William Nasser

Characterization of the Erwinia chrysanthemi expI–expR locus directing the synthesis of two N-acyl-homoserine lactone signal molecules

The plant pathogen Erwinia chrysanthemi produces three acyl‐homoserine lactones (acyl‐HSLs). One has been identified as N‐(3‐oxohexanoyl)‐homoserine lactone (OHHL), and the two others were supposed to be N‐(hexanoyl)‐homoserine lactone (HHL) and N‐(decanoyl)‐homoserine lactone (DHL). The genes for a quorum‐sensing signal generator (expIu200a) and a response regulator (expRu200a) were cloned. These genes are convergently transcribed and display high similarity to the expI–expR genes of Erwinia carotovora. ExpI is responsible for both OHHL and HHL production. Inactivation of expI had little effect on pectinase synthesis in E. chrysanthemi, as expression of only two of the pectate lyase genes, pelA and pelB, was decreased. E. chrysanthemi expR mutants still produced acyl‐HSL and pectinases. However, gel shift and DNAse I footprinting experiments showed that the purified E. chrysanthemi ExpR protein binds specifically to the promoter regions of the five major pel genes. Addition of OHHL modified the ExpR‐DNA bandshift profiles, indicating that ExpR interacts with OHHL and binds to DNA in different ways, depending on the OHHL concentration. Localization of the ExpR binding sites just upstream of promoter regions suggests that ExpR functions as an activator of pel expression in the presence of OHHL. The absence of a phenotype in expR mutants strongly suggests that at least an additional interchangeable ExpR homologue exists in E. chrysanthemi. Finally, transcription of expIu2003::uidA and expRu2003::uidA fusions is dependent on the population density, suggesting the existence of a quorum‐sensing hierarchy in E. chrysanthemi. These results suggest that the expI–expR locus is part of a complex autoregulatory system that controls quorum sensing in E. chrysanthemi.

pecS: a locus controlling pectinase, cellulase and blue pigment production in Erwinia chrysanthemi

Erwinia chrysanthemi mutants (designated as pecS) displaying derepressed pectate lyase and cellulose synthesis were isolated. In addition, the pecS mutation is responsible for production of an extracellular insoluble blue pigment whose synthesis is cryptic in the wild‐type 3937 strain. Transduction analysis indicates that the phenotype is due to a single mutation located near the xyl marker on the strain 3937 chromosome. This mutation was complemented by an R‐prime plasmid carrying the xyl and argG genes of E. chrysanthemi, suggesting that the pecS product acts in trans to modulate pectinase, cellulase and blue pigment production. Insertion mutagenesis of the cloned region and recombination of the corresponding mutations in the bacterial chromosome by marker exchange revealed the existence of two divergently transcribed genes, pecS and pecM, that are both involved in the pectate lyase and cellulase regulation. The nucleotide sequences of pecS and pecM were determined. The pecS gene encodes a 166 amino acid polypeptide that shows similarity to the MprA regulatory protein of Escherichia coli whereas the pecM gene encodes a 297 amino acid polypeptide that was shown to be an integral membrane protein. The possible functions of the PecS and PecM proteins derived from the mutant phenotype and sequence analysis are discussed in terms of signal transduction and transcription regulation.

Characterization of Indigoidine Biosynthetic Genes in Erwinia chrysanthemi and Role of This Blue Pigment in Pathogenicity

In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain, E. chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant. These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM. Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions. The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E. chrysanthemi pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha. Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response.

Characterization of kdgR, a gene of Erwinia chrysanthemi that regulates pectin degradation

Erwinia chrysanthemi is a phytopathogenic enterobacterium able to degrade the pectic fraction of plant cell walls. The kdgR negative regulatory gene controls all the genes involved in pectin catabolism, including the pel genes encoding pectate lyases. The E. chrysanthemi kdgR regulatory gene was subcloned in Escherichia coli where it was shown to be functional, since it repressed the expression of a pelE::uidA fusion. The nucleotide sequence of kdgR contained an open reading frame of 918bp preceded by classical transcriptional initiation signals. KdgR shows similarity to two other regulatory proteins, namely GylR, encoding an activator protein of the glycerol operon in Streptomyces coelicolor, and IclR, encoding a repressor of the acetate operon in Salmonella typhimurium and in Escherichia coli. Previously, comparison of regulatory regions of several genes controlled by kdgR revealed the existence of a conserved region which was proposed as a KdgR‐binding site. The 25 bp oligonucleotide AAAAAAGAAACATTG‐TTTCATTTGT corresponding to this consensus was substituted to the lac operator, at the beginning of transcription of the lacZ gene. This construct functioned as an operator for binding of the KdgR protein in vivo.

Purification and functional characterization of PecS, a regulator of virulence‐factor synthesis in Erwinia chrysanthemi

The Erwinia chrysanthemi pecS gene encodes a repressor that negatively regulates the expression of virulence factors such as pectinases or cellulases. The cloned pecS gene was overexpressed using a phage T7 system. The purification of PecS involved DEAE‐anion exchange and TSK‐heparin columns and delivered the PecS protein that was purified to homogeneity. The purified repressor displayed an 18 kDa apparent molecular mass and an isoelectric point near to neutrality (PI = 6.5). Gel‐filtration experiments revealed that the PecS protein is a dimer. Bandshift assays demonstrated that the PecS protein could specifically bind in vitro to the regulatory sites of the in vivo PecS‐regulated genes. The interaction between the PecS protein and its DNA‐binding site was characterized by a relatively low affinity (about 10−8 M). DNase I footprintings revealed short protected sequences only with the most in vivo PecS‐regulated genes. Alignment of these PecS‐binding sites did not show a well‐conserved consensus sequence. lmmunoblotting demonstrated that the copy number of the PecS protein was approximately 50 dimers per cell. The low affinity of the PecS repressor for its DNA targets and the low cellular PecS content suggest the existence of E. chrysanthemi‐specific factors able to potentiate PecS protein activity in vivo.

Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti‐repressor function for the PecS regulator

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes including several pectate lyases encoded by the pel genes. We characterized a novel cluster of pectinolytic genes consisting of the three adjacent genes pehV, pehW and pehX, whose products have polygalacturonase activity. The high similarity between the three genes suggests that they result from duplication of an ancestral gene. The transcription of pehV, pehW and pehX is dependent on several environmental conditions. They are induced by pectin catabolic products and this induction results from inactivation of the KdgR repressor which controls almost all the steps of pectin catabolism. The presence of calcium ions strongly reduced the transcription of the three peh genes. Their expression was also affected by growth phase, osmolarity, oxygen limitation and nitrogen starvation. In addition, the pehX transcription is affected by catabolite repression and controlled by the activator protein CRP. PecS, which was initially isolated as a repressor of virulence factors, acts as an activator of the peh transcription. We showed that the three regulators KdgR, PecS and CRP act by direct interaction with the promoter regions of the peh genes. Analysis of simultaneous binding of KdgR, PecS, CRP and RNA polymerase indicated that the activator effect of PecS results from a competition between PecS and KdgR for the occupation of overlapping binding sites. Thus, to activate peh transcription, PecS behaves as an anti‐repressor against KdgR.

Positive co‐regulation of the Escherichia coli carnitine pathway cai and fix operons by CRP and the CaiF activator

Activation of the two divergent Escherichia coli cai and fix operons involved in anaerobic carnitine metabolism is co‐dependent on the cyclic AMP receptor protein (CRP) and on CaiF, the specific carnitine‐sensitive transcriptional regulator. CaiF was overproduced using a phage T7 system, purified on a heparin column and ran as a 15u2003kDa protein on SDS–PAGE. DNase I footprinting and interference experiments identified two sites, F1 and F2, with apparently comparable affinities for the binding of CaiF in the cai–fix regulatory region. These sites share a common perfect inverted repeat comprising two 11u2003bp half‐sites separated by 13u2003bp, and centred at −70 and −127 from the fix transcription start site. They were found to overlap the two low‐affinity binding sites, CRP2 and CRP3, determined previously for CRP. Gel shift assays and footprinting experiments suggest that CaiF and CRP bind co‐operatively to the F1/CRP2 and F2/CRP3 sites of the intergenic cai–fix region. Moreover, they appeared to serve the simultaneous binding of each other, giving rise to an original multiprotein CRP–CaiF complex enabling RNA polymerase recruitment and local DNA untwisting, at least at the fix promoter. Using random mutagenesis, two CaiF mutants impaired in transcription activation were isolated. The N‐terminal A27V mutation affected the structural organization of the activator, whereas the central I62N mutation was suggested to interfere with DNA binding.

Mutual control of the PecS/PecM couple, two proteins regulating virulence-factor synthesis in Erwinia chrysanthemi.

The Erwinia chrysanthemi pecS mutant displays constitutive production of virulence factors, such as pectinases or cellulases. Complementation of the pecS mutation can be obtained in the presence of the pecS wild‐type gene on a low‐copy‐number plasmid. Moreover, the resulting plasmid decreases the expression of a pecS::uidA chromosomal fusion, indicating the existence of an autoregulation mechanism. This negative autoregulation was confirmed and quantified by analysis of the pecS transcripts using primer‐extension experiments. Band‐shift assays and DNase I footprinting experiments demonstrated that the PecS protein could bind to the intergenic regulatory region, located between the pecS and pecM genes, with a relatively high affinity (apparent dissociation constant (K′d) close to 4u2003nM). These PecS‐binding sites overlap the pecS and pecM promoters. The comparison of these new PecS‐binding sites with those previously characterized on the target genes confirms the absence of a consensus. This observation was in accordance with the results of the missing‐contact experiments performed on the pecS–pecM intergenic regulatory region and the celZ operator. Concurrently, we demonstrated that the PecS protein negatively controls the expression of the divergently transcribed pecM gene located 400u2003bp upstream from the pecS gene. By following the efficiency of pecS autoregulation in a double E. chrysanthemi pecM–pecS mutant, we established that the PecM protein potentiates PecS activity in vivo.

PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-alpha-D-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.

Crystallization and preliminary X-ray studies of the pectate lyase from Bacillus subtilis

The pectate lyase (EC 4.2.2.9) from Bacillus subtilis has been crystallized. Crystals of form 1, grown by the hanging drop method using polyethylene glycol as precipitant, diffract to at least 2.4 A resolution. They belong to the spacegroup P2(1) with a = 132.9 A, b = 41.2 A, c = 156.8 A and beta = 114.9 degrees with probably four molecules in the asymmetric unit. A second crystal form grown from 2-methyl-2,4-pentandiol also belongs to the spacegroup P2(1) with a = 55.0 A, b = 88.1 A, c = 50.2 A and beta = 109.0 degrees. These crystals diffract to at least 2.0 A and have one molecule in the asymmetric unit. Both crystal forms are suitable for the determination of high-resolution structures.