5 research outputs found
Genome sequence of Pseudomonas aeruginosa PAO1161, a PAO1 derivative with the ICEPae1161 integrative and conjugative element
Background: Pseudomonas aeruginosa is a cause of nosocomial infections, especially in patients with cystic fibrosis
and burn wounds. PAO1 strain and its derivatives are widely used to study the biology of this bacterium, however
recent studies demonstrated differences in the genomes and phenotypes of derivatives from different laboratories.
Results: Here we report the genome sequence of P. aeruginosa PAO1161 laboratory strain, a leu-, Rif R , restriction-
modification defective PAO1 derivative, described as the host of IncP-8 plasmid FP2, conferring the resistance to
mercury. Comparison of PAO1161 genome with PAO1-UW sequence revealed lack of an inversion of a large
genome segment between rRNA operons and 100 nucleotide polymorphisms, short insertions and deletions. These
included a change in leuA, resulting in E108K substitution, which caused leucine auxotrophy and a mutation in
rpoB, likely responsible for the rifampicin resistance. Nonsense mutations were detected in PA2735 and PA1939
encoding a DNA methyltransferase and a putative OLD family endonuclease, respectively. Analysis of revertants in
these two genes showed that PA2735 is a component of a restriction-modification system, independent of PA1939.
Moreover, a 12 kb RPG42 prophage and a novel 108 kb PAPI-1 like integrative conjugative element (ICE)
encompassing a mercury resistance operon were identified. The ICEPae1161 was transferred to Pseudomonas putida
cells, where it integrated in the genome and conferred the mercury resistance.
Conclusions: The high-quality P. aeruginosa PAO1161 genome sequence provides a reference for further research
including e.g. investigation of horizontal gene transfer or comparative genomics.
The strain was found to carry ICEPae1161, a functional PAPI-1 family integrative conjugative element, containing loci
conferring mercury resistance, in the past attributed to the FP2 plasmid of IncP-8 incompatibility group. This
indicates that the only known member of IncP-8 is in fact an ICE
The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa
Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections
that are especially dangerous for immunocompromised patients. The eradication of P.
aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and
genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire
of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins,
mainly acting as repressors, which are involved in response to various environmental signals. In
this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa.
Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the
mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function
of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in
P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the
PA3459–PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase,
a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of
N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We
showed that PA3458-mediated control of PA3459–PA3461 expression is required for the adaptation
of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in
osmoadaptation control in P. aeruginosa
The AraC-Type Transcriptional Regulator GliR (PA3027) Activates Genes of Glycerolipid Metabolism in Pseudomonas aeruginosa
Pseudomonas aeruginosa encodes a large set of transcriptional regulators (TRs) that modulate
and manage cellular metabolism to survive in variable environmental conditions including that of
the human body. The AraC family regulators are an abundant group of TRs in bacteria, mostly acting
as gene expression activators, controlling diverse cellular functions (e.g., carbon metabolism, stress
response, and virulence). The PA3027 protein from P. aeruginosa has been classified in silico as a
putative AraC-type TR. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3027
revealed a spectacular increase in the mRNA levels of PA3026-PA3024 (divergent to PA3027), PA3464,
and PA3342 genes encoding proteins potentially involved in glycerolipid metabolism. Concomitantly,
chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that at least 22 regions are
bound by PA3027 in the PAO1161 genome. These encompass promoter regions of PA3026, PA3464,
and PA3342, showing the major increase in expression in response to PA3027 excess. In Vitro DNA
binding assay confirmed interactions of PA3027 with these regions. Furthermore, promoter-reporter
assays in a heterologous host showed the PA3027-dependent activation of the promoter of the
PA3026-PA3024 operon. Two motifs representing the preferred binding sites for PA3027, one localized
upstream and one overlapping with th
Functional Characterization of TetR-like Transcriptional Regulator PA3973 from Pseudomonas aeruginosa
Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial
infections. Its ability to survive under different conditions relies on a complex regulatory network
engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use
the available resources. P. aeruginosa PA3973 encodes an uncharacterized TetR family transcriptional
regulator. In this study, we applied a transcriptome profiling (RNA-seq), genome-wide identification
of binding sites using ChIP-seq, as well as the phenotype analyses to unravel the biological role of
PA3973. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes
in the mRNA level of 648 genes. Concomitantly, ChIP-seq analysis identified more than 300 PA3973
binding sites in the P. aeruginosa genome. A 13 bp sequence motif was indicated as the binding site of
PA3973. The PA3973 regulon encompasses the PA3972-PA3971 genes encoding a probable acyl-CoA
dehydrogenase and a thioesterase. In vitro analysis showed PA3973 binding to PA3973p. Accordingly,
the lack of PA3973 triggered increased expression of PA3972 and PA3971. The DPA3972-71 PAO1161
strain demonstrated impaired growth in the presence of stress-inducing agents hydroxylamine
or hydroxyurea, thus suggesting the role of PA3972-71 in pathogen survival upon stress. Overall
our results showed that TetR-type transcriptional regulator PA3973 has multiple binding sites in
the P. aeruginosa genome and influences the expression of diverse genes, including PA3972-PA3971,
encoding proteins with a proposed role in stress response
The AraC-Type Transcriptional Regulator GliR (PA3027) Activates Genes of Glycerolipid Metabolism in Pseudomonas aeruginosa
Pseudomonas aeruginosa encodes a large set of transcriptional regulators (TRs) that modulate and manage cellular metabolism to survive in variable environmental conditions including that of the human body. The AraC family regulators are an abundant group of TRs in bacteria, mostly acting as gene expression activators, controlling diverse cellular functions (e.g., carbon metabolism, stress response, and virulence). The PA3027 protein from P. aeruginosa has been classified in silico as a putative AraC-type TR. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3027 revealed a spectacular increase in the mRNA levels of PA3026-PA3024 (divergent to PA3027), PA3464, and PA3342 genes encoding proteins potentially involved in glycerolipid metabolism. Concomitantly, chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that at least 22 regions are bound by PA3027 in the PAO1161 genome. These encompass promoter regions of PA3026, PA3464, and PA3342, showing the major increase in expression in response to PA3027 excess. In Vitro DNA binding assay confirmed interactions of PA3027 with these regions. Furthermore, promoter-reporter assays in a heterologous host showed the PA3027-dependent activation of the promoter of the PA3026-PA3024 operon. Two motifs representing the preferred binding sites for PA3027, one localized upstream and one overlapping with the −35 promoter sequence, were identified in PA3026p and our data indicate that both motifs are required for full activation of this promoter by PA3027. Overall, the presented data show that PA3027 acts as a transcriptional regulator in P. aeruginosa, activating genes likely engaged in glycerolipid metabolism. The GliR name, from a glycerolipid metabolism regulator, is proposed for PA3027 of P. aeruginosa