Guy Condemine

Functional characterization of the Erwinia chrysanthemi Outs protein, an element of a type II secretion system

Secretion of pectate lyases and a cellulase occurs in Erwinia chrysanthemi through a type II secretion machinery, the Out system. Proper insertion of the secretin OutD in the outer membrane requires the presence of OutS. OutS is an outer-membrane lipoprotein that interacts directly with OutD. Using ligand-blotting experiments, it has been shown that this interaction requires at least the 62 C-terminal amino acids of OutD. When this domain was added to the C-terminal extremity of the secreted pectate lyase PelD, the construct was stabilized by OutS but not inserted into the outer membrane. Thus, this domain is sufficient to interact with OutS but it is unable to confer the ability to be inserted into the outer membrane in the presence of OutS. A screen for outS mutants unable to secrete pectate lyases gave only mutants unable to properly localize OutD in the outer membrane and no mutant in the protection function. Thus, the interaction between OutS and OutD can probably not be abolished by the mutation of a single amino acid, and the insertion of OutD in the outer membrane may require additional proteins.

Function and Expression of an N-Acetylneuraminic Acid-Inducible Outer Membrane Channel in Escherichia coli

The Escherichia coli yjhA (renamed nanC) gene encodes a protein of the KdgM family of outer membrane-specific channels. It is transcribed divergently from fimB, a gene involved in the site-specific inversion of the region controlling transcription of the fimbrial structural genes but is separated from it by one of the largest intergenic regions in E. coli. We show that nanC expression is induced by N-acetylneuraminic acid and modulated by N-acetylglucosamine. This regulation occurs via the NanR and NagC regulators, which also control fimB expression. nanC expression is also activated by the regulators cyclic AMP-catabolite activator protein, OmpR, and CpxR. When the NanC protein was reconstituted into liposomes, it formed channels with a conductance of 450 pS at positive potential and 300 to 400 pS at negative potential in 800 mM KCl. The channels had a weak anionic selectivity. In an ompR background, where the general porins OmpF and OmpC are absent, NanC is required for growth of E. coli on N-acetylneuraminic acid as the sole carbon source. All these results suggest that NanC is an N-acetylneuraminic acid outer membrane channel protein.

Regulatory mutations affecting the synthesis of pectate lyase in Erwinia chrysanthemi

Pectate-lyases (PL) are extracellular enzymes secreted by Erwinia chysanthemi. By their attack of pectin, a constituent of plant cell walls, they are the major factor involved in the phytopathogenicity of this bacterial species. We described the isolation and characterization of mutants in the regulatory systems controlling the synthesis of PL. The regulation of these proteins appeared complex and submitted to at least two important controls. Three classes of mutants constitutively synthesizing PL were found after a chemical mutagenesis of the wild-type strain B374. The cri mutations (class IV) conferred a resistance to the catabolite repression exerted by glucose to various proteins of the cell such as PL, cellulases, proteases, B-galactosidase and the hexuronate transport system. In the cri mutants the synthesis of PL remained inducible by pectin degradative products but were no longer repressible in presence of glucose. In contrast, the gpiR mutants (class I) were always sensitive to glucose repression but no longer inducible in presence of pectin derivatives. In these mutants, the regulatory gene responsible for the induction by pectin derivatives was inactivated. This gene is probably gpiR itself. Moreover the gpiR mutation led to a synthesis of PL independent of the growth phase. In liquid cultures of E. chrysanthemi, PL synthesis increases only at the end of the exponential growth, while in the gpiR mutants the PL synthesis is proportional to the cell density.

Rhamnogalacturonate Lyase RhiE Is Secreted by the Out System in Erwinia chrysanthemi

Supernatants of rhamnose-induced Erwinia chrysanthemi strain 3937 cultures contain a principal secreted protein named RhiE. A rhiE mutant has been found among a set of rhamnose-induced MudI1681 lacZ fusions. RhiE is a 62-kDa protein that has rhamnogalacturonate lyase activity on rhamnogalacturonan I (RG-I). It does not require a divalent cation for its activity and has an optimal pH of 6.0. rhiE expression is strongly induced in the presence of rhamnose but is also regulated by PecT and Crp, two regulators of the transcription of pectinolytic enzyme genes. RhiE is secreted through the type II Out secretion pathway. RhiE has no disulfide bond. The absence of RhiE secretion in a dsb mutant indicated that disulfide bond formation is required for the biogenesis of the secretion apparatus. RhiE was searched for in several E. chrysanthemi strains by using antibodies, and it was found to be present in one-third of the strains tested. However, the reduced virulence of the rhiE mutant indicates that degradation of the RG-I region of pectin is important for full virulence of E. chrysanthemi.

Differential Regulation of Two Oligogalacturonate Outer Membrane Channels, KdgN and KdgM, of Dickeya dadantii (Erwinia chrysanthemi)

The entry of oligogalacturonates into Dickeya dadantii occurs through the specific channel KdgM. The genome of the bacterium encodes a second member of this family of outer membrane proteins, KdgN. We showed that this protein is also involved in the uptake of oligogalacturonates. When KdgN was reconstituted in proteoliposomes, it formed channels with a conductance of about 450 pS at a positive potential. These channels had weak anionic selectivity. The regulation of kdgN is complex, and five genes controlling the expression of kdgN have been identified: kdgR, pecS, ompR, hns, and crp. Moreover, kdgN was regulated by growth phase but only when bacteria were grown in rich medium. Most of these regulators of kdgN also control kdgM expression, but some of them regulate kdgM in the opposite manner: while PecS and OmpR are repressors of kdgM, they are activators of kdgN. This pattern resembles the regulation of the Escherichia coli general porins OmpF and OmpC, but such opposite regulation of two specific outer membrane channels has never been described before. KdgN may allow the bacteria to collect oligogalacturonates under saprophytic conditions, when virulence genes, including kdgM, are not expressed.

Characterization of the nucM gene coding for a nuclease of the phytopathogenic bacteria Erwinia chrysanthemi

The gene nucM encoding a nuclease was cloned from a genomic library of Erwinia chrysanthemi. The nucM gene was subcloned, and mutagenized by insertion of a uidA‐KanR cartridge. This mutation was introduced by recombination into the Erwinia chrysanthemi chromosome. The nucM mutant lost NucM activity when tested on a DNA plate after 24 hours, but still possessed secondary weak nuclease activity. The nucleotide sequence of nucM was determined. It presents a 798 bp open reading frame, coding for a 266‐amino‐acid protein, with a predicted molecular mass of 29 910 Da. The deduced NucM protein shows 59% sequence identity with the DNasel precursor from Vibrio cholerae. It contains a typical leader sequence. Experiments of cell fractionation showed that NucM is periplasmic In E. chrysanthemi. The transcription start has been determined by S1 mapping. The −10 and −35 regions do not show homology with consensus sequence of the promoters recognized by σ;70. In fact, The promoter seems to be dependent on the σ;70, but the first transcription nucleotide is unusually far from the −10 region. nucM seems to be expressed constitutively.

Nouvelle synthese de l'acide 3-desoxy-D-erythro-2-hexulosonique (KDG). A partir de la D-glucono-1,5-lactone synthese et étude de RMN de derives O-methyles du KDG

ABSTRACT 3-Deoxy-D-erythro-2-hexulosonic acid (KDG), an important metabolite of bacterial polysaccharide degradation, was prepared from D-glucono-1,5-lactone through a six-step sequence, with a 45% overall yield. Using suitable intermediates. KDG methyl ester and its 5- and 6-O-methylated derivatives were also synthesized. 1H and 13C NMR studies of 5- and 6-O-methylated derivatives (pyranoid and furanoid forms respectively) compared to those of KDG and its methyl ester allowed us to conclude that these two latter compounds exist in equilibrium as forms whose percentages were determined.

Characterization of SotA and SotB, two Erwinia chrysanthemi proteins which modify isopropyl-beta-D-thiogalactopyranoside and lactose induction of the Escherichia coli lac promoter.

The expression, in Escherichia coli, of variants of the Erwinia chrysanthemi secretion genes outB and outS under the Ptac promoter is toxic to the cells. During attempts to clone E. chrysanthemi genes able to suppress this toxicity, I identified two genes, sotA and sotB, whose products are able to reduce the isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of the E. coli lac promoter. SotA and SotB belong to two different families of the major facilitator superfamily. SotA is a member of the sugar efflux transporter family, while SotB belongs to the multidrug efflux family. The results presented here suggest that SotA and SotB are sugar efflux pumps. SotA reduces the intracellular concentration of IPTG, lactose, and arabinose. SotB reduces the concentration of IPTG, lactose, and melibiose. Expression of sotA and sotB is not regulated by their substrates, but sotA is activated by the cyclic AMP receptor protein (CRP), while sotB is repressed by CRP. Lactose is weakly toxic for E. chrysanthemi. This toxicity is increased in a sotB mutant which cannot efficiently efflux lactose. This first evidence for a physiological role of sugar efflux proteins suggests that their function could be to reduce the intracellular concentration of toxic sugars or sugar metabolites.

Synthesis of methyl esters of 3-deoxy-d-erythro-2-hexulosonic acid (KDG) analogs, inducers of the expression of pectinase genes in bacteria Erwinia chrysanthemi

Abstract Two title compounds, methyl 3-deoxy- l -threo-2-hexulosonate 3 and methyl 3,5-dideoxy- d -glycero-2-hexulosonate 4, were prepared from d -glucono-1,5-lactone and proved to be gratuitous inducers of the expression of pectinase genes in the phytopathogenic bacteria Erwinia chrysanthemi.

Purification and characterization of the nuclease NucM of Erwinia chrysanthemi.

The major periplasmic nuclease of Erwinia chrysanthemi strain 3937, NucM, has been purified near to homogeneity by a one step purification procedure, using chromatography on a sulfopropyl column. NucM cleaves randomly single and double-stranded DNA and RNA. It does not need divalent cations for its action, and is more active in low salt buffers. A serine and a histidine residue could be present in the catalytic site. Formation of disulfide bonds is necessary for NucM activity. NucM is probably synthesized as a reduced inactive polypeptide and becomes active in the periplasm once disulfide bonds are formed.