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NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens.

Therit B, Cheung JK, Rood JI, Melville SB - PLoS ONE (2015)

Bottom Line: Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion.For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively.Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America.

ABSTRACT
Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell.

No MeSH data available.


Related in: MedlinePlus

The nanI promoter from strain 13(pSM230) shows sialic acid-inducible activity.β-glucuronidase activity was measured for each strain indicated. Strain AH1 is a β-glucuronidase mutant derived from strain 13 [31]. Values shown are the mean and SEM of triplicate biological samples. The P value, calculated using the student's two-tailed t-test, is shown in the figure.
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pone.0133217.g004: The nanI promoter from strain 13(pSM230) shows sialic acid-inducible activity.β-glucuronidase activity was measured for each strain indicated. Strain AH1 is a β-glucuronidase mutant derived from strain 13 [31]. Values shown are the mean and SEM of triplicate biological samples. The P value, calculated using the student's two-tailed t-test, is shown in the figure.

Mentions: The intergenic region upstream of the nanI gene (i.e., the nanI promoter region) was cloned upstream of a promoterless gusA gene from E. coli in the shuttle plasmid pSM218 [14] to give pSM230. Each plasmid was then used to transform C. perfringens strain AH1, a derivative of strain 13 in which a mutation was introduced in the major β-glucuronidase-encoding gene [31]. Strain AH1(pSM218) showed low levels of β-glucuronidase activity, while AHI(pSM230) exhibited significantly higher levels of activity (Fig 4). The addition of Neu5Ac to the growth medium stimulated activity from the nanI promoter by 35%, similar to what was observed with the nanE promoter under similar conditions [14].


NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens.

Therit B, Cheung JK, Rood JI, Melville SB - PLoS ONE (2015)

The nanI promoter from strain 13(pSM230) shows sialic acid-inducible activity.β-glucuronidase activity was measured for each strain indicated. Strain AH1 is a β-glucuronidase mutant derived from strain 13 [31]. Values shown are the mean and SEM of triplicate biological samples. The P value, calculated using the student's two-tailed t-test, is shown in the figure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4509764&req=5

pone.0133217.g004: The nanI promoter from strain 13(pSM230) shows sialic acid-inducible activity.β-glucuronidase activity was measured for each strain indicated. Strain AH1 is a β-glucuronidase mutant derived from strain 13 [31]. Values shown are the mean and SEM of triplicate biological samples. The P value, calculated using the student's two-tailed t-test, is shown in the figure.
Mentions: The intergenic region upstream of the nanI gene (i.e., the nanI promoter region) was cloned upstream of a promoterless gusA gene from E. coli in the shuttle plasmid pSM218 [14] to give pSM230. Each plasmid was then used to transform C. perfringens strain AH1, a derivative of strain 13 in which a mutation was introduced in the major β-glucuronidase-encoding gene [31]. Strain AH1(pSM218) showed low levels of β-glucuronidase activity, while AHI(pSM230) exhibited significantly higher levels of activity (Fig 4). The addition of Neu5Ac to the growth medium stimulated activity from the nanI promoter by 35%, similar to what was observed with the nanE promoter under similar conditions [14].

Bottom Line: Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion.For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively.Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America.

ABSTRACT
Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell.

No MeSH data available.


Related in: MedlinePlus