Microbial manganese reduction mediated by bacterial strains isolated from aquifer sediments

One hundred and five strains isolated from aquifer sediments andEscherichia coli ML30S were tested for their ability to reduce manganese oxides. Eighty-two strains, includingE. coli, reduced manganese. In most cases the bacterial activity decreased the pH and Eh below 6.75 and 350 mV, respectively, enhancing a spontaneous and nonspecific reduction of manganese. However, for 12 strains the reduction was specifically catalyzed by bacteria; the high pH and Eh values would not permit a spontaneous reduction of manganese. Some of the most active strains were identified as genera common in soils and waters, i.e.,Pseudomonas, Bacillus, Corynebacterium, andAcinetobacter. Two strains were studied in detail. One of the strains, identified asPseudomonas fluorescens, required contact between the cells and the manganese oxides for reduction to occur. The reduction was inhibited by 15 mM of sodium azide. The other strain, identified asAcinetobacter johnsonii, catalyzed manganese reduction by an inductive and dialyzable substance which was excreted by the bacteria. The mechanism involved has not been previously demonstrated.     

Bacterial feeders, the nematode Caenorhabditis elegans and the flagellate Cercomonas longicauda, have different effects on outcome of competition among the Pseudomonas biocontrol strains CHA0 and DSS73

How bacterial feeding fauna affects colonization and survival of bacteria in soil is not well understood, which constrains the applicability of bacterial inoculants in agriculture. This study aimed to unravel how food quality of bacteria and bacterial feeders with different feeding habits (the selective feeding flagellate Cercomonas longicauda versus the non-selective feeding nematode Caenorhabditis elegans) influence the abundance of two bacteria that compete for resources in simple model communities. Microcosms consisted of either one gfp-tagged bacterial strain (Pseudomonas fluorescens DSM50090 or one of two biocontrol strains P. fluorescens CHA0 or Pseudomonas sp. DSS73) or combinations of two bacterial strains. DSM50090 is a suitable food bacterium, DSS73 is of intermediate food quality, and CHA0 is inedible to the bacterial feeders. Bacterial and protozoan cell numbers were measured by flow cytometry. In the presence of flagellates, CHA0 increased its abundance as compared to the other biocontrol strain DSS73 or to DSM50090, which were both eaten by the flagellates. In contrast, the number of CHA0 declined as compared to DSS73 when the model community was subjected to nematode predation pressure. Hence, the results suggested that the outcome of competition among bacteria depended on their ability to cope with the prevailing bacterial predator.     

药用植物青蒿不同种类的内生菌抑菌活性分析

为了研究青蒿不同种类的内生菌抑制细菌和抑制真菌的活性,该研究采用组织块法和研磨法从青蒿的根、茎、叶中分离内生细菌、放线菌和真菌,以大肠埃希菌(Escherichia coli)(CICC 23657)、枯草芽孢杆菌(Bacillus subtilis)(CICC 10275)、金黄色葡萄球菌(Staphylococcus aureus)(CICC 10384)、黑曲霉(Aspergillus niger)(CICC 2487)、酿酒酵母(Saccharomyces cerevisiae)(CICC 33032)为指示菌,采用琼脂块法和双层平板法检测内生菌的抑菌活性。结果表明:(1)从青蒿植株中共分离到76株内生菌,其中内生细菌19株、内生放线菌34株、内生真菌23株。从分离部位来看,56株来自于茎段、17株来自于根段、3株来自叶片。(2)内生细菌中抑菌活性菌株占总菌株的比例最高,为95%,内生放线菌和内生真菌中抑菌活性菌株的比例分别为41%、35%。(3)内生细菌的抗菌谱较广;虽然内生放线菌的抗菌谱较窄,但其中高抗菌株较多,尤其对酿酒酵母的抑菌效果好。综上结果显示,药用植物青蒿中存在着丰富的有抑菌活性的内生菌,且不同种类的内生菌抑菌活性不同。     

Influence of Host Plant Genotype, Presence of a Pathogen, and Coinoculation with Pseudomonas fluorescens Strains on the Rhizosphere Expression of Hydrogen Cyanide- and 2,4-Diacetylphloroglucinol Biosynthetic Genes in P. fluorescens Biocontrol Strain CHA0

The production of hydrogen cyanide (HCN) and 2,4-diacetylphloroglucinol (DAPG) is a major factor in the control of soil-borne diseases by Pseudomonas fluorescens CHA0. We investigated the impact of different biotic factors on the expression of HCN–in comparison to DAPG biosynthetic genes in the rhizosphere. To this end, the influence of plant cultivar, pathogen infection, and coinoculation with other biocontrol strains on the expression of hcnA-lacZ and phlA-lacZ fusion in strain CHA0 was monitored on the roots of bean. Interestingly, all the tested factors influenced the expression of the two biocontrol traits in a similar way. For both genes, we observed a several-fold higher expression in the rhizosphere of cv. Derakhshan compared with cvs. Goli and Naz, although bacterial rhizosphere colonization levels were similar on all cultivars tested. Root infection by Rhizoctonia solani stimulated total phlA and hcnA gene expression in the bean rhizosphere. Coinoculation of strain CHA0 with DAPG-producing P. fluorescens biocontrol strains Pf-68 and Pf-100 did neither result in a substantial alteration of hcnA nor of phlA expression in CHA0 on bean roots. To our best knowledge, this is the first study investigating the impact of biotic factors on HCN production by a bacterial biocontrol strain in the rhizosphere.     

Investigation of extracellular phospholipase and proteinase activities of Candida species isolated from individuals denture wearers and genotypic distribution of Candida albicans strains

In order to determine the relationship between the development of denture related stomatitis (DRS) and the production of phospholipase and proteinase by Candida species, 156 Candida isolates isolated from the individuals in the control group and from the individuals different denture wearers were included in this study. According to the results of the study, C. albicans strains were determined to produce high levels of phospholipase and proteinase. It was also determined that the prevalence of phospholipase and proteinase activities in C. albicans strains isolated from individuals with DRS and from the individuals without DRS was not different. In order to determine genotypic variation of 109 C. albicans strains isolated, CA-INT-L and CA-INT-R primers specific to the site of the transposable group I intron of the 25S rRNA gene (rDNA) region were used. As a result, it was considered that, there were several other virulence factors belonging to the microorganism which played a role in the development mechanisms of the infection caused by C. albicans. In addition, according to the results of microbial genotyping, it was determined that there were no C. albicans strains specifically responsible for the development of DRS.     

Systemic Resistance in Tomato Induced by Biocontrol Bacteria Against the Root-Knot Nematode, Meloidogyne javanica is Independent of Salicylic Acid Production

Salicylic acid (SA)‐mediated induction of systemic resistance by Pseudomonas aeruginosa strain 7NSK2 and P. fluorescens strain CHA0 against soil‐borne fungi and viruses have been reported. The role of SA biosynthesis in the enhancement of defence mechanism against plant‐parasitic nematodes by these bacterial strains in tomato is not known. To better understand the importance of SA in rhizobacteria‐mediated suppression of root‐knot nematodes, biocontrol potential of SA‐negative or SA‐overproducing mutants against Meloidogyne javanica was evaluated with their respective wild type counter parts. Culture supernatant of 7NSK2, CHA0 and their respective mutants caused significant mortality of M. javanica juveniles in vitro. SA deletion in 7NSK2 and SA overproduction in CHA0 did not influence bacterial efficacy to cause nematode deaths. Similarly, culture supernatants resulting from King's B liquid medium amended with FeCl₃ did not influence nematicidal activity of the bacterial strains. Strain CHA0 induced juvenile deaths more than 7NSK2 did. In pot experiments, the bacterial strains applied in unsterilized sandy loam soil markedly reduced final nematode population densities in roots and subsequent root‐knot infection in tomato seedlings. SA‐negative or overproducing derivatives prevented tomato roots in kinetics similar to those with their respective wild types. When soil iron concentration was lowered by the addition of ethylenediamine di(o‐hydroxyphenylacetic acid), nematode biocontrol by the bacterial strains (both wild type and mutants) remained unaltered. To understand the mechanism involved in rhizobacteria‐mediated suppression of root‐knot nematode in tomato, bacterial performance was assessed in a split root trial in which one‐half of the root system was treated with bacterium while the other inoculated with nematode. Compared with the controls, application of the bacterial cell suspension to one‐half of the root system lowered the populations of root‐knot nematode in non‐bacterized nematode‐treated sections indicating enhanced defence in the non‐bacterized half. With respect to nematode infection, mutants induced systemic resistance to a similar extent as that caused by the wild types in both wild type tomato and NahG tomato plants. It is concluded that fluorescent pseudomonads induce systemic resistance against root‐knot nematode via a signal transduction pathway, which is independent of SA accumulation in roots.     

Engineering of Klebsiella oxytoca for production of 2,3‐butanediol using mixed sugars derived from lignocellulosic hydrolysates

2,3‐Butanediol (2,3‐BDO) is a promising bulk chemical owing to its high potential in industrial applications. Here, we engineered Klebsiella oxytoca for the economic production of 2,3‐BDO using mixed sugars from renewable biomass. First, to improve xylose consumption, the xylose transporter gene (xylE) was integrated into the methylglyoxal synthase A (mgsA)‐coding gene loci, and the engineered CHA004 strain showed much faster consumption of xylose than wild‐type (WT) strain with 1.4‐fold increase of overall sugar consumption rate. To further improve sugar utilization, we performed adaptive laboratory evolution for 90 days. The evolved strain (CHA006) was evaluated by cultivating it in the media containing single‐ or mixed‐sugars, and it was clearly observed that CHA006 has improved sugar consumption and 2,3‐BDO production than those of the parental strain. Finally, we demonstrated the superiority of CHA006 by culturing in two lignocellulosic hydrolysates derived from sunflower or pine tree. Particularly, in the pine tree hydrolysate containing xylose, glucose, galactose, and mannose, the CHA006 strain showed much improved consumption rates for all sugars, and 2,3‐BDO productivity (0.73 g L^?1 hr^?1) increased by 3.2‐fold compared to WT strain. We believe that the engineered CHA006 strain can be a potential host in the development of economic bioprocess for 2,3‐BDO through efficient utilization of mixed sugars derived from lignocellulosic biomass.     

Physiological and pathogenic characteristics of Nocardia brasiliensis isolated from human mycetomas

A previous analysis of the physiological properties of Nocardia brasiliensis strains isolated from soil of Tucumán proves that non-pathogenic strains have a different behaviour pattern from the pathogenic strains.In the present paper, 16 Nocardia brasiliensis strains isolated from human mycetomas were studied in the same way. The object is to determine if any of the Nocardia brasiliensis present in soil can produce human mycetomas.The macro and micromorphological, biochemical (17 tests), physiological (4 tests) and pathological characteristics were determined for each of the strains. Experimental pathogenicity was determined using albino Swiss mice by inoculation into the footpads.The strains of Nocardia brasiliensis that cause human mycetomas have the same physiological pattern and experimental pathogenicity as the virulent strains present in soil.     

Drug Resistance of Enteric Bacteria

Drug resistance of 3,000 Shigella strains isolated in 1965 were investigated. These strains originated from 10 City Hospitals and 4 Prefectural Health Centers, which are located in different parts of Japan. One hundred and seventy strains which were resistant to 4 drugs, chloramphenicol (CM), tetracycline (TC), dihydrostreptomycin (SM), and sulfanilamide (SA), were selected at random from these stock cultures in this laboratory and the distribution of R factors in these isolates was examined. It was found that the strains all harbored R factors which were capable of transferring drug resistance by usual conjugal process. Among the strains carrying R factors, 85 per cent harbored a single type of R factor and 15 per cent carried two types of R factor in a cell. The latter is called the hetero-R state. Among the strains in the hetero-R state, isolation of strains harboring both R (SM.SA) and R (TC.CM.SM.SA) factors was most frequent. It was found that 25 R (SM.SA) factors isolated from strains in hetero-R had the genetic determinant iR^?, while most of the R (TC.CM.SM.SA) factors isolated from natural sources were iR⁺. When two types of R factor, R (SM.SA) and R (TC.CM.SM.SA) derived from the same host cells, were brought together in a host cell by superinfection with both factors, they were found to exist stably in a host bacterium. These results confirmed the stable existence of both factors in Shigella strains isolated from dysenteric patients.     

Phylogenetic analysis of manganese-oxidizing fungi isolated from manganese-rich aquatic environments in Hokkaido, Japan

Three strains of Mn-oxidizing fungi were isolated from manganese-rich aquatic environments: sediment in a stream (Komanoyu) in Mori-machi and inflow to an artificial wetland in Kaminokuni-cho, Hokkaido, Japan. The characteristics of each strain were then established. Genetic analysis based on the ribosomal RNA (rRNA) gene was performed to clarify their classification. The sequences of the 18S rRNA and internal transcribed spacer (ITS1)-5.8S rRNA-ITS2 genes showed that all three strains are Ascomycetes. Based on its morphology, it seems probable that the KY-1 strain from Mori-machi belongs to the genus Phoma or Ampelomyces. The phylogenetic analysis indicates that this strain belongs to Phoma rather than Ampelomyces. Morphological identification of WL-1 and WL-2 strains from Kaminokuni-cho was impossible because of the lack of a sexual stage and specific organs. Phylogenetic analysis of the sequence in the ITS1-5.8S rRNA-ITS2 gene suggests that the WL-1 strain corresponds to Paraconyothyrium sporulosum and that WL-2 also belongs to the genus Paraconiothyrium. Because the ability to oxidize Mn has not been evaluated for most species of Phoma or Paraconiothyrium (Coniothyrium), further study is needed to confirm the status of these three strains.     

Selective isolation of bacteria from soil with hydrophobic materials

Bacterial strains having a hydrophobic cell surface have often been considered as degraders of hydrophobic organic pollutants in soil. In this study, bacterial strains were isolated using hydrophobic materials from 12 soil samples, and their cell surface hydrophobicity was determined by evaluating their adherence to n-hexane. Bacterial strains isolated using polytetrafluoroethylene (PTFE) membrane were more hydrophobic on an average than those isolated with styrene–divinylbenzene (DVB) particles or octadecylsilyl silica gel (ODS) particles. Strains closely related to Burkholderia cepacia could be selectively isolated using the PTFE membrane; those closely related to Ralstonia pickettii, using ODS and DVB particles; and those closely related to B. fungorum, using DVB. These results indicate that bacterial strains having a hydrophobic cell surface or within certain phyla can be selectively isolated from soils using hydrophobic materials, and that this isolation method would be useful for collecting candidates for bioremediation of hydrophobic pollutants.     

海南西海岸真红树内生细菌多样性及其延缓衰老活性研究

该文设计9种分离培养基,采用稀释涂布法从14份真红树植物的46份组织样品中分离纯化内生细菌。并基于菌株形态学特征和16S rRNA基因序列确定分离菌株的种属及分析其物种多样性,采用秀丽隐杆线虫模型筛选菌株延缓衰老活性。结果表明:(1)通过基因序列去重复后从46份真红树植物组织样品中获得32株海洋细菌,基于菌株16S rRNA基因序列信息分析,覆盖12科17属,其中芽孢杆菌属(Bacillus)为优势菌属,并获得1株疑似橙单胞菌属(Aurantimonas)新种,16S rRNA基因序列相似性低于97%;(2)经过秀丽隐杆线虫粗筛发现3株海洋细菌具有显著延缓线虫衰老的活性(P0.05)。以上结果表明海南西海岸真红树内生细菌具有物种多样性,部分菌株具有延缓线虫衰老活性。     

Subclasses of Adenosine Receptors in Brain Membranes from Adult Tissue and from Primary Cultures of Chick Embryo

Abstract: Membranes from adult chicken brain have high-affinity binding sites for N⁶-cyclohexyl[³H]adenosine (CHA) (K_D= 4 nM, Bmax = 0.6 pmol/mg protein). This CHA binding could be attributed to adenosine receptors of the A₁ type, since substituted adenosine analogs, e.g. N⁶-(l -2-phenylisopropyl)adeno sine (IC50 = 60 nM), were very potent displacers. Binding sites for 1,3-diethyl- 8-[³H]phenylxanthine (DPX) in adult brain membranes have a moderate affinity (K_D= 50 nM, Bmax = 1.5 pmol/mg). The association of DPX with these sites could be completely displaced by 8-phenyltheophylline (IC₅₀= 300 nM) and other xanthines, but only 45% of specific DPX binding could be displaced by phenylisopropyladenosine. This suggests that about half of DPX sites are putative A₁ receptors and the other half are of the A₂ type. Primary cultures of pure glial and neuronal cells from chick embryo brain were also examined for adenosine receptors. Specific binding of CHA could not be detected in these preparations, but both glial and neuronal membranes have specific sites for DPX. At a [³H]DPX concentration of 20 nM, specific binding was 50% higher (per mg protein) in glial than in neuronal membranes. The maximum binding of DPX to glial membranes (B_max= 1.6 pmol/mg) was comparable to values for adult brain, but the glial affinity (K_D= 90 nM) was somewhat less. Phenylisopropyladenosine was able to displace less than 20% of the total glial sites for DPX. This finding was in accord with the lack of CHA sites and demonstrates that A₁ receptors make little contribution to DPX binding in glial membranes. In decreasing order of potency, 8-phenyltheophylline, CHA, theophylline, caffeine, and 3-isobutyl-I-methylxanthine completely displace DPX association with glia. DPX binding to glial membranes thus appears due to a single class of receptors, which may prove to be of the A₂ type.     

Epidemiology and molecular typing of Candida isolates from burn patients

This study, spread over a span of 2 years describes Candida infections in burn patients of an Indian hospital. A total of 220 burn patients were monitored and Candida could be isolated from 138 patients. A total of 228 different Candida species were obtained from various body locations of these patients. Species identification revealed that Candida albicans was the most predominant (45) followed by Candida tropicalis(33), Candida glabrata (13.5), C. parapsilosis (4), C. krusei (2.75) and C. kefyr (1.75). DNA fingerprinting of all C. albicans isolates was done by using CARE-2 probe. Fingerprinting analyses of all the C. albicans strains revealed that strains collected from different patients were different. It is noteworthy that patients with disseminated candidiasis had a similar, but unique strain isolated from all body locations, suggesting a possibility that commensal isolates might be turning pathogenic. Taken together, this is probably the first ever detailed survey of Candidainfections in burn patients in India and is expected to lead to better clinical management of this group of patients.     

Phenotypic and Genetic Diversity of Erwinia carotovora ssp. carotovora Strains from Asia

A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR‐restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S‐23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α‐methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia.     

Physiological and Biochemical Characteristics of the Halophilic Bacteria Vibrio parahaemolyticus and V. alginolyticus Isolated from Marine Invertebrates of Peter the Great Bay, Sea of Japan

One hundred strains of halophilic vibrios were isolated from 16 species of marine invertebrates of Peter the Great Bay. Based on their morphological and biochemical characteristics, the bacteria were identified as Vibrio parahaemolyticus and Vibrio alginolyticus. Bacterial isolates possessed virulence enzymes (DNAase, lecithinase, catalase) and were characterized by a high enterotoxigenicity. It was determined that 76% of the V. parahaemolyticus strains and 43% of the V. alginolyticus strains were Kanagawa-positive. The isolates showed a high adhesive capability, the average adhesion index was 18.06 cells per erythrocyte for V. parahaemolyticus and 12.55 for V. alginolyticus. The results of this study suggest a high pathogenic potential of the isolated halophilic vibrios, which are an epidemic hazard to marine invertebrates and to humans.     

Identification of Arcanobacterium haemolyticum isolated from postcastrational complications of a horse

An Arcanobacterium haemolyticum strain isolated from a postcastrational lesion of a horse was identified phenotypically and genotypically. The latter was performed by sequencing the 16S–23S rDNA intergenic spacer region (ISR), by amplification of the gene encoding A. haemolyticum phospholipase D, by amplification of A. haemolyticum specific parts of ISR-23S rDNA and by amplification of the newly described CAMP factor family protein encoding gene of A. haemolyticum. This indicates (as described previously for seven additional A. haemolyticum strains; Hassan et al. 2009) that A. haemolyticum seems to occur also in infections of horses.     

Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus

While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from α-linoleic acid by the enzyme linoleate isomerase (LI, EC 5.2.1.5) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers.     

Isolation of myo-Inositol from Solutions by Enzymatic Biotransformation

The transferase reaction between phospholipids and inositol catalyzed by phospholipase D was studied at interfaces in water–organic solvent systems. Optimum conditions were determined for phosphatidylinositol synthesis in heterogeneous water–organic solvent systems. Hydrophobic components (phospholipids) were readily separated from water-soluble products (alcohols) in systems with organic solvents. In the hexane–water system, addition of methanol (an alcohol substrate) to the reaction medium displaced myo-inositol from the molecule of phosphatidylinositol. myo-Inositol was isolated from the mixture of its isomers using a two-step transferase reaction catalyzed by phospholipase D.