Phage Restriction Research Articles

Physiological and molecular characterization of locally adapted Rhizobium strains of lentil (Lens culinaris Medik.) having restricted phage sensitivity

Indigenous rhizobia were isolated from root nodules of lentil plants collected from various agro-climatic regions of India. These isolates together with four standard lentil Rhizobium strains were screened for sensitivity against eight phages. Four strains, USDA 2431, BHULR 104, BHULR 113 and BHULR 115 having restricted sensitivity to lytic phages LRP-1, LRP-4, LRP-13 and LRP-15 respectively, were characterized for both physiological and molecular characters. All strains had a generation time of between 3.8 and 5.6 h in yeast extract–mannitol (YM) broth, and colonies on YM agar plates showed an acidic reaction. Compared to other strains, strain USDA 2431 grew poorly when sucrose was the sole carbon source and showed maximum growth in arabinose-containing medium. The intrinsic antibiotic resistance level in all strains was tested against seven antibiotics and found to be high with ampicillin and kanamycin (50–60 μg ml-1) but very low with neomycin (0.03 μg ml-1). With the exception of strain BHULR 113, all strains expressed ex planta nitrogenase activity, with strain USDA 2431 showing the maximum activity (26.8 nmol C2H4 h-1 mg-1 protein) after 6 h of incubation. Genomic and phylogenetic relationships among the strains were examined by randomly amplified polymorphic DNA and 16S rRNA sequence analysis. Genetic distance varied from 0.09 to 0.23 among the strains, and the primer OPL-11 was found to be suitable for the discrimination of these strains. The 16S rRNA sequence analysis revealed 99–100% similarity with Rhizobium leguminosarum bv. viciae. These results clearly indicate that phage sensitivity is a useful marker for discriminating locally soil-adapted rhizobial strains forming effective nodules in lentil.

Bacteriophages and Viruses as a Support for Organic Synthesis and Combinatorial Chemistry

Display of polypeptide on the coat proteins of bacteriophages and viruses is a powerful tool for selection and amplification of libraries of great diversity. Chemical diversity of these libraries, however, is limited to libraries made of natural amino acid side chains. Bacteriophages and viruses can be modified chemically; peptide libraries presented on phage thus can be functionalized to yield moieties that cannot be encoded genetically. In this review, we summarize the possibilities for using bacteriophage and viral particles as support for the synthesis of diverse chemically modified peptide libraries. This review critically summarizes the key chemical considerations for on-phage syntheses such as selection of reactions compatible with protein of phage, modification of phage "support" that renders it more suitable for reactions, and characterization of reaction efficiency.

Survival of the fittest: a role for phage‐encoded eukaryotic‐like kinases

Phages are often thought of as mortal enemies of bacteria. This dynamic relationship has led to the evolution of a number of processes in bacteria designed to defeat these attacks. Examples of these include blocking phage attachment, CRISPR, and restriction modification systems. Temperate phages provide another source of protection by excluding infection of heterologous phage, thwarting phage production and further infection. This strategy protects the rest of the bacterial population from attack. The lambdoid phage 933W, a source of the genes encoding Shiga toxin in the highly pathogenic O157:H7 enterohemorrhagic E. coli strain, also carries a gene encoding a eukaryotic-like tyrosine kinase, Stk. In this issue of Molecular Microbiology, Friedman et al. (2011) show that Stk, through its kinase activity, excludes infection by another lambdoid phage HK97. This exclusion is very specific as it does not affect a number of other lambdoid phages. HK97 contributes to its own demise by expressing the product of an open reading frame, orf41, which is required for Stk activation. The authors further show that autophosphorylation increases the stability of Stk and suggest that autophosphorylation contributes to Stk activity. Whether or not this exclusion activity provides a selective advantage through maintenance of Stk activity is yet to be explored.

The Afipia toolbox and its use to isolate flagellar mutants

Afipia felis, a Gram-negative alphaproteobacterium, has been implicated as one of the causative agents of cat scratch disease. To identify and begin to examine the virulence traits of this organism, we developed and tested a highly efficient transposon delivery system and a stable plasmid vector expressing green fluorescent protein. The transposome system is based on a Tn5-derived transposon and a phage restriction endonuclease type I inhibitor. Electroporation of this construct produced a library of >2600 mutants, which were screened for flagella biosynthesis mutants using a monoclonal antibody to Afipia flagellin. Insertion loci for two selected mutants were located in the genes for flagellin and flagellin biosynthesis FlhA, confirming the validity of the approach.

Characterization of the NgoAXP: phase-variable type III restriction–modification system inNeisseria gonorrhoeae

Methyltransferases associated with type III restriction-modification (RM) systems are phase-variably expressed in a variety of pathogenic bacteria. NgoAXP, the type III RM system encoded by Neisseria gonorrhoeae, was characterized in this study. The cloned resngoAXP and ngoAXPmod genes were expressed in Escherichia coli strains. The restriction and modification activities of NgoAXP were confirmed in vivo by the lambda phage restriction and modification test and in vitro by the methylation of DNA substrates in the presence of [methyl-(3)H]AdoMet. As in all known type III systems, the restriction activity needed the presence of both genes, while the presence of the ngoAXPmod gene was sufficient for DNA methylation. Following its overexpression, the DNA methyltransferase M.NgoAXP was purified to apparent homogeneity using metal affinity chromatography. The specific sequence recognized by this enzyme was determined as a nonpalindromic sequence: 5'-CCACC-3', in which the adenine residue is methylated. We observed that in E. coli cells, the expression of the restriction phenotype associated with NgoAXP switched randomly. This phase variation was associated with the change in the number of pentanucleotide repeats (5'-CCAAC/G-3') present at the 5'-end of the coding region of the ngoAXPmod gene.

Morphological and Genetic Diversity of Temperate Phages in Clostridium difficile

Eight temperate phages were characterized after mitomycin C induction of six Clostridium difficile isolates corresponding to six distinct PCR ribotypes. The hypervirulent C. difficile strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) was among these prophage-containing strains. Observation of the crude lysates by transmission electron microscopy (TEM) revealed the presence of three phages with isometric capsids and long contractile tails (Myoviridae family), as well as five phages with long noncontractile tails (Siphoviridae family). TEM analyses also revealed the presence of a significant number of phage tail-like particles in all the lysates. Southern hybridization experiments with restricted prophage DNA showed that C. difficile phages belonging to the family Myoviridae are highly similar and most likely related to previously described prophages phiC2, phiC5, and phiCD119. On the other hand, members of the Siphoviridae phage family are more genetically divergent, suggesting that they originated from distantly related ancestors. Our data thus suggest that there are at least three genetically distinct groups of temperate phages in C. difficile; one group is composed of highly related myophages, and the other two groups are composed of more genetically heterogeneous siphophages. Finally, no gene homologous to genes encoding C. difficile toxins or toxin regulators could be identified in the genomes of these phages using DNA hybridization. Interestingly, each unique phage restriction profile correlated with a specific C. difficile PCR ribotype.

HOST DENSITY IMPACTS RELATIVE FITNESS OF BACTERIOPHAGE Φ6 GENOTYPES IN STRUCTURED HABITATS

Spatially structured environments may impact evolution by restricting population sizes, limiting opportunities for genetic mixis, or weakening selection against deleterious genotypes. When habitat structure impedes dispersal, low-productivity (less virulent) infectious parasites may benefit from their prudent exploitation of local hosts. Here we explored the combined ability for habitat structure and host density to dictate the relative reproductive success of differentially productive parasites. To do so, we allowed two RNA bacteriophage Phi6 genotypes to compete in structured and unstructured (semi-solid versus liquid) habitats while manipulating the density of Pseudomonas hosts. In the unstructured habitats, the more-productive phage strain experienced a relatively constant fitness advantage regardless of starting host density. By contrast, in structured habitats, restricted phage dispersal may have magnified the importance of local productivity, thus allowing the relative fitness of the less-productive virus to improve as host density increased. Further data suggested that latent period (duration of cellular infection) and especially burst size (viral progeny produced per cell) were the phage "life-history" traits most responsible for our results. We discuss the relevance of our findings for selection occurring in natural phage populations and for the general evolutionary epidemiology of infectious parasites.

Restriction and modification of SP10 phage by BsuM of Bacillus subtilis Marburg

Bacillus subtilis Marburg has only one intrinsic restriction and modification system BsuM that recognizes the CTCGAG ( XhoI site) sequence. It consists of two operons, BsuMM operon for two cytosine DNA methyltransferases, and BsuMR operon for a restriction nuclease and two associated proteins of unknown function. In this communication, we analyzed the BsuM system by utilizing phage SP10 that possesses more than twenty BsuM target sequences on the phage genome. SP10 phages grown in the restriction and modification-deficient strain could not make plaques on the restriction-proficient BsuMR + indicator strain. An enforced expression of the wild type BsuMM operon in the BsuMR + indicator strain, however, allowed more than thousand times more plaques. DNA extracted from SP10 phages, thus, propagated became more but not completely refractory to XhoI digestion in vitro. Thus, the SP10 phage genome DNA is able to be nearly full-methylated but some BsuM sites are considered to be unmethylated.

Alleviation of Type I Restriction in Escherichia coli K12 Carrying the arsR Gene from pKW301 of Acidiphilium multivorum AIU 301

INTRODUCTIONConjugal plasmids of various incompatibilitygroups avoid restriction when transferred among var-ious bacterial species and genera. This is mainly dueto the ard ( a lleviation of r estriction of D NA) geneswhich are in the leader region of a plasmid and codefor antirestriction proteins [1–5]. The Ard proteins(ArdA, ArdB, ArdC, ArdU, etc.) are small (140–180amino acid residues), highly acidic (total charge –7 to–30), and nonhomologous to each other, sharing onlyan antirestriction motif [6–9]. The motif is commonlyin the C domain, 30–35 residues away from the C end[10]. Its core consists of nine amino acid residues,with a certain arrangement of negatively charged ones(commonly D/E, highly polar N/Q in one site of ArdBand ArdC) and hydrophobic residues located in thecenter and at the ends (Table 1).We have previously shown that restriction allevia-tion induced by conjugal plasmid R64 (incI1) in Escherichia coli K12 is due to arsR , which codes fora suppressor of the ars operon conferring resistance toarsenite and arsenate [13]. ArsR consists of 117 aminoacid residues, is nonhomologous to the Ard proteins,and contains the antirestriction motif 25–35 residuesaway from the N end [13]. Its antirestriction activity islower than in the Ard proteins: ArsR alleviates restric-tion of nonmodified phage λ DNA by a factor of 20,whereas the factor is about 10

Characterization of a Novel Plasmid-Encoded HsdS Subunit, S.LlaW12I, from Lactococcus lactis W12

A novel type I restriction-modification specificity subunit, S.LlaW12I, has been identified on the naturally occurring 8.0-kb plasmid pAW122 in the lactic acid bacterium Lactococcus lactis subsp. cremoris W12. Presence of the HsdS protein together with a complete type I restriction-modification system conferred increased phage restriction to the host, indicating exchange of specificity subunits. Sequence analysis showed that the S.LlaW12I subunit is most probably of type IC. Presumably, the hsdS gene is organized together with the repB gene on one transcriptional unit.

Phage Restriction and the Presence of Small Plasmids in Salmonella enteritidis

Between 1990-1994, a total of 16,505 S. enteritidis strains of human, animal and food origin were phage-typed, using the Hungarian scheme and the changes of incidence of the dominant phage types were monitored. The incidence of PT1 (corresponding to Ward's PT1 was very high between 1990 and 1992 (67.9-71.0% of the total S. enteritidis isolates), later, it decreased. The prevalence of PT6 (corresponding to Ward's PT4) was rare until 1992, then it gradually increased. The phage type and plasmid content of 78 Salmonella enteritidis strains were determined. Small plasmids were present in 59% of the isolates, together with a serotype-specific (38 MDa) plasmid. A correlation was found between the presence of the small plasmid and phage restriction to two phages used for subdividing the Hungarian phage types 1 (PT1) and 6 (PT6) of S. enteritidis (corresponding to PT1 and PT4 in Ward's typing scheme, respectively).

IncI1 plasmid R64 encodes the ArsR protein that alleviates type I restriction

The host-controlled EcoK restriction of unmodified phage λ was five-fold alleviated in the wild-type Escherichia coli strain K12 carrying the R64 plasmid of the incompatibility group I1. The relevant gene was mapped between the origin of vegetative replication ( rep, oriV) and the tet r gene about 60 kbp downstream from the origin of transfer, oriT. We cloned this gene inside the 613 bp long EcoRI- PstI fragment and sequenced it. Only one 351 bp long open reading frame (ORF) starting at 124 bp from the beginning of the insert was found in the sequence. Computer search in the current databases revealed that the putative protein is identical to the ArsR protein specified by the IncFI plasmid R773. ArsR is a repressor of the arsenical resistance ( ars) operon, arsRDABC. There are no arsABC genes in the R64 plasmid since plasmid R64- (or pSR8)-mediated resistance of E. coli K12 cells to the arsenicals arsenate and arsenite was not detected. The gene arsR and the antirestriction genes ard ( ardA and ardB) are non-homologous. However, comparison of the deduced amino acid sequence of ArsR with the ArdA and ArdB sequences revealed only one small region of similarity, a 9 amino acid motif found in different antirestriction proteins that is hypothesized to be an interaction site for antirestriction proteins with restriction endonucleases.

Characterization of LlaCI, a new restriction-modification system from Lactococcus lactis subsp. cremoris W15.

The genes encoding the restriction-modification (R/M) system LlaCI have been found on the naturally occurring 7.0 kb plasmid pAW153 in L. lactis subsp. cremoris W15. The R/M system was isolated on a chloramphenicol resistant derivative of the wild type plasmid (pAW153cat). Plasmid pAW153cat and a 2.4 kb HincII-SphI fragment cloned into a high- and a low-copy vector conferred decreased sensitivity in L. lactis LM2301 and L. lactis SMQ86 against small isometric-headed phages of the 936 or P335 species, respectively. Increased plasmid copy number enhanced the level of phage restriction. Sequencing the 2.4 kb HincII-SphI fragment revealed two open reading frames arranged convergently with a 94 bp separation. IlaCIM showed 66% identity to hindIIIM, and IlaCIR showed 45% identity to hindIIIR. The organization of the LlaCI operon differs from the HindIII operon, where the endonuclease and methylase genes overlap and are transcribed in the same direction. The LlaCI methylase is predicted to be 296 amino acids long, with 63% identity to the HindIII methylase, while the LlaCI endonuclease is predicted to consist of 324 or 332 amino acids, depending on the position of the start codon. It shows 24% identity to the HindIII endonuclease.

Temperature-sensitive mutants of the EcoRI endonuclease

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30°C and 42°C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 Å resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses.

Molecular characterization of the restriction endonuclease gene (scrFIR) associated with the ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503.

The nucleotide sequence of the chromosomally encoded type II ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503 was completed. The ScrFI restriction endonuclease (ENase) has previously been shown to specifically recognize 5' CCNGG 3' sites, cleaving after the second cytosine and the degenerate central base. The ENase gene (scrFIR; 362 bp) was located between, and co-directionally transcribed with, two formerly characterized 5-methylcytosine methyltransferase genes, which encodes proteins that independently confer protection against ScrFI digestion. scrFIR codes for a protein of 272 amino acids with a predicted molecular mass of 31470 Da, which agrees favourably with a previously estimated molecular mass of 34 kDa for this enzymes. The deduced sequence of this protein did not show any significant homology with known protein sequences, including the isoschizomeric Ssoll ENase from Shigella sonnei. The ENase gene was cloned and expressed in Escherichia coli and Lactococcus; however, no in vivo restriction of phage was observed, suggesting that expression of the ENase gene may be repressed, or that the appropriate expression signals may be absent in the cloned constructs. The ability of ScrFI to cleave non-canonically modified 5' CCNGG 3' sequences suggested that some ScrFI sites may require complex modifications to fully impair digestion by this enzyme.

Distribution of restriction endonucleases among some entomopathogenic strains of Bacillus sphaericus

V. ZAHNER AND F.G. PRIEST. 1997. The restriction enzyme BspTI, an isoschizomer of HaeIII (recognition site GGCC), has been detected in eight strains of serotype 5a5b and two serotype 3 strains of the entomopathogenic bacterium Bacillus sphaericus. Strains from other serotypes contained the enzymes BspTII and BspTIII, which digested pBR322 DNA into similar banding patterns after agarose gel electrophoresis but differed in their susceptibility to methylation of the substrate. Strains from serotypes 9, 25 and 26a26b were lacking in restriction enzyme activity. There was little correlation between phage typing and restriction enzyme activity, suggesting that restriction and modification are not responsible for phage specificity among entomopathogenic B. sphaericus strains.

Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme.

Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.

Isolation of temperature-sensitive mutants of the BamHI restriction endonuclease

Two heat-sensitive R.BamHI mutants, T1571 and P173L, and one cold-sensitive R.BamHI mutant, T1141, were isolated after chemical mutagenesis of the bamhIR gene that codes for the restriction endonuclease BamHI (R.BamHI). The thermosensitivity of T1141, T1571 and P173L is revealed by the 10 2−10 3 lower plating efficiency at the non-permissive temperature of strains bearing these alleles. The conditional-lethal phenotype can be rescued by introduction of the cognate bamhIM gene into the same cell. The mutant enzymes induce the SOS response in vivo and display reduced phage restriction activity. The P173L protein, when expressed at 30°C and purified, shows reduced thermostability at 65°C. T1571 and P173L mutants yield different intermediates during partial trypsin digestion. The conditional-lethal BamHI mutants could be used to deliver in vivo DNA cleavage and for further isolation of relaxed-specificity mutants.

Molecular genetics of bacteriophage and natural phage defence systems in the genus Lactococcus

Bacteriophage infection of starter cultures used in a range of milk fermentation processes, particularly those involving Lactococcus lactis, poses a significant problem in industrial practice. The application of genetic and molecular technologies to the study of lactococcal bacteriophages has proven to be very rewarding in terms of understanding the nature of phage with respect to their physical and genetic organisation. The availability of the full genomic sequence of a number of phages provides an unambiguous basis for determining the relationship between them, for elucidating their evolutionary progression and will also yield strategies for obstructing successful phage proliferation on previously sensitive hosts. The genetic analysis of phage/host interactions has also highlighted the presence of natural defence systems (e.g. adsorption blocking, inhibition of phage DNA entry, restriction modification and abortive infection) in lactococci. A number of restriction modification systems and abortive infection mechanisms have been characterized at a molecular level and the genes involved have been cloned and sequenced. Plasmid-encoded phage resistance mechanisms can be exploited to generate strains which can successfully counter phage proliferation and will provide a basis for understanding the complex interactions between phages and their target hosts at a molecular level.

Evaluation of an antibiogram-resistogram typing scheme for methicillin-resistant Staphylococcus aureus.

Between Dec. 1992 and Aug. 1993, the MRSA population in the Federated Dublin Voluntary Hospitals and St James's Hospital group was studied with an antibiogram-resistogram (AR) typing scheme in which AR patterns were determined by testing susceptibility to 22 antibiotics and chemicals by a modified Stokes' disk diffusion technique. The typing scheme divided this MRSA population into 31 AR types but 90% of isolates belonged to seven types. Isolates belonging to the most frequently occurring types (AR types 13 and 14) differed only in their reaction to lincomycin (or clindamycin) and could not be distinguished by phage typing, plasmid profiling or restriction endonuclease analysis. The AR typing scheme showed that the incidence of different AR types varied in different hospitals and changed during the study period. This typing method differentiated a strain of MRSA responsible for a nosocomial outbreak in an intensive care unit from other MRSA isolated in the unit, and has distinguished imported strains from local ones. In one hospital, AR typing showed that, although a major outbreak occurred with one AR type, there was also a series of smaller outbreaks with other AR types. The technique can be performed in the diagnostic laboratory and results were available within 24 h.