RESULTS:
1 - 6 of 6 for "Matthew J Wargo"
The Pseudomonas aeruginosa sphBC genes are important for growth in the presence of sphingosine by promoting sphingosine metabolism
Sphingoid bases including sphingosine are important components of the antimicrobial barrier at epithelial surfaces where they can cause growth inhibition and killing of susceptible bacteria. Pseudomonas aeruginosa is a common opportunistic pathogen that is less susceptible to sphingosine than many Gram-negative bacteria. Here we determined that the deletion of the sphBCD operon reduced growth in the presence of sphingosine. Using deletion mutants complementation and growth assays in P. aeruginosa PAO1 we determined that the sphC and sphB genes encoding a periplasmic oxidase and periplasmic cytochrome c respectively were important for growth on sphingosine while sphD was dispensable under these conditions. Deletion of sphBCD in P. aeruginosa PA14 Pseudomonas protegens Pf-5 and Pseudomonas fluorescens Pf01 also showed reduced growth in the presence of sphingosine. The P. aeruginosa sphBC genes were also important for growth in the presence of two other sphingoid bases phytosphingosine and sphinganine. In WT P. aeruginosa sphingosine is metabolized to an unknown non-inhibitory product as sphingosine concentrations drop in the culture. However in the absence of sphBC sphingosine accumulates pointing to SphC and SphB as having a role in sphingosine metabolism. Finally the metabolism of sphingosine by WT P. aeruginosa protected susceptible cells from full growth inhibition by sphingosine pointing to a role for sphingosine metabolism as a public good. This work shows that the metabolism of sphingosine by P. aeruginosa presents a novel pathway by which bacteria can alter host-derived sphingolipids but it remains an open question whether SphB and SphC act directly on sphingosine.
Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR
Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined but the full scope of GATR ligands and regulatory networks are still unknown. Here we characterize Pseudomonas putida ’s detection of the animal-derived amine compound creatine a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR PP_3665 here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putida’s ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small and an electrophoretic mobility shift assay demonstrates strong and specific CahR binding only at the creA promoter supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologues associated with cahR orthologues highlights a strain distribution and organization supporting probable horizontal gene transfer particularly evident within the genus Acinetobacter . This study identifies and characterizes the GATR that transcriptionally controls P. putida ’s metabolism of creatine broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.
Characterizing species interactions that contribute to biofilm formation in a multispecies model of a potable water bacterial community
Microbial biofilms are ubiquitous in drinking water systems yet our understanding of drinking water biofilms lags behind our understanding of those in other environments. Here a six-member model bacterial community was used to identify the interactions and individual contributions of each species to community biofilm formation. These bacteria were isolated from the International Space Station potable water system and include Cupriavidus metallidurans Chryseobacterium gleum Ralstonia insidiosa Ralstonia pickettii Methylorubrum (Methylobacterium) populi and Sphingomonas paucimobilis but all six species are common members of terrestrial potable water systems. Using reconstituted assemblages from pairs to all 6 members community biofilm formation was observed to be robust to the absence of any single species and only removal of the C. gleum / S. paucimobilis pair out of all 15 possible 2-species subtractions led to loss of community biofilm formation. In conjunction with these findings dual-species biofilm formation assays supported the view that the contribution of C. gleum to community biofilm formation was dependent on synergistic biofilm formation with either R. insidiosa or C. metallidurans . These data support a model of multiple partially redundant species interactions to generate robustness in biofilm formation. A bacteriophage and multiple predatory bacteria were used to test the resilience of the community to the removal of individual members in situ but the combination of precise and substantial depletion of a single target species was not achievable. We propose that this assemblage can be used as a tractable model to understand the molecular bases of the interactions described here and to decipher other functions of drinking water biofilms.
Differential requirements for processing and transport of short-chain versus long-chain O-acylcarnitines in Pseudomonas aeruginosa
The opportunistic pathogen Pseudomonas aeruginosa can metabolize carnitine and O-acylcarnitines which are abundant in host muscle and other tissues. Acylcarnitines are metabolized to carnitine and a fatty acid. The liberated carnitine and its catabolic product glycine betaine can be used as osmoprotectants to induce the secreted phospholipase C PlcH and as sole carbon nitrogen and energy sources. P. aeruginosa is incapable of de novo synthesis of carnitine and acylcarnitines therefore they must be imported from an exogenous source. In this study we present the first characterization of bacterial acylcarnitine transport. Short-chain acylcarnitines are imported by the ABC transporter CaiX-CbcWV. Medium- and long-chain acylcarnitines (MCACs and LCACs) are hydrolysed extracytoplasmically and the free carnitine is transported primarily through CaiX-CbcWV. These findings suggest that the periplasmic protein CaiX has a binding pocket that permits short acyl chains on its carnitine ligand and that there are one or more secreted hydrolases that cleave MCACs and LCACs. To identify the secreted hydrolase(s) we used a saturating genetic screen and transcriptomics followed by phenotypic analyses but neither led to identification of a contributing hydrolase supporting but not conclusively demonstrating redundancy for this activity.
Carnitine in bacterial physiology and metabolism
Carnitine is a quaternary amine compound found at high concentration in animal tissues particularly muscle and is most well studied for its contribution to fatty acid transport into mitochondria. In bacteria carnitine is an important osmoprotectant and can also enhance thermotolerance cryotolerance and barotolerance. Carnitine can be transported into the cell or acquired from metabolic precursors where it can serve directly as a compatible solute for stress protection or be metabolized through one of a few distinct pathways as a nutrient source. In this review we summarize what is known about carnitine physiology and metabolism in bacteria. In particular recent advances in the aerobic and anaerobic metabolic pathways as well as the use of carnitine as an electron acceptor have addressed some long-standing questions in the field.
Identification of genes required for Pseudomonas aeruginosa carnitine catabolism
Carnitine is a quaternary amine compound prevalent in animal tissues and a potential carbon nitrogen and energy source for pathogens during infection. Characterization of activities in Pseudomonas aeruginosa cell lysates has previously shown that carnitine is converted to 3-dehydrocarnitine (3-dhc) which is in turn metabolized to glycine betaine (GB) an intermediate metabolite in the catabolism of carnitine to glycine. However the identities of the enzymes required for carnitine catabolism were not known. We used a genetic screen of the P. aeruginosa PA14 transposon mutant library to identify genes required for growth on carnitine. We identified two genomic regions and their adjacent transcriptional regulators that are required for carnitine catabolism. The PA5388–PA5384 region contains the predicted P. aeruginosa carnitine dehydrogenase homologue along with other genes required for growth on carnitine. The second region identified PA1999–PA2000 encodes the α and β subunits of a predicted 3-ketoacid CoA-transferase an enzymic activity hypothesized to be involved in the first step of deacetylation of 3-dhc. Furthermore we confirmed that an intact GB catabolic pathway is required for growth on carnitine. The PA5389 and PA1998 transcription factors are required for growth on carnitine. PA5389 is required for induction of the PA5388–PA5384 transcripts in response to carnitine and the PA1999–PA2000 transcripts are induced in a PA1998-dependent manner and induction appears to depend on a carnitine catabolite possibly 3-dhc. These results provide important insight into elements required for carnitine catabolism in P. aeruginosa and probably in other bacteria.