BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits. RESULTS: In this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes. CONCLUSIONS: Contrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed.
Title: A conserved inverted repeat, the LipR box, mediates transcriptional activation of the Streptomyces exfoliatus lipase gene by LipR, a member of the STAND class of P-loop nucleoside triphosphatases Evangelista-Martinez Z, Gonzalez-Ceron G, Servin-Gonzalez L Ref: Journal of Bacteriology, 188:7082, 2006 : PubMed
Expression of the Streptomyces exfoliatus lipA gene, which encodes an extracellular lipase, depends on LipR, a transcriptional activator that belongs to the STAND class of P-loop nucleoside triphosphatases. LipR is closely related to activators present in some antibiotic biosynthesis clusters of actinomycetes, forming the LipR/TchG family of regulators. In this work we showed that purified LipR protein is essential for activation of lipA transcription in vitro and that this transcription depends on the presence of a conserved inverted repeat, the LipR box, located upstream of the lipA promoter. Mutagenesis of the lipA promoter region indicated that most transcription depends on LipR binding to the proximal half-site of the LipR box in close proximity to the -35 region of the promoter. Our experiments also indicated that LipR establishes contact with the RNA polymerase on both sides of the LipR box, since some activation was observed when only the distal half-site was present or when the entire LipR box was moved further upstream. We also showed that the LipR proteins of S. exfoliatus and Streptomyces coelicolor are functionally interchangeable both in vitro and in vivo, revealing the functional conservation of the regulatory elements in these two species.
Title: The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator Valdez F, Gonzalez-Ceron G, Kieser HM, Servin-Gonzalez L Ref: Microbiology, 145 ( Pt 9):2365, 1999 : PubMed
A region of the Streptomyces coelicolor A3(2) chromosome was identified and cloned by using as a probe the lipase gene from Streptomyces exfoliatus M11. The cloned region consisted of 6286 bp, and carried a complete lipase gene, lipA, as well as a gene encoding a transcriptional activator (lipR). The S. coelicolor A3(2) lipA gene encodes a functional extracellular lipase 82% identical to the S. exfoliatus M11 lipase; the partially purified S. coelicolor enzyme showed a preference for substrates of short to medium chain length. Transcription of lipA was completely dependent on the presence of lipR, and occurred from a single promoter similar to the lipA promoters of S. exfoliatus M11 and Streptomyces albus G. These three Streptomyces lipA promoters have well-conserved -10 and -35 regions, as well as additional conserved sequences upstream of the -35 region, which could function as targets for transcriptional activation by the cognate LipR regulators. The Streptomyces LipR activators are related to other bacterial regulators of a similar size, constituting a previously unidentified family of proteins that includes MalT, AcoK, AlkS, AfsR, five mycobacterial proteins of unknown function and some Streptomyces regulators in antibiotic synthesis clusters. A lipase-deficient strain of S. coelicolor was constructed and found to be slightly affected in production of the polyketide antibiotic actinorhodin.
Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases.
RESULTS:
The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases.
CONCLUSIONS:
The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.
Title: bldA-dependent expression of the Streptomyces exfoliatus M11 lipase gene (lipA) is mediated by the product of a contiguous gene, lipR, encoding a putative transcriptional activator Servin-Gonzalez L, Castro C, Perez C, Rubio M, Valdez F Ref: Journal of Bacteriology, 179:7816, 1997 : PubMed
Extracellular lipase synthesis by Streptomyces lividans 66 carrying the cloned lipase gene (lipA) from Streptomyces exfoliatus M11 was found to be growth phase dependent, since lipase was secreted into the medium mainly during the stationary phase; S1 nuclease protection experiments revealed abundant lipA transcripts in RNA preparations obtained during the stationary phase but not in those obtained during exponential growth. Transcription from the lipA promoter was dependent on the presence of lipR, a contiguous downstream gene with a very high guanine-plus-cytosine content (80.2%). The deduced lipR product consists of a protein of 934 amino acids that shows similarity to known transcriptional activators and has a strong helix-turn-helix motif at its C terminus; this motif is part of a domain homologous to DNA-binding domains of bacterial regulators of the UhpA/LuxR superfamily. The lipR sequence revealed the presence of a leucine residue, encoded by the rare TTA codon, which caused bldA dependence of lipA transcription in Streptomyces coelicolor A3(2); replacement of the TTA codon by the alternate CTC leucine codon alleviated bidA dependence but not the apparent growth phase-dependent regulation of lipA transcription. When lipR expression was induced in a controlled fashion during the exponential growth phase, by placing it under the inducible tipA promoter, lipase synthesis was shifted to the exponential growth phase, indicating that the timing of lipR expression, and not its bldA dependence, is the main cause for stationary-phase transcription of lipA.
An extracellular lipase (Lip)-encoding gene from Streptomyces albus G has been cloned and sequenced. It encodes a Lip with 82% sequence identity to another previously cloned Lip from a Streptomyces species not closely related. These two sequences can be aligned with 33% identity to the sequence of Lip1 from the antarctic psychrotroph Moraxella TA144 [G. Feller et al., Nucleic Acids Res. 18 (1990) 6431]. An alignment of the three sequences revealed amino-acid substitutions which might be responsible for the greater thermal stability of the Streptomyces lipases. The presence of this lip gene family in several members of the Streptomyces genus was also shown.
A gene encoding an extracellular lipase from Streptomyces sp. M11 was cloned in the high-copy-number vector pIJ486, using S. lividans 66 as host. A 28-kDa protein was secreted by S. lividans carrying pB13, which harbors a 6-kb insert, and identified as the product of the cloned gene. Comparison of the N-terminal amino acid (aa) sequence of the purified extracellular lipase with the nucleotide (nt) sequence of the lip gene revealed the presence of a 48 aa long signal peptide. The nucleotide sequence also revealed the presence of a motif, Gly-His-Ser-Met-Gly, similar to the one found surrounding the active-site Ser in other lipases. The gene is most likely monocistronic. Subcloning experiments indicated that another gene might be required for high-level expression, since subcloning of the structural gene alone resulted in diminished extracellular lipase activity. The lipase gene promoter was identified by S1 mapping experiments, and found to be similar to other Streptomyces vegetative promoters.
