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Comparative analysis of argK-tox clusters and their flanking regions in phaseolotoxin-producing Pseudomonas syringae pathovars.

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181-10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064-11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named "tox islands." The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437-457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands.

3' Flanking Region↗

A high-throughput, near-saturating screen for type III effector genes from Pseudomonas syringae.

Pseudomonas syringae strains deliver variable numbers of type III effector proteins into plant cells during infection. These proteins are required for virulence, because strains incapable of delivering them are nonpathogenic. We implemented a whole-genome, high-throughput screen for identifying P. syringae type III effector genes. The screen relied on FACS and an arabinose-inducible hrpL sigma factor to automate the identification and cloning of HrpL-regulated genes. We determined whether candidate genes encode type III effector proteins by creating and testing full-length protein fusions to a reporter called Delta79AvrRpt2 that, when fused to known type III effector proteins, is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 plant disease resistance protein. Delta79AvrRpt2 is thus a marker for type III secretion system-dependent translocation, the most critical criterion for defining type III effector proteins. We describe our screen and the collection of type III effector proteins from two pathovars of P. syringae. This stringent functional criteria defined 29 type III proteins from P. syringae pv. tomato, and 19 from P. syringae pv. phaseolicola race 6. Our data provide full functional annotation of the hrpL-dependent type III effector suites from two sequenced P. syringae pathovars and show that type III effector protein suites are highly variable in this pathogen, presumably reflecting the evolutionary selection imposed by the various host plants.

Arabidopsis↗

Diversifying selection drives the evolution of the type III secretion system pilus of Pseudomonas syringae.

The plant pathogenic bacterium Pseudomonas syringae uses a type III secretion system to inject virulence proteins directly into the cytoplasm of its hosts. The P. syringae type III secretion apparatus is encoded, in part, by the HrpZ operon, which carries the hrpA gene encoding the pilin subunit of the pilus, various components of the structural apparatus, and the HrpZ harpin protein that is believed to produce pores in the host cell membrane. The pilus of the type III system comes into direct contact with the host cell and is, therefore, a likely target of the host's pathogen surveillance systems. We sequenced and analyzed 22 HrpZ operons from P. syringae strains spanning the diversity of the species. Selection analyses, including K(a)/K(s) tests and Tajima's D, revealed strong diversifying selection acting on the hrpA gene. This form of selection enables pathogens to maintain genetic diversity within their populations and is often driven by selection imposed by host defense systems. The HrpZ operon also revealed a single significant recombination event that dramatically changed the evolutionary relationships among P. syringae strains from 2 quite distinct phylogroups. This recombination event appears to have introduced genetic diversity into a clade of strains that may now be undergoing positive selection. The identification of diversifying selection acting on the Hrp pilus across the whole population sample and positive selection within one P. syringae lineage supports a trench warfare coevolutionary model between P. syringae and its plant hosts.

Amino Acid Sequence↗

Pseudomonas syringae pv. phaseolicola can be separated into two genetic lineages distinguished by the possession of the phaseolotoxin biosynthetic cluster.

The bean (Phaseolus spp.) plant pathogen Pseudomonas syringae pv. phaseolicola is characterized by the ability to produce phaseolotoxin (Tox(+)). We recently reported that the majority of the Spanish P. syringae pv. phaseolicola population is unable to synthesize this toxin (Tox(-)). These Tox(-) isolates appear to lack the entire DNA region for the biosynthesis of phaseolotoxin (argK-tox gene cluster), as shown by PCR amplification and DNA hybridization using DNA sequences specific for separated genes of this cluster. Tox(+) and Tox(-) isolates also showed genomic divergence that included differences in ERIC-PCR and arbitrarily primed-PCR profiles. Tox(+) isolates showed distinct patterns of IS801 genomic insertions and contained a chromosomal IS801 insertion that was absent from Tox(-) isolates. Using a heteroduplex mobility assay, sequence differences were observed only among the intergenic transcribed spacer of the five rDNA operons of the Tox(-) isolates. The techniques used allowed the unequivocal differentiation of isolates of P. syringae pv. phaseolicola from the closely related soybean (Glycine max) pathogen, P. syringae pv. glycinea. Finally, a pathogenicity island that is essential for the pathogenicity of P. syringae pv. phaseolicola on beans appears to be conserved among Tox(+), but not among Tox(-) isolates, which also lacked the characteristic large plasmid that carries this pathogenicity island. It is proposed that the results presented here justify the separation of the Tox(+) and Tox(-) P. syringae pv. phaseolicola isolates into two distinct genetic lineages, designated Pph1 and Pph2, respectively, that show relevant genomic differences that include the pathogenicity gene complement.

Base Sequence↗

Divergent potentials for cytoplasmic inheritance within the genus Syringa. A new trait associated with speciogenesis.

Epifluorescence microscopic detection of organelle DNA in the mature generative cell is a rapid method for determining the potential for the mode of cytoplasmic inheritance. We used this method to examine 19 of the known 22 to 27 species in the genus Syringa. Organelle DNA was undetectable in seven species, all in the subgenus Syringa, but was detected in the 12 species examined of the subgenera Syringa and Ligustrina. Therefore, species within the genus Syringa display differences in the potential cytoplasmic inheritance. Closer examination revealed that the mature generative cells of the species in which organelle DNA was detected contained both mitochondria and plastids, but cells of the species lacking detectable organelle DNA contained only mitochondria, and the epifluorescent organelle DNA signals from the mature generative cells corresponded to plastid DNA. In addition, semiquantitative analysis was used to demonstrate that, during pollen development, the amount of mitochondrial DNA decreased greatly in the generative cells of the species examined, but the amount of plastid DNA increased remarkably in the species containing plastids in the generative cell. The results suggest that all Syringa species exhibit potential maternal mitochondrial inheritance, and a number of the species exhibit potential biparental plastid inheritance. The difference between the modes of potential plastid inheritance among the species suggests different phylogenies for the species; it also supports recent conclusions of molecular, systematic studies of the Syringa. In addition, the results provide new evidence for the mechanisms of maternal mitochondrial inheritance in angiosperms.

Cytoplasm↗

Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.

Bacterial Proteins↗

Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae.

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.

Base Sequence↗

Phenotypic heterogeneity of Pseudomonas syringae van Hall.

The study of phenotypic properties of 108 strains of Pseudomonas syringae pv. syringae van Hall isolated from Cherry laurel (50 strains) and various host plants (58 strains) and 53 strains of other pathovars of P. syringae and fluorescent Pseudomonas showed that the majority of the strains (91/108) were clustered in one phenon (phenon 14) containing strains commonly considered as P.s. pv. syringae. The present type strain of P.s. pv. syringae was distantly related to phenon 14. Other pathovars of P. syringae constituted 13 discrete phenons.

In Vitro Techniques↗

The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants.

The plant pathogenic bacterium Pseudomonas syringae is divided into pathovars differing in host specificity, with P. syringae pv. syringae (Psy) and P. syringae pv. tomato (Pto) representing particularly divergent pathovars. P. syringae hrp/hrc genes encode a type III protein secretion system that appears to translocate Avr and Hop effector proteins into plant cells. DNA sequence analysis of the hrp/hrc regions in Psy 61, Psy B728a, and Pto DC3000 has revealed a Hrp pathogenicity island (Pai) with a tripartite mosaic structure. The hrp/hrc gene cluster is conserved in all three strains and is flanked by a unique exchangeable effector locus (EEL) and a conserved effector locus (CEL). The EELs begin 3 nt downstream of the stop codon of hrpK and end, after 2.5-7.3 kb of dissimilar intervening DNA with tRNA(Leu)-queA-tgt sequences that are also found in Pseudomonas aeruginosa but without linkage to any Hrp Pai sequences. The EELs encode diverse putative effectors, including HopPsyA (HrmA) in Psy 61 and proteins similar to AvrPphE and the AvrB/AvrC/AvrPphC and AvrBsT/AvrRxv/YopJ protein families in Psy B728a. The EELs also contain mobile genetic element sequences and have a G + C content significantly lower than the rest of the Hrp Pai or the P. syringae genome. The CEL carries at least seven ORFs that are conserved between Psy B728a and Pto DC3000. Deletion of the Pto DC3000 EEL slightly reduces bacterial growth in tomato, whereas deletion of a large portion of the CEL strongly reduces growth and abolishes pathogenicity in tomato.

Bacterial Proteins↗

Identification of a new Arabidopsis disease resistance locus, RPs4, and cloning of the corresponding avirulence gene, avrRps4, from Pseudomonas syringae pv. pisi.

A molecular genetic approach was used to study the interaction between Arabidopsis and an avirulence gene from Pseudomonas syringae pv. pisi. P. syringae pv. pisi strain 151 induces a hypersensitive response (HR) when inoculated on the Arabidopsis accession Po-1. A genomic cosmid library was constructed from DNA from P. syringae pv. pisi strain 151 and a cosmid was identified that causes the normally virulent P. syringae pv. tomato strain DC3000 to induce an HR on Po-1. The cosmid was subcloned and a 1.2-kb DNA fragment conferring avirulence activity was sequenced. The single significant open reading frame within the 1.2-kb fragment was designated avrRps4. An avrRps4-specific probe hybridizes to DNA from all P. syringae pv. pisi strains tested. P. syringae strains carrying avrRps4 induces an HR on specific accessions of both Arabidopsis and soybean. Arabidopsis accession Ws-0 is resistant to DC3000(avrRps4), whereas accession RLD is susceptible. Resistance segregates as a single dominant locus in a genetic cross between Ws-0 and RLD. This disease resistance locus, RPS4, was mapped to chromosome 5 between the molecular markers sAT2105 and KG-8.

Amino Acid Sequence↗

Phenotypic expression of Pseudomonas syringae avr genes in E. coli is linked to the activities of the hrp-encoded secretion system.

The specific recognition of elicitors produced by plant pathogenic bacteria carrying avirulence (avr) genes is postulated to initiate cellular defense responses in plants expressing corresponding resistance genes. The biochemical functions of most avr genes, however, are not known. A heterologous system was developed to phenotypically express Pseudomonas syringae avr genes in Escherichia coli cells that required the P. syringae hrp cluster. E. coli MC4100 transformants carrying the plasmic-borne P. syringae pv. syringae Pss61 hrp cluster and p. syringae pv. glycinea avrB expressed from a triple lacUV5 promoter gained the ability to elicit the hypersensitive response in soybean cultivars expressing Rpg1 and in an Arabidopsis thaliana accession expressing RPM1. Inactivation of energy transducing or outer membrane components of the hrp-encoded secretion system blocked phenotypic expression expression of avrB in E. coli, but deletions abolishing harpinPSS production had little effect on the production of the AvrB phenotype by the E. coli transformants. Phenotypic expression of avrA, AvrPto, avrRpm1, avrRpt2, and avrPph3 in E. coli was also shown to require the hrp cluster. The results indicate that generation of the Avr phenotype in P. syringae strains is specifically dependent on the secretion activities of the hrp cluster.

Base Sequence↗

Expression of avrPphB, an avirulence gene from Pseudomonas syringae pv. phaseolicola, and the delivery of signals causing the hypersensitive reaction in bean.

Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola was examined. Incorporation of [35S]-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product. The 28-kDa native peptide was isolated from E. coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves. Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E. coli and P. syringae pv. phaseolicola. Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY- strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time. Expression of the hrp gene cluster of P. syringae pv. phaseolicola from plasmid pPPY430 in E. coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031). Despite constitutive expression of the hrp and avr genes in E. coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes. The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.

Amino Acid Sequence↗

dnaK and the heat stress response of Pseudomonas syringae pv. glycinea.

The dnaK gene from Pseudomonas syringae pv. glycinea PG4180 was cloned and sequenced. The dnaK coding region was 1,917 bp and contained a putative sigma 32 heat shock promoter 86 bp upstream of the translational start site. grpE, another heat shock gene, was found immediately upstream of the putative dnaK promoter. The predicted amino acid sequence of dnaK showed relatedness to the ATPase and substrate binding domains commonly found in heat shock proteins, as well as the highly conserved signature sequence motifs belonging to the Hsp70 protein family. Furthermore, the PG4180 dnaK gene complemented an Escherichia coli dnaK mutant for growth at temperatures above 37 degrees C, indicating that a fully functional dnaK homologue had been cloned from P. syringae pv. glycinea. All attempts to eliminate dnaK function by insertion mutagenesis failed, possibly because DnaK performs essential functions in P. syringae pv. glycinea. Expression of dnaK in P. syringae pv. glycinea PG4180 was investigated by constructing dnaK::uidA transcriptional fusions; expression of dnaK increased markedly when cells were preincubated at 18 degrees C and then shifted to 35 degrees C. An anti-DnaK monoclonal antibody was used to detect DnaK; in P. syringae pv. glycinea race 4, DnaK levels followed cell density during a 6-h incubation at 26 degrees C. When cells were shifted from 26 degrees C to either 32 or 38 degrees C, DnaK levels increased transiently, and then decreased rapidly. Although the cells continued to grow when incubated at 32 degrees C, growth was not supported at 38 degrees C. Our results indicate that P. syringae pv. glycinea responds to heat shock by producing DnaK, but DnaK does not aid in acclimation to sustained elevated temperatures.

Amino Acid Sequence↗

Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.

Catalase↗

Restriction fragment length polymorphism evidence for genetic homology within a pathovar of Pseudomonas syringae.

Pseudomonas syringae pv. phaseolicola NPS3121 hrp sequences were used as hybridization probes in a restriction fragment length polymorphism (RFLP) analysis of 24 P. syringae pv. tabaci strains as a means to evaluate the genetic and taxonomic relationship of pathovars of P. syringae. Southern blot analyses of genomic restriction digests, with hrpA-S sequences as hybridization probes, and restriction analyses of PCR-amplified DNA of regions within hrpD were conducted. The resulting RFLP patterns were uniform for 23 of the 24 isolates tested, with strain BR2R having a unique pattern. BR2R is a pathogen of bean which was classified as pathovar tabaci because of its ability to produce tabtoxin, but unlike the other 23 tabaci strains in this study, it does not incite disease symptoms on tobacco. When a DNA fragment containing hrpM sequences was used as a hybridization probe, the tabaci isolates could be divided into three groups on the basis of the RFLP patterns : BR2R, Pt11528R and Pt113R, and the remaining strains. For all of the above analyses, BR2R shared identical RFLP patterns with P. syringae pv. phaseolicola NPS3121, also a bean pathogen which does not cause disease on tobacco. However, BR2R AND NPS3121 could be differentiated from each other on the basis of the RFLP patterns from restriction analysis of PCR-amplified DNA of argF, while the remaining tabaci strains had a third pattern. These studies indicate that hrp genes and argF are conserved in strains of P. syringae pathogenic to tobacco, suggesting that P. syringae strains pathogenic to specific hosts may have a high level of genetic similarity. We believe that these analyses have shown that distinct identifiable genetic differences may be correlated with host range and suggest that such information may be useful for assigning pathovar designations.

Bacterial Proteins↗

Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.

Base Sequence↗

Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene.

The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P.aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.

Amino Acid Sequence↗

Suppression of a sensor kinase-dependent phenotype in Pseudomonas syringae by ribosomal proteins L35 and L20.

The lemA gene of Pseudomonas syringae pv. syringae encodes the sensor kinase of a bacterial two-component signal transduction system. Phenotypes that are lemA dependent in P. syringae include lesion formation on bean and production of extracellular protease and the antibiotic syringomycin. Recently, the gacA gene has been identified as encoding the response regulator of the lemA regulon. To identify additional components that interact with LemA, suppressors of a lemA mutation were sought. A locus was identified that, when present in multiple copies, restores extracellular protease production to a lemA insertion mutant of P. syringae pv. syringae. This locus was found to encode the P. syringae homologs of translation initiation factor IF3 and ribosomal proteins L20 and L35 of Escherichia coli and other bacteria. Deletion analysis and data from Western immunoblots with anti-IF3 antiserum suggest that protease restoration does not require IF3. Deletion of both the L35 and L20 genes resulted in loss of protease restoration, whereas disruption of either gene alone increased protease restoration. Our results suggest that overexpression of either L20 or L35 is sufficient for protease restoration. It is unclear how alteration of ribosomal protein expression compensates in this instance for loss of a transcriptional activator, but a regulatory role for L20 and L35 apart from their function in the ribosome may be indicated.

Amino Acid Sequence↗