Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.Cysteine thiols in proteins fulfill an important and diverse set of cellular functions. In particular, they participate in enzymatic catalysis; in metal coordination, such as in the generation of Fe-S-clusters; and in determining the spatial structure of proteins via disulfide bond formation (3, 22, 23, 38). Cysteines are strong nucleophiles amenable to posttranslational modifications by reactive oxygen species (ROS) and reactive nitrogen species, leading to disulfides; to sulfenic, sulfinic, or sulfonic acids; mixed disulfides with low-molecular-weight (LMW) thiols (S thiolations); and S nitrosylations (7, 16, 17, 27).The redox status of the cytoplasm is under physiological conditions in a reduced state. Thus, most cysteines are present as free thiols (6). Because aerobic organisms have to cope with oxidative stress caused by ROS, such as superoxide anions, hydrogen peroxide, or hydroxyl radicals, they need to employ effective mechanisms that maintain the reduced state. In gram-negative bacteria, the thiol-disulfide balance is accomplished by the glutathione (GSH) system, a thiol-based redox buffer. The GSH system consists of glutaredoxin (Grx), GSH (γ-glutamylcysteinyl glycine), GSH reductase, and GSH peroxidase (34). Reduction of disulfides occurs via sequential electron transfer from glutaredoxin and reduced GSH; oxidized GSH (GSSG) is reduced by the NADPH-dependent GSH reductase. GSH peroxidase enables the direct detoxification of ROS by GSH oxidation.However, many gram-positive bacteria lack genes for GSH biosynthesis. Actinomycetes instead use a thiol redox buffer based on mycothiol (50). Bacillus subtilis, Staphylococcus aureus, and other gram-positive bacteria rely on different thiol redox buffers based on cysteine, the novel 398-Da bacillithiol (BSH), or coenzyme A (CoA) (15, 52). To maintain the reduced state of the cytoplasm, most bacteria use enzymatic systems for disulfide bond reduction such as the thioredoxin (Trx) system, which is highly conserved in gram-negative bacteria (3, 10). The Trx system consists of thioredoxin (TrxA) and the NADPH-dependent thioredoxin reductase (TrxB).Any imbalance in the cellular redox state caused by ROS elicits expression of a repertoire of different proteins, commonly under the control of a redox-sensing regulator: for example, OxyR in Escherichia coli and PerR, OhrR, SarZ, and Spx in B. subtilis and S. aureus, respectively (11, 12, 41, 55, 58, 64-66). The subsequently induced proteins detoxify ROS and restore and protect the normal physiological redox state in the cell.Besides ROS and reactive nitrogen species, so-called “reactive electrophilic species” (RES) affect the thiol redox balance. RES include different chemical compounds such as aldehydes, quinones, and the azo compound diamide (2, 43, 45, 46, 53, 66). Quinones and aldehydes have electron-deficient centers that result in thiol-(S) alkylation of cysteine. Exposure of cells to diamide induces the oxidative as well as the electrophile stress response in B. subtilis (43, 45, 53). The toxicity of diamide is based on disulfide bond formation (40), which was recently visualized in B. subtilis and S. aureus by the fluorescence alkylation of oxidized thiols (FALKO) assay (32, 64). It was thought that the formation of nonnative inter- and intramolecular disulfide bonds results in damage of proteins.However, more recent findings demonstrate that diamide stress leads also to S thiolations: formation of disulfide bonds between proteins and LMW thiols (8, 13, 33). S thiolations prevent protein thiols from irreversible oxidation to sulfinic and sulfonic acids, but also affect enzyme activity (35, 47) and signal transduction (39, 42). In B. subtilis, we have identified a few cytoplasmic proteins that are S cysteinylated (33). In addition, the organic peroxide sensor OhrR was inactivated by an S bacillithiolation in B. subtilis (42).Cysteine, BSH, and CoA are also dominant LMW thiols in S. aureus (52). In this study, we have investigated in more detail the extents of S thiolations and inter- and intramolecular disulfide bond formation of B. subtilis and S. aureus in response to disulfide stress. The results showed that exposure to diamide leads to S thiolations in S. aureus. Using a nonreducing/reducing sodium dodecyl sulfate (SDS) diagonal electrophoresis approach, proteins with intermolecular disulfide bonds could be distinguished from proteins with intramolecular disulfide bonds (57). The results support that the majority of reversible thiol oxidations are based on S thiolations rather than disulfide bonds between proteins. Depletion of the free cysteine pool in B. subtilis after exposure to diamide supports this finding. To assess if GSH may have a bearing on the thiol redox buffer of B. subtilis, the gshF gene of Listeria monocytogenes (gshF_Lm) was expressed in B. subtilis, enabling GSH biosynthesis (29). Although GSH production does not enhance the resistance to oxidative stress in B. subtilis, it participates in the formation of S thiolations.     

Homologues of the Bacillus subtilis SpoVB Protein Are Involved in Cell Wall Metabolism

Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of Escherichia coli MviN, to flip the lipid II peptidoglycan precursor. Bacillus subtilis SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the B. subtilis genome. Mutant strains lacking any or all of these genes (yabM, ykvU, and ytgP) in addition to spoVB are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in B. subtilis. Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.The Bacillus subtilis spoVB gene was first identified as a locus in which a mutation could produce a block at a late stage of spore development (14, 30). Analysis of this locus revealed that it encoded an apparent integral membrane protein (33), and a detailed analysis of a spoVB null mutant demonstrated a block at a very early step in synthesis of the spore cortex peptidoglycan (PG) (40). The mutant synthesized essentially no cortex and accumulated cytoplasmic PG precursors, the same phenotype found in other mutant strains blocked in functions known to be directly involved in PG polymerization (40). These results suggested that SpoVB plays a direct role in assembly or function of the spore PG synthesis apparatus.PG synthesis is a highly conserved and complex process that must span the cell membrane (reviewed in reference 38). Soluble nucleotide-linked PG precursors are synthesized in the cytoplasm. N-Acetylmuramic acid with a pentapeptide side chain is then transferred to an undecaprenol lipid carrier to produce lipid I, with subsequent addition of N-acetylglucosamine to produce lipid II, undecaprenyl-pyrophosphoryl-N-acetylmuramic acid (pentapeptide)-N-acetylglucosamine. Lipid II is then flipped across the membrane via an unknown mechanism. Two families of proteins have been postulated to perform this function: the SEDS family of integral membrane proteins, including FtsW, RodA, and SpoVE (13), and, more recently, the COG2244 family (23), which includes SpoVB and the MviN (MurJ) protein of Escherichia coli (35). In both cases, loss of a protein within one of these families has been shown to result in a block in PG synthesis and the accumulation of lipid-linked and/or soluble PG precursors (16, 20, 35, 40).In the standard model of PG synthesis, flippase activity brings the disaccharide-pentapeptide moieties to the penicillin-binding proteins (PBPs), which polymerize the PG macromolecule on the outer surface of the membrane (39). The class A, high-molecular-weight PBPs possess an N-terminal glycosyl transferase domain that polymerizes the disaccharides into polysaccharide chains (38). These chains are cross-linked via the transpeptidase activity within the penicillin-binding, C-terminal domains of both the class A and the class B PBPs. The N-terminal domains of the class A PBPs and the closely related monofunctional glycosyl transferases found in some species are the only defined PG glycan strand polymerases, and in several species the presence of at least one of these enzymes is essential. However, in B. subtilis (26) and Enterococcus faecalis (3), strains lacking all of these known glycosyl transferases are viable and produce PG walls, indicating the presence of another glycosyl transferase capable of this activity. This alternate glycosyl transferase is distinct in that it is relatively resistant to moenomycin (3, 26), an inhibitor of the class A PBP glycosyl transferase activity (6).Given the strong and early block in cortex PG polymerization observed to occur in a spoVB mutant (40), we wished to further analyze the potential role of this class of protein. SpoVB is a member of a relatively large family of proteins, COG2244 (23), some of which are involved in polymerization of other polysaccharides in bacteria, archaea, and eukaryotes. Bioinformatic analysis has generally predicted that these proteins span the membrane 12 to 14 times, and in some cases experimental evidence has supported this structure (7, 24). A role generally ascribed to these proteins is the flipping of lipid-linked oligosaccharides, produced on the inner face of a membrane, to the outside, where the oligosaccharides are then further polymerized or transferred to other substrates. Some prominent members of this family include Wzx, which functions in O-antigen synthesis in gram-negative bacteria (41); TuaB, which functions in teichuronic acid synthesis in B. subtilis (36); and Rft1, which functions in protein glycosylation in eukaryotes (12). MviN is essential in some gram-negative species, including Burkholderia pseudomallei, E. coli, and Sinorhizobium meliloti (22, 34), and has been shown to play a role in E. coli PG synthesis (16, 35). A Rhizobium tropici mutation that truncates mviN approximately 50% into the coding sequence was not lethal (29). However, it is not known whether this was the sole mviN homolog in the genome or whether the truncated gene product might be functional.We have analyzed the phenotypic properties of B. subtilis strains lacking other proteins within the COG2244 family that are most closely related to SpoVB. Results suggest that these proteins also play roles in PG synthesis and that, in one case, this role is in a synthetic system that is relatively moenomycin resistant. We postulate that these proteins function in an alternate pathway for PG synthesis that may involve the flipping of lipid-linked PG oligosaccharides rather than lipid II disaccharides.     

Structural Evidence of Glycoprotein Assembly in Cellular Membrane Compartments prior to Alphavirus Budding

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.Semliki Forest virus (SFV) is an enveloped alphavirus belonging to the family Togaviridae. This T=4 icosahedral virus particle is approximately 70 nm in diameter (30) and consists of 240 copies of E1/E2 glycoprotein dimers (3, 8, 24). The glycoproteins are anchored in a host-derived lipid envelope that encloses a nucleocapsid, made of a matching number of capsid proteins and a positive single-stranded RNA molecule. After entry of the virus via receptor-mediated endocytosis, a low-pH-induced fusion of the viral envelope with the endosomal membrane delivers the nucleocapsid into the cytoplasm, where the replication events of SFV occur (8, 19, 30). Replication of the viral genome and subsequent translation into structural and nonstructural proteins followed by assembly of the structural proteins and genome (7) lead to budding of progeny virions at the plasma membrane (18, 20). The synthesis of viral proteins shuts off host cell macromolecule synthesis, which allows for efficient intracellular replication of progeny virus (7). The expression of viral proteins leads to the formation of cytopathic vacuolar compartments as the result of the reorganization of cellular membrane in the cytoplasm of an infected cell (1, 7, 14).Early studies using electron microscopy (EM) have characterized the cytopathic vacuoles (CPVs) in SFV-infected cells (6, 13, 14) and identified two types of CPV, namely, CPV type I (CPV-I) and CPV-II. It was found that CPV-I is derived from modified endosomes and lysosomes (18), while CPV-II is derived from the trans-Golgi network (TGN) (10, 11). Significantly, the TGN and CPV-II vesicles are the major membrane compartments marked with E1/E2 glycoproteins (9, 11, 12). Inhibition by monensin results in the accumulation of E1/E2 glycoproteins in the TGN (12, 26), thereby indicating the origin of CPV-II. While CPV-II is identified as the predominant vacuolar structure at the late stage of SFV infection, the exact function of this particular cytopathic vacuole is less well characterized than that of CPV-I (2, 18), although previous observations have pointed to the involvement of CPV-II in budding, because an associated loss of viral budding was observed when CPV-II was absent (9, 36).In this study, we characterized the structure and composition of CPV-II in SFV-infected cells in situ with the aid of electron tomography and immuno-electron microscopy after physical fixation of SFV-infected cells by high-pressure freezing and freeze substitution (21, 22, 33). The results revealed a helical array of E1/E2 glycoproteins within CPV-II and indicate that CPV-II plays an important role in intracellular transport of glycoproteins prior to SFV budding.     

Multiple Interaction Domains in FtsL,a Protein Component of the Widely Conserved Bacterial FtsLBQ Cell Division Complex

A bioinformatic analysis of nearly 400 genomes indicates that the overwhelming majority of bacteria possess homologs of the Escherichia coli proteins FtsL, FtsB, and FtsQ, three proteins essential for cell division in that bacterium. These three bitopic membrane proteins form a subcomplex in vivo, independent of the other cell division proteins. Here we analyze the domains of E. coli FtsL that are involved in the interaction with other cell division proteins and important for the assembly of the divisome. We show that FtsL, as we have found previously with FtsB, packs an enormous amount of information in its sequence for interactions with proteins upstream and downstream in the assembly pathway. Given their size, it is likely that the sole function of the complex of these two proteins is to act as a scaffold for divisome assembly.The division of an Escherichia coli cell into two daughter cells requires a complex of proteins, the divisome, to coordinate the constriction of the three layers of the Gram-negative cell envelope. In E. coli, there are 10 proteins known to be essential for cell division; in the absence of any one of these proteins, cells continue to elongate and to replicate and segregate their chromosomes but fail to divide (29). Numerous additional nonessential proteins have been identified that localize to midcell and assist in cell division (7-9, 20, 25, 34, 56, 59).A localization dependency pathway has been determined for the 10 essential division proteins (FtsZ→FtsA/ZipA→FtsK→FtsQ→FtsL/FtsB→FtsW→FtsI→FtsN), suggesting that the divisome assembles in a hierarchical manner (29). Based on this pathway, a given protein depends on the presence of all upstream proteins (to the left) for its localization and that protein is then required for the localization of the downstream division proteins (to the right). While the localization dependency pathway of cell division proteins suggests that a sequence of interactions is necessary for divisome formation, recent work using a variety of techniques reveals that a more complex web of interactions among these proteins is necessary for a functionally stable complex (6, 10, 19, 23, 24, 30-32, 40). While numerous interactions have been identified between division proteins, further work is needed to define which domains are involved and which interactions are necessary for assembly of the divisome.One subcomplex of the divisome, composed of the bitopic membrane proteins FtsB, FtsL, and FtsQ, appears to be the bridge between the predominantly cytoplasmic cell division proteins and the predominantly periplasmic cell division proteins (10). FtsB, FtsL, and FtsQ share a similar topology: short amino-terminal cytoplasmic domains and larger carboxy-terminal periplasmic domains. This tripartite complex can be divided further into a subcomplex of FtsB and FtsL, which forms in the absence of FtsQ and interacts with the downstream division proteins FtsW and FtsI in the absence of FtsQ (30). The presence of an FtsB/FtsL/FtsQ subcomplex appears to be evolutionarily conserved, as there is evidence that the homologs of FtsB, FtsL, and FtsQ in the Gram-positive bacteria Bacillus subtilis and Streptococcus pneumoniae also assemble into complexes (18, 52, 55).The assembly of the FtsB/FtsL/FtsQ complex is important for the stabilization and localization of one or more of its component proteins in both E. coli and B. subtilis (11, 16, 18, 33). In E. coli, FtsB and FtsL are codependent for their stabilization and for localization to midcell, while FtsQ does not require either FtsB or FtsL for its stabilization or localization to midcell (11, 33). Both FtsL and FtsB require FtsQ for localization to midcell, and in the absence of FtsQ the levels of full-length FtsB are significantly reduced (11, 33). The observed reduction in full-length FtsB levels that occurs in the absence of FtsQ or FtsL results from the degradation of the FtsB C terminus (33). However, the C-terminally degraded FtsB generated upon depletion of FtsQ can still interact with and stabilize FtsL (33).While a portion of the FtsB C terminus is dispensable for interaction with FtsL and for the recruitment of the downstream division proteins FtsW and FtsI, it is required for interaction with FtsQ (33). Correspondingly, the FtsQ C terminus also appears to be important for interaction with FtsB and FtsL (32, 61). The interaction between FtsB and FtsL appears to be mediated by the predicted coiled-coil motifs within the periplasmic domains of the two proteins, although only the membrane-proximal half of the FtsB coiled coil is necessary for interaction with FtsL (10, 32, 33). Additionally, the transmembrane domains of FtsB and FtsL are important for their interaction with each other, while the cytoplasmic domain of FtsL is not necessary for interaction with FtsB, which has only a short 3-amino-acid cytoplasmic domain (10, 33).In this study, we focused on the interaction domains of FtsL. We find that, as with FtsB, the C terminus of FtsL is required for the interaction of FtsQ with the FtsB/FtsL subcomplex while the cytoplasmic domain of FtsL is involved in recruitment of the downstream division proteins. Finally, we provide a comprehensive analysis of the presence of FtsB, FtsL, and FtsQ homologs among bacteria and find that the proteins of this complex are likely more widely distributed among bacteria than was previously thought.     

Bacillus subtilis SpoIIIJ and YqjG Function in Membrane Protein Biogenesis

In all domains of life Oxa1p-like proteins are involved in membrane protein biogenesis. Bacillus subtilis, a model organism for gram-positive bacteria, contains two Oxa1p homologs: SpoIIIJ and YqjG. These molecules appear to be mutually exchangeable, although SpoIIIJ is specifically required for spore formation. SpoIIIJ and YqjG have been implicated in a posttranslocational stage of protein secretion. Here we show that the expression of either spoIIIJ or yqjG functionally compensates for the defects in membrane insertion due to YidC depletion in Escherichia coli. Both SpoIIIJ and YqjG complement the function of YidC in SecYEG-dependent and -independent membrane insertion of subunits of the cytochrome o oxidase and F₁F_o ATP synthase complexes. Furthermore, SpoIIIJ and YqjG facilitate membrane insertion of F₁F_o ATP synthase subunit c from both E. coli and B. subtilis into inner membrane vesicles of E. coli. When isolated from B. subtilis cells, SpoIIIJ and YqjG were found to be associated with the entire F₁F_o ATP synthase complex, suggesting that they have a role late in the membrane assembly process. These data demonstrate that the Bacillus Oxa1p homologs have a role in membrane protein biogenesis rather than in protein secretion.The YidC/OxaI/Alb3 protein family plays a crucial role in membrane protein biogenesis by facilitating the insertion of a specific subset of membrane proteins (for reviews, see references 20 and 24). In mitochondria, the OxaI protein is essential for insertion of both nucleus- and mitochondrion-encoded proteins into the inner membrane (39). The OxaI homolog of Escherichia coli, designated YidC, is known to play a role in two different membrane protein insertion pathways. Some proteins, such as subunit c of the rotary domain of the F₁F_o ATP synthase (F_oc) (47), MscL (10), M13 (34), and Pf3 (5), insert via the YidC-only pathway. YidC also functions in concert with the protein-conducting channel SecYEG in membrane insertion of subunit a of cytochrome o oxidase (CyoA) (8, 44) and subunit a of the F₁F_o ATP synthase (23, 53, 54). In addition, YidC has been implicated in the folding of a membrane-inserted lactose permease (30) and the binding protein-dependent maltose ABC transporter (50).Members of the YidC/OxaI/Alb3 protein family are found in all three domains of life, and the number of paralogs per cell or organelle ranges from one (most gram-negative bacteria) to six (Arabidopsis thaliana). The length of Oxa1p-like proteins varies considerably, from just over 200 amino acids (in most gram-positive bacteria) to 795 amino acids (Chlamydophila pneumoniae) (52). However, in all Oxa1p proteins, a conserved region consisting of about 200 amino acids can be recognized, which comprises five putative transmembrane segments, as experimentally demonstrated for E. coli YidC (33). Overall, the amino acid sequence conservation among Oxa1p homologs is low (17). Bacillus subtilis contains two membrane proteins, SpoIIIJ and YqjG, with significant similarity to proteins belonging to the YidC/OxaI/Alb3 family. Previous gene inactivation analysis showed that a single paralog is sufficient for cell viability during vegetative growth of B. subtilis, while a double knockout led to a lethal phenotype (29, 41). SpoIIIJ is essential for activation of a prespore-specific sigma factor (9, 36), and cells with spoIIIJ deleted are incapable of spore formation. Sporulation is blocked at stage III, directly after completion of prespore engulfment (9). YqjG cannot complement SpoIIIJ in this process, but the exact reason for the specific requirement for SpoIIIJ is unknown. Previous studies indicated that the stability of various secretory proteins (e.g., LipA and PhoA) was strongly affected under YqjG- and SpoIIIJ-limiting conditions, while the insertion or stability of a number of membrane proteins tested appeared to be unaffected (41). These data suggested that YqjG and SpoIIIJ, unlike the other Oxa1p-like proteins, play a role in protein secretion. Here we show that both YidC homologs in B. subtilis complement the E. coli growth defect due to a YidC depletion and functionally replace YidC in Sec-dependent and -independent membrane protein insertion. In vitro insertion assays demonstrated that membrane insertion of F_oc of both E. coli and B. subtilis is mediated by SpoIIIJ and YqjG. In addition, the entire F₁F_o ATP synthase of B. subtilis was found to copurify with both SpoIIIJ and YqjG, suggesting that these proteins have a role in a late stage of the assembly of this membrane protein complex.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Mapping of the Saccharomyces cerevisiae Oxa1-Mitochondrial Ribosome Interface and Identification of MrpL40, a Ribosomal Protein in Close Proximity to Oxa1 and Critical for Oxidative Phosphorylation Complex Assembly

The Oxa1 protein plays a central role in facilitating the cotranslational insertion of the nascent polypeptide chains into the mitochondrial inner membrane. Mitochondrially encoded proteins are synthesized on matrix-localized ribosomes which are tethered to the inner membrane and in physical association with the Oxa1 protein. In the present study we used a chemical cross-linking approach to map the Saccharomyces cerevisiae Oxa1-ribosome interface, and we demonstrate here a close association of Oxa1 and the large ribosomal subunit protein, MrpL40. Evidence to indicate that a close physical and functional relationship exists between MrpL40 and another large ribosomal protein, the Mrp20/L23 protein, is also provided. MrpL40 shares sequence features with the bacterial ribosomal protein L24, which like Mrp20/L23 is known to be located adjacent to the ribosomal polypeptide exit site. We propose therefore that MrpL40 represents the Saccharomyces cerevisiae L24 homolog. MrpL40, like many mitochondrial ribosomal proteins, contains a C-terminal extension region that bears no similarity to the bacterial counterpart. We show that this C-terminal mitochondria-specific region is important for MrpL40''s ability to support the synthesis of the correct complement of mitochondrially encoded proteins and their subsequent assembly into oxidative phosphorylation complexes.The mitochondrial genome encodes a small, but important, number of proteins (8). These proteins are predominantly essential components of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. In the yeast Saccharomyces cerevisiae the proteins encoded by the mitochondrial DNA (mtDNA) include cytochrome c oxidase subunits Cox1, Cox2, and Cox3, cytochrome b of the cytochrome bc₁ complex, F₁F_o-ATP synthase subunits Atp6, Atp8, and Atp9, and the small ribosomal subunit component Var1. With the exception of Var1, these mitochondrially encoded proteins are integral membrane proteins which become inserted into the inner membrane during their synthesis on mitochondrial ribosomes tethered to the inner membrane (11, 19, 29, 32, 34). The cotranslational membrane insertion of these proteins is achieved by maintaining a close physical association of the ribosomes to the inner membrane at sites where the insertion machinery exists (19, 31, 32).Oxa1 is an inner membrane protein that forms a central component of the insertion machinery, whose presence is required for the cotranslational membrane insertion of the mitochondrially encoded proteins (4-6, 15-17). The Oxa1 protein has been shown to physically associate with the ribosomes and more specifically with the large ribosomal subunit. Matrix-exposed elements of the Oxa1 protein, such as its hydrophilic C-terminal tail, support this Oxa1-ribosome interaction (19, 32). Furthermore, in intact mitochondria we have previously demonstrated that Oxa1 can be chemically cross-linked to Mrp20, a component of the large ribosomal subunit (19). Mrp20 is homologous to the bacterial ribosomal protein L23, a component known from the structural analysis of the ribosomes to be located next to the polypeptide exit site of the large ribosomal subunit (3, 10, 23, 27, 30). Thus, it was concluded that Oxa1, the site of membrane insertion into the inner membrane, exists in close physical proximity to the large ribosomal subunit and specifically to that region of the ribosomes where the nascent chain emerges. This close physical relationship between ribosomal components and the Oxa1 insertion site has been proposed to support a tight coordination between the protein translation and membrane insertion events (19, 31, 32). Given the strong hydrophobicity of the OXPHOS complex subunits which are encoded by the mitochondrial DNA and synthesized by these ribosomes, a close coupling of the translation and insertion events is proposed to ensure that the hydrophobic nascent chains are directly inserted into the membrane during their synthesis. The exposure of hydrophobic nascent chains to the hydrophilic matrix space may promote their aggregation and thus incompetency for subsequence membrane insertion.In bacteria, the L23 protein has been implicated to play a direct role in the cotranslational insertion of proteins into the membrane (7, 13, 24, 33). Thus, it is possible that proteins adjacent to the polypeptide exit site of mitochondrial ribosomes may be directly involved in targeting ribosomes to specific regions of the inner membrane where the membrane insertion and subsequent assembly events occur. The mitochondrial ribosomes resemble their prokaryotic ancestors in some respects, e.g., antibiotic sensitivity, but they differ in a number of important ways (1, 12, 22, 30). In general, the protein content of the mitochondrial ribosomes is greater than their bacterial counterparts. This increase in protein content is largely attributed to the fact that the mitochondrial ribosomal proteins are larger in size than their bacterial homologs. Over the course of evolution, many of the mitochondrial ribosomal proteins have acquired novel extensions, new domains, in addition to their bacterial homology domains. These acquired extensions not only include N-terminal (often cleavable) signals to target these proteins (nuclear encoded) to the mitochondria but also in many instances large C-terminal extensions, which are unique to the mitochondrial ribosomal proteins and have thus been termed “mitospecific domains” (12, 30). Largely uncharacterized, the functional relevance of these various mitospecific domains of the ribosomal proteins remains unknown. It is speculated that some (or all) of these mitospecific domains serve to ensure that the ribosome becomes assembled and is translationally active while bound to the inner membrane surface.In the present study we sought to further characterize the interaction of the mitochondrial ribosome with the Oxa1 protein. We show here that MrpL40, a large ribosomal subunit component, is physically close to both the Mrp20 and Oxa1 proteins, demonstrating the proximity of MrpL40 to both the ribosomal polypeptide exit site and the Oxa1 membrane insertion site. MrpL40 contains a large C-terminal mitospecific domain, which includes a predicted α-helical region at its extreme C-terminal end. The results presented here highlight that the integrity of this domain of MrpL40 is crucial to ensure ribosome translational fidelity and subsequent OXPHOS complex assembly.     

Protein Phosphatase Type 1-Interacting Protein Ysw1 Is Involved in Proper Septin Organization and Prospore Membrane Formation during Sporulation

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of YSW1 partially suppresses the sporulation defect of a temperature-sensitive allele of gip1. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in ysw1Δ mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in ysw1Δ cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.Diploid cells of Saccharomyces cerevisiae subjected to nitrogen limitation in the presence of a nonfermentable carbon source undergo the developmental process of sporulation (14, 23, 35). Four nuclei produced by two rounds of nuclear division, meiosis I and II, are encapsulated by newly formed double-membrane structures, called prospore membranes, and are finally packaged into spores covered with layered spore walls (35).In this process, prospore membrane formation is one of the most dynamic events. Early in meiosis II, the cytoplasmic surface of the meiotic spindle pole body (SPB) is modified by the recruitment of sporulation-specific protein complex that acts as a site of vesicle recruitment (2, 22, 39). Post-Golgi secretory vesicles dock to the surface of the SPBs and fuse with each other, generating prospore membranes (33, 34). The prospore membranes then grow to engulf daughter nuclei through a series of stages that are categorized by the membranes'' appearance in the fluorescence microscope (12). Initially, the membranes appear as small horseshoes that enlarge to become small round membrane structures. The prospore membranes then extend into a tube-like shape, engulfing the nucleus, as well as some cytosol and organelles (12). After this extension, prospore membrane undergoes a rapid change to a mature round form. This rounding of the membrane is coordinated with membrane closure (12). Spore wall materials are then deposited into the luminal space created by closure of the prospore membrane (9).In addition to the meiotic plaque of the SPB, two protein complexes are associated with the prospore membrane as it forms. One is the leading edge protein complex, which exists at the lip of the prospore membranes and consists of three components: Ssp1, Ady3, and Don1 (27, 30, 38). Ssp1 is the most important of the three and is required for proper extension of the prospore membrane (30). The second complex is a sporulation-specific septin structure. The septins are a family of cytoskeletal proteins, which form filaments (18, 50). Septins are conserved from yeast to mammals. They were originally found and have been extensively studied in S. cerevisiae. In vegetatively growing S. cerevisiae cells, five septin proteins—Cdc3, Cdc10, Cdc11, Cdc12, and Shs1—form a ring at the bud neck that serves as a scaffold for many additional proteins, as well as a barrier to diffusion of proteins between the mother and the bud (19, 29, 50). In sporulating cells, the set of septin proteins is changed. Cdc3 and Cdc10, along with two sporulation-specific septins, Spr3 and Spr28, form a pair of parallel bars or sheets associated with each prospore membrane (11, 15, 29). Although deletion of sporulation-specific septins has only modest effects on sporulation (11, 15), their specific localization suggests that they have some function during prospore membrane formation. Septin organization in vegetatively growing cells is regulated by phosphorylation and dephosphorylation of septin components and septin-associated proteins (29). In sporulating cells, a sporulation-specific protein phosphatase type 1 (PP1) complex Gip1-Glc7 is required for the formation of septin structures (46), although whether this phosphatase acts directly on the septin proteins is unknown.The PP1 catalytic subunit is highly conserved in eukaryotes and is involved in a variety of cellular processes (8, 44). In S. cerevisiae it is encoded by an essential gene, GLC7, and functions in glycogen synthesis, glucose repression, chromosome segregation, cell wall organization, endocytosis, mating, and sporulation (3, 17, 24, 42, 44, 47, 53). The specificity of this enzyme is determined by targeting subunits. GIP1 was originally isolated in a two-hybrid screen by using GLC7 as a bait, and this interaction was confirmed by coimmunoprecipitation of the two proteins (48). GIP1 is a sporulation-specific gene required for sporulation. Further analysis revealed that Gip1 and Glc7 colocalize with septins during sporulation and are required for both septin organization and spore wall formation (46). The specific targets or cofactors of this PP1 complex are unknown.To elucidate the role of Gip1-Glc7 phosphatase, we screened for high-copy suppressors of a temperature-sensitive allele of gip1 and isolated YSW1. Ysw1 interacts with Gip1 and colocalizes with septins similar to Gip1. Furthermore, a ysw1Δ mutant displays aberrant septin structures and prospore membrane extension. These results suggest that Ysw1 may function with Gip1-Glc7 to regulate proper septin organization and prospore membrane formation.     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

Identification of Chlamydia trachomatis Outer Membrane Complex Proteins by Differential Proteomics

The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.Bacteria in the phylum Chlamydiae are characterized by their complex intracellular developmental cycles. Chlamydiae must assume at least two functionally distinct morphotypes, the intracellular, replicative reticulate body (RB) and the extracellular, infectious elementary body (EB), to replicate and be transmitted to new hosts (50). The divergence of distinct RB and EB forms may have been driven by the different pressures these pathogens face inside host cells during replication and outside host cells during transmission. For example, the outer membrane of EB contains a poorly immunogenic truncated lipopolysaccharide (LPS) (14, 30) and immunodominant epitopes of the major outer membrane protein (MOMP) vary substantially among closely related chlamydial strains (13). EB also lack detectable peptidoglycan (2, 20, 60), although functional murein biosynthetic enzymes (2, 5, 16, 21, 32, 43, 45, 46) are expressed in RB during productive and persistent infection (44). To compensate for the loss of murein, EB are enveloped by a protein P-layer, which lends osmotic stability to the infectious particle (29).Attempts to identify components of the P-layer and outer membrane proteins of Chlamydia were advanced by the observation that these layers can be separated from many soluble EB proteins using the detergent N-lauroyl sarcosine (Sarkosyl). Caldwell et al. dubbed the Sarkosyl-insoluble fraction the chlamydial outer membrane complex (COMC) and noted that purified COMC maintained the shape of intact EB and contained a complete outer membrane, and they reported that a single outer membrane protein, MOMP, accounted for more than 60% of total COMC protein content (15). Other studies revealed that the COMC is stabilized by extensive disulfide bonds between MOMP monomers (26, 27, 53) and between MOMP and two abundant cysteine-rich COMC components (26, 28). Other studies revealed that the COMC is stabilized by extensive disulfide bonds between MOMP monomers (18, 29, 52) and the EB surface (3, 19, 47, 67). More recent data suggest that not all EB outer membrane (OM) proteins are disulfide cross-linked to the COMC. For example, polymorphic membrane protein D localizes to the surface of EB but can be extracted from intact EB with gentle detergents in the absence of reducing agents (17, 61). Thus, not all COMC proteins are exposed on the EB surface, nor are all EB OM proteins components of the COMC.Beyond these well-described and abundant COMC components, other studies have indicated that additional proteins localize to the EB surface and/or COMC of Chlamydia trachomatis (7, 28, 36, 51, 57, 64, 67, 70). However, confirming that specific proteins localize to the COMC or OM of EB can be challenging due to factors such as the contamination of EB preparations with RB proteins and technical limitations of proteomic and surface-labeling protein identification methods (29, 56).Here, we used differential proteomics to identify proteins specifically enriched in the COMC. Isolated COMC were dissolved in 8 M urea, and the extracted proteins were digested with trypsin. The resulting peptides were analyzed by high-sensitivity liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to identify low-abundance proteins. Sarkosyl-soluble fractions and whole EB were analyzed in parallel with COMC, and protein assignments were compared among three replicate runs of each fraction. In total, peptides from 329 L2 proteins were identified. The differential analysis of protein abundance indicated the enrichment of 17 proteins in the COMC. Our results define the cadre of low-abundance COMC proteins, provide a starting point for the identification of surface-exposed EB proteins, and identify EB proteins that are likely to be recognized by innate immunity receptors and/or capable of eliciting neutralizing antibodies in vivo. Finally, our findings and data from other recent studies permit the refinement of existing models of EB and COMC structure.     

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.Replication sliding clamps are essential cellular proteins imparting a spectacular degree of processivity to DNA polymerases during genome replication (24, 39-41). Encoded by the dnaN gene, the β clamp is a highly conserved bacterial sliding clamp found in virtually all eubacterial species (reviewed in reference 7). The β clamp is a head-to-tail, ring-shaped homodimer that encircles double-stranded DNA (1, 39). In eukaryotes and archaea, the analog of the β clamp is proliferating cell nuclear antigen (PCNA) (15, 28, 40, 41). Eukaryotic PCNA is a ring-shaped homotrimer that also acts to encircle DNA, increasing the processivity of the replicative DNA polymerases (40, 41). Although the primary structures of the β clamp and PCNA are not conserved, the tertiary structures of these proteins are very similar, demonstrating structural conservation among bacterial, archaeal, and eukaryotic replication sliding clamps (28, 39-41; reviewed in reference 6).The function of the β clamp is not limited to its well-defined role in genome replication. The Escherichia coli β clamp binds Hda, which also binds the replication initiation protein DnaA, regulating the active form of DnaA complexed with ATP (19, 37, 43). This allows the β clamp to regulate replication initiation through the amount of available DnaA-ATP. In Bacillus subtilis, the β clamp binds YabA, a negative regulator of DNA replication initiation (12, 29, 52). It has also been suggested that the B. subtilis β clamp sequesters DnaA from the replication origin during the cell cycle through the binding of DnaA to YabA and the binding of YabA to the β clamp (70). Thus, it is hypothesized that in E. coli and B. subtilis, the β clamp influences the frequency of replication initiation through interactions with Hda and YabA, respectively.The E. coli and B. subtilis β clamp has an important role in translesion DNA synthesis during the replicative bypass of noncoding bases by specialized DNA polymerases belonging to the Y family (20, 33). The roles of the E. coli β clamp in translesion synthesis are well established (5, 8, 30, 31). Binding sites on the E. coli β clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD₂′C) have been identified, and the consequence of disrupting their association with the β clamp has illustrated the critical importance of the β clamp to the activity of both of these polymerases (4, 5, 8, 26, 30, 31, 48, 49).In addition to the involvement of the β clamp in replication initiation, DNA replication, and translesion synthesis, the E. coli and B. subtilis β clamp also functions in DNA mismatch repair (MMR) (45, 46, 64). The MMR pathway recognizes and repairs DNA polymerase errors, contributing to the overall fidelity of the DNA replication pathway (reviewed in references 42 and 60). In both E. coli and B. subtilis, deletion of the genes mutS and mutL increases the spontaneous mutation frequency several hundredfold (13, 25, 63). In E. coli, MutS recognizes and binds mismatches, while MutL functions as a “matchmaker,” coordinating the actions of other proteins in the MMR pathway, allowing the removal of the mismatch and resynthesis of the resulting gap (reviewed in references 42 and 60). MutS and MutL of E. coli and B. subtilis physically interact with the β clamp (45, 46, 51, 64). Interaction between the B. subtilis β clamp and MutS is important for efficient MMR and organization of MutS-green fluorescent protein (GFP) into foci in response to replication errors, while the function of MutL binding to the β clamp is unknown (64).These studies show that the β clamp is critical for several aspects of DNA metabolism in E. coli and B. subtilis. In E. coli, many dnaN alleles have been examined and used to define the mechanistic roles of the β clamp in vivo (5, 18, 24, 30, 31, 48, 49, 73). A limitation in studying the mechanistic roles of the B. subtilis β clamp is that only two dnaN alleles (β clamp) are available, dnaN5 and dnaN34 (36) (www.bgsc.org/), and both of these alleles do not support growth at temperatures above 49°C, suggesting that they may cause similar defects (36) (www.bgsc.org/). Of these two dnaN alleles, only dnaN5 has been investigated in any detail (36, 53, 64). The mutant β clamp encoded by dnaN5 contains a G73R substitution [dnaN5(G73R)] in a surface-exposed residue located on the outside rim of the β clamp (53, 64). Our previous studies with this allele showed that dnaN5(G73R) confers an increase in mutation frequency at 30°C and 37°C (64). Further characterization of dnaN5(G73R) showed that the increased mutation frequency is caused by a partial defect in MMR (64). Additionally, dnaN5(G73R)-containing cells have a reduced ability to support MutS-GFP focus formation in response to mismatches (64). These results support the hypothesis that G73R in the β clamp causes a defect in DNA replication at 49°C (36) and impaired MMR manifested by a defect in establishing the assembly of MutS-GFP foci in response to replication errors (64).To understand the roles of the B. subtilis β clamp in MMR and DNA replication, we examined the dnaN5 and dnaN34 alleles. We found that the nucleotide sequences of dnaN5 and dnaN34 and the phenotypes they produce were identical, both producing the G73R missense mutation. We analyzed in vivo β clamp^G73R protein levels and found that the β clamp^G73R protein accumulated to wild-type levels at elevated temperatures. To identify amino acid residues that would restore DNA replication at elevated temperatures, we isolated three intragenic suppressors of dnaN5(G73R) that conferred growth of B. subtilis cells at 49°C. Epistasis analysis and determination of the mutation spectrum showed that each dnaN allele isolated in this study caused an MMR-dependent increase in mutation frequency. Additionally, we found that the β clamp binding protein YabA can reduce the efficiency of MMR in vivo when yabA expression is induced. Thus, we have identified residues in the β clamp that are critical for DNA replication and MMR in B. subtilis. We also found that a β clamp binding protein, YabA, can reduce the efficiency of MMR in vivo.