Isolation and characterization of entomopathogenic bacteria from soil samples from the western region of Cuba

The use of insect pathogens is a viable alternative for insect control because of their relative specificity and lower environmental impact. The search for wild strains against dipterans could have an impact on mosquito control programs. We have made an extensive screening of soil in western Cuba to find bacteria with larvicidal activity against mosquitoes. A total of 150 soil samples were collected and isolates were identifying using the API 50 CHB gallery. Phenotypic characteristics were analyzed by hierarchical ascending classification. Quantitative bioassays were conducted under laboratory conditions following the World Health Organization protocol in order to ascertain the toxicity and efficacy of isolates. The protein profiles of the crystal components were determined by SDS‐PAGE. Eight hundred and eighty‐one bacterial isolates were obtained, and 13 isolates with entomopathogenic activity were isolated from nine samples. Nine isolates displayed higher entomopathogenic activity against both Cx. quinquefasciatus and Ae. aegypti compared with the reference strain 266/2. All toxic isolates showed higher biological potency than the 266/2 strain. These isolates with high entomopathogenic activity displayed a protein pattern similar to the B. thuringiensis var. israelensis IPS‐82 and 266/2 strains. These results are a valuable tool for the control of Diptera of medical importance.     

Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria

Genetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc-alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane-spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane-spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram-negative and Gram-positive bacteria possess a similar protein export apparatus.     

Isolation and identification of quorum quenching bacteria from environmental samples

A large number of Gram-negative pathogens produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Therefore, strategies to inhibit QS are promising for the control of infectious diseases. The aim of the present study was to develop a high-throughput method for the isolation and identification of AHL-degrading bacteria from environmental samples. Samples were cultured in a microtitre plate in a minimal medium containing 1 mM N-(3-oxo-dodecanoyl)-l-homoserine lactone and 2 mM N-(3-oxo-hexanoyl)-l-homoserine lactone as the sole sources of carbon and nitrogen. Isolates growing on this minimal medium were subcultured and identified by partial 16S rRNA gene sequencing. Subsequently, the AHL-degrading capacity of each isolate was evaluated in the Pseudomonas aeruginosa QSIS2 biosensor assay, as such or after treatment with heat or proteinase K. The 16 samples tested yielded a total of 59 isolates which are, either alone or as part of a consortium, able to use AHL signal molecules as sole sources of carbon and nitrogen. Follow-up experiments have shown that in each sample there is at least one isolate with quorum quenching (QQ) activity, and that for all samples combined, 41 isolates have QQ activity. Furthermore, heat treatment did not fully inhibit QQ activity in all isolates. In some isolates, QQ activity was lost after proteinase K treatment, while others remained able to quench QS. Therefore, it is likely that some isolates produce and secrete (a) heat-stable, low molecular weight inhibitory compound(s).     

Isolation of antimicrobial producing Actinobacteria from soil samples

Emergence of multidrug resistant bacteria has made the search for novel bioactive compounds from natural and unexplored habitats a necessity. Actinobacteria have important bioactive substances. The present study investigated antimicrobial activity of Actinobacteria isolated from soil samples of Egypt. One hundred samples were collected from agricultural farming soil of different governorates. Twelve isolates have produced activity against the tested microorganisms (S. aureus, Bacillus cereus, E. coli, K. pneumoniae, P. aeruginosa, S. Typhi, C. albicans, A. niger and A. flavus). By VITEK 2 system version: 07.01 the 12 isolates were identified as Kocuria kristinae, Kocuria rosea, Streptomyces griseus, Streptomyces flaveolus and Actinobacteria. Using ethyl acetate extraction method the isolates culture’s supernatants were tested by diffusion method against indicator microorganisms. These results indicate that Actinobacteria isolated from Egypt farms could be sources of antimicrobial bioactive substances.     

Isolation of denitrifying bacteria from hydrocarbon-contaminated Antarctic soil

In this study, we report the isolation of denitrifiers from hydrocarbon-contaminated Antarctic soils. Seventy-two isolates were obtained from soils that had received a fertilizer treatment to stimulate hydrocarbon degradation. All isolates, except one, belonged to the genus Pseudomonas. The one exception was a member of the Microbacteriaceae, which was also, coincidentally, the only isolate negative for the nirS gene. The diversity of the 16S rRNA and nosZ genes was assessed by denaturing gradient gel electrophoresis and sequencing. There was a slight correlation between the 16S rRNA and nosZ operational taxonomic units. Surprisingly, many isolates contained nosZ on plasmids and, to the best of our knowledge, this is the first report of nosZ being extra-chromosomally present in Pseudomonas spp.     

红树林土壤中产聚羟基脂肪酸酯细菌的分离及其评估

[背景] 细菌能通过合成聚羟基脂肪酸酯（Polyhydroxyalknoates,PHA）在细胞内储存物质和能量,提高对环境的适应能力。在红树林中,由于土壤周期性受海水浸没,形成营养物质种类丰富和含量波动大的特殊生境,为细菌进化出特殊的PHA合成途径提供了条件。[目标] 为了增加对红树林产PHA细菌资源的了解,获得产PHA细菌,使用纯培养方法分离和鉴定细菌,并评估菌株的产PHA能力。[方法] 采集红树植物海桑根系和红树滩涂土壤样品,连续5周培养、分离纯化获得细菌菌株;通过16S rRNA基因相似性及系统进化分析鉴定细菌分类地位,利用PHA合成酶基因（phaC）鉴定细菌合成PHA的能力;通过基因组草图测序,分析细菌的phaC基因种类、代谢通路及系统进化关系;通过气相色谱分析细菌产PHA的累积量及组成。[结果] 从红树林土壤样品中分离得到97株细菌,其中13株带有phaC基因,包括坚强芽孢杆菌（Cytobacillus firmus）、弯曲芽孢杆菌（Bacillus flexus）、除烃海杆菌（Marinobacter hydrocarbonoclasticus）和酯香微杆菌（Microbacterium esteraromaticum）。B. flexus MN15-19以丙酮酸盐为碳源,可累积细胞干重11%的PHA,同时具有固碳功能的还原性三羧酸循环通路,有开发成为固碳产PHA工程菌株的潜力。酯香微杆菌可产PHA,但是其phaC基因结构特殊,基因组注释未能识别出任何已知phaC基因。[结论] 研究发现红树林土壤可培养细菌中存在未知的PHA合成途径,说明红树林生态系统中的细菌具有资源挖掘的重要价值。     

盐碱土壤PAHs 降解菌的筛选鉴定及其降解特性

采用富集培养的方法,从天津大港油田PAHs污染盐碱化土壤中分离出一株能以菲、芘为唯一碳源和能源的优势菌TJB5。经形态观察和16S rDNA序列分析结果表明,该菌株为成团泛菌(Pantoea agglomerans)。采用液体培养的方法,研究了pH、盐度、菲芘的初始浓度对TJB5菌株降解菲芘效果的影响,确定了最佳降解条件。结果表明,该菌对菲、芘的降解具有较广泛的pH、盐度范围和良好的降解效果。在菲、芘浓度分别为50 mg/L、pH 6.8-9.5、盐度2%-3%、温度30°C条件下,接种15 d后菲降解率在93.3%以上,芘降解率在20%以上。     

Isolation of lightning-competent soil bacteria

Artificial transformation is typically performed in the laboratory by using either a chemical (CaCl(2)) or an electrical (electroporation) method. However, laboratory-scale lightning has been shown recently to electrotransform Escherichia coli strain DH10B in soil. In this paper, we report on the isolation of two "lightning-competent" soil bacteria after direct electroporation of the Nycodenz bacterial ring extracted from prairie soil in the presence of the pBHCRec plasmid (Tc(r), Sp(r), Sm(r)). The electrotransformability of the isolated bacteria was measured both in vitro (by electroporation cuvette) and in situ (by lightning in soil microcosm) and then compared to those of E. coli DH10B and Pseudomonas fluorescens C7R12. The electrotransformation frequencies measured reached 10(-3) to 10(-4) by electroporation and 10(-4) to 10(-5) by simulated lightning, while no transformation was observed in the absence of electrical current. Two of the isolated lightning-competent soil bacteria were identified as Pseudomonas sp. strains.     

Isolation and properties of barophilic and barotolerant bacteria from deep-sea mud samples

Several barophilic and barotolerant bacteria were isolated from deep-sea mud samples of Suruga Bay (2485 m depth), the Ryukyu Trench (5110 m depth), and the Japan Trench (land-side 6356 m, and sea-side 6269 m depth, respectivelys. The barophilic bacteria, strains DB5501, DB6101, DB6705 and DB6906, were albe to grow better under high hydrostatic pressures than under atmospheric pressure (0.1 megapascals; MPa). The optimal growth pressures for the barophilic bacteria were approximately 50 MPa at 10°C. The barotolerant strains DSK1 and DSS12 were determined to be psychrophilic, and had optimal growth temperatures of 10°C and 8°C, respectively. The degree of barophily and barotolerance was shown to be very dependent on temperature. For example, at 4°C the barophilic strains were indistinguishable from barotolerant bacteria, whereas at 15°C the barotolerant strains behaved more like the barophilic strains. Based on sequence analysis of 16S ribosomal DNA, all of the strains included in this study belong to the gamma subgroup of the Proteobacteria. Phylogenetic relations between the isolated strains and the known gamma subgroup bacteria suggested that the isolated strains belong to a new sub-branch of this group.     

一株红壤溶磷菌的分离、鉴定及溶磷特性

【目的】为了提高红壤磷素利用率,探讨溶磷菌溶磷机理。【方法】利用难溶性无机盐培养基从花生根际土壤样品中分离到一株溶磷菌C5-A,结合菌落形态特征、生理生化和16S rRNA序列确定该菌株的系统发育地位;通过菌株C5-A在NBRIP液体培养基培养过程中培养液pH变化确定其溶磷能力;利用液体发酵实验测定不同的碳源、氮源对菌株C5-A溶磷的影响;通过高效液相色谱检测C5-A在不同氮源培养液中有机酸的种类和浓度。【结果】菌株C5-A鉴定为洋葱伯克霍尔德氏菌(Burkholderia cepacia),遗传稳定性较好。在FePO4和AlPO4培养液中,菌株C5-A的溶磷量和pH变化呈显著负相关;菌株C5-A对磷酸三钙、磷酸铝、磷酸铁、磷矿粉均有较强的溶解能力,最高溶磷量分别为125.79、227.34、60.02和321.15 mg/L;菌株C5-A对不同浓度的两种磷矿粉有较强的溶解能力;分别以麦芽糖和草酸铵为碳源和氮源时溶磷量最高。高效液相色谱检测出10种有机酸,分别为草酸(葡萄糖酸)、乙酸、苹果酸、琥珀酸和5种未知有机酸,然而,乙酸而非草酸似乎是影响C5-A溶磷的重要有机酸。【结论】从红壤花生根际土壤中筛选到一株对难溶性无机盐具有较强溶解能力溶的菌株C5-A,有望为开发高效红壤微生物磷肥提供种质资源。     

Isolation and study of azobenzene-transforming soil bacteria

Heterotrophic bacteria were isolated from soil and glass slides and classified as Bacillus cereus SNK12, Paenibacillus polymyxa SNK2, Azotobacter chroococcum ANKII, and Ochrobactrum intermedium ANKI. Their cultures could degrade azobenzene under the conditions of co-metabolism. A rapid test for the ability of bacteria to transform azobenzenes is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 185–188.Original Russian Text Copyright © 2005 by Wackerow-Kouzova.     

Isolation and characterization of polyphenols-degrading bacteria from olive-mill wastewaters polluted soil

In this study 28 bacterial strains, isolated from greenwaters-polluted-soil, were investigated for their ability to grow in presence of phenols added to Mineral Basal Medium (MBM) in aerobic conditions. In particular, three of them were found to be able to use as sole carbon source phenol, cathecol, caffeic acid and ferulic acid with efficiency ranging from 76% (phenol in 5 days, millimolar concentration from 3.7 10^?2 to 9 10^?3) to 95% (ferulic acid in 2 days millimolar concentration from 6.8 10^?1 to 3 10^?2). For these strains the taxonomic position was studied by amplification and sequencing of 16S rRNA genes. The isolated strains were classified belonging to Arthrobacter sulfureus, Pseudomonas synxantha and Pseudomonas oryzihabitans. Noteworthy, for the first time such Pseudomonas strains have been shown to be able to use polyphenols as the only carbon source in vitro. In fact, to the best of our knowledge, this kind of study were not done on Ps. Synxantha, while it was recently shown the ability of P. oryzihabitans to degrade catechol. These findings may open to new biotechnological applications for the degradation of polyphenols.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

Isolation of Gemmata-like and Isosphaera-like planctomycete bacteria from soil and freshwater.

New cultured strains of the planctomycete division (order Planctomycetales) of the domain Bacteria related to species in the genera Gemmata and Isosphaera were isolated from soil, freshwater, and a laboratory ampicillin solution. Phylogenetic analysis of the 16S rRNA gene from eight representative isolates showed that all the isolates were members of the planctomycete division. Six isolates clustered with Gemmata obscuriglobus and related strains, while two isolates clustered with Isosphaera pallida. A double-membrane-bounded nucleoid was observed in Gemmata-related isolates but not in Isosphaera-related isolates, consistent with the ultrastructures of existing species of each genus. Two isolates from this study represent the first planctomycetes successfully cultivated from soil.     

海州香薷（Elsholtzia splendens）根际铜抗性细菌的筛选及生物多样性

摘要：【目的】重金属耐性植物海州香薷根际铜抗性细菌的筛选及生物多样性研究将有助于了解微生物－超富集植物相互关系和植物修复机理、开发微生物－香薷重金属修复新技术。【方法】采用稀释平板涂布法从海州香薷根际筛选铜抗性菌株,测定菌株溶磷和产生吲哚乙酸、铁载体、1-氨基环丙烷-1-羧酸（ACC）脱氨酶的特性,采用16S rDNA限制性酶切多态性分析（amplified rDNA restriction analysis, ARDRA）研究铜抗性细菌的遗传多样性,根据16S rDNA相似性对产ACC脱氨酶的菌株进行了     

Isolation of antibiotic-producing Actinomycetes from soil samples exposed to UV light

The use of nonroutine means in isolation of microorganisms from natural substrates extended the possibilities of detecting new cultures which often appear to be producers of previously unknown antibiotics. A new procedure for isolating actinomyces of definite groups was developed. It implies preliminary exposure of soil suspensions to UV light. With the use of the procedure, 2539 strains of actinomycetes belonging to different genera were isolated. There was a marked decrease after the irradiation in isolation of cultures belonging to Streptomyces, a genus most widely distributed in nature and studied in detail while isolation of cultures belonging to other genera, promising as sources of novel antibiotics, increased. Micromonospora, Amycolatopsis and Nocardia proved to be the most stable to the effect of UV light. With the use of the procedure it is possible to increase 2-3-fold isolation of cultures belonging to Micromonospora, a genus known as a producer of many antibiotics including those used clinically.     

Isolation and study of azobenzene converting soil bacteria

Heterotrophic bacteria were isolated from soil and glass slides and classified as Bacillus cereus SNK12, Paenibacillus polymyxa SNK2, Azotobacter chroococcum ANKII, and Ochrobacterium intermedium ANKI. Their cultures could degrade azobenzene under the conditions of co-metabolism. A rapid test for the ability of bacteria to convert azobenzenes is proposed.