Experimental testing of hypotheses for temperature- and pH-based niche specialization of ammonia oxidizing archaea and bacteria.

Investigation of niche specialization in microbial communities is important in assessing consequences of environmental change for ecosystem processes. Ammonia oxidizing bacteria (AOB) and archaea (AOA) present a convenient model for studying niche specialization. They coexist in most soils and effects of soil characteristics on their relative abundances have been studied extensively. This study integrated published information on the influence of temperature and pH on AOB and AOA into several hypotheses, generating predictions that were tested in soil microcosms. The influence of perturbations in temperature was determined in pH 4.5, 6 and 7.5 soils and perturbations in pH were investigated at 15°C, 25°C and 35°C. AO activities were determined by analysing changes in amoA gene and transcript abundances, stable isotope probing and nitrate production. Experimental data supported major predictions of the effects of temperature and pH, but with several significant discrepancies, some of which may have resulted from experimental limitations. The study also provided evidence for unpredicted activity of AOB in pH 4.5 soil. Other discrepancies highlighted important deficiencies in current knowledge, particularly lack of consideration of niche overlap and the need to consider combinations of factors when assessing the influence of environmental change on microbial communities and their activities.

Archaea predominate among ammonia-oxidizing prokaryotes in soils.

Ammonia oxidation is the first step in nitrification, a key process in the global nitrogen cycle that results in the formation of nitrate through microbial activity. The increase in nitrate availability in soils is important for plant nutrition, but it also has considerable impact on groundwater pollution owing to leaching. Here we show that archaeal ammonia oxidizers are more abundant in soils than their well-known bacterial counterparts. We investigated the abundance of the gene encoding a subunit of the key enzyme ammonia monooxygenase (amoA) in 12 pristine and agricultural soils of three climatic zones. amoA gene copies of Crenarchaeota (Archaea) were up to 3,000-fold more abundant than bacterial amoA genes. High amounts of crenarchaeota-specific lipids, including crenarchaeol, correlated with the abundance of archaeal amoA gene copies. Furthermore, reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology demonstrated the activity of the archaea in situ and supported the numerical dominance of archaeal over bacterial ammonia oxidizers. Our results indicate that crenarchaeota may be the most abundant ammonia-oxidizing organisms in soil ecosystems on Earth.

Molecular analysis of enrichment cultures of marine ammonia oxidisers.

Marine ammonia oxidising bacteria were enriched by incubation of sea water, amended with ammonium sulphate, and subsequent subculture in liquid inorganic medium. PCR primers were designed to be specific for rDNA sequences from ammonia oxidisers belonging to the beta-sub-group of the proteobacteria. These primers were then used to amplify rRNA genes from ammonia oxidiser enrichment cultures containing heterotrophs. PCR products were recovered from all cultures in which complete ammonia oxidation occurred. Subsequent rDNA sequence analysis indicated the presence of three new lineages within the clade defined by sequences of cultured beta-sub-group ammonia oxidisers. Two of the new lineages showed moderate similarity to sequences from pure cultures of ammonia oxidisers previously isolated from marine and brackish environments. The third lineage (AEM-3) was deep branching and occupied an intermediate position between clades defined by Nitrosomonas or Nitrosospira, which were isolated from soil or sewage. The phylogenetic analysis suggests that, in enrichment cultures, the primers are specific for members of the target group, the beta-proteobacteria ammonia oxidisers. The results also indicate the presence of previously unknown ammonia oxidisers in marine samples. The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolation of pure cultures, significantly reducing the time required and facilitating quantitative assessment of relatedness of strains.

Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil.

Genetically modified cells of Pseudomonas fluorescens, chromosomally marked with genes for bioluminescence, were inoculated into sterile soil microcosms. During incubation for 90 days, viable cell concentration did not change significantly but light output, measured by luminometry, decreased, indicating reduced metabolic activity due to lack of substrates. Amendment with nutrients resulted in parallel increases in both luminescence and dehydrogenase activity. Luminometry therefore enables rapid monitoring of the activity of populations of luminescence-marked microbial inocula in the soil, with greater sensitivity and selectivity than traditional techniques.

Spatial structure in soil chemical and microbiological properties in an upland grassland.

We characterised the spatial structure of soil microbial communities in an unimproved grazed upland grassland in the Scottish Borders. A range of soil chemical parameters, cultivable microbes, protozoa, nematodes, phospholipid fatty acid (PLFA) profiles, community-level physiological profiles (CLPP), intra-radical arbuscular mycorrhizal community structure, and eubacterial, actinomycete, pseudomonad and ammonia-oxidiser 16S rRNA gene profiles, assessed by denaturing gradient gel electrophoresis (DGGE) were quantified. The botanical composition of the vegetation associated with each soil sample was also determined. Geostatistical analysis of the data revealed a gamut of spatial dependency with diverse semivariograms being apparent, ranging from pure nugget, linear and non-linear forms. Spatial autocorrelation generally accounted for 40-60% of the total variance of those properties where such autocorrelation was apparent, but accounted for 97% in the case of nitrate-N. Geostatistical ranges extending from approximately 0.6-6 m were detected, dispersed throughout both chemical and biological properties. CLPP data tended to be associated with ranges greater than 4.5 m. There was no relationship between physical distance in the field and genetic similarity based on DGGE profiles. However, analysis of samples taken as close as 1 cm apart within a subset of cores suggested some spatial dependency in community DNA-DGGE parameters below an 8 cm scale. Spatial correlation between the properties was generally weak, with some exceptions such as between microbial biomass C and total N and C. There was evidence for scale-dependence in the relationships between properties. PLFA and CLPP profiling showed some association with vegetation composition, but DGGE profiling did not. There was considerably stronger association between notional sheep urine patches, denoted by soil nutrient status, and many of the properties. These data demonstrate extreme spatial variation in community-level microbiological properties in upland grasslands, and that despite considerable numeric ranges in the majority of properties, overarching controlling factors were not apparent.

Quantification of the presence and activity of specific microorganisms in nature.

Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.

Species Diversity of Uncultured and Cultured Populations of Soil and Marine Ammonia Oxidizing Bacteria.

Although molecular techniques are considered to provide a more comprehensive view of species diversity of natural microbial populations, few studies have compared diversity assessed by molecular and cultivation-based approaches using the same samples. To achieve this, the diversity of natural populations of ammonia oxidising bacteria in arable soil and marine sediments was determined by analysis of 16S rDNA sequences from enrichment cultures, prepared using standard methods for this group, and from 16S rDNA cloned from DNA extracted directly from the same environmental samples. Soil and marine samples yielded 31 and 18 enrichment cultures, respectively, which were compared with 50 and 40 environmental clones. There was no evidence for selection for particular ammonia oxidizer clusters by different procedures employed for enrichment from soil samples, although no culture was obtained in medium at acid pH. In soil enrichment cultures, Nitrosospira cluster 3 sequences were most abundant, whereas clones were distributed more evenly between Nitrosospira clusters 2, 3, and 4. In marine samples, the majority of enrichment cultures contained Nitrosomonas, whereas Nitrosospira sequences were most abundant among environmental clones. Soil enrichments contained a higher proportion of identical sequences than clones, suggesting laboratory selection for particular strains, but the converse was found in marine samples. In addition, 16% of soil enrichment culture sequences were identical to those in environmental clones, but only 1 of 40 marine enrichments was found among clones, indicating poorer culturability of marine strains represented in the clone library, under the conditions employed. The study demonstrates significant differences in species composition assessed by molecular and culture-based approaches but indicates also that, employing only a limited range of cultivation conditions, 7% of the observed sequence diversity in clones of ammonia oxidizers from these environments could be obtained in laboratory enrichment culture. Further studies and experimental approaches are required to determine which approach provides better representation of the natural community.

Luminescence-based detection of Erwinia carotovora associated with rotting potato tubers.

A luminescence-based marker system has been used to follow rotting of potato tubers by Erwinia carotovora. The early stages of infection could be detected by visual observation of areas of luminescence developing from the point of inoculation. During later stages luminescence was limited to peripheral regions and to central regions where the structure of the potato was destroyed. Luminometry demonstrated reduction in aerobic activity of infecting cells due to oxygen limitation. The system enables rapid and sensitive detection of active marked populations and distinction between aerobic and fermentative activity.

Bacterial diversity promotes community stability and functional resilience after perturbation.

The relationships between bacterial community diversity and stability were investigated by perturbing soils, with naturally differing levels of diversity, to equivalent toxicity using copper sulfate and benzene. Benzene amendment led to large decreases in total bacterial numbers and biomass in both soils. Benzene amendment of an organo-mineral/improved pasture soil altered total soil bacterial community structure but, unlike amendment of the mineral/arable soil, maintained genetic diversity, based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis targeting DNA and RNA, until week 9 of the perturbation experiment. Assuming equivalent toxicity, the genetic diversity of the naturally more diverse soil was more resistant to benzene perturbation than the less diverse soil. The broad scale function (mineralization of 14C-labelled wheat shoot) of both benzene- and copper-treated soil communities was unaffected. However, narrow niche function (mineralization of 14C-labelled 2,4-dichlorophenol) was impaired for both benzene-polluted soils. The organo-mineral soil recovered this function by the end of the experiment but the mineral soil did not, suggesting greater resilience in the more diverse soil. Despite a large reduction in bacterial numbers and biomass in the copper-treated soils, only small differences in bacterial community diversity were observed by week 9 in the copper-polluted soils. The overall community structure was little altered and functionality, measured by mineralization rates, remained unchanged. This suggested a non-selective pressure and a degree of genetic and functional resistance to copper perturbation, despite a significant reduction in bacterial numbers and biomass. However, initial shifts in physiological profiles of both copper-polluted soils were observed but rapidly returned to those of the controls. This apparent functional recovery, accompanied by an increase in culturability, possibly reflects adaptation by the surviving communities to perturbation. The findings indicate that, although soil communities may be robust, relationships between diversity and stability need to be considered in developing a predictive understanding of response to environmental perturbations.

Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions.

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.

Protection of Nitrosomonas europaea colonizing clay minerals from inhibition by nitrapyrin.

Nitrate production by Nitrosomonas europaea in inorganic liquid medium containing ammonium was limited by reduction in pH. In the presence of montmorillonite and vermiculite, expanding clays with high cation-exchange-capacity (CEC), nitrite yield was increased, ammonia oxidation continued at pH values below those which inhibited growth in the absence of clays and growth was biphasic. The first phase was similar to that in the absence of clays, while the second was characterized by a lower rate of nitrite production. Illite, a non-expanding clay with low CEC, had no significant effect on ammonia oxidation, while oxidation of ammonia-treated vermiculite (ATV) occurred with no significant change in the pH of the medium. ATV, montmorillonite and vermiculite, but not illite, protected cells from inhibition by nitrapyrin at concentrations inhibitory to cells growing in suspended culture. This protection was maintained in ATV homo-ionic to Al3+, but montmorillonite made homo-ionic to Al3+ did not provide protection from inhibition. Attachment of cells to clays with high CEC is therefore advantageous in providing exchange at the clay surface of NH+4 and H+ produced by ammonia oxidation, in reducing pH toxicity, and in protecting cells from inhibition.

Growth mechanisms and growth kinetics of filamentous microorganisms.

Filamentous microorganisms are of major biotechnological importance, being responsible for production of the majority of secondary metabolites, particularly antibiotics. Two main groups are involved, filamentous fungi and filamentous actinomycetes, particularly the streptomycetes. In terms of cellular growth mechanisms, these groups differ greatly. Eukaryotic fungi possess subcellular organelles and cytoskeletal structures directing growth while prokaryotic streptomycetes have no such cellular organization. Despite these fundamental differences, both groups exhibit similar morphologies, growth patterns, growth forms, and hyphal and mycelial growth kinetics on solid media and in liquid culture both grow as dispersed mycelia and pellets. The article therefore discusses the relationship between cellular growth mechanisms and vegetative growth in both filamentous fungi and actinomycetes, the conceptual and theoretical models applicable to both groups, and the significance of such models in industrial fermentation processes.

Surface attachment of nitrifying bacteria and their inhibition by potassium ethyl xanthate.

Ion exchange resins and glass microscope slides were used to investigate factors affecting attachment of nitrifying bacteria to solid surfaces and the effect of attachment on inhibition ofNitrobacter by potassium ethyl xanthate. The ammonium oxidizerNitrosomonas attached preferentially to cation exchange resins while the nitrite oxidizerNitrobacter colonized anion exchange resins more extensively. Colonization was always associated with growth, and the site of substrate (NH4 (+) or NO2 (-)) adsorption was the major factor in attachment and colonization. The specific growth rate of cells colonizing either ion exchange resin beads or glass surfaces was greater than that of freely suspended cells, butNitrobacter populations colonizing glass surfaces were more sensitive to the inhibitor potassium ethyl xanthate. The findings indicate that surface growth alone does not protect soil nitrifying bacteria from inhibition by potassium ethyl xanthate and explain different patterns of inhibition for ammonium and nitrite oxidizers in the soil.

Inhibition of ammonium oxidation by nitrapyrin in soil and liquid culture.

Inhibition of growth of axenic cultures of Nitrosomonas europaea by nitrapyrin was investigated in liquid culture and in soil. In liquid culture, exponentially growing cells were more sensitive than stationary-phase cells, possibly due to a requirement for uptake of nitrapyrin, metabolism of nitrapyrin, or both before inhibition. Differences in sensitivity were observed between the parent strain and two strains, sp1 and sp2, that were selected through repeated subculturing. These differences were reflected in the length of the lag period induced by nitrapyrin and in the specific growth rate and were due to different bactericidal and bacteriostatic effects. Soil provided significant protection from inhibition, with concentrations of nitrapyrin approximately one order of magnitude greater than those required for equivalent inhibition in liquid culture. The data show that strain differences alone do not explain differences in sensitivity between nitrification in soil and in liquid culture and suggest that the inhibitor may be more effective against actively nitrifying soils.

Inhibition of biofilm populations of Nitrosomonas europaea.

The influence of surface attachment and growth on inhibition of the ammonia oxidizing bacterium, Nitrosomonas europaea, by nitrapyrin was investigated in liquid culture in the presence and absence of glass slides. Significant attachment to glass slides occurred in the absence of ammonia, but the extent of attachment was not affected by nitrapyrin, nor by previous culture of cells in medium containing nitrapyrin. The presence of glass slides affected neither the specific growth rate of N. europaea, measured by changes in nitrite concentration, nor inhibition by nitrapyrin. Inhibitory effects of nitrapyrin on increases in nitrite concentration and in free cell concentration were similar, but greater effects were observed on changes in attached cell concentration. Established biofilms on glass slides grew at a lower specific growth rate than freely suspended cells. Both biofilm cells, and those detached from the biofilm, were protected from inhibition. A mechanism for protection of biofilm populations is proposed involving reduced sensitivity of slowly growing cells producing extracellular polymeric material.

Experimental verification of a mathematical model for pelleted growth of Streptomyces coelicolor A3(2) in submerged batch culture.

A published mathematical model for growth of pellets of filamentous microorganisms has been tested by comparison of model predictions with experimental data on growth of Streptomyces coelicolor in liquid batch culture. The original model considered the classification of pellets into a range of size classes. Growth resulted in movement of pellets to classes of increasing size, while shear forces produced mycelial fragments which entered the smallest size class, from which they grew to form further pellets. This model did not correctly describe changes in pellet size distributions during growth and was therefore modified in two ways. In the first, new pellets were assumed to be formed by the break-up, by shear forces, of existing pellets into two pellets of equal size, rather than removal of small hyphal fragments from the pellet surface. The second modification assumed that the outer shell of active mycelial biomass had a density less than 1 g cm-3 and that hyphal density within this shell decreased with distance from the pellet centre. The modified model generated predictions which agreed closely with experimental data on biomass concentration, pellet size distribution, pellet number and pellet radius during batch growth, thereby supporting the assumptions on which the model was based. The model did not accurately describe final biomass concentration, through lack of consideration of autolysis of mycelia at the centre of larger pellets in which growth was limited by diffusion of nutrients. Attempts to incorporate autolysis into the model improved prediction of biomass concentration but were not based on sound biological assumptions and increased the complexity of the model. Further experimental work is required for accurate description of the effects of autolysis on pellet growth.

[Extracellular factors affecting the adhesion of Pseudomonas fluorescens cells to glass surface]. / Vnekletochnye faktory, vliiaiushchie na adgeziiu Pseudomonas fluorescens na stekle.

Two factors affecting the adhesion of Pseudomonas fluorescens to glass surfaces were revealed in the culture liquid (CL) of this bacterium. One of these factors, adhesin, which is responsible for cell adhesion, was found to be a protein substance located both at the cell surface and in the CL. Bacterial cells grown in rich LB medium were less adhesive than cells grown in minimal M9 medium. The adhesive capacity of cells was independent of the growth phase. The other factor, anti-adhesion (AA), which reduces cell adhesion, was found only in the CL. AA concentration in the CL increased with the culture age.