RecX facilitates homologous recombination by modulating RecA activities.

The Bacillus subtilis recH342 strain, which decreases interspecies recombination without significantly affecting the frequency of transformation with homogamic DNA, carried a point mutation in the putative recX (yfhG) gene, and the mutation was renamed as recX342. We show that RecX (264 residues long), which shares partial identity with the Proteobacterial RecX (<180 residues), is a genuine recombination protein, and its primary function is to modulate the SOS response and to facilitate RecA-mediated recombinational repair and genetic recombination. RecX-YFP formed discrete foci on the nucleoid, which were coincident in time with RecF, in response to DNA damage, and on the poles and/or the nucleoid upon stochastic induction of programmed natural competence. When DNA was damaged, the RecX foci co-localized with RecA threads that persisted for a longer time in the recX context. The absence of RecX severely impaired natural transformation both with plasmid and chromosomal DNA. We show that RecX suppresses the negative effect exerted by RecA during plasmid transformation, prevents RecA mis-sensing of single-stranded DNA tracts, and modulates DNA strand exchange. RecX, by modulating the "length or packing" of a RecA filament, facilitates the initiation of recombination and increases recombination across species.

Bacillus subtilis hlpB encodes a conserved stand-alone HNH nuclease-like protein that is essential for viability unless the hlpB deletion is accompanied by the deletion of genes encoding the AddAB DNA repair complex.

The HNH domain is found in many different proteins in all phylogenetic kingdoms and in many cases confers nuclease activity. We have found that the Bacillus subtilis hlpB (yisB) gene encodes a stand-alone HNH domain, homologs of which are present in several bacterial genomes. We show that the protein we term HlpB is essential for viability. The depletion of HlpB leads to growth arrest and to the generation of cells containing a single, decondensed nucleoid. This apparent condensation-segregation defect was cured by additional hlpB copies in trans. Purified HlpB showed cooperative binding to a variety of double-stranded and single-stranded DNA sequences, depending on the presence of zinc, nickel, or cobalt ions. Binding of HlpB was also influenced by pH and different metals, reminiscent of HNH domains. Lethality of the hlpB deletion was relieved in the absence of addA and of addAB, two genes encoding proteins forming a RecBCD-like end resection complex, but not of recJ, which is responsible for a second end-resectioning avenue. Like AddA-green fluorescent protein (AddA-GFP), functional HlpB-YFP or HlpB-FlAsH fusions were present throughout the cytosol in growing B. subtilis cells. Upon induction of DNA damage, HlpB-FlAsH formed a single focus on the nucleoid in a subset of cells, many of which colocalized with the replication machinery. Our data suggest that HlpB plays a role in DNA repair by rescuing AddAB-mediated recombination intermediates in B. subtilis and possibly also in many other bacteria.

The three-layered DNA uptake machinery at the cell pole in competent Bacillus subtilis cells is a stable complex.

Many bacteria possess the ability to actively take up DNA from the environment and incorporate it into the chromosome. RecA protein is the key protein achieving homologous recombination. Several of the proteins involved in the transport of DNA across the cell envelope assemble at a single or both cell poles in competent Bacillus subtilis cells. We show that the presumed structure that transports DNA across the cell wall, the pseudopilus, also assembles at a single or both cell poles, while the membrane receptor, ComEA, forms a mobile layer throughout the cell membrane. All other known Com proteins, including the membrane permease, localize again to the cell pole, revealing that the uptake machinery has three distinct layers. In cells having two uptake machineries, one complex is occasionally mobile, with pairs of proteins moving together, suggesting that a complete complex may lose anchoring and become mobile. Overall, the cell pole provides stable anchoring. Only one of two uptake machineries assembles RecA protein, suggesting that only one is competent for DNA transfer. FRAP (fluorescence recovery after photobleaching) analyses show that in contrast to known multiprotein complexes, the DNA uptake machinery forms a highly stable complex, showing little or no exchange with unbound molecules. When cells are converted into round spheroplasts, the structure persists, revealing that the assembly is highly stable and does not require the cell pole for its maintenance. High stability may be important to fulfill the mechanical function in pulling DNA across two cell layers.

Lambda Red-mediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.