The use of filamentous phage M13 in protein engineering

M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.     

Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display

A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency. Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat. The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation. Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix. In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes. Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display. Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display. The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display. The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions. These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology.     

High rates of conjugation in bacterial biofilms as determined by quantitative in situ analysis.

Quantitative in situ determination of conjugative gene transfer in defined bacterial biofilms using automated confocal laser scanning microscopy followed by three-dimensional analysis of cellular biovolumes revealed conjugation rates 1,000-fold higher than those determined by classical plating techniques. Conjugation events were not affected by nutrient concentration alone but were influenced by time and biofilm structure.     

Insertion of the cos-site into DNA of phage M13 and its packing in proteins of phage lambda

The cos-site of lambda phage from pHC79 cosmide is transferred to DNA from M13 mp18 phage. The recombinant DNA thus obtained (MC18) is efficiently packaged into lambda proteins in vitro. The BamHI-HindIII fragment of pGP588 (a pBR322 derivatives containing fragment of human DNA) is subcloned into MC18. Although this pGP588 fragment contains numerous Alu repeats, no essential rearrangements of the insert were revealed. The efficiency infection by recombinant DNA packaged with lambda proteins is about 1 X 10(5) pfu/microgram DNA, whereas in the similar conditions the efficiency of lambda EMBL3A was 1 X 10(6) pfu/microgram. It is assumed that the MC vectors might be suitable for cloning and sequencing large fragments either with cohesive or blunt ends. It opens also the way to construct genomic libraries in single-stranded phages.     

Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage

The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions.     

Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.

BACKGROUND: Infection of male Escherichia coli cells by filamentous Ff bacteriophages (M13, fd, and f1) involves interaction of the phage minor coat gene 3 protein (g3p) with the bacterial F pilus (primary receptor), and subsequently with the integral membrane protein TolA (coreceptor). G3p consists of three domains (N1, N2, and CT). The N2 domain interacts with the F pilus, whereas the N1 domain--connected to N2 by a flexible glycine-rich linker and tightly interacting with it on the phage--forms a complex with the C-terminal domain of TolA at later stages of the infection process. RESULTS: The crystal structure of the complex between g3p N1 and TolA D3 was obtained by fusing these domains with a long flexible linker, which was not visible in the structure, indicating its very high disorder and presumably a lack of interference with the formation of the complex. The interface between both domains, corresponding to approximately 1768 A2 of buried molecular surface, is clearly defined. Despite the lack of topological similarity between TolA D3 and g3p N2, both domains interact with the same region of the g3p N1 domain. The fold of TolA D3 is not similar to any previously known protein motifs. CONCLUSIONS: The structure of the fusion protein presented here clearly shows that, during the infection process, the g3p N2 domain is displaced by the TolA D3 domain. The folds of g3p N2 and TolA D3 are entirely different, leading to distinctive interdomain contacts observed in their complexes with g3p N1. We can now also explain how the interactions between the g3p N2 domain and the F pilus enable the g3p N1 domain to form a complex with TolA.     

Sequence analysis of ultraviolet-induced mutations in M13lacZ hybrid phage DNA

We have studied the specificity of ultraviolet (u.v.) mutagenesis in single-stranded DNA phage by analyzing u.v.-induced forward mutations in the lac insert of M13mp2 hybrid phage. Sequence analysis of 114 lac mutants derived from u.v.-irradiated phage grown in u.v.-irradiated cells showed that ultraviolet induces mainly single-nucleotide substitutions and deletions in progeny phage DNA. A total of 74% of the single-base substitution mutations occurred at sites of adjacent pyrimidines in the single-stranded DNA, with both T----C and C----T transitions predominating in the u.v. spectrum. Single-nucleotide deletion mutations occurred preferentially in tracts of repeated pyrimidine nucleotides. Tandem, double-base substitutions did not represent a major class of u.v.-induced mutations, but nearly 10% of mutant clones contained multiple, non-tandem nucleotide changes.     

Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p

Appropriate control of the chromosome end-replicating enzyme telomerase is crucial for maintaining telomere length and genomic stability. The essential telomeric DNA-binding protein Cdc13p both positively and negatively regulates telomere length in budding yeast. Here we test the effect of purified Cdc13p on telomerase action in vitro. We show that the full-length protein and its DNA-binding domain (DBD) inhibit primer extension by telomerase. This inhibition occurs by competitive blocking of telomerase access to DNA. To further understand the requirements for productive telomerase 3′-end access when Cdc13p or the DBD is bound to a telomerase substrate, we constrained protein binding at various distances from the 3′-end on two sets of increasingly longer oligonucleotides. We find that Cdc13p inhibits the action of telomerase through three distinct biochemical modes, including inhibiting telomerase even when a significant tail is available, representing a novel ‘action at a distance’ inhibitory activity. Thus, while yeast Cdc13p exhibits the same general activity as human POT1, providing an off switch for telomerase when bound near the 3′-end, there are significant mechanistic differences in the ways telomere end-binding proteins inhibit telomerase action.     

Genomic fingerprinting of microorganisms: its use as a hybridization probe of phage M13 DNA

Hypervariable nucleotide sequences detected by hybridization with the phage M13 DNA probe were found in the chromosomal DNAs of certain pathogenic microbial species. DNA fingerprinting, based on hybridization of M13-probe with hypervariable chromosomal DNA sequences, opens new approaches to epidemiological analysis, epidemiological prognosis, taxonomy, and other theoretical and applied fields of bacteriology.     

Inhibition of bacterial segregation by early functions of phage Mu and association of replication protein B with the inner cell membrane

Summary Infection of Mu-sensitive bacteria with a recombinant phage that carries the EcoRI·C fragment from the immunity end of wild type Mu DNA causes filamentous growth. Transmission electron microscopy revealed that the cell-division cycle was inhibited at, or prior to, the initiation of septation. The filamentation does not occur after infection of Mu-immune bacteria or after infection with a phage carrying the same EcoRI·C fragment, but with an IS1 insertion in gene B of Mu, showing that either gpB and/or some non-essential functions (e.g. kil) mapping downstream from the insertion are required for the inhibition of cell division. These data and previously published evidence suggest that in the killing of E. coli K12 by early Mu functions expressed from the cloned EcoRI·C fragment, two components have to be distinguished: one, a highly efficient elimination of plasmid DNA carrying the early Mu genes, and second, a series of interactions with host functions conducent to an inhibition of cell division. It is suggested that functions normally involved in the SOS reaction participtate in the inhibition of cell division by early Mu functions. Infected bacteria synthesize the replication protein B (M_R 33000) of Mu, which was found by cell fractionation experiments to be associated with the inner cell membrane. The role of this association for filamentous growth and for the integrative replication of the phage is discussed. The recombinant phage might be useful as a tool for the study of the E. coli cell division cycle.     

Inhibition of atrazine degradation by cyanazine and exogenous nitrogen in bacterial isolate M91-3

A variety of s-triazine herbicides and nitrogen fertilizers frequently occur as co-contaminants at pesticide manufacturing and distribution facilities. The degradation of atrazine and cyanazine by the bacterial isolate M91-3 was investigated in washed-cell suspensions and crude cellular extracts. Cyanazine competitively inhibited atrazine degradation. The maximum atrazine degradation rate (V _max) was 41 times higher and the half-saturation constant for the inhibitor (K _i) was 1.3 times higher in the crude cellular extract than in the washed-cell suspension, suggesting that cellular uptake influenced degradation of the s-triazines. Cultures that had received prior exposure to atrazine and simazine exhibited comparable atrazine degradation rates, while cells exposed to cyanazine, propazine, ametryne, cyanuric acid, 2-hydroxyatrazine, biuret, and urea exhibited a lack of atrazine-degradative activity. Growth in the presence of exogenous inorganic nitrogen inhibited subsequent atrazine-degradative activity in washed-cell suspensions, suggesting that regulation of s-triazine and nitrogen metabolism are linked in this bacterial isolate. These findings have significant implications for the environmental fate of s-triazines in agricultural settings since these herbicides are frequently applied to soils receiving N fertilizers. Furthermore, these results suggest that bioremediation of s-triazine-contaminated sites (common at pesticide distribution facilities in the cornbelt) may be inhibited by the presence of N fertilizers that occur as co-contaminants. Received: 3 March 1998 / Received revision: 24 September 1998 / Accepted: 11 October 1998     

Cut and move: protein machinery for DNA processing in bacterial conjugation

Conjugation is a paradigmatic example of horizontal or lateral gene transfer, whereby DNA is translocated between bacterial cells. It provides a route for the rapid acquisition of new genetic information. Increased antibiotic resistance among pathogens is a troubling consequence of this microbial capacity. DNA transfer across cell membranes requires a sophisticated molecular machinery that involves the participation of several proteins in DNA processing and replication, cell recruitment, and the transport of DNA and proteins from donor to recipient cells. Although bacterial conjugation was first reported in the 1940s, only now are we beginning to unravel the molecular mechanisms behind this process. In particular, structural biology is revealing the detailed molecular architecture of several of the pieces involved.     

Inhibition of bacterial transglutaminase by its heat-treated pro-enzyme

Transglutaminases form a unique family of cross-linking enzymes which may be interesting for pharmaceutical and technical purposes. Bacterial transglutaminase, differing from the eucaryotic counterparts in being independent from Ca2+ ions, is excreted by several Streptomyces species. Until now an endogenous factor regulating activated transglutaminase could not be detected. Here, we investigated whether an inhibitor of transglutaminase is excreted into the culture fluid of Streptomyces mobaraensis. We could demonstrate that heat-resistant inhibitory activity is produced after 24h of growth reaching a maximum after 72h. A two-step ion exchange chromatography purification procedure revealed co-elution of the heat-treated inhibitor with pro-transglutaminase. Experiments with wild-type and recombinant pro-transglutaminase confirmed that the precursor protein indeed inhibits the activity of the mature enzyme.     

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.     

The mechanism of bacterial infection by filamentous phages involves molecular interactions between TolA and phage protein 3 domains

The early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized.     

A simple and rapid method to isolate purer M13 phage by isoelectric precipitation

M13 virus (phage) has been extensively used in phage display technology and nanomaterial templating. Our research aimed to use M13 phage to template sulfur nanoparticles for making lithium ion batteries. Traditional methods for harvesting M13 phage from Escherichia coli employ polyethylene glycol (PEG)-based precipitation, and the yield is usually measured by plaque counting. With this method, PEG residue is present in the M13 phage pellet and is difficult to eliminate. To resolve this issue, a method based on isoelectric precipitation was introduced and tested. The isoelectric method resulted in the production of purer phage with a higher yield, compared to the traditional PEG-based method. There is no significant variation in infectivity of the phage prepared using isoelectric precipitation, and the dynamic light scattering data indirectly prove that the phage structure is not damaged by pH adjustment. To maximize phage production, a dry-weight yield curve of M13 phage for various culture times was produced. The yield curve is proportional to the growth curve of E. coli. On a 200-mL culture scale, 0.2 g L^?1 M13 phage (dry-weight) was produced by the isoelectric precipitation method.