Members of the genus Pseudomonas inhabit a wide variety of environments, which is reflected in their versatile metabolic capacity and broad potential for adaptation to fluctuating environmental conditions. Here, we examine and compare the genomes of a range of Pseudomonas spp. encompassing plant, insect and human pathogens, and environmental saprophytes. In addition to a large number of allelic differences of common genes that confer regulatory and metabolic flexibility, genome analysis suggests that many other factors contribute to the diversity and adaptability of Pseudomonas spp. Horizontal gene transfer has impacted the capability of pathogenic Pseudomonas spp. in terms of disease severity (Pseudomonas aeruginosa) and specificity (Pseudomonas syringae). Genome rearrangements likely contribute to adaptation, and a considerable complement of unique genes undoubtedly contributes to strain- and species-specific activities by as yet unknown mechanisms. Because of the lack of conserved phenotypic differences, the classification of the genus has long been contentious. DNA hybridization and genome-based analyses show close relationships among members of P. aeruginosa, but that isolates within the Pseudomonas fluorescens and P. syringae species are less closely related and may constitute different species. Collectively, genome sequences of Pseudomonas spp. have provided insights into pathogenesis and the genetic basis for diversity and adaptation.

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We have developed a stable isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria Rhodobacter sphaeroides and Paracoccus denitrificans. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the lacI q gene from pYanni3 and the lac promoter, P A1/04/03 , from pJBA24.Rhodobacter sphaeroides and Paracoccus denitrificans are often used for the study of bacterial metabolism, bioenergetics (8), and signal transduction (11). Although inducible-expression plasmids are available for these organisms, e.g., pRKSK1 (5) and pRECTX (9), these plasmids suffer from one or more of the following problems. First, continuous antibiotic selection is essential for maintaining the plasmid in the population, e.g., plasmid pRK415 (7), which is the vector backbone for most of the available expression vectors for these species, is retained by only approximately 10% of the population after 40 generations without antibiotic selection (6) (segregational instability). Second, the inducer affects the expression of many endogenous genes; for example, several R. sphaeroides vectors use either light-or oxygeninducible promoters to deliver high levels of protein expression (5). However, light and oxygen affect the expression of over 35% of the endogenous genes in this organism (2), which limifts the use of these vectors in functional studies.The plasmid developed in this study, pIND4 ( Fig. 1), uses the pMG170 vector backbone (6), the lacI q gene from pYanni3 (4), and the isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible-expression cassette from pJBA24, which includes the P A1/04/03 promoter, a ribosome binding site, a polylinker, and two transcriptional terminators (1). pMG170 confers kanamycin resistance and is an Escherichia coli shuttle cloning vector derived from naturally occurring plasmid pMG160 of Rhodobacter blasticus, which replicates and is segregationally stable in several other members of the Rhodobacteraceae (6). In E. coli, pMG170 has a high copy number, replicating using the ColE1 origin, while in R. sphaeroides, the pMG160 origin delivers a copy number of 18 to 23 (6).Vector construction. The construction of pIND4 is described in the supplemental material.Testing of the expression plasmid. The coding sequence for the R. sphaeroides cheY6 gene was cloned into pIND4, generating pIND4-Y6. The plasmid was introduced into R. sphaeroides strain JPA1336 (⌬cheY6 derivative of WS8N) and P. denitrificans strain PD1222 (wild type) via conjugation with the E. coli donor strain S17-1 pir (10). Cells containing the plasmid were grown from single colonies under aerobic conditions with shaking (225 rpm) in succinate medium containing 25 g/ml kanamycin. The effects of different concentrations of IPTG (Fig. 2) and different induction times (Fig. 3) on CheY6 protein accumulation were investigated.At IPTG concentrations of less than 1 M, no expression of CheY6 was detectable in R. sphaeroides (the minimum detection l...