Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.     

OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.     

Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae.

Metabolic changes have been investigated during continuous growth of yeast cells inoculated in glucose-containing medium until the cells entered the stationary phase in response to glucose exhaustion. Well in advance of glucose exhaustion, a transition phase was observed, characterized by a decrease in the growth rate and a progressive reduction of protein and RNA accumulation. Two-dimensional gel analysis of the proteins synthesized during this stage showed that the pattern of proteins remained similar to that of log-phase cells. When the cells entered the stationary phase, protein accumulation was 10% of that in log-phase cells, and incorporation of labeled RNA precursor was undetectable. Analysis of protein synthesis gave evidence that the synthesis of 95% of the proteins present in log-phase cells was arrested in stationary-phase cells. Among the 20 proteins whose synthesis continues throughout the stationary phase were identified actin, aldehyde dehydrogenase, enolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and five heat shock proteins. In addition, the synthesis of six new proteins was observed. The occurrence of these new proteins in stationary-phase cells is presumed to result from the release of carbon catabolite repression due to glucose exhaustion.     

A low-pH-inducible, stationary-phase acid tolerance response in Salmonella typhimurium.

Acid is an important environmental condition encountered by Salmonella typhimurium during its pathogenesis. Our studies have shown that the organism can actively adapt to survive potentially lethal acid exposures by way of at least three possibly overlapping systems. The first is a two-stage system induced in response to low pH by logarithmic-phase cells called the log-phase acid tolerance response (ATR). It involves a major molecular realignment of the cell including the induction of over 40 proteins. The present data reveal that two additional systems of acid resistance occur in stationary-phase cells. One is a pH-dependent system distinct from log-phase ATR called stationary-phase ATR. It was shown to provide a higher level of acid resistance than log-phase ATR but involved the synthesis of fewer proteins. Maximum induction of stationary-phase ATR occurred at pH 4.3. A third system of acid resistance is not induced by low pH but appears to be part of a general stress resistance induced by stationary phase. This last system requires the alternative sigma factor, RpoS. Regulation of log-phase ATR and stationary-phase ATR remains RpoS independent. Although the three systems are for the most part distinct from each other, together they afford maximum acid resistance for S. typhimurium.     

The induction of mitotic gene conversion in the yeast,Saccharomyces cerevisiae JD1 by 4-chloromethylbiphenyl (4CMB), Benzyl Chloride (BC) and 4-hydroxymethylbiphenyl (4HMB)

The induction of mitotic gene conversion by 4CMB, BC and 4HMB was studied in both log-phase and stationary-phase cultures of the yeast, Saccharomyces cerevisiae JD1. Assays were performed both in the presence and in the absence of S9 microsomal fraction obtained from a liver homogenate from rats pretreated with Aroclor 1254.Exposure of both stationary-phase and log-phase cultures to 4CMB and BC resulted in an increase in mitotic gene conversion, both in the presence and in the absence of a microsomal activation system; the magnitude of response was greater in stationary-phase cultures. 4HMB did not increase the gene conversion frequency in log-phase or stationary-phase cultures.     

Luciferase inactivation in the luminous marine bacterium Vibrio harveyi.

Luciferase was rapidly inactivated in stationary-phase cultures of the wild type of the luminous marine bacterium Vibrio harveyi, but was stable in stationary-phase cultures of mutants of V. harveyi that are nonluminous without exogenous aldehyde, termed the aldehyde-deficient mutants. The inactivation in the wild type was halted by cell lysis and was slowed or stopped by O2 deprivation or by addition of KCN and NaF or of chloramphenicol. If KCN and NaF or chloramphenicol were added to a culture before the onset of luciferase inactivation, then luciferase inactivation did not occur. However, if these inhibitors were added after the onset of luciferase inactivation, then luciferase inactivation continued for about 2 to 3 h before the inactivation process stopped. The onset of luciferase inactivation in early stationary-phase cultures of wild-type cell coincided with a slight drop in the intracellular adenosine 5'-triphosphate (ATP) level from a relatively constant log-phase value of 20 pmol of ATP per microgram of soluble cell protein. Addition of KCN and NaF to a culture shortly after this drop in ATP caused a rapid decrease in the ATP level to about 4 pmol of ATP per microgram whereas chloramphenicol added at this same time caused a transient increase in ATP level to about 25 pmol/microgram. The aldehyde-deficient mutant (M17) showed a relatively constant log-phase ATP level identical with that of the wild-type cells, but rather than decreasing in early stationary phase, the ATP level increased to a value twice that in log-phase cells. We suggest that the inactivation of luciferase is dependent on the synthesis of some factor which is produced during stationary phase and is itself unstable, and whose synthesis is blocked by chloramphenicol or cyanide plus fluoride.     

Impact of pulsed Nd:YAG laser on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora: significance of physiological status

The impact of pulsed Nd:YAG (neodymium-doped yttrium/aluminium garnet) laser irradiation on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora during two growth stages (log phase and stationary phase) and under two stresses (reduced temperature and nutrient limitation) was investigated. Bacteria were exposed to a laser fluence of 0.1 J x cm(-2) for 5, 10, and 15 min with a peak power of 20 MW x cm(-2), a pulse width of 5 ns, and an average power of 1 W x cm(-2) with a repetition rate of 10 Hz. The mortality of bacteria immediately after the irradiation as well as after a set period of time was determined. Mortality was higher among log-phase bacteria (72%) than bacteria in the stationary phase (51%) and those grown under nutrient limitation (51%). Bacteria grown at reduced temperature had a mortality of 49%. However, the differences in cell density of log-phase, stationary-phase, nutrient-limited, and low-temperature irradiated samples compared with controls after 5 h of incubation were 96, 93, 94, and 86%, respectively. The mortality values suggest that the same laser fluence has different degrees of effectiveness, depending on the physiological state of the bacteria.