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Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ(M) and cells lacking MurJBs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.

Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acids precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria. How do bacteria distribute the use of the lipid carrier, UndP, to the various pathways that require it? This study shows that the Bacillus subtilis transcription factor sigma-M and its membrane-anchored anti-sigma factor monitor UndP levels and prioritize its use for cell wall synthesis. [ABSTRACT FROM AUTHOR]

Author(s): Megan Sjodt [1]; Kelly Brock [2]; Genevieve Dobihal [3]; Patricia D. A. Rohs [3]; Anna G. Green [2]; Thomas A. Hopf [2]; Alexander J. Meeske [3]; Veerasak Srisuknimit [4]; [...]

Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (known as steric exclusion). By contrast, large T antigen, the replicative DNA helicase of the simian virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here we use single-molecule and ensemble assays to show that large T antigen assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3'-to-5' direction. Unexpectedly, large T antigen unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the large T antigen ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative helicase mechanisms, and reveal a new level of plasticity in the interactions of replicative helicases with DNA damage., An essential step in DNA replication is the unwinding of the double helix by a DNA helicase (1,2). In general, these enzymes form a hexameric ring that encircles and translocates [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.04.044 Byline: Nora L. Sullivan (1), Kathleen A. Marquis (1), David Z. Rudner (1) Keywords: CELLBIO; MICROBIO; CELLCYCLE Abstract: Organization and segregation of replicated chromosomes are essential processes during cell division in all organisms. Similar to eukaryotes, bacteria possess centromere-like DNA sequences (parS) that cluster at the origin of replication and the structural maintenance of chromosomes (SMC) complexes for faithful chromosome segregation. In Bacillus subtilis, parS sites are bound by the partitioning protein Spo0J (ParB), and we show here that Spo0J recruits the SMC complex to the origin. We demonstrate that the SMC complex colocalizes with Spo0J at the origin and that insertion of parS sites near the replication terminus targets SMC to this position leading to defects in chromosome organization and segregation. Consistent with these findings, the subcellular localization of the SMC complex is disrupted in the absence of Spo0J or the parS sites. We propose a model in which recruitment of SMC to the origin by Spo0J-parS organizes the origin region and promotes efficient chromosome segregation. Author Affiliation: (1) Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA Article History: Received 20 August 2008; Revised 16 February 2009; Accepted 20 April 2009 Article Note: (miscellaneous) Published: May 14, 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.11.009 Byline: Briana M. Burton (1), Kathleen A. Marquis (2), Nora L. Sullivan (2), Tom A. Rapoport (1)(3), David Z. Rudner (2) Keywords: MICROBIO; CELLCYCLE; PROTEINS Abstract: 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. Author Affiliation: (1) Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA (2) Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA (3) Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA Article History: Received 16 September 2007; Revised 18 October 2007; Accepted 2 November 2007 Article Note: (miscellaneous) Published: December 27, 2007

The proteolytic activation of the mother cell transcription factor pro-[[sigma].sup.K] is controlled by a signal transduction pathway during sporulation in the bacterium Bacillus subtilis. The pro-[[sigma].sup.K] processing enzyme SpoIVFB, a membrane-embedded metalloprotease, is held inactive by two other integral membrane proteins, SpoIVFA and BofA, in the mother cell membrane that surrounds the forespore. Two signaling serine proteases, SpoIVB and CtpB, trigger pro-[[sigma].sup.K] processing by cleaving the regulatory protein SpoIVFA. The SpoIVB signal is absolutely required to activate pro-[[sigma].sup.K] processing and is derived from the forespore compartment. CtpB is necessary for the proper timing of [[sigma].sup.K] activation and was thought to be a mother cell signal. Here, we show that the ctpB gene is expressed in both the mother cell and forespore compartments but that synthesis in the forespore under the control of [[sigma].sup.G] is both necessary and sufficient for the proper timing of pro-[[sigma].sup.K] processing. We further show that SpoIVB cleaves CtpB in vitro and in vivo but that this cleavage does not appear to be necessary for CtpB activation. Thus, both signaling proteins are made in the forespore and independently target the same regulatory protein.

Gene expression late during the process of sporulation in Bacillus subtilis is governed by a multistep, signal transduction pathway involving the transcription factor [[sigma].sup.K] which is derived by regulated proteolysis from the inactive proprotein pro-[[sigma].sup.K]. Processing of pro-[[sigma].sup.k] is triggered by a signaling protein known as SpoIVB, a serine protease that contains a region with similarity to the PDZ family of protein-protein interaction domains. Here we report the discovery of a second PDZ-containing serine protease called CtpB that contributes to the activation of the pro-[[sigma].sup.K] processing pathway. CtpB is a sporulation-specific, carboxyl-terminal processing protease and shares several features with SpoIVB. We propose that CtpB acts to fine-tune the regulation of pro-[[sigma].sup.K] processing, and we discuss possible models by which CtpB influences the [[sigma].sup.K] activation pathway.

Research has been conducted on the developmental transcription factor sigma (super)K derived from precursor protein pro-sigma (super)K during sporulation process in Bacillus subtilis. Results indicate that the processing enzyme and its regulators exist in a multimeric complex localized to the surrounding the developing spore membrane.