Isolation and characterization of a new bacteriophage, Cp-1, infecting Streptococcus pneumoniae.

Several pneumococcal phages showing a morphology completely different from those of all other previously found pneumococcal bacteriophages have been isolated. Bacteriophage Cp-1, one of the phages isolated, showed an irregular hexagonal structure and a short tail of 20 nm. The virion density was 1.46 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of nine polypeptides. The polypeptide showing a molecular weight of 39,000 accounted for more than the 90% of the total protein. The nucleic acid of Cp-1 was linear, double-stranded DNA with a mean length of 6.3 microns and a guanine-plus-cytosine content of 41%; its buoyant density was 1.699 and 1.422 g/cm3 in CsCl and CS2SO4, respectively. Its sedimentation coefficient (S20,w) was 19S. Cp-1 DNA showed a remarkable resistance to a large number of restriction endonucleases. A total of 12 fragments, ranging in molecular weight from 1.3 X 10(6) to 0.09 X 10(6), were produced by AluI, two fragments (molecular weight, 5.5 X 10(6) and 0.9 X 10(6)) were generated by HindIII, and two fragments (molecular weight, 6.0 X 10(6) and 5.7 X 10(6)) were produced by HaeIII. The easy visualization of th plaques produced by Cp-1, the small size of Cp-1 DNA (12 X 10(6) daltons), and other biological and physiochemical properties make this phage potentially useful for genetic studies.     

Novel segregation patterns of infecting-mutant genotypes in plate complementation tests among amber mutants of bacteriophage BF23

Amber mutants of bacteriophage BF23 were classified into two functional groups, types I and II, by the yields of the infecting-mutant genotypes in plate complementation tests. Type I mutants produced their genotypes at levels more than 20% of the total progeny phages, and type II mutants did so at levels of less than 5%. Comparison of the results of plate complementation tests with those of extract complementation tests revealed that all the type I mutants were defective in the tail formation, while most type II mutants were defective in the formation of either mature heads (type IIa) or both mature heads and tails (type IIb). Since in extract complementation tests the activated phages are always of genotypes corresponding to mutations defective in only the tail formation, the plate complementation test is comparable with the extract complementation test when judged on the basis of the yield of the mutant genotypes. Of 29 complementation groups, 8 type I, 14 type IIa, and 5 type IIb mutants were identified. Previously, amber mutations of BF23 were mapped on four genetic segments. These segments were ordered in one linkage map by crosses between deletion and amber mutants.     

Biosynthesis of riboflavin. An aliphatic intermediate in the formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphate

6,7-Dimethyl-8-ribityllumazine synthase deficient mutants of Candida guilliermondii were divided into two groups on the basis of in vitro complementation. Mutants of complementation group I produce an intermediate X from ribose 5-phosphate in a reaction requiring Mg++ ions. Compound X was partially purified and was shown to be a phosphoric acid ester. 6,7-Dimethyl-8-ribityllumazine can be formed from Compound X by cell extracts from mutants of complementation group II. The reaction requires 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5'-phosphate as second substrate. No divalent cations are required.     

Isolation and characterization of mycoplasma virus L3 temperature-sensitive mutants

Mycoplasma virus L3 virions are morphologically similar to coliphage T7, contain linear double-stranded DNA of about 39 kilobase pairs, and produce a nonlytic cytocidal infection in Acholeplasma laidlawii host cells. Following nitrous acid mutagenesis, ninety-eight L3 temperature-sensitive (ts) mutants were isolated from a total of 57,000 plaque-forming units (PFU), using 37 degrees C as the permissive temperature and 41 degrees C as the nonpermissive temperature, with reversion frequencies of 10(-5) to 10(-8). Complementation tests allowed fifty-seven of the L3 ts mutants to be placed into twenty-one complementation groups. In mixed infections, recombination frequencies between mutants in different complementation groups were 10(-2) to less than 10(-6). Studies of protein synthesis in L3-infected cells showed synthesis of about twenty virus-specific proteins, including ten L3 virion proteins. After infection with L3 ts mutants from each complementation group, several different patterns of cell- and virus-specific protein synthesis were observed.     

Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

Membrane mutants of animal cells have been isolated by several laboratories, using a variety of selection protocols. The majority are lectin receptor mutants arising from altered glycosylation of membrane molecules. They have been obtained by selection for resistance to cytotoxic plant lectins or by alternative protocols designed, in many cases, to isolate different classes of receptor mutants. The identification of most membrane mutants expressing altered surface carbohydrates is rapidly achieved by determining their resistance to several lectins of different carbohydrate-binding specificities. For Chinese hamster ovary mutants, genetic novelty may subsequently be determined by complementation analysis with selected members of 10 recessive, glycosylation-defective complementation groups defined by this laboratory. In an attempt to identify new complementation groups, 11 Chinese hamster ovary membrane mutants independently isolated in different laboratories have been investigated for their lectin resistance and complementation properties. Only one new complementation group was defined by these studies. The remaining 10 mutants fell into complementation group 1, 2, 3, or 8. Although no evidence for intragenic complementation was observed, indirect evidence for different mutations within some genes was obtained. Seven of the independent isolates fell into complementation group 1, reflecting the high probability of isolating the Lec1 phenotype from Chinese hamster ovary populations. The results emphasize the importance of performing a genetic analysis before biochemical characterization of putative new membrane mutants.     

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad52, rad55 and rad57 were found amoung these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups.     

Morphology of the Bacteriophages of Lactic Streptococci

Electron microscope studies have been made of a number of phages of lactic streptococci, seven of which were phages of Streptococcus lactis C10. Two of the phages are thought to be identical; five have been classified by the method of Tikhonenko as belonging to group IV (phages with noncontractile tails) with type III tail plates; one belongs to group V (phages with tails possessing a contractile sheath). Both prolate polyhedral heads and isometric polyhedral heads are represented among the group IV phages. The phage drc3 of S. diacetilactis DRC3 has been shown to have similar structure to the group IV phages of S. lactis C10 with prolate polyhedral heads. The phages ml1, hp, c11, and z8 of the S. cremoris strains ML1, HP, C11, and Z8, respectively, were shown to belong to the group IV phages with type III tail plates by the method of Tikhonenko. All had octahedral heads and tended to be larger than most of the other phages studied.     

Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion.

We have begun an analysis in Escherichia coli of the conjugal transfer functions of the broad-host-range plasmid RP4. We have isolated 19 tra mutants of RP4, generated by insertion of transposon 7, and mapped their insertion sites by restriction endonuclease analysis. These sites fall into two separate regions on either side of the kanamycin resistance determinant. The transfer rates of the mutants range from 10% of that of RP4 to an undetectable level. Spot tests with the P-1 pilus-specific phages PRR1, Pf3, and PR4 and electron microscopic examination for pili have classified the mutants into several phenotypes consistent with their having normal, retracted, or no pili. Analysis of transient plasmid heterozygotes, created by P1 transduction, divided the tra mutants into a minimum of five complementation groups. Some of these groups contain more than one phenotypic class and may represent more than one gene because of the possible polar and deletion effects of Tn7 insertion.     

New antibiotic resistance Loci in the ribosomal region of yeast mitochondrial DNA

A large number of mitochondrial antibiotic-resistant mutants have been isolated following mutagenesis with manganese. These include several different phenotypic classes of mutants, as distinguished by cross-resistance patterns, that have been found to be allelic at cap1 or ery1; some have been found to be heteroallelic.--Seven chloramphenicol-resistant mutants have been identified that are nonallelic by recombination tests with the three loci (cap1, spi1 and ery1) previously identified in the ribosomal region. Four of these are allelic with each other and define a new locus, cap3; two others are allelic and define another new locus, cap2; the seventh maps at yet a different locus, cap4. One new spiramycin-resistant mutant has been identified that defines still another new locus, spi2. A variety of genetic techniques have been used to map these loci within the ribosomal region of the mitochondrial genome.-Manganese has been shown to be effective in inducing the mutation from omega(-) to omega(n) in many mutants that experience a simultaneous mutation at the closely linked cap1 locus. The omega(n) mutation has also been described in the cap4 mutant, and this locus has been shown to be more closely linked to omega than cap1 is to omega.     

Bacteriophages of methanotrophic bacteria

Bacteriophages of methanotrophic bacteria have been found in 16 out of 88 studied samples (underground waters, pond water, soil, gas and oil installation waters, fermentor cultural fluids, bacterial paste, and rumen of cattle) taken in different geographic zones of the Soviet Union. Altogether, 23 phage strains were isolated: 10 strains that specifically lysed only Methylosinus sporium strains, 2 strains that each lysed 1 of 5 Methylosinus trichosporium strains studied, and 11 strains that lysed Flavobacterium gasotypicum and, at the same time, 1 M. sporium strain. By fine structure, the phages were divided into two types (with very short or long noncontractile tails); by host range and serological properties, they fell into three types. One-step growth characteristics of the phages differed only slightly; the latent period varied from 6 to 8 h, the rise period varied from 4 to 6 h, and the average burst size was 100. All phages had guanine- and cytosine-rich double-stranded deoxyribonucleic acid consisting of common nitrogen bases. The molecular mass of the deoxyribonucleic acid as determined by restriction endonuclease analysis was 29.4 X 10(6) for M. sporium phages and 44 X 10(6) for F. gasotypicum phages. By all of the above-mentioned properties, all phages within each of the groups were completely identical to one another, but differed from phages of other groups. Bacteriophages lysing M. sporium and M. trichosporium GB2 were identical to phages M1 and M4, respectively, which were isolated earlier in the German Democratic Republic on the same methanotrophic species.     

Genetics of Bacillus subtilis chemotaxis: isolation and mapping of mutations and cloning of chemotaxis genes

We isolated a collection of chemotaxis mutants and characterized them for chemotactic phenotype and genotype. The mutations of most of these mutants mapped in the region between pyrD and thyA. However, the mutation in the gene specifying the chemotactic methyltransferase mapped very close to aroF. From a bank of phages containing Bacillus subtilis DNA we identified two lambda charon4 phages that contained genes specifying chemotactic functions. The inserted DNAs were removed by digestion with restriction endonuclease EcoRI and were found to share a 4.0-kilobase (kb) fragment. One of these DNAs also contained a 7.7-kb fragment, and the other also contained a 10.9-kb fragment. We identified mutants that were complemented by each fragment. The fragments were each ligated into plasmid pFH7 and were incorporated into lysogenic SP beta c2 or a deletion mutant of SP beta c2 in order to form transducing phages. The mutants in the collection containing mutations that mapped in the region between pyrD and thyA were tested for complementation by transducing phages containing the 4.0-kb fragment, the 7.7-kb fragment, the 4.0-kb fragment plus the 7.7-kb fragment, and the 10.9-kb fragment. A total of 24 mutants were complemented by the 4.0-kb fragment, 7 were complemented by the 7.7-kb fragment, 9 were complemented by the 4.0-kb fragment plus the 7.7-kb fragment, 15 were complemented by the 10.9-kb fragment, and 25 were complemented by none of the fragments.     

Bacterial rep- mutations that block development of small DNA bacteriophages late in infection.

Several related mutants of Escherichia coli C have been isolated that block the growth of the small icosahedral DNA phages phiX174 and S13 late in infection. Phage G6 is also blocked, at a stage not yet known. Growth of the filamentous phage M13, though not blocked, is affected in these strains. These host mutations co-transduce with ilv at high frequency, as do rep- mutations. However, the new mutants, designated groL-, differ from previously studied rep- mutants in that they permit synthesis of progeny replicative-form DNA. The groL- mutants are blocked in synthesis of stable single-stranded DNA of phiX174 and related phages. They are gro+ for P2. Evidence that groL- mutations and rep- mutations are in the same gene is presented. Spontaneous mutants (ogr) of phiX174, S13, and the G phages can grow on groL- strains. The ogr mutations are located in the phage's major capsid gene, F, as determined by complementation tests. There are numerous sites for mutation to ogr. Some mutations in genes A and F interfere with the ogr property when combined with an ogr mutation on the same genome. The ogr mutations are cis acting in a groL- cell; i.e., an ogr mutant gives very poor rescue of a non-ogr mutant. The wild-type form of each G phage appears to be naturally in the ogr mutant state for one or more groL- strains. It is suggested that a complex between F and rep proteins is involved in phage maturation. The A protein appears to interact with this complex.     

Receptor specificity of the Escherichia coli T-even type phage Ox2. Mutational alterations in host range mutants

The T-even type Escherichia coli phage Ox2 uses the outer membrane protein OmpA as a receptor. The protein is recognized with the ends of the virion's long tail fibers. The 266 residue protein 38 is located at this site and acts as an adhesin. Host-range mutants had previously been isolated from Ox2. Mutant Ox2h5 is able to infect cells possessing an altered OmpA protein, which renders the cell resistant to Ox2. Ox2h10 was selected from Ox2h5. This phage recognizes the OmpC protein in addition to the OmpA protein. Ox2h12, which stems from Ox2h10, binds to OmpC with high affinity, but has lost efficient binding to OmpA. The mutational alterations caused in genes 38 are: Asp231----Asn(h5) and His170----Arg(h10). The triple mutant Ox2h12 possesses an insertion of a Gly residue next to Gly121. The three mutants have additionally acquired mutations affecting their base plate, making them "trigger-happy". When protein 38 was compared with the same protein derived from other E. coli phages, it was found to contain two constant and one variable domains, the latter harboring four hypervariable regions flanked by a largely conserved glycine-rich sequence. The h5 and h10 mutations occurred within two hypervariable areas, while the additional Gly residue was present in one of the flanking conserved sequences. On the basis of these results, as well as those obtained from host-range mutants analyzed previously, a model for such adhesins is proposed. Receptor recognition is most likely performed via the hypervariable regions, which may form loops held together in close proximity by the oligoglycine sequences. The latter may achieve this by being part of highly compact omega loops.     

Morphology of Bacteriophages of Lactobacillus bulgaricus, L. lactis and L. helveticus

Eleven virulent phages isolated from cheese or yoghurt factories and active on thermophilic lactobacilli used as starters were examined by electron microscopy. Five phages active on Lactobacillus bulgaricus belonged to Bradley's group B and can be divided into two groups with different host specificity. The three phages of the first group (two isolated in France, one in the USA) differ in size; the heads are either icosahedrons or octahedrons and the tails end in short-pronged base plates. The two phages of the second group, isolated in the USA. appear very similar. These are similar in length, have collars and octahedral heads. The non-contractile tails end in clusters of short fibres. An L. lactis phage, isolated in Finland, belongs to the same morphological group. It is similar in overall appearance to the two latter phages of the second group of L. bulgaricus , and there were numerous ghosts with polytails. Five phages. active on L. helveticus belong to Bradley's group A and may be divided into two groups with different host specificity. The first group contains a phage isolated in Finland. The second group is composed of four similar phages isolated in France. The Finnish phage is the largest, but the five phages show similar morphology. They have octahedral heads, firmly attached to the tails by connector devices, and they possess necks. The contractile sheaths have a helicoidal arrangement of hollow tubular subunits. They appear contracted either in a distal or a cervical position, revealing axial cores. Short tail fibres are probably present at the tail tip.     

New rapid and simple methods for detection of bacteria and determination of their antibiotic susceptibility by using phage mutants

Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria ("infectious centers"). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.     

Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.

Nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra were generated with N-methyl-N-nitro-N'-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A total of 16 nonpathogenic mutants were isolated from 2,000 colonies. One mutant, NP1, was chosen for further study. NP1 did not multiply in soybean cotyledons. A genomic library of strain 8ra was constructed in the cosmid pLAFR3, and the cosmids were tested for complementation in NP1. One cosmid clone, pIH1, which contained a 31-kb insert, complemented mutant NP1. A restriction map of pIH1 was constructed, and deletion analyses identified a 10-kb HindIII fragment that restored pathogenicity to NP1. Southern hybridization analysis indicated that DNA sequences in the 10-kb HindIII fragment are conserved among other X. campestris pathovars tested. Three regions responsible for restoring pathogenicity have been identified by Tn3-HoHo1 mutagenesis. A 2.7-kb ClaI fragment was sequenced, and two possible open reading frames (ORF1 and ORF2) were found. Results indicated that ORF2 but not ORF1 may be expressed in Escherichia coli and in X. campestris pv. glycines. The carboxy terminus of the potential polypeptide encoded by ORF2 has an amino acid sequence similar to that of the gamma subunit of oxaloacetate decarboxylase, which is involved in sodium ion transport in Klebsiella pneumoniae.     

Genetic analysis of adenovirus type 2. I. Isolation and genetic characterization of temperature-sensitive mutants.

Temperature-sensitive mutants which replicate normally at 33 C but poorly at 39 C were isolated from nitrosoguanidine- or nitrous acid-mutagenized adenovirus 2 by (i) testing the cytopathic effect or inclusion body-forming capacity of random plaque isolates, or (ii) reduced plaque enlargement upon shifting from 33 to 39 C. Thirty-six mutants were isolated with 33 C/39 C plaque ratios varying from 20 to 10-5. Some of these mutants could be arranged into 13 groups by the complementation test. By means of recombination analysis a provisional linear genetic map was constructed.     

Degrees of relatedness of T-even type E. coli phages using different or the same receptors and topology of serologically cross-reacting sites

The relatedness of a series of T-even like phages which use the Escherichia coli outer membrane protein OmpA as a receptor, and the classical phages T2, T4 and T6 has been investigated. Immunoelectron microscopy and the pattern of phage resistance in bacterial mutants revealed that: (i) phages of this morphology do not necessarily cross-react serologically; (ii) phages using different receptors may bind heterologous IgG everywhere except to the tip (comprising approximately 10% of one fiber polypeptide) of the long tail fibers; (iii) cross-reacting OmpA-specific phages may bind heterologous IgG only to the tip of these fibers: (iv) OmpA-specific phages not cross-reacting at the tip of the tail fibers use different receptor sites on the protein. Absence of cross-reactivity appears to reflect high degrees of dissimilarity. A DNA probe consisting of genes encoding the two most distal tail fiber proteins of T4 detected homologies only in DNA from phages serologically cross-reacting at this fiber. Even under conditions of low stringency, allowing the formation of stable hybrids with almost 30% base mismatch, no such homologies could be found in serologically unrelated phages. Thus, in the collection of phages examined, there are sets of very similar and very dissimilar tail fiber genes and even of such gene segments.     

Comparative Genetics of the T-Even Bacteriophages

A system of amber mutants has been developed for each of the T-even bacteriophages T2 and T6, to complement those already available in T4. In T2 these mutants identify 52 genes, of which 49 are homologous with T4 genes; in T6 they identify 45 genes, of which 42 have T4 homologs, and an additional one which is homologous to a T2 gene not yet identified in T4. In both T2 and T6, recombination between mutants is characterized by considerable negative interference, which is correctable by a mapping function designed for T4. Recombinational maps of T2 and T6 constructed with these mutants have the same gene order and nearly the same gene spacings as in T4, with the exception of the tail fiber region; T2 and T6 appear to lack a localized recombinational expansion of this region found in T4. Homologous gene products from all three phages are in general interchangeable, with the exception of those from two apparently "co-adapted" tail fiber genes, 37 and 38. The general genetic similarity of all three phages suggests that they are analogous to races of higher organisms, retaining the capacity for genetic exchange despite some clear genetic differences and some incipient isolating mechanisms.     

Some Properties of Five New Salmonella Bacteriophages

Five bacteriophages were isolated from lysogenic strains of Salmonella potdam. On the basis of plaque morphology, thermostability, serology, host range, one-step growth parameters, and phage morphology, they were divided into three groups: group A, phages P4 and P9c; group B, phages P3 and P9a; and group C, phage P10. Group A phages had a hexagonal head 55 nm in diameter with a short tail 15 nm long. These phages were particularly characterized by high thermostability, lack of serological relationship with any of the other phages, and restriction of lysis to other Salmonella strains of Kauffmann-White group C(1). Group B phages had a head identical in size and shape to that of the A phages, but they possessed a tail 118 nm long with a contractile sheath. A unique feature was the occurrence of tail fibers at the end of the core rather than at the base of the sheath. These phages were considerably less thermostable, had extended host ranges, and were serologically distinct from each other but unrelated to the A phages. The group C phage, P10, had a head identical to that of the A and B phages. It had a tail 95 nm in length, with tail fibers attached to a base plate at the end of a contractile sheath. P10 was highly sensitive to heat, lysed only smooth strains of Salmonella, and showed a degree of serological relationship to both B phages. The relationship of these phage groups to previous Salmonella phage grouping schemes is discussed.