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• Segrosome assembly at the pliable parH centromere.

  Authors: Meiyi Wu, Massimiliano Zampini, Malte Bussiek, Christian Hoischen, Stephan Diekmann, Finbarr Hayes

  Nucleic acids research.

  The segrosome of multiresistance plasmid TP228 comprises ParF, which is a member of the ParA ATPase superfamily, and the ParG ribbon-helix-helix factor that assemble jointly on the parH centromere.The segrosome of multiresistance plasmid TP228 comprises ParF, which is a member of the ParA ATPase superfamily, and the ParG ribbon-helix-helix factor that assemble jointly on the parH centromere. Here we demonstrate that the distinctive parH site (∼100-bp) consists of an array of degenerate tetramer boxes interspersed by AT-rich spacers. Although numerous consecutive AT-steps are suggestive of inherent curvature, parH lacks an intrinsic bend. Sequential deletion of parH tetramers progressively reduced centromere function. Nevertheless, the variant subsites could be rearranged in different geometries that accommodated centromere activity effectively revealing that the site is highly elastic in vivo. ParG cooperatively coated parH: proper centromere binding necessitated the protein's N-terminal flexible tails which modulate the centromere binding affinity of ParG. Interaction of the ParG ribbon-helix-helix domain with major groove bases in the tetramer boxes likely provides direct readout of the centromere. In contrast, the AT-rich spacers may be implicated in indirect readout that mediates cooperativity between ParG dimers assembled on adjacent boxes. ParF alone does not bind parH but instead loads into the segrosome interactively with ParG, thereby subtly altering centromere conformation. Assembly of ParF into the complex requires the N-terminal flexible tails in ParG that are contacted by ParF.

• Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites.

  Authors: Massimiliano Zampini, Andrew Derome, Simon E S Bailey, Daniela Barillà, Finbarr Hayes

  Journal of bacteriology.

  The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. Additional to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. Additional to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of the parFG partition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallel beta-strands in this fold are presumed to insert into the OF operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The OF locus comprises eight degenerate tetramer boxes arranged in a combination of direct and inverted orientation. Each tetramer motif likely recruits one ParG dimer, implying that the fully-bound operator is cooperatively coated by up to eight dimers. OF was subdivided experimentally into four overlapping 20-bp sites (A-D) each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A-D individually are bound with different affinities by ParG (C > A approximately B >> D). Moreover, comprehensive scanning mutagenesis revealed the contribution of each position in the site core and flanking sequences to ParG binding. Natural variations in the tetramer box motifs, in the interbox spacers, as well as in flanking sequences each influence ParG binding. The OF operator apparently has evolved with sites that bind ParG dissimilarly to produce a nucleoprotein complex fine-tuned for optimal interaction with the transcription machinery. The association of other ribbon-helix-helix proteins with complex recognition sites similarly may be modulated by natural sequence variations between subsites.

• Vibrios in association with sedimentary crustaceans in three beaches of the northern Adriatic Sea (Italy).

  Authors: A Covazzi Harriague, Marco Di Brino, Massimiliano Zampini, Giancarlo Albertelli, Carla Pruzzo, Cristina Misic

  Marine pollution bulletin. 56(3):574-9.

  In the marine environment, vibrios adhere to a number of substrates including chitin-rich organisms such as crustaceans. Their wide diffusion in coastal waters and pathogenic potential requireIn the marine environment, vibrios adhere to a number of substrates including chitin-rich organisms such as crustaceans. Their wide diffusion in coastal waters and pathogenic potential require knowledge of the lifestyle and environmental reservoirs of these bacteria. To test the presence of culturable vibrios in coastal areas and their association with benthic crustaceans, vibrios were isolated from water, sediments and crustaceans (copepods and anphipods) at three stations placed in front of heavily used tourist beaches of the Adriatic Sea. We observed significant correlations between vibrios and temperature. Benthic and planktonic copepods harboured vibrios in summer, while benthic amphipods harboured these bacteria in spring and autumn. Vibrio alginolyticus and Vibrio parahaemolyticus strains gave positive results using primers for Vibrio cholerae toxR and toxS. Sedimentary crustaceans may extend Vibrio persistence in seawater and may represent an additional aquatic reservoir of these bacteria.

• Vibrio cholerae persistence in aquatic environments and colonization of intestinal cells: involvement of a common adhesion mechanism.

  Authors: Massimiliano Zampini, Carla Pruzzo, Vijay P Bondre, Renato Tarsi, Mariangela Cosmo, Alessandro Bacciaglia, Arvind Chhabra, Renjana Srivastava, Brahm S Srivastava

  FEMS microbiology letters. 244(2):267-73.

  Forty-one Tnpho A mutants of Vibrio cholerae O1 classical strain CD81 were analyzed for their ability to interact with chitin particles, Tigriopus fulvus copepods and the Intestine 407 cell lineForty-one Tnpho A mutants of Vibrio cholerae O1 classical strain CD81 were analyzed for their ability to interact with chitin particles, Tigriopus fulvus copepods and the Intestine 407 cell line compared to the parent strain. Thirteen mutants were less adhesive than CD81; in particular, T21, T33 and T87 were less adhesive towards all substrates and insensitive to inhibition by N-acetyl glucosamine (GlcNAc). By SDS-PAGE analysis of sarkosyl-insoluble membrane proteins (siMPs) isolated from mutants and parent, it was found that a 53 kDa siMP is missing in T21, T33 and T87 mutants. It is hypothesized that this protein might have the function to mediate adherence to GlcNAc-containing substrates both in the aquatic environment and in human intestine.

• Adhesion of Enterococcus faecalis in the nonculturable state to plankton is the main mechanism responsible for persistence of this bacterium in both lake and seawater.

  Authors: Caterina Signoretto, Gloria Burlacchini, Maria del Mar Lleò, Carla Pruzzo, Massimiliano Zampini, Luigi Pane, Giorgio Franzini, Pietro Canepari

  Applied and environmental microbiology. 70(11):6892-6.

  The presence of enterococci in lake and seawater in an 18-month survey comparing molecular (PCR and quantitative PCR) and culture methods was evaluated, as well as the possibility that zooplanktonThe presence of enterococci in lake and seawater in an 18-month survey comparing molecular (PCR and quantitative PCR) and culture methods was evaluated, as well as the possibility that zooplankton could act as reservoirs for enterococci. Samples of both water and zooplankton were collected monthly from a Lake Garda site and an Adriatic Sea site. In lake water, the positive samples numbered 13 of 54 (24%) by culture and 32 of 54 (59%) when PCR was applied. In seawater, they numbered 0 of 51 by culture and 18 of 51 (35%) by PCR. Enterococci were found either totally bound to plankton or totally in water, depending on the presence or absence of plankton, respectively. These results clearly indicate that the PCR assay is a powerful tool for detecting fecal indicators and pathogens in the environment, thus providing a much more sensitive method than culture.

• Environmental estrogens can affect the function of mussel hemocytes through rapid modulation of kinase pathways.

  Authors: Laura Canesi, Lucia Cecilia Lorusso, Caterina Ciacci, Michele Betti, Massimiliano Zampini, Gabriella Gallo

  General and comparative endocrinology. 138(1):58-69.

  Estrogens and estrogenic chemicals can affect several vertebrate non-reproductive functions, the immune response in particular. We have previously shown that in the hemocytes of the marine molluscEstrogens and estrogenic chemicals can affect several vertebrate non-reproductive functions, the immune response in particular. We have previously shown that in the hemocytes of the marine mollusc Mytilus the natural estrogen 17beta-estradiol (E(2)) can affect the immune function through rapid tyrosine kinase-mediated signalling pathways converging on phosphorylation of both mitogen activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs), whose activation plays a key role in the immune response. In this work the effects of synthetic estrogens (such as DES), estrogenic chemicals (such as Bisphenol A, Nonylphenol), and plant estrogens (genistein) on mussel hemocytes were evaluated. The results demonstrate that all the EDCs tested exert in vitro effects similar to those of E(2) on lysosomal membrane stability, although at concentrations 1000 times higher than those of the natural estrogen. When the effects of DES, BPA, and NP on tyrosine kinase-mediated cell signalling were investigated, estrogenic compounds showed distinct effects on the phosphorylation state of MAPK and STAT members. In particular, only DES, like E(2), induced p38 MAPK phosphorylation, whereas BPA and NP seem to have opposite effects. Moreover, different EDCs significantly decreased the tyrosine phosphorylation state of STAT3 and STAT5, showing a distinct effect with respect to E(2). Experiments with specific kinase inhibitors showed that activation of p38 MAPK, but also of ERK MAPK and PI3-kinase, plays a key role in mediating the effect of DES. On the other hand, the effects of NP were partly mediated by ERK MAPK activation. BPA-induced lysosomal membrane destabilisation was unaffected by either MAPK or PI3-K inhibitors. However, hemocyte pre-treatment with the PKC inhibitor GF109203X prevented the effects of both BPA and NP, this indicating that kinase pathways other than those involving MAPKs are also responsible for mediating the effects of certain EDCs. Overall, the results support the hypothesis that EDCs may rapidly modulate the function of mussel hemocytes through activation of transduction pathways involving different kinase-mediated cascades. Moreover, the effects of EDCs on the phosphorylation state of transcription factor STATs suggest that these compounds may lead to changes in gene expression secondary to modulation of kinase/phosphatases. Our data address to the importance of investigating full range responses to estrogenic chemicals and may help understanding their basic mechanisms of action in ecologically relevant invertebrate species.

• Effetto del siero sull’adesività di streptococchi orali

  Authors: R. Tarsi, F. Biavasco, M. Zampini, A. Crippa, C. Pruzzo

  L’Igiene Moderna. 121:96.

• Role for mannose-sensitive hemagglutinin in promoting interactions between Vibrio cholerae El Tor and mussel hemolymph.

  Authors: Massimiliano Zampini, Laura Canesi, Michele Betti, Caterina Ciacci, Renato Tarsi, Gabriella Gallo, Carla Pruzzo

  Applied and environmental microbiology. 69(9):5711-5.

  The role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied. Bacterial adherence to and associationThe role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied. Bacterial adherence to and association with hemocytes were evaluated at 4 and 18 degrees C, respectively. In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA. In artificial seawater (ASW), no significant differences between the two strains were observed. N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively. The addition of D-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant. A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria. In contrast, serum adsorbed with either wild-type V. cholerae El Tor or wild-type Escherichia coli carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes. The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins.

• Persistence of adhesive properties in Vibrio cholerae after long-term exposure to sea water.

  Authors: Carla Pruzzo, Renato Tarsi, Maria Mar Lleò, Caterina Signoretto, Massimiliano Zampini, Luigi Pane, Rita R Colwell, Pietro Canepari

  Environmental microbiology. 5(10):850-8.

  The effect of exposure to artificial sea water (ASW) on the ability of classical Vibrio cholerae O1 cells to interact with chitin-containing substrates and human intestinal cells was studied.The effect of exposure to artificial sea water (ASW) on the ability of classical Vibrio cholerae O1 cells to interact with chitin-containing substrates and human intestinal cells was studied. Incubation of vibrios in ASW at 5 degrees C and 18 degrees C resulted in two kinds of cell responses: the viable but non-culturable (VBNC) state (i.e. <0.1 colony forming unit ml-1) at 5 degrees C, and starvation (i.e. maintenance of culturability of the population) at 18 degrees C. The latter remained rod shaped and, after 40 days' incubation, presented a 47-58% reduction in the number of cells attached to chitin, a 48-53% reduction in the number of bacteria adhering to copepods, and a 48-54% reduction in the number of bacteria adhering to human cultured intestinal cells, compared to control cells not suspended in ASW. Bacteria suspended in ASW at 5 degrees C became coccoid and, after 40 days, showed 34-42% fewer cells attached to chitin, 52-55% fewer adhering to copep-ods, and 45-48% fewer cells adhering to intestinal cell monolayers, compared to controls. Sarkosyl-insoluble membrane proteins that bind chitin particles were isolated and analysed by SDS-PAGE. After 40 days incubation in ASW at both 5 degrees C and 18 degrees C vibrios expressed chitin-binding ligands similar to bacteria harvested in the stationary growth phase. It is concluded that as vibrios do not lose adhesive properties after long-term exposure to ASW, it is important to include methods for VBNC bacteria when testing environmental and clinical samples for purposes of public health safety.

• In vitro adhesion to human cells by viable but nonculturable Enterococcus faecalis.

  Authors: Carla Pruzzo, Renato Tarsi, Maria Mar Lleò, Caterina Signoretto, Massimiliano Zampini, Rita R Colwell, Pietro Canepari

  Current microbiology. 45(2):105-10.

  The ability of viable but nonculturable (VBNC) Enterococcus faecalis to adhere to Caco-2 and Girardi heart cultured cells and to urinary tract epithelial cells (ECs) was studied. Enterococci wereThe ability of viable but nonculturable (VBNC) Enterococcus faecalis to adhere to Caco-2 and Girardi heart cultured cells and to urinary tract epithelial cells (ECs) was studied. Enterococci were harvested during the vegetative growth phase (early exponential and stationary), in the VBNC state, and after recovery of the ability to divide. VBNC bacteria maintained their adherence capability but the efficiency of attachment was reduced by about 50 to 70%, depending on the target cell employed. The decrease was transient, since enterococci that regained their culturability showed adherence values similar to those observed for actively growing cells. Analysis of the invasive properties of E. faecalis revealed that the VBNC state caused a decrease in the number of bacteria that entered the cultured HEK cells as a result of the reduction in the number of adhering bacteria. These results highlight the importance of studies of the VBNC phenomenon, with respect to both microbial survival in the environment and the impact on human health.