The production of extracellular enzymes such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt) is activated by the cell density (quorum)-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (HSL); plant signals; and aep genes during postexponential growth of Erwinia carotovora subsp. carotovora 71. Studies with mutants of E. carotovora subsp. carotovora 71 derepressed in exoenzyme production led to the identification of a negative regulator gene, rsmA (rsm, repressor of secondary metabolites). Nucleotide sequencing, transcript assays, and protein analysis established that a 183-bp open reading frame encodes the 6.8-kDa RsmA. rsmA has extensive homology with the csrA gene of Escherichia coli, which specifies a negative regulator of carbon storage. Moreover, the suppression of glycogen synthesis in E. coli by rsmA indicates that the Erwinia gene is functionally similar to csrA. Southern hybridizations revealed the presence of rsmA homologs in soft-rotting and non-soft-rotting Erwinia spp. and in other enterobacteria such as Enterobacter aerogenes, E. coli, Salmonella typhimurium, Shigella flexneri, Serratia marcescens, and Yersinia pseudotuberculosis. rsmA suppresses production of Pel, Peh, Cel, and Prt, plant pathogenicity, and synthesis of HSL in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, and E. chrysanthemi. In the E. carotovora subsp. carotovora 71, rsmA reduces the levels of transcripts of hslI, a luxI homolog required for HSL biosynthesis. This specific effect and the previous finding that HSL is required for extracellular enzyme production and pathogenicity in soft-rotting Erwinia spp. support the hypothesis that rsmA controls these traits by modulating the levels of the cell density (quorum)-sensing signal.

The soft-rotting bacterium, Erwinia carotovora subsp. carotovora 71, produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh), and protease (Prt). While the extracellular levels of these enzymes are extremely low when the bacterium is grown in salts-yeast extract-glycerol (SYG) medium, the enzymatic activities are highly induced in SYG medium supplemented with celery extract. By transposon (mini-Tn5) mutagenesis, we isolated a RsmA ؊ mutant, AC5070, which overproduces extracellular enzymes; the basal levels of Pel, Peh, and Cel in AC5070 are higher than the induced levels in the RsmA ؉ parent, AC5047. While Peh production is mostly constitutive in AC5070, Pel, Cel, and Prt production is still inducible with celery extract. The high basal levels of pel-1, pel-3, and peh-1 mRNAs in AC5070 demonstrate that overproduction of the pectolytic enzymes is due to the stimulation of transcription. Using chromosomal DNA flanking mini-Tn5 as a probe, we cloned the wild-type rsmA ؉ allele, which suppresses Pel, Peh, Cel, and Prt production in both RsmA ؉ and RsmA ؊ strains. The RsmA ؊ mutant, like its parent, produces N-(3oxohexanoyl)-L-homoserine lactone (HSL), a starvation/cell density-sensing signal required for extracellular enzyme production. To examine the role of HSL, we constructed HSL-deficient strains by replacing hslI, a locus required for HSL production, with hslI::Tn3HoHo1-Spc. While the basal levels of Pel, Peh, Cel, and Prt are comparable in the RsmA ؊ mutant and its HSL ؊ derivative, these enzymes are barely detectable in the Hsl ؊ derivative of the RsmA ؉ parent strain. The Hsl ؊ RsmA ؉ strain fails to elicit soft rot, whereas the Hsl ؊ RsmA ؊ strain, like its Hsl ؉ RsmA ؊ parent, remains hypervirulent. These findings demonstrate that the RsmA ؊ mutant produces extracellular enzymes and macerates plant tissue in the absence of HSL. We conclude that overproduction of extracellular enzymes in an HSL-independent manner occurs because of the inactivation of a global repressor locus, rsmA.

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)-~-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-softrotting Erwinia spp. such as E. amylovora, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. cerotovora subsp. carotovora, E. chrysanthemi, E. henbicola and E. stewaflii. 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. henbicola 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 Entvinia species.

Xylella fastidiosa is a xylem-limited, nutritionally fastidious bacterium that causes several plant diseases including Pierce's disease (PD) in grape and leaf scorch in almond (ALS) and oleander (OLS). OLS strains belong to X. fastidiosa subsp. sandyi, PD strains belong to X. fastidiosa subsp. fastidiosa, and strains from almond designated as ALS strains are of two general types belonging either to X. fastidiosa subsp. multiplex or X. fastidiosa subsp. fastidiosa. The ALS strains assigned to X. fastidiosa subsp. multiplex belong to two different genotypes (ALSI and ALSII) below the subspecies level. The OLS strains do not infect grape or almond. PD strains produce diseases in grape, alfalfa, almond, and some weeds, but they do not infect oleander, oak, peach, or citrus. ALS strains that belong to X. fastidiosa subsp. multiplex do not produce disease on grape. In this study, a relatively simple polymerase chain reaction (PCR) based method was developed to distinguish among PD, OLS, and ALS strains. PCR performed with primers XF1968-L and XF1968-R amplified a 638-bp fragment from OLS strains but not from PD strains or ALS strains that belong to X. fastidiosa subsp. fastidiosa. PCR with primers XF2542-L and XF2542-R amplified a 412-bp fragment from PD strains, but not from OLS strains. PCR with primers ALM1 and ALM2 produced a fragment of 521 bp from strains isolated from almond that belong to X. fastidiosa subsp. multiplex. The combination of the three primer sets allowed the distinction of the two ALS genotypes of X. fastidiosa subsp. multiplex. These results are in agreement with those obtained from analysis of sequences of 16S-23S rDNA intergenic spacer regions sequence analysis and with previous results based on randomly amplified polymorphic DNA analysis.

Exopolysaccharides (EPS) synthesized by plant-pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared with the wild type. Furthermore, gas chromatography-mass spectrometry showed that both mutant strains did not produce oligosaccharides. Biologically, the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects: they were very poorly transmitted by and retained within vectors. The gene expression profile indicated upregulation of genes implicated in cell-to-cell signaling and adhesins while downregulation in genes was required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in X. fastidiosa interactions with both plants and insects.

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Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease (PD) of grapevines. A variety of plant species found near a severe outbreak of PD in vineyards in the Temecula Valley of California were tested using enzyme-linked immunosorbent assay, culture on media, and polymerase chain reaction to identify potential inoculum sources in the area. Species that consistently tested positive for X. fastidiosa were the known hosts, grape, almond, and oleander, and two new hosts, Spanish broom (Spartium junceum) and wild mustard (Brassica spp). Sequence analysis of the 16S-23S rRNA spacer region found that strains isolated from grapevine, Spanish broom, wild mustard, and almond clustered with previously sequenced PD strains. Thus, these species could serve as sources of inoculum for infection of grapevines and should be removed or monitored for signs of infection. Sequences from oleander isolates from Temecula formed another cluster with a previously published oleander strain sequence. Oleander strains do not infect grapevines and thus do not appear to cause a direct threat to grapevines. Two additional isolates from almond were determined to be genetically different from PD strains, and the ability of these strains to infect grapevine is not known. Greenhouse transmission studies indicate that the glassy-winged sharpshooter was able to transmit a PD strain of X. fastidiosa to Spanish broom, black mustard, and other hosts.

The xylem-limited, insect-transmitted bacterium Xylella fastidiosa causes Pierce's disease in grapes through cell aggregation and vascular clogging. GacA controls various physiological processes and pathogenicity factors in many gram-negative bacteria, including biofilm formation in Pseudomonas syringae pv. tomato DC3000. Cloned gacA of X. fastidiosa was found to restore the hypersensitive response and pathogenicity in gacA mutants of P. syringae pv. tomato DC3000 and Erwinia amylovora. A gacA mutant of X. fastidiosa (DAC1984) had significantly reduced abilities to adhere to a glass surface, form biofilm, and incite disease symptoms on grapevines, compared with the parent (A05). cDNA microarray analysis identified 7 genes that were positively regulated by GacA, including xadA and hsf, predicted to encode outer membrane adhesion proteins, and 20 negatively regulated genes, including gumC and an antibacterial polypeptide toxin gene, cvaC. These results suggest that GacA of X. fastidiosa regulates many factors, which contribute to attachment and biofilm formation, as well as some physiological processes that may enhance the adaptation and tolerance of X. fastidiosa to environmental stresses and the competition within the host xylem.