YdiV is an EAL-like protein that acts as a post-transcriptional, negative regulator of the flagellar master transcriptional activator complex, FlhD4C2, in Salmonella enterica to couple flagellar gene expression to nutrient availability. Mutants defective in ClpXP protease no longer exhibit YdiV-dependent inhibition of FlhD4C2-dependent transcription under moderate YdiV expression conditions. ClpXP protease degrades FlhD4C2, and this degradation is accelerated in the presence of YdiV. YdiV complexed with both free and DNA-bound FlhD4C2; and stripped FlhD4C2 from DNA. A L22H substitution in FlhD was isolated as insensitive to YdiV inhibition. The FlhD L22H substitution prevented the interaction of YdiV with free FlhD4C2 and the ability of YdiV to release FlhD4C2 bound to DNA. These results demonstrate that YdiV prevents FlhD4C2-dependent flagellar gene transcription and acts as a putative adaptor to target FlhD4C2 for ClpXP-dependent proteolysis. Our results suggest that YdiV is an EAL-like protein that has evolved from a dicyclic-GMP phosphodiesterase into a dual-function regulatory protein that connects flagellar gene expression to nutrient starvation.
A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the Red proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.
Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.
The yycF1(Ts) mutation in Staphylococcus aureus conferred hypersensitivity to macrolide-lincosamide-streptogramin B (MLS B ) antibiotics on strains either containing or lacking ermB. The overexpression of the S. aureus Ssa protein restored the yycF1 mutant to wild-type levels of susceptibility. Inactivation of ssa in an unmutagenized strain dramatically reduced ermB-based resistance. Conditional loss of function or expression of ssa in the yycF1 mutant is proposed to result in the observed hypersensitivity to MLS B antibiotics.One of several phenotypic consequences of the yycF1(Ts) mutation in Staphylococcus aureus was a hypersensitivity to macrolide-lincosamide-streptogramin B (MLS B ) antibiotics (9). This hypersensitivity was returned to wild-type levels when the mutant was cultured under anoxic conditions at neutral pH. Accordingly, when the ermB-containing transposon Tn917lac was transduced into the original yycF1(Ts) mutant (strain NT372), erythromycin-resistant (Em r ) transductants could be selected only anaerobically. An isogenic set of strains, SAM1010 [yycF1(Ts) Tn917lac::purA571] and SAM1011 (Tn917lac::purA571), was constructed by using this strategy (Table 1). Even though both strains were marked with ermB, the SAM1010 mutant expressed only an intermediate level of resistance to MLS B -class antibiotics compared to that expressed by SAM1011 (Table 2). In the absence of resistance genes, the NT372 temperature-sensitive mutant was fourfold more sensitive to erythromycin (ERM) than the unmutagenized parental strain (9). Because the yycF response regulator was reported to effect changes at the level of transcription (3, 4), it is possible that the conditional underexpression of one or more chromosomally carried genes in the yycF1(Ts) mutant of S. aureus could account for this observed hypersensitivity.The NT372 mutant grew very poorly on plating media like Trypticase soy agar (TSA) at 39°C and did not grow at higher temperatures. Thus, by selecting plasmid-based genomic clones that restored high-level Em r (10 g/ml) on TSA at this semipermissive growth temperature, it was thought that one or more related genes affecting this partial MLS B resistance phenotype might be revealed. With a plasmid library (9) constructed from the unmutagenized parental strain (SAM23), a total of 21 Em r transductants of NT372 were isolated. Under identical selection conditions, over 50,000 Em r transductants for the isogenic wild type (SAM23) were obtained. Of the 21 Em r transductants of NT372, 5 could be reselected at 43°C and contained plasmids bearing the original yycFG locus (complementing clones). The remaining 16 clones, represented by strain SAM1287 (Table 1), bore equivalently sized genomic inserts (5.2 kb) that conferred high-level ERM resistance (MIC Ͼ 512 g/ml) but did not fully restore the mutant's ability to grow at the higher temperature (43°C).The inserts of all 16 clones were sequenced and found to be identical. Subcloning and recomplementation of the NT372 mutant (Fig. 1a) correlated the selection of high-l...
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