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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: 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.

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

Extracellular sialidase activity of wild-type C. perfringens, BT1 (nanI), BT2 (nanJ), and BT3 (nanInanJ).Cells were grown overnight in 5 ml of PY with (+) or without (-) 1 mg/ml sialic acid as indicated. Sialidase specific activity was defined as μmoles of 4-methylumbelliferyl-α-D-N-acetylneuraminic acid hydrolyzed per minute per milligram of protein. Values shown are the mean and SEM of triplicate biological samples. The P values, calculated using the student's two-tailed t-test, are shown in the figure.
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pone.0133217.g003: Extracellular sialidase activity of wild-type C. perfringens, BT1 (nanI), BT2 (nanJ), and BT3 (nanInanJ).Cells were grown overnight in 5 ml of PY with (+) or without (-) 1 mg/ml sialic acid as indicated. Sialidase specific activity was defined as μmoles of 4-methylumbelliferyl-α-D-N-acetylneuraminic acid hydrolyzed per minute per milligram of protein. Values shown are the mean and SEM of triplicate biological samples. The P values, calculated using the student's two-tailed t-test, are shown in the figure.

Mentions: To determine if production of the extracellular sialidase enzymes encoded by the nanI and nanJ genes in strain 13 was inducible by Neu5Ac, we used insertion mutagenesis (i.e., homologous recombination) of internal fragments of the nanI and nanJ genes in a suicide plasmid to mutate each gene separately and together. The sialidase activity of strain BT1 (nanI mutant) was 30% of the wild-type level in the presence of Neu5Ac (Fig 3), and 33% of the activity of the wild-type strain in the absence of Neu5Ac. In comparison to strain 13, strain BT2 (nanJ mutant) activity was 81% and 39% in the presence and the absence of Neu5Ac, respectively. Measurement of the sialidase activity of the double mutant BT3 indicated the strain lacked all sialidase activity in the presence or absence of sialic acid (Fig 3). Chiarezza et al. [12] showed that NanI was the major sialidase while NanJ was the minor sialidase in strain 13 in the absence of sialic acid. The levels of activity detected in the nanI and nanJ mutants grown in the absence of sialic acid in this study were actually similar (Fig 3). These differences may be due to the composition of the growth media used in each study, PY here and Todd-Hewitt broth in Chiarezza et al. [12]. In the current study the NanI-dependent sialidase activity (i.e., that seen with the nanJ mutant) was induced about 4-fold by the addition of Neu5Ac, whereas NanJ-dependent activity (i.e., that seen with the nanI mutant) was not statistically different in the presence or absence of Neu5Ac (Fig 3).


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)

Extracellular sialidase activity of wild-type C. perfringens, BT1 (nanI), BT2 (nanJ), and BT3 (nanInanJ).Cells were grown overnight in 5 ml of PY with (+) or without (-) 1 mg/ml sialic acid as indicated. Sialidase specific activity was defined as μmoles of 4-methylumbelliferyl-α-D-N-acetylneuraminic acid hydrolyzed per minute per milligram of protein. Values shown are the mean and SEM of triplicate biological samples. The P values, calculated using the student's two-tailed t-test, are shown in the figure.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133217.g003: Extracellular sialidase activity of wild-type C. perfringens, BT1 (nanI), BT2 (nanJ), and BT3 (nanInanJ).Cells were grown overnight in 5 ml of PY with (+) or without (-) 1 mg/ml sialic acid as indicated. Sialidase specific activity was defined as μmoles of 4-methylumbelliferyl-α-D-N-acetylneuraminic acid hydrolyzed per minute per milligram of protein. Values shown are the mean and SEM of triplicate biological samples. The P values, calculated using the student's two-tailed t-test, are shown in the figure.
Mentions: To determine if production of the extracellular sialidase enzymes encoded by the nanI and nanJ genes in strain 13 was inducible by Neu5Ac, we used insertion mutagenesis (i.e., homologous recombination) of internal fragments of the nanI and nanJ genes in a suicide plasmid to mutate each gene separately and together. The sialidase activity of strain BT1 (nanI mutant) was 30% of the wild-type level in the presence of Neu5Ac (Fig 3), and 33% of the activity of the wild-type strain in the absence of Neu5Ac. In comparison to strain 13, strain BT2 (nanJ mutant) activity was 81% and 39% in the presence and the absence of Neu5Ac, respectively. Measurement of the sialidase activity of the double mutant BT3 indicated the strain lacked all sialidase activity in the presence or absence of sialic acid (Fig 3). Chiarezza et al. [12] showed that NanI was the major sialidase while NanJ was the minor sialidase in strain 13 in the absence of sialic acid. The levels of activity detected in the nanI and nanJ mutants grown in the absence of sialic acid in this study were actually similar (Fig 3). These differences may be due to the composition of the growth media used in each study, PY here and Todd-Hewitt broth in Chiarezza et al. [12]. In the current study the NanI-dependent sialidase activity (i.e., that seen with the nanJ mutant) was induced about 4-fold by the addition of Neu5Ac, whereas NanJ-dependent activity (i.e., that seen with the nanI mutant) was not statistically different in the presence or absence of Neu5Ac (Fig 3).

Bottom Line: 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.

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