Comparative genomics of Pseudomonas syringae pathovar tomato reveals novel chemotaxis pathways associated with motility and plant pathogenicity.

The majority of bacterial foliar plant pathogens must invade the apoplast of host plants through points of ingress, such as stomata or wounds, to replicate to high population density and cause disease. How pathogens navigate plant surfaces to locate invasion sites remains poorly understood. Many bacteria use chemical-directed regulation of flagellar rotation, a process known as chemotaxis, to move towards favorable environmental conditions. Chemotactic sensing of the plant surface is a potential mechanism through which foliar plant pathogens home in on wounds or stomata, but chemotactic systems in foliar plant pathogens are not well characterized. Comparative genomics of the plant pathogen Pseudomonas syringae pathovar tomato (Pto) implicated annotated chemotaxis genes in the recent adaptations of one Pto lineage. We therefore characterized the chemosensory system of Pto. The Pto genome contains two primary chemotaxis gene clusters, che1 and che2. The che2 cluster is flanked by flagellar biosynthesis genes and similar to the canonical chemotaxis gene clusters of other bacteria based on sequence and synteny. Disruption of the primary phosphorelay kinase gene of the che2 cluster, cheA2, eliminated all swimming and surface motility at 21 °C but not 28 °C for Pto. The che1 cluster is located next to Type IV pili biosynthesis genes but disruption of cheA1 has no observable effect on twitching motility for Pto. Disruption of cheA2 also alters in planta fitness of the pathogen with strains lacking functional cheA2 being less fit in host plants but more fit in a non-host interaction.

Global analysis of the HrpL regulon in the plant pathogen Pseudomonas syringae pv. tomato DC3000 reveals new regulon members with diverse functions.

The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen Pseudomonas syringae pv. tomato DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a promoter sequence, often designated as the "hrp promoter." Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely hrp promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new hrp promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. PSPTO_5633 is the only new HrpL regulon member that is potentially an effector and is now designated HopBM1. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or ΔhopQ1-1 backgrounds, do not affect the hypersensitive response or in planta growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new hrp promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors.

Oligonucleotide recombination enabled site-specific mutagenesis in bacteria.

Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligonucleotide recombination is one type of recombineering that uses ssDNA oligonucleotides to direct chromosomal mutations. Oligo recombination occurs without addition of any exogenous functions, making this approach potentially useful in many different bacteria. Here we describe the basic technique for constructing a site-specific genomic mutation in Pseudomonas syringae.

A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha.

Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB(-)4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4x10(3) Km-resistance colonies/mug DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor alpha-galactosidase in R. eutropha.

Isolation of novel Pseudomonas syringae promoters and functional characterization in polyhydroxyalkanoate-producing pseudomonads.

A library of genomic DNA fragments of Pseudomonas syringae pv. tomato DC3000 was constructed in a lacZalpha-containing plasmid, pBS29. The library was used in a preliminary alpha-complementation-based screen to identify clones with promoter activity in Escherichia coli. Ten positive clones were sequenced and their locations in the chromosomal DNA of DC3000 strain were mapped. Five positive clones (P2, P3, P4, P6 and P8) were further assayed for promoter activity in three polyhydroxyalkanoate-producing pseudomonads: Pseudomonas resinovorans, P. corrugata and P. chlororaphis. To this end, a green-fluorescent-protein gene (gfp) was cloned downstream from the putative (DC3000) promoter in a shuttle plasmid. We found that only Pseudomonas transformants harboring the gfp-containing plasmid driven by putative promoter P2 showed fluorescence, indicating that this promoter is functioning in the three tested pseudomonads. Results of an in silico analysis of the P2 sequence further support the assignment of P2 as a bona fide promoter by the localization of putative -10 and -35 promoter regions and a transcription-factor-binding site, rpoD17, in this sequence. We successfully applied promoter P2 to drive the expression in P. chlororaphis of a recombinant alpha-galactosidase gene of Streptomyces coelicolor, which should be useful for the utilization of oligosaccharides of soy molasses for the production of polyhydroxyalkanoate biopolymer or rhamnolipid biosurfactant.