Neisseria gonorrhoeae, 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. Changes occur during DNA replication and are commonly referred to as """"""""illegitimate recombination"""""""" since the mechanism for change is not dependent on the cells homologous recombination pathways. 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. Each of the eleven opa loci phase varies independently resulting in expression of none to several proteins simultaneously by a single bacterium. 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. Neisserial homologues of the genes identified in E. coli as influencing illegitimate recombination have been identified and cloned from Ng MS11mk. Each of the genes has been mutated in E. coli and will be transferred back to the Ng MS11mk reporter strain using the shuttle mutagenesis system developed by Seifert et al. (1990). 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 (as shown for other reporter systems tested in E. coli mutants e.g. pilC::phoA). The possibility exists that deregulation of the variation rates of certain genes may be detrimental to the host strain. 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. 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.

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