Selective methylation changes on the Bacillus subtilis chemotaxis receptor McpB promote adaptation

The Bacillus subtilis McpB is a class III chemotaxis receptor, from which methanol is released in response to all stimuli. McpB has four putative methylation sites based upon the Escherichia coli consensus sequence. To explore the nature of methanol release from a class III receptor, all combinations of putative methylation sites Gln(371), Gln(595), Glu(630), and Glu(637) were substituted with aspartate, a conservative substitution that effectively eliminates methylation. McpB((Q371D,E630D,E637D)) in a Delta(mcpA mcpB tlpA tlpB)101::cat mcpC4::erm background failed to release methanol in response to either the addition or removal of the McpB-mediated attractant asparagine. In the same background, McpB((E630D,E637D)) produced methanol only upon asparagine addition, whereas McpB((Q371D,E630D)) produced methanol only upon asparagine removal. Thus methanol release from McpB was selective. Mutants unable to methylate site 637 but able to methylate site 630 had high prestimulus biases and were incapable of adapting to asparagine addition. Mutants unable to methylate site 630 but able to methylate site 637 had low prestimulus biases and were impaired in adaptation to asparagine removal. We propose that selective methylation of these two sites represents a method of adaptation novel from E. coli and present a model in which a charged residue rests between them. The placement of this charge would allow for opposing electrostatic effects (and hence opposing receptor conformational changes). We propose that CheC, a protein not found in enteric systems, has a role in regulating this selective methylation.     

Chemotaxis towards sugars by Bacillus subtilis.

Many sugars and derivatives were tested in the capillary assay for their attraction of Bacillus subtilis. The major attractants were 2-deoxy-D-glucose, D-fructose, gentiobiose, D-glucose, maltose, D-mannitol, D-mannose, N-acetylglucosamine, alpha-methyl-D-glucoside, beta-methyl-D-glucoside, N-acetylmannosamine, alpha-methyl-D-mannoside, D-sorbitol, L-sorbose, sucrose, trehalose and D-xylose. Only glucose chemotaxis was completely constitutive. Competition experiments were carried out to determine the specificities of chemoreceptors. There were 25 instances of no influence of two sugars on each other's taxis, 92 instances of one sugar interfering non-reciprocally with chemotaxis towards another and 49 instances of two sugars reciprocally competing. However, in most of the last instances, other sugars were identified that interfered with chemotaxis towards one member of the pair but not the other. Thus, nearly all sugars and related compounds appear to be detected by their own chemoreceptors, but many secondary interactions exist.     

CheY-dependent methylation of the asparagine receptor, McpB, during chemotaxis in Bacillus subtilis

For the Gram-positive organism Bacillus subtilis, chemotaxis to the attractant asparagine is mediated by the chemoreceptor McpB. In this study, we show that rapid net demethylation of B. subtilis McpB results in the immediate production of methanol, presumably due to the action of CheB. We also show that net demethylation of McpB occurs upon both addition and removal of asparagine. After each demethylation event, McpB is remethylated to nearly prestimulus levels. Both remethylation events are attributable to CheR using S-adenosylmethionine as a substrate. Therefore, no methyl transfer to an intermediate carrier need be postulated to occur during chemotaxis in B. subtilis as was previously suggested. Furthermore, we show that the remethylation of asparagine-bound McpB requires the response regulator, CheY-P, suggesting that CheY-P acts in a feedback mechanism to facilitate adaptation to positive stimuli during chemotaxis in B. subtilis. This hypothesis is supported by two observations: a cheRBCD mutant is capable of transient excitation and subsequent oscillations that bring the flagellar rotational bias below the prestimulus value in the tethered cell assay, and the cheRBCD mutant is capable of swarming in a Tryptone swarm plate.     

Bacillus subtilis SepF Binds to the C-Terminus of FtsZ

Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.     

Transmembrane organization of the Bacillus subtilis chemoreceptor McpB deduced by cysteine disulfide crosslinking

The Bacillus subtilis chemoreceptor McpB is a dimer of identical subunits containing two transmembrane (TM) segments (TM1, residues 17-34: TM2, residues 280-302) in each monomer with a 2-fold axis of symmetry. To study the organization of the TM domains, the wild-type receptor was mutated systematically at the membrane bilayer/extracytoplasmic interface with 15 single cysteine (Cys) substitutions in each of the two TM domains. Each single Cys substitution was capable of complementing a null allele in vivo, suggesting that no significant perturbation of the native tertiary or quaternary structure of the chemoreceptor was introduced by the mutations. On the basis of patterns of disulfide crosslinking between subunits of the dimeric receptor, an alpha-helical interface was identified between TM1 and TM1' (containing residues 32, 36, 39, and 43) and between TM2 and TM2' (containing residues 276, 277, 280, 283 and 286). Pairs of cysteine substitutions (positions 34/280 and 38/273) in TM1 and TM2 were used to further elucidate specific contacts within a monomer subunit, enabling a model to be constructed defining the organization of the TM domain. Crosslinking of residues that were 150-180 degrees removed from position 32 (positions 37, 41, and 44) suggested that the receptors may be organized as an array of trimers of dimers in vivo. All crosslinking was unaffected by deletion of cheB and cheR (loss of receptor demethylation/methylation enzymes) or by deletion of cheW and cheV (loss of proteins that couple receptors with the autophosphorylating kinase). These findings indicate that the organization of the transmembrane region and the stability of the quaternary complex of receptors are independent of covalent modifications of the cytoplasmic domain and conformations in the cytoplasmic domain induced by the coupling proteins.     

Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.     

Chemotaxis toward amino acids by Bacillus subtilis.

Conditions for assaying chemotaxis in Bacillus subtilis are described. The chemotaxis medium we used afforded excellent motility for hours. In it, chemotaxis measured by capillary assays was insensitive to pH between 5.5 and 9, and to temperature between 28 degrees C and 42 degrees C. Chemotaxis was observed toward all 20 common amino acids, with thresholds varying from 3nM for alanine to 0.1 mM for glutamate, in the capillary assay, and from 0.1 muM for alanine to 0.32 mM for glutamate in the microscope assay.     

Chemotaxis of Azotobacter vinelandii and Bacillus subtilis in a mixed culture

In the mixed culture of Azotobacter vinelandii and Bacillus subtilis, chemotaxis of Azotobacter to glucose remained unchanged, while that of bacilli decreased. Microelectrophoresis demonstrated adhesion of the A. vinelandii polysaccharide on the surface of B. subtilis cells. In the presence of 0.05–1.0 g/L of this biopolymer, the chemotaxis of bacilli to glucose decreased 2.6 to 6.8 times. A. vinelandii polysaccharide molecules adherent on the surface of B. subtilis cells were suggested to block bacillary chemotactic receptors, resulting in a decrease in their directed motility in the mixed culture.     

Unique Regulation of Carbohydrate Chemotaxis in Bacillus subtilis by the Phosphoenolpyruvate-Dependent Phosphotransferase System and the Methyl-Accepting Chemotaxis Protein McpC

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for E. coli, a B. subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An mcpC null mutant showed no response to the stepwise addition or removal of d-glucose or d-mannitol, both of which are PTS substrates. Therefore, in contrast to E. coli PTS carbohydrate chemotaxis, B. subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate d-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that B. subtilis EnzymeI-P has the ability to inhibit B. subtilis CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of d-glucose, a decrease in d-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.  In enteric bacteria, chemotaxis toward many carbohydrate attractants is dependent upon components of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (1, 9, 15). This carbohydrate transport system consists of an autophosphorylating histidine kinase, EnzymeI, a common phosphocarrier protein, HPr, and a number of substrate-specific transporters, the EnzymeII complexes. At the expense of PEP, EnzymeI autophosphorylates on a histidine residue and transfers this phosphoryl group to a histidine residue on HPr. HPr-P then donates this phosphoryl group to a carbohydrate-specific EnzymeII complex. The carbohydrate substrate is the final phosphoryl group acceptor, as it is transported into the cell and is concomitantly phosphorylated by EnzymeII (13).Chemotaxis is also controlled by a phosphoryl transfer cascade. CheA, in response to an attractant- or repellent-bound receptor (methyl-accepting chemotaxis protein [MCP]), alters its rate of autophosphorylation appropriately to transiently increase or decrease the intracellular CheY-P pool and thereby modulate swimming behavior (4, 16). In enteric bacteria, increased CheY-P leads to tumbling (19). In Bacillus subtilis, increased CheY-P leads to smooth swimming (3). In enteric bacteria, chemotaxis toward PTS substrates requires CheA, CheY, EnzymeI, and HPr but does not depend on the presence of an MCP (12, 18). These observations have led investigators to suggest that the changes in the phosphorylation state of PTS components that accompany carbohydrate transport regulate CheA activity (10).Recent work has provided the following model for the role of the PTS in chemotaxis toward its substrates in Escherichia coli. As the bacteria encounter a PTS carbohydrate, HPr dephosphorylates EnzymeI faster than the latter protein can be rephosphorylated. The resulting increase in unphosphorylated EnzymeI and the resulting decrease in PEP both function to decrease the rate of CheA autophosphorylation. This is believed to lead to a transient decrease in the CheY-P pool that suppresses tumbling, allowing the bacteria to move up the carbohydrate gradient (10).This article describes studies on the process of carbohydrate chemotaxis in B. subtilis. In particular, we provide evidence that McpC is absolutely required for any response to all of the PTS carbohydrates tested. This is surprising considering the fact that McpC has previously been shown to also mediate chemotaxis toward eight different amino acids (11). McpA has previously been shown to partially mediate chemotaxis toward glucose (7). This result is confirmed in the present study with the use of direct behavioral assays. Our results suggest the existence of a multidimensional signaling mechanism involving both the PTS and specific MCPs, an unprecedented finding in the study of the molecular control of bacterial carbohydrate chemotaxis.     

一种展示SARS冠状病毒受体结合区的重组枯草杆菌芽孢的制备及免疫原性分析

目的：利用枯草杆菌芽孢呈递技术制备表达SARS冠状病毒S蛋白受体结合区（RBD）的重组芽孢。方法：将枯草杆菌 CotB 基因构建到基因组整合质粒pDG1664中,再将 RBD 基因连接到 CotB 基因的下游,构建成重组质粒pDG1664-CotB-RBD,通过同源重组整合到PY-79枯草杆菌基因组中;利用红霉素抗性筛选重组菌并进行PCR和DNA测序鉴定,Western印迹鉴定重组菌芽孢表面RBD蛋白的表达情况;用表达RBD的重组芽孢以口服方式免疫小鼠,通过ELISA和流式细胞术检测重组芽孢的免疫原性。结果：制备出枯草杆菌基因组整合了RBD抗原基因的重组菌株RS1931,形成的重组芽孢表达相对分子质量约62×103的CotB-RBD融合蛋白;重组芽孢免疫的小鼠血清RBD抗原特异性IgG抗体滴度在末次免疫后2周可达1∶10880,重组芽孢初免后18周的小鼠脾细胞中IFN-γ＋CD4^＋、IL-4＋CD4^＋和IFN-γ＋CD8^＋T细胞比例上调,表明重组芽孢经口服免疫产生良好的体液免疫和细胞免疫应答。结论：针对SARS冠状病毒S蛋白RBD建立了枯草杆菌芽孢呈递技术方法,制备出在枯草杆菌芽孢表面稳定表达外源RBD蛋白的重组株,获得的重组芽孢具有良好的免疫原性,为开发芽孢呈递型SARS疫苗奠定了基础。     

Chemotaxis in Bacillus subtilis requires either of two functionally redundant CheW homologs.

We have characterized mutants in a novel gene of Bacillus subtilis, cheV, which encodes a protein homologous to both CheW and CheY. A null mutant in cheV is only slightly defective in capillary and tethered cell assays. However, a double mutant lacking both CheV and CheW has a strong tumble bias, does not respond to addition of attractant, and shows essentially no accumulation in capillary assays. Thus, CheV and CheW appear in part to be functionally redundant. A strain lacking CheW and expressing only the CheW domain of CheV is chemotactic, suggesting that the truncated CheV protein retains in vivo function. We speculate that CheV and CheW function together to couple CheA activation to methyl-accepting chemotaxis protein receptor status and that possible CheA-dependent phosphorylation of CheV contributes to adaptation.     

嗜铬粒蛋白N端片段基因在枯草杆菌中的表达及表达产物的活性分析

嗜铬粒蛋白(CGA)是存在于分泌细胞的由439个氨基酸组成的可溶性蛋白。近年的研究发现CGA的N端具有抗血管收缩、抗细菌和抗真菌的功能。为了寻找高效低毒的抗真菌片段,利用PCR技术扩增了编码人嗜铬粒蛋白N端1-76位氨基酸（CGA1-76）的DNA片段,并将之克隆进本实验构建的枯草杆菌诱导型表达载体pSBPTQ,获得含CGA1-76基因的重组质粒pSVTQ,转化蛋白酶三缺陷的枯草杆菌DB403。经蔗糖诱导后,CGA1-76片段在枯草杆菌DB403（pSVTQ）中获得表达,产物分泌到细胞外,分泌量约为5mg/L,占总分泌蛋白的133% 。测试了表达产物对几种丝状真菌和酵母的抑制作用,发现在4μmol/L的浓度下,枯草杆菌表达的重组CGA1-76对镰刀菌、链格孢霉及白假丝酵母有明显的抑制作用。     

General Stress Protein CTC from Bacillus subtilis Specifically Binds to Ribosomal 5S RNA

Two recombinant proteins of the CTC family were prepared: the general stress protein CTC from Bacillus subtilis and its homolog from Aquifex aeolicus. The general stress protein CTC from B. subtilis forms a specific complex with 5S rRNA and its stable fragment of 60 nucleotides, which contains internal loop E. The ribosomal protein TL5 from Thermus thermophilus, which binds with high affinity to 5S rRNA in the loop E region, was also shown to replace the CTC protein from B. subtilis in its complexes with 5S rRNA and its fragment. The findings suggest that the protein CTC from B. subtilis binds to the same site on 5S rRNA as the protein TL5. The protein CTC from A. aeolicus, which is 50 amino acid residues shorter from the N-terminus than the proteins TL5 from T. thermophilus and CTC from B. subtilis, does not interact with 5S rRNA.     

Residues in the N-Terminal Domain of MutL Required for Mismatch Repair in Bacillus subtilis

Mismatch repair is a highly conserved pathway responsible for correcting DNA polymerase errors incorporated during genome replication. MutL is a mismatch repair protein known to coordinate several steps in repair that ultimately results in strand removal following mismatch identification by MutS. MutL homologs from bacteria to humans contain well-conserved N-terminal and C-terminal domains. To understand the contribution of the MutL N-terminal domain to mismatch repair, we analyzed 14 different missense mutations in Bacillus subtilis MutL that were conserved with missense mutations identified in the human MutL homolog MLH1 from patients with hereditary nonpolyposis colorectal cancer (HNPCC). We characterized missense mutations in or near motifs important for ATP binding, ATPase activity, and DNA binding. We found that 13 of the 14 missense mutations conferred a substantial defect to mismatch repair in vivo, while three mutant alleles showed a dominant negative increase in mutation frequency to wild-type mutL. We performed immunoblot analysis to determine the relative stability of each mutant protein in vivo and found that, although most accumulated, several mutant proteins failed to maintain wild-type levels, suggesting defects in protein stability. The remaining missense mutations located in areas of the protein important for DNA binding, ATP binding, and ATPase activities of MutL compromised repair in vivo. Our results define functional residues in the N-terminal domain of B. subtilis MutL that are critical for mismatch repair in vivo.     

Protein Connectivity in Chemotaxis Receptor Complexes

The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.     

Engineering a More Thermostable Blue Light Photo Receptor Bacillus subtilis YtvA LOV Domain by a Computer Aided Rational Design Method

The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the T _m of the flavin mononucleotide based fluorescent protein Bacillus Subtilis YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.