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Category: Bacterial Pathogenesis
Regulation of Exopolysaccharide Biosynthesis in Pseudomonas aeruginosa, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555818524/9781555816766_Chap09-1.gif /docserver/preview/fulltext/10.1128/9781555818524/9781555816766_Chap09-2.gifAbstract:
Alginate is arguably the best-characterized exopolysaccharide produced by Pseudomonas aeruginosa, and several excellent reviews have been written on the molecular biology of its production and clinical ramifications. This chapter reviews the transcriptional factor and posttranscriptional factor involved in controlling and inducing alginate production. The gene encoding the cognate histidine kinase for the response regulator AlgB is kinB (PA5484 on the PAO1 chromosome), located directly downstream of algB on the PAO1 chromosome. Deletion of mucR also affected other known cyclic dimeric-GMP (c-di-GMP) processes in P. aeruginosa, including swarming motility, biofilm formation, and alginate production. It additionally showed, through the use of lacZ and phoA fusions, that the predicted amino-terminal MHYT domain of MucR resides on the inner membrane. MHYT domains are proposed to bind O2, NO, or CO. A model for c-di-GMP regulation of alginate production was proposed whereby the guanylate cyclase of MucR is stimulated by a yet-to-be-identified signal, which binds to a predicted MHYT domain in the amino terminus of MucR. The current state of knowledge indicates that Psl and Pel are likely involved with the initial stages of biofilm development, whereas alginate is the stress response exopolysaccharide. The levels of alginate produced by newly generated MucA, MucB, MucC, or MucD mutants of P. aeruginosa PAO1 and other nonmucoid clinical isolates (i.e., non-CF isolates) was found to be inversely related to biologically relevant concentrations (e.g., <5 to 100 μM) of iron present in the media used in this study.
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Biochemical pathways that leads to Pel, alginate, and Psl exopolysaccharide production. Alginate production requires fructose-6-phosphate as the precursor for GDP-mannuronic acid. AlgA, AlgC, and AlgD perform this conversion. Since P. aeruginosa lacks enzymes required for glycolysis, carbon sources must be converted into tricarboxylic acid (TCA) cycle intermediates before being processed to fructose-6-phosphate via gluconeogenesis. GDP-mannuronic acid is polymerized (Alg8, Alg44, AlgX, and AlgK) and modified (AlgI, AlgJ, AlgF, AlgG, and AlgL) before being exported (AlgE) out to the extracellular space. Several enzymes required for alginate production overlap with Psl and Pel production. The precursors for Psl are sugar nucleotides, including GDP-mannose, UDP-glucose, and dTDP-l-rhamnose, which are derived from fructose-6-phosphate and glucose-6- phosphate from the activity of AlgC, a Psl-specific enzyme PslB, the AlgA homolog WbpW, and an enzyme involved with rhamnose production, RmlC. These precursors are polymerized (PslA, PslE, PslF, PslC, PslH, and PslI), modified (PslG), and exported (PslD). Pel is thought to consist of linear chains of sugar moieties. Sugar nucleotides produced by the metabolic pathway are, again, polymerized (PelF), modified (PelA), and exported (PelB). Currently, the roles of PelG, PelE, and PelC have not been elucidated. c-di-GMP also activates alginate and Pel production. MucR contains a GGDEF domain to synthesize c-di-GMP. c-di-GMP, in turn, binds to the PilZ domain of Alg44 to enhance alginate polymerization. PelD also contains a PilZ domain. Acetyl-CoA, acetyl coenzyme A; IM, inner membrane; OM, outer membrane; PDG, peptidoglycan layer. doi:10.1128/9781555818524.ch9f1
Biochemical pathways that leads to Pel, alginate, and Psl exopolysaccharide production. Alginate production requires fructose-6-phosphate as the precursor for GDP-mannuronic acid. AlgA, AlgC, and AlgD perform this conversion. Since P. aeruginosa lacks enzymes required for glycolysis, carbon sources must be converted into tricarboxylic acid (TCA) cycle intermediates before being processed to fructose-6-phosphate via gluconeogenesis. GDP-mannuronic acid is polymerized (Alg8, Alg44, AlgX, and AlgK) and modified (AlgI, AlgJ, AlgF, AlgG, and AlgL) before being exported (AlgE) out to the extracellular space. Several enzymes required for alginate production overlap with Psl and Pel production. The precursors for Psl are sugar nucleotides, including GDP-mannose, UDP-glucose, and dTDP-l-rhamnose, which are derived from fructose-6-phosphate and glucose-6- phosphate from the activity of AlgC, a Psl-specific enzyme PslB, the AlgA homolog WbpW, and an enzyme involved with rhamnose production, RmlC. These precursors are polymerized (PslA, PslE, PslF, PslC, PslH, and PslI), modified (PslG), and exported (PslD). Pel is thought to consist of linear chains of sugar moieties. Sugar nucleotides produced by the metabolic pathway are, again, polymerized (PelF), modified (PelA), and exported (PelB). Currently, the roles of PelG, PelE, and PelC have not been elucidated. c-di-GMP also activates alginate and Pel production. MucR contains a GGDEF domain to synthesize c-di-GMP. c-di-GMP, in turn, binds to the PilZ domain of Alg44 to enhance alginate polymerization. PelD also contains a PilZ domain. Acetyl-CoA, acetyl coenzyme A; IM, inner membrane; OM, outer membrane; PDG, peptidoglycan layer. doi:10.1128/9781555818524.ch9f1
Transcriptional regulation of alginate biosynthetic genes. The transcription of the major operon (algD, alg8, alg44, algK, algE, algG, algX, algL, algI, algJ, algF, and algA) encoding the biosynthetic enzymes and membrane-associated polymerization, modification, and export proteins is regulated by the promoter region of algD (A). Transcriptional regulators (AlgR, AlgB, AmrZ, CysB, and CRP [from E. coli]), histone-like proteins (Hp-1 and IHF), and two sigma factors (AlgU/T and RpoN) associate with the DNA and are involved with PalgD transcription. Numbers underneath the regulator name indicate the regions of the DNA (relative to the transcriptional start site) that have been found to bind the regulator through experimental evidence. Binding sites of the regulators are shaded with their respective colors. Hp-1 binding regions are underlined. Sigma factor consensus sequences are boxed. (B and C) Models showing AlgU/T- and RpoN-dependent transcriptional activation. DNA bending is thought to occur with the aid of IHF and Hp-1. Transcriptional activators in the far upstream region, such as AlgR (B) or AlgB (C), are then able to activate transcription near the +1 site by interaction with the AlgU-RNAP or RpoN-RNAP complexes, respectively. PalgD transcription is thought to be regulated by sigma factor competition (D). RpoN and AlgU binding sites overlap, and alginate production is activated depending on the type of stress encountered by the cell (nitrogen-related stress versus cell wall stress). doi:10.1128/9781555818524.ch9f2
Transcriptional regulation of alginate biosynthetic genes. The transcription of the major operon (algD, alg8, alg44, algK, algE, algG, algX, algL, algI, algJ, algF, and algA) encoding the biosynthetic enzymes and membrane-associated polymerization, modification, and export proteins is regulated by the promoter region of algD (A). Transcriptional regulators (AlgR, AlgB, AmrZ, CysB, and CRP [from E. coli]), histone-like proteins (Hp-1 and IHF), and two sigma factors (AlgU/T and RpoN) associate with the DNA and are involved with PalgD transcription. Numbers underneath the regulator name indicate the regions of the DNA (relative to the transcriptional start site) that have been found to bind the regulator through experimental evidence. Binding sites of the regulators are shaded with their respective colors. Hp-1 binding regions are underlined. Sigma factor consensus sequences are boxed. (B and C) Models showing AlgU/T- and RpoN-dependent transcriptional activation. DNA bending is thought to occur with the aid of IHF and Hp-1. Transcriptional activators in the far upstream region, such as AlgR (B) or AlgB (C), are then able to activate transcription near the +1 site by interaction with the AlgU-RNAP or RpoN-RNAP complexes, respectively. PalgD transcription is thought to be regulated by sigma factor competition (D). RpoN and AlgU binding sites overlap, and alginate production is activated depending on the type of stress encountered by the cell (nitrogen-related stress versus cell wall stress). doi:10.1128/9781555818524.ch9f2
Posttranslational regulatory system for alginate production. The cell wall stress-sensing mechanism that is intimately linked with alginate is encoded by the genes algUmucABCD. MucA is a membrane-spanning anti-sigma factor that represses the AlgU sigma-factor regulon by sequestering AlgU to the membrane. MucB protects MucA from degradation. MucD is a general protease that scans the periplasm for misfolded or excess proteins to be degraded. Increase in membrane stress results in MucE or other proteins to be degraded in a way to reveal a WVF or YVF triple-residue motif, which is recognized by the protease AlgW. An unidentified signal activates the MucP and ClpP proteases. AlgW, MucP, and ClpP cleave MucA at either the cytoplasmic domain, the inner membrane, or the periplasmic domain, which results in AlgU/T release and activation of PalgD. IM, inner membrane; OM, outer membrane; PDG, peptidoglycan layer. doi:10.1128/9781555818524.ch9f3
Posttranslational regulatory system for alginate production. The cell wall stress-sensing mechanism that is intimately linked with alginate is encoded by the genes algUmucABCD. MucA is a membrane-spanning anti-sigma factor that represses the AlgU sigma-factor regulon by sequestering AlgU to the membrane. MucB protects MucA from degradation. MucD is a general protease that scans the periplasm for misfolded or excess proteins to be degraded. Increase in membrane stress results in MucE or other proteins to be degraded in a way to reveal a WVF or YVF triple-residue motif, which is recognized by the protease AlgW. An unidentified signal activates the MucP and ClpP proteases. AlgW, MucP, and ClpP cleave MucA at either the cytoplasmic domain, the inner membrane, or the periplasmic domain, which results in AlgU/T release and activation of PalgD. IM, inner membrane; OM, outer membrane; PDG, peptidoglycan layer. doi:10.1128/9781555818524.ch9f3