Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA.

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Restriction and modification activities from Streptococcus lactis ME2 are encoded by a self-transmissible plasmid, pTN20, that forms cointegrates during mobilization of lactose-fermenting ability.

A self-transmissible (Tra+) plasmid encoding determinants for restriction and modification activities (R+/M+) from Streptococcus lactis ME2 was isolated and characterized. The 28-kilobase (kb) plasmid (pTN20) was detected in lactose-fermenting (Lac+) transconjugants generated from matings between S. lactis N1, and ME2 variant, and a plasmid-free recipient, S. lactis LM2301. The plaquing efficiencies of prolate- and small isometric-headed phages were reduced on transconjugants containing either pTN20 (R+/M+ Tra+) or 100-kb plasmids encoding Lac+, R+/M+, and Tra+. Lac+ transconjugants which harbored pTR1040 (Lac+) and pTN20 (R+/M+) were phenotypically R-/M- and transferred Lac+ at low frequency in subsequent matings to give rise to 100-kb R+/M+ plasmids. R+/M+ activities and high-frequency conjugal transfer ability were detected in Lac+ transconjugants that contained pTR1041 (Lac+) and pTN20 (R+/M+). No 100-kb R+/M+ plasmids were recovered after these matings, suggesting that pTR1041 was mobilized by pTN20 through a process that resembled plasmid donation. pTR1041 was identical to pTR1040 but contained an additional 3.3-kb DNA fragment. These data suggested that phenotypic expression of R+/M+ and Tra+ is affected by coresident Lac+ plasmids. Restriction enzyme analysis and hybridization reactions demonstrated that the 100-kb R+/M+ plasmid was formed by a cointegration event between pTR1040 (Lac+) and pTN20 (R+/M+ Tra+) during conjugal transfer via a conductive-type process. This is the first report that defines self-transmissible restriction and modification plasmids in the lactic streptococci.     

Study of the transmissibility of multiple drug resistance and the capacity to ferment lactose in a clinical strain of Kl. pneumoniae]

The donor properties of K. pneumoniae PI 220 with multiple drug resistance were studied. It was shown that the above strain carried 2 plasmids, i.e. R-plasmid pPI 220 controling resistance to ampicillin, streptomycin, tetracycline, chloramphenicol, kanamycin and sulphanylamides and plasmid pPI 221 controlling lactose fermentation. Both plasmids can be transfered on conjugation to strain E. coli P678 at a temperture of 28 degrees C at a rate of 10(-5) for pPI 220 and 10(-4) for pPI 221. The drug resistance controlled by pPI 221 was transfered mainly in a "blocks" simultaneously to 6 drugs. Deletion of plasmid pPI 220 was observed rarely. The donor properties of the strain were defined by the conjugative plasmid pPI 220 controlling the self-transfer and mobilization of plasmid pPI 221 incapable of the self-transfer. E. coli P678 (pPI 220) (PPI 221) acquired the donor properties and transfered both plasmids to E. coli J62 on crossing simultaneously at a rate of 10(-2), as well as to S. typhimurium LT2 and P. rettgeri at a rate of 10(-5). In all the recipient strains studied the transfered plasmids were unstable and segregated also simultaneously at a rate being the highest for P. retgari PI 230. The clones with stable preservation of the plasmids could be obtained by selection.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages.

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Plasmids in Streptococcus lactis: evidence that lactose metabolism and proteinase activity are plasmid linked.

Populations of lactose positive (Lac+) and proteinase positive (Prt+) cells from Streptococcus lactis M18, C10, and ML3 grown at 39 degrees C gave rise to increasing proportions of Lac- Prt- clones. The deficiencies did not appear until after a number of generations at the elevated temperature, and the rate depended on the strain.Lac- Prt+ and Lac+ Prt- mutants were isolated after treatment with ethidium bromide. Plasmid deoxyribonucleic acid was isolated by cesium chloride-ethidium bromide equilibrium density gradient centrifugation from the parent cultures as well as from their Lac- Prt-, Lac- Prt+, and Lac+ Prt- mutants. Five distinct plasmid sizes of approximate molecular weights of 2,4, 8, 21, and 27 million were found in S. lactis C10, whereas the Lac- Prt- derivative lacked the 8- and 21-million-dalton plasmids, but the 8-million-dalton plasmid was present in the Lac-Att mutant. In S. lactis m18 five plasmids possessing molecular weights of about 2, 4, 10, 18 and 27 million were observed. The 10- and 18-million-dalton plasmids were not detected in the Lac- Prt- mutants, whereas the Lac- Prt+ derivative lacked only the 18-million-dalton plasmid and the Lac+ Prt- mutant lacked only the 10-million-dalton plasmid. In S. lactis ML3 five distinct plasmids, with approximate molecular weights of 2, 4, 8, 22, and 30 million, were present. The 8- and 22-million-dalton plasmids were not detected in the Lac- Prt- derivative, but the 8-million-dalton plasmid was present in the Lac- Prt+ mutant. The evidence suggests that lactose-fermenting ability and proteinase activity in these organisms are mediated through two distinct plasmids having molecular weights of 8 x 10(6) to 10 x 10(6) for proteinase activity and 18 x 10(6) to 22 x 10(6) for lactose metabolism.     

Effect of conjugative R plasmids on the virulence of antibiotic-sensitive Salmonella strains and their streptomycin-resistant mutants

The pathogenicity level of antibiotic sensitive and streptomycin resistant strains of S. typhimurium, S. paratyphi B and S. kottbus changed under the effect of identical R plasmids more frequently in contrary directions. The conjugative plasmids of antibiotic resistance widened the ranges of the virulence changes in the Salmonella serovars for albino mice. It was found that 7 out of 8 plasmids studied significantly decreased and increased the virulence of the antibiotic sensitive Salmonella strains. As a rule, R plasmids of various origin decreased the virulence of all the tested streptomycin chromosome resistant causative agents of salmonellosis.     

Characteristics of new pKMR-plasmids detected in natural strains of Enterobacteriaceae

pKMR-plasmids controlling the antibiotic resistance and adhesive properties were isolated from clinical strains of E. coli O26 and O124, and Sh. sonnei. Two of them, i.e. pKMR 207 and pKMR 208 were conjugative. On conjugation they jointly transferred the features of the antibiotic resistance and capacity for production of the colonization antigen. The studies on transformation of E. coli K 12 802 with the plasmid DNA of E. coli O124 showed that the antibiotic resistance and colonization properties in E. coli O124 were controlled by the nonconjugative plasmid pKMR 209. It was found that plasmids pKMR 207 and pKMR 208 had the fi(-)-phenotype. None of the plasmids allotted the host cells sensitivity to the donor specific phages of the incompatibility groups F, N, P, W, and I. Probably, the plasmids did not belong to these incompatibility groups. When the cells of E. coli K 12 802 were transformed with the plasmid DNA of the wild strain to the hemolytic strain of S. typhimurium with multiple antibiotic resistance, 3 pKMR 210 plasmids with different markers of the antibiotic resistance were detected in the transformants. One of the plasmids controlled both the drug resistance and the capacity for production of hemolysin. The ability of the detected pKMR plasmids to inhibit fertility and relation to the donor specific phages was studied.     

Inorganic salts resistance associated with a lactose-fermenting plasmid in Streptococcus lactis.

Present evidence indicates that lactose metabolism in group N streptococci is linked to plasmid deoxyribonucleic acid. Lactose-positive (Lac+) Streptococcus lactis and lactose-negative (Lac-) derivatives were examined for their resistance to various inorganic ions. Lac+ S. lactis strains ML3, M18, and C2 were found more resistant to arsenate (7.5- to 60.2-fold), arsenite (2.25- to 3.0-fold), and chromate (6.6- to 9.4-fold), but more sensitive to copper (10.0- to 13.3-fold) than their Lac- derivatives. These results suggested that genetic information for resistance and/or sensitivity to these ions resides on the "lactose plasmid." Kinetics of ultraviolet irradiation inactivation of transducing ability for lactose metabolism and arsenate resistance confirmed the plasmid location of the two markers. Lac+ transductants from S. lactis C2 received genetic determinants for resistance to arsenate, arsenite, and chromate but not for copper sensitivity. In this case, resistance markers were lost when the transductants became Lac- but the derivatives remained copper resistant. The resistant markers for arsenate and arsenite could not be identified as separate genetic loci, but chromate resistance and copper sensitivity markers were found to be independent genetic loci. The "lactose plasmid" from S. lactis C10 possessed the genetic loci for arsenate and arsenite resistance but not for chromate resistance or copper sensitivity.     

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.     

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.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

Study of the inactivation of antibiotics by bacterial colonies

A procedure of bacteria application to disks from the colonies was used for determining antibiotic inactivation in the disks by the bacteria colonies after the disk direct contact with the colonies. Changes in the antibiotic activity in the disks were registered after incubation at 37 degrees C for 2 hours. It was shown that ampicillin resistant strains of E. coli K12 carrying R plasmids and strains of S. typhimurium and S. aureus inactivated the antibiotics in the disks and their population were homogenous in this respect. It is advisable to use the procedure in assaying drug resistance of bacterial populations.     

Transforming activity of R- and Lac-plasmids of clinical strains of Gram-negative bacteria]

The isolated plasmid DNA of clinical strains of Gram-negative bacteria were shown to have transforming activity when E. coli strain 0600 and S. typhimurium strain LT-2 were used as recipients. The frequency of transformation depended on the recipient strain and the character of the plasmids. The presence of deletion mutants was revealed among the transformants. Such mutants occurred with varying frequency, most often in S. typhimurium strain LT-20; the reason for this phenomenon is at present under discussion. The transformation of plasmids controlling lactose splitting and their conjugation transfer into recipient S. typhimurium strain LT-2 is possible only under condition of using recipient (R+). The possibility of the formation of the cointegrate (R and lac plasmids) in recipient S. typhimurium strain LT-2 is discussed.     

Influence of lipopolysaccharide and protein in the cell envelope on recipient capacity in conjugation of Salmonella typhimurium.

In crosses of Salmonella typhimurium FfinP301 lac+ to F- strains of S. typhimurium in broth, recipient strains which were rough mutants affected in the outer core region of the lipopolysaccharide gave an average of 1.4 Lac+ transconjugants per donor cell and over 50% of the donor and recipient cells in mating aggregates, whereas smooth recipient strains gave 0.08 Lac+ transconjugants and few cells in mating aggregates. Strains with mutations affecting the inner core of the lipopolysaccharide were usually poor recipients. When cells were mated on Millipore membrane filters, both smooth and rough strains gave ca. 1.0 Lac+ transconjugants per donor cell. Plasmids in Inc groups FI, FII, M, J, and I beta gave more transconjugants with rough than smooth strains, but there were no difference in crosses with plasmids in Inc groups T, L, P, N, and W. Strains with mutations in the ompA gene (deficient in Omp Ap = 33K = II* = conjugation protein) yielded only 0.02 Lac+ transconjugants per donor cell and few cells in mating aggregates. There was no indication of a deficiency of Omp Ap in smooth strains compared with rough strains. Reduced fertility of smooth recipients may occur because the O side chains of the lipopolysaccharide shield the recipient and reduce the frequency of stabilization of mating aggregates. However, gradient-of-transmission experiments indicated that once these mating aggregates are stabilized, they are equally stable in both smooth and rough recipients. Fertility was high in crosses of S. typhimurium Flac+ to Escherichia coli K-12 F- (0.75 Lac+ transconjugants per donor cell; over 50% of the cells in mating aggregates). In crosses of E. coli K-12 Flac+ to S. typhimurium smooth F-, ca. 10(-5) Lac+ transconjugants per donor cell were obtained; in crosses to rough recipient strains, fertility was increased 14-fold, and when the recipient was defective in the SA and LT host restriction systems, fertility was increased in additional 100-fold. Thus, both the lipopolysaccharide and the protein in the cell envelope of S. typhimurium were shown to be important in the recipient function in F-mediated conjugation.     

Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli.

One of the most studied examples of adaptive mutation is a strain of Escherichia coli, FC40, that cannot utilize lactose (Lac-) but that readily reverts to lactose utilization (Lac+) when lactose is its sole carbon source. Adaptive reversion to Lac+ occurs at a high rate when the Lac- allele is on an F' episome and conjugal functions are expressed. It was previously shown that nonselected mutations on the chromosome did not appear in the Lac- population while episomal Lac+ mutations accumulated, but it remained possible that nonselected mutations might occur on the episome. To investigate this possibility, a second mutational target was created on the Lac- episome by mutation of a Tn1O element, which encodes tetracycline resistance (Tetr), to tetracycline sensitivity (Tets). Reversion rates to Tetr during normal growth and during lactose selection were measured. The results show that nonselected Tetr mutations do accumulate in Lac- cells when those cells are under selection to become Lac+. Thus, reversion to Lac+ in FC40 does not appear to be adaptive in the narrow sense of the word. In addition, the results suggest that during lactose selection, both Lac+ and Tetr mutations are created or preserved by the same recombination-dependent mechanism.     

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.     

Characterization of Lac+ Transductants of Streptococcus lactis

A phage-mediated transducing system was used in studying certain physiological characteristics of S. lactis C2 wild type, lactose-negative mutants, and lactose-positive transductants. Lac(-) mutants, obtained by acriflavine treatment of the wild type, were similar to the wild type in all characteristics tested except they lacked beta-D-phosphogalactoside galactohydrolase (beta-Pgal) and could not transport [(14)C]lactose; they also had approximately 10% of the proteolytic ability than wild-type cells. The lactose-fermenting characteristic was transduced from the wild type to Lac(-) mutants. The Lac(+) transductants obtained were similar to the wild-type parent with respect to lactose fermentation and level of beta-Pgal activity (0.186 U of protein per mg). These transductants, however, had not regained full proteolytic ability and were similar to the Lac(-) mutant in this respect. Lactic acid production of the transductants in milk was approximately two-thirds that of the wild type. Data suggest that both the lactose-fermenting and proteolytic characters are carried on extrachromasomal particles (plasmids).     

Effect of Ca2+ ions on plasmid transformation of Streptococcus lactis protoplasts

The effects of Mg2+ and Ca2+ ions on the efficiency of the plasmid transformation of lysozyme-treated Streptococcus lactis protoplasts were compared. A 33-megadalton plasmid, pLP712, coding for lactose fermentation and a 6.5-megadalton plasmid, pGB301, coding for erythromycin and chloramphenicol resistance were used as model plasmids, and S. lactis MG1614 was the recipient. Replacing Mg2+ with Ca2+ in the transformation buffer was found to increase transformant frequency more than 10-fold with both plasmids.