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L L McKay

Publications and source records attributed to L L McKay.

At least 73 records · Page 4Linked to original sources
PubMed1983

Functional properties of plasmids in lactic streptococci.

Plasmid biology has become an important area of investigation in dairy starter cultures since it now appears that some properties, vital for successful milk fermentations, are coded by genes located on plasmid DNA. Some of these metabolic properties observed in lactic streptococci have been clearly established as being plasmid-mediated. Examples would be lactose utilization and in Streptococcus lactis subsp. diacetylactis the ability to produce a bacteriocin-like substance. Phenotypic and physical evidence for plasmid linkage has been obtained for other traits such as citrate, sucrose, galactose, glucose, mannose, and xylose utilization, proteinase activity, modification/restriction systems, as well as for nisin production. Further genetic evidence is now needed to confirm plasmid association to these properties. For some characteristics the association with plasmids is highly speculative and is solely based on the phenotypic loss of a metabolic property. In this category would be sensitivity to agglutinins, sensitivity to the lactoperoxidase-thiocyanate-hydrogen peroxide inhibitory system, arginine hydrolysis, and slime production. Other properties which appear plasmid-mediated in lactic streptococci and which will be discussed include inorganic ion resistance, drug resistance, and diplococcin production.

Agglutinins↗
PubMed1983

Plasmid linkage of a bacteriocin-like substance in Streptococcus lactis subsp. diacetylactis strain WM4: transferability to Streptococcus lactis.

Streptococcus lactis subsp. diacetylactis strain WM4 transferred lactose-fermenting and bacteriocin-producing (Bac+) abilities to S. lactis LM2301, a lactose-negative, streptomycin-resistant (Lac- Strr), plasmid-cured derivative of S. lactis C2. Three types of transconjugants were obtained: Lac+ Bac+, Lac+ Bac-, and Lac-Bac+.S. diacetylactis WM4 possessed plasmids of 88, 33, 30, 5.5, 4.8, and 3.8 megadaltons (Mdal). In Lac+ Bac+ transconjugants, lactose-fermenting ability was linked to the 33-Mdal plasmid and bacteriocin-producing ability to the 88-Mdal plasmid. Curing the 33-Mdal plasmid from Lac+ Bac+ transconjugants resulted in loss of lactose-fermenting ability but not bacteriocin-producing ability (Lac- Bac+). These strains retained the 88-Mdal plasmid. Curing of both plasmids resulted in a Lac- Bac- phenotype. The Lac+ Bac- transconjugant phenotype was associated with a recombinant plasmid of 55 or 65 Mdal. When these transconjugants were used as donors in subsequent matings, the frequency of Lac transfer was about 2.0 X 10(-2) per recipient plated, whereas when Lac+ Bac+ transconjugants served as donors, the frequency of Lac transfer was about 2.0 X 10(-5) per recipient plated. Also, Lac- Bac+ transconjugants were found to contain the 88-Mdal plasmid. The data indicate that the ability of WM4 to produce bacteriocin is linked to an 88-Mdal conjugative plasmid and that lactose-fermenting ability resides on a 33-Mdal plasmid.

Conjugation, Genetic↗
PubMed1983

Simple and rapid method for isolating large plasmid DNA from lactic streptococci.

A procedure for the rapid isolation of plasmid DNA larger than 30 megadaltons from lactic streptococci is described. This protocol can be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments. A scaled-down protocol is very useful for rapidly screening the plasmid content of streptococcal strains. With this methodology, previously undetected large plasmids were observed.

DNA, Bacterial↗
PubMed1983

Isolation of a recombination-deficient mutant of Streptococcus lactis ML3.

A recombination-deficient mutant of Streptococcus lactis ML3 designated MMS36 was isolated on the basis of its sensitivity to methyl methanesulfonate. This mutant also displayed sensitivity to UV irradiation. The inability of MMS36 to mediate homologous recombination was demonstrated by transduction of plasmid-linked lactose fermenting ability but not chromosomally mediated streptomycin resistance.

Lactococcus lactis↗
PubMed1982

Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids.

We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.

Base Sequence↗
PubMed1982

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA.

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization.

Journal Article↗
PubMed1982

Distinct galactose phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus lactis.

Lactose-negative (Lac-) mutants were isolated from a variant of Streptococcus lactis C2 in which the lactose plasmid had become integrated into the chromosome. These mutants retained their parental growth characteristics on galactose (Lac- Gal+). This is in contrast to the Lac- variants obtained when the lactose plasmid is lost from S. lactis, which results in a slower growth rate on galactose (Lac- Gal+). The Lac- Gal+ mutants were defective in [14C]thiomethyl-beta-D-galactopyranoside accumulation, suggesting a defect in the lactose phosphoenolpyruvate-dependent phosphotransferase system, but still possessed the ability to form galactose-1-phosphate and galactose-6-phosphate from galactose in a ratio similar to that observed from the parental strain. The Lac- Gald variant formed only galactose-1-phosphate. The results imply that galactose is not translocated via the lactose phosphoenolpyruvate-dependent phosphotransferase system, but rather by a specific galactose phosphoenolpyruvate-dependent phosphotransferase system for which the genetic locus is also found on the lactose plasmid in S. lactis.

Disaccharides↗
PubMed1981

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage.

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types-those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.

Journal Article↗
PubMed1981

Conjugal Transfer of Lactose-Fermenting Ability Among Streptococcus cremoris and Streptococcus lactis Strains.

Streptococcus cremoris C3 was found to transfer lactose-fermenting ability to LM2301, a Streptococcus lactis C2 lactose-negative streptomycin-resistant (Lac Str) derivative which is devoid of plasmid deoxyribonucleic acid (DNA); to LM3302, a Lac erythromycin-resistant (Ery) derivative of S. lactis ML3; and to BC102, an S. cremoris B(1) Lac Ery derivative which is devoid of plasmid DNA. S. cremoris strains R1, EB(7), and Z8 were able to transfer lactose-fermenting ability to LM3302 in solid-surface matings. Transduction and transformation were ruled out as mechanisms of genetic transfer. Chloroform treatment of donor cells prevented the appearance of recombinant clones, indicating that viable cell-to-cell contact was responsible for genetic transfer. Transfer of plasmid DNA was confirmed by agarose gel electrophoresis. Transconjugants recovered from EB(7) and Z8 matings with LM3302 exhibited plasmid sizes not observed in the donor strains. Transconjugants recovered from R1, EB(7), and Z8 matings with LM3302 were able to donate lactose-fermenting ability at a high frequency to LM2301. In S. cremoris R1, EB(7), and Z8 matings with LM2301, streptomycin resistance was transferred from LM2301 to the S. cremoris strains. The results confirm genetic transfer resembling conjugation between S. cremoris and S. lactis strains and present presumptive evidence for plasmid linkage of lactose metabolism in S. cremoris.

Journal Article↗
PubMed1981

Recombinant plasmid associated cell aggregation and high-frequency conjugation of Streptococcus lactis ML3.

Lactose-positive (Lac+) transconjugants resulting from matings between Streptococcus lactic ML3 and S. lactis LM2301 possess a single plasmid of approximately 60 megadaltons (Mdal) which is nearly twice the size of the lactose plasmid of the donor. The majority of these Lac+ transconjugants aggregated in broth and were able to transfer lactose-fermenting ability at a frequency higher than 10(-1) per donor on milk agar plates or in broth. Lac+ transconjugants which did not clump conjugated at a much lower frequency. Lactose-negative derivatives of Lac+ clumping transconjugants did not aggregate in broth and were missing the 60-Mdal plasmid. The ability to aggregates in broth was very unstable. Strains could lose the ability to clump but retain lactose-fermenting ability. The majority of these Lac+ nonclumping derivatives of clumping transconjugants contained a plasmid of approximately 33 Mdal, the size of the lactose plasmid of the original donor ML3. These strains transferred lactose-fermenting ability at a frequency of approximately 10(-6) per donor, resulting in both Lac+ clumping transconjugants which contained a 60-Mdal plasmid and Lac+ nonclumping transconjugants which possessed a 33-Mdal plasmid. Our results suggest that the genes responsible for cell aggregation and high-frequency conjugation are on the segment of deoxyribonucleic acid which recombined with the 33-Mdal lactose plasmid in S. lactis ML3.

Conjugation, Genetic↗
PubMed1980

Conjugal transfer of genetic information in group N streptococci.

Streptococcus lactis strains ML3 and C(2)O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM(4) 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 WM(4), 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 C(2)O 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 WM(4). In S. lactis C2 x 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.

Conjugation, Genetic↗
PubMed1979

Characterization of Plasmid Deoxyribonucleic Acid in Streptococcus lactis subsp. diacetylactis: Evidence for Plasmid-Linked Citrate Utilization.

The use of Streptococcus diacetylactis as a flavor producer in dairy fermentations is dependent upon its ability to produce diacetyl from citrate. Treatment of S. diacetylactis strains 18-16 and DRC1 with acridine orange resulted in the conversion of approximately 2% of the DRC1 population and 20% of the 18-16 population to citrate negative, which is indicative of the involvement of plasmid deoxyribonucleic acid (DNA). Growth in the presence of acridine orange also resulted in the appearance of 2% lactose-negative derivatives in S. diacetylactis 18-16 and 99% lactose-defective, proteinase-negative derivatives in S. diacetylactis DRC1. Cesium chloride-ethidium bromide equilibrium density gradients of cleared lysate material from each strain revealed the presence of covalently closed circular DNA. Samples of this covalently closed circular DNA were subjected to agarose gel electrophoresis to determine the plasmid composition of each strain. S. diacetylactis 18-16 was found to possess six plasmids, of approximately 41, 28, 6.4, 5.5, 3.4, and 3.0 megadaltons (Mdal). S. diacetylactis DRC1 contained six plasmids, of approximately 41, 31, 18, 5.5, 4.5, and 3.7 Mdal. Variants of S. diacetylactis 18-16 which failed to produce acetoin plus diacetyl from citrate (citrate negative) were missing a 5.5-Mdal plasmid. Lactose-negative mutants of the same strain were devoid of a 41-Mdal plasmid. Lactose-defective, proteinase-negative mutants of S. diacetylactis DRC1 were missing a 31-Mdal plasmid. The citrate-negative mutants of S. diacetylactis DRC1 isolated in this study did not possess a 5.5-Mdal plasmid. Thus, we have evidence that there is a correlation between the ability to utilize citrate and the presence of a 5.5-Mdal plasmid. A relationship was also noted between lactose fermentation and proteinase activity and plasmid DNA in S. diacetylactis.

Journal Article↗
PubMed1979

Genetic Evidence for Plasmid-Linked Lactose Metabolism in Streptococcus lactis subsp. diacetylactis.

It has been previously observed that loss of plasmid pGK4101 occurred concomitantly with loss of lactose-fermenting ability in Streptococcus lactis subsp. diacetylactis 18-16. Transfer of this 41-megadalton plasmid to LM0230, a lactosenegative (Lac) strain of S. lactis, required cell-to-cell contact and resulted in a conversion of LM0230 to the Lac phenotype. This confirms the linkage of lactose-fermenting ability to the 41-megadalton plasmid in S. lactis subsp. diacetylactis and, in addition, demonstrates transfer by a process resembling conjugation in the group N streptococci.

Journal Article↗
PubMed1979

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML(3), and M18.

Lactose- and proteinase-negative (Lac Prt) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac Prt mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.

Journal Article↗
PubMed1978

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.

Bacteriolysis↗
PubMed1978

Stabilization of Lactose Metabolism in Streptococcus lactis C2.

The integration of the lactose plasmid from lactic streptococci into the host chromosome could stabilize this trait for dairy fermentations. Sixty lactose-positive (Lac) transductants of lactose- and proteinase-negative (Lac Prt) LM0220 were induced for temperature phage by UV irradiation or mitomycin C. Four of the transductants, designated KB18, KB21, KB54, and KB58, yielded lysates demonstrating less than one Lac transductant per 0.2 ml of phage lysate. Successive transferring in the presence of acriflavine did not yield Lac segregants from KB18, KB21, KB54, or KB58, whereas Streptococcus lactis C2 (parent culture) and three other Lac transductants showed 12 to 88% conversion from Lac to Lac within 6 to 10 repetitive transfers. When grown in continuous culture, KB21 did not show any Lac variants in 168 h, while S. lactis C2 had 96% conversion from Lac to Lac in 144 h. Agarose gel electrophoresis of plasmid DNA isolated from KB18, KB21, KB54, and KB58 revealed that the lactose plasmid, pLM2103, normally present in Lac transductants, was missing. This suggested integration of the transferred lactose plasmid into the chromosome. In contrast to phage lysates induced from S. lactis C2, which exhibited an exponential decrease in the number of Lac transductants after exposure to small doses of UV irradiation, the transduction frequency for lactose metabolism was stimulated by UV irradiation of lysates from KB58. The latter indicated chromosomal linkage for lac and that integration of the lactose genes plasmid into the chromosome had occurred.

Journal Article↗
PubMed1978

Isolation and characterization of plasmid DNA in Streptococcus cremoris.

Nine industrially important strains of Streptococcus cremoris (HP, AM(2), ML(1), WC, C(3), R(1), E(8), KH, and Wg(2)) were shown to possess a diversity of plasmid molecules. Molecular weights of plasmids were determined from their relative mobilities after agarose gel electrophoresis and via electron microscopy. To illustrate the varied plasmid sizes, strain HP contained plasmids of 26, 18, 8.5, 3.3, and 2 megadaltons (Mdal); strain ML(1) contained plasmids of 29, 18, 9, 4, 2.2, and 1.8 Mdal; and strain AM(2) had plasmids of 42, 27, 16, and 8.4 Mdal. The numbers of plasmids observed in the other strains were 6, 5, 5, 7, 5, and 4 for C(3), E(8), KH, R(1), WC, and Wg(2), respectively. A spontaneous proteinase-negative (Prt(-)) mutant of HP was missing the 8.5-Mdal plasmid, which suggests that in this strain proteinase activity could be linked to this particular plasmid. A lactose-negative (Lac(-)) Prt(-) mutant of ML(1) lacked the 2.2-Mdal plasmid. Under the conditions employed, antibiotic sensitivity and heavy-metal susceptibility did not correlate with the missing plasmid in Prt(-) HP or in the Lac(-) Prt(-) ML(1). Curing experiments with AM(2), using acridine dyes and elevated temperatures, did not yield Lac(-) variants. AM(2) was also cultured at high dilution rates in a chemostat for 168 h by using a buffered milk or lactic broth at 18 or 32 degrees C with no selection of Lac(-) derivatives. The inability to obtain Lac(-) variants under conditions known to facilitate plasmid elimination suggests that lactose metabolism is not plasmid-mediated in AM(2).

Anti-Bacterial Agents↗