Identification of Novel Genes Associated with Alginate Production in <em>Pseudomonas aeruginosa</em> Using Mini-<em>himar1</em> Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Marshall University

Alginate overproduction by P. aeruginosa strains, also known as mucoidy, is associated with chronic lung infections in cystic fibrosis (CF). It is not clear how alginate induction occurs in the wild-type (wt) mucA strains. When grown on Pseudomonas isolation agar (PIA), P. aeruginosa strains PAO1 and PA14 are non-mucoid, producing minimal amounts of alginate. Here we report the addition of ammonium metavanadate (AMV), a phosphatase inhibitor, to PIA (PIA-AMV) induced mucoidy in both these laboratory strains and early lung colonizing non-mucoid isolates with a wt mucA. This phenotypic switch was reversible depending on the availability of vanadate salts and triclosan, a component of PIA. Alginate induction in PAO1 on PIA-AMV was correlated with increased proteolytic degradation of MucA, and required envelope proteases AlgW or MucP, and a two-component phosphate regulator, PhoP. Other changes included the addition of palmitate to lipid A, a phenotype also observed in chronic CF isolates. Proteomic analysis revealed the upregulation of stress chaperones, which was confirmed by increased expression of the chaperone/protease MucD. Altogether, these findings suggest a model of alginate induction and the PIA-AMV medium may be suitable for examining early lung colonization phenotypes in CF before the selection of the mucA mutants.

Vanadate and triclosan synergistically induce alginate production by Pseudomonas aeruginosa strain PAO1.

&#x94DC;&#x7EFF;&#x5047;&#x5355;&#x80DE;&#x83CC;&#x662F;&#x673A;&#x4F1A;&#x4E3B;&#x4E49;&#x7684;&#x75C5;&#x539F;&#xFF0C;&#x786E;&#x7ACB;&#x4E86;&#x4E2A;&#x4EBA;&#x60A3;&#x56CA;&#x6027;&#x7EA4;&#x7EF4;&#x5316;&#x6162;&#x6027;&#x80BA;&#x90E8;&#x611F;&#x67D3;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x516C;&#x5E03;&#x7684;&#x8349;&#x6848;&#x7EFF;&#x8113;&#x83CC; PAO579&#xFF0C;PAO381 &#x682A;&#x9AD8;&#x4EA7;&#x6D77;&#x85FB;&#x884D;&#x751F;&#x7269;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x3002;

&#x7C98;&#x6DB2;&#x578B; PAO381 &#x5BFC;&#x6570;&#x7684;&#x94DC;&#x7EFF;&#x5047;&#x5355;&#x80DE;&#x83CC;&#x682A; PAO579 &#x57FA;&#x56E0;&#x7EC4;&#x5E8F;&#x5217;&#x8349;&#x56FE;&#x3002;

Pseudomonas aeruginosa is an opportunistic pathogen that establishes a chronic lung infection in individuals afflicted with cystic fibrosis. Here, we announce the draft genome of P. aeruginosa strain PAO579, an alginate-overproducing derivative of strain PAO381.

Draft Genome Sequence for Pseudomonas aeruginosa Strain PAO579, a Mucoid Derivative of PAO381.

Pseudomonas aeruginosa est un pathog&egrave;ne opportuniste qui &eacute;tablit une infection pulmonaire chronique chez les personnes atteintes de fibrose kystique. Ici, nous vous annon&ccedil;ons le g&eacute;nome de projet de P. aeruginosa souche PAO579, un d&eacute;riv&eacute; de l&#39;alginate surproduisant de la souche PAO381.

Projet Genome Sequence pour Pseudomonas aeruginosa souche PAO579, un d&eacute;riv&eacute; muco&iuml;de de PAO381.

Pseudomonas Aeruginosa ist eine opportunistische Erreger, die eine chronische Lungenentz&uuml;ndung bei Patienten mit zystischer Fibrose leiden herstellt. Hier geben wir bekannt das Entwurf-Genom von p. Aeruginosa-Stamm PAO579, ein Alginat-&Uuml;berproduktion Derivat-Stamm PAO381.

Entwurf-Genom-Sequenz f&uuml;r Pseudomonas Aeruginosa Stamm PAO579, eine schleimig-Derivat des PAO381.

Pseudomonas aeruginosa &egrave; un patogeno opportunistico che stabilisce un&#39;infezione polmonare cronica in individui affetti da fibrosi cistica. Qui, annunciamo il genoma del progetto di p. aeruginosa strain PAO579, un derivato dell&#39;alginato-sovrapproduzione di ceppo PAO381.

Progetto Genome Sequence per Pseudomonas aeruginosa Strain PAO579, un derivato di PAO381 mucoide.

&#x7DD1;&#x81BF;&#x83CC;&#x304C;&#x6162;&#x6027;&#x80BA;&#x611F;&#x67D3;&#x75C7;&#x56A2;&#x80DE;&#x6027;&#x7DDA;&#x7DAD;&#x75C7;&#x306B;&#x60A9;&#x3080;&#x4EBA;&#x3005; &#x306E;&#x500B;&#x4EBA;&#x306E;&#x78BA;&#x7ACB;&#x3001;&#x65E5;&#x548C;&#x898B;&#x75C5;&#x539F;&#x4F53;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30C9;&#x30E9;&#x30D5;&#x30C8;&#x7CFB;&#x7D71;&#x30B2;&#x30CE;&#x30E0;&#x306E;&#x7DD1;&#x81BF;&#x83CC; PAO579, &#x30A2;&#x30EB;&#x30AE;&#x30F3;&#x9178;&#x904E;&#x5270;&#x751F;&#x6210;&#x306E;&#x30C7;&#x30EA;&#x30D0;&#x30C6;&#x30A3;&#x30D6;&#x306E;&#x3072;&#x305A;&#x307F; PAO381 &#x3092;&#x767A;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;

&#x7DD1;&#x81BF;&#x83CC;&#x3072;&#x305A;&#x307F; PAO579 &#x306F;&#x4E2D;&#x819C;&#x30E0;&#x30B3;&#x30A4;&#x30C9; PAO381 &#x8A98;&#x5C0E;&#x4F53;&#x306E;&#x30C9;&#x30E9;&#x30D5;&#x30C8; &#x30B2;&#x30CE;&#x30E0; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3002;

&#xB179; &#xB18D; &#xADE0; &#xAC1C;&#xC778; &#xB0AD; &#xC131; &#xC12C;&#xC720; &#xC99D;&#xC5D0; &#xAC78;&#xB9AC;&#xB2E4;&#xC5D0; &#xB9CC;&#xC131; &#xD3D0; &#xAC10;&#xC5FC;&#xC744; &#xC124;&#xC815; &#xD558;&#xB294; &#xAE30;&#xD68C; &#xBCD1;&#xC6D0; &#xCCB4; &#xC774;&#xB2E4;. &#xC5EC;&#xAE30;, P. &#xB18D; &#xADE0; &#xC2A4;&#xD2B8;&#xB808;&#xC778; PAO579, &#xC2A4;&#xD2B8;&#xB808;&#xC778; pao381&#xC758; alginate overproducing &#xD30C;&#xC0DD;&#xC758; &#xCD08;&#xC548; &#xAC8C;&#xB188;&#xC744; &#xBC1C;&#xD45C;&#xD569;&#xB2C8;&#xB2E4;.

&#xB179; &#xB18D; &#xADE0; &#xC2A4;&#xD2B8;&#xB808;&#xC778; PAO579 pao381&#xC758; Mucoid &#xD30C;&#xC0DD; &#xC0C1;&#xD488;&#xC5D0; &#xB300; &#xD55C; &#xCD08;&#xC548; &#xAC8C;&#xB188; &#xC2DC;&#xD000;&#xC2A4;.

Pseudomonas aeruginosa es un pat&oacute;geno oportunista que establece una infecci&oacute;n pulmonar cr&oacute;nica en personas con fibrosis qu&iacute;stica. Aqu&iacute;, anunciamos el genoma de proyecto de p. aeruginosa cepa PAO579, un derivado de la sobreproducci&oacute;n de alginato de cepa PAO381.

Secuencia preliminar del genoma de Pseudomonas aeruginosa cepa PAO579, un derivado de PAO381 de mucoide.

Antimicrobial peptides (AMPs) can cause lysis of target bacteria by directly inserting themselves into the lipid bilayer. This killing mechanism confounds the identification of the intracellular targets of AMPs. To circumvent this, we used a shuttle vector containing the inducible expression of a human cathelicidin-related AMP, LL-37, to examine its effect on Escherichia coli TOP10 under aerobic and anaerobic growth conditions. Induction of LL-37 caused growth inhibition and alteration in cell morphology to a filamentous phenotype. Further examination of the E. coli cell division protein FtsZ revealed that LL-37 did not interact with FtsZ. Moreover, intracellular expression of LL-37 results in the enhanced production of reactive oxygen species (ROS), causing lethal membrane depolarization under aerobic conditions. Additionally, the membrane permeability was increased after intracellular expression of LL37 under both aerobic and anaerobic conditions. Transcriptomic analysis revealed that intracellular LL-37 mainly affected the expression of genes related to energy production and carbohydrate metabolism. More specifically, genes related to oxidative phosphorylation under both aerobic and anaerobic growth conditions were affected. Collectively, our current study demonstrates that intracellular expression of LL-37 in E. coli can inhibit growth under aerobic and anaerobic conditions. While we confirmed that the generation of ROS is a bactericidal mechanism for LL-37 under aerobic growth conditions, we also found that the intracellular accumulation of cationic LL-37 influences the redox and ion status of the cells under both growth conditions. These data suggest that there is a new AMP-mediated bacterial killing mechanism that targets energy metabolism.

Effect of intracellular expression of antimicrobial peptide LL-37 on growth of escherichia coli strain TOP10 under aerobic and anaerobic conditions.

Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU (AlgU(A61V)). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgU(A61V), 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ(70)). Induction of AlgU(A61V) in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgU(A61V) is functional in activating alginate production. Furthermore, the level of AlgU(A61V) was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgU(A61V) had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ(70) orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in all mucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependent promoter (PssrA ) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicate that the anti-σ(70) factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD.

Evidence for sigma factor competition in the regulation of alginate production by Pseudomonas aeruginosa.

A mutation in the mucA gene, which encodes a negative regulator of alginate production in Pseudomonas aeruginosa, is the main mechanism underlying the conversion to mucoidy in clinical isolates from patients with cystic fibrosis (CF). Here, we announce the draft genome sequence of the stable alginate-overproducing mucoid strain P. aeruginosa PAO581 with a mucA25 mutation, a derivative from the nonmucoid strains P. aeruginosa PAO381 and PAO1.

Draft Genome Sequence of a Stable Mucoid Strain of Pseudomonas aeruginosa PAO581 with a mucA25 Mutation.

Alginate overproduction by Pseudomonas aeruginosa, or mucoidy, plays an important role in the pathogenesis of chronic lung infections in cystic fibrosis (CF) patients. Here we report the draft genome sequence of a clinical isolate of mucoid P. aeruginosa strain C7447m from a CF patient with chronic lung infection.

Draft Genome Sequence of a Mucoid Isolate of Pseudomonas aeruginosa Strain C7447m from a Patient with Cystic Fibrosis.

In this study, we used whole genome complementation with a PAO1 cosmid library, coupled with in vitro transposon mutagenesis, to identify gene locus PA1494 as a novel inhibitor of alginate overproduction in P. aeruginosa strains possessing a wild-type mucA. This article is protected by copyright. All rights reserved.

Identification and characterization of a novel inhibitor of alginate overproduction in Pseudomonas aeruginosa.

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.

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

The small envelope protein MucE and the sensor kinase KinB are a positive and negative alginate regulator, respectively. Here, we announce the draft genome sequences of the alginate-overproducing variants Pseudomonas aeruginosa PAO1-VE2 (PAO1 with constitutive expression of mucE) and PAO1-VE13 (PAO1 with kinB inactivated). Both mutants were generated from a transposon mutagenesis screen.

T. Ryan Withers

Department of Biochemistry and Microbiology,
Joan C. Edwards School of Medicine,
Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine

Marshall University

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

T. Ryan Withers

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Microbiology,Joan C. Edwards School of Medicine,Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine

Marshall University

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Department of Biochemistry and Microbiology,
Joan C. Edwards School of Medicine,
Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine