Characterization of the hrp pathogenicity cluster of Erwinia carotovora subsp. carotovora: high basal level expression in a mutant is associated with reduced virulence.

Extracellularly targeted proteins are crucial for virulence of gram-negative phytopathogenic bacteria. Erwinia carotovora subsp. carotovora employs the so-called type II (GSP) pathway to secrete a number of pectinases and cellulases, which cause the typical tissue maceration symptoms of soft-rot disease. The type III (hrp) pathway is the major virulence determinant in the genera Pseudomonas, Ralstonia and Xanthomonas, and in non-macerating species of Erwinia. The hrp cluster was recently partially characterized from E. carotovora sp. carotovora, and shown to affect virulence during early stages of infection. Here we have isolated and characterized 15 hrp genes comprising the remaining part of the cluster. The genes hrpL, hrpXY and hrpS were deduced to be transcribed as separate units, whereas the 11 remaining genes from hrpJ to hrcU form a single large operon. The hrpX gene, which codes for the sensory kinase of the two-component regulatory locus hrpXY was insertionally inactivated by placing a transposon (entranceposon) in the gene. The resulting mutant bacterium expresses the hrp genes at high basal level even in a non-inducing medium. This relative overexpression was shown to be due to the hrpX::entranceposon insertion causing enhanced transcription of the downstream hrpY gene. The hrpX(-)-hrpYC mutant bacterium exhibited a slower growth rate and the appearance of disease symptoms in infected Arabidopsis plants was delayed, as compared to the wild-type strain. The need for hrp gene expression for virulence has been documented in both non-macerating plant pathogens and in soft-rotting Erwinia sp. but this is the first demonstration that high basal-level expression of hrp -regulated genes may actually have a negative impact on disease progress in a susceptible host plant.

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.

Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway.

A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C. A single ORF of 999 nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.

Control of virulence gene expression by plant calcium in the phytopathogen Erwinia carotovora.

Plant calcium can modulate a particular plant-pathogen interaction and have a decisive role in disease development. Enhanced resistance to the phytopathogenic enterobacterium Erwinia carotovora, the causal agent of bacterial soft rot disease, is observed in high-calcium plants. One of the main virulence determinants of E. carotovora, the PehA endopolygalacturonase, is specifically required in the early stages of the infection. Production of PehA was found to be dependent on the calcium concentration in the bacterial environment. An increase in extracellular calcium to mM concentrations repressed pehA gene expression without reducing or even enhancing expression of other extracellular enzyme-encoding genes of this pathogen. An increase in plant calcium levels could be correlated to enhanced resistance to E. carotovora infection and to an inhibition of in planta production of PehA. Ectopic expression of pehA from a calcium-insensitive promoter allowed E. carotovora to overcome this calcium-induced resistance. The results imply that plant calcium can constitute an important signal molecule in plant-pathogen interaction, which acts by modulating the expression of virulence genes of the pathogen.

Isolation and characterization of new C-terminal substitution mutations affecting secretion of polygalacturonase in Erwinia carotovora ssp. carotovora.

An intact C-terminus was previously shown to be required for stability and secretion of the polygalacturonase (PehA) in Erwinia carotovora ssp. carotovora. Here we have analyzed the effects of amino acid (aa) substitutions generated to five C-terminal positions of PehA. Conservation of two hydrophobic and one non-hydrophobic residue (V372, V374 and N371, respectively) was found to be essential for maintenance of the protein stability. As an exception, one of the mutants (V372G) did not show major effects on protein stability, as determined by immunoblots and enzyme activity assay, yet it prevented the secretion completely. We conclude that the C-terminus of PehA is directly involved in the formation or stabilization of a conformation-sensitive structure needed for recognition of the protein as secreted.

The extreme C-terminus is required for secretion of both the native polygalacturonase (PehA) and PehA-Bla hybrid proteins in Erwinia carotovora subsp. carotovora.

A set of gene fusions was constructed between the pehA gene encoding the secreted endopolygalacturonase (PehA) and the bla gene coding for a normally periplasmic beta-lactamase (Bla). The resulting hybrid proteins were specifically and actively routed out of the cells via the Out-terminal branch of the general secretory pathway (GSP) in Erwinia carotovora subsp. carotovora (Ecc), provided that no more than the last two amino acids (aa) of the PehA domain were excluded from the fusion. However, both PehA-Bla hybrid proteins and PehA variants lacking at least four aa from the C-terminus of the PehA accumulated in the periplasm. Also, overexpression of the gene fusions prevented extracellular targeting of the hybrid proteins. Site-directed mutagenesis of the codons -4 and -3 (encoding Asn-373 and Val-374, respectively) from the end of the pehA gene and analysis of the protein products suggested that the Val-374 was important both for the structure and secretion of PehA, while the Asn-373 proved to be insignificant. We conclude that: (i) the GSP of Ecc is capable of secreting heterologous proteins; (ii) as the PehA protein can accommodate C-terminal extensions, secretion can occur with no part of the proposed targeting signal lying within the C-terminal extremity of a secreted molecule; and (iii) residues within the C-terminus of PehA play a role in secretion, possibly through stabilization of a structure needed for proper exposition of the proposed targeting motif.

Characterization of polygalacturonases.

Lei et al. [Gene 117 (1992) 119-124] recently published the nucleotide sequence of the peh gene of Erwinia carotovora subsp. carotovora (Ecc) and a characterization of its product endopolygalacturonase (Peh). The gene appears highly similar to previously described peh sequences of Ecc [Hinton et al., Mol. Microbiol. 4 (1990) 1029-1036; Saarilahti et al., Mol. Microbiol. 4 (1990) 1037-1044] which were not cited in the article. Ecc carries a single peh gene whose product, Peh, is here shown to share similarity with the two Pehs characterized thus far and a Peh-like protein of eukaryotic origin at the amino acid (aa) sequence level. Additionally, a highly conserved region within their C-terminal domains was found to share local similarity with two 13-aa segments of an otherwise distinct exo-poly-alpha-D-galacturonosidase (exo-Peh), suggesting that these segments might be required for enzyme activity in both Peh and exo-Peh.

The virulence plasmid does not contribute to growth of Salmonella in cultured murine macrophages.

The virulence plasmid, characteristic of many serovars of Salmonella sp., and specifically its spv genes, promote intracellular growth of the bacteria in the liver and spleen and are essential for the virulence of these Salmonella serovars in the mouse. In an attempt to establish an in vitro model for studying its function, we evaluated its effect on the intracellular growth of the bacteria in macrophages in culture. We used a number of different macrophage-like cell lines (J774-A.1, IC-21 and PU5-1.8), as well as peritoneal or splenic macrophages from genetically Salmonella-sensitive (Itys, BALB/c) or resistant (Ityr, C3H/HeN) mice, and at different states of activation, stimulated in vivo or in vitro with lipopolysaccharide and/or recombinant gamma interferon. These were found to differ in their ability to suppress or sustain intracellular growth of several Salmonella serovars, but in all cases the growth was independent of the spv genes.

Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production.

In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75-2, 75-5 and 75-6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 aa PehA protein, respectively, fused to the mature part of the periplasmic beta-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75-5 and 75-6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp- mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR-), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR- mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR- as well as PehA- mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.

A new alpha-helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid.

A new protein of Salmonella typhimurium was identified and characterized. The gene (tlpA) encoding this protein (TlpA) was isolated from the large virulence-associated plasmid of S. typhimurium and sequenced in order to predict the primary structure of TlpA. tlpA encodes a 371-amino acid soluble protein with a calculated M(r) of 41600 and pI of 4.63. Secondary structure predictions and sequence statistics of TlpA indicated a predominant alpha-helical configuration and presence of heptapeptide repeat motifs characteristic of coiled coil proteins. Purified TlpA was shown to have biochemical properties similar to those of coiled coil proteins, including adoption of an alpha-helical configuration and a tendency to form homodimers. Furthermore, TlpA possessed heat resistance, evidence for a chain register and altered mobility in urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels which are characteristics of tropomyosins. TlpA shows 32% overall sequence similarity with rat cardiac myosin and 36% similarity with horse platelet beta-tropomyosin over 226 residues, whereas selected regions possessed significant sequence identities with myosins, tropomyosins, and alpha-helical surface proteins of Streptococcus pyogenes. Our results indicate that TlpA represents a new member of prokaryotic coiled coil proteins.

Plasmid-mediated serum resistance in Salmonella enterica.

The ability of Salmonella enterica to survive an incubation of 2 h in fresh, pooled guinea-pig serum was determined comparing strain sets of serovariants Dublin and Typhimurium, harbouring or lacking the virulence plasmid. All strains showed marked serum resistance, which was slightly decreased among cured strains of Typhimurium. However, when introduced into a rough Escherichia coli strain, all Typhimurium plasmids substantially increased the resistance of the host strain to guinea-pig serum, whereas the Dublin plasmid did not. The traT gene, previously shown to affect serum resistance, was identified on all Typhimurium plasmids, but not on the Dublin plasmid. Mutational inactivation of the traT gene on the Typhimurium plasmids eliminated the serum resistance mediated by the plasmids.

The mkaC virulence gene of the Salmonella serovar typhimurium 96 kb plasmid encodes a transcriptional activator.

The intracellular growth and virulence of Salmonella serovar Typhimurium for mice is dependent on a plasmid-borne gene cluster termed mka. We studied the regulatory interactions of the genes mkaA, mkaB, mkaC and mkaD using lacZ gene fusions. Complementation experiments with cloned DNA fragments encoding each of the four MKa proteins indicated that mkaC enhances the expression of beta-galactosidase from the mkaA-, mkaB- and mkaC-lacZ gene fusions in trans. An mkaD-lacZ fusion or mkaA-lacZ fusion that did not contain DNA proximal to mkaB was not inducible with MkaC, indicating that at least mkaB and mkaA are induced together as an operon. MkaC is thus the first virulence protein whose function has been resolved.

Molecular cloning of ompRS, a regulatory locus controlling production of outer membrane proteins in Erwinia carotovora subsp. carotovora.

A locus, ompRS, controlling synthesis of outer membrane proteins was cloned from Erwinia carotovora subsp. carotovora (Ecc) by complementation of an Escherichia coli ompR-envZ mutant. The Ecc ompRS locus was both structurally and functionally similar to the ompR-envZ operon controlling porin gene expression in E. coli as shown by DNA hybridization and complementation of E. coli ompR and envZ mutants. Furthermore, introduction of ompRS into E. coli delta (ompR-envZ) strains restored the osmoregulation of the major outer membrane protein genes ompC and ompF. Maxicell analysis of ompRS-carrying plasmids suggested that proteins similar in size to the E. coli ompR and envZ gene products were encoded by the Ecc ompR and ompS genes, respectively. Introduction of an ompRS transposon mutant onto the Ecc chromosome by marker exchange mutagenesis showed that ompRS is essential for production of a major outer membrane porin in Ecc. This mutational defect could be complemented by clones carrying Ecc ompRS or E. coli ompR envZ. The lack of the porin did not, however, compromise the virulence of these Ecc mutants.

Structural analysis of the pehA gene and characterization of its protein product, endopolygalacturonase, of Erwinia carotovora subspecies carotovora.

A clone producing a polygalacturonase (EC 3.2.1.15) in Escherichia coli was isolated from a genomic library of Erwinia carotovora subspecies carotovora constructed in PUC18. The DNA segment carrying the corresponding structural gene, named pehA, contained an open reading frame (ORF) encoding a 402-amino-acid (aa) polypeptide with an Mr of 42,849. In E. carotovora the polygalacturonase was synthesized with a 26-aa cleavable signal peptide. The mature 376-aa PehA had a calculated Mr of 40,064 and a pl of 10.19. The pH optimum of the enzyme was about 5.5 and the temperature optimum was in the range 35-45 degrees C. Analysis of the reaction products of polygalacturonic acid hydrolysis indicated that the PehA protein is an endopolygalacturonase. No similarity was observed between the aa sequences of PehA and other pectic enzymes of erwinias. However, substantial similarity was detected within the C-terminal portions of PehA and a previously described tomato polygalacturonase, suggesting that the bacterial and eukaryotic polygalacturonases may have a common origin.

Analysis of OmpC-beta-lactamase hybrid proteins: OmpC appears not to contain discrete localization signals.

Fusions to the beta-lactamase (bla) gene were employed to analyze the presence of localization information in the mature part of OmpC, a major pore-forming outer membrane protein in Escherichia coli K-12. Six translational ompC-bla gene fusions were constructed, the shortest of them containing only part of the ompC signal sequence and the largest approximately 90% of the sequence encoding mature OmpC protein. Export of the hybrid proteins to a non-cytoplasmic location was a prerequisite for ampicillin resistance. Localization of the hybrid proteins by cell fractionation and solid phase iodination of whole cells suggested that the exported hybrid proteins possibly interacted with the outer membrane in vivo. No specific sequence of the mature OmpC protein, however, was found to promote this interaction.

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.

Fusion of genes encoding Escherichia coli heat-stable enterotoxin and outer membrane protein OmpC.

The OmpC outer membrane protein of Escherichia coli was used as a carrier molecule for the nonimmunogenic heat-stable enterotoxin STa. Two fragments of different lengths of the gene encoding STa were fused in vitro to the 3' terminus of the truncated ompC gene. The resulting OmpC-STa hybrid proteins could be detected by L-[35S]cysteine labeling, and they were processed and thus exported. All synthesized hybrid protein remained cell bound and was found by fractionation mainly in the periplasm. Immunoblot analysis showed that the hybrid proteins reacted in vitro both with anti-OmpC and anti-STa antibodies, and immunization of rabbits evoked an antibody response to either of these proteins.

In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12.

The product of the ompR gene of E. coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E. coli K12. A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene. The resulting clones were transformed into wild type and corresponding mutant backgrounds to analyze their effects on ompC and ompF expression. All of the cloned ompR mutant alleles exhibited a dominant OmpC- phenotype in an ompR+ background. In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles. The results suggest an interaction between different OmpR molecules and thereby support the idea that OmpR can exist as a multimeric protein.