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Multiple FadD acyl-CoA synthetases contribute to differential fatty acid degradation and virulence in Pseudomonas aeruginosa.

Kang Y, Zarzycki-Siek J, Walton CB, Norris MH, Hoang TT - PLoS ONE (2010)

Bottom Line: When compared to the wild type strain, the fadD2 mutant exhibited decreased production of lipase, protease, rhamnolipid and phospholipase, and retardation of both swimming and swarming motilities.Growth analysis of the fadD mutants showed noticeable deficiencies in utilizing FAs and phosphatidylcholine (major components of lung surfactant) as the sole carbon source.This defect translated into decreased in vivo fitness of P. aeruginosa in a BALB/c mouse lung infection model, supporting the role of lipids as a significant nutrient source for this bacterium in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.

ABSTRACT
A close interconnection between nutrient metabolism and virulence factor expression contributes to the pathophysiology of Pseudomonas aeruginosa as a successful pathogen. P. aeruginosa fatty acid (FA) degradation is complicated with multiple acyl-CoA synthetase homologs (FadDs) expressed in vivo in lung tissue during cystic fibrosis infections. The promoters of two genetically linked P. aeruginosa fadD genes (fadD1 and fadD2) were mapped and northern blot analysis indicated they could exist on two different transcripts. These FadDs contain ATP/AMP signature and FA-binding motifs highly homologous to those of the Escherichia coli FadD. Upon introduction into an E. coli fadD(-)/fadR(-) double mutant, both P. aeruginosa fadDs functionally complemented the E. coli fadD(-)/fadR(-) mutant, allowing degradation of different chain-length FAs. Chromosomal mutagenesis, growth analysis, induction studies, and determination of kinetic parameters suggested that FadD1 has a substrate preference for long-chain FAs while FadD2 prefers shorter-chain FAs. When compared to the wild type strain, the fadD2 mutant exhibited decreased production of lipase, protease, rhamnolipid and phospholipase, and retardation of both swimming and swarming motilities. Interestingly, fadD1 mutant showed only increased swarming motility. Growth analysis of the fadD mutants showed noticeable deficiencies in utilizing FAs and phosphatidylcholine (major components of lung surfactant) as the sole carbon source. This defect translated into decreased in vivo fitness of P. aeruginosa in a BALB/c mouse lung infection model, supporting the role of lipids as a significant nutrient source for this bacterium in vivo.

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Transcriptional profile of fadD1 and fadD2 in various FAs.For a short- (C8:0), medium- (C10:0), and long-chain FA (C18:1Δ9), northern blot analysis indicated two possible transcripts for both fadD genes when probed with either fadD1 (A) or fadD2 (B). Gene-fusion studies of strains P518 (PfadD1-lacZ) and P520 (PfadD2-lacZ), grown to mid-log phase, showed differential expression of fadD1 and fadD2 in the presence of different FAs (C and D). (C) fadD1 was up-regulated in the presence of the unsaturated LCFA (C18:1Δ9), while fadD2 expression was significantly increased in the presence of shorter chain FAs (C8:0, C10:0) (D). For (C) and (D), all cultures had identical growth-rates and overall cell densities (data not shown).
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pone-0013557-g003: Transcriptional profile of fadD1 and fadD2 in various FAs.For a short- (C8:0), medium- (C10:0), and long-chain FA (C18:1Δ9), northern blot analysis indicated two possible transcripts for both fadD genes when probed with either fadD1 (A) or fadD2 (B). Gene-fusion studies of strains P518 (PfadD1-lacZ) and P520 (PfadD2-lacZ), grown to mid-log phase, showed differential expression of fadD1 and fadD2 in the presence of different FAs (C and D). (C) fadD1 was up-regulated in the presence of the unsaturated LCFA (C18:1Δ9), while fadD2 expression was significantly increased in the presence of shorter chain FAs (C8:0, C10:0) (D). For (C) and (D), all cultures had identical growth-rates and overall cell densities (data not shown).

Mentions: To gain information on the regulatory regions of the P. aeruginosa fadDs, we mapped their transcriptional start sites to assign putative promoter sequences, and then determined transcription levels of each gene on various carbon sources (Figs. 2 and 3). Promoter mapping experiments indicated that each fadD had an independent transcriptional start site, suggesting that they were independently transcribed; however, northern blot analyses indicated that fadD2 and fadD1 can be co-transcribed on a single larger transcript or as smaller independent transcripts (Fig. 3A and 3B). Both fadD2 and fadD1 can exist as two different transcripts, suggesting some level of regulation by the predicted transcriptional terminator or attenuator sequence within the intergenic region (Fig. 2C). From Figure 3, we hypothesize that the promoter upstream of fadD2 drives the expression of both genes, and the intergenic terminator attenuates the larger fadD1 transcript. The fadD1 promoter immediately downstream of this regulatory element was induced by LCFA (e.g. C18:1Δ9), and initiated the expression of the smaller fadD1 transcript (Fig. 2C, 3A and 3B). Presumably, when there was no termination of transcription from the fadD2 promoter, fadD2 and fadD1 were transcribed together on the larger transcript of the same size observed on both blots (Fig. 3A and 3B). Based on the determination that these fadD genes could be independently transcribed or co-transcribed, it was necessary to determine which chain-length FA induced fadD1 and fadD2. The observed levels of induction showed that fadD1 was mainly induced by LCFA, particularly C18:1Δ9, while fadD2 was specifically induced by short- to medium-chain FA (Fig. 3C and 3D). Both fadD genes showed some level of expression under all conditions tested, indicating low levels of constitutive expression.


Multiple FadD acyl-CoA synthetases contribute to differential fatty acid degradation and virulence in Pseudomonas aeruginosa.

Kang Y, Zarzycki-Siek J, Walton CB, Norris MH, Hoang TT - PLoS ONE (2010)

Transcriptional profile of fadD1 and fadD2 in various FAs.For a short- (C8:0), medium- (C10:0), and long-chain FA (C18:1Δ9), northern blot analysis indicated two possible transcripts for both fadD genes when probed with either fadD1 (A) or fadD2 (B). Gene-fusion studies of strains P518 (PfadD1-lacZ) and P520 (PfadD2-lacZ), grown to mid-log phase, showed differential expression of fadD1 and fadD2 in the presence of different FAs (C and D). (C) fadD1 was up-regulated in the presence of the unsaturated LCFA (C18:1Δ9), while fadD2 expression was significantly increased in the presence of shorter chain FAs (C8:0, C10:0) (D). For (C) and (D), all cultures had identical growth-rates and overall cell densities (data not shown).
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pone-0013557-g003: Transcriptional profile of fadD1 and fadD2 in various FAs.For a short- (C8:0), medium- (C10:0), and long-chain FA (C18:1Δ9), northern blot analysis indicated two possible transcripts for both fadD genes when probed with either fadD1 (A) or fadD2 (B). Gene-fusion studies of strains P518 (PfadD1-lacZ) and P520 (PfadD2-lacZ), grown to mid-log phase, showed differential expression of fadD1 and fadD2 in the presence of different FAs (C and D). (C) fadD1 was up-regulated in the presence of the unsaturated LCFA (C18:1Δ9), while fadD2 expression was significantly increased in the presence of shorter chain FAs (C8:0, C10:0) (D). For (C) and (D), all cultures had identical growth-rates and overall cell densities (data not shown).
Mentions: To gain information on the regulatory regions of the P. aeruginosa fadDs, we mapped their transcriptional start sites to assign putative promoter sequences, and then determined transcription levels of each gene on various carbon sources (Figs. 2 and 3). Promoter mapping experiments indicated that each fadD had an independent transcriptional start site, suggesting that they were independently transcribed; however, northern blot analyses indicated that fadD2 and fadD1 can be co-transcribed on a single larger transcript or as smaller independent transcripts (Fig. 3A and 3B). Both fadD2 and fadD1 can exist as two different transcripts, suggesting some level of regulation by the predicted transcriptional terminator or attenuator sequence within the intergenic region (Fig. 2C). From Figure 3, we hypothesize that the promoter upstream of fadD2 drives the expression of both genes, and the intergenic terminator attenuates the larger fadD1 transcript. The fadD1 promoter immediately downstream of this regulatory element was induced by LCFA (e.g. C18:1Δ9), and initiated the expression of the smaller fadD1 transcript (Fig. 2C, 3A and 3B). Presumably, when there was no termination of transcription from the fadD2 promoter, fadD2 and fadD1 were transcribed together on the larger transcript of the same size observed on both blots (Fig. 3A and 3B). Based on the determination that these fadD genes could be independently transcribed or co-transcribed, it was necessary to determine which chain-length FA induced fadD1 and fadD2. The observed levels of induction showed that fadD1 was mainly induced by LCFA, particularly C18:1Δ9, while fadD2 was specifically induced by short- to medium-chain FA (Fig. 3C and 3D). Both fadD genes showed some level of expression under all conditions tested, indicating low levels of constitutive expression.

Bottom Line: When compared to the wild type strain, the fadD2 mutant exhibited decreased production of lipase, protease, rhamnolipid and phospholipase, and retardation of both swimming and swarming motilities.Growth analysis of the fadD mutants showed noticeable deficiencies in utilizing FAs and phosphatidylcholine (major components of lung surfactant) as the sole carbon source.This defect translated into decreased in vivo fitness of P. aeruginosa in a BALB/c mouse lung infection model, supporting the role of lipids as a significant nutrient source for this bacterium in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.

ABSTRACT
A close interconnection between nutrient metabolism and virulence factor expression contributes to the pathophysiology of Pseudomonas aeruginosa as a successful pathogen. P. aeruginosa fatty acid (FA) degradation is complicated with multiple acyl-CoA synthetase homologs (FadDs) expressed in vivo in lung tissue during cystic fibrosis infections. The promoters of two genetically linked P. aeruginosa fadD genes (fadD1 and fadD2) were mapped and northern blot analysis indicated they could exist on two different transcripts. These FadDs contain ATP/AMP signature and FA-binding motifs highly homologous to those of the Escherichia coli FadD. Upon introduction into an E. coli fadD(-)/fadR(-) double mutant, both P. aeruginosa fadDs functionally complemented the E. coli fadD(-)/fadR(-) mutant, allowing degradation of different chain-length FAs. Chromosomal mutagenesis, growth analysis, induction studies, and determination of kinetic parameters suggested that FadD1 has a substrate preference for long-chain FAs while FadD2 prefers shorter-chain FAs. When compared to the wild type strain, the fadD2 mutant exhibited decreased production of lipase, protease, rhamnolipid and phospholipase, and retardation of both swimming and swarming motilities. Interestingly, fadD1 mutant showed only increased swarming motility. Growth analysis of the fadD mutants showed noticeable deficiencies in utilizing FAs and phosphatidylcholine (major components of lung surfactant) as the sole carbon source. This defect translated into decreased in vivo fitness of P. aeruginosa in a BALB/c mouse lung infection model, supporting the role of lipids as a significant nutrient source for this bacterium in vivo.

Show MeSH
Related in: MedlinePlus