Ethanol stimulates trehalose production through a SpoT-DksA-AlgU dependent pathway in Pseudomonas aeruginosa

Pseudomonas aeruginosa frequently resides among ethanol-producing microbes, making its response to these microbially-produced concentrations of ethanol relevant to understanding its biology. Our ranscriptome analysis found that the genes involved in trehalose metabolism were induced by low concentrations of ethanol, and levels of intracellular trehalose increased significantly upon growth with ethanol. The increase in trehalose was dependent on the TreYZ pathway, but not other trehalose metabolic enzymes TreS or TreA. The sigma factor AlgU (AlgT), a homolog of RpoE in other species, was required for increased expression of the treZ gene and trehalose levels, but induction was not controlled by the well-characterized proteolysis of its antisigma factor MucA. Growth with ethanol led to increased SpoT-dependent (p)ppGpp accumulation, which stimulates AlgU-dependent transcription of treZ and other AlgU-regulated genes through DksA, a (p)ppGpp and RNA polymerase binding protein. Ethanol stimulation of trehalose also required acylhomoserine lactone (AHL)-mediated quorum sensing, as induction was not observed in a ΔlasRΔrhlR strain. A network analysis using a model, eADAGE, built from publicly available P. aeruginosa transcriptome datasets (1) provided strong support for our model that treZ and co-regulated genes are controlled by both AlgU and AHL-mediated QS (QS). Consistent with (p)ppGpp and AHL-mediated quorum sensing regulation, ethanol, even when added at the time of culture inoculation, stimulated treZ transcript levels and trehalose production in cells from post-exponential phase cultures but not from exponential phase cultures. These data highlight the integration of growth and cell density cues in the P. aeruginosa transcriptional response to ethanol.ImportancePseudomonas aeruginosa is often found with bacteria and fungi that produce fermentation products including ethanol. At concentrations similar to those produced by environmental microbes, we found that ethanol stimulated expression of trehalose biosynthetic genes and cellular levels of trehalose, a disaccharide that protects against environmental stresses. The induction of trehalose by ethanol required the alternative sigma factor AlgU through DksA and SpoT-dependent (p)ppGpp. Trehalose accumulation also required AHL quorum sensing and only occurred in post-exponential phase cultures. This work highlights how cells integrate cell-density and growth cues in their responses to products made by other microbes and a reveals a new role for (p)ppGpp in the regulation of AlgU activity.

Translational Control of the SigR-Directed Oxidative Stress Response in Streptomyces via IF3-Mediated Repression of a Noncanonical GTC Start Codon

ABSTRACT The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry. IMPORTANCE In all sigma factor-antisigma factor regulatory switches, the relative abundance of the two proteins is critical to the proper functioning of the system. Many sigma-antisigma operons are cotranscribed and translationally coupled, leading to a generic assumption that the sigma and antisigma factors are produced in a fixed 1:1 ratio. In the case of sigR - rsrA , we show instead that the antisigma factor is produced in excess over the sigma factor, providing a buffer to prevent spurious release of sigma activity. This excess arises in part because sigR has an extremely rare noncanonical GTC start codon, and as a result, SigR translation initiation is repressed by IF3. This finding highlights the potential significance of noncanonical start codons, very few of which have been characterized experimentally. It also emphasizes the limitations of predicting start codons using bioinformatic approaches, which rely heavily on the assumption that ATG, GTG, and TTG are the only permissible start codons.

Structural basis for the sequestration of the anti-σ70factor Rsd from σ70by the histidine-containing phosphocarrier protein HPr

Histidine-containing phosphocarrier protein (HPr) is a general component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) involved in the phosphorylation-coupled transport of numerous sugars called PTS sugars. HPr mainly exists in a dephosphorylated form in the presence of PTS sugars in the medium, while its phosphorylation increases in the absence of PTS sugars. A recent study revealed that the dephosphorylated form of HPr binds and antagonizes the function of the antisigma factor Rsd. This anti-sigma factor sequesters the housekeeping sigma factor σ70to facilitate switching of the sigma subunit on RNA polymerase from σ70to the stress-responsive sigma factor σSin stationary-phase cells. In this study, the structure of the complex of Rsd and HPr was determined at 2.1 Å resolution and revealed that the binding site for HPr on the surface of Rsd partly overlaps with that for σ70. The localization of the phosphorylation site on HPr at the binding interface for Rsd explains why phosphorylation of HPr abolishes its binding to Rsd. The mutation of crucial residues involved in the HPr–Rsd interaction significantly influenced the competition between HPr and σ70for binding to Rsd bothin vitroandin vivo. The results provide a structural basis for the linkage of global gene regulation to nutrient availability in the external environment.

Role of Cell Surface Signaling in Proteolysis of an Alternative Sigma Factor in Pseudomonas aeruginosa

ABSTRACT Alternative sigma factor proteins enable transcription of specific sets of genes in bacterial cells. Their activities can be controlled by posttranslational mechanisms including inhibition by antisigma proteins and proteolytic degradation. PvdS is an alternative sigma factor that is required for expression of genes involved in synthesis of a siderophore, pyoverdine, by Pseudomonas aeruginosa. In the absence of pyoverdine, the activity of PvdS is inhibited by a membrane-spanning antisigma factor, FpvR. Inhibition is relieved by a cell surface signaling pathway. In this pathway, a combination of pyoverdine and a cell surface receptor protein, FpvA, suppresses the antisigma activity of FpvR, enabling transcription of PvdS-dependent genes. In this research, we investigated proteolytic degradation of PvdS in response to the signaling pathway. Proteolysis of PvdS was observed in strains of P. aeruginosa in which FpvR had anti-sigma factor activity due to the absence of pyoverdine or the FpvA receptor protein or overproduction of FpvR. Suppression of antisigma activity by addition of pyoverdine or through the absence of FpvR prevented detectable proteolysis of PvdS. The amounts of PvdS were less in bacteria in which proteolysis was observed, and reporter gene assays showed that this reduction was not due to decreased expression of PvdS. In wild-type bacteria, there was an average of 730 molecules of PvdS per cell in late exponential growth phase. Our results show that proteolysis and amounts of PvdS are affected by the antisigma factor FpvR and that this activity of FpvR is controlled by the cell surface signaling pathway.

Unique Degradation Signal for ClpCP in Bacillus subtilis

ABSTRACT Regulation of the cell-specific transcription factor σF in the spore-forming bacterium Bacillus subtilis involves the antisigma factor SpoIIAB. Contributing to the activation of σF is the degradation of SpoIIAB in a manner that depends on the protease ClpCP. Here we show that the three residues (LCN) located at the extreme C terminus of SpoIIAB are both necessary and sufficient for this degradation. We also report that the use of the LCN extension as a degradation signal for ClpCP is unique to SpoIIAB.