Phage Restriction Research Articles

Pleiotropic effects of the rpoC10 mutation affecting the RNA polymerase β′ subunit of Escherichia coli on factor‐dependent transcription termination and antitermination

Escherichia coli RNA polymerase is composed of four different subunits, 2 alpha, beta, beta' and sigma. Among these subunits, the role of beta' is poorly understood. The rpoC10 mutation affecting beta' has been isolated as a suppressor mutation of the temperature-sensitive nusA11 mutant. DNA sequence analysis revealed that the rpoC10 mutant is a substitution of Lys for Glu-402. This increased positive charge appears to compensate for the increased negative charge present in the nusA11 protein (Asp for Gly-181). In vivo measurements of reporter gene expression have revealed that rpoC10 restores rho-dependent termination but fails to restore rho-independent termination in nusA11. Moreover, the rpoC10 mutation, in combination with any nusA mutation, inhibited lambda Q-mediated antitermination without affecting N antitermination and severely restricted lambda phage development. The inhibition of Q function and lambda growth could be compensated for by overproducing Q. These results suggest that the RNA polymerase beta' subunit plays a crucial role in factor-dependent transcription termination and antitermination.

The utility of restriction endonuclease analysis and phage typing in the epidemiologic investigation of a Staphylococcus aureus outbreak in a neonatal nursery

Outbreaks of Staphylococcus aureus infections in neonatal units require prompt investigation and implementation of control measures. From January to March 1990, a marked increase in the number of S. aureus infections was observed in a neonatal nursery. Twenty-seven S. aureus isolates from 23 patients were analyzed by phage typing and restriction endonuclease analysis (REA). Only nine strains were differentiated by phage type. However, REA with Hind III, Cfo I, and Cla I differentiated 20 strains. The REA results indicated that the outbreak was due to several different S. aureus strains and did not represent transmission of a single epidemic strain. REA may enable more accurate determination of the presence or absence of an epidemic strain during an outbreak than would traditional methods such as phage typing.

Ultraviolet light induction of lambda from dcm host strains alleviates Eco RII restriction of phage

A new form of restriction alleviation is demonstrated for phage induced by ultraviolet light from dcm strains of Escherichia coli K-12. EcoRII restriction of the induced phage is alleviated, which is the first report of Type II restriction alleviation. Unlike previously reported restriction alleviation, the increase in phage-plating efficiency is not dependent upon irradiation of the plating host for its induction.

Ultraviolet light induction of lambda fromdcmhost strains alleviatesEcoRII restriction of phage

A new form of restriction alleviation is demonstrated for phage induced by ultraviolet light from dcm strains of Escherichia coli K-12. EcoRII restriction of the induced phage is alleviated, which is the first report of Type II restriction alleviation. Unlike previously reported restriction alleviation, the increase in phage-plating efficiency is not dependent upon irradiation of the plating host for its induction.

The use of transgenic mice for short-term, in vivo mutagenicity testing

In order to develop a short-term, in vivo assay to study the mutagenic effects of chemical exposure, transgenic mice were generated using a lambda shuttle vector containing a lacZ target gene. Following exposure to mutagens, this target can be rescued efficiently from genomic DNA prepared from tissues of the treated mice using restriction minus, in vitro lambda phage packaging extract and restriction minus Escherichia coli plating cultures. Mutations in the target gene appear as colorless plaques on a background of blue plaques when plated on indicator agar. Spontaneous background levels were ′1 × 10 −5 in each of three mouse lineages analyzed. Exposure of lambda transgenic mice to N-ethyl- N-nitrosourea resulted in as much as a 14-fold induction in detected mutations over background levels. The assay is currently being modified to incorporate lacI as the target for ease of mutation detection as well as in vivo excision properties of the Lambda ZAP vector, facilitating sequence analysis of mutant plaques.

Cloning and expression of the BamHI restriction-modification system in Bacillus subtilis

The genes of the GGATCC-specific BamHI restriction-modification system of Bacillus amyloliquefaciens H have been cloned and expressed in Bacillus subtilis MT-2. B. subtilis MT-2 carrying the recombinant plasmid (pBamHIRM22) produced about 10-fold more BamHI restriction endonuclease and BamHI methylase than B. amyloliquefaciens H did. B. subtilis MT-2 (pBamHIRM22) restricted unmodified phage. Restriction and modification genes were stably maintained in B. subtilis MT-2 on one plasmid and the produced BamHI endonuclease remained stable even in the stationary phase of the culture. BamHI endonuclease from B. subtilis MT-2 (pBamHIRM22) had the same molecular weight and N-terminal amino acid sequence as that from B. amyloliquefaciens H.

Cloning of Breakpoints of a Chromosome Translocation Identifies the AN2 Locus

Chromosome translocations involving 11p13 have been associated with familial aniridia in two kindreds highlighting the chromosomal localization of the AN2 locus. This locus is also part of the WAGR complex (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation). In one kindred, the translocation is associated with a deletion, and probes for this region were used to identify and clone the breakpoints of the translocation in the second kindred. Comparison of phage restriction maps exclude the presence of any sizable deletion in this case. Sequences at the chromosome 11 breakpoint are conserved in multiple species, suggesting that the translocation falls within the AN2 gene.

Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12.

The McrB restriction system in Escherichia coli K12 causes sequence-specific recognition and inactivation of DNA containing 5-methylcytosine residues. We have previously located the mcrB gene near hsdS at 99 min on the E. coli chromosome and demonstrated that it encodes a 51 kDa polypeptide required for restriction of M.AluI methylated (A-G-5mC-T) DNA. We show here, by analysis of maxicell protein synthesis of various cloned fragments from the mcrB region, that a second protein of approximately 39 kDa is also required for McrB-directed restriction. The new gene, designated mcrC, is adjacent to mcrB and located distally to hsdS. The McrB phenotype has been correlated previously with restriction of 5-hydroxy-methyl-cytosine (HMC)-containing T-even phage DNA that lacks the normal glucose modification of HMC, formally designated RglB (for restriction of glucoseless phage). This report reveals a difference between the previously correlated McrB and RglB restriction systems: while both require the mcrB gene product only the McrB system requires the newly identified mcrC-encoded 39-kDa polypeptide.

Cloning, characterization and heterologous expression of theSmaI restriction-modification system

The genes coding for the class-II Serratia marcescens restriction-modification system have been cloned and expressed in E. coli. Recombinant clones, restricted incoming phage only poorly; the recombinant plasmids, however, became fully modified in vivo, i.e. completely resistant against digestion with R.SmaI. The determined nucleotide sequence of the cloned system revealed three open reading frames with lengths of 252 bp, 741 bp, and 876 bp. Through various deletion experiments and an insertion-mutation experiment the 876 bp open reading frame could be assigned to the SmaI DNA modification enzyme and the 741 bp open reading frame to the SmaI restriction endonuclease. Mapping of the transcription start sites of the genes revealed that the SmaI endonuclease is transcribed as polycistronic mRNA together with a 252 bp long preceding open reading frame of unknown function. No homology was found when comparing the amino acid sequence of M.SmaI with the published sequences of m5C-specific DNA modification methyltransferases. On the other hand, a stretch of 14 amino acids in the C-proximal region of M.SmaI shows a significant homology to the C-proximal amino acid sequences of the N6A-methyltransferases M.HinfI and M.DpnIIA and the N4C-methyltransferase M.PvuII.

Recognition sequences of type II restriction systems are constrained by the G + C content of host genomes.

I show that the recognition sequences of Type II restriction systems are correlated with the G + C content of the host bacterial DNA. Almost all restriction systems with G + C rich tetranucleotide recognition sequences are found in species with A + T rich genomes, whereas G + C rich hexanucleotide and octanucleotide recognition sequences are found almost exclusively in species with G + C rich genomes. Most hexanucleotide recognition sequences found in species with A + T rich genomes are A + T rich. This distribution eliminates a substantial proportion of the potential variance in the frequency of restriction recognition sequences in the host genomes. As a consequence, almost all restriction recognition sequences, including those eight base pairs in length (Not I and Sfi I), are predicted to occur with a frequency ranging from once every 300 to once every 5,000 base pairs in the host genome. Since the G + C content of bacteriophage DNA and of the host genome are also correlated, the data presented is evidence that most Type II "restriction systems" are indeed involved in phage restriction.

Cloning and characterization of the dcm locus of Escherichia coli K-12.

The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3'. This sequence is also recognized by the EcoRII restriction-modification system coded by the E. coli plasmid N3. The methylase within the EcoRII system methylates the same cytosine as the dcm protein. We have isolated, from a library of E. coli K-12 DNA, two overlapping clones that carry the dcm locus. We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain. We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease. We found no phage restriction activity associated with the dcm clones.

Epidemic strains of Salmonella agona differentiated by phage restriction and u. v.-protection markers on Collb plasmids

Epidemic strains of Salmonella agona differentiated by phage restriction and u. v.-protection markers on Collb plasmids

Characterization of transferable R plasmids from Aeromonas hydrophila.

The properties and DNA structures of 14 transferable R plasmlds detected in Aeromonas hydrophila strains isolated from various areas were compared. Eight of nine R plasmids with resistance mazkers to tetracycline (TC) and sulfonamide (SA) were classified into the incompatibility (lnc.) Group A-C complex. One R plasmid was classified into Inc. Group C. Two R plasmids with resistance to chloramphenicol (CM), streptoanycin (SM), and SA were classified into Inc. Group U. The remaining R plasmids, pES10 and pES15 encoded with CM, SM, TC, kanamycinand SA, were not classified into any of the tested Inc. Groups. Rplasmids with resistance markersto either TC and SA or CM, SM and SA did not restrict DNA phage T1 growth; however, pES10 and pES15 restricted phage T1 growth. This restriction and modification system was similar to that of R plasmid N-3.1) The endonuclease digestion patterns of the DNA's were identical in R plasmids with the same resistance markers. The DNA of R plasmids with resistance to TC and SA showed a high homology to cach other when compazed by Southern blot hybridization.2) R plasmid DNAs encodcd with CM, SM, and SA showed homology with the probe of pAR323) detected from A. salmonicida. A. hydrophila strains carrying R plasmids having the identical DNA structure are widely distributed in freshwater fish culture ponds in various areas.

Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.

A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.

Transfer of Sucrose-Fermenting Ability and Nisin Production Phenotype among Lactic Streptococci

Transfer of sucrose fermentation ability, nisin production, and nisin resistance from Streptococcus lactis to S. lactis and Streptococcus lactis subsp. diacetylactis occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Transconjugants were able to act as donors to transfer the Suc-Nis phenotype in subsequent mating. No changes in sensitivity to lytic phage c2 were noted in S. lactis transconjugants. However, temperature-independent restriction of lytic phage 18-16 was noted in transconjugants of S. lactis subsp. diacetylactis 18-16. Adsorption studies with phage-resistant transconjugants showed that resistance was not due to lack of adsorption by the lytic phage. Physical evidence for the presence of introduced plasmid DNA was not found in lysates of transconjugants.

Protection of nonmodified phage lambda against EcoK restriction mediated by recA protein.

A study was conducted to establish whether the EcoK-specific restriction, which is alleviated in E. coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level of recA protein without induction of other SOS functions. The host cells used were E. coli K-12, strain AB2497, and its derivatives; the nonmodified phage lambda was a mutant b2b5(vir). An increase of the recA protein level was induced using the plasmid pX02, which is a recombinant of pBR322 carrying the recA gene of E. coli. AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid. The results indicate that the recA protein protects phage DNA during the process of restriction. A further factor affecting restriction is the growth phase of the culture of the restricting host: cells in the late stationary phase exhibit lower restriction than those in the exponential phase of growth. By a combination of these two factors (presence of plasmid pX02 and stationary growth phase) one can reduce the restriction of nonmodified phage about 300 times.

Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products.

Bal31 deletion experiments on clones of the PaeR7 restriction-modification system from Pseudomonas aeruginosa demonstrate that it is arranged as an operon, with the methylase gene preceding the endonuclease gene. The DNA sequence of this operon agrees with in vitro transcription-translation assays which predict proteins of 532 amino acids, Mr = 59,260 daltons, and 246 amino acids, Mr = 27,280 daltons, coincident with the methylase and endonuclease genes, respectively. These predicted values coincide with the measured molecular weights of the purified, denatured PaeR7 endonuclease and methylase proteins. The first twenty amino acids from the amino-terminus of the purified endonuclease exactly match those predicted from the DNA sequence. Finally, potential regulatory mechanisms for the expression of phage restriction are described based on the properties of several PaeR7 subclones.

Phage Resistance in a Phage-Insensitive Strain of Streptococcus lactis : Temperature-Dependent Phage Development and Host-Controlled Phage Replication

Streptococcus lactis ME2 is a dairy starter strain that is insensitive to a variety of phage, including phi18. The efficiency of plating of phi18 on ME2 and N1 could be increased from <1 x 10 to 5.0 x 10 and from 7.6 x 10 to 2.1 x 10, respectively, when the host strains were subcultured at 40 degrees C before plating the phage and the phage assay plates were incubated at 40 degrees C. Host-dependent replication was demonstrated in N1 at 30 degrees C and in N1 and ME2 at 40 degrees C, suggesting the operation of a temperature-sensitive restriction and modification system in ME2 and N1. The increased sensitivity of ME2 and N1 to phi18 at 40 degrees C was also demonstrated by lysis of broth cultures and increased plaque size. ME2 grown at 40 degrees C showed an increased ability to adsorb phi18, indicating a second target for temperature-dependent phage sensitivity in ME2. Challenge of N1 with a phi18 preparation that had been previously modified for growth on N1 indicated that at 40 degrees C phage development was characterized by a shorter latent period and larger burst size than at 30 degrees C. The evidence presented suggests that the high degree of phage insensitivity expressed by ME2 consists of a variety of temperature-sensitive mechanisms, including (i) the prevention of phage adsorption, (ii) host-controlled restriction of phage, and (iii) suppression of phage development. At 30 degrees C these factors appear to act cooperatively to prevent the successful emergence of lytic phage active against S. lactis ME2.

Plasmids of enterotoxigenic Escherichia coli H10407: evidence for two heat-stable enterotoxin genes and a conjugal transfer system.

Three species of plasmids, associated with virulence and conjugal transfer, were identified in a clinically isolated enterotoxigenic Escherichia coli strain, H10407 (serotype O78:H11). pCS1, a non-self-transmissible plasmid species with a molecular weight of 62 X 10(6) and a 47 mol% guanine-plus-cytosine content, specified colonization factor antigen I and heat-stable enterotoxin (ST) production, as reported by others previously. A second non-self-transmissible plasmid species, designated pJY11, with a molecular weight of 42 X 10(6) and a 51 mol% guanine-plus-cytosine content, specified ST and heat-labile enterotoxin production and manifested T5/T6 phage restriction. The third plasmid species, pTRA1, also had a molecular weight of 42 X 10(6) and had a guanine-plus-cytosine content of 51 mol%; this species was self-transmissible and promoted transfer of both pCS1 and pJY11 to other bacterial cells. pCS1 may have originated from species of bacteria with a lower guanine-plus-cytosine content than E. coli. Finally, although demonstrating some heterogeneity with each other, both STs encoded by pCS1 and pJY11 belonged to the STa group.

Drug resistance and R plasmids of Escherichia coli strains isolated from six species of wild birds.

Drug resistance and R plasmids of Escherichia coli strains isolated from 30 wild birds of six species (jungle crows, bamboo partridges, grey starlings, rufous turtle doves, brown-eared bulbuls and azule-winged magpies) were investigated. Resistant strains were isolated from three species of birds: 34 (28.3%) of 120 strains isolated from jungle crows, 2 (8.0%) of 25 strains from bamboo partridges and 7 (70%) of 10 strains from grey starlings. Totally 43 (18.7%) of 230 strains were resistant. These strains were resistant to one or more drugs of oxytetracycline hydrochloride, dihydrostreptomycin and sulfadimethoxine. R plasmids were detected in 5 (14.7%) of the 34 resistant strains isolated from jungle crows. Quadruple-, quintuple- and sextuple-resistant strains were found among the 34 strains on agar plates containing oxytetracycline hydrochloride, chloramphenicol or dihydrostreptomycin and R plasmids were detected in eight of them. Of those 13 R plasmids, half of them showed Fi+ character, most of them showed restriction of phages λ, T2, T4 or T7, and their incompatibility groups were untypable. From these results, it is considered that these wild birds play a less role in spreading R plasmids than do domestic animals.