Comparative study of regulatory mechanisms for pectinase production by Erwinia carotovora subsp. carotovora and Erwinia chrysanthemi

The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors. We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E. chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E. carotovora subsp. carotovora. Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E. carotovora subsp. carotovora. In fact, KdgR and CRP homologues of E. carotovora subsp. carotovora had high amino acid identities to those of E. chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif. However, in Western blot analyses using anti-Pir (E. chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E. carotovora subsp. carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalacturonic acid) in which hyperinduction by Pir has been reported in E. chrysanthemi EC16. When plasmids that contained each of these regulatory genes from E. chrysanthemi were introduced into E. carotovora subsp. carotovora, Pel production was controlled as predicted from their roles in E. chrysanthemi, except for PecS. PecS exerted a positive control in E. carotovora subsp. carotovora, in contrast to a negative control in E. chrysanthemi. DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E. chrysanthemi and KdgR and CRP homologues of E. carotovora subsp. carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E. carotovora subsp. carotovora. Taken together, KdgR and CRP homologues of E. carotovora subsp. carotovora may regulate Pel and Peh production as in E. chrysanthemi. However, the presence of Pir and PecS homologues in E. carotovora subsp. carotovora was not identified in this study, though these proteins of E. chrysanthemi were functional on the promoter regions of the pectinase genes of E. carotovora subsp. carotovora.     

Identification of cfxR, an activator gene of autotrophic CO2 fixation in Alcaligenes eutrophus

A regulatory gene, cfxR, involved in the carbon dioxide assimilation of Alcaligenes eutrophus H16 has been characterized through the analysis of mutants. The function of cfxR is required for the expression of two cfx operons that comprise structural genes encoding Calvin cycle enzymes. CfxR (34.8 kDa) corresponds with an open reading frame of 954 bp, with a translational initiation codon 167 bp upstream of the chromosomal cfx operon. The cfx operon and cfxR are transcribed divergently. The N-terminal sequence of CfxR is very similar to those of bacterial regulatory proteins belonging to the LysR family. Heterologous expression of cfxR in Escherichia coli was achieved using the pT7-7 system. Mobility shift experiments demonstrated that CfxR is a DNA-binding protein with a target site upstream of both the chromosomal and the plasmid-encoded cfx operons.     

2-keto-3-deoxygluconate transport system in Erwinia chrysanthemi.

In Erwinia chrysanthemi, the gene kdgT encodes a transport system responsible for the uptake of ketodeoxyuronates. We studied the biochemical properties of this transport system. The bacteria could grow on 2,5-diketo-3-deoxygluconate but not on 2-keto-3-deoxygluconate. The 2-keto-3-deoxygluconate entry reaction displayed saturation kinetics, with an apparent Km of 0.52 mM (at 30 degrees C and pH 7). 5-Keto-4-deoxyuronate and 2,5-diketo-3-deoxygluconate appeared to be competitive inhibitors, with Kis of 0.11 and 0.06 mM, respectively. The 2-keto-3-deoxygluconate permease could mediate the uptake of glucuronate with a low affinity. kdgT was cloned on an R-prime plasmid formed by in vivo complementation of a kdgT mutation of Escherichia coli. After being subcloned, it was mutagenized with a mini-Mu-lac transposable element able to form fusions with the lacZ gene. We introduced a kdgT-lac fusion into the E. chrysanthemi chromosome by marker exchange recombination and studied its regulation. kdgT product synthesis was not induced by external 2-keto-3-deoxygluconate in the wild-type strain but was induced by galacturonate and polygalacturonate. Two types of regulatory mutants able to grow on 2-keto-3-deoxygluconate as the sole carbon source were studied. Mutants of one group had a mutation in the operator region of kdgT; mutants of the other group had a mutation in kdgR, a regulatory gene controlling kdgT expression.     

KdgR, an IClR family transcriptional regulator, inhibits virulence mainly by repression of hrp genes in Xanthomonas oryzae pv. oryzae

KdgR has been reported to negatively regulate the genes involved in degradation and metabolization of pectic acid and other extracellular enzymes in soft-rotting Erwinia spp. through direct binding to their promoters. The possible involvement of a KdgR orthologue in virulence by affecting the expression of extracellular enzymes in Xanthomonas oryzae pv. oryzae, the causal agent of rice blight disease, was examined by comparing virulence and regulation of extracellular enzymes between the wild type (WT) and a strain carrying a mutation in putative kdgR (ΔXoo0310 mutant). This putative kdgR mutant of X. oryzae pv. oryzae showed increased pathogenicity on rice without affecting the regulation of extracellular enzymes, such as amylase, cellulase, xylanase, and protease. However, the mutant carrying a mutation in an ortholog of xpsL, which encodes the functional secretion machinery for the extracellular enzymes, showed a dramatic decrease in pathogenicity on rice. Both mutants of kdgR and of xpsL orthologs showed higher expression of two major hrp regulatory genes, hrpG and hrpX, and the genes in the hrp operons when grown in hrp-inducing medium. Thus, both genes were shown to be involved in repression of hrp genes. The kdgR ortholog was thought to suppress virulence mainly by repressing the expression of hrp genes without affecting the expression of extracellular enzymes, unlike findings for the kdgR gene in soft-rotting Erwinia spp. On the other hand, xpsL was confirmed to be involved in virulence by promoting the secretion of extracellular enzymes in spite of repressing the expression of the hrp genes.     

Nucleotide sequence of the Erwinia chrysanthemi gene encoding 2-keto-3-deoxygluconate permease

The phytopathogenic bacterium Erwinia chrysanthemi produces a group of pectolytic enzymes able to depolymerise the pectic compounds in plant cell walls. The resulting tissue maceration is known as soft rot disease. The degraded pectin products are transported by 2-keto-3-deoxygluconate permease into the bacterial cell, where they serve as carbon and energy sources. This H+ coupled transport system is encoded by the kdgT gene; we report the nucleotide sequence of kdgT. It is encoded by an open reading frame (ORF) of 1194 bp, which is preceded by an Escherichia coli-type promoter region. The ORF encodes a protein with 398 amino acid (aa) residues and a predicted Mr of 48,550. As would be expected for a membrane protein, it is very hydrophobic, containing 63% nonpolar aa. However, the kdgT gene has no apparent evolutionary relationship to other genes encoding sugar transport proteins, such as lacY, melB or the E. coli citrate transport gene. Southern hybridization experiments indicate a strong homology between the Er. chrysanthemi and E. coli kdgT genes; there is also a second region on the E. coli chromosome with homology to kdgT. The kdgT gene is located near the ade-377 marker on the Er. chrysanthemi chromosome (equivalent to the region between 20 and 30 min in E. coli), whereas the E. coli kdgT gene is located at 88 min. Thus, these two enterobacteria show some significant differences in their genomic organization.     

Lactose metabolism in Erwinia chrysanthemi.

Wild-type strains of the phytopathogenic enterobacterium Erwinia chrysanthemi are unable to use lactose as a carbon source for growth although they possess a beta-galactosidase activity. Lactose-fermenting derivatives from some wild types, however, can be obtained spontaneously at a frequency of about 5 X 10(-7). All Lac+ derivatives isolated had acquired a constitutive lactose transport system and most contained an inducible beta-galactosidase. The transport system, product of the lmrT gene, mediates uptake of lactose in the Lac+ derivatives and also appears to be able to mediate uptake of melibiose, raffinose, and galactose. Two genes encoding beta-galactosidase enzymes were detected in E. chrysanthemi strains. That mainly expressed in the wild-type strains was the lacZ product. The other, the lacB product, is very weakly expressed in these strains. These enzymes showed different affinities for the substrates o-nitrophenyl-beta-D-galactopyranoside and lactose and for the inhibitors isopropyl-beta-D-thiogalactopyranoside and galactose. The lmrT and lacZ genes of E. chrysanthemi, together with the lacI gene coding for the regulatory protein controlling lacZ expression, were cloned by using an RP4::miniMu vector. When these plasmids were transferred into Lac- Escherichia coli strains, their expression was similar to that in E. chrysanthemi. The cloning of the lmrT gene alone suggested that the lacZ or lacB gene is not linked to the lmrT gene on the E. chrysanthemi chromosome. One Lac+ E. chrysanthemi derivative showed a constitutive synthesis of the beta-galactosidase encoded by the lacB gene. This mutation was dominant toward the lacI lacZ cloned genes. Besides these mutations affecting the regulation of the lmrT or lacB gene, the isolation of structural mutants unable to grow on lactose was achieved by mutagenic treatment. These mutants showed no expression of the lactose transport system, the lmrT mutants, or the mainly expressed beta-galactosidase, lacZ mutants. The lacZ mutants retained a very low beta-galactosidase level, due to the lacB product, but this level was low enough to permit use of the lacZ mutants for the construction of gene fusions with the Escherichia coli lac genes.