Induction of Cellular Differentiation and Single Cell Imaging of <em>Vibrio parahaemolyticus </em>Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Max Planck Institute for Terrestrial Microbiology

Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions without the involvement of partition-specific host cell receptors and is thus consistent with the striking observation that partition loci can function in heterologous host organisms.

Regular cellular distribution of plasmids by oscillating and filament-forming ParA ATPase of plasmid pB171.

The double par locus of Escherichia coli virulence factor pB171 consists of two adjacent and oppositely oriented par loci of different types, called par1 and par2. par1 encodes an actin ATPase (ParM), and par2 encodes an oscillating, MinD-like ATPase (ParA). The par loci share a central cis-acting region of approximately 200 bp, called parC1, located between the two par loci. An additional cis-acting region, parC2, is located downstream of the parAB operon of par2. Here we show that ParR of par1 and ParB of par2 bind cooperatively to unrelated sets of direct repeats in parC1 to form the cognate partition and promoter repression complexes. Surprisingly, ParB repressed transcription of the noncognate par operon, indicating cross-talk and possibly epistasis between the two systems. The par promoters, P1 and P2, affected each other negatively. The DNA binding activities of ParR and ParB correlated well with the observed transcriptional regulation of the par operons in vivo and in vitro. Integration host factor (IHF) was identified as a novel factor involved in par2-mediated plasmid partitioning.

Regulatory cross-talk in the double par locus of plasmid pB171.

The par2 locus of Escherichia coli plasmid pB171 encodes two trans-acting proteins, ParA and ParB, and two cis-acting sites, parC1 and parC2, to which ParB binds cooperatively. ParA is related to MinD and oscillates in helical structures and thereby positions ParB/parC-carrying plasmids regularly over the nucleoid. ParB ribbon-helix-helix dimers bind cooperatively to direct repeats in parC1 and parC2. Using four different assays we obtain solid evidence that ParB can pair parC1- and parC2-encoding DNA fragments in vitro. Convincingly, electron microscopy revealed that ParB mediates binary pairing of parC fragments. In addition to binary complexes, ParB mediated the formation of higher order complexes consisting of several DNA fragments joined by ParB at centromere site parC. N-terminal truncated versions of ParB still possessing specific DNA binding activity were incompetent in pairing, hence identifying the N terminus of ParB as a requirement for ParB-mediated centromere pairing. These observations suggest that centromere pairing is an important intermediate step in plasmid partitioning mediated by the common type I loci.

Centromere pairing by a plasmid-encoded type I ParB protein.

Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA-binding protein ParR and its cognate centromere site parC on the DNA. The partition complex is recognized by a second partition protein, the actin-like ATPase ParM, which forms filaments required for the active bidirectional movement of DNA replicates. Here, we present the 2.8 A crystal structure of ParR from E. coli plasmid pB171. ParR forms a tight dimer resembling a large family of dimeric ribbon-helix-helix (RHH)2 site-specific DNA-binding proteins. Crystallographic and electron microscopic data further indicate that ParR dimers assemble into a helix structure with DNA-binding sites facing outward. Genetic and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition.

Structural analysis of the ParR/parC plasmid partition complex.

Bacterial plasmids encode partitioning (par) loci that confer stable plasmid inheritance. We showed previously that, in the presence of ParB and parC encoded by the par2 locus of plasmid pB171, ParA formed cytoskeletal-like structures that dynamically relocated over the nucleoid. Simultaneously, the par2 locus distributed plasmids regularly over the nucleoid. We show here that the dynamic ParA patterns are not simple oscillations. Rather, ParA nucleates and polymerizes in between plasmids. When a ParA assembly reaches a plasmid, the assembly reaction reverses into disassembly. Strikingly, plasmids consistently migrate behind disassembling ParA cytoskeletal structures, suggesting that ParA filaments pull plasmids by depolymerization. The perpetual cycles of ParA assembly and disassembly result in continuous relocation of plasmids, which, on time averaging, results in equidistribution of the plasmids. Mathematical modeling of ParA and plasmid dynamics support these interpretations. Mutational analysis supports a molecular mechanism in which the ParB/parC complex controls ParA filament depolymerization.

Movement and equipositioning of plasmids by ParA filament disassembly.

Stochastic processes are thought to mediate localization of membrane-associated chemotaxis signaling clusters in peritrichous bacteria. Here, we identified a new family of ParA-like ATPases (designated ParC [for partitioning chemotaxis]) encoded within chemotaxis operons of many polar-flagellated γ-proteobacteria that actively promote polar localization of chemotaxis proteins. In Vibrio cholerae, a single ParC focus is found at the flagellated old pole in newborn cells, and later bipolar ParC foci develop as the cell matures. The cell cycle-dependent redistribution of ParC occurs by its release from the old pole and subsequent relocalization at the new pole, consistent with a "diffusion and capture" model for ParC dynamics. Chemotaxis proteins encoded in the same cluster as ParC have a similar unipolar-to-bipolar transition; however, they reach the new pole after the arrival of ParC. Cells lacking ParC exhibit aberrantly localized foci of chemotaxis proteins, reduced chemotaxis, and altered motility, which likely accounts for their enhanced colonization of the proximal small intestine in an animal model of cholera. Collectively, our findings indicate that ParC promotes the efficiency of chemotactic signaling processes. In particular, ParC-facilitated development of a functional chemotaxis apparatus at the new pole readies this site for its development into a functional old pole after cell division.

A family of ParA-like ATPases promotes cell pole maturation by facilitating polar localization of chemotaxis proteins.

The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology.

Studies of dynamic protein-protein interactions in bacteria using Renilla luciferase complementation are undermined by nonspecific enzyme inhibition.

&#x7EC6;&#x80DE;&#x4E24;&#x6781;&#x6784;&#x6210;&#x5173;&#x952E;&#x7684;&#x4E9A;&#x7EC6;&#x80DE;&#x57DF;&#xFF0C;&#x5F80;&#x5F80;&#x662F;&#x52A8;&#x529B;&#x3001; &#x8D8B;&#x5316;&#x6027;&#x548C;&#x6746;&#x5F62;&#x7EC6;&#x83CC;&#x67D3;&#x8272;&#x4F53;&#x5206;&#x79BB;&#x7684;&#x5173;&#x952E;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x5728;&#x51E0;&#x4E4E;&#x6240;&#x6709;&#x7684;&#x68D2;&#xFF0C;&#x4F5C;&#x4E3A;&#x57FA;&#x7840;&#x5F62;&#x6210;&#x3001; &#x8BC6;&#x522B;&#x548C;&#x6C38;&#x4E45;&#x5316;&#x7684;&#x6781;&#x5730;&#x57DF;&#x7684;&#x8FC7;&#x7A0B;&#x662F;&#x5F88;&#x5927;&#x7A0B;&#x5EA6;&#x4E0A;&#x672A;&#x77E5;&#x7684;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x970D;&#x4E71;&#x5F27;&#x83CC;&#xFF0C;&#x6211;&#x4EEC;&#x786E;&#x5B9A;&#x4E86; HubP &#xFF08;&#x6781;&#x7684;&#x96C6;&#x7EBF;&#x5668;&#xFF09;&#x3001; &#x6781;&#x5730;&#x8DE8;&#x819C;&#x86CB;&#x767D;&#x5B88;&#x6052;&#x5728;&#x6240;&#x6709;&#x5F27;&#x83CC;&#x548C;&#x951A;&#x5B9A;&#x5230;&#x7684;&#x5355;&#x5143;&#x683C;&#x7684;&#x4E09;&#x6BB5;&#x5F0F;&#x4E09;&#x6746;&#xFF0C;&#x5E76;&#x901A;&#x8FC7;&#x5B83;&#x4EEC;&#x63A7;&#x5236;&#x7684;&#x67D3;&#x8272;&#x4F53;&#x8D77;&#x6E90;&#x3001; &#x8D8B;&#x5316;&#x7684;&#x673A;&#x68B0;&#x548C;&#x97AD;&#x6BDB;&#x7684;&#x6781;&#x5730;&#x672C;&#x5730;&#x5316;&#x3002;&#x5728;&#x6CA1;&#x6709; HubP &#x7684;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;oriCI &#x4E0D;&#x9488;&#x5BF9;&#x7684;&#x662F;&#x5355;&#x5143;&#x683C;&#x7535;&#x6746;&#x3001; &#x8D8B;&#x5316;&#x6027;&#x662F;&#x53D7;&#x635F;&#xFF0C;&#x548C;&#x7EC6;&#x80DE;&#x867D;&#x5C0F;&#x4F46;&#x589E;&#x52A0;&#x5206;&#x6570;&#x4EA7;&#x751F;&#x591A;&#x4E2A;&#x800C;&#x4E0D;&#x662F;&#x5355;&#x4E00;&#x7684;&#x97AD;&#x6BDB;&#x3002;HubP &#x5185;&#x7684;&#x4E0D;&#x540C;&#x80DE;&#x8D28;&#x57DF;&#x6240;&#x9700;&#x7684;&#x6781;&#x5730;&#x9488;&#x5BF9;&#x4E09;&#x4E09;&#xFF0C;&#x867D;&#x7136; HubP &#x5468;&#x8D28;&#x90E8;&#x5206;&#x662F;&#x5176;&#x672C;&#x5730;&#x5316;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;HubP &#x90E8;&#x5206; relocalizes &#x4ECE;&#x4E24;&#x6781;&#x5230;&#x7EC6;&#x80DE;&#x5206;&#x88C2;&#x524D; mid-cell &#x4ECE;&#x800C;&#x4F7F;&#x6781;&#x5730;&#x57DF;&#x4E2D;&#x5355;&#x5143;&#x683C;&#x7684;&#x5973;&#x513F;&#x4ECA;&#x540E;&#x7684;&#x5EF6;&#x7EED;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x4E00;&#x4E2A;&#x5355;&#x4E00;&#x7684;&#x6781;&#x5730;&#x4E2D;&#x5FC3;&#x662F;&#x6709;&#x52A9;&#x4E8E;&#x5EFA;&#x7ACB;&#x6781;&#x5730;&#x7684;&#x8EAB;&#x4EFD;&#x548C;&#x7EC4;&#x7EC7;&#x3002;

&#x591A;&#x57DF;&#x7684;&#x67A2;&#x7EBD;&#x5B9A;&#x4F4D;&#x7684;&#x67D3;&#x8272;&#x4F53;&#x5206;&#x79BB;&#x548C;&#x8D8B;&#x5316;&#x673A;&#x68B0;&#x5230;&#x7EC6;&#x83CC;&#x5357;&#x6781;&#x3002;

The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization.

A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole.

P&ocirc;les de la cellule constituent les principaux domaines subcellulaires qui sont souvent essentiels pour la motilit&eacute;, le chimiotactisme et la s&eacute;gr&eacute;gation des chromosomes chez les bact&eacute;ries en forme de b&acirc;tonnet. Cependant, dans presque toutes les tiges, les processus qui sous-tendent la formation, la reconnaissance et la perp&eacute;tuation des domaines polaires sont largement inconnus. Ici, &agrave; Vibrio cholerae, nous avons identifi&eacute; HubP (moyeu du p&ocirc;le), une prot&eacute;ine transmembranaire polaire conserv&eacute;e chez tous les vibrions, qui ancre les ATPases ParA trois ressemblant &agrave; la cellule de polonais et, &agrave; travers eux, contr&ocirc;le localisation polaire de l&#39;origine chromosomique, la machinerie chimiotactique et le flagelle. En l&#39;absence de HubP, oriCI n&#39;est pas destin&eacute; aux p&ocirc;les de la cellule, la chimiotaxie est alt&eacute;r&eacute;e, et une fraction faible mais une augmentation des cellules produit multiple, plut&ocirc;t que de simples, flagelles. Domaines cytoplasmiques distincts au sein de l&#39;HubP sont requises pour polar cibler les trois ATPases, lorsqu&#39;une partie p&eacute;riplasmique de HubP est requise pour sa localisation. HubP relocalizes partiellement des p&ocirc;les &agrave; le mid-cell avant la division cellulaire, permettant ainsi &agrave; la perp&eacute;tuation du domaine polaire dans les futures cellules-filles. Ainsi, un seul hub polaire est instrumental permettant d&#39;&eacute;tablir l&#39;organisation et l&#39;identit&eacute; polaire.

Un hub multidomain d&#39;ancrer la s&eacute;gr&eacute;gation des chromosomes et machines chimiotactique au p&ocirc;le bact&eacute;rien.

Poli cella costituiscono chiave domini sottocellulari che spesso sono fondamentali per la motilit&agrave;, la chemiotassi e segregazione del cromosoma in batteri a forma di bastoncello. Tuttavia, in quasi tutte le canne, i processi che sottendono la formazione, il riconoscimento e la perpetuazione dei domini polar sono in gran parte sconosciuti. Qui, in Vibrio cholerae, abbiamo identificato HubP (hub del Polo), una proteina transmembrana polar conservata in tutti vibrione, che ancore tre ParA-come ATPasi nella cella poli e, attraverso di loro, controlla la localizzazione polare di origine cromosomica, il macchinario chemiotattico e il flagello. In assenza di HubP, oriCI non &egrave; rivolta verso i poli della cellula, chemiotassi &egrave; compromessa e una frazione piccola ma aumentata delle cellule produce pi&ugrave;, piuttosto che singoli, flagelli. Distinti domini citoplasmatici all&#39;interno di HubP sono necessari per polar targeting della tre ATPasi, mentre una parte periplasmica di HubP &egrave; necessaria per la sua localizzazione. HubP parzialmente relocalizes dai poli a mid-cell prima della divisione cellulare, consentendo la perpetuazione del dominio polare in future cellule figlie. Cos&igrave;, un unico hub polar &egrave; strumentale per stabilire identit&agrave; polare e organizzazione.

Un hub multidominio ancore il cromosoma segregazione e chemiotattica macchinari al Polo batterico.

&#x7D30;&#x80DE;&#x6975;&#x904B;&#x52D5;&#x3068;&#x8D70;&#x5316;&#x6027;&#x3001;&#x68D2;&#x72B6;&#x7D30;&#x83CC;&#x306E;&#x67D3;&#x8272;&#x4F53;&#x5206;&#x96E2;&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x306A;&#x3053;&#x3068;&#x304C;&#x591A;&#x3044;&#x30AD;&#x30FC;&#x306E;&#x7D30;&#x80DE;&#x5185;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3092;&#x69CB;&#x6210;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x307B;&#x307C;&#x3059;&#x3079;&#x3066;&#x306E;&#x68D2;&#x3001;&#x5F62;&#x6210;&#x3001;&#x8A8D;&#x8B58;&#x3001;&#x304A;&#x3088;&#x3073;&#x6975;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x6C38;&#x7D9A;&#x5316;&#x306E;&#x6839;&#x5E95;&#x306B;&#x3042;&#x308B;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306F;&#x4E3B;&#x3068;&#x3057;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30B3;&#x30EC;&#x30E9;&#x83CC;&#x3001;&#x6211;&#x3005; HubP&#x3001;&#x30DD;&#x30FC;&#x30EB;&#x306E; &#xFF08;&#x30CF;&#x30D6;&#xFF09; &#x3092;&#x8B58;&#x5225;&#x3001;3 &#x30D1;&#x30E9;&#x306E;&#x3088;&#x3046;&#x306A; Atpase &#x306E;&#x30BB;&#x30EB;&#x3092;&#x30A2;&#x30F3;&#x30AB;&#x30FC;&#x3059;&#x308B;&#x3059;&#x3079;&#x3066;&#x30D3;&#x30D6;&#x30EA;&#x30AA;&#x3067;&#x3001;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x3066;&#x3001;&#x6975;&#x306E;&#x819C;&#x8CAB;&#x901A;&#x578B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30DD;&#x30FC;&#x30EB;&#x3057;&#x3001;&#x305D;&#x308C;&#x3089;&#x3092;&#x67D3;&#x8272;&#x4F53;&#x306E;&#x8D77;&#x6E90;&#x3001;&#x8D70;&#x5316;&#x6027;&#x6A5F;&#x68B0;&#x30FB;&#x97AD;&#x6BDB;&#x306E;&#x5C40;&#x5728;&#x3092;&#x5236;&#x5FA1;&#x3057;&#x307E;&#x3059;&#x3002;HubP &#x304C;&#x306A;&#x3044;&#x5834;&#x5408;&#x3001;oriCI &#x306F;&#x3001;&#x7D30;&#x80DE;&#x306E;&#x6975;&#x3092;&#x5BFE;&#x8C61;&#x306B;&#x306A;&#x3089;&#x306A;&#x3044;&#x3001;&#x8D70;&#x5316;&#x6027;&#x306F;&#x640D;&#x306A;&#x308F;&#x308C;&#x308B;&#x3068;&#x8907;&#x6570;&#x306E;&#x30BB;&#x30EB;&#x306E;&#x5C0F;&#x3055;&#x306A;&#x304C;&#x5897;&#x52A0;&#x5272;&#x5408;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B; 1 &#x3064;&#x306F;&#x3001;&#x97AD;&#x6BDB;&#x306E;&#x3067;&#x306F;&#x306A;&#x304F;&#x3002;HubP &#x5185;&#x306E;&#x7570;&#x306A;&#x308B;&#x7D30;&#x80DE;&#x8CEA;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306F; HubP &#x306E;&#x30DA;&#x30EA;&#x30D7;&#x30E9;&#x30BA;&#x30E0;&#x90E8;&#x5206;&#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x304C;&#x3001;3 &#x3064;&#x306E; Atpase &#x306E;&#x6975;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3092;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;HubP &#x90E8;&#x5206;&#x7684;&#x306B;&#x304B;&#x3089;&#x6975;&#x7D30;&#x80DE;&#x5206;&#x88C2;&#x306E;&#x524D;&#x306B; mid-cell &#x306B;&#x305D;&#x308C;&#x306B;&#x3088;&#x3063;&#x3066;&#x5C06;&#x6765;&#x306E;&#x5A18;&#x7D30;&#x80DE;&#x306B;&#x6975;&#x6027;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x6C38;&#x7D9A;&#x5316;&#x3092;&#x6709;&#x52B9;&#x306B;&#x3059;&#x308B; relocalizes &#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;1 &#x3064;&#x306E;&#x6975;&#x30CF;&#x30D6;&#x306F;&#x6975;&#x306E;&#x30A2;&#x30A4;&#x30C7;&#x30F3;&#x30C6;&#x30A3;&#x30C6;&#x30A3;&#x3068;&#x7D44;&#x7E54;&#x3092;&#x78BA;&#x7ACB;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5C3D;&#x529B;&#x3067;&#x3059;&#x3002;

&#x30DE;&#x30EB;&#x30C1; &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x30CF;&#x30D6;&#x306F;&#x3001;&#x67D3;&#x8272;&#x4F53;&#x5206;&#x914D;&#x3068;&#x8D70;&#x5316;&#x6027;&#x6A5F;&#x68B0;&#x7D30;&#x83CC;&#x306E;&#x30DD;&#x30FC;&#x30EB;&#x306B;&#x56FA;&#x5B9A;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC140; &#xADF9; &#xAD6C;&#xC131; &#xD0A4; subcellular &#xB3C4;&#xBA54;&#xC778;&#xC758; &#xC6B4;&#xB3D9; &#xC131;, chemotaxis, &#xBC0F; &#xB9C9;&#xB300; &#xBAA8;&#xC591;&#xC758; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xC5D0;&#xC11C; &#xC5FC;&#xC0C9;&#xCCB4; &#xBD84;&#xB9AC;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xACBD;&#xC6B0;&#xAC00; &#xB9CE;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xAC70;&#xC758; &#xBAA8;&#xB4E0; &#xBD09; &#xD615;&#xC131;, &#xC778;&#xC2DD; &#xBC0F; &#xC601;&#xAD6C; &#xD654; &#xADF9; &#xB3C4;&#xBA54;&#xC778;&#xC758; &#xAE30;&#xBC18;&#xC774; &#xB418;&#xB294; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB294; &#xD06C;&#xAC8C; &#xC54C;&#xB824;&#xC9C4; &#xD558;&#xC9C0;. &#xC5EC;&#xAE30;, &#xBE44; &#xBE0C;&#xB9AC; &#xC624;&#xC758; cholerae HubP (&#xADF9;&#xC758; &#xD5C8;&#xBE0C;) &#xC2DD;&#xBCC4;, &#xADF8; &#xC575;&#xCEE4; &#xC140;&#xC744; &#xC138; &#xAC00;&#xC9C0; &#xD30C;&#xB791; &#xC0C8; &#xAC19;&#xC740; ATPases &#xBAA8;&#xB4E0; &#xBC00;&#xC5B4;&#xB0B4;&#xC5D0; &#xBCF4;&#xC874; &#xADF9; &#xB9C9; &#xD6A1;&#xB2E8; &#xB2E8;&#xBC31;&#xC9C8; &#xAE30;&#xB465; &#xD558; &#xACE0;, &#xADF8;&#xB4E4;&#xC744; &#xD1B5;&#xD574; &#xC5FC;&#xC0C9;&#xCCB4; &#xAE30;&#xC6D0;, &#xD608; &#xAE30;&#xACC4;&#xACFC;&#xB97C; &#xC2E0;&#xC124;&#xC758; &#xADF9; &#xC9C0; &#xC9C0;&#xC5ED;&#xD654;&#xB97C; &#xC81C;&#xC5B4; &#xD569;&#xB2C8;&#xB2E4;. &#xD558;&#xC9C0; Hubp&#xC758; &#xBD80;&#xC7AC;, Orici&#xC740; &#xC140; &#xADF9;&#xC744; &#xD0C0;&#xAC9F;&#xC73C;&#xB85C;, chemotaxis&#xC740; &#xC7A5;&#xC560;&#xC778;, &#xADF8;&#xB9AC;&#xACE0; &#xC140;&#xC758; &#xC791;&#xC9C0;&#xB9CC; &#xC99D;&#xAC00; &#xBD84;&#xC218; &#xC0DD;&#xC0B0; &#xB2E4;&#xC911;, &#xB2E8;&#xC77C;, flagella &#xBCF4;&#xB2E4;. HubP &#xB0B4;&#xC5D0;&#xC11C; &#xB69C;&#xB837;&#xD55C; &#xC138;&#xD3EC;&#xC9C8; &#xB3C4;&#xBA54;&#xC778; periplasmic &#xBD80;&#xBD84;&#xC758; Hubp&#xC758; &#xC9C0;&#xC5ED;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694;&#xB294; 3 Atpases&#xC758; &#xADF9; &#xC9C0; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xD544;&#xC694; &#xD558;&#xB2E4;. HubP &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; relocalizes &#xD3F4;&#xB780;&#xB4DC;&#xC5D0;&#xC11C; &#xC138;&#xD3EC; &#xBD84;&#xC5F4; &#xC804;&#xC5D0; mid-cell &#xD558; &#xBBC0;&#xB85C; &#xBBF8;&#xB798;&#xC758; &#xB538; &#xC138;&#xD3EC;&#xC5D0; &#xADF9; &#xC9C0; &#xB3C4;&#xBA54;&#xC778;&#xC758; &#xC601;&#xAD6C; &#xD654;. &#xB530;&#xB77C;&#xC11C;, &#xB2E8;&#xC77C; &#xADF9; &#xD5C8;&#xBE0C; &#xD3F4;&#xB77C; &#xC815;&#xCCB4;&#xC131;&#xACFC; &#xC870;&#xC9C1; &#xAD6C;&#xCD95;&#xC744; &#xC704;&#xD55C; &#xC218;&#xB2E8; &#xC774;&#xB2E4;.

&#xB2E4;&#xC911; &#xB3C4;&#xBA54;&#xC778; &#xD5C8;&#xBE0C; &#xC5FC;&#xC0C9;&#xCCB4; &#xBD84;&#xB9AC; &#xBC0F; &#xC138;&#xADE0; &#xADF9; &#xD608; &#xAE30;&#xACC4;&#xC5D0; &#xACE0;&#xC815;&#xD569;&#xB2C8;&#xB2E4;.

Los polos celulares constituyen dominios subcelulares claves que a menudo son cruciales para la motilidad, Quimiotaxis y segregaci&oacute;n cromos&oacute;mica en bacterias en forma de bastoncillos. Sin embargo, en casi todas las barras, los procesos que subyacen a la formaci&oacute;n, el reconocimiento y la perpetuaci&oacute;n de los dominios polares son en gran parte desconocidos. Aqu&iacute;, en Vibrio cholerae, identificamos HubP (eje del poste), una prote&iacute;na transmembrana polar conservada en los vibriones, que ancla ParA tres-como ATPasas a la celda postes y, a trav&eacute;s de ellos, controla la localizaci&oacute;n polar del origen del cromosoma, la maquinaria quimiot&aacute;ctica y el flagelo. En la ausencia de HubP, oriCI no est&aacute; dirigido a los polos celulares, quimiotaxis se deteriora, y una fracci&oacute;n peque&ntilde;a pero creciente de c&eacute;lulas produce m&uacute;ltiples, en lugar de flagelos solos,. Distintos dominios citopl&aacute;smicos en HubP son necesarios para la orientaci&oacute;n polar de las tres ATPasas, mientras que una porci&oacute;n periplasmic de HubP se requiere para su localizaci&oacute;n. HubP parcialmente relocalizes desde los polos a la mid-cell antes de la divisi&oacute;n celular, permitiendo la perpetuaci&oacute;n del dominio polar en futuras c&eacute;lulas hijas. As&iacute;, un solo eje polar es fundamental para el establecimiento de la organizaci&oacute;n e identidad polar.

Un centro multidominio anclas de la segregaci&oacute;n cromos&oacute;mica y quimiot&aacute;ctica maquinaria al Polo bacteriana.

Bacterial chemotaxis proteins are organized into ordered arrays. In peritrichous organisms, such as Escherichia coli, stochastic assembly processes are thought to account for the placement of chemotaxis arrays, which are nonuniformly distributed. In contrast, we previously found that chemotactic signaling arrays in polarly flagellated vibrios are uniformly polar and that array localization is dependent on the ParA-like ATPase ParC. However, the processes that enable ParC to facilitate array localization have not been described. Here, we show that a previously uncharacterized protein, ParP, interacts with ParC and that ParP is integral to array localization in Vibrio parahaemolyticus. ParC's principal contribution to chemotaxis appears to be via positioning of ParP. Once recruited to the pole by ParC, ParP sequesters arrays at this site by capturing and preventing the dissociation of chemotactic signaling protein (CheA). Notably, ParP also stabilizes chemotactic protein complexes in the absence of ParC, indicating that some of its activity is independent of this interaction partner. ParP recruits CheA via CheA's localization and inheritance domain, a region found only in polarly flagellated organisms that encode ParP, ParC, and CheA. Thus, a tripartite (ParC-ParP-CheA) interaction network enables the polar localization and sequestration of chemotaxis arrays in polarly flagellated organisms. Localization and sequestration of chemotaxis clusters adjacent to the flagella--to which the chemotactic signal is transmitted--facilitates proper chemotaxis as well as accurate inheritance of these macromolecular machines.

ParP prevents dissociation of CheA from chemotactic signaling arrays and tethers them to a polar anchor.

PprA is known to contribute to Deinococcus radiodurans' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA's role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans's capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB.

PprA contributes to Deinococcus radiodurans resistance to nalidixic acid, genome maintenance after DNA damage and interacts with deinococcal topoisomerases.

Vibrio cholerae, the agent of cholera, is a motile non-invasive pathogen that colonizes the small intestine (SI). Most of our knowledge of the processes required for V. cholerae intestinal colonization is derived from enumeration of wt and mutant V. cholerae recovered from orogastrically infected infant mice. There is limited knowledge of the distribution of V. cholerae within the SI, particularly its localization along the villous axis, or of the bacterial and host factors that account for this distribution. Here, using confocal and intravital two-photon microscopy to monitor the localization of fluorescently tagged V. cholerae strains, we uncovered unexpected and previously unrecognized features of V. cholerae intestinal colonization. Direct visualization of the pathogen within the intestine revealed that the majority of V. cholerae microcolonies attached to the intestinal epithelium arise from single cells, and that there are notable regiospecific aspects to V. cholerae localization and factors required for colonization. In the proximal SI, V. cholerae reside exclusively within the developing intestinal crypts, but they are not restricted to the crypts in the more distal SI. Unexpectedly, V. cholerae motility proved to be a regiospecific colonization factor that is critical for colonization of the proximal, but not the distal, SI. Furthermore, neither motility nor chemotaxis were required for proper V. cholerae distribution along the villous axis or in crypts, suggesting that yet undefined processes enable the pathogen to find its niches outside the intestinal lumen. Finally, our observations suggest that host mucins are a key factor limiting V. cholerae intestinal colonization, particularly in the proximal SI where there appears to be a more abundant mucus layer. Collectively, our findings demonstrate the potent capacity of direct pathogen visualization during infection to deepen our understanding of host pathogen interactions.

Insights into Vibrio cholerae intestinal colonization from monitoring fluorescently labeled bacteria.

The diarrheal pathogen Vibrio cholerae contains three gene clusters that encode chemotaxis-related proteins, but only cluster II appears to be required for chemotaxis. Here, we present the first characterization of V. cholerae's 'cluster III' chemotaxis system. We found that cluster III proteins assemble into foci at bacterial poles, like those formed by cluster II proteins, but the two systems assemble independently and do not colocalize. Cluster III proteins are expressed in vitro during stationary phase and in conjunction with growth arrest linked to carbon starvation. This expression, as well as expression in vivo in suckling rabbits, is dependent upon RpoS. V. cholerae's CAI-1 quorum sensing (QS) system is also required for cluster III expression in stationary phase and modulates its expression in vivo, but is not required for cluster III expression in response to carbon starvation. Surprisingly, even though the CAI-1 and AI-2 QS systems are thought to feed into the same signaling pathway, the AI-2 system inhibited cluster III gene expression, revealing that the outputs of the two QS systems are not always the same. The distinctions between genetic determinants of cluster III expression in vitro and in vivo highlight the distinctive nature of the in vivo environment.

RpoS and quorum sensing control expression and polar localization of Vibrio cholerae chemotaxis cluster III proteins in vitro and in vivo.

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.

Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM.

When encountering new environments or changes to their external milieu, bacteria use elaborate mechanisms to respond accordingly. Here, we describe how Vibrio parahaemolyticus coordinates two such mechanisms - differentiation and chemotaxis. V. parahaemolyticus differentiates between two distinct cell types: short rod-shaped swimmer cells and highly elongated swarmer cells. We show that the intracellular organization of chemotactic signaling arrays changes according to the differentiation state. In swimmer cells chemotaxis arrays are strictly polarly localized, but in swarmer cells arrays form both at the cell poles and at irregular intervals along the entire cell length. Furthermore, the formation of lateral arrays increases with cell length of swarmer cells. Occurrence of lateral signaling arrays is not simply a consequence of the elongated state of swarmer cells, but is instead differentiation state-specific. Moreover, our data suggest that swarmer cells employ two distinct mechanisms for localization of polar and lateral signaling arrays, respectively. Furthermore, cells show a distinct differentiation and localization pattern of chemosensory arrays, depending on their location within swarm colonies, which likely allows for the organism to simultaneously swarm across surfaces while sustaining a pool of swimmers immediately capable of exploring new liquid surroundings.

Simon Ringgaard

Department of Ecophysiology

Max Planck Institute for Terrestrial Microbiology

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Simon Ringgaard

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ecophysiology

Max Planck Institute for Terrestrial Microbiology

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Department of Ecophysiology