Sigma 38

Sigma factors are bacterial proteins that help control how genes are used (expressed). In particular, sigma factors are used when genes are "turned on", eg if a bacterium is growing in a liquid (growth medium) that is poor in food, and a rich food like glucose (sugar) is added, the genes needed for glucose utilization will be turned on.Sigma factors (σ factor) are prokaryotic transcription initiation factors that regulate the expression of several genes. Several sigma factors are activated in response to different environmental conditions. The gene RpoS transcribes Sigma 38 (σ38), a 37.8 kD protein in E. Coli, which is transcribed in late-exponential phase and is the primary regulator of stationary-inducible genes. It is a central regulator of the general stress response and operates in both a retroactive and a proactive manner: not only does it allow the cell to survive environmental challenges, but it also prepares the cell for subsequent stresses (cross-protection) (Hengge-Aronis, 2002). The gene RpoS most likely originated in the γ branch of the proteobacteria (as reviewed in Hengge-Aronis, 2002).

Environmental Signal to activation: Regulation of RpoS

Regulatory mechanisms that control RpoS exist at various levels of gene and protein organization: transcription, translation, degradation and protein activity. These processes occur in response to stresses such as near-UV radiation, acid, temperature or osmotic shock, oxidative stress and nutrient deprivation. While many key regulatory entities have been identified in these areas, the precise mechanisms by which they signal rpoS transcription, translation, proteolysis or activity remain largely un characterized.

Transcriptional Control of rpoS

RpoS transcription is mainly regulated by the rpoSp promoter on the E. coli chromosome (Lange "et al.", 1995). rpoSp produces rpoS mRNA and is induced upon entry into stationary-phase in cells growing on rich media (Takayanagi "et al.", 1994) via an unknown mechanism. Flanking rpoSp are two putative cAMP-CRP (cyclic AMP-cAMP receptor protein) binding sites that seem to control rpoS transcription in an antagonistic manner. The position of the first site upstream of the major rpoS promoter corresponds to a “classical activator” similarly found in the lac promoter thereby suggesting that its effects on transcription are activating (Lange and Hengge-Aronis, 1994); in contrast, the location of the second cAMP-CRP site is indicative of inhibitory action. In exponential phase, crp mutants exhibit high levels of rpoS expression, suggesting that cAMP-CRP inhibits rpoS transcription. Upon entry into stationary phase, on the other hand, cAMP-CRP may upregulate rpoS transcription (Hengge-Aronis, 2002). While these observations may explain the seemingly dual nature of the cAMP-CRP binding sites, they require an explanation of phase-dependent selection of cAMP-CRP site activation to fully account for the contradictory data. Additional regulatory controls for rpoS transcription include: BarA, a histidine sensor kinase which can activate OmpR and thereby promote porin synthesis; levels of small molecules such as ppGppp which may hinder transcriptional elongation or stability in response to amino acid limitations or carbon, nitrogen or phosphorus starvation (Gentry "et al.", 1993). Despite the numerous controls on rpoS transcription, cellular rpoS mRNA levels remain high during exponential phase and the majority of extra-cellular stimuli do not significantly affect rpoS transcription.

Translational Control of rpoS

Instead, most RpoS expression is determined at the translational level (Repoila "et al.", 2003). sRNAs (small noncoding RNAs) sense environmental changes and in turn increase rpoS translation to allow the cell to accordingly adjust to the external stress. The promoter of the 85 nucleotide sRNA DsrA contains a temperature-sensitive transcription initiation thermocontrol as it is repressed at high (42 ˚ C) temperatures, but induces (perhaps by complementary binding to) rpoS at low (25 ˚C) temperatures (Sledjeski "et al.", 1996). Another sRNA, RprA stimulates rpoS translation in response to cell surface stress signaled via the RcsC sensor kinase (Sledjeski "et al.", 1996). A third type of sRNA, OxyS is regulated by OxyR, the primary sensor of oxidative shock (Altuvia "et al.", 1997). The mechanism by which OxyS interferes with rpoS translational efficiency is not known. However, the RNA-binding protein Hfq is implicated in the process (Brown and Elliott, 1996). Hfq binds to rpoS mRNA "in vitro" and may thereby modify rpoS mRNA structure for optimal translation. Hfq activates both DsrA and RprA. In contrast, LeuO inhibits rpoS translation by repressing dsrA expression and the histone-like protein HN-S (and its paralog StpA) inhibits rpoS translation via an unknown mechanism. In addition, H-NS, LeuO, Hfq and DsrA form an interconnected regulatory network that ultimately controls rpoS translation (Figure 2).

Figure 2. Regulatory mechanisms of rpoS translational control. Inactive rpoS mRNA consists of a closed structure with the translation initiation region base paired to an upstream internal antisense element. Various environmental stimuli (depicted in ovals) signal activation (arrows) or repression (line ending in a perpendicular bar) of the translationally-competent open rpoS mRNA via translation-stimulating factors Hfq, HU, and DsrA. rpoS can then be translated to RpoS (σS). Source: (Hengge-Aronis, 2002a).

RpoS Degradation

RpoS proteolysis forms another level of the sigma factor’s regulation. Degradation occurs via ClpXP, a barrel-shaped protease comprised of two six-subunit rings of the ATP-dependent ClpX chaperone that surround two seven-subunit rings of ClpP (Repoila "et al.", 2003). The response regulator RssB has been identified as a σS-specific recognition factor crucial for RpoS degradation. Additional factors known to regulate RpoS proteolysis but via incompletely characterized mechanisms include: RssA which is found on the same operon as RssB; H-NS and DnaK, both of which also regulate rpoS translation and LrhA; and acetyl phosphate affects RpoS proteolysis by possibly acting as a phosphoryl donor to RssB.

The RpoS Regulon

Consistent with its role as the master controller of the bacterial stress response, RpoS regulates the expression of stress-response genes that fall into various functional categories: stress resistance, cell morphology, metabolism, virulence and lysis.

tress Resistance

Many genes under RpoS control confer stress resistance to assaults such as DNA damage, presence of reactive oxygen species and osmotic stress. The product of xthA is an exonuclease that participates in DNA repair by recognizing and removing 5’ monophosphates near abasic sites in damaged DNA (Demple "et al.", 1983). Likewise, catalases HPI and HPII, encoded by katG and katE convert harmful hydrogen peroxide molecules to water and oxygen (Loewen, 1992). The otsBA gene product trehalose functions as an osmoprotectant and is needed for desiccation resistance (Kaasen "et al.", 1992). Additional RpoS-dependent factors involved in oxidative stress include glutathione reductase (encoded by gor), and superoxide dimutase (encoded by sodC) (Beckerhapak and Eisenstark, 1995).

Morphology

RpoS-dependent genes involved in changes in cell membrane permeability and general cell morphology mostly belong to the osm family of genes. osmB encodes an outer membrane lipoprotein that may play a role in cell aggregation (Jung "et al.", 1990), whereas osmY encodes a periplasmic protein. Additional RpoS-dependent factors that determine the size and shape of the cell include the morphogene bolA and products of the ftsQAZ operon that play a role in the timing of cell division (Lange "et al.", 1995). Control of cell shape, cell division and cell-cell interaction are likely to be important in inhibiting cell proliferation and thus allocating resources to cell survival during periods of stress.

Metabolism

Metabolically-optimal survival conditions include RpoS-dependent decreased Krebs cycle activity and increased glyocolytic activity to limit the reactive oxygen species that are byproduced as a result of essential cellular processes. Pyruvate entry into the Krebs cycle is inhibited by the product of the RpoS-dependent gene poxB. An overall slowdown in metabolic activity is consistent with energy conservation and reduced growth during periods of stress.Virulence

As a defense mechanism, the host environment is hostile to invading pathogens. Therefore, infection can be a stressful event for pathogenic bacteria and control of virulence genes may be temporally correlated with the timing of infection by pathogens (reviewed in Hengge-Aronis, 2000). Discovery of RpoS-dependent virulence genes in Salmonella are consistent with RpoS as a general regulator of the stress response: the spv gene found on a virulence plasmid in this bacterium is controlled by RpoS, and interestingly, required for growth in deep lymphoid tissue such as the spleen and liver (Gulig "et al.", 1993).

Lysis

RpoS also plays an important role in regulating cell lysis. Along with OmpR, it upregulates the entericidin (ecnAB) locus which encodes a lysis-inducing toxin (Bishop "et al.", 1998). In contrast, ssnA is negatively controlled by RpoS but it also promotes lysis. Paradoxically, lysis is seen as a survival process in certain contexts.