Isolation and characterization of actinobacteria ectosymbionts from Acromyrmex subterraneus brunneus (Hymenoptera, Formicidae)

The ectosymbiont actinobacterium Pseudonocardia was isolated from the integument of Acromyrmex leaf-cutter ants and seems to play a crucial role in maintaining asepsis of the nest. Currently, there has been an intensive search for Pseudonocardia associated with several attine species, but few studies have indicated that other actinobacteria may be associated with these ants as well. We therefore characterized the culturable actinobacteria community associated with the integument of the fungus-growing ant Acromyrmex subterraneus brunneus Forel, 1893 (Hymenoptera: Formicidae). Ectosymbionts were isolated using four different media and characterized by morphological and molecular (16S rDNA) methods. A total of 20 strains were isolated, of which 17 were characterized as Streptomyces spp., and one isolate each as Pseudonocardia, Kitassatospora and Propionicimonas. Unlike other Acromyrmex species, A. subterraneus brunneus is associated with a diversity of actinobacteria. Even though Pseudonocardia is present on this leaf-cutting ant`s integument, the number and diversity of Streptomyces spp. found differs from those of previous studies with other attine ants and suggest that different culturing approaches are needed to characterize the true diversity of microbes colonizing the integument of attine ants. Moreover, understanding the diversity of the culturable actinobacteria associated with A. subterraneus brunneus should increase our knowledge of the evolutionary relationship of this intricate symbiotic association. (C) 2010 Elsevier GmbH. All rights reserved.

Effects of the herbicides acetochlor and metolachlor on antioxidant enzymes in soil bacteria

The aim of this study was to investigate the antioxidant responses of three bacteria (SD1. KD and K9) isolated from soil previously treated with the herbicides metolachlor and acetochlor. By 165 rRNA gene sequencing, we determined that SD1 is phylogenetically related to Enterobacter asburiae, while KD and K9 have divergent genomes that more closely resemble that of Enterobacter amnigenus. Decreased levels of lipid peroxidation were observed in SD1 and KD following treatment with 34 mM metolachlor or 62 mM acetochlor, respectively, indicating that both bacteria were able to adapt to an increase in ROS production. In the presence of 34 mM metolachlor or 62 mM acetochlor, all bacterial isolates exhibited increases in total catalase (CAT) activity (81% for SDI, 53% for KD and 59% for K9), whereas total SOD activity (assessed based on the profile and intensity of the bands) was slightly reduced when the bacteria were exposed to high concentrations of the herbicides (340 mM metolachlor or 620 mM acetochlor). This effect was due to a specific reduction in SOD IV (K9 and KD isolates) by 45% and 90%, respectively, and SOD V (SD1 isolate) isoenzymes by 60%. The most striking result was obtained in the SD1 isolate, where two novel isoenzymes of glutathione reductase (GR) that responded specifically to metolachlor were identified. In addition, acetochlor was shown to induce the expression of a new 57 kDa protein band in the K9 and KD isolates. The bacteria isolated from the herbicide-contaminated soil exhibited an efficient antioxidant system response at herbicide concentrations of up to 34 mM metolachlor or 62 mM acetochlor. These data suggest a mechanism for tolerance that may include the control of an imbalance in ROS production versus scavenging. The data suggest that specific isoenzymes of CAT and GR could be involved in this herbicide tolerance mechanism. (C) 2011 Elsevier Ltd. All rights reserved.

Bacterial community in the rhizosphere and rhizoplane of wild type and transgenic eucalyptus

The rhizosphere is a niche exploited by a wide variety of bacteria. The expression of heterologous genes by plants might become a factor affecting the structure of bacterial communities in the rhizosphere. In a greenhouse experiment, the bacterial community associated to transgenic eucalyptus, carrying the Lhcb1-2 genes from pea (responsible for a higher photosynthetic capacity), was evaluated. The culturable bacterial community associated to transgenic and wild type plants were not different in density, and the Amplified Ribosomal DNA Restriction Analysis (ARDRA) typing of 124 strains revealed dominant ribotypes representing the bacterial orders Burkholderiales, Rhizobiales, and Actinomycetales, the families Xanthomonadaceae, and Bacillaceae, and the genus Mycobacterium. Principal Component Analysis based on the fingerprints obtained by culture-independent Denaturing Gradient Gel Electrophoresis analysis revealed that Alphaproteobacteria, Betaproteobacteria and Actinobacteria communities responded differently to plant genotypes. Similar effects for the cultivation of transgenic eucalyptus to those observed when two genotype-distinct wild type plants are compared.

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of Methylobacterium in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism-plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR-DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.

Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium-plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant cultivation.

Changes in the genetic structure of Bacteria and microbial activity in an agricultural soil amended with tannery sludge

The application of tannery sludge to soils is a form of recycling; however, few studies have examined the impacts of this practice on soil microbial properties. We studied effects of two applications (2006 and 2007) of tannery sludge (with a low chromium content) on the structure of the bacterial community and on the microbial activity of soils. We fertilized an agricultural area in Rolandia, Parana state, Brazil with different doses of sludge based on total N content, which ranged from 0 to 1200 kg N ha(-1). Sludge remained on the soil surface for three months before being plowed. Soils were sampled seven times during the experiment. Bacterial community structure, assessed by denaturing gradient gel electrophoresis (DGGE), was modified by the application of tannery sludge. Soon after the first application, there was clear separation between the bacterial communities in different treatments, such that each dose of sludge was associated with a specific community. These differences remained until 300 days after application and also after the second sludge application, but 666 days after the beginning of the experiment no differences were found in the bacterial communities of the lowest doses and the control. The principal response curve (PRC) analysis showed that the first sludge application strongly stimulated biological activity even 300 days after application. The second application also stimulated activity, but at a lower magnitude and for a shorter time, given that 260 days after the second application there was no difference in biological activity among treatments. PRC also showed that the properties most influenced by the application of tannery sludge were enzymatic activities related to N cycling (asparaginase and urease). The redundancy analysis (RDA) showed that tannery sludge`s influence on microbial activity is mainly related to increases in inorganic N and soil pH. Results showed that changes in the structure of the bacterial community in the studied soils were directly related to changes of their biological activity. (C) 2010 Elsevier Ltd. All rights reserved.

Bacterial stimulation of copper phytoaccumulation by bioaugmentation with rhizosphere bacteria

Copper contaminated areas pose environmental health risk to living organisms. Remediation processes are thus required for both crop production and industrial activities. This study employed bioaugmentation with copper resistant bacteria to improve phytoremediation of vineyard soils and copper mining waste contaminated with high copper concentrations. Oatmeal plant (Avena sativa L) was used for copper phytoextraction. Three copper resistant bacterial isolates from oatmeal rhizosphere (Pseudomonas putida A1 Stenotrophomonas maltophilia A2 and Acinetobacter calcoaceticus A6) were used for the stimulation of copper phytoextraction. Two long-term copper contaminated vineyard soils (Mollisol and Inceptisol) and copper mining waste from Southern Brazil were evaluated. Oatmeal plants substantially extracted copper from vineyard soils and copper mining waste. As much as 1549 mg of Cu kg(-1) dry mass was extracted from plants grown in Inceptisol soil. The vineyard Mollisol copper uptake (55 mg Cu kg(-1) of dry mass) in the shoots was significantly improved upon inoculation of oatmeal plants with isolate A2 (128 mg of Cu kg(-1) of shoot dry mass). Overall oatmeal plant biomass displayed higher potential of copper phytoextraction with inoculation of rhizosphere bacteria in vineyard soil to the extent that 404 and 327 g ha(-1) of copper removal were respectively observed in vineyard Mollisol bioaugmented with isolate A2 (S. maltophilia) and isolate A6 (A. calcoaceticus). Results suggest potential application of bacterial stimulation of phytoaccumulation of copper for biological removal of copper from contaminated areas. (C) 2010 Elsevier Ltd. All rights reserved.

Real-time Polymerase Chain Reaction Quantification of Porphyromonas gingivalis and Tannerella forsythia in Primary Endodontic Infections

Introduction: Porphyromonas gingivalis and Tannerella forsythia are anaerobic bacteria commonly involved in root canal infections. Although previous investigations have assessed these species by strictly qualitative approaches, accurate determination of their cell levels by a sensitive quantitative technique may contribute with additional information regarding relevance in pain of endodontic origin. Method: The root canal levels of P gingivalis, T forsythia, and total bacteria were investigated by a quantitative polymerase chain reaction (PCR) assay based on unique copy molecular markers. A total of 32 symptomatic (n = 14) and asymptomatic (n = 18) cases of endodontic infections were analyzed. Root canal samples were collected; genomic DNA was extracted and submitted to SYBR Green I real-time PCR targeting the rgpB (P gingivalis), bspA (T forsythia), and rpoB (total bacteria) single copy genes. Results: Overall, R gingivalis, T forsythia, and the coexistence of both species were encountered in 28%, 66%, and 22% of the subjects, respectively. P gingivalis and T forsythia levels ranged from 5.65 x 10(-6) to 1.20 x 10(-2) and from 5.76 x 10(-6) to 1.35 x 10(-1). T forsythia was highly prevalent and numerous in the study groups, whereas P gingivalis was moderately frequent and less abundant, displaying 19-fold lower average levels than the former. Conclusions: The endodontic levels of P gingivalis and T forsythia, individually or in conjunction, did not display significant associations with the manifestation of pain of endodontic origin. (J Endod 2009,35:1518-1524)

Effect of the Gamma Radiation Dose Rate on Psychrotrophic Bacteria, Thiobarbituric Acid Reactive Substances, and Sensory Characteristics of Mechanically Deboned Chicken Meat

Frozen samples of mechanically deboned chicken meat (MDCM) with skin were irradiated with gamma radiation doses of 0.0 kGy (control) and 3 kGy at 2 different radiation dose rates: 0.32 kGy/h (3 kGy) and 4.04 kGy/h (3 kGy). Batches of irradiated and control samples were evaluated during 11 d of refrigerated (2 +/- 1 degrees C) storage for the following parameters: total psychrotrophic bacteria count, thiobarbituric acid reactive substances (TBARS), evaluation of objective color (L*, a*, and b*) and a sensory evaluation (irradiated odor, oxidized odor, pink and brown colors). No statistical difference (P > 0.05) was found amongst the TBARS values obtained for the MDCM samples irradiated with dose rates of 0.32 and 4.04 kGy/h. There was a significant increase (P < 0.05) in the psychrotrophic bacterial count as from the 7th day of refrigerated storage, for the MDCM samples irradiated at the dose rate of 4.04 kGy/h. With respect to the attribute of oxidized odor, the samples irradiated with a dose rate of 0.32 kGy/h showed a stronger intensity and were significantly different (P < 0.05) from the sample irradiated with a dose rate of 4.04 kGy/h on days 0 and 2 of refrigerated storage. Irradiation with a dose rate of 4.04 kGy/h (3 kGy) was shown to be the best condition for the processing of MDCM according to the evaluation of all the variables, under the conditions of this study. Practical Application The results obtained for the application of different dose rates of ionizing radiation to mechanically deboned chicken meat will provide the food industry with information concerning the definition of the best processing conditions to maximize the sensory and food quality.

Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry.

Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within Gram-negative bacteria

Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of Gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway that converts tryptophan to PRN is composed of four genes, prnA through D, whose diversity, genomic context and spread over bacterial genomes are poorly understood. Therefore, we launched an endeavour aimed at retrieving, by in vitro and in silico means, diverse bacteria carrying the prnABCD biosynthetic loci in their genomes. Analysis of polymorphisms of the prnD gene sequences revealed a high level of conservation between Burkholderia, Pseudomonas and Serratia spp. derived sequences. Whole-operon- and prnD-based phylogeny resulted in tree topologies that are incongruent with the taxonomic status of the evaluated strains as predicted by 16S rRNA gene phylogeny. The genomic composition of c. 20 kb DNA fragments containg the PRN operon varied in different strains. Highly conserved and distinct transposase-encoding genes surrounding the PRN biosynthetic operons of Burkholderia pseudomallei strains were found. A prnABCD-deprived genomic region in B. pseudomallei strain K96243 contained the same gene composition as, and shared high homology with, the flanking regions of the PRN operon in B. pseudomallei strains 668, 1106a and 1710b. Our results strongly suggest that the PRN biosynthetic operon is mobile. The extent, frequency and promiscuity of this mobility remain to be understood.

Bacterial communities and biogeochemical transformations of iron and sulfur in a high saltmarsh soil profile

Microbial community structure in saltmarsh soils is stratified by depth and availability of electron acceptors for respiration. However, the majority of the microbial species that are involved in the biogeochemical transformations of iron (Fe) and sulfur (S) in such environments are not known. Here we examined the structure of bacterial communities in a high saltmarsh soil profile and discuss their potential relationship with the geochemistry of Fe and S. Our data showed that the soil horizons Ag (oxic-suboxic), Bg (suboxic), Cri (anoxic with low concentration of pyrite Fe) and Cr-2 (anoxic with high concentrations of pyrite Fe) have distinct geochemical and microbiological characteristics. In general, total S concentration increased with depth and was correlated with the presence of pyrite Fe. Soluble + exchangable-Fe, pyrite Fe and acid volatile sulfide Fe concentrations also increased with depth, whereas ascorbate extractable-Fe concentrations decreased. The occurrence of reduced forms of Fe in the horizon Ag and oxidized Fe in horizon Cr-2 suggests that the typical redox zonation, common to several marine sediments, does not occur in the saltmarsh soil profile studied. Overall, the bacterial community structure in the horizon Ag and Cr-2 shared low levels of similarity, as compared to their adjacent horizons, Bg and Cr-1, respectively. The phylogenetic analyses of bacterial 16S rRNA gene sequences from clone libraries showed that the predominant phylotypes in horizon Ag were related to Alphaproteobacteria and Bacteroidetes. In contrast, the most abundant phylotypes in horizon Cr-2 were related to Deltaproteo-bacteria, Chloroflexi, Deferribacteres and Nitrospira. The high frequency of sequences with low levels of similarity to known bacterial species in horizons Ag and Cr-2 indicates that the bacterial communities in both horizons are dominated by novel bacterial species. (c) 2008 Elsevier Ltd. All rights reserved.

Diversity of endophytic bacteria from Eucalyptus species seeds and colonization of seedlings by Pantoea agglomerans

The diversity and beneficial characteristics of endophytic microorganisms have been studied in several host plants. However, information regal-ding naturally, occurring seed-associated endophytes and vertical transmission among different life-history stages of hosts is limited. Endophytic bacteria were isolated from seeds and seedlings of 10 Eucalyptus species and two hybrids. The results showed that endophytic bacteria, Such as Bacillus, Enterococcus, Paenibacillus and Methylobacterium, are vertically transferred from seeds to seedlings. In addition, the endophytic bacterium Pantoea agglomerans was tagged with the gfp gene, inoculated into seeds and further reisolated from seedlings. These results suggested it novel approach to change the profile of the plants, where the bacterium is a delivery vehicle for desired traits. This is the first report of an endophytic bacterial community residing in Eucalyptus seeds and the transmission of these bacteria from seeds to seedlings. The bacterial species reported ill this work have been described as providing benefits to host plants. Therefore, we Suggest that endophytic bacteria can be transmitted vertically from seeds to seedlings, assuring the support of the bacterial community in the host plant.

Quorum sensing detected by atomic force microscopy imaging of corrals surrounding multicellular arrangement of bacteria

Connectivity of the glycocalyx covering of small communities of Acidithiobacillus ferrooxidans bacteria deposited on hydrophilic mica plates was imaged by atomic force microscopy. When part of the coverage was removed by water rinsing, an insoluble structure formed by corrals surrounding each individual bacterium was observed. A collective ring structure with clustered bacteria (>= 3) was observed, which indicates that the bacteria perceived the neighborhood in order to grow a protective structure that results in smaller production of exopolysaccharides material. The most surprising aspect of these collective corral structures was that they occur at a low bacterial cell density. The deposited layers were also analyzed by confocal Raman microscopy and shown to contain polysaccharides, protein, and glucoronic acid.

Potential beneficial properties of bacteriocin-producing lactic acid bacteria isolated from smoked salmon

Aims: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. Methods and Results: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell-free supernatants containing bacteriocins, added to 3-h-old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto-aggregation was strain-specific, and values ranged from 7 center dot 2% for ET35 to 12 center dot 1% for ET05. Various degrees of co-aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco-2 cells was within the range reported for Lactobacillus rhamnosus GG, a well-known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61 center dot 9-64 center dot 6%), Lact. fermentum (78 center dot 9%), Lact. delbrueckii (43 center dot 7%) and Ped. acidilactici (51 center dot 3%), which are higher than the one recorded for Lact. rhamnosus GG (53 center dot 3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain-dependent manner. Conclusions: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco-2 cells was within the range reported for Lact. rhamnosus GG, a well-known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Smoked salmon contains a number of different probiotic LAB and could be marketed as having a potential beneficial effect.