Massimiliano Zampini

Publications (13) View all

• Article: Structural mechanism of ATP-induced polymerization of the partition factor ParF: implications for DNA segregation.

  Maria A Schumacher, Qiaozhen Ye, Madhuri T Barge, Massimiliano Zampini, Daniela Barillà, Finbarr Hayes
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  ABSTRACT: Segregation of the bacterial multidrug resistance plasmid TP228 requires the centromere-binding protein ParG, the parH centromere, and the Walker box ATPase ParF. The cycling of ParF between ADP- and ATP-bound states drives TP228 partition; ATP binding stimulates ParF polymerization, which is essential for segregation, whereas ADP binding antagonizes polymerization and inhibits DNA partition. The molecular mechanism involved in this adenine nucleotide switch is unclear. Moreover, it is unknown how any Walker box protein polymerizes in an ATP-dependent manner. Here, we describe multiple ParF structures in ADP- and phosphomethylphosphonic acid adenylate ester (AMPPCP)-bound states. ParF-ADP is monomeric but dimerizes when complexed with AMPPCP. Strikingly, in ParF-AMPPCP structures, the dimers interact to create dimer-of-dimer "units" that generate a specific linear filament. Mutation of interface residues prevents both polymerization and DNA segregation in vivo. Thus, these data provide insight into a unique mechanism by which a Walker box protein forms polymers that involves the generation of ATP-induced dimer-of-dimer building blocks.
  Journal of Biological Chemistry 06/2012; 287(31):26146-54. · 4.77 Impact Factor
• Source

  Available from: PubMed Central

  Article: Combinatorial targeting of ribbon-helix-helix artificial transcription factors to chimeric recognition sites.

  Massimiliano Zampini, Finbarr Hayes
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  ABSTRACT: Artificial transcription factors (ATFs) are potent synthetic biology tools for modulating endogenous gene expression and precision genome editing. The ribbon-helix-helix (RHH) superfamily of transcription factors are widespread in bacteria and archaea. The principal DNA binding determinant in this family comprises a two-stranded antiparallel β-sheet (ribbons) in which a pair of eight-residue motifs insert into the major groove. Here, we demonstrate that ribbons of divergent RHH proteins are compact and portable elements that can be grafted into a common α-helical scaffold producing active ATFs. Hybrid proteins cooperatively recognize DNA sites possessing core tetramer boxes whose functional spacing is dictated by interactions between the α-helical backbones. These interactions also promote combinatorial binding of chimeras with different transplanted ribbons, but identical backbones, to synthetic sites bearing cognate boxes for each protein either in vitro or in vivo. The composite assembly of interacting hybrid proteins offers potential advantages associated with combinatorial approaches to DNA recognition compared with ATFs that involve binding of a single protein. Moreover, the new class of RHH ATFs may be utilized to re-engineer transcriptional circuits, or may be enhanced with affinity tags, fluorescent moieties or other elements for targeted genome marking and manipulation in bacteria and archaea.
  Nucleic Acids Research 04/2012; 40(14):6673-82. · 8.03 Impact Factor
• Source

  Available from: PubMed Central

  Article: Segrosome assembly at the pliable parH centromere.

  Meiyi Wu, Massimiliano Zampini, Malte Bussiek, Christian Hoischen, Stephan Diekmann, Finbarr Hayes
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  ABSTRACT: 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.
  Nucleic Acids Research 03/2011; 39(12):5082-97. · 8.03 Impact Factor
• Article: Recruitment of the ParG segregation protein to different affinity DNA sites.

  Massimiliano Zampini, Andrew Derome, Simon E S Bailey, Daniela Barillà, Finbarr Hayes
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  ABSTRACT: The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. In addition 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 O(F) operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The O(F) 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. O(F) was subdivided experimentally into four overlapping 20-bp sites (A to D), each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A to 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 and in the interbox spacers, as well as in flanking sequences, each influence ParG binding. The O(F) 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.
  Journal of bacteriology 04/2009; 191(12):3832-41. · 3.94 Impact Factor
• Article: Vibrios in association with sedimentary crustaceans in three beaches of the northern Adriatic Sea (Italy).

  A Covazzi Harriague, Marco Di Brino, Massimiliano Zampini, Giancarlo Albertelli, Carla Pruzzo, Cristina Misic
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  ABSTRACT: 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 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.
  Marine Pollution Bulletin 04/2008; 56(3):574-9. · 2.50 Impact Factor