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PubMed2000

Aggregation substance promotes colonic mucosal invasion of Enterococcus faecalis in an ex vivo model.

BACKGROUND: Bacterial translocation through the gastrointestinal tract is the crucial step in the pathogenesis of intraabdominal infections. We assessed whether aggregation substance (AS), a bacterial adhesin and virulence factor of Enterococcus faecalis, promotes bacterial translocation and colonic mucosal invasion in an ex vivo experiment. METHODS: Colonic mucosa of male Wistar rats was placed in a modified Ussing system. The mucosal side of the chamber was filled with a suspension of E. faecalis OG1X:pAM721 (AS-positive) or E. faecalis OG1X (AS-negative). The serosal side was filled with sterile Dulbecco's modified Eagle's medium. For assessment of colonic mucosal invasion the mucosal side was incubated for 2.5 h with a suspension of AS-positive or AS-negative enterococci. After being washed, a solution of gentamicin and penicillin G in tissue culture medium was added on both sides in order to kill extracellular bacteria. Subsequently, the mucosa was removed from the system, washed, lysed with Triton X-100, and homogenized. Viable intramural bacteria were quantified by plating serial dilutions of the homogenate on Todd-Hewitt broth agar plates. To quantify bacterial translocation samples which were taken at various time points from the serosal side were plated on Todd-Hewitt broth agar plates and colony forming units (CFU) were determined. RESULTS: Invasion of the AS-positive E. faecalis strain OG1X:pAM721 into the colonic mucosa was significantly higher than invasion rates of the AS-negative strain OG1X (2.88 log(10) CFU/ml vs 1.73 log(10) CFU/ml; P = 0.02). However, none of the tested strains was found to translocate from the mucosal to the serosal side within the incubation time of 4 h. CONCLUSIONS: The aggregation substance promotes invasion of E. faecalis into colonic mucosa.

Animals↗

PubMed1992

Cis-acting, orientation-dependent, positive control system activates pheromone-inducible conjugation functions at distances greater than 10 kilobases upstream from its target in Enterococcus faecalis.

The prgB gene encodes the surface protein, Asc10, which mediates cell aggregation, resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline-resistance plasmid pCF10 in Enterococcus faecalis. Messenger RNA analysis by Northern blot hybridization and primer extension indicates that prgB transcription is pheromone-inducible and monocistronic. Previous transposon mutagenesis and sequencing analysis of a 12-kilobase (kb) region of pCF10 indicated that several genes including prgR and prgS are required to activate expression of prgB. The distance (3-4 kb) between these regulatory genes and prgB suggested that the activation might function in trans. To test this, a promoterless lacZ gene fusion to prgB was constructed and cloned without some or all of the regulatory genes. Several restriction fragments of the regulatory region were cloned in a higher copy-number plasmid, and numerous complementation studies were carried out in E. faecalis. Complementation in trans was not observed in any of these experiments. However, when the regulatory region and target genes were cloned in different sites of the same plasmid, separated by as much as 12 kb, activation of prgB was observed. Interestingly, this activation occurred only when the regions were cloned in the same relative orientation in which they exist on wild-type pCF10. These results suggest that one or more regulatory molecules may bind to an upstream cis-acting site and track along the DNA to reach a target site to activate prgB transcription.

Bacterial Proteins↗

PubMed2004

Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis.

AIM: To determine the resistance of microorganisms associated with refractory endodontic infections to sodium hypochlorite used as a root canal irrigant. METHODOLOGY: Two strains each of Actinomyces naeslundii, Candida albicans and Enterococcus faecalis were tested as late logarithmic phase inocula, against sodium hypochlorite adjusted to 0.5, 1.0, 2.5 and 5.25% w/v. Contact times used were 0, 10, 20, 30, 60 and 120 s. In the case of E. faecalis, additional experiments used contact times of 1.0, 2.0, 5.0, 10.0 and 30.0 min. Anti-microbial action was halted by sodium thiosulphate addition. Survivors were measured primarily using viable counts on drop plates. Additionally, pour plates were used to count low colony-forming units (cfu) and dilutions to 10(-6) were used to count high cfu. RESULTS: All concentrations of NaOCl lowered cfu below the limit of detection after 10 s in the case of A. naeslundii and C. albicans. However, E. faecalis proved to be more resistant to NaOCl. Using 0.5% NaOCl for 30 min reduced cfu to zero for both strains tested. This compares with 10 min for 1.0%, 5 min for 2.5% and 2 min for 5.25% (P < 0.001). Regression analysis for the dependent variable log(e)(count + 1) with log(e)(time + 1) and concentration as explanatory variables gave rise to a significant interaction between time and concentration (P < 0.001). CONCLUSION: The published association of E. faecalis with refractory endodontic infection may result, at least partially, from high resistance of this species to NaOCl. This does not appear to be the case with A. naeslundii or C. albicans.

Actinomyces↗

PubMed2003

In vitro activities of garenoxacin (BMS-284756) against Streptococcus pneumoniae, viridans group streptococci, and Enterococcus faecalis compared to those of six other quinolones.

The activity of garenoxacin, a new quinolone, was determined in comparison with other quinolones against different strains of S. pneumoniae, viridans group streptococci (VGS), and Enterococcus faecalis. Strains were quinolone-susceptible clinical isolates and quinolone-resistant strains with defined mechanisms of resistance obtained from either clinical isolates or derivatives of S. pneumoniae R6. Clinical quinolone-susceptible strains of S. pneumoniae, VGS and E. faecalis showed garenoxacin MICs within a range of 0.03 microg/ml to 0.25 micro g/ml. Garenoxacin MICs increased two- to eightfold when one mutation was present in the ParC quinolone resistance-determining region (QRDR), fourfold when one mutation was present in the GyrA QRDR (S. pneumoniae), 8- to 64-fold when two or three mutations were associated in ParC and GyrA QRDR, and 2,048-fold when two mutations were present in both the GyrA and ParC QRDRs (Streptococcus pneumoniae). Increased active efflux had a moderate effect on garenoxacin MICs for S. pneumoniae and VGS. Against S. pneumoniae, garenoxacin behaved like moxifloxacin and sparfloxacin, being more affected by a single gyrA mutation than by a single parC mutation. Although garenoxacin was generally two- to fourfold more active than moxifloxacin against the different wild-type or mutant strains of S. pneumoniae, VGS, and E. faecalis, it was two- to fourfold less active than gemifloxacin. At four times the respective MIC for each strain, the bactericidal effect of garenoxacin, observed at 6 h for S. pneumoniae and at 24 h for S. oralis and E. faecalis, was not influenced by the presence of mutation either in the ParC or in both the ParC and GyrA QRDRs.

Anti-Bacterial Agents↗

PubMed2003

Enterococcus faecalis plasmid pAD1-encoded Fst toxin affects membrane permeability and alters cellular responses to lantibiotics.

Fst is a peptide toxin encoded by the par toxin-antitoxin stability determinant of Enterococcus faecalis plasmid pAD1. Intracellular overproduction of Fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. Cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. Extracellular addition of synthetic Fst had no effect on cell growth. Spontaneous Fst-resistant mutants had a phenotype consistent with changes in membrane composition. Interestingly, overproduction of Fst sensitized cells to the lantibiotic nisin, and Fst-resistant mutants were cross-resistant to nisin and the pAD1-encoded cytolysin.

Amino Acid Sequence↗

PubMed1996

Effect of orally administered heat-killed Enterococcus Faecalis FK-23 preparation on neutropenia in dogs treated with cyclophosphamide.

Dogs injected intravenously for 3 days with cyclophosphamide (CY) at a dose of 10 mg/kg were given 100 mg/kg of Enterococcus faecalis FK-23 preparation (FK-23) perorally for 14 days to confirm the beneficial effects of the latter drug in neutropenic dogs when orally administered. Although FK-23 treatment did not inhibit CY-induced neutropenia, it augmented neutrophil-reconstituting capacity in these dogs. Increases in the myeloid/ erythroid ratio and neutrophilic lineages were found in the bone marrow of FK-23 administered dogs. The oral administration significantly restored the reduced activity of neutrophil phagocytosis and chemiluminescence in dogs treated with CY. These findings indicate that FK-23 administered perorally not only augments neutrophil reconstitution through the activation of bone marrow but also functions in dogs treated with CY. It may thus be a useful supportive agent to reduce the adverse side-effects associated with the administration of chemotherapeutic agents such as CY.

Administration, Oral↗

PubMed2002

Enterococcus faecalis induces inflammatory bowel disease in interleukin-10 knockout mice.

Germ-free interleukin-10 knockout (IL-10 KO) mice developed inflammatory bowel disease (IBD) after they were colonized with a pure culture of Enterococcus faecalis. E. faecalis not only induced IBD (primarily in colon and rectum) but rectal dysplasia and adenocarcinoma was also found in the IL-10 KO mice. Conventional (complex-intestinal flora) IL-10 KO mice developed IBD within 10 to 15 weeks of age and showed more pathology in the cecum (typhlitis) than we observed with E. faecalis-induced IBD in gnotobiotic IL-10 KO mice. Conversely, neither germ-free IL-10 mice nor IL-10 KO mice colonized as adults, with a pure culture of Candida albicans, Escherichia coli, Lactobacillus casei, L. reuteri, L. acidophilus, a Bifidobacterium sp., Lactococcus lactis, or a Bacillus sp. developed IBD during the 25- to 30-week study. E. faecalis is a common intestinal microbe of man and animals that can trigger IBD, dysplasia, and carcinoma in a genetically susceptible murine host.

Animals↗

PubMed1992

Infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis.

OBJECTIVE: To investigate the risk factors, clinical features, molecular epidemiology, and treatment outcomes associated with an outbreak of infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis. DESIGN: Case-control and molecular genetics study. SETTING: Tertiary care Veterans Affairs hospital. PATIENTS: Sixty-five patients infected or colonized with beta-lactamase-producing high-level gentamicin-resistant E. faecalis (case patients) were matched and compared with 65 randomly selected patients infected or colonized with beta-lactamase-negative, gentamicin-susceptible E. faecalis. MEASUREMENTS AND MAIN RESULTS: During the 20-month study period, 124 of 1506 isolates (8.2%) of E. faecalis from 70 patients were found to produce beta-lactamase. Univariate analysis showed older age, higher APACHE II score, nosocomial acquisition, recent surgical procedure, total parenteral nutrition, and antibiotic treatment to be significantly associated with the acquisition of beta-lactamase-producing, high-level gentamicin-resistant E. faecalis. A multivariate analysis, done using stepwise multiple logistic regressions, showed that only two variables remained significant: an APACHE II score greater than 6 (odds ratio, 9.5; 95% CI, 4.4 to 20.3) and antibiotic treatment (odds ratio, 10.2; CI, 4.5 to 23.2). The mortality rate for case patients was 7.7% (5 of 65 patients; CI, 1.2% to 14.2%); no control patient died. Of 23 infections occurring in case patients, 13 were treated with "inappropriate" antibiotics (regimens that included a beta-lactamase-unstable antibiotic); 2 patients improved and 11 had complete resolution of disease. "Appropriate" treatments (regimens that included a beta-lactamase-stable antibiotic) were used in 10 patients; 5 of 10 infections were fatal. Restriction enzyme digests of total chromosomal DNA showed nearly identical patterns for selected isolates of beta-lactamase-producing, high-level gentamicin-resistant E. faecalis, suggesting dissemination through the hospital of a single strain of E. faecalis. CONCLUSIONS: Fatal infections were observed despite treatment with beta-lactamase-stable antibiotics. The risk for infection or colonization with beta-lactamase-producing, high-level gentamicin-resistant E. faecalis was strongly associated with severe underlying disease (acute physiology and chronic health evaluation [APACHE] II score, greater than 6) and previous antibiotic treatment. These results may be useful in targeting high-risk patients for infection-control interventions.

Case-Control Studies↗

PubMed1988

Aortic endocarditis caused by gentamicin-resistant Enterococcus faecalis.

A case is reported of a patient who developed aortic endocarditis after protracted hospitalization during which time the patient received multiple courses of aminoglycoside and cephalosporin therapy. The infecting organism, an Enterococcus faecalis strain, was resistant to gentamicin and to the combination of ampicillin with gentamicin. Valve replacement was performed after four weeks of therapy with ampicillin, and enterococci were isolated from cardiac vegetations. Valve replacement may be necessary to achieve cure of endocarditis due to enterococci highly resistant to gentamicin.

Aged↗

PubMed2002

Adhesion to bile drain materials and physicochemical surface properties of Enterococcus faecalis strains grown in the presence of bile.

The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of Enterococcus faecalis strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for E. faecalis not expressing Agg or Esp and E. faecalis with Esp on its surface. E. faecalis expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of E. faecalis.

Adhesins, Bacterial↗

PubMed1993

The role of lipoic acid in product formation by Enterococcus faecalis NCTC 775 and reconstitution in vivo and in vitro of the pyruvate dehydrogenase complex.

The role of the pyruvate dehydrogenase complex (PDC) in the formation of different fermentation products by Enterococcus faecalis was studied. This organism was grown on a semi-defined medium under various conditions in the presence or absence of lipoic acid, an essential cofactor of the enzyme complex. When grown on a medium without added lipoic acid, a very low activity, both in vivo and in vitro, of the PDC was observed. When pyruvate served as the energy source, lipoic acid was found to be essential for growth under anaerobic conditions at low culture pH values. The presence of lipoic acid in the culture medium had a marked effect on the production of acetoin: in the presence of lipoic acid, acetoin was produced only when the intracellular pyruvate concentration was relatively high, whereas in the absence of lipoic acid, acetoin was a common product. Under potassium-limited conditions, lactate was the main product and culture pH significantly affected the bacterial dry weight. After instantaneous addition of lipoic acid to a glucose+pyruvate-limited chemostat culture, an immediate activation of the PDC took place as deduced from the change in fermentation pattern. Reconstitution of the PDC by the addition of lipoic acid was also possible in cell-free extracts, although pre-incubation with ATP and lipoic acid for 90 min was necessary for maximal activation. The effects of an active PDC on product formation and the physiological role of the complex under anaerobic growth conditions are discussed.

Acetoin↗

PubMed2006

Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubules.

AIM: To evaluate the efficacy of 0.5%, 2.5% and 5.25% sodium hypochlorite (NaOCl) as intracanal irrigants associated with hand and rotary instrumentation techniques against Enterococcus faecalis within root canals and dentinal tubules. METHODOLOGY: A total of 180 extracted human premolar teeth were infected for 21 days with E. faecalis. The specimens were divided into 12 groups, as follows: group 1: 5.25% NaOCl + Hybrid technique (Valdrighi et al. 1998); group 2: 5.25% NaOCl + nickel-titanium (NiTi) rotary technique 4 mm shorter than the apex (by FOP-UNICAMP); group 3: 5, 25% NaOCl + NiTi rotary technique (Hero 642); group 4: 2.5% NaOCl +Hybrid technique; group 5: 2.5% NaOCl + NiTi rotary technique 4 mm shorter than the apex; group 6: 2.5% NaOCl + NiTi rotary technique (Hero 642); group 7: 0.5% NaOCl + Hybrid technique; group 8: 0.5% NaOCl + NiTi rotary technique 4 mm shorter than the apex; group 9: 0.5% NaOCl + NiTi rotary technique (Hero 642); group 10: sterile saline solution + Hybrid technique; group 11: sterile saline solution + NiTi rotary technique 4 mm shorter than the apex; group 12: sterile saline solution + NiTi rotary technique (Hero 642). Canals were sampled before and after preparation. After serial dilution, samples were plated onto brain heart infusion (BHI) agar, and the colony forming units (CFU) that were grown were counted. The teeth were sectioned into three thirds and dentine chips were removed from the canals with conical burs. The samples obtained with each bur were immediately collected into test tubes containing BHI broth, and were incubated at 37 degrees C and plated onto BHI agar. The CFU were counted and analysed. RESULTS: At all depths and thirds of the root canals and for all techniques used, 5.25% NaOCl was shown to be the most effective irrigant solution tested when dentinal tubules were analysed, followed by 2.5% NaOCl. No differences among concentrations in cleaning the canals were found. CONCLUSIONS: Especially at higher concentrations, NaOCl, was able to disinfect the dentinal tubules, independent of the canal preparation technique used.

Dental Pulp Cavity↗

PubMed1990

Evidence for the translocation of Enterococcus faecalis across the mouse intestinal tract.

The pathogenesis of Enterococcus (Streptococcus) faecalis was studied in mice with E. faecalis intestinal overgrowth (10(9) - 10(10) per gram of cecum) induced by metronidazol and streptomycin treatment coupled with oral inoculation of E. faecalis. E. faecalis was recovered from the mesenteric lymph nodes, liver, and spleen; mortality was noted in 8 (13%) of 62 mice after 14 days of E. faecalis intestinal overgrowth. Light, immunofluorescent, and electron (scanning and transmission) microscopy of ileal tissue was used in an attempt to localize E. faecalis translocating across intestinal tissue. Dense coccal bacteria were observed in the intestinal lumen, and the epithelium appeared intact. Coccal bacteria were observed adherent to the microvillus border of the entire villous epithelium, including the deeper regions of the intestinal crypts. Immunofluorescence localized E. faecalis within columnar epithelial cells, lamina propria, submucosa, and muscularis externa (including the lumen of small vessels). Transmission electron microscopy localized coccal bacteria within vacuoles in the cytoplasm of intact epithelial cells. These results indicated that E. faecalis could translocate across an intact intestinal tract and cause systemic infection and death. In this model, the intestinal epithelial cell appeared to be a portal of entry in the pathogenesis of systemic E. faecalis infection.

Animals↗

PubMed2005

The tcrB gene is part of the tcrYAZB operon conferring copper resistance in Enterococcus faecium and Enterococcus faecalis.

The plasmid-localized tcrB (transferable copper-resistance gene B) gene from Enterococcus faecium was identified to be part of an operon called the tcrYAZB operon, which has a genetic organization similar to the copYZAB copper-homeostasis gene cluster from Enterococcus hirae. Putative promoter (P(tcr))- and repressor-binding sites highly similar to the E. hirae cop-promoter region were identified upstream of the tcrYAZB genes. The P(tcr) promoter was cloned in both the absence and the presence of the proximal repressor-encoding tcrY gene into a promoter-probe vector. Induction of the promoter was shown in liquid growth medium containing increasing concentrations of copper sulphate. To determine the growth advantage conferred by the tcrYAZB genes in a copper environment, a tcr-deletion mutant was isolated, and its growth was compared with that of its copper-resistant ancestor (strain A17sv1) in sublethal concentrations of copper sulphate. A competition assay using these two isogenic strains showed that copper sulphate concentrations of 3 mmol l(-1) and above are sufficient to select for copper resistance.

Bacterial Proteins↗

PubMed1994

Identification and characterization of an Enterococcus faecalis plasmid pAD1-encoded stability determinant which produces two small RNA molecules necessary for its function.

A determinant, designated par, essential for stable maintenance for an autonomously replicating fragment of the Enterococcus faecalis plasmid pAD1, was identified by transposon mutagenesis, and its DNA sequence was determined. The position of flanking transposon inserts with no effect on stability indicates that par is encoded on no more than approximately 720 bp of DNA. This region contains no large open reading frames (> 62 amino acids) but does contain a number of direct and inverted repeats and GC- and AT-rich boxes. Disruption of these elements by transposon insertion, or deletion of the entire cluster of elements, resulted in a loss of plasmid stability but did not affect replication or copy number in E. faecalis. Characterization of selected mutants suggested that some manipulations of par may interfere with essential plasmid replication functions and/or be lethal to the host cell. Northern blot analysis revealed that two small RNA molecules of approximately 250 and 145 nucleotides homologous to par are produced by cells containing the complete pAD1 replicon. Mutations affecting par that resulted in a decrease in plasmid stability also resulted in changes in the pattern of production of the par RNAs, suggesting that these RNAs are important for par function.

Base Sequence↗

PubMed2005

Enterococcus faecalis mammalian virulence-related factors exhibit potent pathogenicity in the Arabidopsis thaliana plant model.

Some pathogenic bacteria belong to a large, diverse group of species capable of infecting plants, animals, and humans. Enterococcus faecalis is an opportunistic human pathogen capable of infecting patients with a deficient immune system. Here we report that three E. faecalis strains (FA-2-2, V583, and OG1RF) are capable of infecting the leaves and roots of the model plant species Arabidopsis thaliana, causing plant mortality 7 days postinoculation. We found that E. faecalis pathogenesis in A. thaliana leaves is determined by the following series of events: attachment to leaf surface, entry through stomata or wounds, and colonization in intercellular spaces, leading to rotting and to the disruption of plant cell wall and membrane structures. The three E. faecalis strains colonize the roots of A. thaliana by forming a mosaic of large clusters of live bacteria on the root surface, as observed by scanning electron microscopy, phase-contrast microscopy, and fluorescence microscopy. To dissect the involvement of mammalian virulence-related factors in plant pathogenicity, we tested E. faecalis mutant strains DeltafsrA (TX5240), DeltafsrB (TX5266), DeltafsrC (TX5242), DeltagelE (TX5264), and DeltasprE (TX5243), which correspond to virulence factors involved in pathogenesis in different animal models. Two E. faecalis virulence-related factors that play an important role in mammalian and nematode models of infection, a putative quorum-sensing system (DeltafsrB) and serine protease (DeltasprE), were also found to be important for plant pathogenesis. The development of an E. faecalis-A. thaliana model system could potentially be used to circumvent certain inherent limitations that an animal model imposes on the identification and study of virulence factors. Furthermore, our study suggests an evolutionary crossover of virulence factors in plant, animal, and nematode pathogenesis.

Arabidopsis↗

PubMed2006

Overexpression, crystallization and preliminary X-ray crystallographic analysis of phosphopantetheine adenylyltransferase from Enterococcus faecalis.

Phosphopantetheine adenylyltransferase, an essential enzyme in the coenzyme A biosynthetic pathway, catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine, yielding 3'-dephospho-CoA and pyrophosphate. Enterococcus faecalis PPAT has been overexpressed in Escherichia coli as a fusion with a C-terminal purification tag and crystallized at 297 K using a reservoir solution consisting of 0.1 M sodium HEPES pH 7.5, 0.8 M sodium dihydrogen phosphate and 0.8 M potassium dihydrogen phosphate. X-ray diffraction data were collected to 2.70 A at 100 K. The crystals belong to the primitive tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = b = 160.81, c = 225.68 A. Four copies of the hexameric molecule are likely to be present in the asymmetric unit, giving a crystal volume per protein weight (V(M)) of 3.08 A(3) Da(-1) and a solvent content of 60.1%.

Bacterial Proteins↗

PubMed1991

Factors influencing the synthesis of an extracellular proteinase by Enterococcus faecalis subsp. liquefaciens.

Studies were conducted on optimum temperature, pH and the requirement for an energy source, amino acids, casein, Zn2+ and Ca2+ during the synthesis of an extracellular acid proteinase by Enterococcus faecalis var. liquefaciens. Synthesis was monitored using cells grown to mid-logarithmic phase and resuspended at high density in fresh growth medium. Proteinase production was optimal at 30 degrees C and pH 7.0. Proteinase synthesis, being energy-dependent, occurred only in glycolysing cells. The synthesis was high when lactose but not glucose was utilized as a source of energy, indicating that phospho-beta-galactosidase gene might probably be located directly upstream the proteinase gene on a plasmid. Good induction of proteinase synthesis could be achieved by 0.2-0.5% of either yeast extract or tryptic digested casein, perhaps due to its content of a wide variety of free amino acids. Casein was essential for preventing proteinase autolysis and sustaining the enzyme production. Zn2+ and Ca2+ were required for the formation of an active extracellular proteinase. The synthesis immediately ceased after addition of chloramphenicol or EDTA. EDTA inactivated the preformed proteinase as well. Sodium chloride at a concentration of 6.5% inhibited both proteinase synthesis and glycolysis.

Caseins↗