Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea, exhibits high frequency phase and antigenic variation during the course of disease. The predominant genetic mechanism responsible for variable expression of surface constituents (i.e. contingency genes) involves the deletion or insertion of tandem DNA repeats. The opa gene family of N. gonorrhoeae is the best known example of a structurally related group of contingency genes. Neisserial opa genes encode a family of outer-membrane proteins that show phase variable expression in the laboratory and in vivo (as shown in natural and male-volunteer infection studies). Phase variation results from changes in the number of pentameric ([CTTCT]) repeat elements within each opa gene in the region encoding the processed signal-peptide. The number of repeats within a given gene serves to either align (in frame) or misalign (out- of-frame) the start codon with the sequence encoding the mature Opa protein. Our goal is to understand the mechanisms for illegitimate recombination by determining the genetic and environmental influences on phase variation. Reporter systems have been used to measure phase variation frequencies.A. Genetic Influences on Phase Variation.Previous studies have shown that the DNA repair pathways of the bacterial cell are important in the genetic switching that occurs in hypermutable genes that utilize tandem DNA repeats. We have mutated genes in N. gonorrhoeae involved in the major DNA repair pathways. The genes identified and mutated at this time are involved in Mismatch Repair (mutS/hexA, mutL/hexB), Transcription Coupled Repair (tcrF), Nucleotide Excision Repair (uvrA, uvrB and uvrC) and Oxidative Damage Control and Repair (fpg, oxyR and katF). Introduction of these mutations into Ng should increase the phase variation frequency of the opa gene reporter but, at the same time, increase the phase change rates of all of the genes that utilize illegitimate recombination, resulting in strains that express all of the variable components simultaneously. These strains will help us understand the role of variation in bacterial pathogenesis and may also be useful in the generation of attenuated strains with broad immunological reactivity.B. Environmental Influences on Phase Variation.The Ng opa::phoA reporter strain was used to measure phase variation frequencies under a number of environmental conditions. We have expanded our repertoire of reporter costructions by making opa::bla reporter strains, allowing us to directly select for specific gene configurations. Frequencies were measured during the course of the growth phase by simply comparing the number of phase variants (either ?on to off? or ?off to on?) present in the population. Environmental factors which increased the frequency of phase variation included i) growth phase and starvation, ii) eukaryotic cell contact, iii) DNA transformation and iv) DNA damage. - Neisseria gonorrhoeae, gonorrhea, Sexually Transmitted Diseases, Phase Variation, Antigenic Variation, Opa, Illegitimate Recombination

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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