Mechanisms of lactose utilization by lactic acid streptococci: enzymatic and genetic analyses

The apparent instability of beta-galactosidase in toluene-treated cells or cell-free extracts of lactic streptococci is explained by the fact that these organisms do not contain the expected enzyme. Instead, various strains of Streptococcus lactis, S. cremoris, and S. diacetilactis were shown to hydrolyze o-nitrophenyl-beta-d-galactoside-6-phosphate (ONPG-6-P), indicating the presence of a different enzyme. In addition, lactose metabolism in S. lactis C(2)F was found to involve enzyme I (EI), enzyme II (EII), factor III (FIII), and a heat-stable protein (HPr) of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system analogous to that of Staphylococcus aureus. Mutants of S. lactis C(2)F, defective in lactose metabolism, possessed the phenotype lac(-) gal(-). These strains were unable to accumulate (14)C-thiomethyl-beta-d-galactoside, to hydrolyze ONPG, or to utilize lactose when grown in lactose or galactose broth. In addition, these mutants contained EI and HPr, but lacked EII, FIII, and the ability to hydrolyze ONPG-6-P. This suggested that the defect was in the phosphorylation step. Lactose-negative mutants of S. lactis 7962, a strain containing beta-galactosidase, could be separated into several classes, which indicated that this organism is not dependent upon the PEP-phosphotransferase system for lactose metabolism.     

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).     

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.     

Involvement of phosphoenolpyruvate in lactose utilization by group N streptococci

The effect of sodium fluoride on lactose metabolism and o-nitrophenyl-beta-d-galactopyranoside (ONPG) hydrolysis by Streptococcus lactis strains 7962 and C(2)F suggested that different mechanisms of lactose utilization existed in the two strains. Sodium fluoride prevented lactose utilization and ONPG hydrolysis by whole cells of S. lactis C(2)F but had no effect on S. lactis 7962. Although hydrolysis of ONPG by toluene-treated cells of S. lactis 7962 occurred without addition of phospho-enolpyruvate (PEP), toluene-treated cells of S. lactis C(2)F required the presence of this cofactor. Concentrated cell extracts of S. lactis C(2)F hydrolyzed ONPG; this hydrolysis was inhibited by NaF, but the addition of PEP, in the presence of NaF, restored maximal activity. Addition of acetyl-phosphate, carbamyl-phosphate, adenosine-5'-triphosphate, guanosine-5'-triphosphate, or uridine-5'-triphosphate did not stimulate activity. The presence of cofactors did not stimulate and NaF did not inhibit the hydrolysis in extracts of S. lactis 7962. To confirm the operation of two mechanisms, S. lactis 7962 was shown to hydrolyze lactose to glucose and galactose, whereas S. lactis C(2)F was unable to split the disaccharide. In addition, whole cells of S. lactis C(2)F rapidly accumulated a phosphorylated derivative of thiomethyl-beta-d-galactoside (TMG) which behaved chromatographically and electrophoretically like TMG-PO(4). Unexpectedly, S. lactis 7962 also accumulated a TMG derivative, although the rate was extremely low. These data indicate that different mechanisms of lactose utilization exist in the two strains, with a phosphorylation step dependent on PEP involved in S. lactis C(2)F.     

Simultaneous Loss of Proteinase- and Lactose-Utilizing Enzyme Activities in Streptococcus lactis and Reversal of Loss by Transduction

During studies on spontaneous loss of lactose metabolism in Streptococcus lactis C2, it was found that the lactose-negative (lac(-)) mutants were also proteinase negative (prt(-)). This pleiotropic effect was observed in S. diacetilactis 18-16, but not in S. cremoris B1. The lac(-)prt(-) mutants from S. lactis C2 were able to grow in milk, but no pH change or measurable protein breakdown occurred. When the milk was supplemented with glucose, a slow decline in pH occurred. Addition of a protein hydrolysate to milk did not stimulate acid production. When both supplements were added to milk, normal growth and pH change were obtained. When the lac(-)prt(-) mutant of S. lactis C2 was transduced with the temperate phage from the lac(+)prt(+) parent culture, approximately equal numbers of lac(+)prt(-) and lac(+)prt(+) transductants were obtained. When the spontaneous lac(+)prt(-) strain of S. lactis C2 was converted to a lac(-)prt(-) derivative and transduced, similar results were obtained. The co-transduction of the lactose and proteinase markers suggest they are closely associated. The findings indicate that the transducing phage from S. lactis C2 can be used to examine the causes of instability in both the lactose and proteinase enzyme systems of this organism.     

Characterization of lactose-fermenting revertants from lactose-negative Streptococcus lactis C2 mutants

Partial lactose-fermenting revertants from lactose-negative (lac(-)) mutants of Streptococcus lactis C2 appeared on a lawn of lac(-) cells after 3 to 5 days of incubation at 25 C. The revertants grew slowly on lactose with a growth response similar to that for cryptic cells. In contrast to lac(+)S. lactis C2, the revertants were defective in the accumulation of [(14)C]thiomethyl-beta-d-galactoside, indicating that they were devoid of a transport system. Hydrolysis of o-nitrophenyl-beta-d-galactoside-6-phosphate by toluene-treated cells confirmed the presence of phospho-beta-d-galactosidase (P-beta-gal) in the revertant. However, this enzyme was induced only when the cells were grown in the presence of lactose; galactose was not an inducer. In lac(+)S. lactis C2, enzyme induction occurred in lactose- or galactose-grown cells. The revertants were defective in EII-lactose and FIII-lactose of the phosphoenolpyruvate-dependent phosphotransferase system. Galactokinase activity was detected in cell extracts of lac(+)S. lactis C2, but the activity was 9 to 13 times higher in extracts from the revertant and lac(-), respectively. This suggested that the lac(-) and the revertants use the Leloir pathway for galactose metabolism and that galactose-1-phosphate rather than galactose-6-phosphate was being formed. This may explain why lactose, but not galactose, induced P-beta-gal in the revertants. Because the revertant was unable to form galactose-6-phosphate, induction could not occur. This compound would be formed on hydrolysis of lactose phosphate. The data also indicate that galactose-6-phosphate may serve not only as an inducer of the lactose genes in S. lactis C2, but also as a repressor of the Leloir pathway for galactose metabolism.     

Selected properties of lactose-fermenting and non-fermenting Salmonella agona strains isolated from specimens from hospitalized infants

The aim of this study was to compare some of the properties of 28 lactose-positive and 28 lactose-negative Salmonella agona strains isolated from faeces of infants hospitalized in the same hospital. Some of biochemical properties, sensitivity to 14 antibiotics and chemotherapeutic agents and sensitivity to bacteriophages used for typing of this Salmonella genus were tested. Results of biochemical examinations revealed that lactose-fermenting strains retain the remaining of Salmonella of subspecies I. Two biochemical features are of particular importance: the ability to ferment lactose on all lactose containing media and a lack of the ability to produce H2S on Kligler medium. These two features differentiate lactose-fermenting strains of Salmonella from non-lactose fermenting ones. Antibiotic sensitivity pattern differed between lactose-positive and lactose-negative strains. Lactose-positive strains showed higher degree of resistance than lactose-negative strains. The differences in resistance were seen in the case of chloramphenicol, doxycycline, gentamicin and tetracycline. Both lactose-positive and lactose-negative strains were sensitive to colistin, neomycin, nitrofurantoin and nalidixic acid. They were resistant to ampicillin, cloxacillin, rifampicin, streptomycin, sulfatiazol and biseptol. Bacteriophage typing revealed that all lactose-negative strains isolated in this study from clinical samples belonged to the same phage pattern V. Lactose-positive strains belonged to two phage types VB and XI. Type VB prevailed.     

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.     

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.     

Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities.

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose-phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of [14C]lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution 31P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM. We conclude from our data that phosphorylation of glucose by S. lactis 133 can be mediated by only two mechanisms: (i) via ATP-dependent glucokinase, and (ii) by the phosphoenolpyruvate-dependent mannose-PTS system.     

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway.     

Presence of Lactose Genes and Insertion Sequences in Plasmids of Minor Species of the Genus Lactococcus

The type strains of all known species and biovars of the Lactococcus genus were tested for the presence of plasmids, lactose genes, and insertion sequences cloned from the lactose plasmid of Lactococcus lactis subsp. lactis. Only the biovar xylosus of this subspecies is plasmid free. The lactose plasmid is present only in lactose-positive strains except in Lactococcus plantarum. The distribution of insertion sequences varies within the type strains of the Lactococcus genus.     

Selection of methods of detecting lactose-fermenting Salmonella strains and evaluating their usefulness for diagnostic studies

Salmonella rods of subspecies I, lactose-fermenting were first isolated in Poland in 1980. They were isolated from a plus sample taken from a brain abscess of a child. Next strains were isolated from faeces of newborn and hospitalized children. Growth characteristic of colonies of lactose-fermenting Salmonella strains on selective-differentiating media (Mac Conkey's Levine, SS, So?tys) recommended for inoculation of clinical material resembled Escherichia coli. So far these type of colonies were omitted in diagnostic examinations. Lactose-fermenting variants showed on Bismuth sulfate agar "Difco" (WB) typical for Salmonella growth pattern. They grew on this medium after 48 hr of incubation in a form of black, medium sized colonies, with some metallic brilliance and characteristic blackening of the medium undercolonies. Precise knowledge of biochemical properties of lactose-fermenting Salmonella allows to supplement so far used diagnostic scheme with additional tests permitting differentiation of lactose-fermenting variants of Salmonella from the other members of Enterobacteriaceae family. Taking into consideration biochemical variants in diagnostic procedure i.e. lactose-fermenting Salmonella, allowedns to isolate in the years 1983-1985 lactose-positive strains in 1305 out of 2773 (47%) individuals positive for S. agona. In 1987, 246 persons (28.3%) out of 869 with lactose-fermenting Salmonella of various serotypes were simultaneously infected with lactose-negative variant. Lactose-fermenting strains of Salmonella belonged most frequently to the following genera: S. agona, S. enteritidis, S. oranienburg, S. typhimurium, and S. goldcoast. It was found that the modified diagnostic procedure makes possible the isolation and the identification of lactose-positive varians of Salmonella.     

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.     

Transduction of Lactose Metabolism in Streptococcus lactis C2

Ultraviolet (UV)-induced phage lysates, from lactose-positive (lac(+)) Streptococcus lactis C2, transduced lactose fermenting ability to lac(-) recipient cells of this organism. Although the phage titer could not be determined due to the absence of an appropriate indicator strain, the number of transductants was proportional to the amount of phage lysate added. Treatment of the lysate with deoxyribonuclease had no effect on this conversion, indicating the observed genetic change was not mediated by free deoxyribonucleic acid. When the lac(+) transductants were isolated and exposed to UV irradiation, lysates with higher transducing ability were obtained. The transducing ability of this lysate was about 100-fold higher than that observed in the original lysates. The lac(+) transductants were unstable since lac(-) segregants occurred at high frequency. The phage lysate from S. lactis C2 also transduced maltose and mannose metabolism to the respective negative recipient cells. The results demonstrate the transduction of carbohydrate markers by a streptococcal phage and establish a genetic transfer system in group N streptococci.     

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.     

Transductional evidence for plasmid linkage of lactose metabolism in streptococcus lactis C2.

A lactose-negative (Lac-), proteinase-negative (Prt-) mutant, designated C145 was isolated from Streptococcus lactis C2 after treatment with nitrosoguanidine and ultraviolet irradiation. The mutant appeared to be cured of the prophage(s) present in S. lactis C2 based on non-inducibility by ultraviolet irradiation or mitomycin C. When cleared lysate material from C145 was subjected, to cesium chloride-ethidum bromide (EB) density gradient centrifugation, no plasmid peak was observed, suggesting that C145 was cured of plasmid deoxyribonucleic and (DNA). A histogram showing distribution of contour lengths of circular molecules of DNA from C145, however, revealed the presence of a greatly diminished number of DNA molecules as compared with the parent culture and indicated the absence of the 30 x 10(6) plasmid. Cesium chloride-ethidium bromide gradient profiles from Lac+, Prt- and Lac+ Prt+ transductants of C145 revealed no plasmid peak, but electron microscopy of the fractions normally possessing the satellite band of DNA showed the presence of a new plasmid species having a molecular weight from 20 x 10(6) to 22 x 10(6). This plasmid was lost when the transductants became Lac-. Examination of a plasmid histogram from a spontaneous Lac- Prt- mutants of S. lactis C2 resembled that of C145, with the absence of the 30 x 10(6) plasmid and the presence of the 22 x 10(6) plasmid in Lac+ Prt+ transductants. The results suggest that lactose metabolism is mediated through the 30 x 10(6) plasmid in S. lactis C2 and that the transducing bacteriophage, which is too small to accommodate the entire plasmid, is transferring about two-thirds of the original plasmid through a process termed transductional shortening.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

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.     

Detection of streptococcal mutants presumed to be defective in sugar catabolism.

The tetrazolium method for detection of bacterial mutants defective in sugar catabolism was modified for use with streptococci. The critical factors were (i) the concentration of tetrazolium, which must be titrated to determine the optimum concentration for each species or even strain, and (ii) anaerobic incubation of tetrazolium-containing agar plates. When used with standard mutagenesis protocols, this method yielded lactose-negative mutants of nine streptococcal strains representing six species. A collection of lactose-negative mutants of streptococcus, sanguis Challis was characterized and contained phospho-beta-galactosidase, lactose phosphotransferase, and general phosphotransferase mutants.