Complementation of Rickettsia rickettsii RelA/SpoT Restores a Nonlytic Plaque Phenotype

Abstract

Spotted fever group rickettsiae are known to produce distinct plaque phenotypes. Strains that cause lytic infections in cell culture form clear plaques, while nonlytic strains form opaque plaques in which the cells remain intact. Clear plaques have historically been associated with more-virulent species or strains of spotted fever group rickettsiae. We have selected spontaneous mutant pairs from two independent strains of Rickettsia rickettsii, the virulent R strain and the avirulent Iowa strain. A nonlytic variant of R. rickettsii R, which typically produces clear plaques, was isolated and stably maintained. A lytic variant of the Iowa strain, which characteristically produces opaque plaques, was also selected and maintained. Genomic resequencing of the variants identified only a single gene disrupted in each strain. In both cases, the mutation was in a gene annotated as relA/spoT-like. In the Iowa strain, a single mutation introduced a premature stop codon upstream from region encoding the predicted active site of RelA/SpoT and caused the transition to a lytic plaque phenotype. In R. rickettsii R, the nonlytic plaque phenotype resulted from a single-nucleotide substitution that shifted a tyrosine residue to histidine near the active site of the enzyme. The intact relA/spoT gene thus occurred in variants with the nonlytic plaque phenotype. Complementation of the truncated relA/spoT gene in the Iowa lytic plaque variant restored the nonlytic phenotype. The relA/spoT mutations did not affect the virulence of either strain in a Guinea pig model of infection; R strain lytic and nonlytic variants both induced fever equally, and the mutation in Iowa to a lytic phenotype did not cause them to become virulent.

Bacteriophage Ecology in a Commercial Cucumber Fermentation

“…During infection, a portion of the phages may enter the lysogenic cycle instead of the lytic cycle, resulting in turbid plaques. Clark et al (6) found that rickettsial phages that cause lytic infections in cell culture form clear plaques, while nonlytic strains form opaque plaques in which the cells remain intact. Plaque size may correlate with virulence: the plaques produced by foot-and-mouth disease viruses are generally larger than the plaques from attenuated strains and those causing milder clinical illness (8).…”

Transformation Frequency of a mariner-Based Transposon in Rickettsia rickettsii

“…required for their manipulation. The cumulative results of analyzing insertion sites of pMW1650 indicate that transposition is random in the genomes of R. rickettsii (3,9) and R. prowazekii ( Fig. 2; see also Tables S1 and S2 in the supplemental material).…”

“…The development of mariner-based transposon systems for rickettsiae (10) has offered unprecedented opportunities in the field. Although the obligate intracellular nature of rickettsiae remains a limiting factor in the study of rickettsial pathogenesis, the application of molecular genetics has led to a number of recent advances (3,5,9). The development of transformation systems for rickettsiae was accomplished in R. prowazekii but necessitated extensive cell culture passage and selection for clonal isolation by limiting dilution (10,16,17).…”

“…Diagrams of R. rickettsii and R. prowazekii chromosomes depicting the insertion sites for each of the mutants generated by transposon mutagenesis in several independent experiments. The R. rickettsii map was derived from three independent experiments using either the R or Iowa strain (3,9). The map was constructed using R. rickettsii Iowa coordinates, and insertion sites determined in experiments 1 (9) (arrowheads labeled "1"), 2 (3) (arrowheads labeled "2"), and 3 (arrowheads labeled "3") are indicated.…”

GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8 + T Cell Immunology in R. typhi Infection

“…Transposon mutagenesis has also been successfully applied to R. rickettsii (6, 7) and Rickettsia monacensis transformed to express GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light) and a chloramphenicol resistance marker (8). Furthermore, targeted gene knockout by homologous recombination has been achieved in R.…”