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PubMed2003

Crystal structure of carbapenem synthase (CarC).

The proposed biosynthetic pathway to the carbapenem antibiotics proceeds via epimerization/desaturation of a carbapenam in an unusual process catalyzed by an iron- and 2-oxoglutarate-dependent oxygenase, CarC. Crystal structures of CarC complexed with Fe(II) and 2-oxoglutarate reveal it to be hexameric (space group C2221), consistent with solution studies. CarC monomers contain a double-stranded beta-helix core that supports ligands binding a single Fe(II) to which 2-oxoglutarate complexes in a bi-dentate manner. A structure was obtained with l-N-acetylproline acting as a substrate analogue. Quantum mechanical/molecular mechanical modeling studies with stereoisomers of carbapenams and carbapenems were used to investigate substrate binding. The combined work will stimulate further mechanistic studies and aid in the engineering of carbapenem biosynthesis.

Binding Sites↗
PubMed2003

Carboxymethylproline synthase (CarB), an unusual carbon-carbon bond-forming enzyme of the crotonase superfamily involved in carbapenem biosynthesis.

Carboxymethylproline synthase (CarB) catalyzes the committed step in the biosynthesis of (R)-1-carbapen-2-em-3-carboxylate, the simplest member of the carbapenem family of beta-lactam antibiotics, some of which are used clinically. CarB displays sequence homology with members of the crotonase family including enoyl-CoA hydratase (crotonase) and methylmalonyl-CoA decarboxylase. The CarB reaction has been proposed to comprise condensation of acetyl coenzyme A (AcCoA) and glutamate semi-aldehyde to give (2S,5S)-carboxymethylproline ((2S,5S)-CMP). (2S,5S)-CMP is then cyclized in an ATP-driven reaction catalyzed by CarA to give a carbapenam, which is subsequently epimerized and desaturated to give a carbapenem in a CarC-mediated reaction. Here we report the purification of recombinant CarB and that it exists predominantly in a trimeric form as do other members of the crotonase family. AcCoA was not found to be a substrate for CarB. Instead malonyl-CoA was found to be a substrate, efficiently producing (2S,5S)-CMP in the presence of glutamate semi-aldehyde. In the absence of glutamate semi-aldehyde, mass spectrometric analysis indicated that CarB catalyzed the decarboxylation of malonyl-CoA to AcCoA. The reactions of CarB, CarA, and CarC were coupled in vitro demonstrating the viability of malonyl-CoA as a carbapenem precursor. CarB was also shown to accept methylmalonyl CoA as a substrate to form 6-methyl-(2S,5S)CMP, which in turn is a substrate for CarA. The implications of the results for the biosynthesis of both carbapenem-3-carboxylate and C-2/C-6-substituted carbapenems, such as thienamycin, are discussed.

Acetyl Coenzyme A↗
PubMed2005

Altering substrate chain length specificity of an acylhomoserine lactone synthase in bacterial communication.

Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), which are central to the regulation of virulence in many plant and animal pathogens. Variation of the acyl side chain of the AHSLs underlies the observed species specificity of this communication system. Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl chain length specificity of distinct AHSL synthases. Under physiological concentrations, only the cognate AHSL with the "right" acyl chain is recognized as a signal that will switch on virulence genes. Mutagenesis of the AHSL synthase gene expI(SCC1) identified the changes M127T and F69L as sufficient to effectively alter ExpI(SCC1) (an N-3-oxohexanoyl-l-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-l-homoserine lactone producer. Our data identify critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help in understanding and manipulating this bacterial language.

Amino Acid Sequence↗
PubMed2001

Phenotypic and genotypic characterization of antagonistic bacteria associated with roots of transgenic and non-transgenic potato plants.

Rhizobacteria obtained during a risk assessment study from parental and transgenic T4 lysozyme-expressing potato plants were investigated to determine whether or not the strains could be grouped based on the source of isolation, transgenic or non-transgenic plants, respectively. A total of 68 representative bacterial strains of the group of enterics and pseudomonads were investigated by phenotypic profiling (the antagonistic activity towards bacterial and fungal plant pathogens, the production of the plant growth hormone indole-3-acetic acid [auxin], and the sensitivity to T4 lysozyme in vitro) and genotypic profiling by PCR fingerprints using BOX primers. All isolates were identified by fatty acid methyl ester (FAME) analysis. Computer-based analysis of the phenotypic characteristics showed that both, enterics and Pseudomonas strains clustered into six to seven groups at an Euclidian distance of 10. According to their BOX-PCR-generated fingerprints the Pseudomonas strains clustered into seven groups and the enterobacteria into two groups at the same genetic distance level of 10. The majority of groups were heterogeneous and contained isolates from all plant lines. In conclusion, cluster analysis of the phenotypic and genotypic features did not reveal correlations between bacterial isolates and transgenic character of plants.

Cluster Analysis↗
PubMed2001

Overexpresssion of a Legionella pneumophila homologue of the E. coli regulator csrA affects cell size, flagellation, and pigmentation.

Legionella pneumophila is an inhabitant of the aquatic environment and the causative agent of a bacterial pneumonia. We identified the presence of an L. pneumophila homologue of csrA of E. coli and rsmA of Erwinia carotovora, genes which regulate gene expression by destabilising mRNA and which have been shown to relate to environmental fitness and pathogenicity. The Legionella csrA was able to complement a csrA-negative mutant of E. coli. Overproduction of csrA in L. pneumophila lead to a reduction of flagellation and pigmentation and an increase in bacterial cell size. csrA overproduction was associated with a reduction of fliA and flaA transcripts. This suggests that similar to E. coli and Erwinia, L. pneumophila csrA is a regulator of gene expression and may contribute to the capability of the pathogen to rapidly adapt to changing environments.

Amino Acid Sequence↗
PubMed1992

Construction of a recombinant bacterial human CD4 expression system producing a bioactive CD4 molecule.

The CD4 protein expressed on helper T lymphocytes is a restriction element for major histocompatibility class II immune responses. This molecule is also used by the human immunodeficiency virus as its specific cellular receptor facilitating binding of virus to cells. As soluble forms of CD4 inhibit HIV infection in tissue culture, attention has focused on this molecule. Bacterially produced CD4 would facilitate studies of the biology of the CD4 molecule. However, bacterially expressed CD4 must be refolded for assumption of its interaction with conformationally dependent anti-CD4 monoclonal antibodies as well as the HIV-1 envelope protein gp120. We report here the engineering of an external domain construct of the CD4 gene into a novel expression vector containing the nucleotide sequence encoding the pelB leader peptide of Erwinia carotovara (pDABL), to facilitate correct folding of CD4 in bacteria. Monoclonal antibodies specific for important conformational epitopes of the CD4 molecule were able to bind bacterial colonies containing the pDABL/CD4 vector but not colonies with vector alone. Importantly, recombinant gp120 produced in baculovirus bound specifically to bacterial colonies expressing the CD4 recombinant molecule. This system presents a simple screening mechanism for molecules that bind to the external domain of the CD4 glycoprotein. Vectors such as pDABL will also facilitate the production of large amounts of biologically active proteins in bacteria.

Base Sequence↗
PubMed2002

Altering plant-microbe interaction through artificially manipulating bacterial quorum sensing.

Many bacteria regulate diverse physiological processes in concert with their population size. Bacterial cell-to-cell communication utilizes small diffusible signal molecules, which the bacteria both produce and perceive. The bacteria couple gene expression to cell density by eliciting a response only when the signalling molecules reach a critical threshold (a point at which the population is said to be 'quorate'). The population as a whole is thus able to modify its behaviour as a single unit. Amongst Gram-negative bacteria, the quorum sensing signals most commonly used are N-acylhomoserine lactones (AHLs). It is now apparent that AHLs are used for regulating diverse behaviours in epiphytic, rhizosphere-inhabiting and plant pathogenic bacteria and that plants may produce their own metabolites that interfere with this signalling. Transgenic plants that produce high levels of AHLs or which can degrade bacterial-produced AHLs have been made. These plants have dramatically altered susceptibilities to infection by pathogenic Erwinia species. In addition, such plants will prove useful tools in determining the roles of AHL-regulated density-dependent behaviour in growth promoting, biological control and pathogenic plant-associated bacterial species.

4-Butyrolactone↗
PubMed1997

Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins.

OutD is an outer membrane component of the main terminal branch of the general secretory pathway (GSP) in Erwinia chrysanthemi. We analyzed the interactions of OutD with other components of the GSP (Out proteins) and with secreted proteins (PelB, EGZ and PemA). OutD is stabilized by its interaction with another GSP component, OutS. The 62 C-terminal amino acids of OutD are necessary for this interaction. In vivo formation of OutD multimers, up to tetramers, was proved after the dissociation in mild conditions of the OutD aggregates formed in the outer membrane. Thus, OutD could form a channel-like structure in the outer membrane. We showed that OutD is stabilized in vivo when co-expressed with Out-secreted proteins. This stabilization results from the formation of complexes that were detected in experiments of co-immunoprecipitation and co-sedimentation in sucrose density gradients. The presence of the N-terminal part of OutD is required for this interaction. The interaction between OutD and the secreted protein PelB was confirmed in vitro, suggesting that no other component of the GSP is required for this recognition. No interaction was observed between the E. carotovora PelC and the E. chrysanthemi OutD. Thus, the interaction between GspD and the secreted proteins present in the periplasm could be the key to the specificity of the secretion machinery and a trigger for that process.

Bacterial Outer Membrane Proteins↗
PubMed2000

N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia.

Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 microM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.

Bacterial Proteins↗
PubMed2005

Identification of a new quorum-sensing-controlled virulence factor in Erwinia carotovora subsp. atroseptica secreted via the type II targeting pathway.

Two-dimensional polyacrylamide gel electrophoresis of the secreted proteins of Erwinia carotovora subsp. atroseptica revealed a low-abundance protein that was identified by mass spectrometry as a homologue of a Xanthomonas campestris avirulence protein with unknown function. The predicted Svx protein has an N-terminal signal sequence and zinc binding-region signature, and the mature protein is post-translationally modified. A 2D difference gel electrophoresis (DIGE) showed that the protein is secreted by the type II (out) secretion apparatus, which is also responsible for the secretion of the major known virulence factors, PelC and CelV. Transcription of the svx gene is under N-acyl-homoserine lactone-mediated quorum-sensing control. The svx gene was inactivated by transposon insertion. The mutant showed a decrease in virulence in potato plant assays, demonstrating a role for Svx in the pathogenicity of E. carotovora subsp. atroseptica. These results show that Svx is a previously unidentified virulence determinant which is secreted by the out machinery and is regulated by quorum sensing, two systems employed by several other virulence factors. Thus, the type II secretory machine is a conduit for virulence factors other than the main pectinnases and cellulase in E. carotovora subsp. atroseptica.

Bacterial Proteins↗
PubMed2005

Novel quorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium.

Seven new genes controlled by the quorum-sensing signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) have been identified in Erwinia carotovora subsp. carotovora. Using TnphoA as a mutagen, we enriched for mutants defective in proteins that could play a role in the interaction between E. carotovora subsp. carotovora and its plant hosts, and identified NipEcc and its counterpart in E. carotovora subsp. atroseptica. These are members of a growing family of proteins related to Nep1 from Fusarium oxysporum which can induce necrotic responses in a variety of dicotyledonous plants. NipEcc produced necrosis in tobacco, NipEca affected potato stem rot, and both affected virulence in potato tubers. In E. carotovora subsp. carotovora, nip was shown to be subject to weak repression by the LuxR family regulator, EccR, and may be regulated by the negative global regulator RsmA.

4-Butyrolactone↗
PubMed2006

GacS-dependent production of 2R, 3R-butanediol by Pseudomonas chlororaphis O6 is a major determinant for eliciting systemic resistance against Erwinia carotovora but not against Pseudomonas syringae pv. tabaci in tobacco.

Root colonization by a plant-beneficial rhizobacterium, Pseudomonas chlororaphis O6, induces disease resistance in tobacco against leaf pathogens Erwinia carotovora subsp. carotovora SCC1, causing soft-rot, and Pseudomonas syringae pv. tabaci, causing wildfire. In order to identify the bacterial determinants involved in induced systemic resistance against plant diseases, extracellular components produced by the bacterium were fractionated and purified. Factors in the culture filtrate inducing systemic resistance were retained in the aqueous fraction rather than being partitioned into ethyl acetate. Fractionation on high-performance liquid chromatography followed by nuclear magnetic resonance mass spectrometry analysis identified the active compound as 2R, 3R-butanediol. 2R, 3R butanediol induced systemic resistance in tobacco to E. carotovora subsp. carotovora SCC1, but not to P. syringae pv. tabaci. Treatment of tobacco with the volatile 2R, 3R-butanediol enhanced aerial growth, a phenomenon also seen in plants colonized by P. chlororaphis O6. The isomeric form of the butanediol was important because 2S, 3S-butandiol did not affect the plant. The global sensor kinase, GacS, of P. chlororaphis O6 was a key regulator for induced systemic resistance against E. carotovora through regulation of 2R, 3R-butanediol production. This is the first report of the production of these assumed fermentation products by a pseudomonad and the role of the sensor kinase GacS in production of 2R, 3R-butanediol.

Bacterial Proteins↗
PubMedDate not supplied

Cloning and characterization of a pectate lyase gene from Erwinia carotovora EC153.

A pel gene cloned from strain EC153 of Erwinia carotovora encoded a pectate lyase that macerated plant tissue with moderate efficiency. This gene, called pel153, was sequenced and found to possess considerable homology with a pectate lyase gene from Yersinia pseudotuberculosis. The Yersinia protein, however, was truncated at the carboxyl terminal end relative to the Erwinia gene product and had a lower isoelectric point. The Erwinia pel153 gene was overexpressed in cells of Escherichia coli, and a 56-kDa protein was observed on sodium dodecyl sulfate-polyacrylamide gels. This compares with a molecular weight of 61 kDa for the mature, secreted protein as determined from sequencing data. Southern blot analysis disclosed the presence of the pel153 gene in three different strains of E. carotovora, but mutation of the gene in strain EC153 did not affect its ability to soft-rot potato tubers.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of a novel regulatory gene aepA that controls extracellular enzyme production in the phytopathogenic bacterium Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora strain Ecc71 produces an array of extracellular enzymes including pectate lyase (Pel), polygalacturonase, cellulase, and protease. In strain Ecc71, these enzymes are coregulated by aepA, which encodes an activator of extracellular protein production (H. Murata, J. L. McEvoy, A. Chatterjee, A. Collmer, and A. K. Chatterjee, Mol. Plant-Microbe Interact, 4:239-246, 1991). The nucleotide sequence of a 2.7-kb aepA+ DNA segment revealed an open reading frame (ORF) of 1,395 bp which matches with the size of the aepA transcript determined by Northern blot analysis. aepA is predicted to encode a protein of 465 amino acid residues with a molecular mass of approximately 51 kDa and a pI of 6.52. The occurrence of a putative signal sequence and several hydrophobic domains suggest membrane localization of AepA. An aepA-lacZ operon fusion was constitutively expressed in E. coli (DH5 alpha) but inducible by pectate and celery extract in E. c. subsp. carotovora (AC5006). These findings suggest that aepA expression may be negatively regulated in E. c. subsp. carotovora. By assaying for the transcript of pel-1, which species a major secreted Pel species in strain Ecc71, and by following the expression of a pel1-lacZ operon fusion we determined that AepA activates pel-1 transcription. The characteristics of aepA including the lack of homology with other prokaryotic regulatory genes indicate that aepA encodes a novel regulatory protein required for extracellular protein production. Whereas homologs of Ecc71 aepA occur in E. c. subsp. carotovora and E. c. subsp. atroseptica strains, activation of exoenzyme production is markedly stimulated by aepA in E. c. subsp. carotovora than in E. c. subsp. atroseptica.

Amino Acid Sequence↗
PubMedDate not supplied

Nucleotide sequence of a pectate lyase structural gene, pel1 of Erwinia carotovora subsp. carotovora strain 71 and structural relationship of pel1 with other pel genes of Erwinia species.

Of the various exoproteins secreted by Erwinia carotovora subsp. carotovora strain 71, Pel1 is the major pectate lyase species with tissue macerating activity. Nucleotide sequencing of a 2.2-kb pel1+ DNA segment revealed a 1,122 base pair open reading frame which could encode pre-Pel1 of 374 amino acid residues. A signal peptide of 22 amino acid residues is present within the NH2-terminal region of pre-Pel1. Transcription of pel1 was initiated at the guanine residue 111 base pairs upstream of the start codon. Consensus sequences for the binding of KdgR, a negative regulatory factor known to control some of the E. chrysanthemi pectinases, flank the promoter of pel1. Although pel1 belongs to the pelBC family, it is more closely related to the pel genes of E. carotovora than to the pelBC genes of E. chrysanthemi.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of a novel pectate lyase from Erwinia carotovora subsp. carotovora.

The pectate lyase (Pel, EC 4.2.2.2) isoenzyme profile of Erwinia carotovora subsp. carotovora was characterized by isoelectric focusing, and the corresponding genes coding for four different exported Pels were cloned. The nucleotide sequence of the pelB gene encoding one of these isoenzymes was determined and was shown to contain 1,040-bp open reading frame coding for a 37,482-Da protein with a putative cleavable amino terminal signal peptide. Overexpression and selective labeling experiments with the pelB clone demonstrated the synthesis of a 35-kDa polypeptide, which is in accordance with the deduced size of the processed PelB. The predicted amino acid sequence of PelB was very similar to that of Pel-3 of another E.c. subsp. carotovora strain 71, but showed no similarity to other previously characterized pectinolytic enzymes. The pelB gene is located next to the previously characterized pehA gene encoding an endopolygalacturonase. The two genes are divergently transcribed from a common control region and are subject to similar global regulation by the central virulence regulator expI. Inactivation of pelB did not appear to reduce the virulence of the mutant strain, suggesting that pelB does not have a major role in pathogenicity. Unlike other Pels, PelB required partially methyl esterified pectin as substrate suggesting that PelB represents a novel isoform of pectate lyase.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of a pathogen-induced potato catalase and its systemic expression upon nematode and bacterial infection.

We have isolated a cDNA encoding a catalase (Cat2St) by differential screening of a cDNA library constructed from potato roots infected with the cyst nematode Globodera pallida. Expression analysis confirmed the local induction of Cat2St and showed that it was highest at the adult stage of the parasite. It also revealed that Cat2St was induced in uninfected roots, stems, and leaves of infected plants. Localized and systemic induction of Cat2St was also observed upon root-knot nematode (Meloidogyne incognita) and root bacteria (Erwinia carotovora, Corynebacterium sepedonicum) infections. Based on sequence and expression analysis, Cat2St was found to belong to the recently described class II of dicotyledonous catalases, suggesting that these catalase isoforms could also be pathogen induced. Plant-parasitic nematodes are known to induce, in the roots of their hosts, highly metabolic feeding cells that function as nutritional sinks. Whereas the local induction of Cat2St is probably a consequence of an oxidative stress of metabolic nature, the systemic induction of Cat2St shows striking similarities with the induction of systemic acquired resistance (SAR) genes. The possible role of catalase in compatible plant-pathogen interactions is discussed.

Amino Acid Sequence↗