Biogenesis of a Bacteriophage Long Non-Contractile Tail.

Siphoviruses are main killers of bacteria. They use a long non-contractile tail to recognize the host cell and to deliver the genome from the viral capsid to the bacterial cytoplasm. Here, we define the molecular organization of the Bacillus subtilis bacteriophage SPP1 ~ 6.8 MDa tail and uncover its biogenesis mechanisms. A complex between gp21 and the tail distal protein (Dit) gp19.1 is assembled first to build the tail cap (gp19.1-gp21Nter) connected by a flexible hinge to the tail fiber (gp21Cter). The tip of the gp21Cter fiber is loosely associated to gp22. The cap provides a platform where tail tube proteins (TTPs) initiate polymerization around the tape measure protein gp18 (TMP), a reaction dependent on the non-structural tail assembly chaperones gp17.5 and gp17.5* (TACs). Gp17.5 is essential for stability of gp18 in the cell. Helical polymerization stops at a precise tube length followed by binding of proteins gp16.1 (TCP) and gp17 (THJP) to build the tail interface for attachment to the capsid portal system. This finding uncovers the function of the extensively conserved gp16.1-homologs in assembly of long tails. All SPP1 tail components, apart from gp22, share homology to conserved proteins whose coding genes' synteny is broadly maintained in siphoviruses. They conceivably represent the minimal essential protein set necessary to build functional long tails. Proteins homologous to SPP1 tail building blocks feature a variety of add-on modules that diversify extensively the tail core structure, expanding its capability to bind host cells and to deliver the viral genome to the bacterial cytoplasm.

Crosstalk between vertical and horizontal gene transfer: plasmid replication control by a conjugative relaxase.

Horizontal gene transfer is a key process in the evolution of bacteria and also represents a source of genetic variation in eukaryotes. Among elements participating in gene transfer, thousands of small (<10 kb) mobile bacterial plasmids that replicate by the rolling circle mechanism represent a driving force in the spread of antibiotic resistances. In general, these plasmids are built as genetic modules that encode a replicase, an antibiotic-resistance determinant, and a relaxase that participates in their conjugative mobilization. Further, they control their relatively high copy number (∼30 copies per genome equivalent) by antisense RNAs alone or combined with a repressor protein. We report here that the MobM conjugative relaxase encoded by the promiscuous plasmid pMV158 participates in regulation of the plasmid copy number by transcriptional repression of the antisense RNA, thus increasing the number of plasmid molecules ready to be horizontally transferred (mobilization) and/or vertically inherited (replication). This type of crosstalk between genetic modules involved in vertical and horizontal gene flow has not been reported before.

Functional amyloids as inhibitors of plasmid DNA replication.

DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is 'handcuffing', i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation.

Two p53 tetramers bind one consensus DNA response element.

p53 tumor suppressor is a transcription factor that controls cell cycle and genetic integrity. In response to genotoxic stress p53 activates DNA repair, cell cycle arrest, apoptosis or senescence, which are initiated via p53 binding to its specific DNA response elements (RE). The consensus p53 DNA RE consists of two decameric palindromic half-site sequences. Crystallographic studies have demonstrated that two isolated p53 DNA-binding core domains interact with one half-site of the p53 DNA REs suggesting that one p53 tetramer is bound to one RE. However, our recent 3D cryo-EM studies showed that the full-length p53 tetramer is bound to only one half-site of RE.Here, we have used biochemical and electron microscopy (EM) methods to analyze DNA-binding of human and murine p53 tetramers to various p53 DNA REs. Our new results demonstrate that two p53 tetramers can interact sequence-specifically with one DNA RE at the same time. In particular, the EM structural analysis revealed that two p53 tetramers bind one DNA RE simultaneously with DNA positioned between them. These results demonstrate a mode different from that assumed previously for the p53-DNA interaction and suggest important biological implications on p53 activity as a transcriptional regulator of cellular response to stress.

A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI.

DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine (AdoMet or SAM) to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase from Thermus aquaticus (M.TaqI) is also capable of coupling synthetic N-adenosylaziridine cofactor analogues to its target adenine within the double-stranded 5'-TCGA-3' sequence. This M.TaqI-mediated coupling reaction was exploited to sequence-specifically deliver fluorophores and biotin to DNA using N-adenosylaziridine derivatives carrying reporter groups at the 8-position of the adenine ring. However, these 8-modified aziridine cofactors were poor substrates for the DNA cytosine-C5 MTase from Haemophilus haemolyticus (M.HhaI). Based on the crystal structure of M.HhaI in complex with a duplex oligodeoxynucleotide and the cofactor product, we synthesized a stable 7-deazaadenosylaziridine derivative with a biotin group attached to the 7-position via a flexible linker. This 7-modified aziridine cofactor can be efficiently used by M.HhaI for the direct, quantitative and sequence-specific delivery of biotin to the second cytosine within 5'-GCGC-3' sequences in short duplex oligodeoxynucleotides and plasmid DNA. In addition, we demonstrate that biotinylation by M.HhaI depends on the methylation status of the target cytosine and, thus, could provide a method for cytosine-C5 DNA methylation detection in mammalian DNA.

Aß42-oligomer Interacting Peptide (AIP) neutralizes toxic amyloid-ß42 species and protects synaptic structure and function.

The amyloid-ß42 (Aß42) peptide is believed to be the main culprit in the pathogenesis of Alzheimer disease (AD), impairing synaptic function and initiating neuronal degeneration. Soluble Aß42 oligomers are highly toxic and contribute to progressive neuronal dysfunction, loss of synaptic spine density, and affect long-term potentiation (LTP). We have characterized a short, L-amino acid Aß-oligomer Interacting Peptide (AIP) that targets a relatively well-defined population of low-n Aß42 oligomers, rather than simply inhibiting the aggregation of Aß monomers into oligomers. Our data show that AIP diminishes the loss of Aß42-induced synaptic spine density and rescues LTP in organotypic hippocampal slice cultures. Notably, the AIP enantiomer (comprised of D-amino acids) attenuated the rough-eye phenotype in a transgenic Aß42 fly model and significantly improved the function of photoreceptors of these flies in electroretinography tests. Overall, our results indicate that specifically "trapping" low-n oligomers provides a novel strategy for toxic Aß42-oligomer recognition and removal.

ParAB Partition Dynamics in Firmicutes: Nucleoid Bound ParA Captures and Tethers ParB-Plasmid Complexes.

In Firmicutes, small homodimeric ParA-like (δ2) and ParB-like (ω2) proteins, in concert with cis-acting plasmid-borne parS and the host chromosome, secure stable plasmid inheritance in a growing bacterial population. This study shows that (ω:YFP)2 binding to parS facilitates plasmid clustering in the cytosol. (δ:GFP)2 requires ATP binding but not hydrolysis to localize onto the cell's nucleoid as a fluorescent cloud. The interaction of (δ:CFP)2 or δ2 bound to the nucleoid with (ω:YFP)2 foci facilitates plasmid capture, from a very broad distribution, towards the nucleoid and plasmid pairing. parS-bound ω2 promotes redistribution of (δ:GFP)2, leading to the dynamic release of (δ:GFP)2 from the nucleoid, in a process favored by ATP hydrolysis and protein-protein interaction. (δD60A:GFP)2, which binds but cannot hydrolyze ATP, also forms unstable complexes on the nucleoid. In the presence of ω2, (δD60A:GFP)2 accumulates foci or patched structures on the nucleoid. We propose that (δ:GFP)2 binding to different nucleoid regions and to ω2-parS might generate (δ:GFP)2 gradients that could direct plasmid movement. The iterative pairing and unpairing cycles may tether plasmids equidistantly on the nucleoid to ensure faithful plasmid segregation by a mechanism compatible with the diffusion-ratchet mechanism as proposed from in vitro reconstituted systems.

The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site.

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\(_{\rm{R}}\)3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\(_{\rm{R}}\)3) upstream of the prophage repressor gene. The O\(_{\rm{R}}\)3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages.

Structural rearrangements in the phage head-to-tail interface during assembly and infection.

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal-phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein-DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein-protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere.

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids.

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail.

Nuclear translocation uncovers the amyloid peptide Aß42 as a regulator of gene transcription.

Although soluble species of the amyloid-ß peptide Aß42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-ß (Aß). Here, we show that Aß peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear Aß42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled Aß in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of Aß in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for Aß42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that Aß42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic Aß42 wild-type peptide. Shorter peptides (Aß38 or Aß40) and other longer peptides (nontoxic Aß42 G33A substitution or Aß43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of Aß42 impacts gene regulation, and deleterious effects of Aß42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes.

A touch of glue to complete bacteriophage assembly: the tail-to-head joining protein (THJP) family.

Bacteriophage SPP1 is a nanomachine built to infect the bacterium Bacillus subtilis. The phage particle is composed of an icosahedric capsid, which contains the viral DNA, and a long non-contractile tail. Capsids and tails are produced in infected cells by two distinct morphogenetic pathways. Characterization of the suppressor-sensitive mutant SPP1sus82 showed that it produces DNA-filled capsids and tails but is unable to assemble complete virions. Its purified tails have a normal length but lack a narrow ring that tapers the tail end found at the tail-to-head interface. The mutant is defective in production of gp17. The gp17 ring is exposed in free tails competent for viral assembly but becomes shielded in the final virion structure. Recombinant gp17 is active in an in vitro assay to stick together capsids and tails present in extracts of SPP1sus82-infected cells, leading to formation of infectious particles. Gp17 thus plays a fundamental role in the tail-to-head joining reaction, the ultimate step of virus particle assembly. This is the conserved function of gp17 and its structurally related proteins like lambda gpU. This family of proteins can also provide fidelity to termination of the tail tube elongation reaction in a subset of phages including coliphage lambda.

The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation.

The replicative mini-chromosome-maintenance 2-7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the 'initial' complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.

Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components.

Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.

Crystal structure of pb9, the distal tail protein of bacteriophage T5: a conserved structural motif among all siphophages.

The tail of Caudovirales bacteriophages serves as an adsorption device, a host cell wall-perforating machine, and a genome delivery pathway. In Siphoviridae, the assembly of the long and flexible tail is a highly cooperative and regulated process that is initiated from the proteins forming the distal tail tip complex. In Gram-positive-bacterium-infecting siphophages, the distal tail (Dit) protein has been structurally characterized and is proposed to represent a baseplate hub docking structure. It is organized as a hexameric ring that connects the tail tube and the adsorption device. In this study, we report the characterization of pb9, a tail tip protein of Escherichia coli bacteriophage T5. By immunolocalization, we show that pb9 is located in the upper part of the cone of the T5 tail tip, at the end of the tail tube. The crystal structure of pb9 reveals a two-domain protein. Domain A exhibits remarkable structural similarity with the N-terminal domain of known Dit proteins, while domain B adopts an oligosaccharide/oligonucleotide-binding fold (OB-fold) that is not shared by these proteins. We thus propose that pb9 is the Dit protein of T5, making it the first Dit protein described for a Gram-negative-bacterium-infecting siphophage. Multiple sequence alignments suggest that pb9 is a paradigm for a large family of Dit proteins of siphophages infecting mostly Gram-negative hosts. The modular structure of the Dit protein maintains the basic building block that would be conserved among all siphophages, combining it with a more divergent domain that might serve specific host adhesion properties.

The pneumococcal MgaSpn virulence transcriptional regulator generates multimeric complexes on linear double-stranded DNA.

The MgaSpn transcriptional regulator contributes to the virulence of Streptococcus pneumoniae. It is thought to be a member of the Mga/AtxA family of global regulators. MgaSpn was shown to activate in vivo the P1623B promoter, which is divergent from the promoter (Pmga) of its own gene. This activation required a 70-bp region (PB activation region) located between both promoters. In this work, we purified an untagged form of the MgaSpn protein, which formed dimers in solution. By gel retardation and footprinting assays, we analysed the binding of MgaSpn to linear double-stranded DNAs. MgaSpn interacted with the PB activation region when it was placed at internal position on the DNA. However, when it was positioned at one DNA end, MgaSpn recognized preferentially the Pmga promoter placed at internal position. In both cases, and on binding to the primary site, MgaSpn spread along the adjacent DNA regions generating multimeric protein-DNA complexes. When both MgaSpn-binding sites were located at internal positions on longer DNAs, electron microscopy experiments demonstrated that the PB activation region was the preferred target. DNA molecules totally or partially covered by MgaSpn were also visualized. Our results suggest that MgaSpn might recognize particular DNA conformations to achieve DNA-binding specificity.

Nicking activity of the pMV158 MobM relaxase on cognate and heterologous origins of transfer.

The MobM relaxase (494 amino acids) encoded by the promiscuous streptococcal plasmid pMV158 recognizes the plasmid origin of transfer, oriTpMV158, and converts supercoiled pMV158 DNA into relaxed molecules by cleavage of the phosphodiester bond of a specific dinucleotide within the sequence 5'-GTGTG/TT-3' ("/" being the nick site). After cleavage, the protein remains stably bound to the 5'-end of the nick site. Band-shift assays with single-stranded oligonucleotides and size-exclusion chromatography allowed us to show that MobM was able to generate specific complexes with one of the inverted repeats of the oriTpMV158, presumably extruded as stem-loop structure. A number of tests have been performed to attain a better characterization of the nicking activity of MobM and its linkage with its target DNA. The optimal pH for DNA relaxation was found to be 6.5. Upon nicking, gel retardation assays showed that MobM formed stable complexes with its target DNA. Moreover, MobM bound to relaxed pMV158 molecules were visualized by electron microscopy. The staphylococcal plasmids pUB110 and pE194, and the streptococcal plasmid pDL287 harbour putative oriTs and may encode Mob proteins homologous to MobM. The oriTpUB110, oriTpDL287, and oriTpE194 sequences share 100%, 70%, and 67% (in a 43-nucleotide stretch and allowing a 3-bp gap) identity to oriTpMV158, respectively. Nicking assays using supercoiled DNAs from pUB110, pDL287, and pE194 showed that MobM was able to relax, to differing degrees, all plasmid DNAs. Our results suggest that cross-recognition of heterologous oriTs by Mob proteins could play an important role in the plasmid spreading between bacteria.

In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization.

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2-7 onto DNA. Helicase loading involves two MCM2-7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2-7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC-Cdc6 interaction and MCM2-7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2-7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2-7. To determine whether Cdc6 regulates MCM2-7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2-7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2-7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2-7 recruitment, show that ATPase activity is required for MCM2-7 hexamer dimerization and demonstrate that MCM2-7 hexamers are recruited to origins in a consecutive process.

LGALS3BP regulates centriole biogenesis and centrosome hypertrophy in cancer cells.

Centrosome morphology and number are frequently deregulated in cancer cells. Here, to identify factors that are functionally relevant for centrosome abnormalities in cancer cells, we established a protein-interaction network around 23 centrosomal and cell-cycle regulatory proteins, selecting the interacting proteins that are deregulated in cancer for further studies. One of these components, LGALS3BP, is a centriole- and basal body-associated protein with a dual role, triggering centrosome hypertrophy when overexpressed and causing accumulation of centriolar substructures when downregulated. The cancer cell line SK-BR-3 that overexpresses LGALS3BP exhibits hypertrophic centrosomes, whereas in seminoma tissues with low expression of LGALS3BP, supernumerary centriole-like structures are present. Centrosome hypertrophy is reversed by depleting LGALS3BP in cells endogenously overexpressing this protein, supporting a direct role in centrosome aberration. We propose that LGALS3BP suppresses assembly of centriolar substructures, and when depleted, causes accumulation of centriolar complexes comprising CPAP, acetylated tubulin and centrin.

Inducible Clostridium perfringens bacteriophages ΦS9 and ΦS63: Different genome structures and a fully functional sigK intervening element.

Two inducible temperate bacteriophages ΦS9 and ΦS63 from Clostridium perfringens were sequenced and analyzed. Isometric heads and long non-contractile tails classify ΦS9 and ΦS63 in the Siphoviridae family, and their genomes consist of 39,457 bp (ΦS9) and 33,609 bp (ΦS63) linear dsDNA, respectively. ΦS63 has 3'-overlapping cohesive genome ends, whereas ΦS9 is the first Clostridium phage featuring an experimentally proven terminally redundant and circularly permuted genome. A total of 50 and 43 coding sequences were predicted for ΦS9 and ΦS63, respectively, organized into 6 distinct lifestyle-associated modules typical for temperate Siphoviruses. Putative functions could be assigned to 26 gene products of ΦS9, and to 25 of ΦS63. The ΦS9 attB attachment and insertion site is located in a non-coding region upstream of a putative phosphorylase gene. Interestingly, ΦS63 integrates into the 3' part of sigK in C. perfringens, and represents the first functional skin-element-like phage described for this genus. With respect to possible effects of lysogeny, we did not obtain evidence that ΦS9 may influence sporulation of a lysogenized host. In contrast, interruption of sigK, a sporulation associated gene in various bacteria, by the ΦS63 prophage insertion is more likely to affect sporulation of its carrier.