A DNA-dependent ATPase from Bacillus subtilis.

A DNA-dependent ATPase (molecular weight 68000) has been purified from extracts of $\text{B. subtilis}$. The enzyme shows specificity for single-stranded DNA and for hydrolysis of ATP (Km 0.4 mM). Similarities with the $\text{rep}$ gene product from $\text{E.coli}$ are discussed.     

Bacillus subtilis SecA ATPase exists as an antiparallel dimer in solution

SecA ATPase promotes the biogenesis of membrane and secretory proteins into and across the cytoplasmic membrane of Eubacteria. SecA binds to translocon component SecYE and substrate proteins and undergoes ATP-dependent conformational cycles that are coupled to the stepwise translocation of proteins. Our recent crystal structure of B. subtilis SecA [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] showed two different dimer interactions in the lattice which both buried significant solvent-accessible surface area in their interface and could potentially be responsible for formation of the physiological dimer in solution. In this paper, we utilize fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues to determine the oligomeric structure of SecA protein in solution. Our results show that of the two dimers interactions observed in the crystal structure, SecA forms an antiparallel dimer in solution that maximizes the buried solvent-accessible surface area and intermolecular contacts. At the submicromolar protein concentrations used in the fluorescence experiments, we saw no evidence for the formation of higher-order oligomers of SecA based on either the alternative dimer or the 3(1) helical fiber observed in the crystal lattice. Our studies are consistent with previous ones demonstrating the existence of a dimerization determinant within the C-domain of SecA as well as those documenting the interaction of N- and C-domains of SecA. Our results also provide a valuable starting point for a determination of whether the subunit status of SecA changes during the protein translocation as well as studies designed to elucidate the conformational dynamics of this multidomain protein during its translocation cycle.     

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.     

Different nuclease activities in competent and noncompetent Bacillus subtilis.

Competent and noncompetent cells of Bacillus subtilis were separated on the basis of their different buoyant densities. The two types of cells were compared with respect to their interactions with exogenous deoxyribonucleic acid(DNA). After exposure of DNA to the cells, the unadsorbed fraction of DNA molecules was examined. Both types of cells decreased the biological activity of this DNA, the inactiviation exerted by noncompetent cells being more severe than that exerted by competent cells. Sedimentation analysis of the inactivated DNA revealed that fragments of DNA are produced, owing mainly to the introduction of double-strand scissions. In addition to this fragmentation, the competent bacteria extensively digested the DNA exonucleolytically. This type of breakdown was specifically related to the competent state rather than to the state of low density. The exonucleolytic activity is, in all probability, associated with the cell envelope, because most of the activity is released into the medium when the cells are converted to protoplasts. At 37 C the competence-specific exonucleolytic breakdown started 2 to 3 min after the binding of DNA to the cells. In unfractionated cultures, breakdown may proceed until 70% of the total amount of DNA added has been made acid soluble. Nontransforming Escherichia coli DNA was also subject to exonucleolytic degradation; it seems unlikely,therefore, that this type of breakdown occurs as a consequence of recombination. Since ethylenediaminetetraacetate blocked both transformation by native DNA and the exonucleolytic breakdown of bound DNA, we suggest that the breakdown of DNA by competent cells fulfills an essential function in genetic transformation of B. subtilis.     

CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA

The structure of the hypothetical copper-metallochaperone CopZ from Bacillus subtilis and its predicted partner CopA have been studied but their respective contributions to copper export, -import, -sequestration and -supply are unknown. DeltacopA was hypersensitive to copper and contained more copper atoms cell(-1) than wild-type. Expression from the copA operator-promoter increased in elevated copper (not other metals), consistent with a role in copper export. A bacterial two-hybrid assay revealed in vivo interaction between CopZ and the N-terminal domain of CopA but not that of a related transporter, YvgW, involved in cadmium-resistance. Activity of copper-requiring cytochrome caa(3) oxidase was retained in deltacopZ and deltacopA. DeltacopZ was only slightly copper-hypersensitive but deltacopZ/deltacopA was more sensitive than deltacopA, implying some action of CopZ that is independent of CopA. Significantly, deltacopZ contained fewer copper atoms cell(-1) than wild-type under these conditions. CopZ makes a net contribution to copper sequestration and/or recycling exceeding any donation to CopA for export.     

The ATPase SpoIIIE transports DNA across fused septal membranes during sporulation in Bacillus subtilis

The FtsK/SpoIIIE family of ATP-dependent DNA transporters mediates proper chromosome segregation in dividing bacteria. In sporulating Bacillus subtilis cells, SpoIIIE translocates much of the circular chromosome from the mother cell into the forespore, but the molecular mechanism remains unclear. Using a new assay to monitor DNA transport, we demonstrate that the two arms of the chromosome are simultaneously pumped into the forespore. Up to 70 molecules of SpoIIIE are recruited to the site of DNA translocation and assemble into complexes that could contain 12 subunits. The fusion of the septal membranes during cytokinesis precedes DNA translocation and does not require SpoIIIE, as suggested by analysis of lipid dynamics, serial thin-section electron microscopy, and cell separation by protoplasting. These data support a model for DNA transport in which the transmembrane segments of FtsK/SpoIIIE form linked DNA-conducting channels across the two lipid bilayers of the septum.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

A manganese-stimulated endonuclease from Bacillus subtilis

An endonuclease activity has been identified in extracts of $\text{Bacillus subtilis}$. This activity is stimulated by Mn⁺⁺ or Ca⁺⁺ ions but not by Mg⁺⁺ ions. The enzyme catalyzes the breakdown of native DNA of high molecular weight to fragments of molecular weights ranging from 3 × 10⁶ to 20 × 10⁶. A variety of DNA's from sources such as $\text{B. subtilis, Salmonella}$ and T7 phage are attacked. About 61% of the activity of the cells is released into the medium during protoplast formation under conditions where 98% of the glucose 6-P dehydrogenase activity is retained by the cells.     

A Love Affair with Bacillus subtilis

My career in science was launched when I was an undergraduate at Princeton University and reinforced by graduate training at the Massachusetts Institute of Technology. However, it was only after I moved to Harvard University as a junior fellow that my affections were captured by a seemingly mundane soil bacterium. What Bacillus subtilis offered was endless fascinating biological problems (alternative sigma factors, sporulation, swarming, biofilm formation, stochastic cell fate switching) embedded in a uniquely powerful genetic system. Along the way, my career in science became inseparably interwoven with teaching and mentoring, which proved to be as rewarding as the thrill of discovery.     

Expression of a P-type Ca(2+)-transport ATPase in Bacillus subtilis during sporulation

The open reading frame designated yloB in the genomic sequence of Bacillus subtilis encodes a putative protein that is most similar to the typically eukaryotic type IIA family of P-type ion-motive ATPases, including the endo(sarco)plasmic reticulum (SERCA) and PMR1 Ca(2+)-transporters, located respectively in the SERCA and the Golgi apparatus. The overall amino acid sequence is more similar to that of the Pmr1s than to the SERCAs, whereas the inverse is seen for the 10 amino acids that form the two Ca(2+)-binding sites in SERCA. Sporulating but not vegetative B. subtilis cells express the predicted protein, as shown by Western blotting and by the formation of a Ca(2+)-dependent phosphorylated intermediate. Half-maximal activation of phosphointermediate formation occurred at 2.5 microM Ca(2+). Insertion mutation of the yloB gene did not affect the growth of vegetative cells, did not prevent the formation of viable spores, and did not significantly affect 45Ca accumulation during sporulation. However, spores from knockouts were less resistant to heat and showed a slower rate of germination. It is concluded that the P-type Ca(2+)-transport ATPase from B. subtilis is not essential for survival, but assists in the formation of resistant spores. The evolutionary relationship of the transporter to the eukaryotic P-type Ca(2+)-transport ATPases is discussed.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.