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K H Pritchard

Publications and source records attributed to K H Pritchard.

4 recordsLinked to original sources

Differential expression of genes in the vir regulon of Streptococcus pyogenes is controlled by transcription termination.

Streptococcal C5a peptidase (SCP), encoded by scpA in Streptococcus pyogenes, is a surface molecule which is able to cleave and inactivate the chemotactic factor C5a. The scpA gene is part of the vir regulon and subject to positive regulation by the Mga protein. It is down-regulated compared to another Mga-activated gene, emm. A chloramphenicol acetyltransferase (CAT) reporter gene was used to measure scpA promoter activity. Previous work had shown that when a large portion of the scpA promoter region was deleted, expression of CAT increased relative to the wild-type. This deleted region was found to contain an inverted repeat. In this study we show that the inverted repeat in the leader mRNA is the site of transcription termination, which down-regulates expression of scpA. This is a novel mechanism for regulation of gene expression in S. pyogenes. A specific deletion of the inverted repeat in the scpA promoter-CAT reporter construct was made using inverse PCR. Expression was measured from single-copy chromosomal integrants. When the inverted repeat was deleted, expression increased. Furthermore, Northern hybridization confirmed the existence of a truncated transcript, consistent with a transcription termination mechanism.

Adhesins, Bacterial↗

Direct transformation of Neisseria gonorrhoeae by gel-isolated DNA.

The naturally competent organism, Neisseria gonorrhoeae, can efficiently transform a marker carried on DNA purified in low-melting-temperature agarose without prior purification or dilution. Neither the agarose or buffer components inhibit transformation frequencies, but exposure to UV irradiation completely abrogates transformation.

Electrophoresis, Agar Gel↗

A conserved DNA sequence is required for efficient gonococcal pilin antigenic variation.

Antigenic variation of the Neisseria gonorrhoeae pilus occurs when a variant pilin sequence from a silent locus recombines into the expression locus by predominantly unidirectional, homologous recombination. At the 3' end of all pilin loci lies a conserved DNA sequence, called the Sma/Cla repeat, which has sequence similarity to several recombinase-binding sites, and therefore may be involved in pilin recombination. We have developed a novel reverse transcriptase/polymerase chain reaction (RT-PCR) assay for direct monitoring of pilin recombination, and both RT-PCR and phase variation were used to examine pilin recombination in a gonococcal strain that had had the pilE Sma/Cla repeat removed. Results from these experiments showed a decrease in pilin recombination when the Sma/Cla sequence was deleted from the expression locus, showing that a specialized site (Sma/Cla) is involved in efficient pilin recombination.

Antigenic Variation↗