, (ii) fatty acid composition of the cell membrane, or (iii) chromosome organization. Mutations in glycolytic enzymes have been shown to influence both cell division and DNA replication. To measure the effect of WhiA on carbon metabolism, we tested different carbon sources and measured exometabolome fluxes. This revealed that the absence of WhiA does not affect glycolysis but does influence the pool of branched-chain fatty acid precursors. Due to the effect of WhiA on chromosome segregation, we examine chromosome organization in a ∆whiA mutant using chromosome conformation capture (Hi-C) analysis. This revealed a local reduction in short-range chromosome interactions. Together, these findings provide new avenues for future research into how this protein works in the non-actinomycete firmicutes

Cell Size and the Initiation of DNA Replication in Bacteria

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ∼30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA

When simple sequence comparison fails: the cryptic case of the shared domains of the bacterial replication initiation proteins DnaB and DnaD

DnaD and DnaB are essential DNA-replication-initiation proteins in low-G+C content Gram-positive bacteria. Here we use sensitive Hidden Markov Model-based techniques to show that the DnaB and DnaD proteins share a common structure that is evident across all their structural domains, termed DDBH1 and DDBH2 (DnaD DnaB Homology 1 and 2). Despite strong sequence divergence, many of the DNA-binding and oligomerization properties of these domains have been conserved. Although eluding simple sequence comparisons, the DDBH2 domains share the only strong sequence motif; an extremely highly conserved YxxxIxxxW sequence that contributes to DNA binding. Sequence alignments of DnaD alone fail to identify another key part of the DNA-binding module, since it includes a poorly conserved sequence, a solvent-exposed and somewhat unstable helix and a mobile segment. We show by NMR, in vitro mutagenesis and in vivo complementation experiments that the DNA-binding module of Bacillus subtilis DnaD comprises the YxxxIxxxW motif, the unstable helix and a portion of the mobile region, the latter two being essential for viability. These structural insights lead us to a re-evaluation of the oligomerization and DNA-binding properties of the DnaD and DnaB proteins

The Binary Protein Interactome of Treponema pallidum – The Syphilis Spirochete

Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this “minimal” bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions (“interologs”) for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step

The beta-propeller protein YxaL increases the processivity of the PcrA helicase

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Dynamics of Aspen Roots Colonization by Pseudomonads Reveals Strain-Specific and Mycorrhizal-Specific Patterns of Biofilm Formation

Rhizosphere-associated Pseudomonas fluorescens are known plant growth promoting (PGP) and mycorrhizal helper bacteria (MHB) of many plants and ectomycorrhizal fungi. We investigated the spatial and temporal dynamics of colonization of mycorrhizal and non-mycorrhizal Aspen seedlings roots by the P. fluorescens strains SBW25, WH6, Pf0-1, and the P. protegens strain Pf-5. Seedlings were grown in laboratory vertical plates systems, inoculated with a fluorescently labeled Pseudomonas strain, and root colonization was monitored over a period of 5 weeks. We observed unexpected diversity of bacterial assemblies on seedling roots that changed over time and were strongly affected by root mycorrhization. P. fluorescens SBW25 and WH6 stains developed highly structured biofilms with internal void spaces forming channels. On mycorrhizal roots bacteria appeared encased in a mucilaginous substance in which they aligned side by side in parallel arrangements. The different phenotypic classes of bacterial assemblies observed for the four Pseudomonas strains were summarized in a single model describing transitions between phenotypic classes. Our findings also reveal that bacterial assembly phenotypes are driven by interactions with mucilaginous materials present at roots