Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC₄ strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac⁺), lactococcin-producing (Lap⁺), lactococcin-resistant (Lap^r), sucrosefermenting (Suc⁺) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Ery^r) at the frequencies of 10⁴ transformants/g of DNA. PC₄ protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac^–Lap^–Ery^r, Lac⁺Suc⁺Lap⁺Lap^rEry^r, and Lac^–Suc⁺Lap⁺ Lap^rLap^r, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc⁺Lap⁺Lap^r and Lac⁺ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC₄ transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.     

Recognition of Group D Streptococcal Species of Human Origin by Biochemical and Physiological Tests

The speciation of 262 strains of group D streptococci isolated from human sources is described. One hundred forty-two isolates from blood cultures were included; 96 of these were submitted as isolates from clinical cases of subacute bacterial endocarditis. The results show that 98 Streptococcus faecalis, 29 S. faecalis var. zymogenes, 44 S. faecalis var. liquefaciens, 27 S. faecium, 13 S. durans, 44 S. bovis, and 7 unspeciated S. bovis-like group D isolates were identified. No S. faecium var. casseliflavus, S. equinus, or S. avium (group Q streptococci) were identified among the human isolates. The speciation procedures and techniques are detailed. The procedures and limitations of the tests used are discussed. Ninety-eight percent of the 262 strains were speciated by a spectrum of tests that allowed us to recognize atypical as well as typical strains within species.     

Strain-related differences in bacterivory and demography of Diaphanosoma mongolianum (Cladocera) in relation to diet and previous exposure to cyanobacteria in nature

We compared the demographic variables and bacterivory of two strains of Diaphanosoma mongolianum from two water bodies in Spain, one without Microcystis (Maidevera in Zaragoza) and the other with dense Microcystis (La Albufera of Valencia). We hypothesized that the strain rarely exposed to Microcystis would be unable to grow on this cyanobacterial diet. We fed both strains Monoraphidium caribeum and Microcystis aeruginosa, together and separately, and compared their demographic variables. Monoraphidium caribeum was cultured in the laboratory on a defined medium, while the cyanobacteria were collected from La Albufera and sonicated before feeding the cladocerans (at 0.5?×?10⁶ cells ml^?1). We also tested the growth of D. mongolianum on bacterial diets by using seston (0–15 µm), bacterioplankton (0–3 µm) and mixed fractions (3–15 µm), from sieving Lake Albufera. We conducted population growth and life table demography experiments at 25 °C, using the two strains of D. mongolianum. Both strains had r (population growth rate) ranging from 0.05 to 0.3 d^?1, on all diets. The r was higher (0.18 d^?1) on the 0–15 µm seston compared to the mixed fraction (0.12 d^?1) although D. mongolianum also grew well on bacterioplankton (0.16 d^?1) alone. The response of the strains collected from two different water bodies was different to the test diets. We found that both strains of D. mongolianum could effectively utilize Microcystis for survival and growth, regardless of previous exposure to the cyanobacteria. The tested cladocerans could also grow well on small sized food particles (0–3 µm and 0–15 µm). Our results explain why D. mongolianum is common in eutrophic water bodies

     

Kinetics of batch-wise enzymatic cycling system for mass production of chiral compound

Kinetics of batch-wise enzymatic cycling system (oxidoreductase-catalyzed reaction system involving enzyme-coupled cofactor regeneration) has been studied covering a broad range of the conserved total cofactor concentration, [C]₀ (=NAD(P)⁺?+?NAD(P)H), based on reasonable several assumptions. It is composed of two elementary reactions, i.e. product synthesis reaction and cofactor regeneration reaction, both of which have been expressed by Michaelis–Menten type rate equations. A novel dimensionless variable, r, has been introduced, which is defined as the concentration of one of the two cofactor components, [X] (NADH⁺ or NADPH⁺), divided by [C]₀, i.e. r .e[X]/[C]₀. The following results have been obtained. (1) The fundamental equation of the batch-wise enzymatic cycling system has been transformed to a differential equation whose formula is: dr/dT?=?N(r)/D(r) (N(r) and D(r) are quadratic equations of r having different coefficients). (2) It has been elucidated that the batch-wise enzymatic cycling system has two phases, an early short transient phase followed by a long phase in quasi-steady state (QSS). (3) In the enzymatic cycling system, r converges to a definite level regardless of any initial value of r. (4) In QSS, the definite level of r nearly equals the singular solution, r_singular, of the differential equation. (5) The actual rate of the targeted product (chiral compound) formation can be calculated by Michaelis–Menten equation in which the cofactor concentration is [C]₀×r_singular instead of [C]₀. r_singular has been proposed to name “redistribution factor”. (6) It is recommended that the “unit” of the cofactor regeneration enzyme be 2–3 times more used than the “unit” of the synthesis enzyme and that [C]₀ be 15–25 times more than the K_m value. Four special cases relating to the batch-wise enzymatic cycling system have been discussed.     

Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.     

Comparison of Bacteriocins Produced by Lactic-Acid Bacteria Isolated from Boza, a Cereal-Based Fermented Beverage from the Balkan Peninsula

Boza is a low-pH and low-alcohol cereal-based beverage produced in the Balkan Peninsula. From a total population of 9 × 10⁶ colony-forming units ml⁻¹, four isolates (JW3BZ, JW6BZ, JW11BZ, and JW15BZ) produced bacteriocins active against a broad spectrum of Gram-positive bacteria. Bacteriocin JW15BZ inhibited the growth of Klebsiella pneumoniae. The producer strains were identified as Lactobacillus plantarum (strains JW3BZ and JW6BZ) and L. fermentum (strains JW11BZ and JW15BZ). The spectrum of antimicrobial activity, characteristics, and mode of action of these bacteriocins were compared with bacteriocins previously described for lactic-acid bacteria isolated from boza.     

Phosphatidylethanolamine Deficiency Impairs Escherichia coli Adhesion by Downregulating Lipopolysaccharide Synthesis,Which is Reversible by High Galactose/Lactose Cultivation

As the initiation step of bacterial infection or biofouling, bacterial adhesion on cells or substrates is generally an optimal target for antibacterial design. Phosphatidylethanolamine (PE) is the principal phospholipid in bacteria, and its function in bacterial adhesion remains unclear. In this study, four E. coli strains including two PE-deficient mutants (PE^?PC^? and PE^?PC^+?strains) and two PE-containing wild-type controls (PE?⁺?PC^? strains) were recruited to investigate the influence of PE deficiency on bacterial adhesion. We found that PE deficiency could impair E. coli adhesion on macrophages (human THP-1-derived and mouse RAW264.7 macrophages) or glass coverslips by downregulating lipopolysaccharide (LPS) biosynthesis, which could be reversible by high galactose/lactose but not glucose cultivation. The data imply that PE play important role in bacterial adhesion probably via affecting LPS biosynthesis and suggest that targeting PE biosynthesis is also a potential antibacterial strategy.     

Plasmid-associated Bacteriocin Production by and Immunity of Lactobacillus acidophilus TK8912

Lactobacillus acidophilus TK8912 produces an antibacterial substance, designated acidocin 8912, which is active against strains of Lactobacillus and Lactococcus. Of all conditions tested, the production of acidocin 8912 was maximum at 30°C in MRS broth. Acidocin 8912 was stable to heat treatment (120°C for 20min), but completely inactivated by protease treatment. Curing a plasmid pLA103 resulted in the loss of both acidocin 8912 production (Acd⁺) and host immunity (Acd^r). A plasmid-cured strain, TK1–4 (Acd^- Acd⁸), was transformed to Acd⁺Acd^r with the pLA103 plasmid. These results provided the first direct evidence in lactobacilli for involvement of this plasmid in bacteriocin production and immunity.     

Impact of antifungals producing rhizobacteria on the performance of Vigna radiata in the presence of arbuscular mycorrhizal fungi

Plant growth-promoting rhizobacteria (PGPR) that produce antifungal metabolites are potential threats for the arbuscular mycorrhizal (AM) fungi known for their beneficial symbiosis with plants that is crucially important for low-input sustainable agriculture. To address this issue, we used a compartmented container system where test plants, Vigna radiata, could only reach a separate nutrient-rich compartment indirectly via the hyphae of AM fungi associated with their roots. In this system, where plants depended on nutrient uptake via AM symbiosis, we explored the impact of various PGPR. Plants were inoculated with or without a consortium of four species of AM fungi (Glomus coronatum, Glomus etunicatum, Glomus constrictum, and Glomus intraradices), and one or more of the following PGPR strains: phenazine producing (P⁺) and phenazine-less mutant (P⁻), diacetylphloroglucinol (DAPG) producing (G⁺) and DAPG-less mutant (G⁻) strains of Pseudomonas fluorescens, and an unknown antifungal metabolite-producing Alcaligenes faecalis strain, SLHRE425 (D). PGPR exerted only a small if any effect on the performance of AM symbiosis. G⁺ enhanced AM root colonization and had positive effects on shoot growth and nitrogen content when added alone, but not in combination with P⁺. D negatively influenced AM root colonization, but did not affect nutrient acquisition. Principal component analysis of all treatments indicated correlation between root weight, shoot weight, and nutrient uptake by AM fungus. The results indicate that antifungal metabolites producing PGPR do not necessarily interfere with AM symbiosis and may even promote it thus carefully chosen combinations of such bioinoculants could lead to better plant growth.     

Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids

Summary Wild-type strain A454 (Streptococcus pyogenes) transferred en bloc its erythromycin (Em) and tetracycline (Tc) resistance markers into several plasmid-free streptococcal recipients. No plasmid DNA was detected in either the wild-type or the transconjugant strains. Crosses were performed between A454 and S. faecalis Rec⁺ or Rec^- recipients carrying hemolysin-bacteriocin plasmids, pIP964 or pAD1. The Em Tc-resistant transconjugants obtained harbored either the parental plasmid or an Em Tc resistance plasmid derived from pIP964 or pAD1. The restriction endonuclease analysis of 12 derivative plasmids showed insertions of various sizes into different fragments of pIP964 or pAD1. A454 and the Em Tc-resistant plasmid-free transconjugants were found to contain two EcoRI DNA fragments, that shared homology with ³²P-labeled pIP1077, one of the Em Tc resistance derivative plasmids, but not with ³²P-labeled pIP964. No homology was detected between pIP1077 and the cellular DNA of the antibiotic-susceptible recipients.Previously Thea Horodniceanu     

Cation transport and electrogenesis byStreptococcus faecalis

Summary Uptake of the lipid-soluble cations dibenzyldimethylammonium (DDA⁺) and triphenylmethylphosphonium (TPMP⁺) byStreptococcus faecalis is biphasic. The initial phase is a rapid binding of the ions which does not require a source of metabolic energy and apparently consists of cation exchange at the cell surface. Upon addition of glucose further uptake of the cations occurs, by exchange for Na⁺ and H⁺. Evidence is presented suggesting that this metabolic uptake of DDA⁺ and TPMP⁺ is not due to active transport. It rather appears that uptake results from the generation of an electrical potential, interior negative, by the extrusion of H⁺ and, indirectly, of Na⁺. Accumulated DDA⁺ and TPMP⁺ are discharged by proton-conducting uncouplers. The cationconducting antibiotics valinomycin, monactin, nigericin and monensin do not inhibit uptake. Potassium and, under certain conditions, H⁺ displace DDA⁺ and TPMP⁺. Generation of an electrical difference across the membrane was verified by the accumulation of K⁺ in the presence of valinomycin. The concentration ratios achieved correspond to potentials of the order of –150 to –200 mV.     

Characterization of Anti-Listeria monocytogenes Bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis Strains Isolated from Raïb, a Moroccan Traditional Fermented Milk

Seventy-four samples of raïb, a Moroccan traditional fermented milk, were screened for their anti-Listeria monocytogenes activity. Nine lactic acid bacteria with antilisterial activity were isolated and identified as Lactococcus lactis[⁴], Enterococcus faecium[⁴], and E. faecalis[¹]. Antibacterial spectra, determined against 45 target strains, led to the selection of four antibacterial-producing strains, which were further characterized. Their anti-microbial agents, inactivated by one or more proteases, were designed as bacteriocins. Lactococcin R9/2 and R10/1 showed the broadest range of inhibitory action. Anti-bacterial spectra and physico-chemical properties suggest that these bacteriocins were similar to nisin. Enterocin R69 had a specificity of action against Listeria spp., whereas Enterocin R18 had a broad spectrum of activity. Lc. lactis R9/2 and E. faecalis R18 were able to coagulate sterilised UHT milk at 30°C in 24 h and induced a 2 log reduction in L. monocytogenes ATCC 15313 population.     

The action of nitrosoguanidine and other DNA-inactivating agents on Streptococcus pyogenes K56 strains with normal and reduced dark repair ability

Summary The response pattern for ultraviolet light, nitrogen mustard, and ethyl methane sulphonate of Hcr⁺ and Hcr^- strains ofStreptococcus pyogenes K 56 is similar to that observed for analogous strains ofE. coli, whereas repair-apt streptococcal strains are much more sensitive to nitrosoguanidine and mitomycin C thanE. coli. Theuvr gene(s) appear(s) to be without effect upon survival, prophage induction, and mutation to streptomycin resistance caused by nitrosoguanidine and only of little influence on repair of mitomycin C damage.     

Enantioselective biocatalytic hydrolysis of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-mandelonitrile for production of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid by a newly isolated mutant strain

(R)-(−)-Mandelic acid (R-MA) is an important intermediate with broad uses. Recently, R-MA production using nitrilase has been gaining more and more attention due to its higher productivity and enantioselectivity. In this work, a new bacterium WT10, which exhibited favorable nitrilase activity and excellent enantioselectivity for production of R-MA by enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile, was isolated and identified as a strain of Alcaligenes faecalis. In order to improve its nitrilase activity for industrial application, the wild-type strain WT10 was further subjected to mutagenesis using a combined LiCl–ultraviolet irradiation and low energy N⁺ ion beams implantation technique. A valuable mutant strain A. faecalis ZJUTB10 was obtained. The nitrilase specific activity of the mutant strain was greatly improved up to 350.8 U g⁻¹, in comparison with wild-type strain WT10 of 53.09 U g⁻¹. The reaction conditions for R-MA production by mutant strain A. faecalis ZJUTB10 were also optimized. Nitrilase activity in mutant strain showed a broad pH optimum at pH 7.7–8.5. The optimal temperature was 35°C. The highest production rate reached 9.3 mmol h⁻¹ g⁻¹. The results showed that mutant strain A. faecalis ZJUTB10 was a new candidate for efficient R-MA production from (R,S)-mandelonitrile and could potentially be used in industrial production.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

Lactobacillus gasseri LF221 and K7 — from isolation to application

The article presents research findings on two human strains with probiotic activity. On the basis of API 50 CHL fermentation pattern, PCR by species-specific primers and sequencing of the V2–V3 region of 16S rRNA both strains designated as LF221 and K7 were identified as members of the Lactobacillus gasseri species. Two LF221 bacteriocins, acidocin LF221 A and B were purified and sequenced. They were classified as members of the two-component class II bacteriocins. Among basic probiotic properties, the survival under conditions in gastro-intestinal tract, ability to adhere to cultured intestinal enterocytes and pig’s mucosa and stimulation of the immune response were demonstrated. In in vivo study of 24 weaned piglets, the survival rate of K7 Rif^r and LF221 Rif^r was quantified by selective enumeration on MRS agar with rifampicin. The survival of both strains was good (2.9 × 10⁵ cfu of K7 Rif^r /g faeces; 4.8 × 10⁵ cfu of LF221 Rif^r /g) and the LF221 Rif^r /K7 Rif^r viable cells were found either in the mucosa of duodenum, jejunum or in the ileum. The possible effect of K7 to inhibit adhesion of E. coli O8:K88 to enterocytes was studied on Caco-2 cultured cells, on tissue obtained from small intestines of pigs and in vivo on gnotobiotic piglets. Lactobacilli were found to be effective in reducing E. coli adhesion to enterocytes in Caco-2 model, but not on mucosa of pig’s jejunum under ex vivo conditions. Competitive exclusion, production of organic acids and stimulation of immune response, were involved in inhibition of E. coli by K7 strain in gnotobiotic piglets. Any inflammatory change in intestines of piglets treated with K7 was observed, which confirmed its safe use. Among the technological parameters the survival and activity of the strains during cheese-making are presented. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Genetic reidentification of the pectinolytic yeast strain SCPP as Saccharomyces bayanus var. uvarum

Using genetic hybridization analysis, pulsed-field gel electrophoresis of chromosomal DNA and PCR/RFLP analysis of the MET2 gene, we reidentified 11 Champagne yeast strains. Two of them, SCPP and SC4, were found to belong to Saccharomyces bayanus var. uvarum and the remaining strains to S. cerevisiae. Strain SCPP (CLIB 2025) of S. bayanus var. uvarum is known as a producer of three pectinolytic enzymes. Received: 28 April 2000 / Received revision: 20 July 2000 / Accepted: 25 July 2000     

Bacteriocins Pep5 and Epidermin Inhibit Staphylococcus epidermidis Adhesion to Catheters

Pep5 and epidermin bacteriocins were tested on clinical strains of Staphylococcus epidermidis and S. aureus isolated from catheter-related infections. These bacteriocins were inhibitory to several isolates at a concentration of 640 activity units mL⁻¹. The ability of bacteriocins in inhibiting adhesion of S. epidermidis to silicone catheters was evaluated. When Pep5 and epidermin were added to in vitro catheter colonization experiments, there was a significant decrease in the cell number of S. epidermidis adhered to silicone catheters. Bacteriocins used to decrease bacterial attachment to medical devices may represent a novel strategy to control catheter-related infections.     

Antimicrobial efficacy of lactoferrin, its amidated and pepsin-digested derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens

Aims: To evaluate the in vitro bactericidal efficacy of lactoferrin (LF), its amidated (AMILF) and pepsin‐digested (PDLF) derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens. Methods and Results: PDLF exhibited the most potent bactericidal efficacy on E. coli O157:H7 (>2·5 log₁₀ CFU ml^?1 reduction at concentrations ≥1 mg ml^?1), and AMILF on Ser. liquefaciens (1 log₁₀ CFU ml^?1 reduction at 0·25–0·50 mg ml^?1). Some combinations of LF with PDLF or AMILF showed a slight synergy on E. coli O157:H7 and Ser. liquefaciens. However, all combinations of AMILF with PDLF were less active than the sum of the individual effects of the two antimicrobials. Production of capsular polysaccharide by bacteria might be involved in antimicrobial resistance. Conclusions: Escherichia coli O157:H7 and Ser. liquefaciens showed marked differences in the sensitivity to LF and its derivatives. E. coli O157:H7 was strongly inhibited by PDLF, whereas the effect of LF and its derivatives on Ser. liquefaciens was weak to negligible. Significance and Impact of the Study: PDLF was the most promising of the tested antimicrobials on E. coli O157:H7. However, the resistance of Ser. liquefaciens to LF and its derivatives hinders their use in the food industry.     

Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.