Low-temperature adaptation and cryoprotection were studied in the lactic acid bacterium Lactococcus lactis MG1363. An approximately 100-fold increased survival after freezing was observed when cells were shocked to 10 °C for 4 h compared to mid-exponential-phase cells grown at 30 °C, indicating an active protection against freezing. Using two-dimensional gel electrophoresis a group of 7 kDa cold-induced proteins (CSPs) was identified that corresponds to a previously described family of csp genes of L. lactis MG1363 (Wouters et al., 1998 R32 , Microbiology 144, 2885–2893). The 7 kDa CSPs appeared to be the most strongly induced proteins upon cold shock to 10 °C. Northern blotting and two-dimensional gel electrophoresis showed that the csp genes were maximally expressed at 10 °C, while induction was lower at 20 and 4 °C. However, pre-incubation at 20 and 4 °C, as well as stationary-phase conditions, also induced cryoprotection (approx. 30-, 130- and 20-fold, respectively, compared to 30 °C mid-exponential phase). For all treatments leading to an increased freeze survival (exposure to 4, 10 and 20 °C and stationary-phase conditions), increased levels of three proteins (26, 43 and 45 kDa) were observed for which a role in cryoprotection might be suggested. Increased freeze survival coincides with increased CSP expression, except for stationary-phase conditions. However, the level of observed freeze protection does not directly correlate with the csp gene expression levels. In addition, for the first time specific overproduction of a CSP in relation to freeze survival was studied. This revealed that L. lactis cells overproducing CspD at 30 °C show a 2–10-fold increased survival after freezing compared to control cells. This indicates that the 7 kDa cold-shock protein CspD may enhance the survival capacity after freezing but that other factors supply additional cryoprotection.
Crh of Bacillus subtilis exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS). Crh can be phosphorylated by ATP at the regulatory Ser-46 and similar to P-Ser-HPr, P-Ser-Crh plays a role in carbon-catabolite repression. The sequence around the phosphorylatable Ser-46 in Crh exhibits strong similarity to the corresponding sequence of HPr of Gram-positive and a few Gram-negative bacteria. In contrast, the catalytic His-15, the site of PEP-dependent phosphorylation in HPr, is replaced with a glutamine in Crh. When Gln-15 was exchanged for a histidyl residue, in vitro PEP-dependent enzyme I-catalysed phosphorylation of the mutant Crh was observed. However, expression of the crhQ15H mutant allele did not restore growth of a ptsH deletion strain on the PTS sugars glucose, fructose or mannitol or on the non-PTS sugar glycerol. In contrast, Q15H mutant Crh could phosphorylate the transcriptional activator LevR as well as LevD, the enzyme IIA of the fructose-specific lev-PTS, which together with enzyme I, HPr and LevE forms the phosphorylation cascade regulating induction of the lev operon via LevR. As a consequence, the constitutive expression from the lev promoter observed in a ΔptsH strain became inducible with fructose when the crhQ15H allele was expressed in this strain.
The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80·6%) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64·8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Purified glycerol kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, phosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T. flavus glycerol kinase caused only a slight increase in enzyme activity.
The induction of the formation of inclusion vesicles in Leishmania amazonensis by the sterol biosynthesis inhibitors (SBI) ketoconazole and terbinafine has been reported previously. These compartments were recently identified as acidocalcisomes. By the use of electron spectroscopic imaging and energy loss spectroscopy, the presence of calcium, phosphorus and oxygen in the electron-dense inclusions located within the acidocalcisomes has been demonstrated. Endoplasmic reticulum cisternae formed membrane whorls which enclosed large portions of the cytoplasm and sometimes circumscribed acidocalcisomes. In addition, acid phosphatase activity, as well as the endocytic tracers horseradish peroxidase and gold-labelled transferrin and cystatin C were detected within these organelles in both SBI-treated and untreated parasites. These data suggest that impairment of sterol biosynthesis induces the biogenesis of acidocalcisomes and triggers an autophagic process that leads to intersection of the endosomal/lysosomal system with the acidocalcisomes.
Osmotolerance in yeast is regulated by at least two distinct mechanisms. The acquired response occurs following long-term exposure to hypertonic medium and requires the induction of the HOG-MAP (high-osmolarity glycerol mitogen-activated protein) kinase cascade to increase levels of the osmolyte glycerol. The acute response occurs following sudden exposure to high osmotica and appears to be dependent on normal vacuole function. In this study it is reported that the yeast endosomal/prevacuolar Na+/H+ exchanger Nhx1 contributes to osmotolerance following sudden exposure to hyperosmotic media. Vacuolar shrinkage and recovery in response to osmotic shock was altered in the Δnhx1 null mutant. Our results also show that the osmotolerance conferred by Nhx1 contributes to the postdiauxic/stationary-phase resistance to osmotic stress and allows for the continued growth of cells until the acquired osmotolerance response can occur.
To understand the molecular mechanisms induced by stress that contribute to the development of tolerance in eukaryotic cells, the filamentous fungus Aspergillus nidulans has been chosen as a model system. Here, the response of A. nidulans germlings to heat shock is reported. The heat treatment dramatically increased the concentration of trehalose and induced the accumulation of mannitol and mRNA from the catalase gene catA. Both mannitol and catalase function to protect cells from different reactive oxygen species. Treatment with hydrogen peroxide increased A. nidulans germling viability after heat shock whilst mutants deficient in catalase were more sensitive to a 50 °C heat exposure. It is concluded that the defence against the lethal effects of heat exposure can be correlated with the activity of the defence system against oxidative stress.