Expression of the Fis protein is sustained in late-exponential- and stationary-phase cultures of Salmonella enterica serovar Typhimurium grown in the absence of aeration

The classic expression pattern of the Fis global regulatory protein during batch culture consists of a high peak in the early logarithmic phase of growth, followed by a sharp decrease through mid-exponential growth phase until Fis is almost undetectable at the end of the exponential phase. We discovered that this pattern is contingent on the growth regime. In Salmonella enterica serovar Typhimurium cultures grown in non-aerated SPI1-inducing conditions, Fis can be detected readily in stationary phase. On the other hand, cultures grown with standard aeration showed the classic Fis expression pattern. Sustained Fis expression in non-aerated cultures was also detected in some Escherichia coli strains, but not in others. This novel pattern of Fis expression was independent of sequence differences in the fis promoter regions of Salmonella and E. coli. Instead, a clear negative correlation between the expression of the Fis protein and of the stress-and-stationary-phase sigma factor RpoS was observed in a variety of strains. An rpoS mutant displayed elevated levels of Fis and had a higher frequency of epithelial cell invasion under these growth conditions. We discuss a model whereby Fis and RpoS levels vary in response to environmental signals allowing the expression and repression of SPI1 invasion genes.     

Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number.

Microtubule organization in the cytoplasm is in part a function of the number and length of the assembled polymers. The intracellular concentration of tubulin could specify those parameters. Saccharomyces cerevisiae strains constructed with moderately decreased or increased copy numbers of tubulin genes provide an opportunity to study the cellular response to a steady-state change in tubulin concentration. We found no evidence of a mechanism for adjusting tubulin concentrations upward from a deficit, nor did we find a need for such a mechanism: cells with no more than 50% of the wild-type tubulin level were normal with respect to a series of microtubule-dependent properties. Strains with increased copies of both alpha- and beta-tubulin genes, or of alpha-tubulin genes alone, apparently did down regulate their tubulin levels. As a result, they contained greater than normal concentrations of tubulin but much less than predicted from the increase in gene number. Some of this down regulation occurred at the level of protein. These strains were also phenotypically normal. Cells could contain excess alpha-tubulin protein without detectable consequences, but perturbations resulting in excess beta-tubulin genes may have affected microtubule-dependent functions. All of the observed regulation of levels of tubulin can be explained as a response to toxicity associated with excess tubulin proteins, especially if beta-tubulin is much more toxic than alpha-tubulin.     

Leishmania chagasi: the alpha-tubulin intercoding region results in constant levels of mRNA abundance despite protein synthesis inhibition and growth state

The intercoding regions of many Leishmania sp. genes have been implicated in the regulation of mRNA processing, stability, and translation. Herein we show that the intercoding region of the Leishmania chagasi alpha-tubulin gene (alpha-TUB) confers stable beta-galactosidase (beta-GAL) reporter mRNA levels during promastigote growth and development in vitro and during protein synthesis inhibition. The abundance of both endogenous alpha-TUB mRNA and beta-GAL mRNA from a beta-GAL coding region situated upstream of the alpha-TUB intercoding region did not change significantly as promastigotes grew from logarithmic to stationary phase in vitro and the half-life of the beta-GAL mRNA remained constant. The abundance of both the endogenous alpha-TUB and the beta-GAL mRNA increased by less than 2-fold after protein synthesis inhibition corresponding to a moderate increase in mRNA half-life. These data suggest that the alpha-TUB intercoding region is an excellent control for the study of the regulation of other differentially expressed genes.     

A novel step in beta-tubulin folding is important for heterodimer formation in Saccharomyces cerevisiae

Undimerized beta-tubulin is toxic in the yeast S. cerevisiae. It can arise if levels of beta-tubulin and alpha-tubulin are unbalanced or if the tubulin heterodimer dissociates. We are using the toxicity of beta-tubulin to understand early steps in microtubule morphogenesis. We find that deletion of PLP1 suppresses toxic beta-tubulin formed by disparate levels of alpha- and beta-tubulin. That suppression occurs either when alpha-tubulin is modestly underexpressed relative to beta-tubulin or when beta-tubulin is inducibly and strongly overexpressed. Plp1p does not affect tubulin expression. Instead, a significant proportion of the undimerized beta-tubulin in plp1Delta cells is less toxic than that in wild-type cells. It is also less able to combine with alpha-tubulin to form a heterodimer. As a result, plp1Delta cells have lower levels of heterodimer. Importantly, plp1Delta cells that also lack Pac10, a component of the GimC/PFD complex, are even less affected by free beta-tubulin. Our results suggest that Plp1p defines a novel early step in beta-tubulin folding.     

Induction of Hsp104 synthesis in Saccharomyces cerevisiae in the stationary growth phase is inhibited by the petite mutation

The elevation of Hsp104 (heat shock protein) content under heat stress plays a key role in the development of thermotolerance in yeast (Saccharomyces cerevisiae) cells. Hsp104 synthesis is increased under heat stress and in the stationary growth phase. The loss of mitochondrial DNA (petite mutation) was shown to inhibit the induction of Hsp104 synthesis under heat stress (39°C) and during the transition to the stationary growth phase. Also, the petite mutation suppressed the increase in activity of antioxidant enzymes in the stationary phase, which accompanied by decrease in thermotolerance. At the same time, mutation inhibited production of reactive oxygen species and prevented cell death under heat shock in the logarithmic growth phase. The results of this study suggest that disruption of the mitochondrial functional state suppresses the expression of yeast nuclear genes upon upon entry into the stationary growth phase.