Mutations in Prophage φ11 That Impair the Transducibility of Their Staphylococcus aureus Lysogens for Methicillin Resistance

Methicillin resistance (mec) is not transduced into Staphylococcus aureus 8325-4, but is transduced into this host after it has been lysogenized with phage phi11 and has acquired the penicillinase plasmid pI524 by a separate transduction (Cohen and Sweeney, 1970, 1973). Strain 8325-4 is competent for transformation of typical plasmid or chromosomal markers and for mec only if it is lysogenic for phi11 or a related prophage (Sj?str?m et al., 1974, 1975). A mutant strain of phi11 that was temperature sensitive (Ts) for vegetative multiplication did not mediate competence for transformation of its 8325-4 lysogen if the lysogen had been grown at a nonpermissive temperature (Sj?str?m and Philipson, 1974). We isolated four Ts mutants of phi11 that did not mediate transducibility of their 8325-4(pI524) lysogens for mec after growth at nonpermissive temperatures (40 to 42 degrees C). Transduction of typical plasmid or chromosomal markers was not affected. These phi11-Ts mutants mediated normal competence of their lysogens for transformation of a tetracycline resistance plasmid. Similarly, phi11-Ts mutants that rendered their lysogens temperature sensitive for transformation did not depress the frequency of transduction of mec. These two types of phi11-Ts mutants fell into two different genetic complementation groups that differed in the physiology of deoxyribonucleic acid synthesis and in the time of expression of the mutations during a single-burst growth cycle at a nonpermissive temperature. A virulent mutant of phi11, which plaqued with 100% efficiency on 8325(phi11), also failed to condition strain 8325-4 for transducibility of mec but retained the ability to confer competence for transformation of a tetracycline resistance plasmid. Different genetic loci and physiological functions are involved in phi11 mutations that affect transducibility of mec and those that affect competence for transformation of markers generally in S. aureus 8325-4.     

Thymineless bacteriophage induction in Staphylococcus aureus. I. High-frequency transduction with lysates containing a bacteriophage related to bacteriophage phi 11.

A thymine-requiring mutant of Staphylococcus aureus, strain 8325 (PI258)thy, undergoes prophage induction and lysis after thymine starvation. Four different phages were isolated from the lysate in low titers, among which was a phage designated phi 14, which differs from phage phi 11 in its immunity locus. The thymineless induced lysates of strain 8325(PI258)thy transduce the penicillinase plasmid at high frequency (10(-1), whereas transduction of chromosomal markers is inefficient. A plasmic-cured derivative of strain 8325(PI258)thy is also lysed by thymine starvation and be used for high-frequency transduction of other plasmids. Reconstitution of a strain of S. aureus that responds to thymine starvation was only partially successful, but this system can effectively be used to transduce plasmids or plasmid derivatives.     

Staphylococcus aureus chromosomal mutation specifically affecting the copy number of Inc3 plasmids

A chromosomal mutation leading to an important increase in the copy number of plasmid pT181 and its derivatives has been isolated from Staphylococcus aureus strain 8325. The amplification effect in the mutant strain SA1350 was found to be specific for plasmids of the Inc3 group, to which belongs pT181. There are some other differences in the behavior of Inc3 plasmids between SA1350 and 8325, including stable maintenance in SA1350 at high copy number of temperature-sensitive replication mutants at restrictive temperatures, and altered incompatibility properties. Derivatives of SA1350 carrying only Inc3 plasmid mutants with high copy numbers (Cop mutants) could not be obtained, suggesting a lethal runaway plasmid replication in this situation. SA1350 expressed also a temperature-sensitive phenotype. The relationship of this character to the plaC1 mutation determining the amplification of Inc3 plasmids has not yet been elucidated.     

Role of the ø11 Phage Genome in Competence of Staphylococcus aureus

Both phage ?11 and 83A, when present as prophage or when used as helper phage, induce competence for transfection and transformation to the same level in Staphylococcus aureus, strain 8325-4. Cells lysogenized with certain temperature-sensitive (ts) mutants of phage ?11 show competence at the nonpermissive temperature (41 C) without production of infectious phages. Phage ?11ts allele 31 can neither as a prophage nor as a helper phage develop competence under nonpermissive conditions. This mutant appears, therefore, to be mutated in the region of the phage genome controlling competence. The competence level for both transfection and transformation is increased by superinfecting strain 8325-4 (?11) or 8325-4 (83A) at high multiplicities with phage ?11 with some of its mutants or with phage 83A. This superinfection enhancement appears to require protein synthesis but not deoxyribonucleic acid synthesis as judged from studies with inhibitors of macromolecular synthesis. Besides the phage particle, no extracellular or cell-bound factors so far detected can induce competence. The phage-induced product conferring competence is rapidly synthesized by strain 8325-4 (ts?11(31)) after shift to permissive conditions, but requires deoxyribonucleic acid and protein synthesis to be expressed. Recombination between the sus mutants of phage ?11 of Kretschmer and Egan and ts?11(31) indicate that competence is controlled by an early gene in the lytic cycle which may be expressed also in lysogenic cells. The phage product inducing competence appears to have a half-life of 10 to 15 min in the conditional lethal mutant at shift to nonpermissive temperature. Ultraviolet inactivation of phage ?11 infectivity occurs more rapidly than inactivation of competence induction. In fact, the number of transformants is increased at low doses of irradiation. Competence induction is, however, decreased at high does of ultraviolet irradiation.     

DNA-mediated transformation in mutants of phage group 2 Staphylococcus aureus

A large pool of antibiotic resistant and auxotrophic mutants was isolated from the Staphylococcus aureus phage group 2 strains UT0002-19 and UT0017 by (1) antibiotic gradient plates, (2) trimethoprim selection, and (3) nitrosoguanidine mutagenesis, which sometimes was coupled by enrichment with either penicillin or methicillin. Strain UT0002-19 has a chromosomal determinant for exfoliative toxin (ET), which causes "scalded skin syndrome" in man. A few mutants were isolated from the phage group 1 strain UT0080, which also produces ET. Two transformation regimens, called the broth and plate methods, were devised for the phage group 2 strains. They employed 80 alpha as helper phage, and recipient cells were incubated with transforming DNA in the presence of Ca2+. Strain UT0080 was transformed using phage 55 as helper. Maximum competence of the phage group 2 strains occurred during early logarithmic growth in trypticase soy broth, but cells grown overnight on heart infusion agar were also competent. Transformation frequencies of all markers ranged from 10(-6) to 10(-8). For phage 80 alpha, a multiplicity of infection of 4 was optimal in transforming a mutant of strain UT0002-19. Transformation of gly, lin, met, ole, rif, and ser markers in S. aureus is reported for the first time. Ery and ole markers in all three strains exhibited cross-resistance. Mapping studies, similar to those performed by DNA-mediated transformation in the phage group 3 strain 8325, can now be commenced for phage group 2 strains of S. aureus in order to elucidate the molecular genetics of this medically important bacterium.     

Suppression of Amber and Ochre Mutants in Salmonella typhimurium by a Mutant F′-1-gal Factor Carrying an Ochre Suppressor Gene

A Salmonella typhimurium strain was given the amber mutation hisC527 by transduction, made galactose-negative by mutation, then infected with the F'-1-gal factor. Of 107 spontaneous and mutagen-induced histidine-independent mutants tested, 3 proved to result from suppressor mutations within the F' factor. The mutant F' factors, when transferred to S. typhimurium and E. coli auxotrophs, suppressed amber and ochre but not UGA or missense mutants, and are inferred to carry ochre suppressor genes. Attempts to isolate an F' amber suppressor mutant were unsuccessful. A suppressor F' factor was transferred to 14 rough mutants which had been isolated from LT2 hisC527 (amber) by selection for resistance to phage P22.c2. One rough mutant was partly suppressed, as shown by its acquisition of O agglutinability and by alterations in its phage resistance pattern. Phage P22h grown on the suppressed mutant contransduced its rf. gene with cysE(+) and with pyrE(+), and the affected locus is inferred to be rfaL. Both the original and the mutant F' factors conferred resistance to the rough-specific phage Br60, which is therefore "female-specific."     

Competence for Transfection in Staphylococcus aureus

Lysogenicity with phage P11 is a requirement for competence in the presence of calcium ions in Staphylococcus aureus 8325N. The wild-type strain 8325N, lysogenic for the phages P11, P12, and P13, is also competent, but strain 8325-4, a nonlysogenic derivative of strain 8325N, as well as strains 8325-4 (P12) and 8325-4 (P13) could not develop competence. Preincubation of strain 8325-4 with culture filtrates from a competent strain can induce competence, but rabbit anti-P11 serum can neutralize the competence factor. Superinfection of competent strain 8325-4 (P11) with phage P11 at high multiplicities increases the transfection frequency. Uptake of deoxyribonucleic acid by competent cells is dependent on calcium ion concentration, pH, and temperature. Inhibition of energy metabolism or protein synthesis before and during incubation with deoxyribonucleic acid affects the binding and uptake. The ability to develop competence during bacterial growth differs between the wild-type strain (8325N) and a nuclease-deficient mutant (8325N nuc). The wild-type strain has a narrow competence maximum in the early exponential growth phase where no extracellular nuclease activity is produced. The nuc strain shows in addition competence maxima later in the exponential growth phase.     

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes

Staphylococcus aureus 8325-4 has the potential to express two distinct cell wall-associated fibronectin-binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin-coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB . A double fnbA fnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non-conserved N-terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild-type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion-defective phenotype of the 8325-4 fnbA fnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325-4 to adhere to fibronectin-coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea-pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo .     

Two restriction and modification systems in Staphylococcus aureus NCTC8325.

The presence of two distinct host specificities in Staphylococcus aureus strain NCTC8325 was revealed by the isolation of restriction- and modification-deficient mutants. The two host specificity systems, designated S1 and S2, are both active on phage 80mualpha but are not additive in their restricting activity. Restriction-deficient, modification-proficient mutants were invariably affected in both restriction systems. The functional relationship between these two systems is discussed.     

Bacteriophage-resistant mutants of Bacillus thuringiensis with decreased virulence in pupae of Hyalophora cecropia

Starting from a crystal-negative parental strain of Bacillus thuringiensis, we isolated certain bacteriophage-resistant mutants which showed decreased virulence in pupae of the cecropia moth (Hyalophora cecropia). These strains (class I mutants) were highly pleiotropic and showed resistance to seven or eight different phages, sensitivity to methicillin, and loss of flagella. They were also more sensitive to cecropia immune hemolymph in vitro. In addition, the export of at least three proteins was reduced. Revertants (class II mutants) were sensitive to phages, virulent, and resistant to penicillin derivatives. One class II mutant was a complete revertant in all properties examined. The other class II mutant was an incomplete revertant still susceptible to immune hemolymph and with repressed export of proteins. Virulence was not coupled to phage resistance as such or to lack of flagella because other mutants affected in these properties were virulent. Other factors which could be excluded as causes of virulence were production of extracellular protease and hemolysin.     

Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Phenotypic correction of nonsense mutation carrying non-converting PE5 phages in Shigella flexneri with suppressor gene.

(i) Phenotypic suppression by aminoglycoside antibiotics of a polyauxotrophic Shigella flexneri var. Y strain on partially completed minimal medium has shown that its Thr dependence is associated with nonsense mutation. Induced Thr+ revertants selected from the culture yielded clones correcting the lytic cycle of nonsense T4 mutant phages. Transfer of R1am plasmid to these clones carrying a nonsense mutation of ampicillin resistance was performed. In this manner a S. flexneri var. Y derivative was isolated which, on the basis of the phenotypic correction of T4 phages and R1am factor, proved to be a suppressor positive clone. (ii) From phage PE5 responsible for conversion of type antigen V, mutants were isolated that had lost their converting capacity. Selected Sup+ and control Sup- strains were treated with the mutant phages and examined for the appearance of type antigen V. Three phage mutants were found to induce antigen conversion only in Sup+ strains. (iii) The data suggest that, at least with phage PE5, the information for type antigen conversion is carried by phage genome.     

Genetic analysis of a temperature-sensitive Salmonella typhimurium rho mutant with an altered rho-associated polycytidylate-dependent adenosine triphosphatase activity.

A conditional-lethal rho mutant of Salmonella typhimurium LT2 has been isolated. The mutation was selected as a suppressor of the polarity of an insertion sequence (IS)2-induced mutation (gal3) carried on an F' plasmid. In addition to suppression of IS2-induced polarity, the rho-111 mutation suppressed nonsense and frameshift polarity. The rho-associated polycytidylic acid-dependent adenosine triphosphatase activity in the mutant strain was elevated 15-fold above that in the parental strain, and the mutant rho protein was thermally unstable. A temperature-resistant revertant of the mutant strain did not suppress polarity and contained normal levels of polycytidylic acid-dependent adenosine triphosphatase, suggesting that the phenotype of the rho-111-bearing strain is the consequence of a single mutation. The rho-111 mutation was located on the S. typhimurium linkage map midway between the ilv and cya loci by phage P22 cotransduction studies. F' plasmid maintenance was not impaired in the mutant strain, and the mutation was recessive to the wild-type allele. The rho-111 mutation did not alter in vivo expression of either the tryptophan or histidine operons.     

Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Genetic control of maltase synthesis in yeast

Summary A new series of maltase negative mutants have been isolated from yeast strains carrying the MAL4 gene. These mutants are allelic to the MAL4 gene and fail to ferment maltose, sucrose, and alphamethylglucoside. Most revertants isolated from these mutants restore the ability to ferment above sugars, and also produce the same levels of maltase as the parental strains. One of the revertants (NA-520-R1), however, ferments maltose slowly, and produces 24 fold less enzyme than the parental strain. Genetic studies revealed that revertant (NA-520-R1), is not a truc back mutation but is carrying an extra-genic suppressor, which suppresses the mal4 allele in mutant (NA-520). Since several lines of published evidence indicate that the MAL4 gene is a regulatory gene, it is suggested that the MAL4 gene codes for a regulatory protein, which acts as positive regulatory element in maltase synthesis.     

Escherichia coli mutants with an altered sensitivity to cecropin D.

Cecropins are a family of small, basic antibacterial polypeptides which can be isolated from pupae of immunized Lepidoptera. They are active against both gram-negative and gram-positive bacteria. We studied a mutant of Escherichia coli, strain SB1004, which is more sensitive to cecropin D than is the parental strain. The mutant was selected as resistant to a host range mutant of a Serratia marcescens phage. When the protein composition of the outer membrane was examined, strain SB1004 and some other phage-resistant mutants were found to be deficient in the OmpC protein. It was concluded that the OmpC protein is the receptor of the phage. Strain SB1004 was found to differ from other ompC mutants in being especially sensitive to hydrophobic antibiotics and to cecropin D. Furthermore, strain SB1004 has a tendency for spontaneous autolysis. A genetic analysis showed the mutations in strain SB1004 and a suppressor mutant to map in the ompC region. The activity of cecropin D against different strains of E. coli was specifically enhanced when divalent cations were absent. No such effect was found with cecropins A and B, which are less hydrophobic than the D form.     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

Spontaneous mutations in the human immunodeficiency virus type 1 gag gene that affect viral replication in the presence of cyclosporins.

Human immunodeficiency virus type 1 mutants that are resistant to inhibition by cyclosporins arise spontaneously in vitro during propagation in a HeLa-CD4+ cell line in the presence of a nonimmunosuppressive analog of cyclosporin A. Interestingly, the phenotype of all of the mutants examined is drug resistant and drug dependent, with both cyclosporin A and its analog. Four independently isolated mutants have been analyzed genetically by construction of recombinant proviruses in the NL4-3 parental strain background and subsequent testing of the chimeric viruses in HeLa cells. The cyclosporin-resistant, cyclosporin-dependent phenotype consistently transfers with a 1.3-kb fragment of gag, within which the four mutants share one of two possible single amino acid exchanges in a proline-rich stretch in the capsid domain of Pr55gag. These mutants provide the first evidence that mutations in human immunodeficiency virus type 1 gag confer resistance to cyclosporins; however, replication is conditional on the presence of the drug. In the T-cell line CEM, replication of the recombinant mutant viruses is also cyclosporin dependent. The drug-dependent replication in HeLa cells is stringent, and in the absence of cyclosporin only revertant viruses with the parental phenotype grow out of cultures infected with cyclosporin-dependent virus. In at least one isolate examined, the revertant phenotype appears to be due to suppressor mutations near the proline-rich region.