Streptococcus Faecium Var Research Articles

Heat-Inactivation of Streptococcus faecium var. casseliflavus in Skim Milk Cultures with Pseudomonas fluorescens

Thermal destruction of Streptococcus faecium var. casseliflavus (SFC) at 57°C in autoclaved skim milk was determined at several pH values, and when skim milk was inoculated with Pseudomonas fluorescens and incubated 3 d at 10°C plus 4 d at room temperature before SFC was added. The pH values between 6.4 and 6.6 had little effect on heat resistance of SFC. At pH 5.6, however, accelerated thermal destruction occurred in sterile skim milk as compared to skim milk at pH 6.5. (D-values were 4.8 min and 10.2 min, respectively). Presence of large populations (log10 bacterial count = 9.8 to 9.9/ml) of P. fluorescens had a protective effect on SFC (D = 7.6 ± .7 min in skim milk preincubated with P. fluorescens and 6.2 min in skim milk without P. fluorescens).

Incidence and relationship of group D streptococci with other indicator organisms in meats.

Raw and processed meats were analyzed for presumptive group D streptococci using KF streptococcus agar. Counts were compared with coliform, presumptive Escherichia coli, and Enterobacteriaceae counts but no meaningful relationships were observed. Results indicated that group D streptococci and E. coli type I were principally contaminants from the packing plant, rather than at retail level. The predominating group D streptococcus in both beef and pork cuts was Streptococcus faecalis, while in processed meat (bologna), the predominating group D streptococci were Streptococcus faecium var. durans and Streptococcus faecium. Streptococcus bovis was not detected among the isolates from any meat samples. Marked differences were noted in numbers of group D streptococci in processed meat from different manufacturers. The results did not support the use of group D streptococci as alternative indicator organisms for meats. However, the association of group D streptococci with packing plant contamination may prove to be of value.

Nuclear magnetic resonance studies on bacterial dihydrofolate reductase containing [methyl-13C]methionine.

(methyl-/sup 13/C)Methionine has been incorporated with high efficiency by Streptococcus faecium var. Durans strain A into dihydrofolate reductase isoenzyme 2. In the /sup 13/C NMR spectrum of the purified enzyme the resonances corresponding to the seven methionine residues are partially resolved into three composite peaks. Denaturation with urea collapses these into a single peak centered at 15.32 ppm, whereas the resonance of free methionine is at 15.04 ppm. Spectra of the free enzyme, its complex with methotrexate, and its complex with methotrexate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) have been simulated, permitting more accurate estimates of line widths and nuclear Overhauser enhancement (NOE) values. These, together with the T/sub 1/ values, cannot be explained solely by the effects of macromolecular tumbling and very rapid rotation of the methionine methyl group about its axis. A model assuming, in addition, the occurrence of free rotation about the methionine CH/sub 2/-S bond is also unsatisfactory, and it is concluded that internal rotation about the CH/sub 2/-S bond is highly restricted so that the methyl group oscillates through a relatively narrow angular range. Complex formation with NADPH produced rather small changes in the spectrum of the native enzyme, probably due to conformational transitionsmore » in the enzyme. However, NADP/sup +/ produced several changes,including movement of one resonance downfield by at least 1.7 ppM.« less

Nucleosides. CIX. 2′‐Deoxy‐ψ‐isocytidine, 2 ‐deoxy‐ψ‐uridine, and 2′‐deoxy‐l‐methyl‐ψ‐uridine. Isosteres of deoxycytidine, deoxyuridine and thymidine

Abstract2′‐Deoxy‐ψ‐isocytidine (VIIβ), a 2′‐deoxy analog of antileukemic ψ‐isocytidine and also a C‐nucleoside analog of deoxycytidine, was synthesized from ψ‐uridine by making use of the newly discovered pyrimidine to pyrimidine transformation reaction [J. Chem. Soc., 14, 537 (1977)]. 2′‐Deoxy‐ψ‐uridine (IIβ) and 2′‐deoxy‐l‐methyl‐ψ‐uridine (V), both C‐nucleoside analogs of deoxyuridine and thymidine, were also synthesized.ψ‐Uridine was converted into the 2′‐chloro analogs (I) which was reduced with tributyltin hydride to give an α,β‐mixture of 2′‐deoxy‐ψ‐uridines. The β‐isomer (11β was trimethylsilylated and the product (III) treated with methyl iodide to afford the 1‐methyl derivative (IV). After hydrolytic removal of the trimethylsilyl groups from IV, the thymidine analog (V) was obtained in good yield.A crude mixture of II was converted in good yield into an α,β‐mixture of 1,3‐dimethyl‐2 ‐deoxy‐ψ‐uridines (VI) by treatment with DMF dimethyl acetal in DMF. Treatment of the β‐isomer (VIβ) with guanidine, however, gave the α,β‐mixture of 2 ‐deoxy‐ψ‐isocytidines (VII). The pure β‐isomer (VIIβ) was obtained by thick layer chromatography. The pure α‐isomer (VIIα) was obtained when VIα was treated with guanidine.2 ‐Deoxy‐ψ‐isocytidine (VIIβ) and 2 ‐deoxy‐l‐methyl‐ψ‐uridine (V) exhibited inhibitory activity against P815 cells (ID5 0 1.2 μg./ml. and 4.9 μg./ml., respectively) and the thymidine analog V was found to be active against Streptococcus faecium var. duran. J. Heterocyclic Chem., 14, 1119 (1977)

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Amino acid sequence of peptide CNBr 7 and complete sequence of the protein.

The complete amino acid sequence of the mutant dihydrofolate reductase from Streptococcus faecium var. Durans strain A has been determined by sequence analysis of peptides produced by tryptic, chymotryptic, thermolytic, and mild acid cleavage of the large peptide CNBr 7 and from previously reported studies. The sequence of the S. faecium enzyme is compared to the reported sequence of dihydrofolate reductase from Escherichia coli and the two are shown to contain two domains of substantial homology. One of these domains consists of the NH2-terminal 60 residues and is considered to contribute the dihydrofolate binding site. The second domain probably contains the dinucleotide binding structure. Comparison of the sequences of the dihydrofolate reductases with those of larger dehydrogenases of known structure failed to show any evidence for homology. Considerations of size and predictions of secondary structure also suggest that the second domain in the reductases has no structural similarity to the nucleotide binding site in the larger dehydrogenases. It is concluded that the two reductases are related, although distantly, but that they have evolved from an ancestral protein different from the primitive predecessor of the other oxidoreductases.

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Partial sequence and order of the limited tryptic and cyanogen bromide peptides.

The major form of dihydrofolate reductase from a methotrexate-resistant mutant (strain A) of Streptococcus faecium var. Durans has been purified on a large scale. Amino acid analysis of this form of the enzyme (isoenzyme 2) reveals an absence of cystine or cysteine, and sedimentation studies indicate a molecular weight of 20,800. The NH2-terminal sequence was determined by Edman degradation of the intact protein and the COOH terminus by selective tritiation and by carboxypeptidase treatment. After the action of trypsin on the citraconylated protein, seven of the expected nine peptides were purified from the digest, and after cyanogen bromide treatment of the unmodified protein, all seven of the anticipated peptides were isolated. The amino acid composition of all of these peptides has been established as well as their complete or partial sequences. From the results it was possible to order these peptides within the sequence and to establish the sequence of the NH2-terminal 60 residues and the COOH-terminal 11 residues.

The structure of dihydrofolate reductase. Identification of methionine residues carboxymethylated by iodoacetate with loss of catalytic activity.

Dihydrofolate reductase from the amethopterin-resistant mutant (strain A) of Streptococcus faecium var. Durans was reacted with iodo[14C]acetate according to three procedures; (a) in the absence of an inhibitor, (b) in the presence of aminopterin, and (c) in absence of inhibitor, but after treatment with unlabeled iodoacetate in presence of aminopterin. The first and last procedures resulted in the loss of approximately 90% of the catalytic activity, whereas in the presence of aminopterin essentially no activity was lost. Peptides were produced from all three labeled proteins by tryptic digestion after citraconylation of the lysine residues. From the amino acid compositions and partial amino acid sequences of these peptides the position of all modified methionines in the sequence was determined. The extent of labeling at each methionine, in enzyme labeled in the different procedures, indicated that methionines 28 and 50 may be at the binding site for inhibitors and that residue 50 is less accessible to iodoacetate than is residue 28. It is likely that carboxymethylation of residue 28 is responsible for the loss of enzyme activity.

The structure of dihydrofolate reductase. I. Inactivation of bacterial dihydrofolate reductase concomitant with modification of a methionine residue at the active site.

Carboxymethylation by iodoacetate of dihydrofolate reductase from the amethopterin-resistant mutant Streptococcus faecium var. Durans strain A leads to a loss of enzymic activity. Amino acid analysis showed that methionine is the only amino acid residue significantly affected by iodoacetate under the experimental conditions, and this was confirmed by the use of [1-14-C]iodoacetate and ion exchange chromatography of the products obtained by acid hydrolysis of the modified enzyme. During loss of 90% of the activity a total of about 2 of the 7 methionine residues present in the enzymes are carboxymethylated. Over this range of activity loss the decrease is proportional to the number of methionine residues modified. Fluorescence-quenching experiments demonstrated that dissociation constants for complexes of inhibitors with the carboxymethylated enzyme were 20 to 30 times greater than dissociation constants for corresponding complexes with native enzyme. Similarly, equilibrium dialysis studies showed that dihydrofolate binding to the modified enzyme was decreased 10-fold compared with binding to the native enzyme. These data suggest that iodoacetate modifies one or more methionine residues at the binding site for dihydrofolate and inhibitors. In accordance with this view it was shown that enzyme can be protected from inactivation by the folate analogue aminopterin and to a lesser extent by folate and dehydrofolate. Enzyme carboxymethylated in the presence of aminopterin, and subsequently freed of the latter, was found to bind inhibitors and dihydrofolate as tightly as the native enzyme. It is concluded that the loss of enzyme activity is caused by carboxymethylation of at least 1 methionine residue which is at or near the binding site of dihydrofolate.

Metabolic control of synthesis of the different forms of dihydrofolate reductase of the amethopterin-resistant Streptococcus faecium var. durans A k

The metabolic regulation of the synthesis of the folate reductase and the specific dihydrofolate reductase of the amethopterin-resistant bacterium Streptococcus faecium var. durans A k was investigated. Under growth conditions for maximum synthesis, the differential rate increased two fold during exponential growth. Synthesis of both reductases was coordinate and air-sensitive. The interrelated growth requirements of the bacterium contribute to the complexity of regulation. Folate acts as a “positive” inducer of both types of reductase. Purines and thymine or their metabolic derivatives are interdependent in exerting an action which may antagonize the folate effect. These regulator agents apparently have little selective effect on any of the electrophoretically distinct forms of dihydrofolate reductase. Proportionate synthesis of the folate reductase (the major form), of the specific dihydrofolate reductase (the minor form) and of a minor component of folate reductase occurred in the presence and absence of the exogenous regulators. Amethopterin appears to act as a protective agent of reductase-active protein synthesized during exponential growth.

Enterococci in insects.

Enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 10(3) to 3 x 10(7)/g of insect. Insects were taken only from nonurban, wild, and cultivated fields and woods. In species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. Enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their role as overwintering agents. They were generally present in species feeding on nectar, succulent plant parts, and on and ir forest litter, but not from insects feeding on less succulent leaves and stems. Streptococcus faecalis was recovered from 32%, Streptococcus faecium from 22.4%, and Streptococcus faecium var. casseliflavus from 43.5% of members of the 37 taxa of insects. S. faecalis and S. faecium var. casseliflavus exhibit a high percent of conformity to the properties published for them. The heterogeneity in properties of S. faecium is similar to that found for the species taken from plants. Many fail to grow in broth at 45 C or in broth containing 6.5% NaCl; 50% of the cultures ferment both melezitose and melibiose, and a few ferment neither sugar. The remainder ferment melibiose only. Failure to reduce methylene blue in milk by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, following the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution.

Enterococci in Insects

Enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 10(3) to 3 x 10(7)/g of insect. Insects were taken only from nonurban, wild, and cultivated fields and woods. In species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. Enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their role as overwintering agents. They were generally present in species feeding on nectar, succulent plant parts, and on and ir forest litter, but not from insects feeding on less succulent leaves and stems. Streptococcus faecalis was recovered from 32%, Streptococcus faecium from 22.4%, and Streptococcus faecium var. casseliflavus from 43.5% of members of the 37 taxa of insects. S. faecalis and S. faecium var. casseliflavus exhibit a high percent of conformity to the properties published for them. The heterogeneity in properties of S. faecium is similar to that found for the species taken from plants. Many fail to grow in broth at 45 C or in broth containing 6.5% NaCl; 50% of the cultures ferment both melezitose and melibiose, and a few ferment neither sugar. The remainder ferment melibiose only. Failure to reduce methylene blue in milk by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, following the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution.

Induction of the dihydrofolate reductase of Streptococcus faecium var. durans by folic and by some of its derivatives

For growth in a defined medium, Streptococcus faecium var. durans requires either folate or some of its derivatives, or a natural purine together with thymine. When folate at 2.3·10 −8–2.3·10 −10 M, was added to S. faecium cells growing in the presence of purines and thymine, a concentration dependent increase in dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP exodoreductase, EC 1.5.1.3) activity occurred. This increase was detectable in cell extracts within 30 min, and reached a maximal level of 6–10 times that of control in approx. 5 h. Dihydrofolate, tetrahydrofolate and folinic acid caused a similar increase in reductase activity. Chloramphenicol or actinomycin D, when added to growing cultures simultaneously with folate, prevented any increase in reductase activity. Furthermore, titration of the reductase with methotrexate showed that the increase in enzyme activity, which occurred in the presence of the cofactors, was accompanied by a parallel increase in the number of enzyme sites. Taken together, these findings indicate that the observed increase in reductase activity was due to new protein synthesis. In the absence of the cofactors no increase in enzyme activity occurred during the entire growth cycle of the culture. Methotrexate itself did not induce the dihydrofolate reductase. In the presence of this analog, folate was ineffective as an inducer, whereas its reduced derivatives brought about some increase in enzyme activity. When cells containing high levels of the induced enzyme were transferred from the folate-containing to the folate-free medium, the enzyme activity decreased slowly. Three passages in the folate-free medium were required before the constitutive enzyme level was restored. The progressive decrease in reductase activity which occurred during these passages, was paralleled by the increasing sensitivity of the subcultures to inhibition of their growth by methotrexate. By affecting the level of dihydrofolate reductase, folate and its derivatives can, thus, regulate the extent of their activation, and thereby, the extent of their cofactor function.

Dihydrofolate Reductase of Streptococcus faecium: II. PURIFICATION AND SOME PROPERTIES OF TWO DIHYDROFOLATE REDUCTASES FROM THE AMETHOPTERIN-RESISTANT MUTANT, STREPTOCOCCUS FAECIUM VAR. DURANS STRAIN A

Abstract From a single amethopterin-resistant organism, Streptococcus faecium var. durans strain A, two different dihydrofolate reductases have been obtained as essentially homogeneous proteins in good yield. One of the reductases has a similar substrate specificity and turnover number (about 8000 moles per min per mole of enzyme) to the single reductase found in the amethopterin-sensitive strain of S. faecium var. durans, ATCC 8043, and has therefore been designated type. The other enzyme, which is distinguished by its ability to catalyze the reduction of folate, in addition to dihydrofolate, and by its lower turnover number (about 900 with dihydrofolate), has been designated type. Since the wild type and mutant type reductases have sedimentation constants (s20,buffer) of 2.58 S and 2.04 S, respectively, they are probably significantly different in molecular weight. Each exhibits a single pH optimum at pH 5.8 and is inactivated by urea. Neither is affected by methylmercuric salts but the wild type reductase is inactivated by phenyl-mercuric acetate and p-mercuribenzoate. Monovalent cations increase the activity of the mutant type reductase but decrease that of the wild type reductase. It is suggested that the amethopterin resistance in vivo of strain A depends at least partly on the folate reductase activity of the mutant type reductase.

Streptococcus faecium var. casselifavus, nov. var.

Streptococcus faecium var. casseliflavus is a gram-positive, spherical cell. The cells occur chiefly as pairs within chains and elongate to ogive-shaped cells during growth. Growth is good on 5% bile salts-agar and in broth at 10 C, and in broth adjusted to pH 9.6 or containing 6.5% NaCl, but many strains fail to grow at 45 C. Litmus is reduced rapidly prior to formation of an acid curd. Few strains release ammonia from arginine or serine. The organism is not proteolytic and does not produce H(2)S or acetylmethylcarbinol, reduce nitrate, decarboxylate tyrosine, or produce slime on sucrose-agar. Most strains survive heating to 60 C for 30 min. It produces gray colonies on potassium tellurite agar, reduces 2,3,5-triphenyltetrazolium-HCl to a pink color, and ferments cellobiose, dextrin, maltose, mannose, and sorbitol, thus resembling S. faecalis. Like S. faecium, it produces peroxidase but not catalase on heated blood media, dissimilates malate, and ferments arabinose, melibiose, and salicin, but not melezitose. Like both species, it ferments dextrose, galactose, lactose, mannitol, sucrose, trehalose, and citrate. Properties peculiar to the variant include the high pH limiting initiation and termination of growth; the fermentation of alpha-methyl-d-glucoside, raffinose, and xylose; motility; and growth without blue button formation in ethyl violet broth. The water-soluble, pale lemon-yellow pigment is released into the aqueous phase only after the cell envelope is altered by fat solvents. The bacterium thrives as an epiphyte on plants.

Strain variation within Streptococcus faecium var. durans.

Strain variation within Streptococcus faecium var. durans.