Extracellular proteinase fromEnterococcus faecalis subsp.liquefaciens

Growth and extracellular proteinase production byEnterococcus faecalis subsp.liquefaciens was studied on several culture media and under different incubation conditions. The organisms grew well and developed extracellular proteinase activity on proteinaceous media, but when it grew on Collins basal medium (lacking of protein), growth was poor and proteinase activity was not detected. The activation energy for growth was estimated to be 116 kJ/mol, the optimum being at 37°C. Proteinase production was not affected by temperature in the range studied (7–45°C). Growth rate was not affected by aeration although a higher amount of microorganisms was observed on shaking the culture during incubation. Likewise, extracellular proteolytic activity was about twice higher in cultures shaken at 2.3 or 3.3 Hz than in those shaken at 0 or 1.3 Hz.     

Stimulation of proteinase biosynthesis by canavanine in streptococcus faecalis var. liquefaciens

l-Canavanine, an analogue of arginine, was found to stimulate the synthesis of an extracellular proteinase in Streptococcus faecalis var. liquefaciens. Cells grown in a synthetic medium containing 10(-4)m arginine and 10(-4)m canavanine produced almost twice as much proteinase as cells grown in 2 x 10(-3)m arginine alone; total growth was the same in both media. Hydrolyzed proteinase samples were analyzed for arginine and canavanine by means of paper chromatography and electrophoresis. Arginine, but not canavanine, was detected in the purified enzyme sample.     

Growth of Streptococcus faecalis var. liquefaciens on Plants

The proliferation of Streptococcus faecalis var. liquefaciens on two varieties of beans, and on corn, rye, and cabbage was investigated. Comparisons were made with growth patterns on these same plants exhibited by S. lactis and Lactobacillus plantarum. The ability of each of the bacteria to multiply and to spread to new plant parts as they developed from seed was studied under several environmental conditions. Plants were grown aseptically in glass culture and in sterilized and non-sterilized soil in the greenhouse. Quantitative estimations of increase in bacterial numbers were made. S. faecalis established commensal growth on each of five plants, although selectivity was noted for some plant parts. The organism increased in numbers on the plants equally as well as did the control bacteria, both alone, and in competition with the control bacteria and the microflora of the soil.     

Carbon Dioxide Fixation by Extracts of Streptococcus faecalis var. liquefaciens

Extracts of cells of Streptococcus faecalis var. liquefaciens strain 31 incorporated (14)CO(2) into aspartate. Dialyzed extracts produced radioactive oxalacetate in the absence of exogenously added glutamate and pyridoxal-5'-phosphate and produced radioactive aspartate in the presence of these components. Reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate could not be substituted for adenosine triphosphate (ATP); phosphoenolpyruvate even in the presence of nucleoside diphosphates could not replace pyruvate plus ATP; propionate plus coenzyme A (CoA) could not replace pyruvate in supporting CO(2) fixation by cell extracts. Fixation by dialyzed cell extracts required pyruvate, ATP, MgSO(4), and was stimulated by biotin, KCl, 2-mercaptoethanol, CoA, and acetyl CoA. Inhibition of fixation occurred when avidin, NaCl, oxalacetate, or aspartate was added to dialyzed extracts. On the basis of the products formed and the effects of substrates and cofactors on the fixation reaction, it was concluded that pyruvate carboxylase is responsible for CO(2) fixation in this microorganism.     

Carbon Dioxide Fixation by Cells of Streptococcus faecalis var. liquefaciens

Fixation of NaH(14)CO(3) by a heavy cell suspension of Streptococcus faecalis var. liquefaciens was studied. Several nutrients, pyridoxal, riboflavine, adenine, uracil, and O(2) stimulated (14)CO(2) incorporation into cells only under conditions that were adequate for synthesis of cell macromolecules. Biotin increased CO(2) incorporation in the absence of extensive synthesis of macromolecules, whereas O(2) inhibited incorporation under these conditions. When (14)CO(2) fixation was occurring during synthesis of macromolecules, 71% of the (14)C was incorporated into cells and 29% occurred extracellularly. Ninety-three per cent of the cellular (14)C was in protein and 5.5% was in nucleic acid. Aspartic acid was the only amino acid in the protein fraction that was radioactive. Eighty-three per cent of the extracellular (14)C was resistant to precipitation by trichloroacetic acid. When (14)CO(2) fixation was occurring in cells that were not carrying on extensive synthesis of macromolecules, 38% of the (14)C was incorporated into cells and 59% occurred in the supernatant fluid. Sixty-nine per cent of the cellular (14)C was in protein, 21% was in low-molecular-weight compounds, and 9% was in nucleic acid. Addition of unlabeled aspartate to the medium inhibited incorporation of (14)CO(2). Based on studies of the rate of (14)CO(2) fixation, the cells fix CO(2) into a pool of intermediates which are either used for synthesis, primarily protein, or are excreted into the medium.     

Occurrence and Distribution of Proteinase of Streptococcus faecalis var. liquefaciens

Shugart, Lee R. (University of Tennessee, Knoxville), and Raymond W. Beck. Occurrence and distribution of proteinase of Streptococcus faecalis var. liquefaciens. J. Bacteriol. 92:338-341. 1966.-The proteolytic enzyme produced by Streptococcus faecalis var. liquefaciens (ATCC 13398) was shown to be an exoenzyme. The production of the proteinase was followed in growing cultures, and its distribution was compared with that of the intracellular enzymes reduced nicotinamide adenine dinucleotide (NADH(2)) peroxidase and lactate dehydrogenase. The proteinase appeared in the culture medium prior to the stationary phase of growth, whereas the other enzymes could be found only in whole cells. Fractionation of whole cells by sonic treatment and by treatment with lysozyme showed the proteinase to be associated primarily with the cell wall and cell membrane, and NADH(2) peroxidase to be associated only with the cytoplasmic fractions.     

A simple method for semi-preparative-scale production and recovery of enterocin AS-48 derived from Enterococcus faecalis subsp. liquefaciens A-48-32

Production of enterocin AS-48 by Enterococcus faecalis A-48-32 was compared between standard and high-cell density batch fermentations. In high-cell density cultures, bacteriocin production was 2.47-fold higher, provided that the pH was controlled during the fermentation. A two-step procedure for recovery of milligram quantities of purified bacteriocin was developed, based on adsorption of the bacteriocin on Carboxymethyl Sephadex CM-25 followed by reversed-phase chromatography on a semi-preparative column. The purified bacteriocin was active on all the Gram-positive bacteria tested (for example, species of Bacillus, Paenibacillus, Staphylococcus, and Listeria). Strains E. coli U-9, E. coli CECT 102, E. coli CECT 104, E. coli CECT 432, E. coli CECT 543, E. coli CECT 877 and Shigella sonnei CECT 542 were sensitive, while seven other E. coli strains as well as Salmonella choleraesuis CECT 722, S. choleraesuis CECT 916, Enterobacter cloacae CECT 194 and Aeromonas hydrophila CECT 398 were resistant.     

Extracellular proteinase fromLactobacillus murinus

Lactobacillus murinus CNRZ 313 produced an extracellular proteinase irrespective of the Ca²⁺ content in the culture medium. Proteinase activity was optimal at 37 °C and pH 7.5 in phosphate buffer (0.2 mol/L). It was stimulated by Mg²⁺ and Mn²⁺ and was inhibited by Zn²⁺. Ca²⁺ did not affect the enzymic activity but the proteinase liberated in the presence of this ion is more stable. The enzyme was purified to homogeneity from cell-free culture medium.     

A new sequence-specific endonuclease from Streptococcus faecalis subsp. zymogenes.

A new sequence-specific endonuclease, SfaI, has been partially purified from Streptococcus faecalis subsp. zymogenes. SfaI recognizes the tetranucleotide sequence 5'G-G-C-C 3' 3' C-C-G-G 5' and cleaves it at the sites indicated by the arrows.     

Draft Genome Sequence of Alcaligenes faecalis subsp. faecalis NCIB 8687 (CCUG 2071)

Alcaligenes faecalis subsp. faecalis NCIB 8687, the betaproteobacterium from which arsenite oxidase had its structure solved and the first "arsenate gene island" identified, provided a draft genome of 3.9 Mb in 186 contigs (with the largest 15 comprising 90% of the total) for this opportunistic pathogen species.     

Alcaligenes faecalis subsp. parafaecalis subsp. nov., a bacterium accumulating poly-beta-hydroxybutyrate from acetone-butanol bioprocess residues

The authors have previously isolated a solvent tolerant bacterium, strain G(T), (T = type strain) capable to convert acetone-butanol bioprocess residues into poly-beta-hydroxybutyrate. Strain G(T) was initially identified as Alcaligenes spp by standard bacteriological tests. In this study the taxonomic position of the bacterium was investigated in detail. The 165 rDNA sequence analysis, the G + C content of DNA (56 mol%) and the presence of ubiquinone Q-8 confirmed strain G(T) as a representative of the genus Alcaligenes. In the polyamine pattern of the bacterium putrescine and cadaverine were detected, but only trace amounts of 2-hydroxyputrescine. The extremely low content of 2-hydroxyputrescine is remarkable, since this unique diamine is a common marker for beta-proteobacteria. Phylogenetic analyses of 16S rDNA demonstrated that Alcaligenes sp. G(T) is most closely related to the species Alcaligenes faecalis (99.6% sequence similarity to A. faecalis HR4 and 98.7% sequence similarity to A. faecalis [ATCC 8750T = DSM 30030T]. On the basis of DNA-DNA relatedness (56% similarity), the unique polyamine pattern, the physiological and biochemical differences strain G(T) could be distinguished from the species A. faecalis. Therefore, a new subspecies for the species Alcaligenes faecalis is proposed; Alcaligenes faecalis subsp. parafaecalis subsp. nov.     

Genetic loci of hemolysin production in Streptococcus faecalis subsp. zymogenes.

Two plasmids corresponding to molecular weights of 38.5 X 10(6) and 3.6 X 10(6) have been identified in Streptococcus faecalis subsp. zymogenes strain X-14. The larger plasmid is required for hemolysin-bacteriolysin production. Strain L2, a nonlytic nitrosoquanidine mutant of strain X-14, still harbors the hemolytic plasmid and produces the lysin component, but not the activator component, of the lytic system. Conjugal transfer of this plasmid from strain L2 to plasmid-free strains and strains cured of the 38.5-megadalton plasmid gives rise to hemolytic recipients. This implicates a gene in hemolysin production at a site other than the 38.5-megadalton plasmid.     

Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor

A Gram (-) coccobacillary bacterium, J(T), was isolated from a graywater bioprocessor. 16S rRNA and biochemical analysis has revealed strain J(T) closely resembles Alcaligenes faecalis ATCC 8750T and A. faecalis subsp. parafaecalis DSM 13975T, but is a distinct, previously uncharacterized isolate. Strain J(T), along with the type strain of A. faecalis and its previously described subspecies share the ability to aerobically degrade phenol. The degradation rates of phenol for strain J(T) and reference phenol degrading bacteria were determined by photometrically measuring the change in optical density when grown on 0.1% phenol as the sole carbon source, followed by addition of Gibb's reagent to measure depletion of substrate. The phenol degradation rates of strain J(T) was found to exceed that of the phenol hydroxylase group III bacterium Pseudomonas pseudoalcaligenes, with isolate J(T) exhibiting a doubling time of 4.5 h. The presence of the large subunit of the multicomponent phenol hydroxylase gene in strain J(T) was confirmed by PCR. The presence of the nirK nitrite reductase gene as demonstrated by PCR as well as results obtained from nitrite media indicated denitrification at least to N2O. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA DNA hybridization, we propose assigning a novel subspecies of Alcaligenes faecalis, to be named Alcaligenes faecalis subsp. phenolicus with the type strain J(T) (= DSM 16503) (= NRRL B-41076).     

Extracellular serine proteinase from Bacillus licheniformis

The serine proteinase from B. licheniformis was purified by affinity chromatography on the sorbent obtained by attachment of p-(omega-aminomethyl)-phenylboronic acid via an amino group to CH-Sepharose. The use of this sorbent specific to the serine proteinases active sites resulted in a 35-fold purification of the enzyme with an apparent activity yield of 288%. Such a high activity yield is due to a removal of the enzyme inhibitors. The N-terminal sequence of B. licheniformis extracellular serine proteinase traced for 35 amino acid residues coincides with that of subtilisin Carlberg, a serine proteinase presumed to be secreted by a B. subtilis strain. Since the amino acid composition as well as the functional properties of these two enzymes did not reveal any noticeable differences, it was assumed that both proteinases are very similar, if not identical. This conclusion leads to reconsideration of the existing concept on an extremely fast rate of subtilisin evolution. Three multiple forms of B. licheniformis extracellular serine proteinase were found to differ only in their net charges, presumably as a result of partial deamidation of Asn or Gln residues within their structure.     

Extracellular alkaline proteinase of Colletotrichum gloeosporioides

The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.     

Carbohydrate utilization affects Lactobacillus delbrueckii subsp. lactis 313 cell-enveloped-associated proteinase production

The effect of different sugars (glucose, glycerol, maltose, galactose and lactose) on cell-membrane-associated proteinase production by Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was investigated. The experimental results showed that aside glycerol and galactose, all the other sugars supported high growth levels of LDL 313, with glucose displaying the maximum biomass concentration of 0.85 mg/mL dry cell weight for cells harvested at the mid-exponential phase of ??12 h after inoculation. The specific proteinase yield, a measure of the rate of proteinase production relative to cell wall biosynthesis, was used to evaluate the preferential degree of proteinase metabolism as induced by the consumption of different sugar substrates by LDL 313. It was found that maltose displayed the highest specific proteinase yield of 12.59 U/mg sugar consumed. Further, molecular differences were observed in the SDS electrophoretic profile of cell surface proteins generated for the different carbon substrates. This is a preliminary study which supports the inference that different sugars stimulate the production of different cell-surface proteins with a significant effect on cell proteinase activity.