Enumeration of Particle-Bound and Unattached Respiring Bacteria in the Salt Marsh Environment

Proportions of respiring bacteria determined with a 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride dye-epifluorescent technique were significantly elevated in the 300-μm surface layer of a salt marsh estuary. Almost all the detectably respiring bacteria in the particle-laden surface layer and a significant proportion in subsurface waters were attached to particles.     

Thymidine Incorporation by Free-Living and Particle-Bound Bacteria in a Eutrophic Dimictic Lake

The percentage of [methyl-³H]thymidine incorporated into samples from a dimictic eutrophic lake and retained on polycarbonate membranes of 3.0-, 1.0-, and 0.2-μm pore size was studied in a lake with filamentous cyanobacteria as the dominant phytoplankton type throughout the period of thermal stratification. Water samples were also examined by epifluorescence microscopy for evidence of algal senescence and bacterial colonization of intact and damaged cyanobacterial filaments. A small percentage (2 to 20%) of bacterial activity was retained by filters with pore sizes ≥ 1 μm in epilimnetic samples. Epilimnetic samples also had a small percentage of cyanobacterial filaments, either intact or damaged, which were visibly colonized by bacteria in summer and fall samples. A significant proportion (20 to 35%) of bacterial activity was retained by filters with pore sizes ≥ 1 μm in samples collected from the metalimnion and hypolimnion during late summer and fall. The proportion of damaged cyanobacterial filaments was higher in these samples than in those from the epilimnion or from those obtained early in the summer. Furthermore, the filaments in these samples were more heavily colonized by bacteria. Overall, particle-bound production accounted for only 2 to 19% of total bacterial production from April to August in all water layers. It appears that the supply of colonizable particles (damaged cyanobacterial filaments) is an important factor affecting the level of particle-bound bacterial activity in this lake.     

Antagonistic Activity of Five Myrothecium Species Against Fungi and Bacteria in Vitro

Antagonistic activity of 69 Myrothecium isolates belonging to M. carmichaelü, M. cinctum, M. roridum, M. tongaense and M. verrucaria against six soil-borne plant pathogenic fungi was investigated by using the streak method. Results clearly showed that M. carmichaelü is a strong antibiont having a very high degree of antifungal efficacy. M. cinctum and M. tongaense isolates were found to have an antagonistic activity either through antibiosis or mycoparasitism or both against the test fungi. M. roridum and M. verrucaria isolates also possessed both kinds of activities. After this first screening (of all Myrothecium isolates), one isolate from each species exhibiting the highest degree of antagonistic activity was selected for further studies. Inhibitory effects of culture filtrates of these representative isolates on the mycelial growth of the test fungi were investigated by incorporating them into PDA at a dilution of 1:8. M. carmichaelü was the most and M. cinctum the least effective species under assay. Moreover, culture filtrates and mycelium extracts of the selected isolates were found to have both antifungal and antibacterial properties using the agar-well method. Among the test fungi, Sclerotinia sclerotiorum proved to be generally the most, and Pythium debaryanum the least sensitive to the antibiotic effects. M. cinctum showed the widest antibacterial spectrum inhibiting five bacterial species except for Pseudomonas syringae Pv. Phaseolicola. Additionally, the observations using light and scanning electron microscopy clearly demonstrated that all of the five Myrothecium species were able to parasitize all of the tested fungi.     

Exoprotease Activity of Two Marine Bacteria during Starvation

Exoprotease activity during 120 h of total energy and nutrient starvation was examined in two marine bacteria, Vibrio sp. strain S14 and Pseudomonas sp. strain S9. The activity was determined by spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder (hide powder azure). Starved cells of both strains (5 h for S14, and 4 or 24 h for S9) showed greater extracellular proteolytic activity than at the onset of starvation. The exoprotease activity of cells starved for longer periods of time then decreased, but was found to be present at significant levels throughout the starvation period studied (120 h). The accumulation of exoprotease activity in the bulk phase during starvation indicated that both strains constitutively excreted extracellular proteases. As deduced from experiments with chloramphenicol, de novo protein synthesis during starvation was required for the production and/or release of the exoproteases into the surrounding environment. The degradation of hide powder azure allowed an immediate increase in respiration rate, also by long-term-starved cells. This suggests that metabolic systems are primed to respond to the availability of substrates, allowing the cells to recover rapidly. The regulation of exoprotease activity was also studied and found to be different in the two strains. Casamino Acids repressed exoprotease activity in Pseudomonas sp. strain S9, whereas a mechanism similar to catabolite repression was found for Vibrio sp. strain S14 in that glucose repressed activity and cyclic AMP reversed this effect. The exoproteases appeared to be metalloproteinases because the addition of EDTA to cell-free starvation supernatants from both strains significantly inhibited the activity of the proteases.     

十八种辛香料对五种食源性致病菌的抑菌研究(英文)

本实验研究18种辛香料对五种食源性病原菌:单增李斯特菌,大肠杆菌O157∶H7,肠炎沙门氏菌,副溶血性弧菌和金黄色葡萄球菌的抑制作用。辛香料以60℃蒸馏水浸提。研究各种辛香料的最低抑菌浓度(80~5 mg/mL)以及经100℃处理15 m in和121℃处理15 m in的稳定性。结果表明,大部分辛香料具有良好的抑菌效果,尤其是丁香和桂皮对副溶血弧菌和金黄色葡萄球菌的最低抑菌浓度为5 mg/mL。经热处理部分香料失去了抑制作用,如八角,茴香,五香粉,海南白和黑胡椒,但大多数香料保持抑菌效果。这些结果表明,香料可抑制感染海产品的病原菌,同时在加工香料的过程中应该避免高温处理。     

Impacts of Organic and Conventional Crop Management on Diversity and Activity of Free-Living Nitrogen Fixing Bacteria and Total Bacteria Are Subsidiary to Temporal Effects

A three year field study (2007–2009) of the diversity and numbers of the total and metabolically active free-living diazotophic bacteria and total bacterial communities in organic and conventionally managed agricultural soil was conducted using the Nafferton Factorial Systems Comparison (NFSC) study, in northeast England. Fertility management appeared to have little impact on both diazotrophic and total bacterial communities. However, copy numbers of the nifH gene did appear to be negatively impacted by conventional crop protection measures across all years suggesting diazotrophs may be particularly sensitive to pesticides. Impacts of crop management were greatly overshadowed by the influence of temporal effects with diazotrophic communities changing on a year by year basis and from season to season. Quantitative analyses using qPCR of each community indicated that metabolically active diazotrophs were highest in year 1 but the population significantly declined in year 2 before recovering somewhat in the final year. The total bacterial population in contrast increased significantly each year. It appeared that the dominant drivers of qualitative and quantitative changes in both communities were annual and seasonal effects. Moreover, regression analyses showed activity of both communities was significantly affected by soil temperature and climatic conditions.     

Contribution of Crenarchaeota and Bacteria to autotrophy in the North Atlantic interior

Marine Crenarchaeota are among the most abundant groups of prokaryotes in the ocean and recent reports suggest that they oxidize ammonia as an energy source and inorganic carbon as carbon source, while other studies indicate that Crenarchaeota use organic carbon and hence, live heterotrophically. We used catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) to determine the crenarchaeal and bacterial contribution to total prokaryotic abundance in the (sub)tropical Atlantic. Bacteria contributed ～50% to total prokaryotes throughout the water column. Marine Crenarchaeota Group I (MCGI) accounted for ～5% of the prokaryotes in subsurface waters (100 m depth) and between 10 and 20% in the oxygen minimum layer (250–500 m depth) and deep waters (North East Atlantic Deep Water). The fraction of both MCGI and Bacteria fixing inorganic carbon, determined by combining microautoradiography with CARD‐FISH (MICRO‐CARD‐FISH), decreased with depth, ranging from ～30% in the oxygen minimum zone to < 10% in the intermediate waters (Mediterranean Sea Outflow Water, Antarctic Intermediate Water). In the deeper water masses, however, MCGI were not taking up inorganic carbon. Using quantitative MICRO‐CARD‐FISH to determine autotrophy activity on a single cell level revealed that MCGI are incorporating inorganic carbon (0.002–0.1 fmol C cell^?1 day^?1) at a significantly lower rate than Bacteria (0.01–0.6 fmol C cell^?1 day^?1). Hence, it appears that MCGI contribute substantially less to autotrophy than Bacteria. Taking the stoichiometry of nitrification together with our findings suggests that MCGI might not dominate the ammonia oxidation step in the mesopelagic waters of the ocean to that extent as the reported dominance of archaeal over bacterial amoA would suggest.     

Relationships between Sediment Microbial Communities and Pollutants in Two California Salt Marshes

Salt marshes are important ecosystems whose plant and microbial communities can alter terrestrially derived pollutants prior to coastal water discharge. However, knowledge regarding relationships between anthropogenic pollutant levels and salt marsh microbial communities is limited, and salt marshes on the West Coast of the United States are rarely examined. In this study, we investigated the relationships between microbial community composition and 24 pollutants (20 metals and 4 organics) in two California salt marshes. Multivariate ordination techniques were used to assess how bacterial community composition, as determined by terminal restriction fragment length polymorphism and phospholipid fatty acid analyses, was related to pollution. Sea urchin embryo toxicity measurements and plant tissue metabolite profiles were considered two other biometrics of pollution. Spatial effects were strongly manifested across marshes and across channel elevations within marshes. Utilizing partial canonical correspondence analysis, an ordination technique new to microbial ecology, we found that several metals were strongly associated with microbial community composition after accounting for spatial effects. The major patterns in plant metabolite profiles were consistent with patterns across microbial community profiles, but sea urchin embryo assays, which are commonly used to evaluate ecological toxicity, had no identifiable relationships with pollution. Whereas salt marshes are generally dynamic and complex habitats, microbial communities in these marshes appear to be relatively sensitive indicators of toxic pollutants.     

Contribution of Fungi and Bacteria to Leaf Litter Decomposition in a Polluted River

The contribution of fungi and bacteria to the decomposition of alder leaves was examined at two reference and two polluted sites in the Ave River (northwestern Portugal). Leaf mass loss, microbial production from incorporation rates of radiolabeled compounds into biomolecules, fungal biomass from ergosterol concentration, sporulation rates, and diversity of aquatic hyphomycetes associated with decomposing leaves were determined. The concentrations of organic nutrients and of inorganic nitrogen and phosphorus in the stream water was elevated and increased at downstream sites. Leaf decomposition rates were high (0.013 day⁻¹ < k < 0.042 day⁻¹), and the highest value was estimated at the most downstream polluted site, where maximum values of microbial production and fungal biomass and sporulation were found. The slowest decomposition occurred at the other polluted site, where, along with the nutrient enrichment, the lowest current velocity and dissolved-oxygen concentration in water were observed. At this site, fungal production, biomass, and sporulation were depressed, suggesting that stimulation of fungal activity by increased nutrient concentrations might be offset by other factors. Although bacterial production was higher at polluted sites, fungi accounted for more than 94% of the total microbial net production. Fungal yield coefficients varied from 10.2 to 13.6%, while those of bacteria were less than 1%. The contribution of fungi to overall leaf carbon loss (29.0 to 38.8%) greatly exceeded that of bacteria (4.2 to 13.9%).     

Seasonal Variations in Survival of Indicator Bacteria in Soil and Their Contribution to Storm-water Pollution

Survival of a fecal coliform (Escherichia coli) and a fecal streptococcus (Streptococcus faecalis var. liquifaciens) was studied through several years at shaded and exposed outdoor soil plots. Death rates for both organisms were calculated for the different seasons at both sites. The 90% reduction times for the fecal coliform ranged from 3.3 days in summer to 13.4 days in autumn. For the fecal streptococcus, 90% reduction times were from 2.7 days in summer to 20.1 days in winter. During summer, the fecal coliform survived slightly longer than the fecal streptococcus; during autumn, survival was the same; and in spring and winter the fecal streptococcus survived much longer than the fecal coliform. Both organisms were isolated from storm-water runoff collected below a sampling site when counts were sufficiently high in soil. Isolation was more frequent during prolonged rains, lasting up to 10 days, than during short rain storms. There was evidence of aftergrowth of nonfecal coliforms in the soil as a result of temperature and rainfall variations. Such aftergrowth may contribute to variations in bacterial count of storm-water runoff which have no relation to the sanitary history of the drainage area.     

三种海水生态养殖池塘细菌数量特征及其与环境因子的相关性

为阐明不同生态养殖池塘浮游细菌数量变化规律及其与环境因子间的相关性,以期为多元立体综合养殖模式中的水质安全与生物疾病防控提供科学依据。利用荧光显微镜细菌计数法(Acridine Orange Direct Counting),研究了“参-虾”“蟹-蛏”和“蜇-蛏”三种海水生态养殖池塘的细菌数量特征,使用典范对应分析(Canonical Correspondence Analysis, CCA)探讨细菌数量与环境因子的相互关系。结果表明,“参-虾”“蟹-蛏”和“蜇-蛏”池塘的细菌平均密度分别为(0.49～3.32)×10⁵ cell/mL、(0.52～2.98)×10⁵ cell/mL和(0.47～2.55)×10⁵ cell/mL,细菌数量有明显的时间差异,夏季密度大于春秋季。三种海水生态养殖池塘的水温、溶解氧和pH变化均不明显,且相差不大,而硝态氮均发生显著变化;其中“蟹-蛏”和“蜇-蛏”池塘的硝态氮、亚硝态氮、活性磷、总氮和总磷均高于“参-虾”池塘,透明度、叶绿素a和化学耗氧量则均低于“参-虾”池塘。典范对应分析(C...     

Arsenic Contamination in Agricultural Soil Reduces Metabolic Activity of Total and Free-Living Nitrogen-Fixing Bacteria as Revealed by Real-Time qPCR

Laboratory microcosms were used to assess the impact of arsenic (As) contamination on agricultural soil bacterial activity focusing free-living nitrogen fixers for three months. Periodically collected microcosm samples were analyzed by RT–qPCR following extraction of total RNA and cDNA preparation for assessing the metabolically active bacterial population. RT–qPCR data showed the gradual increase of 16S rRNA and nifH gene expression, and relative activity of diazotrophs in the enriched soil bacterial consortia under short time As exposure up to 20 ppm and 10 ppm, respectively. A similar trend of these variables was also noticed but up to 1 ppm As when incubating the bulk soil for the same duration. Reduced bacterial activity was noticed at higher concentration of As although in short time exposure. Extending the As exposure time, the bacterial activities in both enriched consortia and bulk soil were decreased. Although, the relative activity of diazotrophs in enriched consortia was increased in presence of 10 ppm As, the same was decreased in bulk soil when exposed to >1 ppm As for long time indicating susceptibility of nitrogen fixer to As contamination in soil. PCA of the data obtained also indicated a negative correlation between As concentrations and diazotrophic activity.     

Thermostability and Refolding of Proteins in Bacteria Is Determined by the Activity of Two Different ATP-Dependent Chaperone Groups

Molecular Biology - The thermal stability of protein enzymes is determined in vitro by measuring the enzymatic activity during incubation at constant temperature. Refolding of thermal inactivated...     

Levels of Total Airborne Bacteria,Gram-Negative Bacteria,and Endotoxin According to Biosafety Levels in Korean Biosafety Laboratories

We assessed in this study the total airborne bacteria (TAB), gram-negative bacteria (GNB), and endotoxin (ET) concentrations in three Korean university laboratories and two hospital diagnostic laboratories categorized as biosafety level 1 (BL 1) or biosafety level 2 (BL 2). We also investigated the concentrations of TAB, GNB, and ET relative to the performance results of biosafety cabinets (BSCs). TAB and GNB were incubated, and ET was analyzed using the kinetic Limulus amebocyte lysate assay. A total of 246 (TAB = 95, GNB = 95, ET = 56) air samples were collected from nine laboratories. TAB, GNB, and ET concentrations were significantly higher in BL 1 laboratories and in laboratories that failed the BSC performance test compared to BL 2 laboratories and laboratories that passed the BSC performance test. Correlation coefficients were calculated to determine the relationships between TAB, GNB, and ET, and it was found that TAB significantly correlated with GNB (r = 0.35, p < .01) and ET (r = 0.29, p < .01). Careful attention needs to be paid to BL 1 laboratories, and BSCs should be managed on a regular basis to improve the air quality of university and hospital laboratories.     

Contribution of Archaea to Total Prokaryotic Production in the Deep Atlantic Ocean

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.     

The Intracellular Water Activity of Bacteria in Relation to the Water Activity of the Growth Medium

Intracellular water activities ( a _w) calculated from the solute composition of various bacterial cells, are in good agreement with values derived from intracellular freezing point data. Further, and confirming literature results based on freezing points, the intracellular a _w was found to be generally equal to or lower than that of the growth medium.     

Optimal Staining and Sample Storage Time for Direct Microscopic Enumeration of Total and Active Bacteria in Soil with Two Fluorescent Dyes

Direct counting techniques, first developed for aquatic samples, can be used to enumerate bacteria in soil and groundwater sediments. Two fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for actively respiring bacteria and 4(prm1),6-diamidino-2-phenylindole (DAPI) for total bacteria, were tested for their usefulness in epifluorescent direct bacterial enumeration in soil. Both dyes can be used for the same soil sample without affecting enumeration results. Staining for 8 h with CTC and for 40 min with DAPI resulted in maximum numbers of stained cells. The optimal DAPI staining concentration is 10 mg liter(sup-1). After preparation, slides should be stored at 4(deg)C and counted within 2 days for CTC and within 24 h for DAPI. Sodium PP(infi) or sodium chloride solutions were used to desorb bacteria from soil prior to counting. Counts were significantly higher when sodium chloride was used.     

Competition between Two Isolates of Denitrifying Bacteria Added to Soil

We examined the competitive relationship between two isolates of denitrifying bacteria, both of which grow well under aerobic conditions but differ in their ability to grow under denitrifying conditions. The growth and persistence of the two isolates, added to sterile soil or added to soil previously colonized by the other isolate, were monitored under aerobic and denitrifying (anaerobic) conditions. When isolates were added together to sterile soil, the isolate added at the higher density reduced the growth of the isolate added at the lower density. The magnitude of the growth reduction varied depending on the competitive abilities of the individual isolates and the aeration state of the soil. Prior colonization of soil with one of the isolates conferred a competitive advantage on the colonized isolate but did not lead to the disappearance of the challenging isolate. Fluctuations in aeration state caused large changes in the population density of one isolate and altered the competitive relationship between the two isolates. The competitive effectiveness of each isolate varied with cell density, the degree of prior colonization of the soil by the other isolate, and the aeration state of the soil.