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PubMed1997

The rap and hor proteins of Erwinia, Serratia and Yersinia: a novel subgroup in a growing superfamily of proteins regulating diverse physiological processes in bacterial pathogens.

The enteric bacterium Serratia marcescens is an opportunistic human pathogen. The strain ATCC39006 makes the red pigment, prodigiosin (Pig), and the beta-lactam antibiotic carbapenem (Car). Mutants were isolated that were concomitantly defective for Pig and Car production. These mutants were found to have a mutation in the rap gene (Regulation of Antibiotic and Pigment). Sequence analysis of the rap gene revealed a predicted protein product showing strong homology to SlyA, originally thought to be a haemolytic virulence determinant in Salmonella typhimurium. Homologues of rap were detected in several bacterial genera, including Salmonella, Yersinia, Enterobacter, and species of the plant pathogen, Erwinia. The Erwinia hoeEr (homologue of rap) and the Yersinia horYe genes were also found to be very similar to rap and slyA. Marker exchange mutagenesis of horEr revealed that it encoded a regulatory protein controlling the production of antibiotic and exoenzyme virulence determinants in the phytopathogen, Erwinia carotovora subspecies carotovora. We have shown that these new homologues of SlyA form a highly conserved subgroup of a growing superfamily of bacterial regulatory proteins controlling diverse physiological processes in human, animal and plant pathogens.

Amino Acid Sequence↗
PubMed1997

Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel beta-lactam resistance mechanism.

Members of two genera of Gram-negative bacteria, Serratia and Erwinia, produce a beta-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have reported previously the cloning and sequencing of the genes responsible for production of this carbapenem in Erwinia carotovora. These genes are organized as an operon, carA--H, and are controlled by a LuxR-type transcriptional activator, encoded by the linked carR gene. We report in this paper the genetic dissection of this putative operon to determine the function of each of the genes. We demonstrate by mutational analysis that the products of the first five genes of the operon are involved in the synthesis of the carbapenem molecule. Three of these, carABC, are absolutely required. In addition, we provide evidence for the existence of a novel carbapenem resistance mechanism, encoded by the CarF and carG genes. Both products of these overlapping and potentially translationally coupled genes have functional, N-terminal signal peptides. Removal of these genes from the Erwinia chromosome results in a carbapenem-sensitive phenotype. We assume that these novel beta-lactam resistance genes have evolved in concert with the biosynthetic genes to ensure 'self-resistance' in the Erwinia carbapenem producer.

Amino Acid Sequence↗
PubMed1998

The hexA gene of Erwinia carotovora encodes a LysR homologue and regulates motility and the expression of multiple virulence determinants.

We have identified a gene important for the regulation of exoenzyme virulence factor synthesis in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc) and virulence and motility in Erwinia carotovora ssp. atroseptica (Eca). This gene, hexA (hyperproduction of exoenzymes), is a close relative of the Erwinia chrysanthemi (Echr) gene pecT and encodes a member of the LysR family of transcriptional regulators. hexA mutants in both Ecc and Eca produce abnormally high levels of the exoenzyme virulence factors pectate lyase, cellulase and protease. In addition, Eca hexA mutants show increased expression of the fliA and fliC genes and hypermotility. Consistent with a role as a global regulator, expression of hexA from even a low-copy plasmid can suppress exoenzyme production in Ecc and Eca and motility in Eca. Production of the quorum-sensing pheromone OHHL in Ecc hexA is higher throughout the growth curve compared with the wild-type strain. Overexpression of Ecc hexA also caused widespread effects in several strains of the opportunistic human pathogen, Serratia. Low-copy hexA expression resulted in repression of exoenzyme, pigment and antibiotic production and repression of the spreading phenotype. Finally, mutations in hexA were shown to increase Ecc or Eca virulence in planta.

4-Butyrolactone↗
PubMed1998

Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites.

The enterobacterium Erwinia carotovora ssp. carotovora strain 71 (hereafter Ecc71) produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh) and protease (Prt). These enzymes degrade plant cell wall components and are largely responsible for the elicitation of soft-rot diseases in plants and plant products. Ecc71 also produces HarpinEcc, the elicitor of hypersensitive reaction (HR) and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (OHL). OHL controls extracellular enzyme and HarpinEcc production. The levels of these enzymes, as well as the expression of hrpNEcc, the structural gene for HarpinEcc, and ohll, the gene specifying OHL synthesis, are negatively regulated by RsmaA. rsmB, formerly aepH, on the other hand, positively regulates extracellular enzyme production. 6His-RsmA recombinant protein purified from E. coli binds rsmB RNA as indicated by gel mobility shift assays. rsmB comprises 547 bp DNA, which is transcribed from a single start site immediately after a sigma70-like promoter. In Ecc71, two rsmB RNA species are detected: a full-length 479 base rsmB RNA and a 259 base rsmB' RNA. rsmB' DNA hybridizes with the 259 base and the 479 base transcripts. A 3' RNase protection assay revealed that the 259 base and the 479 base RNA species end at the same position immediately after the putative rho-independent terminator. The expression of rsmB-lacZ transcriptional fusions established that the rsmB' RNA is not produced because of the activation of an internal promoter. These data strongly suggest that the 259 base rsmB' RNA is derived by processing of the primary rsmB RNA. In Ecc71, rsmB' expression driven by the lac promoter causes overproduction of Pel, Peh, Cel and Prt, and accumulation of pel-1, peh-1, hrpNEcc and ohll transcripts. By contrast, a plasmid with the rsmB' DNA sequence deleted fails to cause overproduction of the extracellular enzymes in Ecc71. The rsmB' effect also occurs in Escherichia coli as glycogen accumulation is stimulated in the presence of rsmB'. In vivo and in vitro translation as well as mutational analysis of rsmB' have established that rsmB' RNA does not yield a translational product. Therefore, we concluded that the rsmB' RNA itself functions as the regulator. Indeed, the expression rsmB' DNA leads to neutralization of the negative effects of the RNA-binding protein, RsmA, in Ecc71 and Serratia marcescens strain SM274. We propose a model that explains how RsmA and rsmB control the expression of genes for extracellular enzymes.

Bacterial Outer Membrane Proteins↗
PubMed1998

Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB.

Csr (carbon storage regulator) is a recently discovered global regulatory system that controls bacterial gene expression post-transcriptionally. Its effector is a small RNA-binding protein referred to as CsrA or, in phytopathogenic Erwinia species, RsmA (repressor of stationary phase metabolites). Numerous genes whose expression occurs in the stationary phase of growth are repressed by csrA/rsmA, and csrA activates certain exponential-phase metabolic pathways. Glycogen synthesis and catabolism, gluconeogenesis, glycolysis, motility, cell surface properties and adherence are modulated by csrA in Escherichia coli, while the production of several secreted virulence factors, the plant hypersensitive response elicitor HrpN(Ecc) and, potentially, other secondary metabolites are regulated by rsmA in Erwinia carotovora. CsrA represses glycogen synthesis by binding to and destabilizing glgCAP mRNA and is hypothesized to repress other genes by a similar mechanism. The second component of the Csr system is CsrB (AepH in Erwinia species), a non-coding RNA molecule that forms a large globular ribonucleoprotein complex with approximately 18 CsrA subunits and antagonizes the effects of CsrA in vivo. Highly repeated sequence elements found within the loops of predicted stem-loops and other single-stranded segments of CsrB RNA may facilitate CsrA binding. Current information supports a model in which CsrA exists in an equilibrium between CsrB and CsrA-regulated mRNAs, which predicts that CsrB levels may be a key determinant of CsrA activity in the cell. The presence of csrA homologues in phylogenetically diverse species further suggests that this novel kind of regulatory system is likely to play a broad role in modulating eubacterial gene expression.

Amino Acid Sequence↗
PubMed1998

Characterization of the Erwinia chrysanthemi expI-expR locus directing the synthesis of two N-acyl-homoserine lactone signal molecules.

The plant pathogen Erwinia chrysanthemi produces three acyl-homoserine lactones (acyl-HSLs). One has been identified as N-(3-oxohexanoyl)-homoserine lactone (OHHL), and the two others were supposed to be N (hexanoyl)-homoserine lactone (HHL) and N-(decanoyl)-homoserine lactone (DHL). The genes for a quorum-sensing signal generator (expI) and a response regulator (expR) were cloned. These genes are convergently transcribed and display high similarity to the expI-expR genes of Erwinia carotovora. ExpI is responsible for both OHHL and HHL production. Inactivation of expl had little effect on pectinase synthesis in E. chrysanthemi, as expression of only two of the pectate lyase genes, pelA and pelB, was decreased. E. chrysanthemi expR mutants still produced acyl-HSL and pectinases. However, gel shift and DNAse I footprinting experiments showed that the purified E. chrysanthemi ExpR protein binds specifically to the promoter regions of the five major pel genes. Addition of OHHL modified the ExpR-DNA bandshift profiles, indicating that ExpR interacts with OHHL and binds to DNA in different ways, depending on the OHHL concentration. Localization of the ExpR binding sites just upstream of promoter regions suggests that ExpR functions as an activator of pel expression in the presence of OHHL. The absence of a phenotype in expR mutants strongly suggests that at least an additional interchangeable ExpR homologue exists in E. chrysanthemi. Finally, transcription of expI::uidA and expR::uidA fusions is dependent on the population density, suggesting the existence of a quorum-sensing hierarchy in E. chrysanthemi. These results suggest that the expI-expR locus is part of a complex autoregulatory system that controls quorum sensing in E. chrysanthemi.

4-Butyrolactone↗
PubMed2000

Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control.

Serratia sp. ATCC 39006 produces the carbapenem antibiotic, carbapen-2-em-3-carboxylic acid and the red pigment, prodigiosin. We have previously reported the characterization of a gene, carR, controlling production of carbapenem in this strain. We now describe further characterization of the carR locus to locate the genes encoding carbapenem biosynthetic and resistance functions. A novel family of diverse proteins showing sequence similarity to the C-terminal domain of CarF (required for carbapenem resistance) is described. We also report the isolation of the locus involved in the biosynthesis of the red pigment, prodigiosin. A cosmid containing approximately 35 kb of the Serratia chromosome encodes synthesis of the pigment in the heterologous host, Erwinia carotovora, demonstrating, for the first time, that the complete prodigiosin biosynthetic gene cluster had been cloned and functionally expressed. We report the isolation of a third locus in Serratia, containing convergently transcribed genes, smaI and smaR, encoding LuxI and LuxR homologues respectively. SmaI directs the synthesis of N-acyl homoserine lactones involved in the quorum sensing process. We demonstrate that biosynthesis of the two secondary metabolites, carbapenem antibiotic and prodigiosin pigment, is under pheromone-mediated transcriptional regulation in this bacterium. Finally, we describe a new prodigiosin-based bioassay for detection of some N-acyl homoserine lactones.

Amino Acid Sequence↗
PubMed2002

A putative three-dimensional targeting motif of polygalacturonase (PehA), a protein secreted through the type II (GSP) pathway in Erwinia carotovora.

Intramolecular information specifying protein secretion through the type II (GSP) pathway of Gram-negative bacteria was investigated. Two regions of the polygalacturonase (PehA) of Erwinia carotovora containing residues proposed to be included in a targeting motif were located, one close to the C-terminus between residues 342 and 369 and another between residues 84 and 135 in the large central loops. The regions were required together to promote secretion. Further residues in the middle of the protein were required for proper positioning of the regions, suggesting that they were both involved in interaction with the GSP. To our knowledge, this is the first time that a possible three-dimensional targeting motif has been defined. At least one of the motifs comprises a cluster on the surface of the protein. The two motifs are structurally dissimilar, suggesting that there are two distinct recognition regions in the GSP apparatus. Finally, we propose that the targeting motifs are of a complex conformational nature with some variability accommodated, as illustrated by the observation that many mutations exhibited no clear phenotype individually but, in combination, severely compromised secretion.

Amino Acid Motifs↗
PubMed2003

The PmrA-PmrB two-component system responding to acidic pH and iron controls virulence in the plant pathogen Erwinia carotovora ssp. carotovora.

Efficient response to environmental cues is crucial to successful infection by plant-pathogenic bacteria such as Erwinia carotovora ssp. carotovora. The expression of the main virulence genes of this pathogen, encoding extracellular enzymes that degrade the plant-cell wall, is subject to complex regulatory machinery where two-component systems play an important role. In this paper, we describe for the first time the involvement of the PmrA-PmrB two-component system in regulation of virulence in a plant-pathogenic bacterium. Disruption of pmrB resulted in reduced virulence both in potato and in Arabidopsis. This is apparently due to reduced production of the extracellular enzymes. In contrast, a pmrA mutant exhibited increased levels of these enzymes implying negative regulation of the corresponding genes by PmrA. Furthermore, the pmrB but not pmrA mutant exhibited highly increased resistance to the cationic antimicrobial peptide polymyxin B suggesting alterations in cell surface properties of the mutant. A similar increase of polymyxin resistance was detected in the wild type at mildly acidic pH with low Mg2+. Functional pmrA is essential for bacterial survival on excess iron at acidic pH, regardless of the Mg2+ concentration. We propose that PmrA-PmrB TCS is involved in controlling of bacterial response to external pH and iron and is crucial for bacterial virulence and survival in planta.

Amino Acid Sequence↗
PubMed1999

The hexY genes of Erwinia carotovora ssp. carotovora and ssp. atroseptica encode novel proteins that regulate virulence and motility co-ordinately.

Mutations located in a new gene, hexY, in Erwinia carotovora ssp. carotovora (Ecc) and ssp. atroseptica (Eca) cause strong upregulation of production of exoenzyme virulence factors and motility. The hexY gene encodes a novel 14.4 kDa protein with no known homologues. The hexY mRNA transcript has an unusually long (525bp) 5' untranslated region, which may be important for post-transcriptional regulation. An elevated level of transcription of two exoenzyme genes, pelCand celV, was observed in the HexY mutant background. The levels of cellulase and protease in a HexY mutant were independent of the presence of PGA, suggesting a role for HexY in the induction of these enzymes seen upon PGA addition. Electron microscopy revealed that HexY cells were hyperflagellated, perhaps contributing to the hypermotility phenotype of this mutant. The HexY mutant M5 exhibited enhanced maceration capacity on potato tubers. Therefore, the hexY gene and its gene product may define another level of regulation of virulence determinants in Ecc and Eca.

Bacterial Proteins↗
PubMed2000

hexA of Erwinia carotovora ssp. carotovora strain Ecc71 negatively regulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone.

The soft-rotting bacterium, Erwinia carotovora ssp. carotovora (E. c. carotovora), produces an array of extracellular enzymes (= exoenzymes), including pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel) and protease (Prt), as well as HarpinEcc, the elicitor of hypersensitive reaction (HR). The production of these exoenzymes and HarpinEcc responds to plant products and the quorum-sensing signal [N-(3-oxohexanoyl)-L-homoserine lactone; OHL] and is subject to both transcriptional and post-transcriptional regulation. hexA of E. c. carotovora strain Ecc71 (hereafter hexA71), like that of another E. c. carotovora strain, negatively controls the production of exoenzymes, OHL and virulence in E. c. carotovora strain Ecc71. In addition to exoenzymes, HexA71 negatively regulates the expression of hrpNEcc, the structural gene for HarpinEcc. Exoenzyme overproduction is abolished by OHL deficiency in a HexA- and Ohll- double mutant, indicating that HexA and OHL are components of a common regulatory pathway controlling exoenzyme production. HexA71 negatively affects RpoS, as the levels of this alternative sigma factor are higher in the HexA- mutant than in the HexA+ strain. However, a HexA- and RpoS double mutant produces higher levels of exoenzymes and transcripts of pel-1, peh-1 and celVgenes than the HexA- and RpoS+ parent. Thus, the elevated levels of RpoS protein in the HexA- mutant do not account for exoenzyme overproduction. The following evidence associates for the first time the phenotypic changes in the HexA mutant to overproduction of rsmB RNA, a global regulator of exoenzymes, HarpinEcc, OHL and secondary metabolites. Analyses of rsmB transcripts and expression of an rsmB-lacZoperon fusion in E. c. carotovora strain Ecc71 revealed that HexA71 negatively regulates transcription of rsmB. Multiple copies of hexA71+ DNA suppress various phenotypes, including exoenzyme production in E. c. carotovora strain Ecc71, and concomitantly inhibit the production of rsmB, pel-1, peh-1, celV and hrpNEcc transcripts. Multiple copies of rsmB+ DNA, on the other hand, stimulate exoenzyme production by relieving the negative effects of a chromosomal copy of hexA+. The occurrence of hexA homologues and the negative effect of the dosage of hexA71 DNA on rsmB transcripts were also detected in other E. c. carotovora strains as well as Erwinia carotovora atroseptica and Erwinia carotovora betavasculorum. Extrapolating from the findings with LrhA, the Escherichia coli homologue of HexA, and the presence of sprE homologues in E. carotovora subspecies, we propose that HexA71 controls several regulatory pathways in E. carotovora including rsmB transcription and the production of SprEEcc which, in turn, affects RpoS levels. A model is presented that integrates the findings presented here and our current knowledge of the major regulatory network that controls exoprotein production in soft-rotting Erwinia carotovora subspecies.

4-Butyrolactone↗
PubMed2000

A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica On potato tubers.

A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.

Colony Count, Microbial↗
PubMed2004

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

Base Sequence↗
PubMed2002

Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner.

Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T(0)) were 2-4-fold higher than the controls, whereas phytoene, lycopene, beta-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T(1) and T(2) generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5-10-fold elevations in total phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into phytoene) were not significantly altered by the presence of the bacterial phytoene synthase.

Alkyl and Aryl Transferases↗
PubMed2000

The phytopathogenic bacteria Erwinia carotovora infects Drosophila and activates an immune response.

Although Drosophila possesses potent immune responses, little is known about the microbial pathogens that infect Drosophila. We have identified members of the bacterial genus Erwinia that induce the systemic expression of genes encoding antimicrobial peptides in Drosophila larvae after ingestion. These Erwinia strains are phytopathogens and use flies as vectors; our data suggest that these strains have also evolved mechanisms for exploiting their insect vectors as hosts. Erwinia infections induce an antimicrobial response in Drosophila larvae with a preferential expression of antibacterial versus antifungal peptide-encoding genes. Antibacterial peptide gene expression after Erwinia infection is reduced in two Drosophila mutants that have reduced numbers of hemocytes, suggesting that blood cells play a role in regulating Drosophila antimicrobial responses and also illustrating that this Drosophila-Erwinia interaction provides a powerful model for dissecting host-pathogen relationships.

Animals↗
PubMed2000

AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora.

N-acylhomoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many gram-negative bacteria. AIs are involved in the regulation of diverse biological functions, including expression of pathogenic genes in the plant pathogens Pseudomonas solanacearum, several Erwinia species, and the human pathogen Pseudomonas aeruginosa. A bacterial isolate, Bacillus sp. 240B1, is capable of enzymatic inactivation of AIs. The gene (aiiA) for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Sequence alignment indicates that AiiA contains a "HXHXDH" zinc-binding motif that is conserved in several groups of metallohydrolases. Site-directed mutagenesis showed that conserved aspartate and most histidine residues are required for AiiA activity. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. Our results indicate that the AI-inactivation approach represents a promising strategy for prevention of diseases in which virulence is regulated by AIs.

4-Butyrolactone↗
PubMed1997

The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli.

The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.

Bacterial Proteins↗
PubMed1998

Crystal structure of polygalacturonase from Erwinia carotovora ssp. carotovora.

The crystal structure of the 40-kDa endo-polygalacturonase from Erwinia carotovora ssp. carotovora was solved by multiple isomorphous replacement and refined at 1.9 A to a conventional crystallographic R-factor of 0.198 and Rfree of 0.239. This is the first structure of a polygalacturonase and comprises a 10 turn right-handed parallel beta-helix domain with two loop regions forming a "tunnel like" substrate-binding cleft. Sequence conservation indicates that the active site of polygalacturonase is between these two loop regions, and comparison of the structure of polygalacturonase with that of rhamnogalacturonase A from Aspergillus aculeatus enables two conserved aspartates, presumed to be catalytic residues, to be identified. An adjacent histidine, in accord with biochemical results, is also seen. A similarity in overall electrostatic properties of the substrate-binding clefts of polygalacturonase and pectate lyase, which bind and cleave the same substrate, polygalacturonic acid, is also revealed.

Amino Acid Sequence↗