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.     

Deoxyribonucleic Acid Homology Among Lactic Streptococci

A comparison was made by deoxyribonucleic acid homology of 45 strains of lactic streptococci, using two strains of Streptococcus cremoris and three strains of Streptococcus lactis as reference strains. All S. cremoris strains were grouped together by deoxyribonucleic acid homology. S. lactis strains formed a second group, except that three strains of S. lactis showed a high degree of homology with S. cremoris strains. The three Streptococcus diacetylactis strains could not be differentiated from S. lactis strains. In spite of these differences between S. lactis and S. cremoris strains, the majority of S. cremoris, S. lactis, and S. diacetylactis strains studied had at least 50% of their base sequences in common. In contrast, Streptococcus thermophilus strains generally showed little relationship with the other strains of lactic streptococci. The relevance of these findings to the selection of starter strains for cheese making is discussed.     

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.     

Long-Term Storage of Bacteriophages of Lactic Streptococci

Four phage strains representing phages of Streptococcus lactis, S. cremoris, and S. diacetilactis were selected for the observation of the effect of cold storage on their viability. Phages were stored at 4 C and at -18 C, or were frozen at approximately -70 C and stored at -18 C. They were found to display a high degree of stability with these storage methods. The same phage strains showed good stability to storage at room temperature for 3 weeks after thawing and also to alternate freezing and thawing eight times. Three series consisting of from 23 to 31 lactic streptococcal phage preparations were observed over periods extending up to 6 years, and with only a few exceptions were found to store satisfactorily at -18 C after quick freezing. Although the same phage preparations stored at 4 C were generally somewhat less stable, many were stable when stored by both methods.     

Loss of Lactose Metabolism in Lactic Streptococci

Lactose-negative mutants occurred spontaneously in broth cultures of Streptococcus lactis C(2)F. Instability of lactose metabolism was noted in other strains of S. lactis, in strains of S. cremoris, and in S. diacetilactis. Colonies of S. lactis C(2)F grown with lactose as the carbohydrate source also possessed lac(-) cells. Treatment of lactic streptococci with the mutagen acriflavine (AF) increased the number of non-lactose-fermenting variants. The effect of AF on growth and on loss of lactose-fermenting ability in S. lactis C(2)F was consequently further examined. The presence of AF appears to favor competitively the growth of spontaneously occurring lactose-negative cells and appears to act in the conversion of lactose-positive to non-lactose-fermenting cells. The lactose-negative mutants partially revert to lactose-positive variants which remain defective in lactose metabolism and remain unable to coagulate milk. The lactose-negative cells become dominant in continuous culture growth and provide evidence that alterations in the characteristics of starter strains can be produced by continuous culture, in this case, the complete loss in ability to ferment lactose.     

Lysogenic Strains of Group N Lactic Streptococci

A temperate bacteriophage, designated r(1)t, was inducible from the group N lactic streptococcus, Streptococcus cremoris R(1), by ultraviolet irradiation or mitomycin C treatment. Induced lysates produced plaques on lawns of three closely related S. cremoris strains, AM(1), SK(11), and US(3). Strain SK(11) was readily lysogenized. S. cremoris AM(1) was the most reliable indicator strain, although the age of the culture used for seeding plates was critical. Zones of lysis but no plaque formation were observed on lawns of nine additional S. cremoris strains. Phage r(1)t could not be detected in filtrates of stationary-phase R(1) cultures and was near the limits of detection in logarithmically growing cultures. Phage levels were still very low (1 plaque-forming unit on AM(1) per 10 induced cells) in induced lysates of R(1) cultures. These low levels of detectable phage may be attributable to an inadequate indicator, lysogenization of the indicator, adsorption of induced phage to cellular debris, concurrent induction of other undetectable phages, or the production of high proportions of defective phages. Electron micrographs of induced R(1) lysates revealed a high incidence of incomplete phage particles, fragments, and ghosts.     

Protoplast Formation and Regeneration in Lactic Streptococci

We cloned the promoter of the 9-cis-epoxycarotenoid dioxygenase gene from Arachis hypogaea L. β-Glucuronidase (GUS) histochemical staining and GUS activity assay indicated that the activity of the promoter was exhibited predominantly in the leaves and enhanced by water and NaCl stresses, and by application of abscisic acid (ABA) and salicylic acid (SA) in transgenic Arabidopsis. Moreover, two novel ABRE-like (abscisic acid response element) elements were identified in the promoter region.     

Tubular Heads in Bacteriophages from Lactic Streptococci

Tubular bacteriophage heads were observed in the lysate of two phages from Streptococcus lactis obtained from single plaques without mutagenesis. The frequency of appearance of the tubular heads was 2.5 and 16%.     

Activator Specificity of Pyruvate Kinase from Lactic Streptococci

The pyruvate kinase from lactic streptococci was activated by 20 structurally dissimilar sugar phosphates and glycolytic intermediates. Nine compounds were more effective activators than fructose-1,6-diphosphate.     

Growth Stimulation of Lactobacillus Species by Lactic Streptococci

Cell extracts of Streptococcus species important in cheese starters stimulated the growth of Lactobacillus species common to Cheddar cheese. All Lactobacillus strains employed, with the exception of a strain of L. casei, were significantly stimulated by a strain of S. diacetilactis. L. casei was highly stimulated by both a strain of S. lactis and a strain of S. diacetilactis. The stimulant(s) was dialyzable and was partially inactivated by heat. The stimulatory principle was active at 10 C, indicating that the stimulatory effect may be influencing the growth of lactobacilli in Cheddar cheese during curing. Viable Streptococcus cells did not inhibit the growth of Lactobacillus species.     

Effect of Oxygen on Lactose Metabolism in Lactic Streptococci

Three strains of Streptococcus lactis, two of Streptococcus cremoris, and one of Streptococcus thermophilus metabolized oxygen in the presence of added carbohydrate primarily via a closely coupled NADH oxidase/NADH peroxidase system. No buildup of the toxic intermediate H₂O₂ was detected with the three S. lactis strains. All six strains contained significant superoxide dismutase activity and are clearly aerotolerant. Lactose- or glucose-driven oxygen consumption was biphasic, with a rapid initial rate followed by a slower secondary rate which correlated with factors affecting the in vivo activation of lactate dehydrogenase. The rate of oxygen consumption was rapid under conditions that led to a reduction in lactate dehydrogenase activity (low intracellular fructose 1,6-bisphosphate concentration). These conditions could be achieved with nongrowing cells by adding lactose at a constant but limiting rate. When the rate of lactose fermentation was limited to 5% of its maximum, nongrowing cells of S. lactis strains ML3 and ML8 carried out an essentially homoacetic fermentation under aerobic conditions. These same cells carried out the expected homolactic fermentation when presented with excess lactose under anaerobic conditions. Homoacetic fermentation leads to the generation of more energy, by substrate-level phosphorylation via acetate kinase, than the homolactic fermentation. However, it was not observed in growing cells and was restricted to slow fermentation rates with nongrowing cells.     

Lysogeny in Lactic Streptococci Producing and Not Producing Nisin

Eighty-seven strains of lactic streptococci (46 of Streptococcus lactis, 24 of S. diacetilactis, and 17 of S. cremoris) were tested for lysogeny; 12 S. lactis strains produced nisin. Lysogeny was found in five S. lactis strains (two of them were nisin producers) and in two S. diacetilactis strains. Four S. lactis and two S. diacetilactis lysogens liberated phages both spontaneously and after ultraviolet treatment, and one S. lactis strain liberated phages spontaneously only. No lysogens were found among the S. cremoris strains tested. An initial characterization of the lysogens and their phages was made. The lytic spectrum of some of the examined phages was very narrow (homospecific), whereas that of others was wide, including strains of the three investigated species.     

Effect of Chloramphenicol on Host-Bacteriophage Relationships in the Lactic Streptococci

Chloramphenicol (CM)-resistant mutants of Streptococcus lactis strain ML3 were obtained either as a consequence of continuous transfer of the bacteria in broth containing increasing amounts of CM or by selecting for high-level resistant derivatives after mutagenic treatment of the bacteria. Some CM-resistant cells obtained by the first method were also resistant to the homologous bacteriophage. Cells trained to grow in the presence of CM developed resistance to some heterologous attacking phages but not to phage ml(3). Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 mug/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml(3) phage in strain ML3-CM(r)I, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phage-resistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one.     

Characterization of Growth Stimulants in Corn Steep for Lactic Streptococci

The production of acid in milk cultures of lactic streptococci was stimulated by the addition of corn steep liquor. Separation by ion-exchange and paper chromatography indicated the presence of four major stimulatory components in the corn steep. Some variation was noted in the response of the lactic streptococci to the individual stimulatory components. The four components were further purified by paper and column chromatography. One of the four stimulatory components stained positively on chromatograms with ninhydrin. The remaining stimulatory components were detectable only by bioautography. All four components were unstable to acid hydrolysis and absorbed ultraviolet light between 230 and 275 nm in aqueous solution. The stimulatory components did not contain pentoses, suggesting that they were not nucleotides or nucleosides; however, they might be purine or pyrimidine bases.     

Conjugal Transfer in Lactic Streptococci of Plasmid-Encoded Insensitivity to Prolate- and Small Isometric-Headed Bacteriophages

Eight of 40 strains of Streptococcus lactis and S. lactis subsp. diacetylactis were able to conjugally transfer a degree of phage insensitivity to Streptococcus lactis LM0230. Transconjugants from one donor strain, S. lactis subsp. diacetylactis 4942, contained a 106-kilobase (kb) cointegrate plasmid, pAJ1106. The plasmid was conjugative (Tra⁺) and conferred phage insensitivity (Hsp) and lactose-fermenting ability (Lac) in S. lactis and Streptococcus cremoris transconjugants. The phage resistance mechanism was effective against prolate- and small isometric-headed phages at 30°C. In S. lactis transconjugants, the phage resistance mechanism was considerably weakened at elevated temperatures. A series of deletion plasmids was isolated from transconjugants in S. cremoris 4854. Deletion plasmids were pAJ2074 (74 kb), Lac⁺, Hsp⁺, Tra⁺; pAJ3060 (60 kb), Lac⁺, Hsp⁺; and pAJ4013 (13 kb), Lac⁺. These plasmids should facilitate mapping Hsp and tra genes, with the aim of constructing phage-insensitive strains useful to the dairy industry.     

Grouping of Lactic Streptococci by Gel Electrophoresis of Soluble Cell Extracts

Soluble cell proteins obtained from 35 strains of lactic streptococci were examined by gel electrophoresis. A mathematical analysis of the densitometer scans of the gels enabled strains to be grouped according to their overall similarity. Strains which were known to be variants of the same parent strain fell into the same group, supporting the validity of the method. It is suggested that strains which are alike according to their gel electrophoretic patterns when grown under standard conditions have an overall phenotypic similarity and that this indicates a similarity in genotype. The relevance of this to selection of strains of lactic streptococci for cheesemaking is discussed.     

Improved Enumeration of Lactic Acid Streptococci on Elliker Agar Containing Phosphate

Enumeration of lactic acid streptococci from dairy starter cultures on Elliker agar was improved by the addition of 0.4% (wt/vol) diammonium phosphate. Agar plate counts were up to 7.75 times greater due to improved buffering by the phosphate salt.