Bacteriocin Production by Transformable Group H Streptococci

Group H streptococci (strain Challis) which are competent for transformation release a bacteriocin into liquid medium which is bacteriocidal for another group H streptococcus (strain Wicky). The streptocin (STH(1)) is resistant to treatment with deoxyribonuclease and ribonuclease but is sensitive to trypsin, phospholipase C, and alkaline phosphatase. Such enzyme sensitivity experiments indicate that the bacteriocin may be a complex molecule (protein and lipid) containing phosphate groups essential for activity. STH(1), which is readily distinguishable from competence factor and bacteriophage activity, appears to have no role in the initiation of the competent state in strain Wicky. The presence of this factor in Challis culture supernatant fluids indicates that a reevaluation of earlier studies performed with the Challis-Wicky transformation system may be necessary.     

Inhibition of Transformation in Group H Streptococci by Lysogeny

Group H streptococcal strains Challis and WE4 were lysogenized with a bacteriophage isolated from strain Channon, after which their capacity for transformation to streptomycin and rifampin resistance was reduced by three orders of magnitude. The probable reason is the inability of the lysogenized strains to bind deoxyribonucleic acid irreversibly, even though they exhibit earlier stages of competence development during a competence regimen.     

Transfection of Group H Streptococci

Transfection of streptococci is reported here for the first time.     

Transformability of Streptomycin-resistant Group H Streptococci

Several resistant mutants of a transformable group H streptococcus, strain Challis, were isolated from media containing high concentrations of streptomycin. Mutants SR5a and SR5 exhibited high and low transformability, respectively, when exposed to deoxyribonucleic acid (DNA) from a novobiocin-resistant Challis strain. With similar exposure, mutant SR30 exhibited loss of transformability. The mutants further differed from the parent strain in time of appearance of optimal competence, and, in the case of SR5 and SR30, total growth was somewhat less than that of the parent. The rapidity with which transformants appeared upon initial exposure to DNA was approximately the same in the mutants and the parent strain. The decrease or loss of transformability of mutants SR5 and SR30 was found to be due to an alteration in capacity to take up DNA. Mutant SR5a (highly transformable) was further differentiated from mutants SR5 and SR30 in that it was somewhat more sensitive to high concentrations of streptomycin. Transformants obtained by treating strain Challis with the three types of mutant DNA, on the other hand, exhibited similar degrees of resistance to increasing concentrations of streptomycin. The additional decrease in transforming ability of mutant SR5a and the loss of transforming ability of mutant SR5 after a second exposure to streptomycin may indicate a stepwise process in the change from transformability to nontransformability. Although streptomycin resistance may not be directly related to inability to transform, results indicate that streptomycin greatly increases the chances of selecting these mutants and also can be of value in serving as a marker in studies of this nature.     

Isolation of Bacteriophages from Group H Streptococci

Temperate phages were isolated from 5 of the 17 strains of group H streptococci tested.     

Growth and Development of Competence in the Group H Streptococci

The growth and development of competence by group H streptococci, strain Challis, were compared in synthetic, semisynthetic, and complex media with respect to the cultural conditions required, time of onset and persistence of competence and transformation efficiency. Provided that cultural conditions were strictly controlled in the synthetic system, transformation frequencies of 1% or above were routinely observed. The initial pH must be between 7.3 and 7.6, and the addition of freshly prepared bicarbonate ion was required. Furthermore, competence was sensitive to the degree of initial agitation of the culture. There was no evidence that "step-down" or "unbalanced" growth conditions were required. Competence could be provoked in the incompetent strain Wicky, growing in complex or semisynthetic media, by the addition of heat-killed or filtered cultures of strain Challis prepared during the competent period of growth in synthetic medium.     

Accumulation of 14C-Streptomycin by Streptomycin-sensitive and Streptomycin-resistant Group H Streptococci

Three streptomycin-resistant mutants of group H streptococcus, strain Challis, were examined for ability to accumulate ¹⁴C-streptomycin. Although the mutants exhibited different levels of transformation, only the streptomycin-sensitive parent Challis strain accumulated significant amounts of ¹⁴C-streptomycin. It appears that impermeability to streptomycin does not necessarily result in reduction or loss of transformability. The amount of label accumulated by strain Challis was correlated with a loss of viability. In addition, accumulation of label was influenced by the concentration of ¹⁴C-streptomycin, the time of exposure, and the type of medium employed.     

Preparation of Protoplasts of Group H Streptococci (Streptococcus sanguis)

Stable protoplasts of several strains of group H streptococci (Streptococcus sanguis) can be prepared by use of group C streptococcal phage-associated lysin in the presence of 30% raffinose. Sucrose cannot be substituted for raffinose. Protoplast formation did not require the addition of Mg²⁺; however, this cation enhanced their stability. Some other strains, also presumptive group H streptococci, were not sensitive to phage-associated lysin.     

Extrachromosomal Elements in Group N Streptococci

The deoxyribonucleic acid (DNA) of Streptococcus lactis C2, S. cremoris B(1), and S. diacetilactis 18-16 was labeled by growing cells in Trypticase soy broth containing (3)H-labeled thymine. The cells were gently lysed with lysozyme, ethylenediaminetetraacetic acid, and sodium lauryl sulfate. The chromosomal DNA was separated from plasmid DNA by precipitation with 1.0 M sodium chloride. The existence of covalently closed circular DNA in the three organisms was shown by cesium chloride-ethidium bromide equilibrium density gradient centrifugation of the cleared lysate material. In an attempt to correlate the loss of lactose metabolism with the loss of plasmid DNA, lactose-negative mutants of these organisms were examined for the presence of extrachromosomal particles. Covalently closed circular DNA was detected in the lactose-negative mutants of S. lactis C2 and S. diacetilactis 18-16. In S. cremoris B(1), however, no covalently closed circular DNA was observed by using cesium chloride-ethidium bromide gradients. Electron micrographs of the satellite band material from S. lactis C2 and its lactose-negative mutant confirmed the presence of plasmid DNA. Three distinct plasmids having approximate molecular weights of 1.3 x 10(6), 2.1 x 10(6), and 5.1 x 10(6) were observed in both organisms.     

Group B Streptococci in Neonatal Deaths

Clinical microbiologists are reminded to look for group B streptococci in neonatal infants. We report four deaths, one with meningitis, occurring among 14 such infected newborn infants at our institution; of the nursing staff, 31% were also carriers of group B streptococci.     

Autolytic Activity and Its Association with the Development of Competence in Group H Streptococci

Seventeen strains of group H streptococci were tested for their ability to develop competence for genetic transformation, either spontaneously or with the addition of competence factor derived from strain Challis supernatant fluids, and for their ability to autolyze. Autolysis was measured as a decline in optical density after washed cells were placed in a buffer at pH 9. Kinetic experiments showed that, in strains Challis, SBE I/II, WE4, SR 30, and a strain (FW 227) cured of its bacteriophage, competence and the ability to autolyze occurred simultaneously. Since autolysis was observed only in (i) competent cells, (ii) cells that passed their peak of competence, and (iii) those cells that exhibit a potential for developing competence but never go on to transform (i.e., lysogenized Challis cells), it is concluded that, in the group H streptococci, autolytic events are associated with the competent state. Strains that transformed but did not autolyze were not found.     

Antigenic Variation in Group A Streptococci: Types 11 and 9

A strain of group A Streptococcus which was virulent but M-nontypable was isolated from patients in a hospital nursery during an epidemic. This strain, Boston 11, reacted in T-agglutination tests with antisera for types 9 and 11, an unusual combination. A comparison of this strain with Lancefield's M-11 strain (NCDC SS-721) and Alabama 11 (Provisional 61) revealed three serologically related but distinct strains. Antiserum produced with the Boston 11 strain exhibited similar reactivity with all three "11" strains as well as with M-9 (SS-501) as demonstrated in precipitin tests. Immunodiffusion studies indicated that the Boston 11 antigen was partially identical with the M-11 and M-9 strains and shared at least one antigen with the Alabama 11 strain. The Boston 11 antiserum could be made specific for precipitin tests, but bactericidal activity for the Alabama 11, M-11, and Boston 11 strains was essentially negative.     

Transduction of Rifampin Resistance in Group A Streptococci

Rifampin-resistant strains of group A streptococci were isolated as spontaneous mutants. Transduction analyses employing phage A25 showed the rifampin marker to be transferred with high frequency. The mutations conferring resistance to rifampin and streptomycin are not co-transducible.     

Competence for Natural Genetic Transformation in the Streptococcus bovis Group Streptococci S. infantarius and S. macedonicus

Natural genetic transformation is common among many species of the genus Streptococcus, but it has never, or rarely, been reported for the Streptococcus pyogenes and S. bovis groups of species, even though many streptococcal competence genes and the competence regulators SigX, ComR, and ComS are well conserved in both groups. To explore the incidence of competence in the S. bovis group, 25 isolates of S. infantarius and S. macedonicus were surveyed by employing culture in chemically defined media devoid of peptide nutrients and treatment with synthetic candidate pheromone peptides predicted from the sequence of the gene comS. Approximately half of strains examined were transformable, many transforming at high rates comparable to those for the well-characterized streptococcal natural transformation systems. In S. infantarius, nanomolar amounts of the synthetic pheromone LTAWWGL induced robust but transient competence in high-density cultures, but mutation of the ComRS locus abolished transformation. We conclude that at least these two species of the S. bovis group retain a robust system of natural transformation regulated by a ComRS pheromone circuit and the alternative sigma factor SigX and infer that transformation is even more common among the streptococci than has been recognized. The tools presented here will facilitate targeted genetic manipulation in this group of streptococci.     

Intraphagocytic Degradation of Group A Streptococci: Electron Microscopic Studies

The morphological changes that occur during intraphagocytic digestion of group A streptococci were studied by electron microscopy The first evidence of degradation of the ingested organism was the appearance of reticular changes in the bacterial endoplasm. This was followed by gradual swelling and dissolution of the bacterial wall, with final degradation of all the constitutuents to electron-dense debris. Accompanying changes in the phagocytic cell were observed; they consisted of vacuole formation, fusion of lysosomes with the wall of the yacuole, release of the lysosomal contents into the vacuole, and aggregation of the lysosomal contents around the ingested organism. Changes in the morphology of the organism similar to those observed during intraphagocytic digestion were also obtained by subjecting streptococcal cells to the action of the phage-associated lysin.     

Presumptive Identification of Group D Streptococci: the Bile-Esculin Test

Six tests commonly used for the presumptive identification of group D streptococci were evaluated. Strains tested included 282 group D streptococci and 366 non-group D. Ratios of percentages of group D to non-group D strains which gave positive reactions for each test are as follows: bile-esculin, 100:2; salt tolerance, 88:24; heat tolerance, 100:80; SF broth, 86:1; KF broth, 99:40; and methylene blue milk reduction, 90:17. These data indicate that the bile-esculin test provided a reliable means of identifying group D streptococci and differentiating them from non-group D streptococci. Methodology for reading and interpreting positive reactions and time of incubation of the bile-esculin medium was defined. Evidence of the need for standardization of salt and heat-tolerance tests was obtained.     

Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.