Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes

Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse effects on potato-associated bacteria which frequently exhibit plant beneficial functions such as plant growth promotion and antagonism towards pathogens invading the plant. Two field experiments were carried out in Spain to analyze the potential effects of conventional and genetically modified T4-lysozyme producing potatoes on shoot-associated bacteria. The first baseline field trial 2002 was performed in Meliana in which three conventional potato lines, Achirana Inta, Desirée, and Merkur, were cultivated and sampled at flowering. The second field trial was conducted in Cella in 2003 in order to compare the effects of a senescent transgenic, T4 lysozyme expressing potato trait, Desirée DL 12, with its isogenic, non-transformed parental line Desirée. Structural characteristics of potato shoot-associated bacteria was assayed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and dominant community members within T-RFLP profiles were identified by sequence analysis of generated 16S rRNA gene libraries. Cultivable bacteria isolated from shoots of potatoes grown in the Meliana field trial were monitored for antibiosis against Ralstonia solanacearum, whereas isolates derived from shoots of potatoes cultivated in the Cella trial were screened for antagonism against Ralstonia solanacearum and Rhizoctonia solani, and for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Determined antagonists were identified by 16S rRNA gene analysis. All potato traits hosted a cultivar-specific community of bacteria with antagonism against the pathogens and/or potential to produce ACC deaminase. Several antagonists obtained from the Cella field potatoes were also observed as ACC deaminase producers. Community profiling revealed a greater diversity differentiation between the senescent T4 lysozyme expressing and parental Desirée lines grown in the Cella field as compared to the variations between the three flowering conventional lines cultivated in the Meliana field trial. Effects of the two varying field sites and different vegetation stages were greater than those of T4 lysozyme when investigating the community composition of bacteria colonizing the shoots of the Desirée line cultivated in both field trials.     

Bacteriolytic activities of the free-living soil amoebae,Acanthamoeba castellanii,Acanthamoeba polyphaga andHartmannella vermiformis

Bacteriolytic activities of axenically grown free-living soil amoebaeAcanthamoeba castellanii, Acanthamoeba polyphaga andHartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of bothAcanthamoeba species were higher as those of the threeHartmannella strains.Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus andPseudomonas fluorescens were more easily lysed than the other bacteria tested.Agrobacterium tumefaciens, Klebsiella aerogenes andSerratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3–10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities inAcanthamoeba andHartmannella were shown. Bacteriolytic activities ofHartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0–9.3 while forAcanthamoeba the range was pI 4.5–8.9.Abbreviations IEF isoelectric focusing - PAA-IEF polyacrylamide-isoelectric focusing - CCAP culture collection of algae and protozoa - AS amoeba saline medium - pI isoelectric points     

Nickel resistance in Escherichia coli V38 is dependent on the concentration used for induction

Strain Escherichia coli V38 resistant to 4 mM NiCl₂ was isolated from the city sewage sludge. It showed low nickel accumulation by cells and nickel ion efflux. Cells were pregrown (induced) overnight in the presence of Ni²⁺, then the culture was kept on ice for 20–30 min and transferred to 37°C for further incubation. When the Ni²⁺ concentration during growth was the same as during incubation, there was no noticeable accumulation of Ni²⁺. When the Ni²⁺ concentration during incubation was higher than that used for induction, uptake of ⁶³Ni²⁺ and delayed efflux were seen. The uptake and delay of both efflux and growth were directly proportional to the difference between the concentrations used for induction and incubation. Active nickel ion uptake was seen in cells taken from cultures in the delayed efflux period.     

Antibacterial activities of the methanol extracts of Canarium schweinfurthii and four other Cameroonian dietary plants against multi-drug resistant Gram-negative bacteria

Bacterial infections are among the major cause of morbidity and mortality worldwide. The present study was designed to evaluate the in vitro antibacterial activities of the methanol extracts of five Cameroonian edible plants namely Colocasia esculenta, Triumfetta pentandra, Hibiscus esculentus, Canarium schweinfurthii and Annona muricata against a panel of 19 multidrug resistant Gram-negative bacterial strains. The liquid broth microdilution was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extracts. The preliminary phytochemical screening of the extracts was conducted according to the standard phytochemical methods. Results showed that all extracts contained compounds belonging to the classes of polyphenols, triterpenes and steroids, other classes of chemicals being selectively distributed. Canarium schweinfurthii extract showed the best activity with MIC values ranging from 64 to 1024 μg/mL against 89.5% of the 19 tested bacteria strains. MIC values below or equal to 1024 μg/mL were also recorded with Triumfetta pentandra, Annona muricata, Colocasia esculenta and Hibiscus esculentus extracts respectively against 15/19 (78.9%), 11/19 (57.9%), 10/19 (52.6%) and 10/19 (52.6%) tested bacteria. Extract from C. schweinfurthii displayed the lowest MIC value (64 μg/mL) against Escherichia coli AG100A_Tet. Finally, the results of this work provide baseline information for the use of C. esculenta, T. pentandra, H. esculentus, C. schweinfurthii and A. muricata in the treatment of bacterial infections including multidrug resistant phenotypes.     

Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli

Abstract A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn 5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum . The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum , including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength.     

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.