Non-specific, general and multiple stress resistance of growth-restricted Bacillus subtilis cells by the expression of the σB regulon

Bacillus subtilis cells respond almost immediately to different stress conditions by increasing the production of general stress proteins (GSPs). The genes encoding the majority of the GSPs that are induced by heat, ethanol, salt stress or by starvation for glucose, oxygen or phosphate belong to the σ^B-dependent general stress regulon. Despite a good understanding of the complex regulation of the activity of σ^B and knowledge of a very large number of general stress genes controlled by σ^B, first insights into the physiological role of this non-specific stress response have been obtained only very recently. To explore the physiological role of this regulon, we and others identified σ^B-dependent general stress genes and compared the stress tolerance of wild-type cells with mutants lacking σ^B or general stress proteins. The proteins encoded by σ^B-dependent general stress genes can be divided into at least five functional groups that most probably provide growth-restricted B. subtilis cells with a multiple stress resistance in anticipation of future stress. In particular, sigB mutants are impaired in non-specific resistance to oxidative stress, which requires the σ^B-dependent dps gene encoding a DNA-protecting protein. Protection against oxidative damage of membranes, proteins or DNA could be the most essential component of σ^B-mediated general stress resistance in growth-arrested aerobic Gram-positive bacteria. Other general stress genes have both a σ^B-dependent induction pathway and a second σ^B-independent mechanism of stress induction, thereby partially compensating for a σ^B deficiency in a sigB mutant. In contrast to sigB mutants, null mutations in genes encoding those proteins, such as clpP or clpC, cause extreme sensitivity to salt or heat.     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.     

Back to log phase: σS as a global regulator in the osmotic control of gene expression in Escherichia coli

It is now well established that the σ^S subunit of RNA polymerase is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-inducible genes in Escherichiacoli. In this review, more recent findings will be summarized that demonstrate that σ^S also acts as a global regulator for the osmotic control of gene expression, and actually does so in exponentially growing cells. Thus, many σ^S-dependent genes are induced during entry into stationary phase as well as in response to osmotic upshift. K⁺ glutamate, which accumulates in hyperosmotically stressed cells, seems to specifically stimulate the activity of σ^S-containing RNA polymerase at σ^S-dependent promoters. Moreover, osmotic upshift results in an elevated cellular σ^S level similar to that observed in stationary-phase cells. This increase is the result of a stimulation of rpoS translation as well as an inhibition of the turnover of σ^S, which in exponentially growing non-stressed cells is a highly unstable protein. Whereas the RNA-binding protein HF-I, previously known as a host factor for the replication of phage Qβ RNA, is essential for rpoS translation, the recently discovered response regulator RssB, and ClpXP protease, have been shown to be required for σ^S degradation. The finding that the histone-like protein H-NS is also involved in the control of rpoS translation and σ^S turnover, sheds new light on the function of this protein in osmoregulation. Finally, preliminary evidence suggests that additional stresses, such as heat shock and acid shock, also result in increased cellular σ^S levels in exponentially growing cells. Taken together, σ^S function is clearly not confined to stationary phase. Rather, σ^S may be regarded as a sigma factor associated with general stress conditions.     

The stationary-phase sigma factor σS (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium

The acid tolerance response (ATR) of log-phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid. Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor σ^S encoded by rpoS. Differences between the acid responses of virulent S. typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts. The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon σ^S for their synthesis. It is predicted that one or more of these ASPs is essential for the sustained system. The sustained ATR also provided cross-protection to a variety of other environmental stresses (heat, H₂O₂ and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance. Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance. Consistent with this model, σ^S proved to be induced by acid shock. Our results also revealed a connection between the transient and sustained ATR systems. Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant. One member of the AtbR regulon, designated atrB, was found to be co-regulated by σ^S and AtbR. Both regulators had a negative effect on atrB expression. The results suggest AtrB serves as a link between the sustained and transient ATR systems. When σ^S concentration are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS-independent system of acid tolerance. Results also suggest different acid-sensitive targets occur in log-phase versus stationary-phase cells.     

Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress

Na⁺ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PP_i-dependent H⁺ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na⁺/H⁺ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na⁺ was mainly accumulated in the roots. Under a more severe salt treatment, Na⁺ was equally distributed in shoots and roots. In contrast to what was observed with Na⁺, all the salt treatments reduced the shoot K⁺ content. Dissipation by Na⁺ of the H⁺ gradient generated by the tonoplast H⁺-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na⁺/H⁺ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na⁺/H⁺ exchange activity, which displayed saturation kinetics for Na⁺ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na⁺/H⁺ antiports. No Na⁺/H⁺ exchange was detected in vesicles from control roots. The activity was specific for Na⁺. since K⁺ added to the assay medium slightly dissipated H⁺ gradients and displayed non-saturating kinetics for all salt treatments. Apparent K_m for Na⁺/H⁺ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, V_max remaining unchanged. The results suggest that the existence of a specific Na⁺/H⁺ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity.