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The widely conserved protein CsrA (carbon storage regulator A) globally regulates bacterial gene expression at the post-transcriptional level. In many species, CsrA activity is governed by untranslated sRNAs, CsrB and CsrC in Escherichia coli, which bind to multiple CsrA dimers, sequestering them from lower affinity mRNA targets. Both the synthesis and turnover of CsrB/C are regulated. Their turnover requires the housekeeping endonuclease RNase E and is activated by the presence of a preferred carbon source via the binding of EIIA(Glc) of the glucose transport system to the GGDEF-EAL domain protein CsrD. We demonstrate that the CsrB 3' segment contains the features necessary for CsrD-mediated decay. RNase E cleavage in an unstructured segment located immediately upstream from the intrinsic terminator is necessary for subsequent degradation to occur. CsrA stabilizes CsrB against RNase E cleavage by binding to two canonical sites adjacent to the necessary cleavage site, while CsrD acts by overcoming CsrA-mediated protection. Our genetic, biochemical and structural studies establish a molecular framework for sRNA turnover by the CsrD-RNase E pathway. We propose that CsrD evolution was driven by the selective advantage of decoupling Csr sRNA decay from CsrA binding, connecting it instead to the availability of a preferred carbon source.

DOI： 10.1093/nar/gkw484

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Biological control, especially with Bacillus-based biocontrol agents, offers an attractive alternative to synthetic pesticides for sustainable management of white mold disease caused by Sclerotinia sclerotiorum. In this study, eight effective Bacillus isolates were isolated from rhizospheric soil samples as potential bacterial biocontrol agents. Cultural, biochemical, and molecular analyses of 16S rDNA and gyrase subunit A (gyrA) confirmed that all isolates were identified as Bacillus amyloliquefaciens subsp. plantarum. The production of hydrolytic enzymes and the plant growth-promotional attributes of these isolates confirmed their multifaceted potential. Molecular analysis of the eight biosynthetic genes, which are related to antibiotic properties of bacilli, revealed that all of the isolates possess five genes: bacA for bacilysin, dfnM for difficidin, fenA for fengycin, ituA for iturin, and sfp for surfactin. The Bacillus isolates inhibited mycelial growth and suppressed formation of sclerotia during an in vitro test against S. sclerotiorum. Deformities and cell-wall lysis of mycelia, abnormalities of apothecia, and germination failure of ascospores through interaction with the Bacillus isolates were observed with light and scanning electron microscopes, suggesting that they have high antagonistic effects against S. sclerotiorum. Seed bacterization with the Bacillus isolates protected mustard seedlings in vitro up to 98 % against S. sclerotiorum. In a pot experiment, damages of mustard plants against the pathogen decreased up to 90 % after foliar spray of the Bacillus isolates. In addition, the isolates increased seed germination and accelerated seedling vigor of mustard, suggesting that they have plant growth-promoting functions.

DOI： 10.1007/s13313-016-0397-4

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Serratia marcescens 2170 produces three different types of chitinases and chitin-binding protein CBP21. We found that transposon insertion into the 5 untranslated region (5 UTR) of chiPQ-ctb led to defective chitinase and CBP21 production. ChiX small RNA possessed the complementary sequence of the 5 UTRs of the chiPQ-ctb and chiR and repressed the expression of chiP and chiR. ChiX was detected in a medium containing glucose, glycerol, GlcNAc, and (GlcNAc)(2), but the expression of both chiP and chiR was only observed in a medium containing (GlcNAc)(2). chiX mutant produced chitinases, CBP21, and chitobiase without induction. chiP transcripts were more abundant than those of chiR or chiX in a medium containing (GlcNAc)(2). These results suggest that the constitutively expressed ChiX binds to the highly abundant chiP 5 UTR, thereby leading to the induction of chiR mRNA translation and the subsequent expression of chitinases and CBP21.

DOI： 10.1080/09168451.2015.1083399

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Chitinase B from Serratia marcescens 2170 is one of the processive chitinases, and it has a linear path of aromatic amino acid residues on the surface and in the catalytic cleft. There are four surface-exposed residues lined-up towards the cleft, Y481, W479, W252, and Y240. The substitution of these residues with alanine causes a decrease in both the extent of the substrate binding and the hydrolytic activity (Katouno et al., 2004). Here, we examine the three mutants without losing the substrate-binding ability, Y240W, Y481W, and Y240W/Y481W. These mutants were prepared for a detailed analysis of the functions of Y240 and Y481, which showed a lower contribution to substrate binding than W479 and W252. The parameters for the binding of the three mutants to crystalline beta-chitin were similar to those for the wild type. The hydrolytic activity of Y240W and Y240W/Y481W against crystalline beta-chitin was significantly decreased. However, the hydrolytic activity of Y481W was similar to that of the wild type, indicating some differences in the roles of Y240 and Y481 during the processive degradation of crystalline beta-chitin. Taken together with the previous results, it was suggested that while Y240 and Y481 were required for the substrate binding, Y240 had additional roles in the processive degradation of crystalline beta-chitin, possibly in guiding a chitin chain into the catalytic cleft.

DOI： 10.2323/jgam.61.255

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Processivity refers to the ability of synthesizing, modifying and degrading enzymes to catalyse multiple successive cycles of reaction with polymeric substrates without disengaging from the substrates. Since biomass polysaccharides, such as chitin and cellulose, often form recalcitrant crystalline regions, their degradation is highly dependent on the processivity of degrading enzymes. Here we employ high-speed atomic force microscopy to directly visualize the movement of two processive glycoside hydrolase family 18 chitinases (ChiA and ChiB) from the chitinolytic bacterium Serratia marcescens on crystalline beta-chitin. The half-life of processive movement and the velocity of ChiA are larger than those of ChiB, suggesting that asymmetric subsite architecture determines both the direction and the magnitude of processive degradation of crystalline polysaccharides. The directions of processive movements of ChiA and ChiB are observed to be opposite. The molecular mechanism of the two-way traffic is discussed, including a comparison with the processive cellobiohydrolases of the cellulolytic system.

DOI： 10.1038/ncomms4975

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In order to elucidate the roles of ChiP, ChiQ, and ChiX in chitin utilization by Serratia marcescens 2170, the construction of single-gene deletion mutants of the chiP, chiQ, and chiX genes was attempted by allelic exchange mutagenesis. Delta chiP formed smaller clearing zones and Delta chiX formed larger ones than wild-type 2170 on an agar plate containing colloidal chitin. Delta chiP grew slowly on the lower concentration of (GlcNAc)(2), and there was essentially no growth on chitin oligosaccharides larger than (GlcNAc)(3). The gene product of chiP was detected in the outer membrane fraction, consistently with the hypothesis that chiP encodes outer membrane chitoporin. Deletion of chiQ decreased and that of chiX increased the growth rates on chitin oligosaccharides. These observations strongly suggest that all three genes are involved in chitin utilization and that the deletion mutants obtained in this study might prove useful tools to clarify the details of the chitin utilization system of this bacterium.

DOI： 10.1080/09168451.2014.882755

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The carbon storage regulator (Csr) global regulatory system is conserved in many eubacteria and coordinates the expression of various genes that facilitate adaptation during the major physiological growth phase. The Csr system in Escherichia coli comprises an RNA-binding protein, CsrA; small non-coding RNAs, CsrB and CsrC; and a decay factor for small RNAs, CsrD. In this study, we identified the Csr system in Serratia marcescens 2170. S. marcescens CsrA was 97% identical to E. coli CsrA. CsrB and CsrC RNAs had typical stem-loop structures, including a GGA motif that is the CsrA binding site. CsrD was composed of N-terminal two times transmembrane region and HAMP-like, GGDEF, and EAL domains. Overexpression of S. marcescens csr genes complemented the phenotype of E. coli csr mutants. S. marcescens CsrD affected the decay of CsrB and CsrC RNAs in E. coli. These results suggest that the Csr system in S. marcescens is composed of an RNA-binding protein, two Csr small RNAs, and a decay factor for Csr small RNAs.

DOI： 10.2323/jgam.60.79

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Chitinase A1 (ChiA1) from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III domains, and a C-terminal chitin-binding domain (ChBD). The ChBD of ChiA1 (ChBD(ChiA1)) belongs to carbohydrate-binding module (CBM) family 12 and specifically binds to insoluble or crystalline chitin. It has been suggested that tryptophan-687 (Trp687) is involved in the chitin-binding activity of this ChBD. Site-directed mutagenesis was used to identify additional amino acid residues required for chitin-binding activity of this domain. Furthermore, a total of 14 amino acid residues in ChBD(ChiA1) were carefully selected, and it was found that mutation of Gln679, which is not well-conserved in CBM family 12, significantly decreased the binding activity to colloidal chitin. A nuclear magnetic resonance study demonstrated that neither the Q679A nor the W687A mutation altered the overall structure of ChBD(ChiA1). Therefore, Gln679 was identified as a new residue that is involved in the chitin-binding activity of ChBD(ChiA1) in addition to Trp687. However, the mechanism of chitin binding by ChBD is still unknown.

DOI： 10.1093/jb/mvt043

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Serratia marcescens 2170 produces three chitinases and the chitin-binding protein CBP21, and efficiently degrades insoluble and crystalline chitin. The three chitinases and CBP21 are induced by N,N'-diacetylchitobiose [(GlcNAc)(2)], the major product of chitin hydrolysis by S. marcescens chitinases. We have found that uptake of both (GlcNAc)(2) and N-acetylglucosamine (GlcNAc) is important for the efficient utilization of (GlcNAc)(2) because (GlcNAc)(2) is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. In order to determine the mechanism of utilization of the degradation products of chitin by S. marcescens, chitobiase deficient transposon mutants were screened. A transposon present in chitobiase-deficient mutants was inserted into the ybfMN-ctb cluster. The mutants produced chitinases, except for TT327, in which a transposon was inserted into the 5'-untranslated region (5'-UTR) of ybfM. Ectopic expression of this region in TT327 restored chitinase production. These results indicate that the 5'-UTR of ybfM is important for chitinase induction in S. marcescens.

DOI： 10.1271/bbb.120403

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Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH2-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity.

DOI： 10.1074/jbc.M112.399667

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Aspergillus oryzae AB390, a derivative of A. oryzae OR101, was found to be suitable for soy sauce production, yielding a product light brown in color. Compared to the parent strain, hemicellulase and cellulase activities in the mutant were higher; however, its amylase activity was found to be much lower. To determine the cause of these differences, the enzymatic profile change, as a function of the carbon source in submerged cultures, was examined. Amylase activity in AB390 was hardly detectable and not affected by the carbon source utilized. In the absence of starch where glucose could not be generated, hemicellulase and cellulase activities in both the parent and mutant were the same. A nonsense mutation was found in the upstream region of the putative transactivation domain of the transcriptional activator of the amylolytic genes, amyR in AB390. Complementation of AB390 with the wild-type amyR reduced hemicellulase and cellulase activities and increased amylase activity in soy sauce koji, the mold responsible for giving soy sauce. Northern analysis and two-dimensional (2-D) electrophoresis indicated that the unique enzymatic profile of AB390 was regulated transcriptionally. The results suggested that the loss of amyR function indirectly affected the production of hemicellulolytic and cellulolytic enzymes, likely through a carbon catabolite repression-mediated control. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2010.12.006

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The rice class I chitinase OsChia1b, also referred to as RCC2 or Cht-2, is composed of an N-terminal chitin-binding domain (ChBD) and a C-terminal catalytic domain (CatD), which are connected by a proline- and threonine-rich linker peptide. Because of the ability to inhibit fungal growth, the OsChia1b gene has been used to produce transgenic plants with enhanced disease resistance. As an initial step toward elucidating the mechanism of hydrolytic action and antifungal activity, the full-length structure of OsChia1b was analyzed by X-ray crystallography and small-angle X-ray scattering (SAXS). We determined the crystal structure of full-length OsChia1b at 2.00-angstrom resolution, but there are two possibilities for a biological molecule with and without interdomain contacts. The SAXS data showed an extended structure of OsChia1b in solution compared to that in the crystal form. This extension could be caused by the conformational flexibility of the linker. A docking simulation of ChBD with tri-N-acetylchitotriose exhibited a similar binding mode to the one observed in the crystal structure of a two-domain plant lectin complexed with a chitoo-ligosaccharide. A hypothetical model based on the binding mode suggested that ChBD is unsuitable for binding to crystalline alpha-chitin, which is a major component of fungal cell walls because of its collisions with the chitin chains on the flat surface of alpha-chitin. This model also indicates the difference in the binding specificity of plant and bacterial ChBDs of GH19 chitinases, which contribute to antifungal activity. Proteins 2010; 78:2295-2305. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/prot.22742

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To improve the transformation efficiency of Zygosaccharomyces rouxii by electroporation, glycerol was added to the electroporation buffer and the cells were frozen at -80 degrees C. These alterations drastically increased transformation efficiency, and we found that competent cells can be preserved at -80 degrees C without decreasing their transformation efficiency for at least 30d.

DOI： 10.1271/bbb.90865

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N,N'-diacetylchitobiose [(GlcNAC)(2)] is the main degradation product from chitin by the action of chitinases of Serratia marcescens 2170. Uptake of (GlcNAC)(2) via a (GlcNAC)(2)-specific enzyme II permease by this bacterium has been demonstrated previously. Here, we report the contribution of chitobiase and N-acetylglucosamine (GlcNAc) uptake to the utilization of (GlcNAC)(2). When S. marcescens 2170 was cultivated in a medium containing chitin, chitobiase activity was detected both inside and outside the cells; intracellular chitobiase was more abundant and suggested to be mainly located in the periplasm. Production of chitobiase was induced by GlcNAc and more effectively by (GlcNAC)(2). For induction of chitobiase, uptake of (GlcNAC)(2) was essential but ChiR, an essential regulator of chitinase induction, was not required. S. marcescens 2170 grew well on both GlcNAc and (GlcNAC)(2) but mutants defective in either chitobiase or NagE, the GlcNAc-specific enzyme II permease, showed reduced growth on (GlcNAc)(2). These results suggest that, in addition to uptake as (GlcNAC)(2), a proportion of the (GlcNAC)(2) is converted to GlcNAc by chitobiase, mainly in the periplasm, and incorporated into the cytoplasm by NagE. The mutant defective in chitobiase grew more slowly on (GlcNAC)(2) than on GlcNAc, indicating that (GlcNAC)(2) is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. Therefore, uptake as both (GlcNAc)(2) and GlcNAc is important for efficient utilization of (GlcNAC)(2) in S. marcescens.

DOI： 10.1099/mic.0.2007/016246-0

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In the three-dimensional structure of a rice class I chitinase (OsChia1b) determined recently, a loop structure (loop II) is located at the end of the substrate-binding cleft, and is thus suggested to be involved in substrate binding. In order to test this assumption, deletion of the loop II region from the catalytic domain of OsChia1b and replacement of Trp159 in loop II with Ala were carried out. The loop II deletion and the W159A mutation increased hydrolytic activity not only towards (GlcNAc)(6) but also towards polysaccharide substrates. Similar results were obtained for k(cat)/K-m values determined for substrate reduced-(GlcNAc)(5). The two mutations shifted the splitting positions in (GlcNAc)(6) to the reducing end side, but the shift was less intensive in the Trp mutant. Theoretical analysis of the reaction time course indicated that sugar residue affinity at the +3 subsite was reduced from -2kcal/mol to +0.5 kcal/mol by loop II deletion. Reduced affinity at the +3 subsite might enhance the release of product fragments, resulting in higher turnover and higher enzymatic activities. Thus, we concluded that loop II is involved in sugar residue binding at the +3 subsite, but that Trp159 itself appears to contribute only partly to sugar residue interaction at the subsite.

DOI： 10.1093/jb/mvn004

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To determine the properties and structure of OsChia1b, a family 19 chitinase from Oryza sativa L. cv. Nipponbare (japonica sp.), recombinant OsChia1b was produced in Esherichia coli cells and purified to homogeneity by chitin affinity column chromatography. OsChia1b was highly active against soluble chitinous substrate, but not against crystalline chitin, and clearly inhibited hyphal extension of Trichoderma reesei.

DOI： 10.1271/bbb.706931

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The barA and uvrY genes of Escherichia coli encode a two-component sensor kinase and a response regulator, respectively. Although this system plays a major role in the regulation of central carbon metabolism, motility, and biofilm formation by controlling the expression of the CsrB and CsrC noncoding RNAs, the environmental conditions and the physiological signal(s) to which it responds remain obscure. In this study, we explored the effect of external pH on the activity of BarA/UvrY. Our results indicate that a pH lower than 5.5 provides an environment that does not allow activation of the BarA/UvrY signaling pathway.

DOI： 10.1128/JB.01052-06

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The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine- scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA'-'lacZ, flhDC'-'lacZ, and pgaA'-'lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (beta(1), residues 2-7) and containing the last (beta(5), residues 40-47) beta-strands of CsrA. The beta(1) and beta(5) strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.

DOI： 10.1074/jbc.M606057200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

In Escherichia coli, the global regulatory protein CsrA (carbon store regulator A) binds to leader segments of target mRNAs, affecting their translation and stability. CsrA activity is regulated by two noncoding RNAs, CsrB and CsrC, which act by sequestering multiple CsrA dimers. Here, we describe a protein (CsrD) that controls the degradation of CsrB/CRNAs. The dramatic stabilization of CsrB/C RNAs in a csrD mutant altered the expression of CsrA-controlled genes in a manner predicted from the previously described Csr regulatory circuitry. A deficiency in RNase E, the primary endonuclease involved in mRNA decay, also stabilized CsrB/C, although the half-lives of other RNAs that are substrates for RNase E (rpsO, rpsT, and RyhB) were unaffected by csrD. Analysis of the decay of CsrB RNA, both in vitro and in vivo, suggested that CsrD is not a ribonuclease. Interestingly, the CsrD protein contains GGDEF and EAL domains, yet unlike typical proteins in this large superfamily, its activity in the regulation of CsrB/C decay does not involve cyclic di-GMP metabolism. The two predicted membrane-spanning regions are dispensable for CsrD activity, while HAMP-like, GGDEF, and EAL domains are required. Thus, these studies demonstrate a novel process for the selective targeting of RNA molecules for degradation by RNase E and a novel function for a GGDEF-EAL protein.

DOI： 10.1101/gad.1461606

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The RNA-binding protein CsrA represses biofilm formation, while the non-coding RNAs CsrB and CsrC activate this process by sequestering CsrA. We now provide evidence that the pgaABCD transcript, required for the synthesis of the polysaccharide adhesin PGA (poly-beta-1,6-N-acetyl-D-glucosamine) of Escherichia coli, is the key target of biofilm regulation by CsrA. csrA disruption causes an approximately threefold increase in PGA production and an approximately sevenfold increase in expression of a pgaA'-'lacZ translational fusion. A Delta csrB Delta csrC mutant exhibits a modest decrease in pgaA'-'lacZ expression, while the response regulator UvrY, a transcriptional activator of csrB and csrC, stimulates this expression. Biofilm formation is not regulated by csrA, csrB or uvrY in a Delta pgaC mutant, which cannot synthesize PGA. Gel mobility shift and toeprint analyses demonstrate that CsrA binds cooperatively to pgaA mRNA and competes with 30S ribosome subunit for binding. CsrA destabilizes the pgaA transcript in vivo. RNA footprinting and boundary analyses identify six apparent CsrA binding sites in the pgaA mRNA leader, the most extensive arrangement of such sites in any mRNA examined to date. Substitution mutations in CsrA binding sites overlapping the Shine - Dalgarno sequence and initiation codon partially relieve repression by CsrA. These studies define the crucial mechanisms, though not the only means, by which the Csr system influences biofilm formation.

DOI： 10.1111/j.1365-2958.2005.04648.x

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CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia colt genomic sequence for other genes that might be regulated by CsrA. cstA contained an exact match that overlapped its Shine-Dalgarno sequence. cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a cstA'-'lacZ translational fusion in wild-type and csrA mutant strains was examined. Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5- to 10-fold higher when LB was supplemented with glucose. It was previously shown that cstA is regulated by the cyclic AMP (CAMP)-cAMP receptor protein complex and transcribed by Esigma(70). We investigated the influence of sigma(S) on cstA expression and found that a sigma(S) deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated cstA regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA Shine-Dalgarno sequence, as predicted. These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding.

DOI： 10.1128/JB.185.15.4450-4460.2003

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Small untranslated RNAs (sRNAs) perform a variety of important functions in bacteria. The 245 nucleotide sRNA of Escherichia coli , CsrC, was discovered using a genetic screen for factors that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post-transcriptionally regulates central carbon flux, biofilm formation and motility in E. coli . CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both sRNAs possess similar imperfect repeat sequences (18 in CsrB, nine in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is indirectly activated by CsrA via the response regulator UvrY. Because CsrB and CsrC antagonize CsrA activity and depend on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. Homologues of csrC are apparent in several Enterobacteriaceae. The regulatory and evolutionary implications of these findings are discussed.

DOI： 10.1046/j.1365-2958.2003.03459.x

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The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E. coli drastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment.

DOI： 10.1128/JB.185.3.843-853.2003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation in Escherichia coli. CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex. CsrA indirectly activates csrB transcription, in an apparent autoregulatory mechanism. In the present study, we elucidate the intermediate regulatory circuitry of this system. Mutations affecting the BarA/UvrY two-component signal transduction system decreased csrB transcription but did not affect csrA'-'lacZ expression. The uvrYdefect was severalfold more severe than that of barA. Both csrA and urrY were required for optimal barA expression. The latter observation suggests an autoregulatory loop for UvrY. Ectopic expression of uvrY suppressed the csrB-lacZ expression defects caused by uvrY, csrA, or barA mutations; csrA suppressed csrA or barA defects; and barA complemented only the barA mutation. Purified UvrY protein stimulated csrB-lacZ expression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY on csrB transcription. Disruption of sdiA, which encodes a LuxR homologue, decreased the expression of uvrY'-'lacZ and csrB-lacZ fusions but did not affect csrA'-'lacZ. The BarA/UvrY system activated biofilm formation. Ectopic expression of uvrY stimulated biofilm formation by a csrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development. Collectively, these results demonstrate that uvrY resides downstream from csrA in a signaling pathway for csrB and that CsrA stimulates UvrY-dependent activation of csrB expression by BarA-dependent and -independent mechanisms.

DOI： 10.1128/JB.184.18.5130-5140.2002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

The carbon storage regulatory system of Escherichia coli controls the expression of genes involved in carbohydrate metabolism and cell motility. CsrA binding to glgCAP transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay. CsrB RNA functions as an antagonist of CsrA by sequestering this protein and preventing its action. In this paper, we elucidate further the mechanism of CsrA-mediated glgC regulation. Results from gel shift assays demonstrate that several molecules of CsrA can bind to each glgC transcript. RNA footprinting studies indicate that CsrA binds to the glgCAP leader transcript at two positions. One of these sites overlaps the glgC Shine-Dalgarno sequence, whereas the other CsrA target is located further upstream in an RNA hairpin. Results from toeprint and cell-free translation experiments indicate that bound CsrA prevents ribosome binding to the glgC Shine-Dalgarno sequence and that this reduces GlgC synthesis. The effect of two deletions in the upstream binding site was examined. Both of these deletions reduced, but did not eliminate, CsrA binding in vitro and CsrA-dependent regulation in vivo . Our findings establish that bound CsrA inhibits initiation of glgC translation, thereby reducing glycogen biosynthesis. This inhibition of translation probably contributes to destabilization of the glgC transcript that was observed previously.

DOI： 10.1046/j.1365-2958.2002.02982.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

To discover the individual roles of the chitinases from Serratia marcescens 2170, chitinases A, B, and C1 (ChiA, ChiB, and ChiC1) were produced by Escherichia coli and their enzymatic properties as well as synergistic effect on chitin degradation were studied. All three chitinases showed a broad pH optimum and maintained significant chitinolytic activity between pH 4 and 10. ChiA was the most active enzyme toward insoluble chitins, but ChiC1 was the most active toward soluble chitin derivatives among the three chitinases. Although all three chitinases released (GlcNAO(2) almost exclusively from colloidal chitin, ChiB and ChiC1 split (GlcNAc)(6) to (GlcNAc)(3), while ChiA exclusively generated (GlcNAc)(2) and (GlcNAc)(4). Clear synergism on the hydrolysis of powdered chitin was observed in the combination between ChiA and either ChiB or ChiC, and the sites attacked by ChiA on the substrate are suggested to be different from those by either ChiB or ChiC1.

DOI： 10.1271/bbb.66.1075

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with gig genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA'-'lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.

DOI： 10.1128/JB.184.1.290-301.2002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity. We have further examined the regulatory interactions of CsrA and CsrB RNA. The 5' end of the CsrB transcript was mapped, and a csrB::cam null mutant was constructed. CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic csrA, csrB, rpoS, or csrA rpoS mutant strains. CsrA levels exhibited modest or negligible effects of these mutations. The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA. In contrast, CsrB levels were drastically decreased (similar to 10-fold) in the csrA mutants. CsrB transcript stability was unaffected by csrA. The expression of a csrB-lacZ transcriptional fusion containing the region from -242 to +4 bp of the csrB gene was decreased similar to 20-fold by a csrA::kanR mutation in vivo but was unaffected by CsrA protein in vitro. These results reveal a significant, though most likely indirect, role for CsrA in regulating csrB transcription. Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB.

DOI： 10.1128/JB.183.20.6017-6027.2001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

To identify the genes required for chitinase production by Serratia marcescens 2170, various Tn5 mutants somehow defective in chitinase production were isolated in a previous study. In order to identify the mutated gene in one of the chitinase-deficient mutants, N1, DNA regions flanking the Tn5 insertion were cloned and sequenced. Sequence comparison showed that the mutation occurred in the ORF located between chiB and cbp, which encode chitinase B and chitin-binding protein CBP21, respectively. The ORF encodes a 313-amino acid polypeptide which has significant similarity with various LysR-type transcriptional regulators, and thus the gene was designated chiR. Targeted mutagenesis confirmed that disruption of the chiR gene results in the phenotype of N1. Gel mobility shift assays using partially purified ChiR protein demonstrated that this protein specifically binds to the intergenic region between chiR and cbp. These results strongly suggest that ChiR is a LysR-type transcriptional regulator which is essential for production of all chitinases and CBP21.

DOI： 10.1271/bbb.65.338

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Language：English   Publishing type：Research paper (international conference proceedings)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS  

The third chitinase gene (chiC) of Serratia marcescens 2170, specifying chitinases C1 and C2, was identified. Chitinase C1 lacks a signal sequence and consists of a catalytic domain belonging to glycoside hydrolase family 18, a fibronectin type III-like domain (Fn3 domain) and a C-terminal chitin-binding domain (ChBD). Chitinase C2 corresponds to the catalytic domain of C1 and is probably generated by proteolytic removal of the Fn3 and ChBDs. The loss of the C-terminal portion reduced the hydrolytic activity towards powdered chitin and regenerated chitin, but not towards colloidal chitin and glycol chitin, illustrating the importance of the ChBD for the efficient hydrolysis of crystalline chitin. Phylogenetic analysis showed that bacterial family 18 chitinases can be clustered in three subfamilies which have diverged at an early stage of bacterial chitinase evolution. Ser. marcescens chitinase C1 is found in one subfamily, whereas chitinases A and B of the same bacterium belong to another subfamily. Chitinase C1 is the only Ser. marcescens chitinase that has an Fn3 domain. The presence of multiple, divergent, chitinases in a single chitinolytic bacterium is perhaps necessary for efficient synergistic degradation of chitin.

DOI： 10.1042/0264-6021:3430587

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PESTICIDE SCI SOC JAPAN  

Synergistic effects of chitinases A, B and C1, chitin binding protein and prodigiosin from Serratia marcescens on the insecticidal activity of delta-endotoxin, CrylC, of Bacillus thuringiensis against the common cutworm, Spodoptera litura, were investigated. Only prodigiosin showed a potent synergistic activity with CrylC on both the lethal and growth inhibitory activity. In the previous paper (Asano et al., 1999) a synergistic effect in the supernatants of S. marcescens culture on the insecticidal activity of CrylC was described. The supernatants from S. marcescens and partially purified prodigiosin showed a similar synergistic activity on the insecticidal activity of CrylC. The content of prodigiosin in the supernatants was estimated as 10% according to the absorbance at 467 nm where prodigiosin showed a peak and the content seemed to be enough to explain the synergistic activity of the supernatants on the activity of CrylC.

DOI： 10.1584/jpestics.24.381

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PESTICIDE SCI SOC JAPAN  

The supernatants of Serratia marcescens culture was shown to possess an enhancing effect on the larvicidal activity of Bacillus thuringiensis delta-endotoxin (Cry1C) against the common cutworm, Spodoptera litura. The synergistic effect was observed in both larval mortality and growth inhibition. The synergistic activity increased in proportion to the increment either of the supernatant or Cry1C concentration. The addition of supernatants of S. marcescens culture to Cry1C was able to enhance the insecticidal activity of delta-endotoxin over 8 fold compared with that of toxin alone. The synergism was very high to the insecticidal activity against S, litura but not in other three lepidopterous insects tested, i.e., Mamestra brassicae, Plutella xylostella and Adoxophyes honmai. Therefore the synergistic activity of the supernatants of S, marcescens culture seemed to be specific to insect species.

DOI： 10.1584/jpestics.24.44

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

A chitin binding protein (CBP21) 21kDa in size, is a major protein in the culture snpernatant when Serratia marcescens 2170 is grown in the presence of chitin, The gene (cbp) for CBP21 was found to be located in a region 1.5 kb downstream of the chiB gene encoding chitinase B. The cbp gene encodes a polypeptide of 197 amino-acids with a calculated size of 21.6 kDa containing a putative signal sequence of 27 amino acids. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins showed that CBP21 is similar (45.3% amino acid identity) to CHB1 of Streptomyces olivaceoviridis. Purified CBP21 prepared from the periplasmic fraction of Escherichia coli carrying the cloned cbp gene showed its highest binding activity to squid chitin (beta-chitin) followed by colloidal chitin and regenerated chitin. Binding of CBP21 to regenerated chitin was affected by pH, in particular, low pH reduced binding activity markedly. The presence of similar chitin binding proteins in the distantly related microorganisms, Streptomyces and Serratia, suggests a wide distribution of this type of chitin binding protein in chitinolytic microorganisms.

DOI： 10.1271/bbb.62.128

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

To carry out a genetic analysis of the degradation and utilization of chitin by Serratia marcescens 2170, various Tn5 insertion mutants with characteristic defects in chitinase production were isolated and partially characterized. Prior to the isolation of the mutants, proteins secreted into culture medium in the presence of chitin were analyzed. Four chitinases, A, B, C1, and C2, among other proteins, were detected in the culture supernatant of S. marcescens 2170. All four chitinases and a 21-kDa protein (CBP21) lacking chitinase activity shelved chitin binding activity. Cloning and sequencing analysis of the genes encoding Chitinases A and B of strain 2170 revealed extensive similarities to those of other strains of S. marcescens described previously. Tn5 insertion mutagenesis of strain 2170 was carried out, and mutants which formed altered clearing zones of colloidal chitin were selected. The obtained mutants were divided into five classes as follows: mutants with (i) no clearing zones, (ii) fuzzy clearing zones, (iii) large clearing zones, (iv) delayed clearing zones, and (v) small clearing zones. Preliminary characterization suggested that some of these mutants have defects in chitinase excretion, a negatively regulating mechanism of chitinase gene expression, an essential factor for chitinase gene expression, and a structural gene for a particular chitinase. These mutants could allow researchers to identify the genes involved in the degradation and utilization of chitin by S. marcescens 2170.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

DOI： 10.1271/bbb.56.682

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The gene (chiD) encoding the precursor of chitinase D was found to be located immediately upstream of the chiA gene, encoding chitinase A1, which is a key enzyme in the chitinase system of Bacillus circulans WL-12. Sequencing analysis revealed that the deduced polypeptide encoded by the chiD gene was 488 amino acids long and the distance between the coding regions of the chiA and chiD genes was 103 bp. Remarkable similarity was observed between the N-terminal one-third of chitinase D and the C-terminal one-third of chitinase A1. The N-terminal 47-amino-acid segment (named ND) of chitinase D showed a 61.7% amino acid match with the C-terminal segment (CA) of chitinase A1. The following 95-amino-acid segment (R-D) of chitinase D showed 62.8 and 60.6% amino acid matches, respectively, to the previously reported type III-like repeating units R-1 and R-2 in chitinase A1, which were shown to be homologous to the fibronectin type III sequence. A 73-amino-acid segment (residues 247 to 319) located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitinases and class III higher-plant chitinases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha-subunit. The evolutionary and functional meanings of these similarities are discussed.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

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Language：English   Publishing type：Research paper (scientific journal)  

Bacillus circulans WL-12, a yeast and fungal cell wall lytic bacterium, secretes a variety of polysaccharide degrading enzymes into the culture medium. When β-l, 3-glucanase was induced with pachyman, a β-1, 3-glucose polymer obtained from the tree fungus Poria cocus Wolf, six distinct active molecules of the enzyme with different molecular weights were detected in the culture supernatant of this bacterium. Molecular cloning of one of the β-l, 3-glucanase genes into E. coli was achieved by transforming E. coli HB101 cells with recombinant plasmids composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pUC 19. A recombinant plasmid containing 4.4 kb of inserted DNA in the Pst I site of pUC 19, designated as pNT003, conferred the ability to degrade pachyman on E. coli cells. The presence of pNT003 was harmful for E. coli cells and caused cell lysis, especially at higher temperatures of cultivation. β-l, 3-Glucanase activity detected in E. coli was mainly recovered in the periplasmic fraction when cell lysis did not occur. SDS-PAGE analysis revealed that the periplasmic fraction contained four active molecules of β-l, 3-glucanase which corresponded to four of the six active molecules produced by B. circulans WL-12. © 1989 Agricultural Chemical Society of Japan.

DOI： 10.1080/00021369.1989.10869584

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Niigata University  

In many species of bacteria, the Csr (carbon storage regulator) global regulatory system coordinates the expression of various genes. In Escherichia coli, the central component of this system, CsrA, is a RNA-binding protein. The CsrA is a homodimer and binds to leader segments of target mRNAs, affecting their translation and stability. CsrA activity is regulated by two small non-coding RNAs, CsrB and CsrC. These RNAs contain multiple CsrA-binding sequences and act by sequestering CsrA. In this system, CsrA indirectly activates transcription of csrB and csrC through a two-component signal transduction system, BarA/UvrY. Another component of this system, CsrD, controls the degradation of CsrB and CsrC RNAs. CsrD contains GGDEF and EAL signaling domains, however, unlike typical GGDEF and EAL domain proteins, its activity does not involve cyclic di-GMP metabolism. The dramatic stabilization of CsrB and CsrC RNAs in a csrD mutant altered the expression of CsrA-regulated genes. The Csr components, CsrA, CsrB and CsrC RNAs, and CsrD, interact within the autoregulatory circuit that provides a homeostatic mechanism for control of CsrA activity.

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Other Link： http://www.agr.niigata-u.ac.jp/study_report/report/59-02/056-063.pdf

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

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Audience： High school students

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Audience： High school students

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Audience： High school students

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