Chaperone–Protein Interactions That Mediate Assembly of the Bacteriophage Lambda Tail to the Correct Length

Bacteriophage λ makes two proteins with overlapping amino acid sequences that are essential for tail assembly. These two proteins, gpG and gpGT, are related by a programmed translational frameshift that is conserved among diverse phages and functions in λ to ensure that gpG and the frameshift product gpGT are made in a molar ratio of approximately 30:1. Although both proteins are required and must be present in the correct ratio for assembly of functional tails, neither is present in mature tails. During λ tail assembly, major tail protein gpV polymerizes to form a long tube whose length is controlled by the tape measure protein gpH. We show that the “G” domains of gpG and gpGT bind to all or parts of tail length tape measure protein gpH and that the “T” domain of gpGT binds to major tail shaft subunit gpV, and present a model for how gpG and gpGT chaperone gpH and direct the polymerization of gpV to form a tail of the correct length.     

Conserved translational frameshift in dsDNA bacteriophage tail assembly genes

A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.     

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.     

Crystal Structure of ORF12 from Lactococcus lactis Phage p2 Identifies a Tape Measure Protein Chaperone

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is detected in the second half of the lytic cycle, indicating that it is a late-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-Å resolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface. Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice was identified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeated motif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex between ORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fitted exquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act as a chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of the lactococcal siphophage p2.During industrial milk fermentation, Lactococcus lactis cells are added to transform milk into an array of fermented products such as cheese. However, this manufacturing process may be impaired by lytic phages present in the factory environment as well as in the milk itself (30). Due to the destructive effects of phage infections on bacterial fermentation, much effort has been undertaken to isolate and study the biodiversity of these bacteriophages. Lactococcal bacteriophages belong to at least 10 different genetically distinct species of double-stranded DNA viruses (9). Of them, three lactococcal phage species, all belonging to the Siphoviridae family, are the major source of problems in milk fermentation, namely, the 936, P335, and c2 species (7, 28, 29). Furthermore, members of the 936 species are by far responsible for the majority of infections (50 to 80%) (1, 24, 41). Numerous phages of the 936 species have been isolated, and several have been characterized at the genome level (25). However, little is known concerning their molecular mechanisms of infection, although we recently solved the structure of the receptor-binding protein (RBP) of our model 936-like phage, namely, the virulent phage p2 (38, 43), and of phages belonging to the P335 species (27, 34, 37, 38).As with all viruses, bacteriophage genomes are quite compact, leaving little room for noncoding sequences (4). In fact, phage genes are disposed in an operon-type organization (4), and the order of genes corresponds to the different phases of the infection cycle. Moreover, genes are often in clusters (referred to as modules), with gene products from adjacent genes generally found to interact with each other. Interestingly, phage genome organization, including individual gene order, is often conserved within a given species, particularly within the Siphoviridae family. In the case of L. lactis virulent phages belonging to the 936 or P335 species, this principle applies particularly to the morphogenesis gene module, which includes all the genes coding for the phage structural protein genes. For the tail assembly, a module comprises a set of genes between the portal protein, which is connecting the tail to the capsid, and the RBP, which is located at the tip of the tail and is involved in host recognition (39, 43).The characterization of tail assembly genes of lactococcal phages has been more extensive for temperate siphophages belonging to the P335 species (27, 34, 37, 38). Because of the similarities in genome organization, the findings in this phage species can, in some cases, be used as clues toward understanding the morphology of 936-like phages. For the temperate phage Tuc2009 (P335 species), all structural proteins required for tail and baseplate assembly have been identified (27, 34, 37, 38). Genes located between those coding for the tape measure protein (TMP) and BppL (RBP) were identified as corresponding to components of the baseplate structure, located at the tail distal end. Furthermore, a gene coding for the major tail protein (MTP) was also identified at a position upstream from tmp. Between the genes coding for the MTP and those coding for the TMP in Tuc2009 are two gene products identified as gpG and gpGT, which are not present in the phage particle. These two proteins were named based on their likely role analogous to the tail assembly proteins present in coliphage lambda, a model virus belonging to the Siphoviridae family (21, 27, 47). gpGT has an essential role in lambda tail assembly, acting prior to tail shaft assembly, while the role of gpG in tail assembly is not known (21). Both gpG and gpGT are also absent from mature lambda virions (21). It has been argued that they may act as assembly chaperones (47).A close examination of 936 genomes indicates the presence of two genes coding for gpG and gpGT-like proteins. Analysis of the phage p2 genome, closely related to that of lactococcal phage sk1 (6), revealed that the putative tail assembly proteins could correspond to gene products ORF12 and ORF13. These two genes are followed by the TMP gene corresponding to orf14, other genes coding for other structural proteins, and the RBP gene orf18. During our ongoing investigation of the structure of phage p2, we report here the cloning, expression, and crystal structure of ORF12 in order to decipher its role in the tail assembly process.     

Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

Mechanism of length determination in bacteriophage lambda tails

The mechanism of length determination in bacteriophage λ tails is discussed as a model for regulation in protein assembly systems.The λ tail is a long flexible tube ending in a conical part and a single tail fiber. Its length is exactly determined in the sense that the number of major tail protein (gpV) molecules, which comprise more than 80% of the mass of the tail, is exactly the same in all tails. Assembly of gpV is regulated by the initiator complex, which contains the tail fiber and the conical part,and by the terminator protein gpU. There are two key points in the assembly of gpV with respect to length determination. (1) Assembly of gpV on the initiator pauses at the correct tail length. Binding of gpU to the tail only fixes the pause firmly. (2) When the tail length is too short, binding of gpU to tails is inhibited.Deletions and a duplication (both in frame) in gene H, which codes for one of the proteins in the initiator, result in production of phage particles with altered tail length. Moreover, the tail length is roughly proportional to the length of the mutated versions of gene H. This shows that the tail length is measured by the length of gene H protein (gpH), which seems to be approximately as long as the tail tube, if extended like a thread, according to secondary structure prediction (α-helices connected by other structures). Various pieces of evidence show that about six molecules of gpH are attached to the remaining portion of the initiator by the C-terminal part and folded into a somewhat compact form, while they are elongated as they are enclosed in the tail tube during assembly of gpV. Unlike interaction between the length-measuring genome RNA and the coat protein of tobacco mosaic virus, the major tail protein gpV does not bind specifically to the ruler protein gpH. Rather, gpH determines the tail length by inhibiting the binding of gpU to short tails and by signalling the pause when the correct tail length is attained.     

Bacteriophage T4 tail assembly: proteins of the sheath, core and baseplate

Structural intermediates in phage tail formation have been isolated by sucrose gradient centrifugation from cells infected with mutants blocked at various stages in tail assembly. The polypeptide chains of these structures containing ¹⁴C-labeled amino acids have been analyzed by sodium dodecyl sulfate—acrylamide gel electrophoresis, enabling us to identify the proteins forming the various morphological components of the tail. Comparison of sheathed tails with corebaseplates shows that the contractile sheath is composed of a single species of subunit, the product of gene 18 (mol.wt 80,000). The site for head attachment terminating the tail is composed of the product of gene 15 (mol.wt 35,000). Comparison of core-baseplates with free baseplates shows that the tail core is composed of a single species of subunit, the product of gene 19 (mol.wt 21,000).Free baseplates are composed of at least twelve species of proteins: the products of genes 6, 7, 8, 9, 10, 11, 12 and 29, and four genetically unidentified species.The incomplete tails which accumulate in cells infected with mutants defective in genes 9, 11 and 12, which specify proteins on the outside of the baseplate, have also been characterized. Tails from 9^? lysates lack only P9. Tails from 11^? lysates lack both Pll and P12. Tails from 12^? infection lack only P12. Incorporation of P12 into the baseplate requires the function of gene 57, which is also required for tail fiber assembly. P57 thus appears to take part in the maturation of three different phage structural proteins.The sequential nature of the protein interactions in tail formation is discussed in terms of the regulation of morphogenesis at the level of assembly.     

Xenopus Actin Depolymerizing Factor/Cofilin (XAC) Is Responsible for the Turnover of Actin Filaments in Listeria monocytogenes Tails

In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.     

Genome wide sequence analysis grants unbiased definition of species boundaries in “Candidatus Phytoplasma”

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species “Ca. P. asteris”, “Ca. P. mali”, “Ca. P. pyri”, “Ca. P. pruni”, and “Ca. P. australiense” were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. “Ca. P. mali” and “Ca. P. pyri” were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.     

Gene switching at Xenopus laevis metamorphosis

During the climax of amphibian metamorphosis many tadpole organs remodel. The different remodeling strategies are controlled by thyroid hormone (TH). The liver, skin, and tail fibroblasts shut off tadpole genes and activate frog genes in the same cell without DNA replication. We refer to this as “gene switching”. In contrast, the exocrine pancreas and the intestinal epithelium dedifferentiate to a progenitor state and then redifferentiate to the adult cell type. Tadpole and adult globin are not present in the same cell. Switching from red cells containing tadpole-specific globin to those with frog globin in the liver occurs at a progenitor cell stage of development and is preceded by DNA replication. Red cell switching is the only one of these remodeling strategies that resembles a stem cell mechanism.     

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

Antenna ring around trimeric Photosystem I in chlorophyll b containing cyanobacterium Prochlorothrix hollandica

Prochlorothrix hollandica is one of the three known species of an unusual clade of cyanobacteria (formerly called “prochlorophytes”) that contain chlorophyll a and b molecules bound to intrinsic light-harvesting antenna proteins. Here, we report the structural characterization of supramolecular complex consisting of Photosystem I (PSI) associated with the chlorophyll a/b-binding Pcb proteins. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the Pcb-PSI supercomplex consists of a central trimeric PSI surrounded by a ring of 18 Pcb subunits. We conclude that the formation of the Pcb ring around trimeric PSI represents a mechanism for increasing the light-harvesting efficiency in chlorophyll b-containing cyanobacteria.     

Dynamics of the glutamic acid 242 side chain in cytochrome c oxidase

In many cytochrome c oxidases glutamic acid 242 is required for proton transfer to the binuclear heme a₃/Cu_B site, and for proton pumping. When present, the side chain of Glu-242 is orientated “down” towards the proton-transferring D-pathway in all available crystal structures. A nonpolar cavity “above” Glu-242 is empty in these structures. Yet, proton transfer from Glu-242 to the binuclear site, and for proton-pumping, is well established, and the cavity has been proposed to at least transiently contain water molecules that would mediate proton transfer. Such proton transfer has been proposed to require isomerisation of the Glu-242 side chain into an “up” position pointing towards the cavity. Here, we have explored the molecular dynamics of the protonated Glu-242 side chain. We find that the “up” position is preferred energetically when the cavity contains four water molecules, but the “down” position is favoured with less water. We conclude that the cavity might be deficient in water in the crystal structures, possibly reflecting the “resting” state of the enzyme, and that the “up/down” equilibrium of Glu-242 may be coupled to the presence of active-site water molecules produced by O₂ reduction.     

Sequential binding of FurA from Anabaena sp. PCC 7120 to iron boxes: Exploring regulation at the nanoscale

Fur (ferric uptake regulator) proteins are involved in the control of a variety of processes in most prokaryotes. Although it is assumed that this regulator binds its DNA targets as a dimer, the way in which this interaction occurs remains unknown. We have focused on FurA from the cyanobacterium Anabaena sp. PCC 7120. To assess the molecular mechanism by which FurA specifically binds to “iron boxes” in P_furA, we examined the topology arrangement of FurA–DNA complexes by atomic force microscopy. Interestingly, FurA–P_furA complexes exhibit several populations, in which one is the predominant and depends clearly on the regulator/promoter ratio on the environment. Those results together with EMSA and other techniques suggest that FurA binds P_furA using a sequential mechanism: (i) a monomer specifically binds to an “iron box” and bends P_furA; (ii) two situations may occur, that a second FurA monomer covers the free “iron box" or that joins to the previously used forming a dimer which would maintain the DNA kinked; (iii) trimerization in which the DNA is unbent; and (iv) finally undergoes a tetramerization; the next coming molecules cover the DNA strands unspecifically. In summary, the bending appears when an “iron box” is bound to one or two molecules and decreases when both “iron boxes” are covered. These results suggest that DNA bending contributes at the first steps of FurA repression promoting the recruitment of new molecules resulting in a fine regulation in the Fur-dependent cluster associated genes.