Resistance to levomycetin and activity of several enzymes in Escherichia coli and the agent of plague]

The authors compared the activity of acetyl-CoA-synthetase and of the enzymes belonging to the group of asparaginic acid in levomycetin sensitive and resistant strains of Y. pestis and E. coli. There were revealed marked differences in the activity of aspartase, fumarase, synthetase and desamidase of L-asparagin, and also of the enzyme activated by acetate in the E. coli strains with plasmide resistance. Transmission of R-factor to the pestis was accompanied by decomposition of L-asparadein, formation of AC-CoA. At the same time transformation of L-asparaginic acid catalyzed by aspartase remained on the same low level in the sensitive pestis cultures and their variants with the R-factor. When the resistance was controlled by chromosomal resistance markers, the activity of the enzymes providing formation of L-asparagic acid, its amide and L-malic acid showed no significant change. In chromosomal type of resistance in the mutants of pestis and E. coli the acetyl-CoA-synthetase reaction was as a rule somewhat increased.     

Peculiarities of ethanol metabolism in an Acinetobacter sp. mutant strain defective in exopolysaccharide synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

Peculiarities of Ethanol Metabolism in an Acinetobacter sp. Mutant Strain Defective in Exopolysaccharide Synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD⁺-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD⁺ and NADP⁺ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C₄-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C₂-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

In vitro antibacterial activity of Chilean red wines against Helicobacter pylori

The antibacterial activity of sixteen Chilean red wines (Cabernet Sauvignon, Cabernet Merlot, Cabernet Organic and Pinot Noir), and the active extracts of two randomly selected wines were assayed for their antibacterial activity on six strains of Helicobacter pylori isolated from gastric biopsies. The active fraction of the wines was obtained by dichloromethane extraction, and the antibacterial activity of the wines and extracts was evaluated by an agar diffusion method. All the red wines studied showed some antibacterial activity on the six strains of H. pylori, although the strains were heterogeneous in their susceptibility to each particular wine. The active fraction of the two wines selected also showed good activity against the strains tested. The main active compound was identified as resveratrol. The results presented indicate that Chilean red wines have antibacterial activity against H. pylori, which depends mainly on the presence of resveratrol.     

Phospho-β-glucosidases and β-Glucoside Permeases in Streptococcus, Bacillus, and Staphylococcus

The presence of phospho-beta-glucosidases and beta-glucoside permeases was found in strains of Streptococcus, Bacillus, and Staphylococcus. In streptococci, the phospho-beta-glucosidase activity depends on the antigenic group. The highest activity was found in strains of group D. In group D strains, phospho-beta-glucosidase activity is induced by beta-methyl glucoside and cellobiose but not by thiophenyl beta-glucoside (TPG). With the exception of four strains isolated in Japan, all strains of B. subtilis tested possess an inducible phospho-beta-glucosidase activity, beta-methyl glucoside, cellobiose, and TPG acting as inducers. S. aureus strains possess phospho-beta-glucosidase A but not phospho-beta-glucosidase B, whereas most S. albus strains show no detectable phospho-beta-glucosidase activity. The prompt fermentation of beta-methyl glucoside by S. aureus strains could serve as an additional criterion for their differentiation from S. albus. A comparative investigation of the active uptake of (14)C-TPG showed that a Streptococcus group D strain and a B. subtilis strain posses two inducible permeases with characteristics similar to the beta-glucoside permeases I and II of Enterobacteriaceae. In S. aureus, TPG is accumulated by a constitutive permease with high affinity for aromatic beta-glucosides and glucose. The active uptake of TPG by S. aureus appears to depend on the activity of the phosphoenol pyruvate-dependent phosphotransferase system.     

Production by fungi of the genera Aspergillus, Acremonium, Verticillium of extracellular proteases which coagulate blood plasma and lyse blood clots

Among 24 fungal strains belonging to the genera Aspergillus, Acremonium and Verticillium, eight strains synthesized proteases with the coagulating activity. The most active strains producing such proteases were found among Aspergillus species. The fibrinolytic activity was detected in 12 strains of the fungi. Proteases with the fibrinolytic activity differed in their sensitivity to plasma inhibitors and the least sensitive proteases were found in A. niger and A. flavus. Some fungal strains synthesized proteases with the fibrinolytic activity exerting an indirect action. Such enzymes activated the conversion of blood profibrinolysin into fibrinolysin in a manner similar to that of staphylokinase, urokinase and trypsin.     

L-Asparaginase of Saccharomyces cerevisiae: an extracellular Enzyme.

During recent studies conducted with suspensions of three strains of Saccharomyces cerevisiae, it was observed that ammonia was rapidly liberated when L-asparagine was added to the medium. Subsequent investigation has revealed that these strains of S. cerevisiae have an externally active asparaginase as well as an internally active one. The appearance of the external asparaginase is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The internal enzyme appears to be constitutive. The external activity is relatively insensitive to para-hydroxymercuribenzoate inhibition, whereas the internal activity is highly inhibited by this compound.     

The study of agglutination of trypsin-treated sheep red cells by Corynebacterium diphtheriae strains

Abstract 620 Corynebacterium diphtheriae strains from 472 sick and healthy persons were studied for their adhesive activity (AA) in direct agglutination of trypsin-treated sheep erythrocytes. Toxigenic strains had more active AA than non-toxigenic ones which was not dependent on the presence of toxin in the culture. Neither biotype nor serotype of the strains correlated with their AA. Several lysotypes among toxigenic and non-toxigenic strains were more active than others. Toxigenic strains from patients had higher AA than those from carriers. Both toxigenic and non-toxigenic strains isolated from the prolonged carriers possessed the highest AA. It was concluded that AA measured in this way was an important colonization factor for all diphtheria strains and a pathogenicity factor for toxigenic strains.     

Antibacterial, antifungal and cytotoxic activity of terrestrial cyanobacterial strains from Serbia

Cyanobacteria are known to be a rich source of biologically active compounds some of which can have pharmaceutical importance. In this work we present the screening results of cyanobacterial strains for their antibacterial, antifungal, and cytotoxic activity. Cyanobacterial strains were isolated from various soil types in province of Vojvodina and Central Serbia, Republic of Serbia. The screening included 9 strains of Anabaena and 9 strains of Nostoc. Both, extracellular products (from the culture liquid) and cellular crude lipophilic extracts were tested against 13 bacterial strains and 8 fungal strains. Cytotoxic activity was tested against three human cell lines. Methanol extracts were prepared according to ?stensvik. Antibacterial and antifungal activities were determined measuring inhibition zone, 48 h after inoculation. The cytotoxic activity was determined by sulforhodamine B (SRB) colorimetric assay. Of all cyanobacterial strains tested, 52% showed some antifungal and 41% antibacterial activity. Two out of six tested strains possessed cytotoxic activity. The cytotoxic activity of Anabaena strain S12 was found both in culture liquid and crude cell extract. It occurred specifically between the 21st and 42nd day of cultivation against HeLa and MCF7 cells, but had no activity against cell line derived from a healthy tissue. A high percentage of the active strains among the tested strains justify the effort of screening cyanobacteria that are isolated from terrestrial environments. The most promising strains for the fur- ther study are Anabaena strain S12 which showed strong cytotoxic and antibacterial activity and Ana- baena strain S20 which produces a potent antifungal compound. The future work, besides further screening and chemical identification of the active compounds, should also include the development of culture techniques that would lead to more efficient production of biologically active compounds.     

Screening of nonpolyenic antifungal metabolites produced by clinical isolates of actinomycetes

The purpose of this work was to screen clinical isolates of actinomycetes producing nonpolyenic antifungals. This choice was made to limit the problem of rediscovery of well-known antifungal families, especially polyenic antifungals. One hundred and ten strains were tested, using two diffusion methods and two test media, against three yeast species and three filamentous fungi. Among 54 strains (49%) showing antifungal activity, five strains belonging to the genus Streptomyces were active against all test organisms and appeared promising. These results indicate that clinical and environmental isolates of actinomycetes could be an interesting source of antifungal bioactive substances. The production of nonpolyenic antifungal substances by these five active isolates was investigated using several criteria: antibacterial activity, ergosterol inhibition, and UV-visible spectra of active extracts. One active strain responded to all three selection criteria and produced potentially nonpolyenic antifungal metabolites. This strain was retained for further investigation, in particular, purification, structure elucidation, and mechanism of action of the active product.     

Study of certain carbohydrate metabolism enzymes in an active oxytetracycline producer strain and in an inactive mutant in relation to antibiotic biosynthesis]

The activity of the glycolysis enzymes, i.e. aldolase and pyruvate decarboxylase and the enzymes of the pentose cycle, i.e. transketolase were investigated in the process of cultivation of an active strain and inactive mutant of Act. rimosus under conditions favourable for oxytetracycline biosynthesis on starch medium and under unfavourable conditions on glucose medium. It was shown that the aldolase and transketolase activity in the inactive mutant was higher on the starch medium as compared to the active strain, while the activity of pyruvate dekarboxylase was lower. The above difference between the both strains was preserved on the glucose medium and the activity of aldolase and transketolase in both strains increased, while the activity of pyruvate dekarboxylase remained at the same level.     

一种真菌对人参皂苷Rg3的转化

[目的]筛选长白山人参土壤中的活性微生物,转化人参总皂苷及单体人参皂苷产生稀有抗肿瘤成份.[方法]从长白山人参根际土壤中分离各类菌株,对人参总皂苷及单体人参皂苷进行微生物转化,并通过硅胶柱层析等方法对转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定;结合菌落形态、产孢结构、孢子形态特征以及菌株ITS rDNA核酸序列分析,对活性菌株进行鉴定.[结果]从长白山人参根际土壤中分离各类真菌菌株68株,有12株菌株对人参总皂苷有转化活性,其中菌株SYP2353对二醇组人参皂苷Rg3具有较强的转化活性.[结论]阳性菌株SYP2353被鉴定为疣孢漆斑菌(Myrothecium verrucaria),能将人参皂苷Rg3转化为稀有人参皂苷Rh2及二醇组人参皂苷苷元PPD,为稀有人参皂苷Rh2的制备提供了新的方法.     

Analysis of the two-peptide bacteriocins lactococcin G and enterocin 1071 by site-directed mutagenesis

The two peptides (Lcn-alpha and Lcn-beta) of the two-peptide bacteriocin lactococcin G (Lcn) were changed by stepwise site-directed mutagenesis into the corresponding peptides (Ent-alpha and Ent-beta) of the two-peptide bacteriocin enterocin 1071 (Ent), and the potencies and specificities of the various hybrid constructs were determined. Both Lcn and, to a lesser extent, Ent were active against all the tested lactococcal strains, but only Ent was active against the tested enterococcal strains. The two bacteriocins thus differed in their relative potencies to various target cells, despite their sequence similarities. The hybrid combination Lcn-alpha+Ent-beta had low potency against all strains tested, indicating that these two peptides do not interact optimally. The reciprocal hybrid combination (i.e., Ent-alpha+Lcn-beta), in contrast, was highly potent, indicating that these two peptides may form a functional antimicrobial unit. In fact, this hybrid combination (Ent-alpha+Lcn-beta) was more potent against lactococcal strains than wild-type Ent was (i.e., Ent-alpha+Ent-beta), but it was inactive against enterococcal strains (in contrast to Ent but similar to Lcn). The observation that Ent-alpha is more active against lactococci in combination with Lcn-beta and more active against enterococci in combination with Ent-beta suggests that the beta peptide is an important determinant of target cell specificity. Especially the N-terminal residues of the beta peptide seem to be important for specificity, since Ent-alpha combined with an Ent-beta variant with Ent-to-Lcn mutations at positions 1 to 4, 7, 9, and 10 was >150-fold less active against enterococcal strains but one to four times more active against lactococcal strains than Ent-alpha+Ent-beta. Moreover, Ent-to-Lcn single-residue mutations in the region spanning residues 1 to 7 in Ent-beta had a more detrimental effect on the activity against enterococci than on that against lactococcal strains. Of the single-residue mutations made in the N-terminal region of the alpha peptide, the Ent-to-Lcn mutations N8Q and P12R in Ent-alpha influenced specificity, as follows: the N8Q mutation had no effect on activity against tested enterococcal strains but increased the activity 2- to 4-fold against the tested lactococcal strains, and the P12R mutation reduced the activity >150-fold and only approximately 2-fold against enterococcal and lactococcal strains, respectively. Changing residues in the C-terminal half/part of the Lcn peptides (residues 20 to 39 and 25 to 35 in Lcn-alpha and Lcn-beta, respectively) to those found in the corresponding Ent peptides did not have a marked effect on the activity, but there was an approximately 10-fold or greater reduction in the activity upon also introducing Lcn-to-Ent mutations in the mid-region (residues 8 to 19 and 9 to 24 in Lcn-alpha and Lcn-beta, respectively). Interestingly, the Lcn-to-Ent F19L+G20A mutation in an Lcn-Ent-beta hybrid peptide was more detrimental when the altered peptide was combined with Lcn-alpha (>10-fold reduction) than when it was combined with Ent-alpha ( approximately 2-fold reduction), suggesting that residues 19 and 20 (which are part of a GXXXG motif) in the beta peptide may be involved in a specific interaction with the cognate alpha peptide. It is also noteworthy that the K2P and A7P mutations in Lcn-beta reduced the activity only approximately 2-fold, suggesting that the first seven residues in the beta peptides do not form an alpha-helix.     

Antimicrobial properties of moderately halotolerant bacteria from cenotes of the Yucatan peninsula

AIMS: Isolation and antimicrobial evaluation of aquatic bacterial strains from two cenotes. METHODS AND RESULTS: A total of 258 bacterial strains were isolated from the water and sediment of two cenotes in the Yucatan peninsula, all of which were screened against six pathogenic micro-organisms. Antimicrobial activity was detected in 46 of the isolated strains against at least one of the target strains tested. Antimicrobially active isolates were identified as: Aeromonas, Bacillus, Burkholderia, Photobacterium, Pseudomonas, Serratia, Shewanella, Stenotrophomonas genera, and 13 remained unidentified. All antimicrobially active strains were able to grow in salt medium at a concentration of 75 g l(-1), thus classifying as moderately halotolerant bacteria. Most of the antimicrobially active strains exhibited a broad action spectrum, where 61% was because of uncharacterized antimicrobial substances, 25% because of bacteriocins and 13% because of siderophores. Ten strains were able to biosynthesize biosurfactant metabolites. CONCLUSIONS: Native bacteria from the Yucatan peninsula showed an interesting antimicrobial activity, diverse mode of action and moderate halotolerance to salt. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on bacterial isolates from cenotes of the Yucatan peninsula and their antimicrobial characterization, with great potential for future biotechnological applications.     

Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains

The human vagina is a complex and dynamic ecosystem containing an abundance of microorganisms. In women of childbearing age, this system is dominated by Lactobacillus spp. In the present work, seventeen newly isolated vaginal strains were identified by 16S rDNA sequencing and were investigated for their antimicrobial properties. Twelve of the isolated Lactobacillus strains showed activity against one or more microorganisms. Six and five of them produced substances that inhibited the growth of two different Klebsiella strains and Staphylococcus aureus, respectively. Two lactobacilli strains were active against an Escherichia coli strain, one isolate was active against an Enterococus faecalis strain and another lactobacilli strain showed antimicrobial activity against a Candida parapsilosis strain. The nature of the active compounds was additionally studied, and the presence of bacteriocin-like substances was proved. The genes related to the bacteriocin production in three of the newly isolated strains were identified and sequenced. The presence of gassericin A operon in the genome of the species Lactobacillus crispatus was described for the first time. The presence of antimicrobial activity contributes to their possible use as potential probiotic strains after further research.     

Properties of the strains<Emphasis Type="Italic">Enterococcus haemoperoxidus</Emphasis> and<Emphasis Type="Italic">E. moraviensis</Emphasis>, new species among enterococci

Antibiotic susceptibility or resistance, urease activity, detection of the structural genes for bacteriocin production, bacteriocin activity as well as sensitivity of the isolates to enterocins (Ent) A and M were determined in 23 isolates of new species Enterococcus haemoperoxidus and E. moraviensis. The majority of the strains were antibiotic sensitive and exhibited low urease activity (< 10 nkat/mL). The most frequently detected genes for Ent were entA and entP. However, only the strain 466 of E. haemoperoxidus produced an antibacterial substance with inhibitory activity against 21 G+ indicators. It was partially purified reaching an activity of up to 12 800 AU/mL. This bacteriocin active strain also possessed the genes for EntA and EntP. The other strains did not inhibit the indicator strains. The substance produced by the 466 strain was active even after a 5-months storage at +4 and -20 degrees C. This substance has proteolytic and hydrophilic character, pH optimum of bacteriocin production by this strain being between 4 and 7. While E. moraviensis strains showed sensitivity to EntA (produced by E. faecium EK13) and to EntM (produced by E. faecium AL41), E. haemoperoxidus strains were sensitive to EntA (except strain 382) but less sensitive to the treatment by EntM.     

鬼臼类植物内生真菌的分离及其抗癌活性研究

通过对三属四种鬼臼类植物地下茎内生真菌的分离,发现鬼臼类植物地下茎中内生真菌的物种类型极其丰富,主要分布在地下茎的表皮层和维管组织中,来源于外界环境.通过对包括鬼臼类植物在内的7种植物内生真菌的抗癌活性测定,发现内生真菌的抗癌活性与宿主有密切有关系,鬼臼类植物地下茎内生真菌含有较高比例的抗癌活性菌株.宿主种类、地理位置都会影响内生真菌的分布,进而影响活性菌株出现的频率.通过对所有鬼臼类植物地下茎内生真菌次生代谢产物的深入分析,并没有发现产生鬼臼毒素的内生真菌,鬼臼类植物地下茎内生真菌的抗癌活性成分是独立产生的.     

Agrocin-producing pathogenic and nonpathogenic biotype-3 strains ofAgrobacterium tumefaciens active against biotype-3 pathogens

Agrocin-producing pathogenic and nonpathogenic biotype-3 strains ofAgrobacterium tumefaciens were isolated from grapevine gall tissue. In vitro activity of the nonpathogenic agrocin producers was restricted to biotype-3 pathogens used. Pathogenic agrocin producers were active in vitro against biotype-3 agrocin-producing nonpathogens, non-agrocin-producing pathogens, and biotype-1 strains when cultivated on a modified Stonier's medium; on a medium designated AB, two strains stested showed no activity against agrocin-producing nonpathogens, but agrocin of one of these strains was active against other agrocin-producing pathogens. In a greenhouse experiment a marked tendency toward decreased gall formation by biotype-3 pathogens on grapevines was obtained when biotype-3 pathogens and nonpathogenic biotype-3 agrocin producers were applied to wounds simultaneously. In this experiment, agrocin-producting pathogens tended to be more virulent than non-agrocin-producing pathogens.     

Insoluble glucan synthesis by mutansucrase as a determinant of the cariogenicity of Streptococcus mutans

Five strains of Streptococcus mutans were grown in continuous culture with either a limited supply or an excess of glucose. Proteins secreted into the extracellular fluid by strains C67-1, 3209 and K1 rapidly catalysed the synthesis of insoluble glucan from sucrose (mutansucrase activity). The culture fluid from strains Ingbritt or C67-25 catalysed the synthesis of soluble glucan (dextransucrase activity) and fructan, but little or no mutansucrase activity was detected. The strains which secreted active mutansucrase readily colonized a smooth hard surface during growth in batch culture and were more cariogenic in pathogen-free rats than those which secreted little mutansucrase activity. There was no similar correlation between fructosyltransferase, dextransucrase or total glucosyltransferase activity and either adherence or cariogenicity. We conclude that the ability to catalyse insoluble glucan synthesis is a major determinant of the cariogenicity of S. mutans strains.     

Morphology of the bifidobacteria by light and electron microscopy and their biological properties]

The morphology and the acid-producing, antagonistic and enzymatic activity of the strains of bifidobacteria isolated from the intestine of adults and children were studied. Bifidobacteria showed a considerable polymorphism, all the strains were antagonistically active. The strains isolated from adults were found to have greater acid-producing activity; the predominant species were B. longum and B. adolescentis, and in children B. bifidum were also isolated. The characteristic feature of B. longum strains was the presence of a slime-like layer and formations resembling bubbles around bacterial cells. The stains with greater physiological activity were found to have an extensive mesosomal complex, as well as a great number of volutin granules.