CcpA mediates proline auxotrophy and is required for Staphylococcus aureus pathogenesis.
ABSTRACT Human clinical isolates of Staphylococcus aureus, for example, strains Newman and N315, cannot grow in the absence of proline, albeit their sequenced genomes harbor genes for two redundant proline synthesis pathways. We show here that under selective pressure, S. aureus Newman generates proline-prototrophic variants at a frequency of 3 x 10(-6), introducing frameshift and missense mutations in ccpA or IS1811 insertions in ptsH, two regulatory genes that carry out carbon catabolite repression (CCR) in staphylococci and other Gram-positive bacteria. S. aureus Newman variants with mutations in rocF (arginase), rocD (ornithine aminotransferase), and proC (Delta(1)-pyrroline 5-carboxylate [P5C] reductase) are unable to generate proline-prototrophic variants, whereas a variant with a mutation in ocd (ornithine cyclodeaminase) is unaffected. Transposon insertion in ccpA also restored proline prototrophy. CcpA was shown to repress transcription of rocF and rocD, encoding the first two enzymes, but not of proC, encoding the third and final enzyme in the P5C reductase pathway. CcpA bound to the upstream regions of rocF and rocD but not to that of proC. CcpA's binding to the upstream regions was greatly enhanced by phosphorylated HPr. The CCR-mediated proline auxotrophy was lifted when nonpreferred carbohydrates were used as the sole carbon source. The ccpA mutant displayed reduced staphylococcal load and replication in a murine model of staphylococcal abscess formation, indicating that carbon catabolite repression presents an important pathogenesis strategy of S. aureus infections.
Article: Isolation and characterization of IS1181, an insertion sequence from Staphylococcus aureus.[show abstract] [hide abstract]
ABSTRACT: The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.Plasmid 06/1994; 31(3):251-64. · 1.52 Impact Factor
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ABSTRACT: PCR amplification of the 531-bp fragment of the Mycobacterium leprae pra gene in fresh biopsy and slit skin smear samples was evaluated for its usefulness in the detection of leprosy bacilli in patients in Thailand. In multibacillary patients, 87.1% (27 of 31) of biopsy specimens and 41.9% (13 of 31) of slit skin smear specimens were positive by PCR, whereas in paucibacillary patients, 36.4% (8 of 22) of biopsy specimens and 18.2% (4 of 22) of slit skin smear specimens yielded detectable PCR amplification. Compared with other diagnostic procedures, PCR showed a clear advantage over both microscopic examination of slit skin smears and serologic detection of anti-phenolic glycolipid 1 antibody, especially in paucibacillary patients when bacterial indexes were 0 and seropositivity was only 6.25%. PCR was also evaluated for its potential to help monitor bacterial clearance in some of these patients during chemotherapeutic treatment. The PCR results on slit skin smear samples at 1, 3, and 6 months of chemotherapy showed that the number of PCR-positive cases of both multibacillary and paucibacillary types decreased sequentially. The results of this study are encouraging. However, investigation of a larger number of clinical specimens with an improvement in PCR methods, especially on slit skin smears, needs to be done before PCR can be established as a diagnostic procedure for leprosy patients and subclinical cases or as a tool for drug assessment.Journal of Clinical Microbiology 02/1995; 33(1):45-9. · 4.15 Impact Factor