capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Biozentrum of the University of Basel

We identified five different putative wav gene cluster types, which are responsible for the synthesis of the core oligosaccharide (OS) region of Vibrio cholerae lipopolysaccharide. Preliminary evidence that the genes encoded by this cluster are involved in core OS biosynthesis came from analysis of the recently released O1 El Tor V. cholerae genome sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of O1 El Tor mutant strains defective in three genes (waaF, waaL, and wavB). Investigations of 38 different V. cholerae strains by Southern blotting, PCR, and sequencing analyses showed that the O1 El Tor wav gene cluster type is prevalent among clinical isolates of different serogroups associated with cholera and environmental O1 strains. In contrast, we found differences in the wav gene contents of 19 unrelated non-O1, non-O139 environmental and human isolates not associated with cholera. These strains contained four new wav gene cluster types that differ from each other in distinct gene loci, providing evidence for horizontal transfer of wav genes and for limited structural diversity of the core OS among V. cholerae isolates. Our results show genetic diversity in the core OS biosynthesis gene cluster and predominance of the type 1 wav gene locus in strains associated with clinical cholera, suggesting that a specific core OS structure could contribute to V. cholerae virulence.

Comparative and genetic analyses of the putative Vibrio cholerae lipopolysaccharide core oligosaccharide biosynthesis (wav) gene cluster.

Since the first occurrence of O139 Vibrio cholerae as a cause of cholera epidemics, this serogroup has been investigated intensively, and it has been found that its pathogenicity is comparable to that of O1 El Tor strains. O139 isolates express a thin capsule, composed of a polymer of repeating units structurally identical to the lipopolysaccharide (LPS) O side chain. In this study, we investigated the role of LPS O side chain and capsular polysaccharide (CPS) in intestinal colonization by with genetically engineered mutants. We constructed CPS-negative, CPS/LPS O side chain-negative, and CPS-positive/LPS O side chain-negative mutants. Furthermore, we constructed two mutants with defects in LPS core oligosaccharide (OS) assembly. Loss of LPS O side chain or CPS resulted in a approximately 30-fold reduction in colonization of the infant mouse small intestine, indicating that the presence of both LPS O side chain and CPS is important during the colonization process. The strain lacking both CPS and LPS O side chain and a CPS-positive, LPS O side chain-negative core OS mutant were both essentially unable to colonize. To characterize the role of surface polysaccharides in survival in the host intestine, resistance to several antimicrobial substances was investigated in vitro. These investigations revealed that the presence of CPS protects the cell against attack of the complement system and that an intact core OS is necessary for survival in the presence of bile.

Role of Vibrio cholerae O139 surface polysaccharides in intestinal colonization.

&#x835A;&#x819C;&#x591A;&#x7CD6; (CPS) &#x7684;&#x7A0B;&#x5E8F;&#x96C6;&#x548C;&#x5176;&#x79FB;&#x4F4D;&#x5230;&#x7EC6;&#x83CC;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x7684;&#x665A;&#x6B65;&#x9AA4;&#x8FD8;&#x4E0D;&#x592A;&#x6E05;&#x695A;&#x3002;Wza &#x86CB;&#x767D;&#x662F;&#x4EE5;&#x524D;&#x6240;&#x793A;&#xFF0C;&#x9700;&#x8981;&#x5F62;&#x6210;&#x7684;&#x539F;&#x578B;&#x7EC4; 1 &#x80F6;&#x56CA;&#x7ED3;&#x6784;&#x8868;&#x9762;&#x7684;&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC;&#x8840;&#x6E05;&#x578B; K30 &#xFF08;Drummelsmith&#xFF0C;J.&#x548C;&#x60E0;&#x7279;&#x83F2;&#x5C14;&#x5FB7;&#xFF0C;C.&#xFF08;2000 &#x5E74;&#xFF09; &#x6069;&#x535A; J.19&#xFF0C;57-66&#xFF09;&#x3002;Wza &#x662F;&#x5B88;&#x6052;&#x7684;&#x5916;&#x819C;&#x8102;&#x86CB;&#x767D;&#x5F62;&#x6210;&#x591A;&#x805A;&#x4F53;&#x91C7;&#x7528;&#x73AF;&#x5F62;&#x7ED3;&#x6784;&#xFF0C;&#x548C;&#x96C6;&#x4F53;&#x7684;&#x8BC1;&#x636E;&#x8868;&#x660E;&#x8FD9;&#x4E9B;&#x7ED3;&#x6784;&#x7684;&#x56CA;&#x888B;&#x805A;&#x5408;&#x7269;&#x7684;&#x51FA;&#x53E3;&#x7684;&#x4F5C;&#x7528;&#x8DE8;&#x5916;&#x819C;&#x3002;Wza &#x88AB;&#x51C0;&#x5316;&#x4EE5;&#x672C;&#x673A;&#x5F62;&#x5F0F;&#x548C;&#x4E0E; C &#x7EC8;&#x7AEF; hexahistidine &#x6807;&#x8BB0;&#x3002;WzaHis6 &#x662F;&#x9170;&#x5316;&#x548C;&#x529F;&#x80FD;&#x5728;&#x80F6;&#x56CA;&#x5927;&#x4F1A;&#xFF0C;&#x867D;&#x7136;&#x5176;&#x6548;&#x7387;&#x4E0E;&#x672C;&#x673A; Wza &#x86CB;&#x767D;&#x76F8;&#x6BD4;&#x7565;&#x6709;&#x51CF;&#x5C11;&#x3002;&#x7EAF;&#x5316;&#x7684;&#x591A;&#x805A;&#x4F53;&#x6210;&#x8840;&#x8102;&#x91CD;&#x7EC4;&#x540E;&#x83B7;&#x5F97;&#x4E86; WzaHis6 &#x7684;&#x6709;&#x5E8F;&#x7684;&#x4E8C;&#x7EF4;&#x6676;&#x4F53;&#x3002;&#x8D1F;&#x67D3;&#x8272;&#x7684;&#x6676;&#x4F53;&#x548C;&#x5085;&#x91CC;&#x53F6;&#x8FC7;&#x6EE4;&#x955C;&#x53D1;&#x73B0;&#x73AF;&#x72B6;&#x4E32;&#x8054;&#x4F53; 8.84 &#x5E73;&#x5747;&#x5916;&#x5F84; nm &#x548C; 2.28 &#x5E73;&#x5747;&#x7684;&#x4E2D;&#x592E;&#x8154;&#x76F4;&#x5F84; nm&#x3002;&#x5355;&#x7C92;&#x5B50;&#x5206;&#x6790;&#x4EA7;&#x751F; 3 &#x4F30;&#x8BA1;&#x5206;&#x8FA8;&#x7387;&#x6295;&#x5F71;&#x7ED3;&#x6784; nm&#xFF0C;&#x6709;&#x5229;&#x4E8E;&#x4E3A; WzaHis6 &#x5305;&#x542B;&#x516B;&#x4E2A;&#x76F8;&#x540C;&#x7684;&#x4E9A;&#x57FA;&#x7ED3;&#x6784;&#x3002;Wza (Wza *) &#x4E2D;&#x7684;&#x539F;&#x59CB;&#x4FE1;&#x53F7;&#x5E8F;&#x5217;&#x88AB;&#x66FF;&#x6362;&#xFF0C;&#x4ECE;&#x5931;&#x8E2A;&#x7684;&#x5BFC;&#x6570;&#x8868;&#x660E; Wza &#x7684;&#x672C;&#x673A;&#x9170;&#x5316; N &#x603B;&#x7AD9;&#x662F;&#x6B63;&#x5E38;&#x7684; multimeric &#x7ED3;&#x6784;&#x7684;&#x5F62;&#x6210;&#xFF0C;&#x5176;&#x6743;&#x9650;&#x4E3A; CPS &#x7A0B;&#x5E8F;&#x96C6;&#xFF0C;&#x800C;&#x4E0D;&#x662F; Wza &#x9776;&#x5411;&#x5916;&#x819C;&#x7684;&#x5173;&#x952E;&#x3002;&#x5728;&#x573A;&#x7684;&#x60C5;&#x51B5;&#x4E0B; Wza * CPS &#x79EF;&#x7D2F;&#x5728; periplasm &#x4E2D;&#xFF0C;&#x4F46;&#x672A;&#x68C0;&#x6D4B;&#x5230;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x3002;&#x5316;&#x5B66;&#x4EA4;&#x8054;&#x7684;&#x5B8C;&#x6574;&#x7EC6;&#x80DE;&#x5EFA;&#x8BAE;&#x5FAE;&#x542B; Wza &#x548C;&#x5185;&#x5728;&#x819C;&#x916A;&#x6C28;&#x9178; autokinase Wzc &#x8DE8;&#x819C;&#x590D;&#x5408;&#x7269;&#x7684;&#x5F62;&#x6210;&#x3002;

&#x79FB;&#x4F4D;&#x7684;&#x7B2C; 1 &#x7EC4;&#x4E2D;&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC;&#x8840;&#x6E05;&#x578B; K30 &#x835A;&#x819C;&#x591A;&#x7CD6;&#x3002;Wza &#x5916;&#x819C;&#x8102;&#x86CB;&#x767D;&#x7684;&#x7ED3;&#x6784;&#x548C;&#x529F;&#x80FD;&#x5206;&#x6790;&#x3002;

The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. WzaHis6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of WzaHis6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the WzaHis6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.

Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza.

Les &#xE9;tapes tardives de l&#x26;#39;assemblage de polysaccharides capsulaires (CPS) et de leur translocation &#xE0; la surface de la cellule bact&#xE9;rienne ne sont pas bien comprises. La prot&#xE9;ine a &#xE9;t&#xE9; montr&#xE9; pr&#xE9;c&#xE9;demment WZA &#xEA;tre requis pour la formation de la structure du prototype du groupe capsule 1 sur la surface de Escherichia coli de s&#xE9;rotype K30 (Drummelsmith, J., et Whitfield, C. (2000) EMBO J. 19, 57-66) . WZA est une lipoprot&#xE9;ine de membrane externe qui forme conserv&#xE9;e multim&#xE8;res adoptant une structure en forme d&#x26;#39;anneau, et ensemble des donn&#xE9;es sugg&#xE8;re un r&#xF4;le pour ces structures dans l&#x26;#39;exportation de polym&#xE8;re capsulaire &#xE0; travers la membrane externe. WZA a &#xE9;t&#xE9; purifi&#xE9; sous la forme native et avec une &#xE9;tiquette hexahistidine C-terminale. WzaHis6 &#xE9;tait acyl&#xE9; et fonctionnel dans l&#x26;#39;assemblage de capsule, m&#xEA;me si son efficacit&#xE9; a &#xE9;t&#xE9; l&#xE9;g&#xE8;rement r&#xE9;duite par rapport &#xE0; la prot&#xE9;ine native WZA. Ordonn&#xE9;es des cristaux bidimensionnels de WzaHis6 ont &#xE9;t&#xE9; obtenues apr&#xE8;s la reconstitution de multim&#xE8;res purifi&#xE9; dans les lipides. La microscopie &#xE9;lectronique de cristaux color&#xE9;s n&#xE9;gativement et filtrage de Fourier a r&#xE9;v&#xE9;l&#xE9; multim&#xE8;res annulaires d&#x26;#39;un diam&#xE8;tre moyen ext&#xE9;rieur de 8,84 nm et un diam&#xE8;tre de la cavit&#xE9; centrale moyenne de 2,28 nm. L&#x26;#39;analyse des particules unique a abouti &#xE0; des structures de projection &#xE0; une r&#xE9;solution estim&#xE9;e de 3 nm, favorisant une structure pour la WzaHis6 contenant huit sous-unit&#xE9;s identiques. Un d&#xE9;riv&#xE9; de WZA (WZA *) dans laquelle la s&#xE9;quence signal d&#x26;#39;origine a &#xE9;t&#xE9; remplac&#xE9; par de OmpF que montr&#xE9; que l&#x26;#39;extr&#xE9;mit&#xE9; N de natif acyl&#xE9; WZA est critique pour la formation de structures de multim&#xE8;res normales et pour leur comp&#xE9;tence pour CPS ensemble, mais non pour le ciblage WZA &#xE0; la membrane externe. En pr&#xE9;sence d&#x26;#39;* WZA, SCP accumul&#xE9;es dans le p&#xE9;riplasme, mais n&#x26;#39;a pas &#xE9;t&#xE9; d&#xE9;tect&#xE9; sur la surface cellulaire. Chimique de r&#xE9;ticulation de cellules intactes sugg&#xE9;r&#xE9; formation d&#x26;#39;un complexe contenant transmembranaire minimale WZA et l&#x26;#39;int&#xE9;rieure de membrane tyrosine autokinase WZC.

La translocation de groupe 1 polysaccharide capsulaire chez Escherichia coli de s&#xE9;rotype K30. Analyse structurale et fonctionnelle de l&#x26;#39;ext&#xE9;rieur des lipoprot&#xE9;ines membrane WZA

Die Translokation der Gruppe 1 Kapselpolysaccharid in Escherichia coli Serotyp K30. Strukturelle und funktionelle Analyse der &#xE4;u&#xDF;eren Membran Lipoprotein WZA.

I tardi passaggi in Assemblea di polisaccaridi capsulari (CPS) e loro traslocazione alla superficie delle cellule batteriche non sono ben compresi. La proteina Wza fu mostrata precedentemente richiesto per la formazione della struttura prototipo gruppo 1 capsula sulla superficie del sierotipo di Escherichia coli K30 (Drummelsmith, J. e Whitfield, c. (2000) EMBO J. 19, 57-66). Wza &egrave; una lipoproteina conservata membrana esterna che si forma multimeri adottando una struttura hanno, e prova collettiva suggerisce un ruolo per queste strutture nell&#39;esportazione del polimero capsula attraverso la membrana esterna. Wza &egrave; stato purificato in forma nativa e con un&#39;etichetta di hexahistidine C-terminale. WzaHis6 era acilata e funzionale in capsule assieme, anche se la sua efficienza &egrave; stata leggermente ridotta rispetto alla proteina nativa Wza. Ordinato cristalli bi-dimensionali di WzaHis6 sono stati ottenuti dopo la ricostituzione dei multimeri purificato in lipidi. La microscopia elettronica di cristalli colorati negativamente e Fourier filtraggio rivelato ridurre multimeri con diametro esterno medio di 8,84 nm e un diametro medio di cavit&agrave; centrale di 2,28 nm. Singola analisi di particelle ha reso le strutture proiezione a una risoluzione stimata di 3 nm, favorendo una struttura per il WzaHis6 contenente otto subunit&agrave; identiche. Un derivato di Wza (Wza *) in cui la sequenza del segnale originale &egrave; stata sostituita con quello da OmpF ha mostrato che il nativo acilata N capolinea della Wza &egrave; critico per la formazione di strutture multimerica normale e per la loro competenza per Assemblea CPS, ma non per il targeting Wza alla membrana esterna. In presenza di Wza *, CPS accumulato nella periplasm ma non &egrave; stato rilevato sulla superficie della cellula. Reticolazione chimica delle cellule intatte suggerito la formazione di un complesso transmembrana contenente minimamente Wza e la membrana interna tirosina autokinase Wzc.

Traslocazione del polisaccaride capsulare gruppo 1 in sierotipo di Escherichia coli K30. Analisi strutturale e funzionale della lipoproteina membrana esterna Wza.

&#x7D30;&#x83CC;&#x306E;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x306B;&#x83A2;&#x819C;&#x591A;&#x7CD6;&#xFF08;CPS&#xFF09;&#x3068;&#x305D;&#x306E;&#x8EE2;&#x5EA7;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x5185;&#x306E;&#x5F8C;&#x534A;&#x306E;&#x624B;&#x9806;&#x304C;&#x3088;&#x304F;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3002; Wza&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306F;&#x5927;&#x8178;&#x83CC;&#x8840;&#x6E05;&#x578B;K30&#xFF08;Drummelsmith&#x3001;J.&#x3068;&#x30DB;&#x30A4;&#x30C3;&#x30C8;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x30C9;&#x3001;C.&#xFF08;2000&#xFF09;EMBO J. 19&#x3001;57&#x304B;&#x3089;66&#xFF09;&#x306E;&#x8868;&#x9762;&#x4E0A;&#x306B;&#x8A66;&#x4F5C;&#x3057;&#x305F;&#x30B0;&#x30EB;&#x30FC;&#x30D7;1&#x30AB;&#x30D7;&#x30BB;&#x30EB;&#x69CB;&#x9020;&#x306E;&#x5F62;&#x6210;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x4EE5;&#x524D;&#x306B;&#x793A;&#x3055;&#x308C;&#x305F;&#x3002; Wza&#x306F;&#x3001;&#x30EA;&#x30F3;&#x30B0;&#x72B6;&#x306E;&#x69CB;&#x9020;&#x3092;&#x63A1;&#x7528;&#x3057;&#x3066;&#x591A;&#x91CF;&#x4F53;&#x3092;&#x5F62;&#x6210;&#x3057;&#x3066;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x5916;&#x819C;&#x30EA;&#x30DD;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3067;&#x3042;&#x308A;&#x3001;&#x96C6;&#x56E3;&#x7684;&#x8A3C;&#x62E0;&#x306F;&#x3001;&#x5916;&#x5074;&#x306E;&#x819C;&#x3092;&#x6A2A;&#x5207;&#x3063;&#x3066;&#x83A2;&#x819C;&#x30DD;&#x30EA;&#x30DE;&#x30FC;&#x306E;&#x8F38;&#x51FA;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x69CB;&#x9020;&#x306E;&#x5F79;&#x5272;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002; Wza&#x306F;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x5F62;&#x5F0F;&#x3067;&#x3001;C&#x672B;&#x7AEF;&#x30D2;&#x30B9;&#x30C1;&#x30B8;&#x30F3;&#x30BF;&#x30B0;&#x3092;&#x6301;&#x3064;&#x7CBE;&#x88FD;&#x3057;&#x305F;&#x3002;&#x305D;&#x306E;&#x52B9;&#x7387;&#x304C;&#x308F;&#x305A;&#x304B;&#x306B;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;Wza&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x6E1B;&#x5C11;&#x3057;&#x305F;&#x3082;&#x306E;&#x306E;WzaHis6&#x306F;&#x3001;&#x30A2;&#x30B7;&#x30EB;&#x5316;&#x3068;&#x30AB;&#x30D7;&#x30BB;&#x30EB;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x6A5F;&#x80FD;&#x7684;&#x3067;&#x3042;&#x3063;&#x305F;&#x3002; WzaHis6&#x306E;&#x9806;&#x5E8F;&#x4ED8;&#x3051;&#x3055;&#x308C;&#x305F;&#x4E8C;&#x6B21;&#x5143;&#x7D50;&#x6676;&#x306F;&#x3001;&#x8102;&#x8CEA;&#x306B;&#x591A;&#x91CF;&#x306E;&#x7CBE;&#x88FD;&#x518D;&#x69CB;&#x6210;&#x5F8C;&#x306B;&#x5F97;&#x3089;&#x308C;&#x305F;&#x3002;&#x96FB;&#x5B50;&#x30CD;&#x30AC;&#x30C6;&#x30A3;&#x30D6;&#x67D3;&#x8272;&#x306E;&#x7D50;&#x6676;&#x306E;&#x9855;&#x5FAE;&#x93E1;&#x304A;&#x3088;&#x3073;&#x30D5;&#x30FC;&#x30EA;&#x30A8;&#x30D5;&#x30A3;&#x30EB;&#x200B;&#x200B;&#x30BF;&#x30EA;&#x30F3;&#x30B0;&#x306F;8.84 nm&#x3068;2.28 nm&#x306E;&#x5E73;&#x5747;&#x5024;&#x3001;&#x4E2D;&#x592E;&#x306E;&#x7A7A;&#x6D1E;&#x5F84;&#x306E;&#x5E73;&#x5747;&#x5916;&#x5F84;&#x3068;&#x30EA;&#x30F3;&#x30B0;&#x72B6;&#x306E;&#x591A;&#x91CF;&#x4F53;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x3002;&#x5358;&#x7C92;&#x5B50;&#x89E3;&#x6790;&#x3067;&#x306F;&#x3001;WzaHis6&#x3092;&#x542B;&#x3080;8&#x3064;&#x306E;&#x540C;&#x4E00;&#x306E;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x69CB;&#x9020;&#x3092;&#x652F;&#x6301;&#x3059;&#x308B;&#x3001;3nm&#x306E;&#x63A8;&#x5B9A;&#x306E;&#x89E3;&#x50CF;&#x5EA6;&#x3067;&#x6295;&#x5F71;&#x69CB;&#x9020;&#x3092;&#x5F97;&#x305F;&#x3002;&#x30AA;&#x30EA;&#x30B8;&#x30CA;&#x30EB;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x914D;&#x5217;&#x304C;OmpF&#x304B;&#x3089;&#x305D;&#x306E;&#x7F6E;&#x304D;&#x63DB;&#x3048;&#x3055;&#x308C;&#x305F;Wza&#x306E;&#x8A98;&#x5C0E;&#x4F53;&#xFF08;Wza *&#xFF09;&#x306F;Wza&#x306E;&#x30A2;&#x30B7;&#x30EB;&#x5316;&#x3055;&#x308C;&#x305F;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x306E;N&#x672B;&#x7AEF;&#x306F;&#x3001;&#x901A;&#x5E38;&#x306E;&#x591A;&#x91CF;&#x4F53;&#x69CB;&#x9020;&#x306E;&#x5F62;&#x6210;&#x304A;&#x3088;&#x3073;CPS&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x5F7C;&#x3089;&#x306E;&#x80FD;&#x529B;&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x660E;&#x3089;&#x304B;&#x306B;&#x306A;&#x3063;&#x305F;&#x3067;&#x306F;&#x306A;&#x304F;&#x3001;Wza&#x3092;&#x6A19;&#x7684;&#x3068;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5916;&#x819C;&#x306B;&#x3002; Wza *&#x306E;&#x5B58;&#x5728;&#x4E0B;&#x3067;&#x306F;&#x3001;&#x30DA;&#x30EA;&#x30D7;&#x30E9;&#x30BA;&#x30E0;&#x306B;&#x84C4;&#x7A4D;&#x3055;&#x308C;&#x307E;&#x3059;&#x304C;&#x3001;CPS&#x306F;&#x3001;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x3067;&#x691C;&#x51FA;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x7121;&#x50B7;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x5316;&#x5B66;&#x7684;&#x67B6;&#x6A4B;&#x306F;&#x3001;&#x6700;&#x5C0F;&#x9650;Wza&#x3068;&#x5185;&#x819C;&#x30C1;&#x30ED;&#x30B7;&#x30F3;autokinase WZC&#x3092;&#x542B;&#x3080;&#x819C;&#x8CAB;&#x901A;&#x8907;&#x5408;&#x4F53;&#x306E;&#x5F62;&#x6210;&#x3092;&#x793A;&#x5506;&#x3057;&#x305F;&#x3002;

&#x5927;&#x8178;&#x83CC;&#x8840;&#x6E05;&#x578B;K30&#x306E;&#x30B0;&#x30EB;&#x30FC;&#x30D7;1&#x306E;&#x83A2;&#x819C;&#x591A;&#x7CD6;&#x306E;&#x8EE2;&#x3002;&#x5916;&#x819C;&#x30EA;&#x30DD;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;Wza&#x306E;&#x69CB;&#x9020;&#x3068;&#x6A5F;&#x80FD;&#x89E3;&#x6790;

&#xC138;&#xADE0;&#xC131; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; capsular &#xB2E4; &#xB2F9; &#xB958; (CPS)&#xC758; &#xC870;&#xB9BD; &#xBC0F; &#xADF8;&#xB4E4;&#xC758; &#xC804; &#xD6C4;&#xBC18; &#xB2E8;&#xACC4; &#xC798; &#xC774;&#xD574; &#xB418;&#xC9C0; &#xC54A;&#xB294;&#xB2E4;. Wza &#xB2E8;&#xBC31;&#xC9C8; (Drummelsmith, &#xC81C;&#xC774;, &#xADF8;&#xB9AC;&#xACE0; &#xC717; &#xD544;&#xB4DC;, C. (2000) EMBO J. 19, 57-66) &#xBCD1;&#xC6D0; &#xC131; &#xB300;&#xC7A5;&#xADE0; serotype K30 &#xD45C;&#xBA74;&#xC5D0; &#xC6D0;&#xD615; &#xADF8;&#xB8F9; 1 &#xCEA1;&#xC290; &#xAD6C;&#xC870; &#xD615;&#xC131;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xC774;&#xC804;&#xC5D0; &#xD45C;&#xC2DC; &#xD588;&#xB2E4;. Wza &#xD615;&#xC131; multimers ringlike &#xAD6C;&#xC870;&#xB97C; &#xCC44;&#xD0DD; &#xD558;&#xB294; &#xBCF4;&#xC874;&#xB41C; &#xC678;&#xBD80; &#xB9C9; &#xB2E8;&#xBC31; &#xC774;&#xBA70; &#xC9D1;&#xB2E8; &#xC99D;&#xAC70; &#xC678;&#xBD80; &#xB9C9;&#xC5D0; &#xAC78;&#xCCD0; capsular &#xD3F4;&#xB9AC;&#xBA38; &#xC218;&#xCD9C;&#xC5D0; &#xC774;&#xB7EC;&#xD55C; &#xAD6C;&#xC870;&#xC5D0; &#xB300; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xC548;. Wza C &#xD130;&#xBBF8;&#xB110; hexahistidine &#xD0DC;&#xADF8;&#xC640; &#xAE30;&#xBCF8; &#xD615;&#xD0DC;&#xB85C; &#xC21C;&#xD654; &#xB418;&#xC5C8;&#xB2E4;. WzaHis6 acylated&#xB294; &#xACE0; &#xAE30;&#xB2A5; &#xCEA1;&#xC290; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0;&#xC11C; &#xD6A8;&#xC728;&#xC131; &#xB124;&#xC774;&#xD2F0;&#xBE0C; Wza &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xBE44;&#xD574; &#xC57D;&#xAC04; &#xAC10;&#xC18C; &#xD588;&#xB2E4;. Wzahis6&#xC758; &#xC21C;&#xC11C;&#xAC00; &#xC9C0;&#xC815; &#xB41C; 2 &#xCC28;&#xC6D0; &#xACB0;&#xC815;&#xC740; &#xC9C0;&#xC9C8;&#xC73C;&#xB85C; &#xC21C;&#xD654; multimers&#xC758; &#xC7AC;&#xAD6C;&#xC131; &#xD6C4; &#xC5BB;&#xC740; &#xD588;&#xB2E4;. &#xBD80;&#xC815;&#xC801; &#xC2A4;&#xD14C;&#xC778;&#xB4DC; &#xD06C;&#xB9AC;&#xC2A4;&#xD0C8; &#xBC0F; &#xD544;&#xD130;&#xB9C1; &#xD478;&#xB9AC;&#xC5D0;&#xC758; &#xC804;&#xC790; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95; &#xACF5;&#xAC1C; 8.84&#xC758; &#xD3C9;&#xADE0; &#xBC14;&#xAE65; &#xC9C0;&#xB984;&#xACFC; ringlike multimers nm&#xC640; 2.28&#xC758; &#xD3C9;&#xADE0; &#xC911;&#xC559; &#xAD6C;&#xBA4D; &#xC9C1;&#xACBD; nm. &#xB2E8;&#xC77C; &#xC785;&#xC790; &#xBD84;&#xC11D; 3&#xC758; &#xC608;&#xC0C1; &#xD574;&#xC0C1;&#xB3C4; &#xD22C;&#xC601; &#xAD6C;&#xC870; &#xB098;&#xC654;&#xACE0; nm, 8 &#xAC1C;&#xC758; &#xB3D9;&#xC77C;&#xD55C; &#xD558;&#xC704; &#xB2E8;&#xC704;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xB294; Wzahis6&#xC5D0; &#xB300; &#xD55C; &#xAD6C;&#xC870;&#xB97C; &#xC560; &#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. Wza (Wza *)&#xB294; &#xC6D0;&#xB798; &#xC2E0;&#xD638; &#xC21C;&#xC11C;&#xB294; Ompf&#xC5D0;&#xC11C; &#xADF8;&#xAC78;&#xB85C; &#xAD50;&#xCCB4; &#xB410;&#xB2E4;&#xC758; &#xD30C;&#xC0DD; Wza &#xB124;&#xC774;&#xD2F0;&#xBE0C; acylated N &#xB9D0;&#xB2E8; &#xC815;&#xC0C1;&#xC801;&#xC778; multimeric &#xAD6C;&#xC870;&#xC758; &#xD615;&#xC131; &#xBC0F; CPS &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0; &#xB300; &#xD55C; &#xD558;&#xC9C0;&#xB9CC; &#xC678;&#xBD80; &#xB9C9; Wza &#xD0C0;&#xAC9F;&#xD305;&#xC5D0; &#xB300; &#xD55C; &#xADF8;&#xB4E4;&#xC758; &#xC5ED;&#xB7C9; &#xC911;&#xC694; &#xBCF4;&#xC5EC;&#xC8FC;&#xC5C8;&#xB2E4;. Wza * &#xC874;&#xC7AC; CPS&#xB294; periplasm&#xC5D0; &#xCD95;&#xC801; &#xD558;&#xC9C0;&#xB9CC; &#xC140; &#xD45C;&#xBA74;&#xC5D0;&#xC11C; &#xAC80;&#xC0C9; &#xB418;&#xC9C0; &#xC54A;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB300;&#xB85C; &#xC140; &#xD654;&#xD559; &#xAD50;&#xAC00; &#xB9C9; &#xD6A1;&#xB2E8; &#xBCF5;&#xC7A1; &#xD55C; &#xCD5C;&#xC18C;&#xD55C;&#xC73C;&#xB85C; &#xD3EC;&#xD568; &#xB41C; Wza &#xB0B4;&#xBD80; &#xB9C9; &#xD2F0;&#xB85C;&#xC2E0; autokinase Wzc&#xC758; &#xD615;&#xC131;&#xC744; &#xC81C;&#xC548; &#xD588;&#xB2E4;.

&#xBCD1;&#xC6D0; &#xC131; &#xB300;&#xC7A5;&#xADE0; serotype k 30&#xC5D0;&#xC5D0;&#xC11C; &#xADF8;&#xB8F9; 1 capsular &#xB2E4; &#xB2F9; &#xB958; &#xC804;. Wza &#xC678;&#xBD80; &#xB9C9; &#xB2E8;&#xBC31;&#xC758; &#xAD6C;&#xC870;&#xC640; &#xAE30;&#xB2A5; &#xBD84;&#xC11D;.

Los pasos finales en el montaje de los polisac&#xE1;ridos capsulares (CPS) y su translocaci&#xF3;n a la superficie celular de la bacteria no se entienden bien. La prote&#xED;na Wza se hab&#xED;a demostrado que se requiere para la formaci&#xF3;n de la estructura del grupo prototipo c&#xE1;psula 1 en la superficie de la Escherichia coli serotipo K30 (Drummelsmith, J., y Whitfield, C. (2000) EMBO J. 19, 57-66) . Wza es una lipoprote&#xED;na de membrana externa conservado que forma mult&#xED;meros la adopci&#xF3;n de una estructura de anillo, y la evidencia colectiva sugiere un papel de estas estructuras en la exportaci&#xF3;n de pol&#xED;mero capsular a trav&#xE9;s de la membrana externa. Wza se purific&#xF3; en la forma nativa y con una etiqueta hexahistidina C-terminal. WzaHis6 era acilado y funcionales en el ensamblaje de c&#xE1;psulas, aunque su eficacia se redujo ligeramente en comparaci&#xF3;n con la prote&#xED;na nativa Wza. Ordenadas cristales bidimensionales de WzaHis6 se obtuvieron despu&#xE9;s de la reconstituci&#xF3;n de mult&#xED;meros purificada en los l&#xED;pidos. La microscop&#xED;a electr&#xF3;nica de cristales te&#xF1;idos negativamente y Filtro de Fourier revel&#xF3; mult&#xED;meros anulares con un di&#xE1;metro medio exterior de 8,84 nm y un di&#xE1;metro medio cavidad central de 2,28 nm. El an&#xE1;lisis de part&#xED;culas solo cedi&#xF3; las estructuras de proyecci&#xF3;n con una resoluci&#xF3;n aproximada de 3 nm, lo que favorece una estructura para el WzaHis6 contiene ocho subunidades id&#xE9;nticas. Un derivado de Wza (Wza *) en el que se sustituye la secuencia se&#xF1;al original con la de OmpF mostr&#xF3; que el t&#xE9;rmino nativo acilado N de Wza es cr&#xED;tica para la formaci&#xF3;n de estructuras normales multim&#xE9;ricas y por su competencia para CPS montaje, pero no para la focalizaci&#xF3;n Wza a la membrana externa. En presencia de * Wza, CPS acumulados en el periplasma, pero no se detect&#xF3; en la superficie celular. Reticulaci&#xF3;n qu&#xED;mica de las c&#xE9;lulas intactas sugiri&#xF3; la formaci&#xF3;n de un complejo que contiene transmembrana m&#xED;nimamente Wza y la membrana interna tirosina autokinase WZC.

La translocaci&#xF3;n de un Grupo de polisac&#xE1;rido capsular en Escherichia coli serotipo K30. An&#xE1;lisis estructural y funcional de la membrana externa de las lipoprote&#xED;nas de Wza

A novel integral membrane lipoprotein, Wza, from Escherichia coli serotype O9a:K30 has been purified and crystallized. Wza is required for the surface expression of the serotype K30 group 1 capsular polysaccharide of E. coli; closely related homologues are found in other bacteria that produce extracellular polysaccharides. The Wza crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 94.6, b = 215.5, c = 218.5 A. A data set to 3.0 A with 99.8% completeness and an R(merge) of 10.5% has been collected from a single crystal.

Crystallization and preliminary X-ray diffraction analysis of Wza outer-membrane lipoprotein from Escherichia coli serotype O9a:K30.

Many types of bacteria produce extracellular polysaccharides (EPSs). Some are secreted polymers and show only limited association with the cell surface, whereas others are firmly attached to the cell surface and form a discrete structural layer, the capsule, which envelopes the cell and allows the bacteria to evade or counteract the host immune system. EPSs have critical roles in bacterial colonization of surfaces, such as epithelia and medical implants; in addition some EPSs have important industrial and biomedical applications in their own right. Here we describe the 2.26 A resolution structure of the 340 kDa octamer of Wza, an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel alpha-helical barrel. The bulk of the Wza structure is located in the periplasm and comprises three novel domains forming a large central cavity. Wza is open to the extracellular environment but closed to the periplasm. We propose a route and mechanism for translocation of the capsular polysaccharide. This work may provide insight into the export of other large polar molecules such as DNA and proteins.

Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein.

The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-alpha subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-alpha for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.

The peptidyl prolyl cis/trans isomerase FKBP38 determines hypoxia-inducible transcription factor prolyl-4-hydroxylase PHD2 protein stability.

The activating transcription factor-4 (ATF-4) is translationally induced under anoxic conditions, mediates part of the unfolded protein response following endoplasmic reticulum (ER) stress, and is a critical regulator of cell fate. Here, we identified the zipper II domain of ATF-4 to interact with the oxygen sensor prolyl-4-hydroxylase domain 3 (PHD3). The PHD inhibitors dimethyloxalylglycine (DMOG) and hypoxia, or proteasomal inhibition, all induced ATF-4 protein levels. Hypoxic induction of ATF-4 was due to increased protein stability, but was independent of the ubiquitin ligase von Hippel-Lindau protein (pVHL). A novel oxygen-dependent degradation (ODD) domain was identified adjacent to the zipper II domain. Mutations of 5 prolyl residues within this ODD domain or siRNA-mediated down-regulation of PHD3, but not of PHD2, was sufficient to stabilize ATF-4 under normoxic conditions. These data demonstrate that PHD-dependent oxygen-sensing recruits both the hypoxia-inducible factor (HIF) and ATF-4 systems, and hence not only confers adaptive responses but also cell fate decisions.

Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor.

Proteins belonging to the Omp85 family are involved in the assembly of beta-barrel outer membrane proteins or in the translocation of proteins across the outer membrane in bacteria, mitochondria, and chloroplasts. The cell envelope of the thermophilic bacterium Thermus thermophilus HB27 is multilayered, including an outer membrane that is not well characterized. Neither the precise lipid composition nor much about integral membrane proteins is known. The genome of HB27 encodes one Omp85-like protein, Omp85(Tt), representing an ancestral type of this family. We overexpressed Omp85(Tt) in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified Omp85(Tt) existed mainly as a monomer, composed of two stable protease-resistant modules. Circular dichroism spectroscopy indicated predominantly beta-sheet secondary structure. Electron microscopy of negatively stained lipid-embedded Omp85(Tt) revealed ring-like structures with a central cavity of approximately 1.5 nm in diameter. Single-channel conductance recordings indicated that Omp85(Tt) forms ion channels with two different conducting states, characterized by conductances of approximately 0.4 nS and approximately 0.65 nS, respectively.

Omp85(Tt) from Thermus thermophilus HB27: an ancestral type of the Omp85 protein family.

The cell envelope of the thermophilic bacterium Thermus thermophilus is multilayered and includes an outer membrane with integral outer membrane proteins that are not well characterized. The hypothetical protein TTC0834 from T. thermophilus HB27 was identified as a 22 kDa outer membrane protein containing eight predicted beta-strands. TTC0834 was expressed with an N-terminal His tag in T. thermophilus HB8 and detected in the S-layer/outer membrane envelope fraction. His-TTC0834 was purified and crystallized under various conditions. Native data sets were collected to 3.2 A resolution and the best diffracting crystals belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 166.67, c = 97.53 A.

Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from Thermus thermophilus HB27.

The thermophilic eubacterium Thermus thermophilus belongs to one of the oldest branches of evolution and has a multilayered cell envelope that differs from that of modern Gram-negative bacteria. Its outer membrane contains integral outer membrane proteins (OMPs), of which only a few are characterized. TtoA, a new beta-barrel OMP, was identified by searching the genome sequence of strain HB27 for the presence of a C-terminal signature sequence. The structure of TtoA was determined to a resolution of 2.8 A, representing the first crystal structure of an OMP from a thermophilic bacterium. TtoA consists of an eight-stranded beta-barrel with a large extracellular part to which a divalent cation is bound. A five-stranded extracellular beta-sheet protrudes out of the membrane-embedded transmembrane barrel and is stabilized by a disulfide bridge. The edge of this beta-sheet forms crystal contacts that could mimic interactions with other proteins. In modern Gram-negative bacteria, the C-terminal signature sequence of OMPs is required for binding to an Omp85 family protein as a prerequisite for its assembly. We present hints that a similar assembly pathway exists in T. thermophilus by an in vitro binding assay, where unfolded TtoA binds to the Thermus Omp85 family protein TtOmp85, while a mutant without the signature sequence does not.

Crystal structure of a major outer membrane protein from Thermus thermophilus HB27.

In Myxococcus xanthus the extracellular matrix is essential for type IV pili-dependent motility and starvation-induced fruiting body formation. Proteins of two-component systems including the orphan DNA binding response regulator DigR are essential in regulating the composition of the extracellular matrix. We identify the orphan hybrid histidine kinase SgmT as the partner kinase of DigR. In addition to kinase and receiver domains, SgmT consists of an N-terminal GAF domain and a C-terminal GGDEF domain. The GAF domain is the primary sensor domain. The GGDEF domain binds the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP) and functions as a c-di-GMP receptor to spatially sequester SgmT. We identify the DigR binding site in the promoter of the fibA gene, which encodes an abundant extracellular matrix metalloprotease. Whole-genome expression profiling experiments in combination with the identified DigR binding site allowed the identification of the DigR regulon and suggests that SgmT/DigR regulates the expression of genes for secreted proteins and enzymes involved in secondary metabolite synthesis. We suggest that SgmT/DigR regulates extracellular matrix composition and that SgmT activity is regulated by two sensor domains with ligand binding to the GAF domain resulting in SgmT activation and c-di-GMP binding to the GGDEF domain resulting in spatial sequestration of SgmT.

The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus.

The second messenger cyclic di-GMP is a near-ubiquitous signaling molecule that globally alters bacterial cell physiology to promote biofilm formation and community behavior. Much progress was made in recent years towards the identification and characterization of diguanylate cyclases and phosphodiersterases, enzymes involved in the synthesis and degradation of this signaling compound. In contrast, our knowledge of the nature and mechanistic details of c-di-GMP effector proteins lags behind, primarily because effective tools for their specific enrichment and rapid analysis are missing. In this report we demonstrate that a novel tri-functional c-di-GMP-specific Capture Compound (cdG-CC) can be effectively used to identify and validate c-di-GMP binding proteins. The cdG-CC was able to specifically and efficiently pull down bona fide c-di-GMP effector proteins. Furthermore, in combination with mass spectrometry (CCMS), this technology robustly identified a substantial fraction of the known c-di-GMP signaling components directly from cell extracts of different model organisms. Finally, we applied the CCMS technique to profile c-di-GMP binding proteins of Pseudomonas aeruginosa and Salmonella enterica serovar typhimurium. Our studies establish CCMS as a powerful and versatile tool to identify and analyze components of the cellular c-di-GMP pathway in a wide range of different organisms.

A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.

Zinc-finger domain transcriptional regulators regulate a myriad of functions in eukaryotes. Interestingly, ancestral versions (MucR) from Alpha-proteobacteria control bacterial virulence/symbiosis. Whether virulence regulators can also control cell cycle transcription is unknown. Here we report that MucR proteins implement a hitherto elusive primordial S→G1 transcriptional switch. After charting G1-specific promoters in the cell cycle model Caulobacter crescentus by comparative ChIP-seq, we use one such promoter as genetic proxy to unearth two MucR paralogs, MucR1/2, as constituents of a quadripartite and homeostatic regulatory module directing the S→G1 transcriptional switch. Surprisingly, MucR orthologues that regulate virulence and symbiosis gene transcription in Brucella, Agrobacterium or Sinorhizobium support this S→G1 switch in Caulobacter. Pan-genomic ChIP-seq analyses in Sinorhizobium and Caulobacter show that this module indeed targets orthologous genes. We propose that MucR proteins and possibly other virulence regulators primarily control bacterial cell cycle (G1-phase) transcription, rendering expression of target (virulence) genes periodic and in tune with the cell cycle.

Article	Year
Comparative and genetic analyses of the putative Vibrio cholerae lipopolysaccharide core oligosaccharide biosynthesis (wav) gene cluster. Infection and immunity\| PubMed ID: 11953379	2002
Role of Vibrio cholerae O139 surface polysaccharides in intestinal colonization. Infection and immunity\| PubMed ID: 12379674	2002
Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza. The Journal of biological chemistry\| PubMed ID: 14522970	2003
Crystallization and preliminary X-ray diffraction analysis of Wza outer-membrane lipoprotein from Escherichia coli serotype O9a:K30. Acta crystallographica. Section D, Biological crystallography\| PubMed ID: 14993692	2004
Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature\| PubMed ID: 17086202	2006
The peptidyl prolyl cis/trans isomerase FKBP38 determines hypoxia-inducible transcription factor prolyl-4-hydroxylase PHD2 protein stability. Molecular and cellular biology\| PubMed ID: 17353276	2007
Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor. Blood\| PubMed ID: 17684156	2007
Omp85(Tt) from Thermus thermophilus HB27: an ancestral type of the Omp85 protein family. Journal of bacteriology\| PubMed ID: 18456816	2008
Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from Thermus thermophilus HB27. Acta crystallographica. Section F, Structural biology and crystallization communications\| PubMed ID: 18540069	2008
Crystal structure of a major outer membrane protein from Thermus thermophilus HB27. Journal of molecular biology\| PubMed ID: 19101566	2009
The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococc... Molecular microbiology\| PubMed ID: 22394314	2012
A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins. Journal of proteomics\| PubMed ID: 22652488	2012
Cell cycle transition from S-phase to G1 in Caulobacter is mediated by ancestral virulence regulators. Nature communications\| PubMed ID: 24939058	2014

Jutta Nesper

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