The Escherichia coli O157 Flagellar Regulatory Gene flhC and Not the Flagellin Gene fliC Impacts Colonization of Cattle

ABSTRACT A virulent European Escherichia coli O157:H − isolate is nonmotile due to a 12-bp deletion in the flagellar regulatory gene flhC. To investigate the contribution of flhC in the relationship between E. coli O157:H7 and cattle, we constructed a similar flhC regulatory mutant in the well-characterized strain ATCC 43894. There was no difference in the growth rate between the wild type and this regulatory mutant, but phenotypic arrays showed substrate utilization differences. Survival in the bovine gastrointestinal tract and colonization of the rectoanal junction mucosa were assessed. Mixtures of both strains were given orally or rectally to steers or administered into the rumen of cattle dually cannulated at the rumen and duodenum. One day post-oral dose, most rectal/fecal isolates (74%) were the regulatory mutant, but by 3 days post-oral dose and throughout the 42-day experiment, ≥80% of the isolates were wild type. Among steers given a rectal application of both strains, wild-type isolates were the majority of isolates recovered on all days. The regulatory mutant survived better than the wild type in both the rumen and duodenum. To test the role of motility, a filament mutant (ΔfliC) was constructed and similar cattle experiments were performed. On all days post-oral dose, the majority of isolates (64% to 98%) were the filament mutant. In contrast, both strains were recovered equally post-rectal application. Thus, the regulatory mutant survived passage through the bovine gastrointestinal tract better than the wild type but failed to efficiently colonize cattle, and the requirement of flhC for colonization was not dependent on a functional flagellum.

A Regulatory Mutant of Hansenula polymorpha Exhibiting Methanol Utilization Metabolism and Peroxisome Proliferation in Glucose

ABSTRACT Mutant LGM-128 of Hansenula polymorpha harbors the recessive mutation glr2-1 which confers a complex pleiotropic phenotype, the major feature of which is the metabolically unnecessary induction of methanol utilization metabolism (C1 metabolism) during growth on glucose, whether or not methanol is in the medium. Therefore, in this mutant, peroxisomes are formed and proliferate upon cultivation in glucose-containing media. In these media, LGM-128 shows induction levels of C1metabolism that are similar to those observed in methanol-containing media. This indicates that GLR2 controls the repression-derepression process stimulated by glucose and that the induction process triggered by methanol plays only a minor role in activating C1 metabolism. Cultivating LGM-128 in methanol and then transferring it to glucose media revealed that active degradative processes occur, leading to the disappearance of C1 metabolism. This observation suggests that, although stimulated by glucose, the two processes are controlled by elements which are, at least in part, distinct. Finally, glr2-1 does not affect ethanol repression, suggesting that in H. polymorpha the two repressing circuits are separated.

Expression of Invasin and Motility Are Coordinately Regulated in Yersinia enterocolitica

ABSTRACT The Yersinia enterocolitica inv gene encodes the primary invasion factor invasin, which has been previously shown to be critical in the initial stages of infection. The expression ofinv is influenced by growth phase and temperature and is maximal during late exponential-early stationary phase at 23°C. In addition, motility of Y. enterocolitica is regulated by temperature. Y. enterocolitica cells are motile when grown at lower temperatures (30°C or below), while bacteria grown at 37°C are nonmotile. This study was initiated to determine the molecular basis for the temperature regulation of inv expression. Two mutants were isolated that both showed a significant decrease in invasin expression but are hypermotile when grown at 23°C. The first mutant (JB1A8v) was a result of a random mTn5Km insertion into the uvrC gene. The uvrC mutant JB1A8v demonstrated a significant decrease in inv and an increase in fleB (encodes flagellin) expression. These results suggest that expression of inv and flagellin genes is coordinated at the level of transcription. The second regulatory mutant, JB16v, was a result of a targeted insertion into a locus similar to sspA which in E. coli encodes a stationary-phase regulator. The E. coli sspA gene was cloned and assayed for complementation in both of the regulatory mutants. It was determined that E. coli sspA restored invasin expression in both the uvrC mutant and thesspA mutant. In addition, the complementing clone decreased flagellin levels in these mutants.