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Cross-species induction and enhancement of antimicrobial activity produced by epibiotic bacteria from marine algae and invertebrates, after exposure to terrestrial bacteria.

Antibiotic producing marine bacteria isolated from surfaces of the marine alga Fucus vesiculosus and the nudibranch Archidoris pseudoargus were exposed to live cells of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and heat-killed cells of Staph. aureus. Twelve out of the 16 marine strains tested showed enhanced antimicrobial activity towards Staph. aureus, E. coli and Ps. aeruginosa following this exposure. Three out of seven strains tested showed enhanced antimicrobial activity when exposed to Ps. aeruginosa and three out of seven strains showed enhanced antimicrobial activity when exposed to E. coli. These results suggest that production of antimicrobial compounds by marine bacteria can be induced by the presence of terrestrial bacteria. This appears to be the first example of cross-species induction and enhancement of antimicrobial activity in marine bacteria and has important implications for the design of antibiotic screening assays and for an understanding of microbial competition in the environment.

Animals↗

Microbial receptor assay for rapid detection and identification of seven families of antimicrobial drugs in milk: collaborative study.

A microbial competitive receptor assay for detecting residues of antibiotic families in milk was studied collaboratively by 13 laboratories. The drugs and levels (ppb) tested in this study include penicillin G, 4.8; cephapirin, 5.0; cloxacillin, 100; tetracycline, 2000; chlortetracycline, 2000; oxytetracycline, 2000; erythromycin, 200; lincomycin, 400; clindamycin, 400; sulfamethazine, 75; sulfamethoxazole, 50; sulfisoxazole, 50; streptomycin, 1000; novobiocin, 50; and chloramphenicol, 800. In this method, microbial cells added to a milk sample provide specific binding sites for which 14C or 3H labeled drug competes with drug residues in the sample. The 14C or 3H binding to the specific binding sites is measured in a scintillation counter and compared with a zero standard milk. If the sample is statistically different from the zero standard, it is positive. The assay takes about 15 min. The binding reaction occurs between the receptor site and the drug functional group, so all members of a drug family are detected. In this case, beta-lactams, tetracyclines, macrolides, aminoglycosides, novobiocin, chloramphenicol, and sulfonamides, including p-aminobenzoic acid (PABA) and its other analogs, are detectable. The incidence of false negative determinations among samples is about 1%; the incidence of false positives is about 3%. For negative cases, the relative standard deviations for repeatability ranged from 0 to 5% and for reproducibility from 0 to 6%. For positive cases, relative standard deviations ranged from 0 to 13% for repeatability and from 0 to 14% for reproducibility. The method has been adopted official first action.

4-Aminobenzoic Acid↗

Simultaneous biodegradation of methyl parathion and carbofuran by a genetically engineered microorganism constructed by mini-Tn5 transposon.

A genetically engineered microorganism (GEM) capable of simultaneous degrading methyl parathion (MP) and carbofuran was successfully constructed by random insertion of a methyl parathion hydrolase gene (mpd) into the chromosome of a carbofuran degrading Sphingomonas sp. CDS-1 with the mini-transposon system. The GEM constructed was relatively stable and cell viability and original degrading characteristic was not affected compared with the original recipient CDS-1. The effects of temperature, initial pH value, inoculum size and alternative carbon source on the biodegradation of MP and carbofuran were investigated. GEM cells could degrade MP and carbofuran efficiently in a relatively broad range of temperatures from 20 to 30 degrees C, initial pH values from 6.0 to 9.0, and with all initial inoculation cell densities (10(5)-10(7) CFU ml(-1)), even if alternative glucose existed. The optimal temperature and initial pH value for GEM cells to simultaneously degrade MP and carbofuran was at 30 degrees C and at pH 7.0. The removal of MP and carbofuran by GEM cells in sterile and non-sterile soil were also studied. In both soil samples, 50 mg kg(-1) MP and 25 mg kg(-1) carbofuran could be degraded to an undetectable level within 25 days even if there were indigenous microbial competition and carbon sources effect. In sterile soil, the biodegradation rates of MP and carbofuran were faster, and the decline of the inoculated GEM cells was slower compared with that in non-sterile soil. The GEM constructed in this study was potential useful for pesticides bioremediation in natural environment.

Biodegradation, Environmental↗

Influence of mycotoxin producing fungi (Fusarium, Aspergillus, Penicillium) on gluten proteins during suboptimal storage of wheat after harvest and competitive interactions between field and storage fungi.

Cereals contaminated by Aspergillus spp., Penicillium spp., and Fusarium spp. and their mycotoxins, for example, ochratoxin A (OTA) and deoxynivalenol (DON), are not only a risk to human and animal health but can also show poor technological properties and baking quality. The influence of these genera on the sulfur speciation of low molecular weight (LMW) subunits of glutenin was characterized by investigating suboptimally stored wheat samples in situ by X-ray absorption near edge structure (XANES) spectroscopy and baking tests. Field fungi of the genus Fusarium have hardly any influence on both the sulfur speciation of wheat gluten proteins and the baking properties, whereas storage fungi of the genera Aspergillus and Penicillium have a direct influence. An increased amount of sulfur in sulfonic acid state was found, which is not available for thiol/disulfide exchange reactions in the gluten network, and thus leads to a considerably reduced baking volume. From changes of the composition of the mould flora during suboptimal storage of wheat and from the mycotoxin contents, it can be concluded that microbial competitive interactions play an important role in the development of the mould flora and the mycotoxin concentrations during (suboptimal) storage of wheat.

Aspergillus↗

Effect of processing time on enteric bacteria survival and on temperature and chemical composition of broiler poultry litter processed by two methods.

This study was conducted to evaluate the effects of deepstack processing broiler poultry litter with or without aeration on enteric bacteria survival (Trial 1), and to determine the optimum aeration period (Trial 2) and aeration frequency (Trial 3) to control excessive heating of litter during the deepstacking process. The effects of deepstacked litter temperature on bacteria survival and on chemical compositions of the litter were monitored. In Trial 1, E. coli, Salmonella enteriticis and Shigella sonnei were intentionally inoculated into the litter and their survival was monitored with time of litter processing. In Trial 2, litter was deepstacked and aerated once a day from the initial day or from the day when maximum stack temperature was reached and started to decrease to the end of processing period. In Trial 3, litter was deepstacked and aerated once or twice a day from the day after maximum stack temperature was attained until constant temperature was attained. During the deepstacking process of litter pathogenic bacteria were eliminated between the 2nd and 4th day of deepstacking. This phenomenon occurred apparently not only by high heat generated, but also by other potential factors, such as ammonia and microbial competition. Aeration resulted in the heat dissipation and early temperature reduction of deepstacked litter. The optimum aeration procedure to avoid excessive heating of deepstacked litter was to aerate once a day after the stack temperature reached its maximum at the early stage of deepstacking and for 3 days or until the stack temperature dropped off and reached constancy. This aerating frequency and duration did not negatively affect the chemical composition of deepstacked litter. These results suggest that poultry litter properly deepstacked, with or without aeration, to ensure the elimination of enteric bacterial pathogens may be fed at least 10 days earlier than the 21-day deepstacking period previously recommended.

Air↗

Bacillus polymyxa stimulates increased Rhizobium etli populations and nodulation when co-resident in the rhizosphere of Phaseolus vulgaris.

Microbial competition for carbon sources is a primary determinant of rhizosphere ecology. We employed the PCR to examine the population fluctuations of a symbiotic nitrogen-fixing bacterium (Rhizobium etli) during the first 11 days following inoculation of Phaseolus vulgaris seedlings grown in the presence or absence of a common asymbiotic rhizosphere resident (Bacillus polymyxa). When B. polymyxa was applied as a co-inoculant, increases in both early rhizobial root populations and final root population densities were observed as compared to single inoculation with R. etli. Modifications to host plant growth (including increased lateral root formation and nodules number) were found concomitant with elevations in R. etli populations on plants co-inoculated with both bacterial genera. In contrast to the in planta results, population enhancements were not observed when R. etli and B. polymyxa were co-cultured in vitro using minimal media in the absence of the seedling. Addition of seed exudate to the growth media also failed to stimulate the population increases observed during co-release in planta. These results suggest that B. polymyxa acts indirectly (i.e., via the plant host) to increase R. etli populations. Our observed synergism among co-resident bacteria supports the hypothesis that microbial communities which colonize the spermosphere may play a significant role in plant development and rhizosphere ecology.

Bacillus↗

Genetics and molecular physiology of the yeast Kluyveromyces lactis.

With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology. In particular, comparative yeast research has been providing insights into the strikingly different physiological strategies that are reflected by dominance of respiration over fermentation in K. lactis versus Saccharomyces cerevisiae. Other than S. cerevisiae, whose physiology is exceptionally affected by the so-called glucose effect, K. lactis is adapted to aerobiosis and its respiratory system does not underlie glucose repression. As a consequence, K. lactis has been successfully established in biomass-directed industrial applications and large-scale expression of biotechnically relevant gene products. In addition, K. lactis maintains species-specific phenomena such as the "DNA-killer system, " analyses of which are promising to extend our knowledge about microbial competition and the fundamentals of plasmid biology.

Aerobiosis↗

Molecular approaches to problems in biogeochemical cycling.

By using molecular probe techniques in combination with activity and expression measurements, it is possible to estimate bacterial populations in nature. This information can be exploited to study a number of important environmental problems. For instance, it will be possible to study ecosystem perturbation and microbial competition, by altering an ecosystem or a laboratory model of an ecosystem, and assessing corresponding changes in key activities and populations. In addition, regulation of activities in the laboratory can be compared to the response of activities and populations in situ, to develop an understanding of the key parameters that control these processes in nature. These types of approaches are important steps for determining the role of microorganisms in geochemical cycling, in both specific habitats and on a global basis.

Antigens, Bacterial↗

Aboveground and belowground responses to quality and heterogeneity of organic inputs to the boreal forest.

Leaf litter and other organic resources returned to the soil are important regulators of ecological processes in forest ecosystems, and their ecological impacts may be strongly influenced both by their quality and by interactions between coexisting resource types. To date, most studies on effects of resource identity and mixing have only involved leaf litter, despite the fact that other resource types constitute a major input to the soil. We investigated how quality and heterogeneity of organic substrates found in boreal forests affects the activity and community structure of soil microbes, and plant growth. Six organic substrates (wood, charcoal, berries, sporocarps, vertebrate faeces and leaf litter) were added singly or in mixtures of two, three and six resource types to pots containing forest soil (with or without tree seedlings of Betula pendula Roth). The largest positive effects of single substrates on microbial basal respiration (BR), substrate-induced respiration (SIR) and microbial metabolic quotient (qCO(2)) were found for nutrient-rich substrates (faeces and sporocarps) or substrates with high sugar-content (berries). Mixing of substrates had no effect on BR or SIR, but decreased qCO(2) or altered the microbial community structure for specific combinations of substrates. In contrast to the niche complementarity hypothesis, microbial catabolic diversity was not stimulated by greater diversity of resources. Seedling growth responses to single substrates were neutral or negative; the inhibition of growth probably resulted largely from microbial competition for nutrients. Substrate mixing enhanced seedling nutrient-uptake and growth for all mixtures containing sporocarps and leaf litter. Overall, plants responded more strongly to resource heterogeneity than microbes, and synergistic effects only occurred when nutrient-rich substrates were present within the substrate mixtures. In particular, our results demonstrate a role for complex and non-additive interactions among previously overlooked resource types returned to the soil in influencing ecosystem functions such as nutrient cycling and plant productivity.

Analysis of Variance↗

Effect of low-dose irradiation on growth of and toxin production by Staphylococcus aureus and Bacillus cereus in roast beef and gravy.

The effect of irradiation (2 kGy) on growth of and toxin production by Staphylococcus aureus and Bacillus cereus in roast beef and gravy during storage at abuse temperatures (15 and 22 degrees C) was assessed by inoculation studies. Irradiation resulted in a 3-4 log10 reduction in numbers of both pathogens. Whenever B. cereus and S. aureus numbers reached 10(6) and 10(7) cfu/g, respectively, during storage their toxins were detectable. As the time taken to attain these levels was longer in irradiated than in unirradiated samples, toxin production by both pathogens was delayed by irradiation. When samples initially containing low levels (10(2)/g) of S. aureus were irradiated no toxin was produced during subsequent storage at 15 or 22 degrees C. Diarrhoeal toxin produced by B. cereus was detected after 2 days at 22 degrees C, but not at 15 degrees C, in samples containing 10(2) cells/g prior to irradiation. When higher numbers (10(6)/g) of either pathogen were present prior to irradiation, toxins were produced by both pathogens at 22 degrees C, but not at 15 degrees C. Microbial competition had an effect on the growth of B. cereus and S. aureus after irradiation when a low initial inoculum was applied. However, when a higher inoculum was used the pathogens outnumbered their competitors and competition effects were less important. It was concluded that low-dose irradiation would improve the microbiological safety of roast beef and gravy.

Animals↗

Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay.

Research on lipase inhibitors could help in the therapy of diseases caused by lipase-producing microorganisms and in the design of novel lipase substrate specificities for biotechnology. Here we report a fast and sensitive colorimetric microassay that is low-cost and suitable for high-throughput experiments for the evaluation of lipase activity and inhibition. Comparison of Candida rugosa activity and inhibition with previous HPLC results validated the method, and revealed the importance of the reaction mixture composition. The assay was used to evaluate the effect of saturated fatty acids on Bacillus-related lipases. Cell-bound esterases were strongly inhibited by fatty acids, suggesting a negative feedback regulation by product, and a role of these enzymes in cell membrane turnover. Bacillus subtilis LipA was moderately activated by low concentrations of fatty acids and was inhibited at greater concentrations. LipB-like esterases were highly activated by myristic and lauric acids and were only slightly inhibited by high capric acid concentrations. Such an activation, reported here for the first time in bacterial lipases, seems to be part of a regulatory system evolved to ensure a high use of carbon sources, and could be related to the successful adaptation of Bacillus strains to nutrient-rich environments with strong microbial competition.

Bacillus↗

Effects of cell motility and chemotaxis on microbial population growth.

A mathematical model is developed to elucidate the effects of biophysical transport processes (nutrient diffusion, cell motility, and chemotaxis) along with biochemical reaction processes (cell growth and death, nutrient uptake) upon steady-state bacterial population growth in a finite one-dimensional region. The particular situation considered is that of growth limitation by a nutrient diffusing from an adjacent phase not accessible to the bacteria. It is demonstrated that the cell motility and chemotaxis properties can have great influence on steady-state population size. In fact, motility effects can be as significant as growth kinetic effects, in a manner analogous to diffusion- and reaction-limited regimes in chemically reacting systems. In particular, the following conclusions can be drawn from our analysis for bacterial populations growing at steady-state in a confined, unmixed region: (a) Random motility may lead to decreased population density; (b) chemotaxis can allow increased population density if the chemotactic response is large enough; (c) a species with superior motility properties can outgrow a species with superior growth kinetic properties; (d) motility effects become greater as the size of the confined growth region increases; and (e) motility effects are diminished by significant mass-transfer limitation of the nutrient from the adjacent source phase. The relationships of these results for populations to previous conclusions for individual cells is discussed, and implications for microbial competition are suggested.

Bacterial Physiological Phenomena↗

Focus on modified microcins: structural features and mechanisms of action.

Microcins are gene-encoded antimicrobial (poly)peptides secreted by Enterobacteriaceae. Produced under conditions of nutrient depletion, they are active against phylogenetically related microbial strains. Therefore, they are considered to play an important role in the microbial competitions within the intestinal flora. Among the limited sample of nine microcins hitherto described, a wide variety of structures and modes of action could be identified. The knowledge on microcins is very uneven, some being extensively studied, and others remaining uncharacterized. In this article, we have focused on a subgroup of highly modified microcins that show very original structures. We present an updated overview on the structures and mechanisms of action of microcins B17, C7 and J25, and on the associated effector proteins, also encoded by the microcin genetic system, which include specific modification enzymes, export proteins, and immunity factors.

Bacteriocins↗

Bioreduction of uranium in a contaminated soil column.

The bioreduction of soluble uranium [U(VI)] to sparingly soluble U(IV) species is an attractive remedial technology for contaminated soil and groundwater due to the potential for immobilizing uranium and impeding its migration in subsurface environments. This manuscript describes a column study designed to simulate a three-step strategy proposed for the remediation of a heavily contaminated site at the U.S. Department of Energy's NABIR Field Research Center in Oak Ridge, TN. The soil is contaminated with high concentrations of uranium, aluminum, and nitrate and has a low, highly buffered pH (approximately 3.5). Steps proposed for remediation are (i) flushing to remove nitrate and aluminum, (ii) neutralization to establish pH conditions favorable for biostimulation, and (iii) biostimulation for U(VI) reduction. We simulated this sequence using a packed soil column containing undisturbed aggregates of U(VI)-contaminated saprolite that was flushed with an acidified salt solution (pH 4.0), neutralized with bicarbonate (60 mM), and then biostimulated by adding ethanol. The column was operated anaerobically in a closed-loop recirculation setup. However, during the initial month of biostimulation, ethanol was not utilized, and U(VI) was not reduced. A bacterial culture enriched from the site groundwaterwas subsequently added, and the consumption of ethanol coupled with sulfate reduction immediately ensued. The aqueous concentration of U(VI) initially increased, evidently because of the biological production of carbonate, a ligand known to solubilize uranyl. After approximately 50 days, aqueous U(VI) concentrations rapidly decreased from approximately 17 to <1 mg/L. At the conclusion of the experiment,the presence of reduced solid phase U(IV) was confirmed using X-ray absorption near edge structure spectroscopy. The results indicate that bioreduction to immobilize uranium is potentially feasible at this site; however, the stability of the reduced U(IV) and its potential reoxidation will require further investigation, as do the effects of groundwater chemistry and competitive microbial processes, such as methanogenesis.

Bacteria, Anaerobic↗

Plastic plants and patchy soils.

Soil nutrients are distributed in a non-uniform or 'patchy' manner. It is well established that the modular nature of root systems allows them to show both morphological and/or physiological plasticity upon encountering nutrient-rich patches. These plastic responses are widely believed to be foraging mechanisms by the plant to enhance nutrient resource capture. Although morphological plasticity has traditionally been viewed as the more expensive option as it requires new root construction, more recent evidence suggests this may not necessarily be the case. Moreover, plants may be able to recapture most of the initial outlay involved in new root construction, again lowering the overall cost to the plant. Under natural conditions the roots of most plant species have an additional nutrient acquisition mechanism namely mycorrhizal symbiosis. However, the impact of these important symbiotic associations upon the host plant's response to nutrient patches has received relatively little attention. The mycorrhizal fungal symbiont should, in theory, be better able to compete directly with the rest of the microbial community for the nutrients in the patch. This could potentially be important to the host plant, as generally, root proliferation responses are more important for interspecific plant, than plant-microbial, competition.

Acclimatization↗

Strategies of mixed substrate utilization in microorganisms.

In natural and man-made environments microorganisms often grow in the presence of a diversity of functionally similar substrates. The pattern of utilization of these mixed substrates is generally dependent upon their concentration. When substrates are present in high (not growth-limiting) concentrations, sequential utilization and diauxic growth is often observed and the substrate that supports the highest growth rate is utilized preferentially from the mixture. When the substrate concentrations are growth-limiting, simultaneous utilization of the various compounds present in the mixture appears to be the general response. Recent studies on mixed substrate utilization in both batch and continuous cultures have thrown light on the strategies of the control mechanisms that, in microbes, govern the utilization of the various substrates. But perhaps more importantly these studies have indicated the possible significance of mixed substrate utilization in microbial competition in nutrient-limited natural ecosystems.

Bacteria↗

Anaerobic and aerobic metabolism of glycogen-accumulating organisms selected with propionate as the sole carbon source.

In the microbial competition observed in enhanced biological phosphorus removal (EBPR) systems, an undesirable group of micro-organisms known as glycogen-accumulating organisms (GAOs) compete for carbon in the anaerobic period with the desired polyphosphate-accumulating organisms (PAOs). Some studies have suggested that a propionate carbon source provides PAOs with a competitive advantage over GAOs in EBPR systems; however, the metabolism of GAOs with this carbon source has not been previously investigated. In this study, GAOs were enriched in a laboratory-scale bioreactor with propionate as the sole carbon source, in an effort to better understand their biochemical processes. Based on comprehensive solid-, liquid- and gas-phase chemical analytical data from the bioreactor, a metabolic model was proposed for the metabolism of propionate by GAOs. The model adequately described the anaerobic stoichiometry observed through chemical analysis, and can be a valuable tool for further investigation of the competition between PAOs and GAOs, and for the optimization of the EBPR process. A group of Alphaproteobacteria dominated the biomass (96 % of Bacteria) from this bioreactor, while post-fluorescence in situ hybridization (FISH) chemical staining confirmed that these Alphaproteobacteria produced poly-beta-hydroxyalkanoates (PHAs) anaerobically and utilized them aerobically, demonstrating that they were putative GAOs. Some of the Alphaproteobacteria were related to Defluvicoccus vanus (16 % of Bacteria), but the specific identity of many could not be determined by FISH. Further investigation into the identity of other GAOs is necessary.

Aerobiosis↗

The purification, crystallization and preliminary structural characterization of PhzF, a key enzyme in the phenazine-biosynthesis pathway from Pseudomonas fluorescens 2-79.

Phenazines produced by members of several bacterial genera are biologically active metabolites that function in microbial competitiveness, the suppression of soil-borne plant diseases and virulence in infectious disease. Despite recent progress towards understanding the biochemistry of phenazine synthesis, the key reactions leading to the formation of the heterocyclic scaffold common to all phenazine compounds remain obscure. Pseudomonas fluorescens 2-79 contains seven phenazine (phz) genes that encode components of the pathway for biosynthesis of phenazine-1-carboxylic acid. A central step in this pathway involves the condensation of two identical precursor molecules derived from chorismic acid and is catalysed by the product of the phzF gene. In this study, recombinant PhzF was purified and crystallized from PEG 4000/ammonium sulfate/sodium citrate pH 5.6. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 56.3, c = 156.4 A. They contain one monomer in the asymmetric unit and diffract to better than 1.7 A on synchrotron beamlines. Crystals of seleno-L-methionine-labelled PhzF have been obtained and SAD data are reported.

Crystallization↗