Regulation of motility in Erwinia carotovora subsp. carotovora: quorum-sensing signal controls FlhDC, the global regulator of flagellar and exoprotein genes, by modulating the production of RsmA, an RNA-binding protein.

Erwinia carotovora subsp. carotovora causes soft-rotting (tissue-macerating) disease in many plants and plant organs. Although pectinases are the primary determinants of virulence, several ancillary factors that augment bacterial virulence have also been identified. One such factor is bacterial motility. Flagellum formation and bacterial movement are regulated in many enterobacteria, including E. carotovora subsp. carotovora, by FlhDC, the master regulator of flagellar genes and FliA, a flagellum-specific σ factor. We document here that motility of E. carotovora subsp. carotovora is positively regulated by the quorum-sensing signal, N-acylhomoserine lactone (AHL), and negatively regulated by RsmA, a post-transcriptional regulator. RsmA, an RNA-binding protein, causes translational repression and promotes RNA decay. Our data show that RsmA negatively regulates flhDC and fliA expression. Moreover, the chemical stabilities of transcripts of these genes are greater in an RsmA- mutant than in RsmA+ bacteria. These observations contrast with positive regulation of flhDC and motility by CsrA (=RsmA) in Escherichia coli. In the absence of AHL, the AHL receptors ExpR1/ExpR2 (=AhlR) in Erwinia carotovora subsp. carotovora negatively regulate motility and expression of flhDC and fliA by activating RsmA production. In the presence of AHL, regulatory effects of ExpR1/ExpR2 are neutralized, resulting in reduced levels of rsmA expression and enhanced motility.

RsmC of Erwinia carotovora subsp. carotovora negatively controls motility, extracellular protein production, and virulence by binding FlhD and modulating transcriptional activity of the master regulator, FlhDC.

RsmC and FlhDC are global regulators controlling extracellular proteins/enzymes, rsmB RNA, motility, and virulence of Erwinia carotovora subsp. carotovora. FlhDC, the master regulator of flagellar genes, controls these traits by positively regulating gacA, fliA, and rsmC and negatively regulating hexA. RsmC, on the other hand, is a negative regulator of extracellular proteins/enzymes, motility, and virulence since the deficiency of RsmC in FlhDC(+) strain results in overproduction of extracellular proteins/enzymes, hypermotility, and hypervirulence. These phenotypes are abolished in an RsmC(-) FlhDC(-) double mutant. We show that RsmC interferes with FlhDC action. Indeed, the expression of all three targets (i.e., gacA, rsmC, and fliA) positively regulated in E. carotovora subsp. carotovora by FlhDC is inhibited by RsmC. RsmC also partly relieves the inhibition of hexA expression by FlhDC. The results of yeast two-hybrid analysis revealed that RsmC binds FlhD and FlhDC, but not FlhC. We propose that binding of RsmC with FlhD/FlhDC interferes with its regulatory functions and that RsmC acts as an anti-FlhD(4)FlhC(2) factor. We document here for the first time that RsmC interferes with activation of fliA and motility in several members of the Enterobacteriaceae family. The extent of E. carotovora subsp. carotovora RsmC-mediated inhibition of FlhDC-dependent expression of fliA and motility varies depending upon enterobacterial species. The data presented here support the idea that differences in structural features in enterobacterial FlhD are responsible for differential susceptibility to E. carotovora subsp. carotovora RsmC action.

Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA(-) FlhDC(-) double mutant and multiple copies of rsmB(+) DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in E. carotovora subsp. carotovora. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, and rsmB RNA in the context of the exoprotein production and virulence of E. carotovora subsp. carotovora.

Parent-to-parent counseling - a gateway for developing positive mental health for the parents of children that have cerebral palsy with multiple disabilities.

Parent-to-parent counseling involves the interaction of two groups of parents, senior and junior, whose children suffer from cerebral palsy with multiple disabilities. The main theme of the interaction is the experience accumulated concerning the progress of the treatment of their child, the understanding of parental responsibility, the scope of rehabilitation, and the change of attitude towards their disabled children during the treatment, management and rehabilitation process, under the supervision of a psychologist or rehabilitation psychologist. The aim of the study is an attempt to minimize the gap between caregivers and the affected child through an exchange of knowledge and experience within the therapeutic group interactions. The parents who have been attending the outpatient department at the National Institute for the Orthopaedically Handicapped for a couple of months take a leading role in this interaction program and newcomers benefit through empathetically realizing the therapeutic aspects, and, simultaneously, releasing their internal tensions and anxiety regarding the rehabilitation of their disabled children. The study has been conducted on 50 Bengali speaking parents, who have attended the parent-to-parent interaction program. They were all from rural, urban and city areas within a 150-km radius of the place of study in Kolkata, West Bengal, situated in the eastern part of India. They were all from lower-income group (60%) and middle-income group (40%) families. Their mean educational qualification was XII standard. All the cases were selected from the mild to moderate range of child disability. All the participants indicated the effectiveness of these therapeutic interaction programs, with an average of 80% of parents favoring this program and acknowledging it as most effective.

Regulation of Erwinia carotovora hrpL(Ecc) (sigma-L(Ecc)), which encodes an extracytoplasmic function subfamily of sigma factor required for expression of the HRP regulon.

In Erwinia carotovora subsp. carotovora (Ecc) strain 71 (Ecc71), HrpL(Ecc), an alternate sigma factor of the extracytoplasmic function subfamily, plays a central role in the expression of the hrp (hypersensitive reaction and pathogenicity) regulon. We document here that sigma-54 (RpoN) is required for full expression of hrpL(Ecc) and that HrpS, in conjunction with sigma-54, activates hrpL(Ecc) transcription. We also made the novel observation that integration host factor is required for the activation of the hrpL(Ecc) promoter. Our findings reveal that the RsmA/rsmB RNA-mediated post-transcriptional system, known to control extracellular enzyme and harpin production, affects hrpL(Ecc) expression as well. For example, hrpL(Ecc) RNA levels are barely detected in an RsmB- strain. Conversely, hrpL(Ecc) mRNA levels are much higher in RsmA- bacteria than in the RsmA+ parent. This effect is due to RsmA-promoted decay of hrpL(Ecc) RNA. Moreover, the following regulators known to control the production of either RsmA, rsmB RNA, or both also affect hrpL(Ecc) expression: GacA (response regulator of a two-component system), KdgR (an IcII type repressor), HexA (a LysR type repressor), RsmC (a putative transcriptional adapter). Based upon the data now available for Ecc and extrapolating from the evidence in other systems, we propose a tentative model that depicts the Hrp regulatory system of Ecc and explains the basis for coregulation of extracellular enzyme production and expression of the Hrp regulon.

GacA, the response regulator of a two-component system, acts as a master regulator in Pseudomonas syringae pv. tomato DC3000 by controlling regulatory RNA, transcriptional activators, and alternate sigma factors.

Concerted investigations of factors affecting host-pathogen interactions are now possible with the model plant Arabidopsis thaliana and its model pathogen Pseudomonas syringae pv. tomato DC3000, as their whole genome sequences have become available. As a prelude to analysis of the regulatory genes and their targets, we have focused on GacA, the response regulator of a two-component system. The DC3000 gene was cloned by testing for the reversal of phenotypes of an Erwinia GacA- mutant. A GacA- mutant of DC3000 constructed by marker exchange produces much-reduced levels of transcripts of three alternate sigma factors: HrpL, required for the production of effector proteins and their translocation via the type III secretion system; RpoS, required for stress responses and secondary metabolite production; and RpoN, required for an assortment of metabolic processes and expression of hrpL. GacA deficiency also reduces the expression of hrpR and hrpS, which specify enhancer-binding proteins of the NtrC family required for hrpL transcription; ahlI and ahlR, the genes for quorum sensing signal; salA, a regulatory gene known to control virulence; CorS, a sensor kinase; CorR, the cognate response regulator that controls coronatine biosynthetic genes; and rsmB and rsmZ, which specify untranslatable regulatory RNA species. gacA expression itself is regulated by environmental conditions in DC3000, since transcript levels are affected by growth phase and media composition. The observations that high levels of gacA RNA occur in the hrp-inducing medium and GacA deficiency reduces the levels of rpoS expression implicate an important role of GacA in stress responses of DC3000. Consistent with the effects on hrpL expression, the GacA- mutant produces lower levels of transcripts of avr, hrp, and hop genes controlled by HrpL. In addition, GacA deficiency results in reduced levels of transcripts of several HrpL-independent genes. As would be expected, these effects on gene expression cause drastic changes in bacterial behavior: virulence towards A. thaliana and tomato; multiplication in planta; efficiency of the induction of the hypersensitive reaction (HR); production of pigment and N-acyl-homoserine lactone (AHL), the presumed quorum-sensing signal; and swarming motility. Our findings establish that GacA, located at the top in a regulatory cascade in DC3000, functions as a central regulator by controlling an assortment of transcriptional and posttranscriptional factors.

Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system.

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.

PsrA, the Pseudomonas sigma regulator, controls regulators of epiphytic fitness, quorum-sensing signals, and plant interactions in Pseudomonas syringae pv. tomato strain DC3000.

Pseudomonas syringae pv. tomato strain DC3000, a pathogen of tomato and Arabidopsis, occurs as an epiphyte. It produces N-acyl homoserine lactones (AHLs) which apparently function as quorum-sensing signals. A Tn5 insertion mutant of DC3000, designated PsrA(-) (Psr is for Pseudomonas sigma regulator), overexpresses psyR (a LuxR-type regulator of psyI) and psyI (the gene for AHL synthase), and it produces a ca. 8-fold-higher level of AHL than does DC3000. The mutant is impaired in its ability to elicit the hypersensitive reaction and is attenuated in its virulence in tomato. These phenotypes correlate with reduced expression of hrpL, the gene for an alternate sigma factor, as well as several hrp and hop genes during early stages of incubation in a Hrp-inducing medium. PsrA also positively controls rpoS, the gene for an alternate sigma factor known to control various stress responses. By contrast, PsrA negatively regulates rsmA1, an RNA-binding protein gene known to function as negative regulator, and aefR, a tetR-like gene known to control AHL production and epiphytic fitness in P. syringae pv. syringae. Gel mobility shift assays and other lines of evidence demonstrate a direct interaction of PsrA protein with rpoS promoter DNA and aefR operator DNA. In addition, PsrA negatively autoregulates and binds the psrA operator. In an AefR(-) mutant, the expression of psyR and psyI and AHL production are lower than those in DC3000, the AefR(+) parent. In an RpoS(-) mutant, on the other hand, the levels of AHL and transcripts of psyR and psyI are much higher than those in the RpoS(+) parent, DC3000. We present evidence, albeit indirect, that the RpoS effect occurs via psyR. Thus, AefR positively regulates AHL production, whereas RpoS has a strong negative effect. We show that AefR and RpoS do not regulate PsrA and that the PsrA effect on AHL production is exerted via its cumulative, but independent, effects on both AefR and RpoS.

Erwinia carotovora subspecies produce duplicate variants of ExpR, LuxR homologs that activate rsmA transcription but differ in their interactions with N-acylhomoserine lactone signals.

The N-acylhomoserine lactone (AHL) signaling system comprises a producing system that includes acylhomoserine synthase (AhlI, a LuxI homolog) and a receptor, generally a LuxR homolog. AHL controls exoprotein production in Erwinia carotovora and consequently the virulence for plants. In previous studies we showed that ExpR, a LuxR homolog, is an AHL receptor and that it activates transcription of rsmA, the gene encoding an RNA binding protein which is a global negative regulator of exoproteins and secondary metabolites. An unusual finding was that the transcriptional activity of ExpR was neutralized by AHL. We subsequently determined that the genomes of most strains of E. carotovora subspecies tested possess two copies of the expR gene: expR1, which was previously studied, and expR2, which was the focus of this study. Comparative analysis of the two ExpR variants of E. carotovora subsp. carotovora showed that while both variants activated rsmA transcription, there were significant differences in the patterns of their AHL interactions, the rsmA sequences to which they bound, and their relative efficiencies of activation of rsmA transcription. An ExpR2- mutant produced high levels of exoproteins and reduced levels of RsmA in the absence of AHL. This contrasts with the almost complete inhibition of exoprotein production and the high levels of RsmA production in an AhlI- mutant that was ExpR1-. Our results suggest that ExpR2 activity is responsible for regulating exoprotein production primarily by modulating the levels of an RNA binding protein.

Comparative analysis of two classes of quorum-sensing signaling systems that control production of extracellular proteins and secondary metabolites in Erwinia carotovora subspecies.

In Erwinia carotovora subspecies, N-acyl homoserine lactone (AHL) controls the expression of various traits, including extracellular enzyme/protein production and pathogenicity. We report here that E. carotovora subspecies possess two classes of quorum-sensing signaling systems defined by the nature of the major AHL analog produced as well as structural and functional characteristics of AHL synthase (AhlI) and AHL receptor (ExpR). Class I strains represented by E. carotovora subsp. atroseptica strain Eca12 and E. carotovora subsp. carotovora strains EC153 and SCC3193 produce 3-oxo-C8-HL (N-3-oxooctanoyl-l-homoserine lactone) as the major AHL analog as well as low but detectable levels of 3-oxo-C6-HL (N-3-oxohexanoyl-l-homoserine lactone). In contrast, the members of class II (i.e., E. carotovora subsp. betavasculorum strain Ecb168 and E. carotovora subsp. carotovora strains Ecc71 and SCRI193) produce 3-oxo-C6-HL as the major analog. ExpR species of both classes activate rsmA (Rsm, repressor of secondary metabolites) transcription and bind rsmA DNA. Gel mobility shift assays with maltose-binding protein (MBP)-ExpR(71) and MBP-ExpR(153) fusion proteins show that both bind a 20-mer sequence present in rsmA. The two ExpR functions (i.e., expR-mediated activation of rsmA expression and ExpR binding with rsmA DNA) are inhibited by AHL. The AHL effects are remarkably specific in that expR effect of EC153, a strain belonging to class I, is counteracted by 3-oxo-C8-HL but not by 3-oxo-C6-HL. Conversely, the expR effect of Ecc71, a strain belonging to class II, is neutralized by 3-oxo-C6-HL but not by 3-oxo-C8-HL. The AHL responses correlated with expR-mediated inhibition of exoprotein and secondary metabolite production.

ExpR, a LuxR homolog of Erwinia carotovora subsp. carotovora, activates transcription of rsmA, which specifies a global regulatory RNA-binding protein.

N-acyl homoserine lactone (AHL) is required by Erwinia carotovora subspecies for the expression of various traits, including extracellular enzyme and protein production and pathogenicity. Previous studies with E. carotovora subsp. carotovora have shown that AHL deficiency causes the production of high levels of RsmA, an RNA binding protein that functions as a global negative regulator of extracellular enzymes and proteins and secondary metabolites (Rsm, regulator of secondary metabolites). We document here that ExpR, a putative AHL receptor belonging to the LuxR family of regulators, activates RsmA production. In the absence of AHL, an ExpR(+) E. carotovora subsp. carotovora strain compared to its ExpR(-) mutant, produces higher levels of rsmA RNA and better expresses an rsmA-lacZ transcriptional fusion. Moreover, the expression of the rsmA-lacZ fusion in Escherichia coli is much higher in the presence of expR(71) (the expR gene of E. carotovora subsp. carotovora strain Ecc71) than in its absence. We also show that purified preparation of MBP-ExpR(71) binds (MBP, maltose binding protein) rsmA DNA. By contrast, MBP-ExpR(71) does not bind ahlI (gene for AHL synthase), pel-1 (gene for pectate lyase), or rsmB (gene for regulatory RNA that binds RsmA), nor does ExpR(71) activate expression of these genes. These observations strongly suggest transcriptional activation of rsmA resulting from a direct and specific interaction between ExpR(71) and the rsmA promoter. Several lines of evidence establish that N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HL), the major AHL analog produced by E. carotovora subsp. carotovora strain Ecc71, inhibits ExpR(71)-mediated activation of rsmA expression. These findings for the first time establish that the expR effect in E. carotovora subsp. carotovora is channeled via RsmA, a posttranscriptional regulator of E. carotovora subspecies, and AHL neutralizes this ExpR effect.

Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition.

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis. Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.

RsmA and the quorum-sensing signal, N-[3-oxohexanoyl]-L-homoserine lactone, control the levels of rsmB RNA in Erwinia carotovora subsp. carotovora by affecting its stability.

RsmA (for regulator of secondary metabolism), RsmC, and rsmB RNA, the components of a posttranscriptional regulatory system, control extracellular protein production and pathogenicity in Erwinia carotovora subsp. carotovora. RsmA, an RNA binding protein, acts as a negative regulator by promoting message decay. rsmB RNA, on the other hand, acts as a positive regulator by neutralizing the effect of RsmA. RsmC modulates the levels of RsmA and rsmB RNA by positively regulating rsmA and negatively controlling rsmB. The level of rsmB RNA is substantially higher in RsmA(+) bacteria than in RsmA(-) mutants. We show that rsmB RNA is more stable in the presence of RsmA than in its absence. RsmA does not stimulate the expression of an rsmB-lacZ transcriptional fusion; in fact, the beta-galactosidase level is somewhat higher in RsmA(-) bacteria than in RsmA(+) bacteria. We also investigated the basis for increased levels of rsmA and rsmB RNAs in the absence of the quorum-sensing signal, N-[3-oxohexanoyl]-L-homoserine lactone (OHL). The absence of OHL activates transcription of rsmA but not of rsmB. Instead, increased stability of rsmB RNA in the presence of RsmA accounts for the elevated levels of the rsmB RNA in OHL(-) bacteria. Mutant studies disclosed that while RsmA, OHL, and RsmC control the levels of rsmB RNA, high levels of rsmB RNA occur in the absence of RsmC or OHL only in RsmA(+) bacteria, indicating a critical role for RsmA in modulating the levels of rsmB RNA. The findings reported here firmly establish that the quorum-sensing signal is channeled in E. carotovora subsp. carotovora via the rsmA-rsmB posttranscriptional regulatory system.

Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnlA: a role for RdgB.

The activation of pectin lyase (Pnl) production in Erwinia carotovora subsp. carotovora strain 71 occurs upon DNA damage via a unique regulatory circuit involving recA, rdgA and rdgB. In a similar Pnl-inducible system reconstituted in Escherichia coli, the rdgB product was found to activate the expression of pnlA, the structural gene for pectin lyase. The kinetic data presented here also show that transcription of pnlA followed that of rdgB in Er. carotovora subsp. carotovora, indicating a temporal order of gene expression. By deletion analysis we have localized the promoter/regulatory region within a 66 bp DNA segment upstream of the pnlA transcriptional start site. This region contains the -10 consensus sequence but not the sequences corresponding to the E. coli -35 region. For DNA-binding studies, rdgB was overexpressed in E. coli and a 14 kDa polypeptide was identified as the gene product. RdgB from crude extracts or a purified preparation caused an identical gel mobility shift of a 164 bp DNA segment containing the pnlA promoter/regulatory region. Utilizing DNase I protection assay the RdgB-binding site was localized between nucleotides -29 and -56, i.e. overlapping the position of the putative -35 box. The findings reported here, taken along with our previous observation that the rdgE product is required for pnlA expression, establishes that rdgB encodes a transcriptional factor which specifically interacts with the pnlA promoter/regulatory region.

Identification of regulators of hrp/hop genes of Erwinia carotovora ssp. carotovora and characterization of HrpL(Ecc) (SigmaL(EccM)), an alternative sigma factor.

Summary Erwinia carotovora ssp. carotovora (Ecc) possesses hrpN(Ecc)[hrp = gene for hypersensitive reaction (HR) and pathogenicity], the structural gene for Harpin(Ecc), the inducer of the HR-like response and genes for the type III secretion system. In Ecc, RsmA, an RNA-binding protein responsible for the accelerated decay of RNA species, tightly controls the expression of the Hrp regulon. We document here that Ecc strain 71 possesses several presumed Hrp regulators: HrpX(Ecc), HrpY(Ecc), HrpS(Ecc), and HrpL(Ecc). Nucleotide sequence data indicate that the regulatory genes occur as a cluster. The data also suggest that HrpX(Ecc) is a putative sensor kinase, HrpY(Ecc) is the cognate response regulator, and HrpS(Ecc) is an NtrC-like regulator. hrpL(Ecc) consists of a 543-bp open reading frame (ORF), which encodes a c. 21-kDa protein product. This protein shares significant homology with members of the extracytoplasmic function (ECF) subfamily of alternative sigma factors, including SigE. To examine the regulatory role of hrpL(Ecc), we constructed by marker exchange an HrpL(Ecc) (-) derivative of an RsmA(-) strain. The levels of transcripts of hrcC(Ecc), a gene for the type III secretion system, and hrpN(Ecc) were lower in the RsmA(-)HrpL(Ecc) (-) mutant compared to its RsmA(-)HrpL(Ecc) (+) parent. The RsmA(-)HrpL(Ecc) (-) strain, like the RsmA(-)HrpL(Ecc) (+) parent, caused maceration of celery petioles and produced extracellular pectinases, cellulase, and protease, indicating that the alternative sigma factor is not required for the type I and type II secretion systems, for tissue maceration, or for the production of proteins secreted by these pathways. However, the RsmA(-)HrpL(Ecc) (-) strain, unlike the RsmA(-)HrpL(Ecc) (+) strain, did not elicit the HR in tobacco leaves. In addition, the RsmA(-)HrpL(-) strain failed to cause symptoms in Arabidopsis thaliana. Genetic and biochemical data revealed that Harpin(Ecc) is required for symptom production in Arabidopsis, but is not sufficient by itself to cause necrosis. These observations raise the possibility that another 'effector' protein of Ecc, secreted by the type III system, acts in conjunction with Harpin(Ecc) to trigger responses resulting in cell death in Arabidopsis.

Global regulation in Erwinia species by Erwinia carotovora rsmA, a homologue of Escherichia coli csrA: repression of secondary metabolites, pathogenicity and hypersensitive reaction.

Our previous studies revealed that rsmA of Erwinia carotovora subsp. carotovora strain 71 suppressed the synthesis of the cell density (quorum) sensing signal N-(3-oxohexanoyl)-L-homoserine lactone, the production of extracellular enzymes and tissue macerating ability in soft-rotting Erwinia species and that homologues of this negative regulator gene were present in other Erwinia species. Northern blot data presented here demonstrate that rsmA and rsmA-like genes are also expressed in soft-rotting and non-soft-rotting Erwinia spp. such as E. amylovora, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. chrysanthemi, E. herbicola and E. stewartii. A low-copy plasmid carrying rsmA of E. carotovora subsp. carotovora strain 71 caused suppression of antibiotic production in E. carotovora subsp. betavasculorum, flagellum formation in E. carotovora subsp. carotovora, carotenoid production in E. herbicola and E. stewartii, and indigoidine production in E. chrysanthemi. In E. amylovora, rsmA of E. carotovora subsp. carotovora suppressed the elicitation of the hypersensitive reaction in tobacco leaves and the production of disease symptoms in apple shoots, in addition to repressing motility and extracellular polysaccharide production. We conclude that rsmA homologues function as global regulators of secondary metabolic pathways as well as factors controlling host interaction of Erwinia species.

Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor.

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N(epsilon)-(indole-3-acetyl)-l-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.

The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000.

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana. The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa, yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.