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PubMed2003

Parallel tempering simulations of HP-36.

We report results from all-atom Monte Carlo simulations of the 36-residue villin headpiece subdomain HP-36. Protein-solvent interactions are approximated by an implicit solvent model. The parallel tempering is used to overcome the problem of slow convergence in low-temperature protein simulations. Our results show that this technique allows one to sample native-like structures of small proteins and points out the need for improved energy functions.

Algorithms↗
PubMed2004

All-atom folding of the three-helix HIV accessory protein with an adaptive parallel tempering method.

All-atom protein structure prediction from the amino acid sequence alone remains an important goal of biophysical chemistry. Recent progress in force field development and validation suggests that the PFF01 free-energy force field correctly predicts the native conformation of various helical proteins as the global optimum of its free-energy surface. Reproducible protein structure prediction requires the availability of efficient optimization methods to locate the global minima of such complex potentials. Here we investigate an adapted version of the parallel tempering method as an efficient parallel stochastic optimization method for protein structure prediction. Using this approach we report the reproducible all-atom folding of the three-helix 40 amino acid HIV accessory protein from random conformations to within 2.4 A backbone RMS deviation from the experimental structure with modest computational resources.

Computer Simulation↗
PubMed2003

Dual isotope and isotopomer ratios of N2O emitted from a temperate grassland soil after fertiliser application.

The N2O and N2 fluxes emitted from a temperate UK grassland soil after fertiliser application (equivalent to 25 and 75 kg N ha(-1)) were simultaneously measured, using a new automated soil incubation system, which replaces soil atmosphere (N2 dominated) with a He+O2 mixture. Dual isotope and isotopomer ratios of the emitted N2O were also determined. Total N2O and N2 fluxes were significantly lower (P<0.001) in the control (0 kg N) than in the 25 and 75 kg N treatments. The total N2O flux was significantly higher (P<0.001) in the 75 kg N than in the 25 kg N treatment. The general patterns of N2O and N2 fluxes were similar for both fertiliser treatments. The total gaseous N loss in the control treatment was nearly all N2, whereas in the fertiliser treatment more N2O than N2 was emitted from the soil. The ratio N2O/N2 fluxes as measured during the experiment suggested three phases in N2O production, in phase 1 nitrification>denitrification, in phase 2 denitrification>nitrification, and in phase 3 denitrification (and total denitrification)>>nitrification. Dual delta15N and delta18O isotope and isotopomer (delta15Nalpha and delta15Nbeta) value ratios of emitted N2O also pointed towards an increasing dominance of the production of N2O by denitrification and total denitrification. The site preference value from the soil-emitted N2O was lower than the troposphere value. This confirmed that the enhanced troposphere N2O site preference could result from back injection of N2O from the stratosphere. The measurements of N2O/N2 flux ratio and the isotopic content of emitted N2O pointed, independently, to similar temporal trends in N2O production processes after fertiliser application to grassland soil. This confirmed that both measurements are suitable diagnostic tools to study the N2O production process in soils.

Agriculture↗
PubMed2000

Genomic sequence and analysis of the atypical temperate bacteriophage N15.

N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli. While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres. Here, we describe the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens. The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms. The left arm contains a long cluster of structural genes that are closely related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB. The right arm likewise contains a mixture of apparently phage- and plasmid-derived genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases. The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized.

Bacteriolysis↗
PubMed1996

Evolution of the genetic switch in temperate bacteriophage. I. Basic theory.

While the molecular mechanisms underlying lysogeny and induction in bacteriophage have been intensely studied, relatively little has been done to relate these findings to their presumed selective functions. To explore the ecological basis for these traits, I have used a resource-based model for competition between bacteriophage with different probabilities of lysogeny and different spontaneous induction rates. In any given habitat the fitness of a phage will depend on the inputs of sensitive cells and nutrient resources. In equable environments (modeled here using chemostats with constant inputs of nutrients and sensitive cells), bacteriophage with low probabilities of lysogeny and low induction rates can always invade when rare and will generally be good competitors. In variable environments (chemostats with seasonal inputs), bacteriophage with higher probabilities of lysogeny and higher induction rates are favored. In both equable and variable environments, the ability of a phage to invade when rare will depend on the properties of the resident phage, and it is possible for phages with divergent parameter values to coexist. The modeling suggests that bacteriophage that have evolved moderately low induction and lysogeny rates will be able to "hedge their bets" against environmental change without sacrificing the ability to compete well in a constant environment. Implications of this theory for understanding the molecular basis of gene regulation in temperate bacteriophage and other viruses are discussed.

Bacteriophages↗
PubMed1995

Characterization of a temperate Streptococcus thermophilus bacteriophage and its genetic relationship with lytic phages.

The temperate Streptococcus thermophilus bacteriophage phi SFi21 showed an 38-kb-long double-stranded DNA genome with cohesive ends. A single integration site was used in lysogens established in three different S. thermophilus strains. The attP and attB sites were localized on the restriction map of phage DNA and by hybridization on pulsed field separated bacterial DNA. All laboratory-established lysogens showed in addition to integrated prophage DNA unintegrated monomer phage DNA with unligated cos sites. The genetic relatedness of phi SFi21 DNA with DNA from lytic phages was studied in dot blot and Southern blot hybridization by using individual restriction fragments of phiSFi21 DNA as probes. Lytic group I phages hybridized with fragments of the central and the right part of the phiSFi21 genome but failed to hybridize with a fragment joining both parts. Lytic group II phages showed hybridization with the right half of the phiSFi21 genome. In lytic group IV phages, biologically a heterogeneous group, many different combinations of cross hybridization were detected in accordance with the hypothesis of the modular evolution of phage genomes.

Attachment Sites, Microbiological↗
PubMed1996

A genomic region of lactococcal temperate bacteriophage TP901-1 encoding major virion proteins.

Two major structural proteins, MHP (major head protein) and MTP (major tail protein), from the lactococcal temperate phage TP901-1 were sequenced at their amino acid termini, and derived degenerate oligonucleotides were used to locate the corresponding genes in the phage genome. This genomic region was sequenced. The sequence characterized includes a total of 11 open reading frames (ORFs) showing an operon structure. Upstream of each ORF, except ORF b2 and ORF x, potential ribosome-binding sites were found, suggesting independent translation. However, coupled translation is suggested for ORF x and as a possibility for ORF b3 and ORF c2, which have ribosome-binding sites located more distant from their start codons. ORF b2 may be translationally fused with mhp at a low frequency. The mhp and mtp genes are transcribed as a 3.7-kb mRNA with at least six additional ORFs. The organization of the genomic region analyzed resembles that of other distantly related phages, providing possible roles for the uncharacterized ORFs.

Amino Acid Sequence↗
PubMed1996

Site-specific spontaneous deletions in three genome regions of a temperate Streptococcus thermophilus phage.

Site-specific spontaneous deletions were observed with high frequency in three regions of the genome of the temperate Streptococcus thermophilus phage phi SFi21. Deletion sizes were 750 bp (type 1), 2.7 kb (type 2), and 1 kb (type 3). Combinations of types 1 and 3 and 2 and 3 were observed. The mutants grew lytically although with reduced burst sizes. Type 2 mutants lost the capacity to lysogenize host cells. Upon serial passage, the deletion mutants overgrew the wild-type phage. No direct or inverted DNA repeats were associated with type 1 or 2 deletion sites. Several independent phage isolates showed deletions at identical nucleotide positions, suggesting a site-specific recombination system. Sequencing of an Xbal restriction fragment covering the type 1 deletion predicted a single long open reading frame (ORF) showing a high degree of amino acid similarity with two proteins from bacteriophage P1 implicated in its immunity control (KiIA, Ant). Type 1 deletion leads to a loss of the conserved C-terminal part of this ORF.

Amino Acid Sequence↗
PubMed1997

The site-specific integration system of the temperate Streptococcus thermophilus bacteriophage phiSfi21.

The temperate bacteriophage phiSfi21 integrates its DNA into the chromosome of Streptococcus thermophilus strains via site-specific recombination. Nucleotide sequencing of the attachment sites identified a 40-bp identity region which surprisingly overlaps both the 18-terminal bp of the phage integrase gene and the 11-terminal bp of a host tRNAArg gene. A 2.4-kb phage DNA segment, covering attP, the phage integrase, and a likely immunity gene contained all the genetic information for faithful integration of a nonreplicative plasmid into the attB site. A deletion within the int gene led to the loss of integration proficiency. A number of spontaneous deletions were observed in plasmids containing the 2.4-kb phage DNA segment. The deletion sites were localized to the tRNA side of the identity region and to phage or vector DNA with 3- to 6-bp-long repeats from the border region. A similar type of deletion was previously observed in a spontaneous phage mutant.

Bacteriophages↗
PubMed1998

Comparison of the lysogeny modules from the temperate Streptococcus thermophilus bacteriophages TP-J34 and Sfi21: implications for the modular theory of phage evolution.

A 7.6-kb DNA segment covering the putative lysogeny module of the pac-site-containing temperate Streptococcus thermophilus bacteriophage TP-J34 was sequenced. Sequence alignment with the lysogeny module from the cos-site-containing S. thermophilus bacteriophage phiSfi21 revealed areas of high sequence conservation (e.g., over the int gene), interspersed with regions of low or no sequence similarity (e.g., over the cro gene). Four of the six sharp transition zones from high to low sequence conservation were found within open reading frames coding for the CI repressor, the Anti-repressor, the Immunity protein, and a protein of unknown function. The transition points in the cI and ant genes appear to separate gene segments coding for distinct functional domains of these proteins. In addition, these two transition points were located at or near the deletion sites observed in spontaneous phage phiSfi21 deletion mutants, thus suggesting these transition points as recombinational hotspots. Furthermore, the sequence at the transition point in the cI gene resembles the attachment site of the phage, suggesting the involvement of the phage integrase in at least some of the exchange reactions. Contrary to the initial formulation of the modular theory of phage evolution the unit of the evolutionary exchange in streptococcal phages is not a group of functional genes, but can be as small as a single gene. Exchange reactions can also occur within genes, possibly between gene segments encoding distinct protein domains.

Amino Acid Sequence↗
PubMed1999

Comparative sequence analysis of the DNA packaging, head, and tail morphogenesis modules in the temperate cos-site Streptococcus thermophilus bacteriophage Sfi21.

The temperate Streptococcus thermophilus bacteriophage Sfi21 possesses 15-nucleotide-long cohesive ends with a 3' overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry. Over the DNA packaging, head and tail morphogenesis modules, the Sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. A nearly one to one gene correlation was found with the phage lambda genes Nu1 to H, except for gene B-to-E complex, where the Sfi21 map resembled that of coliphage HK97. The similarity between Sfi21 and HK97 was striking: both major head proteins showed an N-terminal coiled-coil structure, the mature major head proteins started at amino acid positions 105 and 104, respectively, and both major head genes were preceded by genes encoding a possible protease and portal protein. The purported Sfi21 protease is the first viral member of the ClpP protease family. The prediction of Sfi21 gene functions by reference to the gene map of intensively investigated coliphages was experimentally confirmed for the major head and tail gene. Phage Sfi21 shows nucleotide sequence similarity with Lactococcus phage BK5-T and a lactococcal prophage and amino acid sequence similarity with the Lactobacillus phage A2 and the Staphylococcus phage PVL. PVL is a missing link that connects the portal proteins from Sfi21 and HK97 with respect to sequence similarity. These observations and database searches, which demonstrate sequence similarity between proteins of phage from gram-positive bacteria, proteobacteria, and Archaea, constrain models of phage evolution.

Amino Acid Sequence↗
PubMed2000

Mutational analysis of two structural genes of the temperate lactococcal bacteriophage TP901-1 involved in tail length determination and baseplate assembly.

Two putative structural genes, orf tmp (tape measure protein) and orf bpp (baseplate protein), of the temperate lactococcal phage TP901-1 were examined by introduction of specific mutations in the prophage strain Lactococcus lactic ssp. cremoris 901-1. The adsorption efficiencies of the mutated phages to the indicator strain L. lactic ssp. cremoris 3107 were determined and electron micrographs were obtained. Specific mutations in orf tmp resulted in the production of mostly phage head structures without tails and a few wild-type looking phages. Furthermore, construction of an inframe deletion or duplication of 29% in orf tmp was shown to shorten or lengthen the phage tail by approximately 30%, respectively. The orf tmp is proposed to function as a tape measure protein, TMP, important for assembly of the TP901-1 phage tail and involved in tail length determination. Specific mutations in orf bpp produced phages which were unable to adsorb to the indicator strain and electron microscopy revealed particles lacking the baseplate structure. The orf bpp is proposed to encode a highly immunogenic structural baseplate protein, BPP, important for assembly of the baseplate. Finally, an assembly pathway of the TP901-1 tail and baseplate structure is presented.

Adsorption↗
PubMed2001

Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: evolution, structure, and genome organization of lactococcal bacteriophages.

A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacteriophage TP901-1 proliferation. Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination. Our results indicate that TP901-1 may have evolved by homologous recombination between the host chromosome and a mother phage and support the observation that phage remnants as well as prophages located in the Lactococcus chromosome contribute significantly to bacteriophage evolution. Some proteins encoded in the early transcribed region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L. lactis. This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool.

Bacteriophages↗
PubMed2002

Transcription mapping as a tool in phage genomics: the case of the temperate Streptococcus thermophilus phage Sfi21.

For the lytic growth cycle of the temperate cos-site Streptococcus thermophilus phage Sfi21 a transcription map was developed on the basis of systematic Northern blot hybridizations. All deduced 5' ends were confirmed by primer extension analysis. Three time classes of transcripts were observed. Early transcripts were identified in four different genome regions. One prominent early mRNA of 4.8 kb length covered a group of 12 genes located between the origin of replication and the cos-site. Two short early mRNAs represented a single gene from the direct vicinity of the cos-site and the superinfection immunity gene from the lysogeny module, respectively. A fourth early transcript covered a group of four genes located between the lysin and the integrase gene. Middle transcripts of 2.1 and 5.8 kb length covered cro-like and ant-like repressor genes and the DNA replication module, respectively. Four types of late transcripts were identified. The transcripts covered the likely DNA packaging genes, the head morphogenesis module plus the major tail gene, the remainder of the tail genes, and the putative tail fiber plus lysis genes, respectively. Only the transcript from the head morphogenesis genes yielded defined late mRNA species. The transcription map concurred with most of the in silico predictions for the genome organization of phage Sfi21 except for the separation of the DNA replication module from a possible transcription regulation module. Most 5' ends of the transcripts determined in primer-extension experiments were not preceded by a consensus promoter sequence. The involvement of phage-encoded regulators for middle and late transcription was suggested by chloramphenicol-inhibition experiments.

5' Untranslated Regions↗
PubMed1977

Deletion mutants of temperate Bacillus subtilis bacteriophage phi105.

Six deletion mutants of temperate Bacillus subtilis phage phi105 have been isolated on the basis of their increased resistance to chelating agents. The size and position of the deletions was determined by electronmicroscopy of DNA heteroduplexes. All deletions are located in a region about 55-70% from one end of the DNA molecule, in the right half of the known genetic map of the phage. The segment 55-65% does not contain any genes essential for lytic growth or lysogenization. A gene(s) for immunity is located in a segment 65-70% from the left end. By electronmicroscopy by partially denatured phi105 DNA two A-T rich regions have been localized in the right half of the molecule. One of these regions falls within the non-essential 55-65% DNA segment.

Bacillus subtilis↗
PubMed1995

Isolation and characterization of a temperate bacteriophage from a ruminal acetogen.

Nine acetogenic bacterial cultures recently isolated from the bovine rumen were tested for phage susceptibility by plaque formation. Both clear plaques and plaques with turbid centers were occasionally seen, but could not be used repeatedly to lyse pure cultures of acetogens, suggesting the possibility of a temperate phage. Five of the nine acetogenic isolates showed a response to mitomycin C induction. Acetogenic isolate H3HH was chosen for further study because it produced the greatest lysogenic response to mitomycin C. The bacteriophage was induced with mitomycin C, examined by transmission electron microscopy, and shown to have a hexagonal head (diameter, 59 eta m), a long flexible tail (192 eta m), and a flat collar (diameter, 31 eta m). The bacteriophage was classified within Bradley's group B. Bacteriophage DNA was determined to contain 36.2 kilobases of linear double-stranded DNA.

Acetates↗
PubMed1982

Inverted duplication in the genome of the temperate Streptomyces phage SH3.

The DNA of the temperate Streptomyces phage SH3 contains 100 base-pair long inverted repeats separated by a 940 base-pair long segment of DNA as revealed by electronmicroscopic analysis of snapback structures formed after rapid intrastrand reannealing of denatured DNA. The inverted repeat structure was found preferentially at map unit 22 of the circular physical map, in rare cases also in other positions, suggesting a movable character of this genetic element.

Bacteriophages↗
PubMed1979

Restriction mapping of DNA of temperate Rhizobium meliloti phage 16-3: comparison of genetic and physical maps indicates a long, genetically silent chromosomal arm.

The complete restriction map of DNA (61.57 Kb) of temperate Rhizobium meliloti phage 16-3 has been constructed for enzymes BglII, HindIII, HpaI, KpnI, and a partial map for EcoRI. The strategy employed for mapping included the analysis of double, triple and partial digests; comparison of wild type and deletion mutants; and detailed analysis of subfragments, exploiting the presence of cohesive ends of the phage. Comparison of the genetic and physical maps indicates that one arm of the chromosome is genetically silent and/or contains nonessential genes.

Bacteriophages↗