Creation and analysis of a bank of chromosomal genes of Streptococcus group A

The representative genomic library of chromosomal genes has been constructed for streptococcus group A serotype M48 strain 1/64 on the vector lambda L 47.1. Screening of the obtained genomic library by hybridization and immunological techniques revealed about 50 clones producing the streptococcal antigens (extracellular nonidentified products and non-type specific structural streptococcal proteins). Among the recombinant clones three were found to harbour the genetic determinants for M-protein. One the clones contains a determinant coding for epitopes crossreacting with antisera to M-proteins of other serotypes and a protective epitope. The presence of the latter was tested in an indirect bactericidal test.     

Conditional transduction of Salmonella typhimurium envB mutations.

Joint transduction of the argR and envB genes was observed, at a frequency of 24.5%, when four envB strains were transduced to tetracycline resistance (Tetr) with bacteriophage P22 grown on an argR372::Tn10 envB+ donor. When round-cell argR372::Tn10 derivatives of those envB strains were used as donors, two of them did not produce envB transductants in wild-type LT2 and other envB+ recipients, even though large numbers of Tetr transductants were obtained. This apparent exclusion of envB mutations did not occur when mecillinam-resistant derivatives of those envB+ strains were used as recipients. Mutations conferring partial resistance to mecillinam were found, unlinked to the argR-envB region, in three of the four envB strains studied; envB+ derivatives of the four strains were competent to accept envB mutations excluded by wild-type recipients. It is suggested that some envB mutations are lethal in the absence of suppressor mutations, some of which increase resistance to mecillinam.     

Chromosomal penicillin resistance in Staphylococcus aureus strains of phage group II

The markers coding for serotype B penicillinase (PcB) in wild type strains of group II of S. aureus behaved as chromosal characters in 6 out of 7 cases. The PcB markers could be transduced to group II strains, and with the help of restriction deficient mutants also to some strains of group I. No extrachromosomal DNA could be detected in the transductants. The low frequency of transduction of chromosomal markers as well as the high restriction barrier seem to limit the spread of these markers outside group II.     

Transduction by bacteriophage P22 in nonsmooth mutants of Salmonella typhimurium

The general transducing phage P22 attacks only smooth (S) Salmonella with O antigen 12, determined by the oligosaccharide repeating unit constituting the distal part of the somatic lipopolysaccharide (LPS) side chain; non-S mutants, whose LPS contain few or no O repeating units, appear to be resistant. Auxotrophic non-S mutants of Salmonella typhimurium LT2 were tested as transductional recipients. Some transductants (0.5 to 5% as many as from S recipients) were obtained from most semirough recipients, either of class D (presumed leaky rouA mutants) or of a class due to mutation near his (presumed leaky rouB mutants), and from recipients lacking uridine diphosphogalactose epimerase or phosphomannose isomerase. Transductants were not obtained from several rouA, rouB, "heptose-negative," and glucose-1-transferase mutants, nor from most semirough class C mutants, whose LPS side chains each bear a single O oligosaccharide unit. Most transductants evoked from non-S recipients by temperate (c(+)) phage P22 were nonlysogenic, and virulent P22.c2 phage was about as effective as P22.c(+) in transduction to non-S recipients; probably all P22 transducing particles neither lysogenize nor kill. The extended-host-range mutant P22h gave qualitatively similar results,but evoked 5- to 30-fold more transductants from some non-S recipients than did P22. Probably, the LPS of non-S mutants susceptible to transduction contains a few O-specific oligosaccharide units, conferring a slight ability to adsorb P22 and a greater ability to adsorb P22h.     

The enhancement of the immunogenicity of streptococcal group-A M protein by conjugation with a synthetic polyelectrolyte]

Immunization with the polypeptide fragment of group A streptococcal protein M conjugated with the copolymer of acrylic acid and N-vinylpyrrolidone in complete Freund's adjuvant has been found to lead to a sharp increase in the level of antibodies to the type-specific determinants of protein M, detected in the enzyme immunoassay (EIA). The possibility of the application of such sera to preliminary typing of streptococci in EIA with the use of whole microbial cells as antigens has been shown. The data on high activity of the sera thus obtained in the bactericidal test with streptococci of the homologous type are presented. Recommendations on the use of sera obtained by the above method for highly precise typing of the virulent cultures of group A streptococci in the bactericidal test are given.     

Specialized transduction with lambda plac5: dependence on recB.

Genetically disabled lambda plac5 transducing phage derivatives were used to study the recB dependence of recombination during specialized transduction. The frequency of transduction was normalized to colony-forming units, and the end product of recombination was monitored by scoring for addition and substitution transductants. When a chromosomal lac gene was the recipient DNA substrate molecule, both the normalized transduction frequency and the proportion of addition and substitution transductants showed essentially no recB dependence. There was a pronounced recB dependence for both normalized transduction frequency and recombination end product formation when F42 lac was the recipient DNA substrate. recB appears to have no significant role in the recombination that occurs between the two lac regions in an addition transductant. UV irradiation of the transducing phages increased the absolute level of both addition and substitution transductants obtained with a chromosomal lac gene but resulted in a considerable change in the relative frequency of addition versus substitution transductants.     

Rho and Ribosome Mutation Interaction: Lethality of rho-15 in rpsL or rpsE Strains, and rho-15 Methionine Auxotrophy in rps+ Strains of ESCHERICHIA COLI

The phenotype of Escherichia coli K-12 carrying rho-15 in the genetic background DW319 ilv lacZ::IS1 is described. Seventy-eight percent (70/90) of Ilv+ transductants acquired the following phenotype: temperature-sensitive growth on minimal salts medium, Ts+ growth on complex medium and suppression of the lac polar mutation. At 42 degrees on minimal medium, the rho-15 transductants were cross-fed by a substance diffusing from Rho+ transductants or controls. The requirement for this substance was satisfied by methionine or cystathionine, but not by any other single amino acid or combination of amino acids, by spermidine, or by mono- or divalent cationic salts.--Transduction of rho-15 into four other Ilv- recipients revealed two phenotypic patterns. Recipients with rpsL or rpsE ribosomes yielded rho-15 transductants that were Ts on all media, or Ts on minimal medium whether or not methionine was present. The effect of the ribosome on expression of rho-15 was confirmed by transduction of appropriate rps alleles into DW319, followed by co-transduction of rho-15 with Ilv+. The growth rate of double rho-15 rpsL or rho-15 rpsE strains was severely reduced at 42 degrees in comparison with strains carrying any of these single mutations. Models for rho and ribosome interaction are presented.     

Mapping of the genetic determinant controlling Salmonella K-antigen synthesis. II. Nonmotile mutants of Salmonella typhimurium

According to the preliminary data, S. typhimurium K-antigen is located in the area of minutes 40-44 on the Salmonella chromosome map. The formation of nonmotile mutants from motile Salmonella strains was induced by the action of nitrosoguanidine. Two main groups of mutants differing in their reaction of agglutination with H- and K-antisera were obtained: Mot-H-K- (motA or motB mutants) and Mot-H-K- (H1- or fla- mutants). The transduction transfer of the sign of motility by phage P22HT to H-K- mutants and to H1- and flaE- mutants led to the restoration of agglutination ability with respect to H- and K-antisera in all Mot+ transductants under study simultaneously. The restoration of H+K+ phenotype was also observed in spontaneous motile revertants obtained from H-K- mutants. Thus, the gene controlling the synthesis of K-antigen in Salmonellae was shown to be incorporated into the Fla operon, the regulatory system of the operon controlling the expression of this gene.     

Transduction, Restriction and Recombination Patterns in Escherichia Coli

Chromosomal DNA from several Escherichia coli reference (ECOR) strains was transduced by bacteriophage P1 into E. coli strain K12 W3110 trpA33. Recombination patterns of the transductants were determined by restriction fragment length polymorphism over a 40-kb region centering on a single marker (trpA(+)) in the tryptophan operon. These experiments demonstrate that transduction between different strains of E. coli can result in recombinational replacements that are small in comparison to the entrant molecule (replacements average 8-14 kb, whereas P1 packages ~ 100 kb) often in a series of discrete segments. The transduction patterns generated resemble the natureal mosaic sequence patterns of the ECOR strains described in previous work. Extensive polymorphisms in the restriction-modification systems of the ECOR strains are a possible explanation for the sequence patterns in nature. To test this possibility, two transductants were back-transduced into strain K12 W3110 trpA33. The resulting patterns were strikingly different from the original transductions. The size of the replacements was greater, and no multiple replacements were observed, suggesting a role for restriction-modification systems in the transduction patterns and perhaps for the mosaic sequence patterns in nature.     

A generalised transducing bacteriophage for Rhodococcus erythropolis

Summary Q4, a bacteriophage isolated from soil, mediated the transduction of a number of unlinked markers in Rhodococcus erythropolis. Highest numbers of transductants were obtained at multiplicities of infection of over 100, transductants only being obtained because of the temperate nature of the phage. Under optimal conditions, transduction to prototrophy of auxotrophic markers was over 50 times the spontaneous reversion rate and transduction of some antibitic resistance markers was over 10 times the spontaneous mutation rate. Segregation of unselected, but linked, markers was observed and the phage was used to order loci in a three factor cross. The virus required magnesium ions. Highest phage titres and greatest transduction frequency were obtained with stationary phase cultures.     

Application of Enzyme Production Properties in Subtyping of Group A Streptococci According to T Type

The production of extracellular nicotinamide adenine dinucleotide glycohydrolase (NADG) and the cell-bound lipoproteinase (serum opacity reaction, SOR) by strains of different serological types of group A streptococci, in relation to the T typing, was studied. The production of both NADG and SOR, or only one of them, was found to be characteristic of serotypes, as determined by M and T antigen. No difference in the production of these enzymes was found in relation to M-positive and M-negative variants. Investigation into NADG and SOR production as related to the T type enabled the division of a single agglutination pattern into four main groups, each of which corresponds to one specific M type or more. Of the 370 strains belonging to 12 different T-agglutination patterns, 21% produced both enzymes and 42.5% failed to produce any of them, whereas the remaining 36.5% produced only one out of the two enzymes. Five streptococcal types which did not produce NADG and SOR also failed to synthesize streptolysin S at the early logarithmic phase of growth, indicating that streptolysin S production by young cultures may be also related to serotype. No correlation was found between the production of NADG-SOR as related to serotype and the production of streptolysin O, acid phosphotase, esterase, N-acetylglucosaminidase, hyaluronidase, streptokinase, and the cell-sensitizing factor. The practical and potential usefulness of NADG and SOR production in epidemiological studies is discussed.     

Drug Resistance of Staphylococci VII. Genetic Determinants Responsible for the Resistance to Tetracycline, Streptomycin, Sulfanilamide, and Penicillin

A genetic analysis of resistance to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), and penicillin G was carried out through transduction with phage lysates obtained from a multiply resistant strain of Staphylococcus aureus by ultraviolet irradiation. All transductants acquired resistance to both TC and SA, even when singly selected for either SA or TC resistance. The locus responsible for TC resistance could not be separated genetically from that for SA resistance. On the other hand, in transduction of SM resistance, about 30% of the transductants jointly acquired resistance to both TC and SA. These observations suggest that the loci governing resistance to TC, SA, and SM exist close together on a single genetic unit, this probably being the chromosome.     

High-frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein-mediated invasion and cytoskeletal rearrangements

A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90-226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti-M1 antibody, invasion by an M1- strain (90-226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper-like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal-containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP-1.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

The biochemical and immunological properties of group A type M 29 streptococci cultured in the presence of a bovine blood serum preparation

The influence of the preparation of cattle blood serum on group A streptococcus, type M 29, has been studied. The study has revealed that the addition of 17% of dialysis water obtained from a fraction of cattle blood serum to the standard culture medium (3% Todd-Hewitt broth) produces changes in the amino acid composition of the cell walls of M+ variant without altering the antiphagocytic resistance of the mutant thus obtained. The dialysate of the pepsin digest of the cell walls of the mutant contains Fc-receptors and receptors to fibrinogen, while the initial strain contains only receptors to fibrinogen which are, in this case, the pepsin fragments of M protein. The study has revealed similarity in the amino acid compositions of these proteins (receptors to fibrinogens) of phenotypes M+ and M2+. Thus, our data confirm that the initial strain and the mutant belong to different phenotypes of group A streptococcus, type M 29.     

Unstable generalized transduction in Achromobacter.

Six auxotrophic markers of a halotolerant collagenolytic strain of Achromobacter were transduced by four alpha hages. Abortive transduction was also demonstrated. The generalized transduction system is unusual as the transductants were unstable, characteristic of transduction by lysogeny. The Achromobacter strain is a cryptic lysogen for alpha and purified transductants were either sensitive or resistant to alpha. Purified clones from four resistant transductants and one sensitive transductant liberated phage spontaneously. The host ranges of these spontaneous phage differed from that of the alpha phage used for the transduction experiment. Some initially resistant transductants became simi-sensitive to alpha (efficiency to plating) e.o.p. (10minus-1 to 10minus-2) after repeated cloning.     

Gene Transfer by Transduction in the Marine Environment

To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 × 10⁻⁷ to 5.13 × 10⁻⁹ transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 × 10⁻⁸ to 3.7 × 10⁻⁸ transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 × 10¹⁴ transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.     

Diversity of prophage DNA regions of Streptococcus agalactiae clonal lineages from adults and neonates with invasive infectious disease

The phylogenetic position and prophage DNA content of the genomes of 142 S. agalactiae (group-B streptococcus, GBS) isolates responsible for bacteremia and meningitis in adults and neonates were studied and compared. The distribution of the invasive isolates between the various serotypes, sequence types (STs) and clonal complexes (CCs) differed significantly between adult and neonatal isolates. Use of the neighbor-net algorithm with the PHI test revealed evidence for recombination in the population studied (PHI, P = 2.01 × 10(-6)), and the recombination-mutation ratio (R/M) was 6:7. Nevertheless, the estimated R/M ratio differed between CCs. Analysis of the prophage DNA regions of the genomes of the isolates assigned 90% of the isolates to five major prophage DNA groups: A to E. The mean number of prophage DNA fragments amplified per isolate varied from 2.6 for the isolates of prophage DNA group E to 4.0 for the isolates of prophage DNA group C. The isolates from adults and neonates with invasive diseases were distributed differently between the various prophage DNA groups (P < 0.00001). Group C prophage DNA fragments were found in 52% of adult invasive isolates, whereas 74% of neonatal invasive isolates had prophage DNA fragments of groups A and B. Differences in prophage DNA content were also found between serotypes, STs and CCs (P < 0.00001). All the ST-1 and CC1 isolates, mostly of serotype V, belonged to the prophage DNA group C, whereas 84% of the ST-17 and CC17 isolates, all of serotype III, belonged to prophage DNA groups A and B. These data indicate that the transduction mechanisms, i.e., gene transfer from one bacterium to another by a bacteriophage, underlying genetic recombination in S. agalactiae species, are specific to each intraspecies lineage and population of strains responsible for invasive diseases in adults and neonates.     

Impaired Transduction of R213 and Its Recovery by a Homologous Resident R Factor

Transduction by Plkc of drug-resistance markers of the factor R213 was shown to occur at an exceptionally low frequency (at less than 10(-8) of the input phage), and they could not be transduced by P22. When the recipient cells carried a homologous R factor derived from R213, markers were transduced by Plkc at a normal frequency (at about 10(-5) to 10(-6) of the input phage). Derivative R factors, transducible by Plkc at a normal frequency but being transferred by conjugation at a frequency lower than that of the original R213, were obtained. This type of transductant often segregated R(-) cells. In addition, several transductants contained R factors which were transferred normally by conjugation but were transduced by Plkc at as low a frequency as the original R213. This type of transductant was an effective recipient for transduction by Plkc of R213 when apparently "cured" by acridine treatment. No such effective "cured" recipients were obtained from the transductants with derivatives of R213 transducible at a normal frequency. Two possible interpretations are presented: (i) R213 produces a bacteriocin-like substance upon transduction, or (ii) the genome size of R213 is too large for all of its determinants to be transduced.