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Role of the cAMP-dependent carbon catabolite repression in capsular polysaccharide biosynthesis in Klebsiella pneumoniae.

Lin CT, Chen YC, Jinn TR, Wu CC, Hong YM, Wu WH - PLoS ONE (2013)

Bottom Line: These results were then confirmed by electrophoretic mobility shift assay.In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA).Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, China Medical University, Taichung, Taiwan. Republic of China. gingting@mail.cmu.edu.tw

ABSTRACT
K. pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. Although elevated blood glucose levels cause various immune problems, its effects on K. pneumoniae virulence are unknown. This study investigated the regulation of capsular polysaccharide (CPS) biosynthesis, a major determinant for K. pneumoniae virulence, in response to exogenous glucose. We found that K. pneumoniae produce more CPS in glucose-rich medium via reduction in cyclic AMP (cAMP) levels. Individual deletion of cyaA or crp, which respectively encode adenylate cyclase and cAMP receptor protein in K. pneumoniae, markedly increased CPS production, while deletion of cpdA, which encodes cAMP phosphodiesterase, decreased CPS production. These results indicate that K. pneumoniae CPS biosynthesis is controlled by the cAMP-dependent carbon catabolite repression (CCR). To investigate the underlying mechanism, quantitative real-time PCR and promoter-reporter assays were used to verify that the transcription of CPS biosynthesis genes, which are organized into 3 transcription units (orf1-2, orf3-15, and orf16-17), were activated by the deletion of crp. Sequence analysis revealed putative CRP binding sites located on P(orf3-15) and P(orf16-17), suggesting direct CRP-cAMP regulation on the promoters. These results were then confirmed by electrophoretic mobility shift assay. In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA). The deletion of rcsA in mutation of crp partially reduced CPS biosynthesis and the transcription of orf1-2 but not of orf3-15 or orf16-17. These results suggest that RcsA participates in the CRP-cAMP regulation of orf1-2 transcription and influences CPS biosynthesis. Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing. We here provide evidence that K. pneumoniae increases CPS biosynthesis for successful infection in response to exogenous glucose via cAMP-dependent CCR.

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Identification of the transcriptional start sites of 3 transcriptional units in the K2 cps gene cluster by 5′ RACE.The 5′ RACE experimental design for galF (A), wzi (B), and manC (C). Relative positions of the primers used and the expected size of the PCR product are indicated. The transcriptional start site is marked as +1 and underlined. The potential −10, −35, and ribosomal binding sites (RBS) are underlined. The grey box indicates the predicted RcsAB box. The dashed boxes indicate the predicted CRP binding sites. (D) The predicted CRP binding sites in Pwzi and PmanC are aligned against each other. #, the position is relative to the transcriptional start site.
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pone-0054430-g004: Identification of the transcriptional start sites of 3 transcriptional units in the K2 cps gene cluster by 5′ RACE.The 5′ RACE experimental design for galF (A), wzi (B), and manC (C). Relative positions of the primers used and the expected size of the PCR product are indicated. The transcriptional start site is marked as +1 and underlined. The potential −10, −35, and ribosomal binding sites (RBS) are underlined. The grey box indicates the predicted RcsAB box. The dashed boxes indicate the predicted CRP binding sites. (D) The predicted CRP binding sites in Pwzi and PmanC are aligned against each other. #, the position is relative to the transcriptional start site.

Mentions: Until now, the transcriptional start sites of the cps gene cluster had not been characterized. 5′ rapid amplification of cDNA ends (5′ RACE) was first performed to determine the transcriptional start sites of the 3 transcription units in the K2 cps gene cluster. A single DNA band was obtained for galF, wzi, and manC from the 5′ RACE analysis using either primer pair (data not shown). As shown in Fig 4A, B, and C, sequence analysis of a total of 10 clones each from galF, wzi, and manC revealed a transcriptional start site at the A nucleotide at position −61 relative to the translational start site of galF, at the G nucleotide at position −470 relative to the translational start site of wzi, and at the G nucleotide at position −56 relative to the translational start site of manC. The conserved −10 and −35 promoter sequence of σ70 could be readily identified and is shown in Fig 4.


Role of the cAMP-dependent carbon catabolite repression in capsular polysaccharide biosynthesis in Klebsiella pneumoniae.

Lin CT, Chen YC, Jinn TR, Wu CC, Hong YM, Wu WH - PLoS ONE (2013)

Identification of the transcriptional start sites of 3 transcriptional units in the K2 cps gene cluster by 5′ RACE.The 5′ RACE experimental design for galF (A), wzi (B), and manC (C). Relative positions of the primers used and the expected size of the PCR product are indicated. The transcriptional start site is marked as +1 and underlined. The potential −10, −35, and ribosomal binding sites (RBS) are underlined. The grey box indicates the predicted RcsAB box. The dashed boxes indicate the predicted CRP binding sites. (D) The predicted CRP binding sites in Pwzi and PmanC are aligned against each other. #, the position is relative to the transcriptional start site.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569464&req=5

pone-0054430-g004: Identification of the transcriptional start sites of 3 transcriptional units in the K2 cps gene cluster by 5′ RACE.The 5′ RACE experimental design for galF (A), wzi (B), and manC (C). Relative positions of the primers used and the expected size of the PCR product are indicated. The transcriptional start site is marked as +1 and underlined. The potential −10, −35, and ribosomal binding sites (RBS) are underlined. The grey box indicates the predicted RcsAB box. The dashed boxes indicate the predicted CRP binding sites. (D) The predicted CRP binding sites in Pwzi and PmanC are aligned against each other. #, the position is relative to the transcriptional start site.
Mentions: Until now, the transcriptional start sites of the cps gene cluster had not been characterized. 5′ rapid amplification of cDNA ends (5′ RACE) was first performed to determine the transcriptional start sites of the 3 transcription units in the K2 cps gene cluster. A single DNA band was obtained for galF, wzi, and manC from the 5′ RACE analysis using either primer pair (data not shown). As shown in Fig 4A, B, and C, sequence analysis of a total of 10 clones each from galF, wzi, and manC revealed a transcriptional start site at the A nucleotide at position −61 relative to the translational start site of galF, at the G nucleotide at position −470 relative to the translational start site of wzi, and at the G nucleotide at position −56 relative to the translational start site of manC. The conserved −10 and −35 promoter sequence of σ70 could be readily identified and is shown in Fig 4.

Bottom Line: These results were then confirmed by electrophoretic mobility shift assay.In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA).Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, China Medical University, Taichung, Taiwan. Republic of China. gingting@mail.cmu.edu.tw

ABSTRACT
K. pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. Although elevated blood glucose levels cause various immune problems, its effects on K. pneumoniae virulence are unknown. This study investigated the regulation of capsular polysaccharide (CPS) biosynthesis, a major determinant for K. pneumoniae virulence, in response to exogenous glucose. We found that K. pneumoniae produce more CPS in glucose-rich medium via reduction in cyclic AMP (cAMP) levels. Individual deletion of cyaA or crp, which respectively encode adenylate cyclase and cAMP receptor protein in K. pneumoniae, markedly increased CPS production, while deletion of cpdA, which encodes cAMP phosphodiesterase, decreased CPS production. These results indicate that K. pneumoniae CPS biosynthesis is controlled by the cAMP-dependent carbon catabolite repression (CCR). To investigate the underlying mechanism, quantitative real-time PCR and promoter-reporter assays were used to verify that the transcription of CPS biosynthesis genes, which are organized into 3 transcription units (orf1-2, orf3-15, and orf16-17), were activated by the deletion of crp. Sequence analysis revealed putative CRP binding sites located on P(orf3-15) and P(orf16-17), suggesting direct CRP-cAMP regulation on the promoters. These results were then confirmed by electrophoretic mobility shift assay. In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA). The deletion of rcsA in mutation of crp partially reduced CPS biosynthesis and the transcription of orf1-2 but not of orf3-15 or orf16-17. These results suggest that RcsA participates in the CRP-cAMP regulation of orf1-2 transcription and influences CPS biosynthesis. Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing. We here provide evidence that K. pneumoniae increases CPS biosynthesis for successful infection in response to exogenous glucose via cAMP-dependent CCR.

Show MeSH
Related in: MedlinePlus