Dataset Information

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa.

ABSTRACT:

Background

Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (?(22)) to initiate transcription of the alginate biosynthetic operon.

Results

In the current study, we mapped the mucE transcriptional start site, and determined that P(mucE) activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P(mucE) activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2?algU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE.

Conclusions

Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

SUBMITTER: Yin Y

PROVIDER: S-EPMC3819740 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Publications

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa.

Yin Yeshi Y Damron F Heath FH Withers T Ryan TR Pritchett Christopher L CL Wang Xin X Schurr Michael J MJ Yu Hongwei D HD

BMC microbiology 20131018

<h4>Background</h4>Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ(22)) to initiate transcription of the alginate biosynthetic operon.<h4>Results</h4 ...[more]

PMID: 24138584

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Project description:Pseudomonas aeruginosa isolates from chronic lung infections often overproduce alginate, giving rise to the mucoid phenotype. Isolation of mucoid strains from chronic lung infections correlates with a poor patient outcome. The most common mutation that causes the mucoid phenotype is called mucA22 and results in a truncated form of the anti-sigma factor MucA that is continuously subjected to proteolysis. When a functional MucA is absent, the cognate sigma factor, AlgT, is no longer sequestered and continuously transcribes the alginate biosynthesis operon, leading to alginate overproduction. In this work, we report that in the absence of wild-type MucA, providing exogenous AlgT is toxic. This is intriguing, since mucoid strains endogenously possess high levels of AlgT. Furthermore, we show that suppressors of toxic AlgT production have mutations in mucP, a protease involved in MucA degradation, and provide the first atomistic model of MucP. Based on our findings, we speculate that mutations in mucP stabilize the truncated form of MucA22, rendering it functional and therefore able to reduce toxicity by properly sequestering AlgT.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing chronic lung infections. Phenotypes important for the long-term persistence and adaption to this unique lung ecosystem are largely regulated by the AlgT sigma factor. Chronic infection isolates often contain mutations in the anti-sigma factor mucA, resulting in uncontrolled AlgT and continuous production of alginate in addition to the expression of ∼300 additional genes. Here, we report that in the absence of wild-type MucA, AlgT overproduction is lethal and that suppressors of toxic AlgT production have mutations in the MucA protease, MucP. Since AlgT contributes to the establishment of chronic infections, understanding how AlgT is regulated will provide vital information on how P. aeruginosa is capable of causing long-term infections.

Project description:Overproduction of the exopolysaccharide alginate by Pseudomonas aeruginosa results in mucoid colony morphology and is an important virulence determinant expressed by this organism in cystic fibrosis. Mucoidy is transcriptionally regulated by signal transduction systems and histone-like elements. One point of convergence of regulatory elements controlling mucoidy is the algD promoter. A newly described genetic locus required for algD transcription was characterized in this study. This DNA region, cloned from a nonmucoid PAO strain, was initially isolated on the basis of its ability to suppress mucoidy when present on a plasmid. The suppressing activity was observed in several mucoid PAO derivatives, including strain PAO568, in which the mapped muc-2 mutation is responsible for its mucoid phenotype, and in close to 40% of cystic fibrosis strains tested. Protein expression studies detected two polypeptides with apparent molecular masses of 27.5 and 20 kDa encoded by the region required for the suppression activity. The gene encoding the polypeptide with an apparent molecular mass of 27.5 kDa, termed algU, was further characterized. A functional chromosomal copy of algU was found to be necessary for the expression of mucoidy. Insertional inactivation of algU on the chromosome of the mucoid strain PAO568 abrogated alginate production and algD transcription. DNA sequence analysis revealed sequence similarity of the predicted algU gene product with sigma H (Spo0H), a sigma factor involved in the control of sporulation and competence in Bacillus spp. Physical mapping revealed that algU resided on the same SpeI fragment (F) as did the pruAB locus, known to be tightly linked with genetic determinants (muc) which can confer mucoidy in genetic crosses. When the chromosomal algU copy was tagged with a Tcr cassette (algU::Tcr), a tight genetic linkage of algU with pruAB was demonstrated by F116L-mediated generalized transduction. Moreover, algU::Tcr derivatives of PAO568 (originally carrying the muc-2 marker) lost the ability to transfer mucoidy in genetic crosses. These results suggest that algU, a regulator of algD transcription showing sequence similarity to an alternative sigma factor, and the genes immediately downstream of algU may be associated with a locus participating in the differentiation into the mucoid phenotype.

Project description:The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis and is due to overproduction of an exopolysaccharide called alginate. However, the mucoid phenotype is unstable in vitro, especially when the cells are incubated under low oxygen tension. Spontaneous conversion to the nonmucoid form is typically due to mutations (previously called algS) that are closely linked to the alginate regulatory gene algT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alternate sigma factors in bacteria, suggesting that AlgT (also known as AlgU) interacts directly with RNA polymerase core to activate the promoters of alginate genes. AlgT showed striking sequence similarity (79%) to sigma E of Escherichia coli, an alternate sigma factor involved in high-temperature gene expression. Our analysis of the molecular basis for spontaneous conversion from mucoid to nonmucoid, in the cystic fibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at codons 18 and 29. RNase protection assays showed that spontaneous nonmucoid strains with the algT18 and algT29 alleles have a four- to fivefold reduction in the accumulation of algT transcripts compared with the wild-type mucoid strain. Likewise, a plasmid-borne algT-cat transcriptional fusion was about 3-fold less active in the algT18 and algT29 backgrounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::Tn501 background. These data indicate that algT is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, and the two genes may be cotranscribed. A primer extension analysis showed that algT transcription starts 54 bp upstream of the start of translation. Although the algT promoter showed little similarity to promoters recognized by the vegetative sigma factor, it was similar to the algR promoter. This finding suggests that AlgT may function as a sigma factor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were readily detectable in the typical nonmucoid strain PAO1, which was in contrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-borne algT gene in PAO1 resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that AlgT controls its own gene expression and expression of genes of the alginate regulon.

Project description:Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable with sigma E from Escherichia coli and Salmonella typhimurium. sigma E has been implicated in the expression of systems enhancing bacterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. aeruginosa from multiple promoters (P1 through P5) that fall into three categories: (i) the P1 and P3 promoters, which display strong similarity to the -35 and -10 canonical sequences of sigma E promoters and were found to be absolutely dependent on AlgU; (ii) the P2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tcr cells; and (iii) the transcripts corresponding to P4 and P5, which were not affected by inactivation of algU. Introduction of E. coli rpoE (encoding sigma E) or algU into P. aeruginosa algU::Tcr strains restored P1 and P3 transcription and brought the P2 signal back to the wild-type level. The AlgU-dependent promoters P1 and P3 were inducible by heat shock in wild-type nonmucoid P. aeruginosa PAO1. At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, two-dimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock. Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively, these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AlgU and, by extension, its homologs in other organisms (e.g., sigma E in S. typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions.

Project description:Pseudomonas aeruginosa frequently resides among ethanol-producing microbes, making its response to the microbially produced concentrations of ethanol relevant to understanding its biology. Our transcriptome analysis found that genes involved in trehalose metabolism were induced by low concentrations of ethanol, and biochemical assays showed that 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 anti-sigma 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 (QS), as induction was not observed in a ΔlasR ΔrhlR strain. A network analysis using a model, eADAGE, built from publicly available P. aeruginosa transcriptome data sets (J. Tan, G. Doing, K. A. Lewis, C. E. Price, et al., Cell Syst 5:63-71, 2017, https://doi.org/10.1016/j.cels.2017.06.003) provided strong support for our model in which treZ and coregulated genes are controlled by both AlgU- and AHL-mediated 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 in cells 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 occurred only 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 reveals a new role for (p)ppGpp in the regulation of AlgU activity.

Project description:Alginate overproducition by mucoid Pseudomonas aeruginosa is a critical pathogenic determinant expressed by this organism during chronic infections in cystic fibrosis. Conversion to mucoidy and a subsequent loss of mucoid character can occur via different mutations in the algU mucA mucB gene cluster. The algU gene encodes a 22.2-kDa putative alternative sigma factor required for expression of the critical alginate biosynthetic gene algD. In this work, algU transcription was studied by S1 nuclease protection analysis. Transcription from the promoter proximal to the algU coding region was found to be dependent on AlgU. The -35 and -10 sequences of this newly mapped promoter showed strong similarity ot the promoters of two other critical alg genes: algD and algR. The proximal promoter of algR was also shown to depend on algU. Interestingly, the putative -35 and -10 regions of all three promoters displayed striking similarity to the consensus sequence of the sigma E-dependent promoters in Escherichia coli and Salmonella typhimurium. This 24-kDa sigma factor, controlling genes participating in resistance to high temperatures and oxidative stress, has been previously biochemically characterized, but the gene for sigma E remained unidentified. To examine whether AlgU is related to sigma E, the effect of algU inactivation on the sensitivity of P. aeruginosa to killing by heat and reactive oxygen intermediates was tested. Two isogenic pairs of algU+ and algU mutant strains were compared. The algU mutants, irrespective of the mucoid status of the parental strains, displayed increased sensitivity to killing by paraquat, known to generate intracellular superoxide radicals, and heat. Further lgobal homology searches revealed the presence of a previously unrecognized E. coli gene with the predicted gene product showing a striking 66% identity to AlgU. The corresponding gene from S. typhimurium was cloned and sequenced, and it is displayed one amino acid substitution relative to its E. coli equivalent. AlgU and its close homologs in E. coli and S. typhimurium may be functionally related.

Project description:UnlabelledAdaptation of bacterial pathogens to a host can lead to the selection and accumulation of specific mutations in their genomes with profound effects on the overall physiology and virulence of the organisms. The opportunistic pathogen Pseudomonas aeruginosa is capable of colonizing the respiratory tract of individuals with cystic fibrosis (CF), where it undergoes evolution to optimize survival as a persistent chronic human colonizer. The transcriptome of a host-adapted, alginate-overproducing isolate from a CF patient was determined following growth of the bacteria in the presence of human respiratory mucus. This stable mucoid strain responded to a number of regulatory inputs from the mucus, resulting in an unexpected repression of alginate production. Mucus in the medium also induced the production of catalases and additional peroxide-detoxifying enzymes and caused reorganization of pathways of energy generation. A specific antibacterial type VI secretion system was also induced in mucus-grown cells. Finally, a group of small regulatory RNAs was identified and a fraction of these were mucus regulated. This report provides a snapshot of responses in a pathogen adapted to a human host through assimilation of regulatory signals from tissues, optimizing its long-term survival potential.ImportanceThe basis for chronic colonization of patients with cystic fibrosis (CF) by the opportunistic pathogen Pseudomonas aeruginosa continues to represent a challenging problem for basic scientists and clinicians. In this study, the host-adapted, alginate-overproducing Pseudomonas aeruginosa 2192 strain was used to assess the changes in its transcript levels following growth in respiratory CF mucus. Several significant and unexpected discoveries were made: (i) although the alginate overproduction in strain 2192 was caused by a stable mutation, a mucus-derived signal caused reduction in the transcript levels of alginate biosynthetic genes; (ii) mucus activated the expression of the type VI secretion system, a mechanism for killing of other bacteria in a mixed population; (iii) expression of a number of genes involved in respiration was altered; and (iv) several small regulatory RNAs were identified, some being mucus regulated. This work highlights the strong influence of the host environment in shaping bacterial survival strategies.

Dataset Information

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa.

Background

Results

Conclusions

Publications

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa.

Similar Datasets

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