The development of competence allowing natural transformation of Ralstonia solanacearum was found to occur during exponential growth and not in response to any excreted factors. Linear DNAs were effectively integrated by recombination requiring a minimum of 50 bp of homologous DNA. Therefore, DNA from other genera and species were ineffective.

Molecular MicrobiologyVolume 20, Issue 3 p. 681-683 Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria Adam J. Bogdanove, Adam J. Bogdanove Department of Plant Pathology, 334 Plant Science, Cornell University, Ithaca, New York 14853, USA., Search for more papers by this authorSteven V. Beer, Steven V. Beer Department of Plant Pathology, 334 Plant Science, Cornell University, Ithaca, New York 14853, USA., Search for more papers by this authorUlla Bonas, Ulla Bonas CNRS Institut des Sciences Végétales, Avenue de la Terrasse, Bâtiment 23 911 98, Gif sur Yvette Cedex, France., Search for more papers by this authorChristian A. Boucher, Christian A. Boucher INRA-CNRS Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes, BP27, Chemin de Borde Rouge, Castanet-Tolosan Cedex F-31326, France., Search for more papers by this authorAlan Collmer, Alan Collmer Department of Plant Pathology, 334 Plant Science, Cornell University, Ithaca, New York 14853, USA., Search for more papers by this authorDavid L. Coplin, David L. Coplin Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210-1087, USA., Search for more papers by this authorGuy R. Cornelis, Guy R. Cornelis Microbial Pathogenesis Unit, International Institute of Cellular and Molecular Pathology and University of Louvain Medical Faculty, B-1200 Brussels, Belgium., Search for more papers by this authorHsiou-Chen Huang, Hsiou-Chen Huang Agricultural Biotechnology Laboratories, National Chung-Hsing University, Taichung, Taiwan 40227, Taiwan., Search for more papers by this authorSteven W. Hutcheson, Steven W. Hutcheson Department of Plant Biology, University of Maryland, College Park, Maryland 20742, USA., Search for more papers by this authorNickolas J. Panopoulos, Nickolas J. Panopoulos Institute of Molecular Biology and Biotechnology, F.O.R.T.H. and Department of Biology, University of Crete, PO Box 1527, Heraklion 71110, Crete, Greece.Search for more papers by this authorFrédérique Van Gijsegem, Frédérique Van Gijsegem INRA-CNRS Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes, BP27, Chemin de Borde Rouge, Castanet-Tolosan Cedex F-31326, France., Search for more papers by this author Adam J. Bogdanove, Adam J. Bogdanove Department of Plant Pathology, 334 Plant Science, Cornell University, Ithaca, New York 14853, USA., Search for more papers by this authorSteven V. Beer, Steven V. Beer Department of Plant Pathology, 334 Plant Science, Cornell University, Ithaca, New York 14853, USA., Search for more papers by this authorUlla Bonas, Ulla Bonas CNRS Institut des Sciences Végétales, Avenue de la Terrasse, Bâtiment 23 911 98, Gif sur Yvette Cedex, France., Search for more papers by this authorChristian A. Boucher, Christian A. Boucher INRA-CNRS Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes, BP27, Chemin de Borde Rouge, Castanet-Tolosan Cedex F-31326, France., Search for more papers by this authorAlan Collmer, Alan Collmer Department of Plant Pathology, 334 Plant Science, Cornell University, Ithaca, New York 14853, USA., Search for more papers by this authorDavid L. Coplin, David L. Coplin Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210-1087, USA., Search for more papers by this authorGuy R. Cornelis, Guy R. Cornelis Microbial Pathogenesis Unit, International Institute of Cellular and Molecular Pathology and University of Louvain Medical Faculty, B-1200 Brussels, Belgium., Search for more papers by this authorHsiou-Chen Huang, Hsiou-Chen Huang Agricultural Biotechnology Laboratories, National Chung-Hsing University, Taichung, Taiwan 40227, Taiwan., Search for more papers by this authorSteven W. Hutcheson, Steven W. Hutcheson Department of Plant Biology, University of Maryland, College Park, Maryland 20742, USA., Search for more papers by this authorNickolas J. Panopoulos, Nickolas J. Panopoulos Institute of Molecular Biology and Biotechnology, F.O.R.T.H. and Department of Biology, University of Crete, PO Box 1527, Heraklion 71110, Crete, Greece.Search for more papers by this authorFrédérique Van Gijsegem, Frédérique Van Gijsegem INRA-CNRS Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes, BP27, Chemin de Borde Rouge, Castanet-Tolosan Cedex F-31326, France., Search for more papers by this author First published: May 1996 https://doi.org/10.1046/j.1365-2958.1996.5731077.xCitations: 183 Frédérique Van Gijsegem Tel. 61 28 50 45; Fax 61 28 50 61. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume20, Issue3May 1996Pages 681-683 RelatedInformation

Five transcription units of the Pseudomonas solanacearum hrp gene cluster are required for the secretion of the HR-inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions. Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant-pathogenic bacteria include proteins encoded by hrp genes. For Hrp-related proteins of animal pathogens, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solancearum, include proteins encoded by fli and flh genes of S. typhimurium, Bacillus subtilis and Escherichia coli and by mop genes of Erwinia carotovora. P. solanacearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity.

This paper describes the identification of a new class of extracellular bacterial proteins, typified by PopA1 and its derivative PopA3, which act as specific hypersensitive response (HR) elicitors. These two heat‐stable proteins, with HR‐like elicitor activities on tobacco (non‐host plant) but without activity on tomato (host plant), have been characterized from the supernatant of the plant pathogenic bacterium Pseudomonas solanacearum strain GMI1000. These two proteins induced the same pattern of response on Petunia, as a function of the genotypes tested. popA, the structural gene for PopA1, maps outside of the hrp gene cluster but belongs to the hrp regulon. The amino acid sequence of PopA1 does not show homology to any characterized proteins. Its secretion is dependent on hrp genes and is followed by stepwise removal of the 93 amino‐terminal amino acids, producing the protein PopA3. Petunia lines responsive to PopA3 and its precursors were resistant to infection by strain GMI1000, whereas non‐responsive lines were sensitive, suggesting that popA could be an avirulence gene. A popA mutant remained fully pathogenic on sensitive plants, indicating that this gene is not essential for pathogenicity. While lacking PopA1, this mutant, which remained avirulent on tobacco and on resistant Petunia lines, still produced additional extracellular necrogenic compounds. On the basis of both their structural features and the biological properties of the popA mutant, PopA1 and PopA3 clearly differ from hairpins characterized in other plant pathogenic bacteria.

Beneficial microbes in the microbiome of plant roots improve plant health. Induced systemic resistance (ISR) emerged as an important mechanism by which selected plant growth–promoting bacteria and fungi in the rhizosphere prime the whole plant body for ...Read More

Based on DNA sequencing of 20kb of the left hand of the Pseudomonas solanacearum hrp gene cluster, 19 open reading frames (ORFs) with a high coding probability have been identified. One of these ORFs codes for a positive regulator which controls the expression of at least 5 hrp transcription units in addition to the expression of yet unidentified genes adjacent to the hrp gene cluster. Five other ORFs code for putative proteins which share homology with pathogenicity genes from the animal and human pathogens Yersinia enterocolitica, Y. pestis and Shigella flexneri. These homologies led us to look in the supernatant of bacteria cell cultures for a hrp gene dependant bacterial factor which is able to induce a hypersensitive-like response on tobacco. Preliminary experimental data suggest that such a factor does exist and that it is a heat resistant protein and that it is released in the culture medium via a hrp gene encoded secretion machinery. Cross-hybridizations of a P.solanacearum hrp gene with the hrp gene clusters of P. syringae pv. phaseolicola and Erwinia amylovora are also presented.

SummaryTwenty of the twenty‐two MudII1734 insertions impairing the chrysobactin iron‐assimilation system of Erwinia chrysanthemi 3937 were localized to a 50kbp genomic insert contained in the R‐prime plasmid, R′4 (Enard et al., 1988). Using the conjugative plasmid pULB1 10 (RP4::mini‐Mu) and the generalized transducing phage ΦEC2, we located this iron‐transport region and the two unlinked mutations on the chromosome linkage map. Chrysobactin is a catechol‐type siderophore and, as we have previously observed with the entA locus of Escherichia coli, the E. chrysanthemi‐derived R′4 was found to complement E. coli entB and entE mutations. A 2.9 kb Eco Ri and a 4.8kb BamHI fragment in the R′4 sharing homology with the E. coli entCEBAP15 operon DNA were subcloned. These fragments were used as DNA/DNA hybridization probes to screen a wild‐type gene library, yielding a recombinant cosmid (pEC7) able to complement mutations disrupting the 2,3‐drhydroxy‐benzoic acid biosynthetic pathway in both Erwinia and Escherichia spp. as well as the E. coli entE mutation. Physical mapping of the genomic MudII1734 insertions corresponding to these mutations led to the identification of a cluster of genes confined to a DNA sequence of about 10 kb required for both biosynthetic and receptor functions.

We present a method for identifying plant-inducible genes of Erwinia chrysanthemi 3937. Mutagenesis was done with the Mu dIIPR3 transposon, which carries a promoterless neomycin phosphotransferase gene (nptI), so upon insertion, the truncated gene can fuse to E. chrysanthemi promoters. Mutants containing insertions in plant-inducible genes were selected for their sensitivity to kanamycin on minimal plates and for their acquired resistance to this antibiotic when an S. ionantha plant extract was added to kanamycin minimal plates. The selection allowed the identification of E. chrysanthemi promoters inducible by host factors present in the S. ionantha plant extract. Using this method, we isolated 30 mutants and characterized 10 of them. Two mutants were defective in cation uptake, one was defective in the galacturonate degradation pathway, and another was altered in the production of the acidic pectate lyase. The functions of the other mutated genes are still unknown, but we show that most of them are involved in pathogenicity.

In this paper, we have used filter hybridization and nucleotide sequencing to analyse the relationship between the three genes of the pelADE cluster in the Erwinia chrysanthemi (Ech) strain B374. This cluster encodes for three of the five pectate lyase proteins that are involved in the maceration and soft-rotting of plant tissue, an important trait in Ech pathogenicity. Southern hybridization revealed homology between each of the three pel genes. A 3560 bp DNA fragment containing the pelE and pelD genes was sequenced. These two genes show extensive homology in the coding regions but only low homology in the 5' and 3' non-coding regions. However both genes exhibit sequences homologous to the Escherichia coli CAP-binding site consensus sequence upstream of the start codon and an inverted repeat sequence which may act as a rho-independent transcriptional terminator after the translational stop. The pel genes of Ech B374 were also compared with the already sequenced pel genes of EC16, another Ech strain.

SummaryUsing the RP4::mini‐Mu in vivo cloning technique, van Gijsegem et al. (1985) isolated several pel and cel genes of Erwinia chrysanthemi (Ech) B374 strain. We have localized these genes on the Ech chromosome by co‐transfer mapping of Mud11734 insertion mutants and refined the map by co‐transposition analysis. This analysis has enabled us to identify another cel gene.

A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.

The pelA, pelD and pelE genes encode three of the five major pectate lyase (PL) isoenzymes (PLa, PLd and PLe) in Erwinia chrysanthemi strains B374 and 3937. These genes were previously isolated from genomic libraries or by in vivo cloning as R' factors promoted by the pULB113 plasmid. They are clustered near purE on the chromosomal map of E. chrysanthemi B374 [Van Gijsegem et al., EMBO J. 4 (1985) 787-792]. Genes pelA, pelD and pelE were subcloned separately into pBR322 derivatives, to test their individuality. It then became possible to select specific mutations in each separated gene. Such mutations were obtained using the transposable bacteriophage MudI1734 that allows the construction of lacZ gene fusions. Subcloning experiments and analysis of MudI1734 insertions permitted us to determine the length of each gene, the transcriptional orientation and the location of the promoter. We concluded that the three genes constitute three independent transcriptional units. They are clustered on a 5-kb DNA fragment, in the order: pelD-pelE-pelA. Genes pelD and pelE are transcribed in the same direction, while the transcription of pelA seems to be divergent. Organization of the pel region was very similar in the two strains B374 and 3937. Moreover, lacZ gene fusions were introduced by marker exchange into the chromosome of E. chrysanthemi B374, giving rise to three strains lacking PLa, PLd or PLe. These fusions allowed us to study the regulation of the mutagenized genes.

Structural genes for the five major pectate lyases of Erwinia chrysanthemi, pelA, pelB, pelC, pelD and pelE, have been transferred and expressed in Myxococcus xanthus. These proteins, which are secreted by their original host, are also produced, mainly into the medium, by Myxococcus. These results are discussed in the context of current knowledge about secretion in bacteria.

SUMMARY: The protein content of culture supernatants of three Erwinia chrysanthemi strains, B374, 3937j and 3665, grown on different carbon sources was compared. After growth in presence of polygalacturonate, four new polypeptides, identified as pectinases, were synthesized. These induced proteins, and the pattern of pectate lyase induction, differed among the strains. The proteins present in the supernatants of some mutants known or suspected to be affected in pectinase production (secretion-defective mutants and mutants in the degradative pathway of galacturonate and ketodeoxygluconate) were also analysed.

Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).