Genetic and transcriptional organization of the hrp cluster of Pseudomonas syringae pv. phaseolicola

The hrp cluster of Pseudomonas syringae pv. phaseolicola encodes functions that are essential for pathogenicity on bean plants and for the elicitation of the hypersensitive response on resistant plants. The cluster was saturated with insertions of transposon Tn3-spice that served both as a mutagen and as a sensitive reporter of the expression of the target regions. The mutations covered a 17.5-kb segment in strain NPS3121, in which seven hrp::Tn5 insertions had been previously mapped, and regions outside this segment. The cluster is organized into seven distinct complementation groups (hrpL, hrpAB, hrpC, hrpD, hrpE, hrpF, and hrpSR) on the basis of the analysis of over 100 Tn3-spice insertions in plasmids and 43 similar insertions in the chromosome; it spans nearly 22 kb and is chromosomally located. The transcriptional orientation of all genes in the cluster was established by measuring the level of ice nucleation activity of complemented merodiploids carrying chromosomal hrp::inaZ fusions after inoculation in Red Kidney bean leaves. Although all seven loci were actively expressed in Red Kidney bean leaves, none of them was substantially expressed when the bacteria were grown in King B broth medium. Mutations in all loci, except those in hrpC, greatly reduced the ability of the bacteria to multiply in bean leaves. Mutations in the hrpC locus, although preventing the bacteria from eliciting a hypersensitive reaction on tobacco, allowed the bacteria to produce delayed and attenuated symptoms in Red Kidney bean leaves and to multiply to a level 10(2)- to 10(3)-fold lower than that of the wild-type strain. This is the first comprehensive report of the genetic and transcriptional organization of the hrp gene cluster in a phytopathogenic bacterium.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  J. Vanneste,et al.  Bacteriophage Mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco , 1990, Journal of bacteriology.

[3]  R. Rappuoli,et al.  Families of bacterial signal‐transducing proteins , 1989, Molecular microbiology.

[4]  B. Staskawicz,et al.  Bacterial blight of soybean: regulation of a pathogen gene determining host cultivar specificity. , 1989, Science.

[5]  N. Panopoulos,et al.  The predicted protein product of a pathogenicity locus from Pseudomonas syringae pv. phaseolicola is homologous to a highly conserved domain of several procaryotic regulatory proteins , 1989, Journal of bacteriology.

[6]  S. Hutcheson,et al.  Influence of Pseudomonas syringae culture conditions on initiation of the hypersensitive response of culture tobacco cells , 1989, Applied and environmental microbiology.

[7]  C. Baker,et al.  Elicitation of the hypersensitive response by Pseudomonas syringae , 1989 .

[8]  S. Lindow,et al.  An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola. , 1989, The EMBO journal.

[9]  J. Williams,et al.  Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae , 1988, Journal of bacteriology.

[10]  P. Wolber,et al.  Nonlinear relationship between concentration and activity of a bacterial ice nucleation protein. , 1988, The Journal of biological chemistry.

[11]  C. Baker,et al.  Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants , 1988, Journal of bacteriology.

[12]  P. Shaw,et al.  Isolation and characterization of pathogenicity genes of Pseudomonas syringae pv. tabaci , 1988, Journal of bacteriology.

[13]  L. Sequeira,et al.  Molecular cloning of genes that specify virulence in Pseudomonas solanacearum , 1988, Journal of bacteriology.

[14]  C. Boucher,et al.  Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered , 1987, Journal of bacteriology.

[15]  C. Napoli,et al.  Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea , 1987, Journal of bacteriology.

[16]  N. Panopoulos,et al.  Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants , 1986, Journal of bacteriology.

[17]  R. Green,et al.  Physical and functional repetition in a bacterial ice nucleation gene , 1985, Nature.

[18]  G. An,et al.  A Tn3 lacZ transposon for the random generation of beta‐galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. , 1985, The EMBO journal.

[19]  D. Ish-Horowicz,et al.  Rapid and efficient cosmid cloning , 1981, Nucleic Acids Res..

[20]  S. T. Liu,et al.  Rapid procedure for detection and isolation of large and small plasmids , 1981, Journal of bacteriology.

[21]  G. Ditta,et al.  Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[22]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[23]  D. Vapnek,et al.  Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli. , 1979, Journal of molecular biology.

[24]  A. Riggs,et al.  Genomic Sequencing , 2010 .

[25]  R. Palacios,et al.  The Rhizobium Genome , 1990 .

[26]  L. Sequeira,et al.  A second cluster of genes that specify pathogenicity and host response in Pseudomonas solanacearum. , 1990 .

[27]  M. Mindrinos,et al.  Organization and Expression of the hrp Gene Cluster in Pseudomonas Syringae Pv. Phaseolicola , 1989 .

[28]  A. Osbourn,et al.  Molecular Genetics of Pathogenicity in Phytopathogenic Bacteria , 1988 .

[29]  S. Beer,et al.  Creation and complementation of pathogenicity mutants of Erwinia amylovora , 1988 .

[30]  D. Anderson,et al.  Cloning determinants of pathogenesis from Pseudomonas syringae pathovar syringae. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Amasino,et al.  CROWN GALL: A MOLECULAR AND PHYSIOLOGICAL ANALYSIS , 1984 .

[32]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .

[33]  E. King,et al.  Two simple media for the demonstration of pyocyanin and fluorescin. , 1954, The Journal of laboratory and clinical medicine.