The global response regulator ExpA controls virulence gene expression through RsmA-mediated and RsmA-independent pathways in Pectobacterium wasabiae SCC3193.

ExpA (GacA) is a global response regulator that controls the expression of major virulence genes, such as those encoding plant cell wall-degrading enzymes (PCWDEs) in the model soft rot phytopathogen Pectobacterium wasabiae SCC3193. Several studies with pectobacteria as well as related phytopathogenic gammaproteobacteria, such as Dickeya and Pseudomonas, suggest that the control of virulence by ExpA and its homologues is executed partly by modulating the activity of RsmA, an RNA-binding posttranscriptional regulator. To elucidate the extent of the overlap between the ExpA and RsmA regulons in P. wasabiae, we characterized both regulons by microarray analysis. To do this, we compared the transcriptomes of the wild-type strain, an expA mutant, an rsmA mutant, and an expA rsmA double mutant. The microarray data for selected virulence-related genes were confirmed through quantitative reverse transcription (qRT-PCR). Subsequently, assays were performed to link the observed transcriptome differences to changes in bacterial phenotypes such as growth, motility, PCWDE production, and virulence in planta. An extensive overlap between the ExpA and RsmA regulons was observed, suggesting that a substantial portion of ExpA regulation appears to be mediated through RsmA. However, a number of genes involved in the electron transport chain and oligogalacturonide metabolism, among other processes, were identified as being regulated by ExpA independently of RsmA. These results suggest that ExpA may only partially impact fitness and virulence via RsmA.

Protein neighborhoods in the outer membrane of Salmonella typhimurium.

The organization of proteins in the outer membrane of Salmonella typhimurium was analyzed by cross-linking with cleavable reagents and symmetrical two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The major outer membrane proteins could be cross-linked to form multimeric complexes. The pore-forming 44 000, 36 000 and 34 000 dalton proteins were cross-linked to form dimers and trimers. Lipoprotein was cross-linked to 33 000 and 17 500 dalton proteins. In addition the 33 000, 24 000, 17 500 dalton proteins and the free form of lipoprotein were cross-linked to the peptidoglycan layer of the cell wall. The cross-linked complexes found were similar to those of analogous proteins in the outer membrane of Escherichia coli, thus suggesting a similar organization of outer membrane proteins in these species.

Structure of the lipid-containing bacteriophage phi 6. Disruption by Triton X-100 treatment.

The structure of the lipid-containing bacteriophage phi 6 was studied by means of controlled Triton X-100 disruption and subsequent isolation of subviral particles. Rate-zonal centrifugation yielded two fractions, a nucleocapsid fraction with RNA, proteins P1, P2, P4, P7, P8, and about half of the protein P5 and a membrane fraction with associated proteins P3, P6, P9, P10, and the rest of the protein P5. Following isopycnic sucrose gradient centrifugation, an empty capsid fraction was obtained which lacked RNA but contained a protein composition similar to the nucleocapsid except for the absence of P5. The membrane fraction isolated after isopycnic centrifugation was morphologically indistinguishable from that isolated after rate-zonal centrifugation but contained only proteins P3, P6, P9 and P10. By treating phi 6 with Triton X-100 prior to isopycnic sucrose gradient centrifugation the viral membrane was further separated into submembrane structures and the attachment protein, P3, could be isolated in rather pure form.

Structure and expression of the ompB operon, the regulatory locus for the outer membrane porin regulon in Salmonella typhimurium LT-2.

The ompB operon of Salmonella typhimurium encodes a positive transcriptional regulator OmpR and an inner membrane protein EnvZ. Both proteins are needed for the proper expression of the outer membrane proteins OmpC and OmpF. We have determined the nucleotide sequence of the ompB locus and its adjacent regions. A comparison between the S. typhimurium and Escherichia coli sequences revealed that the ompB locus is highly conserved. The sequence data also showed that ompR and envZ form an operon, where the coding regions overlap by four base-pairs. Utilizing ompR-lacZ and envZ-lacZ gene fusions, the translational levels of expression of these two genes were measured, showing that ompR is considerably more efficiently expressed than envZ. Analysis of ompR frameshift mutations showed that translation of envZ is almost totally dependent on the translation of the upstream gene ompR. The mechanism of this translational coupling appears to be a reinitiation of the ribosome at the overlapping region of the two genes. The characteristics of the OmpR and EnvZ proteins were in agreement with the known functions and cellular locations of these proteins. OmpR was found to contain a putative DNA binding site, while EnvZ contained two hydrophobic stretches typical of transmembrane regions. Both OmpR and EnvZ show extensive homologies with many proteins from a number of different origins, all of which function in pairs and through which environmental signals modulate gene expression. Hence, the tightly coupled synthesis of these proteins seems to be essential in eliciting a proper response in the transmembrane regulation of gene expression.

Role of Abscisic Acid in Drought-Induced Freezing Tolerance, Cold Acclimation, and Accumulation of LT178 and RAB18 Proteins in Arabidopsis thaliana.

To study the role of abscisic acid (ABA) in development of freezing tolerance of Arabidopsis thaliana, we exposed wild-type plants, the ABA-insensitive mutant abi1, and the ABA-deficient mutant aba-1 to low temperature (LT), exogenous ABA, and drought. Exposure of A. thaliana to drought stress resulted in a similar increase in freezing tolerance as achieved by ABA treatment or the initial stages of acclimation, suggesting overlapping responses to these environmental cues. ABA appears to be involved in both LT- and drought-induced freezing tolerance, since both ABA mutants were impaired in their responses to these stimuli. To correlate enhanced freezing tolerance with the presence of stress-specific proteins, we characterized the accumulation of RAB18 and LTI78 in two ecotypes, Landsberg erecta and Coimbra, and in the ABA mutants during stress response. LT- and drought-induced accumulation of RAB18 coincided with the increase in freezing tolerance and was blocked in the cold-acclimation-deficient ABA mutants. In contrast, LT178 accumulated in all genotypes in response to LT and drought and was always present when the plants were freezing tolerant. This suggests that development of freezing tolerance in A. thaliana requires ABA-controlled processes in addition to ABA-independent factors.

Alterations in Water Status, Endogenous Abscisic Acid Content, and Expression of rab18 Gene during the Development of Freezing Tolerance in Arabidopsis thaliana.

Treatments as diverse as exposure to low temperature (LT), exogenous abscisic acid (ABA), or drought resulted in a 4 to 5[deg]C increase in freezing tolerance of the annual herbaceous plant Arabidopsis thaliana. To correlate the increase in freezing tolerance with the physiological changes that occur in response to these treatments, we studied the alterations in water status, endogenous ABA levels, and accumulation of rab18 (V. Lang and E.T. Palva [1992] Plant Mol Biol 20: 951-962) mRNA. Exposure to LT and exogenous ABA caused only a minor decline in total water potential ([psi]w), in contrast to a dramatic decrease in [psi]w during drought stress. Similarly, the endogenous ABA levels were only slightly and transiently increased in LT-treated plants in contrast to a massive increase in ABA levels in drought-stressed plants. The expression of the ABA-responsive rab18 gene was low during the LT treatment but could be induced to high levels by exogenous ABA and drought stress. Taken together, these results suggest that the moderate increases in freezing tolerance of A. thaliana might be achieved by different mechanisms. However, ABA-deficient and ABA-insensitive mutants of A. thaliana have impaired freezing tolerance, suggesting that ABA is, at least indirectly, required for the development of full freezing tolerance.

Global regulators ExpA (GacA) and KdgR modulate extracellular enzyme gene expression through the RsmA-rsmB system in Erwinia carotovora subsp. carotovora.

The production of the main virulence determinants, the extracellular plant cell wall-degrading enzymes, and hence virulence of Erwinia carotovora subsp. carotovora is controlled by a complex regulatory network. One of the global regulators, the response regulator ExpA, a GacA homolog, is required for transcriptional activation of the extracellular enzyme genes of this soft-rot pathogen. To elucidate the mechanism of ExpA control as well as interactions with other regulatory systems, we isolated second-site transposon mutants that would suppress the enzyme-negative phenotype of an expA (gacA) mutant. Inactivation of kdgR resulted in partial restoration of extracellular enzyme production and virulence to the expA mutant, suggesting an interaction between the two regulatory pathways. This interaction was mediated by the RsmA-rsmB system. Northern analysis was used to show that the regulatory rsmB RNA was under positive control of ExpA. Conversely, the expression of rsmA encoding a global repressor was under negative control of ExpA and positive control of KdgR. This study indicates a central role for the RsmA-rsmB regulatory system during pathogenesis, integrating signals from the ExpA (GacA) and KdgR global regulators of extracellular enzyme production in E. carotovora subsp. carotovora.

Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora.

We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.

The response regulator expM is essential for the virulence of Erwinia carotovora subsp. carotovora and acts negatively on the sigma factor RpoS (sigma s).

The main virulence factors of Erwinia carotovora subsp. carotovora, the secreted, extracellular cell-wall-degrading enzymes, are controlled by several regulatory mechanisms. We have isolated transposon mutants with reduced virulence on tobacco. One of these mutants, with a mutation in a gene designated expM, was characterized in this study. This mutant produces slightly reduced amounts of extracellular enzymes in vitro and the secretion of the enzymes is also affected. The expM wild-type allele was cloned together with an upstream gene, designated expL, that has an unknown function. The expM gene was sequenced and found to encode a protein with similarity to the RssB/SprE protein of Escherichia coli and the MviA protein of Salmonella typhimurium. These proteins belong to a new type of two-component response regulators that negatively regulate the stability of the Sigma factor RpoS (sigma s) at the protein level. The results of this study suggest that ExpM has a similar function in E. carotovora subsp. carotovora. We also provide evidence that the overproduction of RpoS in the expM mutant is an important factor for the reduced virulence phenotype and that it partly causes the observed phenotype seen in vitro. However, an expM/rpoS double mutant is still affected in secretion of extracellular enzymes, suggesting that ExpM in addition to RpoS also acts on other targets.

Oligogalacturonide-mediated induction of a gene involved in jasmonic acid synthesis in response to the cell-wall-degrading enzymes of the plant pathogen Erwinia carotovora.

Identification of Arabidopsis thaliana genes responsive to plant cell-wall-degrading enzymes of Erwinia carotovora subsp. carotovora led to the isolation of a cDNA clone with high sequence homology to the gene for allene oxide synthase, an enzyme involved in the biosynthesis of jasmonates. Expression of the corresponding gene was induced by the extracellular enzymes from this pathogen as well as by treatment with methyl jasmonate and short oligogalacturonides (OGAs). This suggests that OGAs are involved in the induction of the jasmonate pathway during plant defense response to E. carotovora subsp. carotovora attack.

Two-component regulators involved in the global control of virulence in Erwinia carotovora subsp. carotovora.

Production of extracellular, plant cell wall degrading enzymes, the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, is coordinately controlled by a complex regulatory network. Insertion mutants in the exp (extracellular enzyme production) loci exhibit pleiotropic defects in virulence and the growth-phase-dependent transcriptional activation of genes encoding extracellular enzymes. Two new exp mutations, designated expA and expS, were characterized. Introduction of the corresponding wild-type alleles to the mutants complemented both the lack of virulence and the impaired production of plant cell wall degrading enzymes. The expA gene was shown to encode a 24-kDa polypeptide that is structurally and functionally related to the uvrY gene product of Escherichia coli and the GacA response regulator of Pseudomonas fluorescens. Functional similarity of expA and uvrY was demonstrated by genetic complementation. The expA gene is organized in an operon together with a uvrC-like gene, identical to the organization of uvrY and uvrC in E. coli. The unlinked expS gene encodes a putative sensor kinase that shows 92% identity to the recently described rpfA gene product from another E. carotovora subsp. carotovora strain. Our data suggest that ExpS and ExpA are members of two-component sensor kinase and response regulator families, respectively. These two proteins might interact in controlling virulence gene expression in E. carotovora subsp. carotovora.

Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora.

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.

Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster.

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative sigmaL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora: the role of expR(Ecc).

The production of the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the extracellular cell wall-degrading enzymes, is partly controlled by the diffusible signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). OHHL is synthesized by the product of the expI/carI gene. Linked to expI we found a gene encoding a putative transcriptional regulator of the LuxR-family. This gene, expR(Ecc), is transcribed convergently to the expI gene and the two open reading frames are partially overlapping. The ExpR(Ecc) protein showed extensive amino acid sequence similarity to the repressor EsaR from Pantoea stewartii subsp. stewartii (formerly Erwinia stewartii subsp. stewartii) and to the ExpR(Ech) protein of Erwinia chrysanthemi. Inactivation of the E. carotovora subsp. carotovora expR(Ecc) gene caused no decrease in virulence or production of virulence determinants in vitro. In contrast, there was a slight increase in the maceration capacity of the mutant strain. The effects of ExpR(Ecc) were probably mediated by changes in OHHL levels. Inactivation of expR(Ecc) resulted in increased OHHL levels during early logarithmic growth. In addition, overexpression of expR(Ecc) caused a clear decrease in the production of virulence determinants and part of this effect was likely to be caused by OHHL binding to ExpR(Ecc). ExpR(Ecc) did not appear to exhibit transcriptional regulation of expI, but the effect on OHHL was apparently due to other mechanisms.

A two-component regulatory system, pehR-pehS, controls endopolygalacturonase production and virulence in the plant pathogen Erwinia carotovora subsp. carotovora.

Genes coding for the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the plant cell wall-degrading enzymes, are under the coordinate control of global regulator systems including both positive and negative factors. In addition to this global control, some virulence determinants are subject to specific regulation. We have previously shown that mutations in the pehR locus result in reduced virulence and impaired production of one of these enzymes, an endopolygalacturonase (PehA). In contrast, these pehR strains produce essentially wild-type levels of other extracellular enzymes including pectate lyases and cellulases. In this work, we characterized the pehR locus and showed that the DNA sequence is composed of two genes, designated pehR and pehS, present in an operon. Mutations in either pehR or pehS caused a Peh-negative phenotype and resulted in reduced virulence on tobacco seedlings. Complementation experiments indicated that both genes are required for transcriptional activation of the endopolygalacturonase gene, pehA, as well as restoration of virulence. Structural characterization of the pehR-pehS operon demonstrated that the corresponding polypeptides are highly similar to the two-component transcriptional regulators PhoP-PhoQ of both Escherichia coli and Salmonella typhimurium. Functional similarity of PehR-PehS with PhoP-PhoQ of E. coli and S. typhimurium was demonstrated by genetic complementation.

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.

A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and is coregulated with class I endochitinase expression.

A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h postinoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.

In vivo transfer of chromosomal mutations onto multicopy plasmids by transduction with bacteriophage P1.

A technique is presented by which chromosomal mutations may be efficiently transferred onto chimeric multicopy plasmids in vivo. The technique employs the transduction of plasmids using bacteriophage P1 as vector. The utility of this method was demonstrated by cloning a chromosomal ompR mutation of Escherichia coli K-12. The high-frequency transduction of the chimeric plasmid appeared to be dependent on its integration into the chromosome by homologous recombination. The results also suggest that the plasmid was transduced as part of the chromosome and resolved from its integrated state in the recipient cell, resulting in a high yield of mutant plasmid segregants.

CelS: a novel endoglucanase identified from Erwinia carotovora subsp. carotovora.

A plasmid clone expressing a beta(1,4)-glucan glucanohydrolase (EC 3.2.1.4; endoglucanase) in Escherichia coli was isolated from a genomic library of Erwinia carotovora subsp. carotovora. The DNA segment carrying the corresponding structural gene, named celS, contained an open reading frame encoding a 264-amino acid (aa) polypeptide. The N-terminal aa sequence of CelS showed that the protein was synthesized with a 32-aa cleavable signal peptide. The mature 232-aa CelS had a calculated Mr of 26,228 and pI of 5.5. The pH optimum was about 6.8 and the temperature optimum was between 45 and 55 degrees C. Comparison of the aa sequence of CelS by hydrophobic cluster analysis with a range of cellulases and other quasi-isofunctional enzymes revealed only very limited sequence similarities, suggesting that the CelS protein may represent the first member of an additional cellulase family.

Cloning, nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816.

We have cloned the naphthalene dioxygenase(ND)-coding genes from Pseudomonas putida strain NCIB9816 based on their ability to convert indole to indigo. The region coding for this enzyme activity was sequenced and three successive open reading frames were found. The corresponding gene products were identified using the T7 polymerase/promoter system. All of them are necessary for the ND activity. A comparison of the ND-coding genes with the ones coding for benzene dioxygenase revealed significant homology which was more pronounced at the nucleotide level than at the amino acid level.