Project description:We report here the draft genome sequence of Pseudomonas putida strain DRA525, isolated from mercury-contaminated soil. This strain shows resistance to mercury and multiple antibiotics, and its genome sequence contains several gene sets known to confer resistance to heavy metals enzymatically and through multidrug efflux pumps.
Project description:Mobile genetic elements (MGE) such as plasmids and transposons mobilise genes within and between species, playing a crucial role in bacterial evolution via horizontal gene transfer (HGT). Currently, we lack data on variation in MGE dynamics across bacterial host species. We tracked the dynamics of a large conjugative plasmid, pQBR103, and its Tn5042 mercury resistance transposon, in five diverse Pseudomonas species in environments with and without mercury selection. Plasmid fitness effects and stability varied extensively between host species and environments, as did the propensity for chromosomal capture of the Tn5042 mercury resistance transposon associated with loss of the plasmid. Whereas Pseudomonas fluorescens and Pseudomonas savastanoi stably maintained the plasmid in both environments, the plasmid was highly unstable in Pseudomonas aeruginosa and Pseudomonas putida, where plasmid-free genotypes with Tn5042 captured to the chromosome invaded to higher frequency under mercury selection. These data confirm that plasmid stability is dependent upon the specific genetic interaction of the plasmid and host chromosome rather than being a property of plasmids alone, and moreover imply that MGE dynamics in diverse natural communities are likely to be complex and driven by a subset of species capable of stably maintaining plasmids that would then act as hubs of HGT.
Project description:Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.
Project description:Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharacterized. A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR. CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase from Escherichia coli. CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems. A related gene, zntR, regulates zntA in E. coli, but it is not contiguous with zntA in the E. coli genome as cadA and cadR were in P. putida. In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the P. aeruginosa genome, the P. putida CadA sequence had a histidine-rich N-terminal extension. CadR and the product of PA3689 of P. aeruginosa also had histidine-rich C-terminal extensions not found in other MerR family response regulators. Mutational analysis indicated that cadA and cadR are fully responsible for cadmium resistance and partially for zinc resistance. However, unlike zntA, they did not confer significant levels of lead resistance. The cadA promoter was responsive to Cd(II), Pb(II), and Zn(II), while the cadR promoter was only induced by Cd(II). CadR apparently represses its own expression at the transcriptional level. However, CadR apparently does not repress cadA. Homologs of the cadmium-transporting ATPase were detected in many other Pseudomonas species.
Project description:In Pseudomonas putida PaW85, the ortho-cleavage pathway is used for catechol degradation. The 11.4-kb XhoI fragment cloned from phenol degradation plasmid pEST1226 into pKT240 (recombinant plasmid pAT1140) contains the inducible pheBA operon that encodes catechol 1,2-dioxygenase (gene pheB) and phenol monooxygenase (gene pheA), the first two enzymes for the phenol degradation pathway. The promoter of the pheBA operon is mapped 1.5 kb upstream of the pheB gene. The plasmid pAT1140, when introduced into P. putida PaW85, enables the bacteria to use the hybrid plasmid-chromosome-encoded pathway for phenol degradation. The synthesis of the plasmid-encoded phenol monooxygenase and catechol 1,2-dioxygenase is induced by cis,cis-muconate. The expression studies of the deletion subclones derived from pAT1140 revealed that the transcription of the pheBA operon is positively controlled by a regulatory protein that is chromosomally encoded in P. putida. cis,cis-Muconate in cooperation with positive transcription factor CatR activates the transcription of the chromosomal ortho-pathway genes catA and catBC in P. putida (R. K. Rothmel, T. L. Aldrich, J. E. Houghton, W. M. Coco, L. N. Ornston, and A. M. Chakrabarty, J. Bacteriol. 172:922-931, 1990). The inability to express the pheBA operon in a P. putida CatR- background and activation of transcription of the pheBA operon in Escherichia coli in the presence of the catR-expressing plasmid demonstrated that the transcription of the pheBA operon in P. putida PaW85 carrying pEST1226 is controlled by the chromosomally encoded CatR.
Project description:Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.