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Genes required for and effects of alginate overproduction induced by growth of Pseudomonas aeruginosa on Pseudomonas isolation agar supplemented with ammonium metavanadate.

ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response, resulting in a colony morphology and phenotype referred to as mucoid. However, how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce alginate. Vanadate is a phosphate mimic and causes protein misfolding by disruption of disulfide bonds. Here we used PIAAMV to characterize the pathways involved in inducible alginate production and tested the global effects of P. aeruginosa growth on PIAAMV by a mutant library screen, by transcriptomics, and in a murine acute virulence model. The PA14 nonredundant mutant library was screened on PIAAMV to identify new genes that are required for the inducible alginate stress response. A functionally diverse set of genes encoding products involved in cell envelope biogenesis, peptidoglycan remodeling, uptake of phosphate and iron, phenazine biosynthesis, and other processes were identified as positive regulators of the mucoid phenotype on PIAAMV. Transcriptome analysis of P. aeruginosa cultures growing in the presence of vanadate showed differential expression of genes involved in virulence, envelope biogenesis, and cell stress pathways. In this study, it was observed that growth on PIAAMV attenuates P. aeruginosa in a mouse pneumonia model. Induction of alginate overproduction occurs as a stress response to protect P. aeruginosa, but it may be possible to modulate and inhibit these pathways based on the new genes identified in this study.

SUBMITTER: Damron FH

PROVIDER: S-EPMC3754734 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Genes required for and effects of alginate overproduction induced by growth of Pseudomonas aeruginosa on Pseudomonas isolation agar supplemented with ammonium metavanadate.

Damron F Heath FH Barbier Mariette M McKenney Elizabeth S ES Schurr Michael J MJ Goldberg Joanna B JB

Journal of bacteriology 20130621 18

Pseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response, resulting in a colony morphology and phenotype referred to as mucoid. However, how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce algi ...[more]

PMID: 23794622

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Project description:Pseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response resulting in a colony morphology and phenotype referred to as mucoid. However how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar (PIA) supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce alginate. Vanadate is a phosphate mimic and causes protein misfolding by disruption of disulfide bonds. Here we used PIAAMV to characterize the pathways involved in inducible alginate production and tested the global effects of P. aeruginosa growth on PIAAMV by a mutant library screen, transcriptomics, and in a murine acute virulence model. The PA14 non-redundant mutant library was screened on PIAAMV to identify new genes that are required for the inducible alginate stress response. A functionally diverse set of genes encoding products involved in cell envelope biogenesis, peptidoglycan, uptake of phosphate and iron, phenazines biosynthesis, and other processes were identified as positive regulators of the mucoid phenotype on PIAAMV. Transcriptome analysis of P. aeruginosa growing in the presence of vanadate caused differential expression of genes involved in virulence, envelope biogenesis, and cell stress pathways. In this study, it was observed that growth on PIAAMV attenuates P. aeruginosa in a mouse pneumonia model. Induction of alginate overproduction occurs as a stress response to protect P. aeruginosa but it may be possible to modulate and inhibit these pathways based on the new genes identified in this study. Strain PAO1 was cultured on both PIA and PIAAMV for 18 hr. Three separate plates were used for each media. The cells from each plate were scraped into 4 ml of RNA Protect (Qiagen) and stored immediately at -80°C. Cells were treated with RNAprotect (Qiagen) and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturer’s instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment. Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. RNA isolation, cDNA preparation, labeling and microarray analysis were performed as previously described in reference: Damron, F. H., J. P. Owings, Y. Okkotsu, J. J. Varga, J. R. Schurr, J. B. Goldberg, M. J. Schurr, and H. D. Yu. 2012. Analysis of the Pseudomonas aeruginosa Regulon Controlled by the Sensor Kinase KinB and Sigma Factor RpoN. J Bacteriol 194:1317-30.

Project description:Mucoid strains of Pseudomonas aeruginosa overproduce alginate, a linear exopolysaccharide Of D-mannuronate and variable amounts of L-guluronate. The mannuronate residues undergo modification by C-5 epimerization to form the L-guluronates and by the addition of acetyl groups at the 0-2 and 0-3 positions. Through genetic analysis, we previously identified algF, located upstream of algA in the 18-kb alginate biosynthetic operon, as a gene required for alginate acetylation. Here, we show the sequence of a 3.7-kb fragment containing the open reading frames termed algI, algJ, and algF. An algI::Tn5O1 mutant, which was defective in algIJFA because of the polar nature of the transposon insertion, produced alginate when algA was provided in trans. This indicated that the algIJF gene products were not required for polymer biosynthesis. To examine the potential role of these genes in alginate modification, mutants were constructed by gene replacement in which each gene (algI, algJ, or algF) was replaced by a polar gentamicin resistance cassette. Proton nuclear magnetic resonance spectroscopy showed that polymers produced by strains deficient in algIJF still contained a mixture of D-mannuronate and L-guluronate, indicating that C-5 epimerization was not affected. Alginate acetylation was evaluated by a colorimetric assay and Fourier transform-infrared spectroscopy, and this analysis showed that strains deficient in algIJF produced nonacetylated alginate. Plasmids that supplied the downstream gene products affected by the polar mutations were introduced into each mutant. The strain defective only in algF expression produced an alginate that was not acetylated, confirming previous results. Strains missing only algJ or algI also produced nonacetylated alginates. Providing the respective missing gene (algI, algJ, or algF) in trans restored alginate acetylation. Mutants defective in algI or algJ, obtained by chemical and transposon mutagenesis, were also defective in their ability to acetylate alginate. Therefore, algI and algJ represent newly identified genes that, in addition to algF, are required for alginate acetylation.

Project description:UNLABELLED:The molecular mechanisms of alginate polymerization/modification/secretion by a proposed envelope-spanning multiprotein complex are unknown. Here, bacterial two-hybrid assays and pulldown experiments showed that the catalytic subunit Alg8 directly interacts with the proposed copolymerase Alg44 while embedded in the cytoplasmic membrane. Alg44 additionally interacts with the lipoprotein AlgK bridging the periplasmic space. Site-specific mutagenesis of Alg44 showed that protein-protein interactions and stability were independent of conserved amino acid residues R17 and R21, which are involved in c-di-GMP binding, the N-terminal PilZ domain, and the C-terminal 26 amino acids. Site-specific mutagenesis was employed to investigate the c-di-GMP-mediated activation of alginate polymerization by the PilZAlg44 domain and Alg8. Activation was found to be different from the proposed activation mechanism for cellulose synthesis. The interactive role of Alg8, Alg44, AlgG (epimerase), and AlgX (acetyltransferase) on alginate polymerization and modification was studied by using site-specific deletion mutants, inactive variants, and overproduction of subunits. The compositions, molecular masses, and material properties of resulting novel alginates were analyzed. The molecular mass was reduced by epimerization, while it was increased by acetylation. Interestingly, when overproduced, Alg44, AlgG, and the nonepimerizing variant AlgG(D324A) increased the degree of acetylation, while epimerization was enhanced by AlgX and its nonacetylating variant AlgX(S269A). Biofilm architecture analysis showed that acetyl groups promoted cell aggregation while nonacetylated polymannuronate alginate promoted stigmergy. Overall, this study sheds new light on the arrangement of the multiprotein complex involved in alginate production. Furthermore, the activation mechanism and the interplay between polymerization and modification of alginate were elucidated. IMPORTANCE:This study provides new insights into the molecular mechanisms of the synthesis of the unique polysaccharide, alginate, which not only is an important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa but also has, due to its material properties, many applications in medicine and industry. Unraveling the assembly and composition of the alginate-synthesizing and envelope-spanning multiprotein complex will be of tremendous significance for the scientific community. We identified a protein-protein interaction network inside the multiprotein complex and studied its relevance with respect to alginate polymerization/modification as well as the c-di-GMP-mediated activation mechanism. A relationship between alginate polymerization and modification was shown. Due to the role of alginate in pathogenesis as well as its unique material properties harnessed in numerous applications, results obtained in this study will aid the design and development of inhibitory drugs as well as the commercial bacterial production of tailor-made alginates.

Dataset Information

Genes required for and effects of alginate overproduction induced by growth of Pseudomonas aeruginosa on Pseudomonas isolation agar supplemented with ammonium metavanadate.

Publications

Genes required for and effects of alginate overproduction induced by growth of Pseudomonas aeruginosa on Pseudomonas isolation agar supplemented with ammonium metavanadate.

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