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The analysis of para-cresol production and tolerance in Clostridium difficile 027 and 012 strains.

Dawson LF, Donahue EH, Cartman ST, Barton RH, Bundy J, McNerney R, Minton NP, Wren BW - BMC Microbiol. (2011)

Bottom Line: It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol.We show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012).The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious & Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

ABSTRACT

Background: Clostridium difficile is the major cause of antibiotic associated diarrhoea and in recent years its increased prevalence has been linked to the emergence of hypervirulent clones such as the PCR-ribotype 027. Characteristically, C. difficile infection (CDI) occurs after treatment with broad-spectrum antibiotics, which disrupt the normal gut microflora and allow C. difficile to flourish. One of the relatively unique features of C. difficile is its ability to ferment tyrosine to para-cresol via the intermediate para-hydroxyphenylacetate (p-HPA). P-cresol is a phenolic compound with bacteriostatic properties which C. difficile can tolerate and may provide the organism with a competitive advantage over other gut microflora, enabling it to proliferate and cause CDI. It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol.

Results: We show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012). Tyrosine conversion to p-HPA was only observed under certain conditions. We constructed gene inactivation mutants in the hpdBCA operon in strains R20291 and 630Δerm which curtails their ability to produce p-cresol, confirming the role of these genes in p-cresol production. The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production.

Conclusions: C. difficile converts tyrosine to p-cresol, utilising the hpdBCA operon in C. difficile strains 630 and R20291. The hypervirulent strain R20291 exhibits increased production of and tolerance to p-cresol, which may be a contributory factor to the virulence of this strain and other hypervirulent PCR-ribotype 027 strains.

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The hpdBCA operon and verification of mutant construction. The hpdBCA operon with insertion sites for the targeted ClosTron mutagenesis, the number refers to the insertion site (bp) and the s/a refers to sense/antisense orientation of the ClosTron insert. B) PCR screen of the mutants (M = mutant; W = wild type; P = plasmid and "-" negative control). Three PCR screens were performed, gene specific forward and reverse primers, intron specific with gene specific primers, and RAM specific primers (Heap et al., 2007). C) Southern blot using a probe specific to the inserted intron. HindIII digests were performed on DNA from M = mutant; W = wild type; P = plasmid. The strains and primer sets are indicated on each figure and in tables 1 and 2. The marker sizes are indicated on the figure and the expected band sizes are as follows for 630Δerm: hpdB 2.8Kb; hpdC 3.4 kb and for R20291: hpdA - 6.3 kb; hpdC - 3.4 kb.
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Figure 3: The hpdBCA operon and verification of mutant construction. The hpdBCA operon with insertion sites for the targeted ClosTron mutagenesis, the number refers to the insertion site (bp) and the s/a refers to sense/antisense orientation of the ClosTron insert. B) PCR screen of the mutants (M = mutant; W = wild type; P = plasmid and "-" negative control). Three PCR screens were performed, gene specific forward and reverse primers, intron specific with gene specific primers, and RAM specific primers (Heap et al., 2007). C) Southern blot using a probe specific to the inserted intron. HindIII digests were performed on DNA from M = mutant; W = wild type; P = plasmid. The strains and primer sets are indicated on each figure and in tables 1 and 2. The marker sizes are indicated on the figure and the expected band sizes are as follows for 630Δerm: hpdB 2.8Kb; hpdC 3.4 kb and for R20291: hpdA - 6.3 kb; hpdC - 3.4 kb.

Mentions: Three co-located genes (hpdB, hpdC and hpdA) are thought to encode the decarboxylase that converts p-HPA to p-cresol in strains 630Δerm and R20291. Gene inactivation mutants were constructed using the ClosTron method [17] in strains 630Δerm (mutants 630ΔermΔhpdB and 630ΔermΔhpdC) and strain R20291 (mutants R20291ΔhpdA and R20291ΔhpdC). The group II intron from the ClosTron system was retargeted using the Sigma TargeTron algorithm to insert into hpdA, hpdB and hpdC in the sense orientation for hpdA and hpdC at position 254 bp and 174 bp, respectively (from the start of the ORF), and in the antisense orientation for hpdB at 748 bp (Figure 3A). Verification of successful mutant construction was performed using three independent PCR screens (Figure 3B). The RAM specific PCR confirmed the loss of the group I intron interrupting the ermB RAM, indicating chromosomal integration of the intron. The gene specific and the intron specific primer revealed insertion of the RAM into the target gene, and the gene specific primers flanking the insertion site revealed an increase in 1.9 kb in size for the mutants compared to the wild-type (Figure 3B). The insertion site was verified by sequencing and by Southern blot analysis of the mutants using the intron specific probe which confirmed insertion of a single site-specific group II intron for all the hpdBCA operon mutants tested (Figure 3C).


The analysis of para-cresol production and tolerance in Clostridium difficile 027 and 012 strains.

Dawson LF, Donahue EH, Cartman ST, Barton RH, Bundy J, McNerney R, Minton NP, Wren BW - BMC Microbiol. (2011)

The hpdBCA operon and verification of mutant construction. The hpdBCA operon with insertion sites for the targeted ClosTron mutagenesis, the number refers to the insertion site (bp) and the s/a refers to sense/antisense orientation of the ClosTron insert. B) PCR screen of the mutants (M = mutant; W = wild type; P = plasmid and "-" negative control). Three PCR screens were performed, gene specific forward and reverse primers, intron specific with gene specific primers, and RAM specific primers (Heap et al., 2007). C) Southern blot using a probe specific to the inserted intron. HindIII digests were performed on DNA from M = mutant; W = wild type; P = plasmid. The strains and primer sets are indicated on each figure and in tables 1 and 2. The marker sizes are indicated on the figure and the expected band sizes are as follows for 630Δerm: hpdB 2.8Kb; hpdC 3.4 kb and for R20291: hpdA - 6.3 kb; hpdC - 3.4 kb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102038&req=5

Figure 3: The hpdBCA operon and verification of mutant construction. The hpdBCA operon with insertion sites for the targeted ClosTron mutagenesis, the number refers to the insertion site (bp) and the s/a refers to sense/antisense orientation of the ClosTron insert. B) PCR screen of the mutants (M = mutant; W = wild type; P = plasmid and "-" negative control). Three PCR screens were performed, gene specific forward and reverse primers, intron specific with gene specific primers, and RAM specific primers (Heap et al., 2007). C) Southern blot using a probe specific to the inserted intron. HindIII digests were performed on DNA from M = mutant; W = wild type; P = plasmid. The strains and primer sets are indicated on each figure and in tables 1 and 2. The marker sizes are indicated on the figure and the expected band sizes are as follows for 630Δerm: hpdB 2.8Kb; hpdC 3.4 kb and for R20291: hpdA - 6.3 kb; hpdC - 3.4 kb.
Mentions: Three co-located genes (hpdB, hpdC and hpdA) are thought to encode the decarboxylase that converts p-HPA to p-cresol in strains 630Δerm and R20291. Gene inactivation mutants were constructed using the ClosTron method [17] in strains 630Δerm (mutants 630ΔermΔhpdB and 630ΔermΔhpdC) and strain R20291 (mutants R20291ΔhpdA and R20291ΔhpdC). The group II intron from the ClosTron system was retargeted using the Sigma TargeTron algorithm to insert into hpdA, hpdB and hpdC in the sense orientation for hpdA and hpdC at position 254 bp and 174 bp, respectively (from the start of the ORF), and in the antisense orientation for hpdB at 748 bp (Figure 3A). Verification of successful mutant construction was performed using three independent PCR screens (Figure 3B). The RAM specific PCR confirmed the loss of the group I intron interrupting the ermB RAM, indicating chromosomal integration of the intron. The gene specific and the intron specific primer revealed insertion of the RAM into the target gene, and the gene specific primers flanking the insertion site revealed an increase in 1.9 kb in size for the mutants compared to the wild-type (Figure 3B). The insertion site was verified by sequencing and by Southern blot analysis of the mutants using the intron specific probe which confirmed insertion of a single site-specific group II intron for all the hpdBCA operon mutants tested (Figure 3C).

Bottom Line: It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol.We show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012).The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious & Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

ABSTRACT

Background: Clostridium difficile is the major cause of antibiotic associated diarrhoea and in recent years its increased prevalence has been linked to the emergence of hypervirulent clones such as the PCR-ribotype 027. Characteristically, C. difficile infection (CDI) occurs after treatment with broad-spectrum antibiotics, which disrupt the normal gut microflora and allow C. difficile to flourish. One of the relatively unique features of C. difficile is its ability to ferment tyrosine to para-cresol via the intermediate para-hydroxyphenylacetate (p-HPA). P-cresol is a phenolic compound with bacteriostatic properties which C. difficile can tolerate and may provide the organism with a competitive advantage over other gut microflora, enabling it to proliferate and cause CDI. It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol.

Results: We show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012). Tyrosine conversion to p-HPA was only observed under certain conditions. We constructed gene inactivation mutants in the hpdBCA operon in strains R20291 and 630Δerm which curtails their ability to produce p-cresol, confirming the role of these genes in p-cresol production. The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production.

Conclusions: C. difficile converts tyrosine to p-cresol, utilising the hpdBCA operon in C. difficile strains 630 and R20291. The hypervirulent strain R20291 exhibits increased production of and tolerance to p-cresol, which may be a contributory factor to the virulence of this strain and other hypervirulent PCR-ribotype 027 strains.

Show MeSH
Related in: MedlinePlus