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.     

Identification of Proteins Involved in the Heat Stress Response of Bacillus cereus ATCC 14579

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42°C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50°C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42°C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.     

NO参与玉米幼苗对盐胁迫的应答

以玉米幼苗为材料,研究盐胁迫下其內源NO含量、NR和NOS活性的变化;NOS专一性抑制剂L-NAME和NR非专一性抑制剂NaN3对玉米幼苗內源NO含量的影响;利用激光共聚焦显微技术观测盐胁迫下玉米幼苗根部NO含量的变化及其分布特点。结果表明,盐胁迫下玉米幼苗根尖和叶片中NO含量有猝发现象,NOS活性也随之显著提高,NR活性则显著降低;L-NAME或NaN3均可降低盐胁迫所引起的玉米幼苗NO水平的增加,L-NAME对NO含量的影响比NaN3更显著。推测,NO参与玉米幼苗对盐胁迫的应答,NOS途径是盐胁迫下玉米幼苗內源NO合成的主要途径。     

Expression of the ςB-Dependent General Stress Regulon Confers Multiple Stress Resistance in Bacillus subtilis

The alternative sigma factor sigmaB of Bacillus subtilis is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation. In this study, we investigated the impact of a null mutation in sigB on the stress and starvation survival of B. subtilis. sigB mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54 degrees C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type. Preloading cells with sigmaB-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt. Exhaustion of glucose or phosphate induced the sigmaB response, but surprisingly, sigmaB did not seem to be required for starvation survival. Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells. The data argue that the expression of sigmaB-dependent genes provides nonsporulated B. subtilis cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.     

A Homolog of Bacillus subtilis Trigger Factor in Listeria monocytogenes Is Involved in Stress Tolerance and Bacterial Virulence

Molecular chaperones play an essential role in the folding of nascent chain polypeptides, as well as in the refolding and degradation of misfolded or aggregated proteins. They also assist in protein translocation and participate in stress functions. We identified a gene, designated tig, encoding a protein homologous to trigger factor (TF), a cytosolic ribosome-associated chaperone, in the genome of Listeria monocytogenes. We constructed a chromosomal Δtig deletion and evaluated the impact of the mutation on bacterial growth in broth under various stress conditions and on pathogenesis. The Δtig deletion did not affect cell viability but impaired survival in the presence of heat and ethanol stresses. We also identified the ffh gene, encoding a protein homologous to the SRP54 eukaryotic component of the signal recognition particle. However, a Δffh deletion was not tolerated, suggesting that Ffh is essential, as it is in Bacillus subtilis and Escherichia coli. Thus, although dispensable for growth, TF is involved in the stress response of L. monocytogenes. The Δtig mutant showed no or very modest intracellular survival defects in eukaryotic cells. However, in vivo it showed a reduced capacity to persist in the spleens and livers of infected mice, revealing that TF has a role in the pathogenicity of L. monocytogenes.     

Collapsin Response Mediator Protein 5 (CRMP5) Induces Mitophagy,Thereby Regulating Mitochondrion Numbers in Dendrites

Degradation of damaged mitochondria by mitophagy is an essential process to ensure cell homeostasis. Because neurons, which have a high energy demand, are particularly dependent on the mitochondrial dynamics, mitophagy represents a key mechanism to ensure correct neuronal function. Collapsin response mediator proteins 5 (CRMP5) belongs to a family of cytosolic proteins involved in axon guidance and neurite outgrowth signaling during neural development. CRMP5, which is highly expressed during brain development, plays an important role in the regulation of neuronal polarity by inhibiting dendrite outgrowth at early developmental stages. Here, we demonstrated that CRMP5 was present in vivo in brain mitochondria and is targeted to the inner mitochondrial membrane. The mitochondrial localization of CRMP5 induced mitophagy. CRMP5 overexpression triggered a drastic change in mitochondrial morphology, increased the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function.     

Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.     

Melanoma Differentiation-Associated Gene 5 (MDA5) Is Involved in the Innate Immune Response to Paramyxoviridae Infection In Vivo

The early host response to pathogens is mediated by several distinct pattern recognition receptors. Cytoplasmic RNA helicases including RIG-I and MDA5 have been shown to respond to viral RNA by inducing interferon (IFN) production. Previous in vitro studies have demonstrated a direct role for MDA5 in the response to members of the Picornaviridae, Flaviviridae and Caliciviridae virus families ((+) ssRNA viruses) but not to Paramyxoviridae or Orthomyxoviridae ((−) ssRNA viruses). Contrary to these findings, we now show that MDA5 responds critically to infections caused by Paramyxoviridae in vivo. Using an established model of natural Sendai virus (SeV) infection, we demonstrate that MDA5^−/− mice exhibit increased morbidity and mortality as well as severe histopathological changes in the lower airways in response to SeV. Moreover, analysis of viral propagation in the lungs of MDA5^−/− mice reveals enhanced replication and a distinct distribution involving the interstitium. Though the levels of antiviral cytokines were comparable early during SeV infection, type I, II, and III IFN mRNA expression profiles were significantly decreased in MDA5^−/− mice by day 5 post infection. Taken together, these findings indicate that MDA5 is indispensable for sustained expression of IFN in response to paramyxovirus infection and provide the first evidence of MDA5-dependent containment of in vivo infections caused by (−) sense RNA viruses.     

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.     

Subcellular Localization of Bacillus subtilis SMC, a Protein Involved in Chromosome Condensation and Segregation

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B. subtilis cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.     

Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis

Purification of poly(3-hydroxybutyrate) depolymerase (EC 3.1.1.75) from Paucimonas lemoignei is complicated because the bacterium produces several isoenzymes which are difficult to separate from each other. The phaZ5 gene of P. lemoignei encoding extracellular poly(3-hydroxybutyrate) depolymerase A was functionally expressed from the constitutive P43 promoter of pWB980 in a multiple protease-negative mutant of Bacillus subtilis (strain WB800) and secreted to the culture medium. The depolymerase (apparent M(r), 42 kDa; 1.9 mg purified protein per liter culture) was purified from cell-free culture fluid to homogenity by applying only one chromatography step in comparison to at least two necessary steps if poly(3-hydroxybutyrate) depolymerases are purified from P. lemoignei. The recombinant depolymerase lacked any carbohydrate content in contrast to the glycosylated depolymerase of the wild-type. Glycosylation was not essential for activity but enhanced the thermal stability of the enzyme at high temperature. Overexpression of poly(3-hydroxybutyrate) depolymerase in B. subtilis is more efficient than in Escherichia coli.     

Long-term Exposure to Low Lithium Concentrations Stimulates Proliferation,Modifies Stress Protein Expression Pattern and Enhances Resistance to Oxidative Stress in SH-SY5Y Cells

SH-SY5Y cells, derived from a human neuroblastoma, were submitted to short- or long-term exposures to lithium carbonate concentrations ranging from 0.5 to 8 mM. Short-term exposures (4 days) to concentrations higher than 6 mM were found to reduce cell growth rate while exposure to 8 mM resulted in significant cell mortality. These ranges of concentrations induced an overexpression of (1) the HSP27 stress protein, (2) a 108 kDa protein (P108) recognized by an anti-phospho-HSP27(Ser78) antibody, and probably corresponding to a phosphorylated HSP27 tetramer, (3) a 105 kDa protein (P105), possible glycosylated or phosphorylated form of the GRP94 stress protein and (4) a phosphorylated (inactivated) form of glycogen synthase kinase (GSK3α/β) SH-SY5Y cells, when cultured in the presence of 0.5 mM lithium for 25 weeks, displayed interesting features as compared to controls: (1) higher cell growth rate, (2) increased resistance toward the inhibitory effects of high lithium concentrations on cell proliferation, (3) lower basal level of lipid peroxidation (TBARS) and improved tolerance to oxidative stress induced by high lithium concentrations, (5) reduced expression of monomeric HSP27 versus an increase of corresponding tetrameric protein (P108) and (6) overexpression of a 105 kDa protein (P105). In conclusion, our study suggests that chronic treatment (over several months) by therapeutic relevant lithium concentrations could favour neurogenesis, decrease the vulnerability of neuronal cells to oxidative stress and induce posttranslational changes of molecular chaperones.     

Structural Changes in the Nucleoid of Bacillus subtilis at Low Temperature

The external shape of the nucleoid of Bacillus subtilis strain w23 was examined with a new electron microscopic technique, the rapid freezing and substitution fixation method. The nucleoid of the log and stationary phase cells was recognized as an area devoid of ribosomes and widely dispersed in the cytoplasm, which was different from that observed in OsO₄-fixed cells. If the bacteria were exposed to low temperatures (0 to 10 C), the nucleoid showed a highly concentrated shape in the middle of the cytoplasm. These structural changes were observed only when the bacteria were maintained in a high-salt buffer. The results are discussed in relation to the membrane fluidity at low temperature.