Automated Ribosomal Intergenic Spacer Analysis Profiles Research Articles

Microbial diversity in grape musts from Austrian and South African grape varieties and regions

Vitis vinifera is one of the most widely planted crops and holds important economic value in South Africa and Austria. Grapes obtained from this plant harbour a complex fungal community which plays a crucial role in the wine fermentation process and influences wine flavour and aroma. For many years the contributions of the natural yeasts has been eclipsed by the use of active dry yeast (ADY) inoculant, mainly of the species Saccharomyces cerevisiae. However, recent studies show a growing interest in deciphering the natural microbial diversity and in promoting its persistence during fermentation in order to enhance wine typicity. The current preliminary study aims to provide a first broad assessment of the fungal community fingerprint of different grape varietals from different wine producing areas in Austria and South Africa through Automated Ribosomal Intergenic Spacer Analysis (ARISA). The ARISA profiles separated the samples according to country of origin, and suggested some regional and varietal separation within each country. Future work will evaluate the contribution of these fungal communities to wine chemical composition and sensorial distinctness.

Assessment of wine microbial diversity using ARISA and cultivation-based methods

This study investigated the microbial diversity present in grape juice and in the early stage of alcoholic fermentation. The grapes were obtained from a South African biodynamic vineyard in three consecutive vintages (2012, 2013, and 2014). Culture-dependent and culture-independent approaches were used to investigate yeast and bacterial diversity. For the culture-independent approach, Automated Ribosomal Intergenic Spacer Analysis (ARISA) was employed. Using basic microbiological analyses, 4, 12, and 15 yeast species were obtained in 2012, 2013, and 2014, respectively. In contrast, ARISA profiles revealed 13 fungal peaks for the year 2013 and 14 for 2012 and 2014. Out of 14 peaks in 2012, two peaks were identified as probable corresponding fungal Operational Taxonomical Units (OTUs) and six peaks were identified for the years 2013 and 2014. The bacterial ARISA revealed ten, seven, and 12 peaks for these three years, respectively. Furthermore, the same technique was used to assess the evolution of the fungal community in the first three days of fermentation of the 2013 grape must. The data show that the yeast population diversity declined rapidly and revealed 12, 10, and 6 peaks for days 1, 2, and 3, respectively. The study demonstrated the suitability of ARISA for studying microbial diversity and dynamics in grape must and during wine fermentation.

Molecular characterization of Pseudo-nitzschia community structure and species ecology in a hydrographically complex estuarine system (Puget Sound, Washington, USA)

Species within the toxic marine diatom genus Pseudo-nitzschia coexist in coastal and estuarine waters globally and are difficult to distinguish by microscopy. Here, we describe a sensitive, high throughput PCR-based Automated Ribosomal Intergenic Spacer Analysis (ARISA) approach to determine the relative abundance of Pseudo-nitzschia species within natural communities over space and time. The method was quantitatively validated using simplified mixtures of DNA or ITS1 standards from isolates of P. pungens, P. multiseries, and P. delicatissima. Relative abundance calculations based on ARISA profiles from these mixtures reflected input ratios, with minor deviations resulting from intraspecific variability. ARISA was used to identify and quantify at least eight species within Puget Sound and the eastern Strait of Juan de Fuca, Washington, USA: P. americana, P. australis/P. seriata, P. cuspidata, P. delicatissima, P. fraudulenta, P. fryxelliana, P. multiseries, and P. pungens; genotypes corresponding to P. pungens var. pungens and P. pungens var. cingulata were identified by environmental sequencing. The different species were significantly correlated with physical (temperature, salinity), biological (chlorophyll a fluorescence, oxygen), and/or chemical (ammonium, nutrient ratios) factors. The ability to determine shifts in the relative abundance of Pseudo-nitzschia species over spatial and temporal scales relevant to dispersion and selection facilitates dissection of the varied mechanisms driving vertical and horizontal species distribution patterns in hydrographically complex systems.

Comparison of the bacterial community structure within the equine hindgut and faeces using Automated Ribosomal Intergenic Spacer Analysis (ARISA)

The horse’s hindgut bacterial ecosystem has often been studied using faecal samples. However few studies compared both bacterial ecosystems and the validity of using faecal samples may be questionable. Hence, the present study aimed to compare the structure of the equine bacterial community in the hindgut (caecum, right ventral colon) and faeces using a fingerprint technique known as Automated Ribosomal Intergenic Spacer Analysis (ARISA). Two DNA extraction methods were also assessed. Intestinal contents and faeces were sampled 3 h after the morning meal on four adult fistulated horses fed meadow hay and pelleted concentrate. Irrespective of the intestinal segment, Principal Component Analysis of ARISA profiles showed a strong individual effect (P<0.0001). However, across the study, faecal bacterial community structure significantly (P<0.001) differed from those of the caecum and colon, while there was no difference between the two hindgut communities. The use of a QIAamp® DNA Stool Mini kit increased the quality of DNA extracted irrespective of sample type. The differences observed between faecal and hindgut bacterial communities challenge the use of faeces as a representative for hindgut activity. Further investigations are necessary to compare bacterial activity between the hindgut and faeces in order to understand the validity of using faecal samples.

Linking community tolerance and structure with low metallic contamination: A field study on 13 biofilms sampled across the Seine river basin

It is difficult to assess the biological consequences of diffuse water contamination by micropollutants which are present in rivers at low, even sublethal levels. River biofilms, which respond quickly to changes of environmental parameters, are good candidates to acquire knowledge on the response of aquatic organisms to diffuse chemical contamination in the field. The study was designed as an attempt to link biofilm metal tolerance and metallic contamination in a field survey covering 13 different sampling sites in the Seine river basin (north of France) with low contamination levels. Cd and Zn tolerance of heterotrophic communities was assessed using a short-term toxicity test based on β-glucosidase activity. Metal tolerance levels varied between sites but there was no obvious correlation between tolerance and corresponding water contamination levels for Cd and Zn. Indeed, metallic contamination at the sampling sites remained subtle when compared to water quality standards (only two sampling sites had either Zn or both Cu and Zn concentrations exceeding the Environmental Quality Standards set by the EU Water Framework Directive). Yet, multivariate analysis of the data using Partial Least Squares Regression revealed that both metallic and environmental parameters were important variables explaining the variability of metal tolerance levels. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was also performed on both bacterial and eukaryotic biofilm communities from the 13 sampling sites. Multivariate analysis of ARISA fingerprints revealed that biofilms with similar tolerance levels have similar ARISA profiles. Those results confirm that river biofilms are potential indicators of low, diffuse contamination levels of aquatic systems.

Molecular monitoring of the bacterial community structure in foal feces pre- and post-weaning

This study assessed the time-scale variability of bacterial community structure in foal feces from birth to 365 days of age using Automated Ribosomal Intergenic Spacer Analysis (ARISA). Fecal samples were collected from five foals 2 h after birth (meconium) and in the morning at days 1, 2, 5, 10, 30, 60, 120, 179, 183, 194 and 365. The ARISA profiles were compared using an analysis of similarity (ANOSIM). Although both the age effect and the foal effect were highly significant (P < 0.010), the R-ANOSIM value for the foal effect was very low (R-ANOSIM = 0.089), while that of the age effect was much higher (R-ANOSIM = 0.309). Significant age-related changes were detected between days 0 and 2 (R-ANOSIM = 0.500), days 2 and 10 (R-ANOSIM = 0.475) and days 10 and 30 (R-ANOSIM = 0.519). No further shifts between consecutive times of sampling were detected in the bacterial community after day 30 and no changes were observed at weaning (day 180). These results show that the establishment of the intestinal bacterial community in foals is a sequential process, which reaches its climax state at around one month of age. Further studies using new generation sequencing based methods could be conducted to identify which bacterial genera are establishing in foals during the first month of life.

Impact of No-Tillage and Conventional Tillage Systems on Soil Microbial Communities

Soil management practices influence soil physical and chemical characteristics and bring about changes in the soil microbial community structure and function. In this study, the effects of long-term conventional and no-tillage practices on microbial community structure, enzyme activities, and selected physicochemical properties were determined in a continuous corn system on a Decatur silt loam soil. The long-term no-tillage treatment resulted in higher soil carbon and nitrogen contents, viable microbial biomass, and phosphatase activities at the 0–5 cm depth than the conventional tillage treatment. Soil microbial community structure assessed using phospholipid fatty acid (PLFA) analysis and automated ribosomal intergenic spacer analysis (ARISA) varied by tillage practice and soil depth. The abundance of PLFAs indicative of fungi, bacteria, arbuscular mycorrhizal fungi, and actinobacteria was consistently higher in the no-till surface soil. Results of principal components analysis based on soil physicochemical and enzyme variables were in agreement with those based on PLFA and ARISA profiles. Soil organic carbon was positively correlated with most of the PLFA biomarkers. These results indicate that tillage practice and soil depth were two important factors affecting soil microbial community structure and activity, and conservation tillage practices improve both physicochemical and microbiological properties of soil.

Spatial heterogeneity of bacterial communities in the mucus of Montastraea annularis

Corals are known to contain a diverse microbiota; however, few studies have explicitly addressed the spatial variability of bacterial communities across individual, healthy coral colonies. This study applied culture-based and culture-independent methods to examine the spatial hetero- geneity in bacterial communities in the mucus of 3 healthy Montastraea annularis colonies from Looe Key Reef, Florida Keys. Automated ribosomal intergenic spacer analysis (ARISA) results showed significant variability (up to 61% dissimilarity) in the composition of the total bacterial community at different locations only centimeters apart on individual coral colonies. Abundances of culturable Vib- rio spp. determined by TCBS plating were highly variable across individual coral colonies, differing by up to 100-fold between different locations on the same colony. ARISA profiles indicated that intra- colony variation rivaled intercolony differences in the composition of the culturable Vibrio commu- nity (i.e. types of culturable Vibrio spp. and their relative abundances). The high degree of spatial heterogeneity in coral-associated bacteria observed across individual colonies has implications for coral microbiology studies and coral restoration projects.

Low exposure levels of urban metals induce heterotrophic community tolerance: a microcosm validation

The biological response of periphyton chronically exposed to metals of urban origin (Cd, Ni and Zn) was investigated with a Pollution-Induced Community Tolerance (PICT) approach using a previously developed short-term toxicity test based on β-glucosidase (heterotrophic) activity. Periphyton was grown on plastic membranes immersed in indoor aquaria contaminated with metals at realistic contamination levels (0.3, 3μg/l for Cd, 5, 50μg/l for Ni, 20, 200μg/l for Zn). After 3weeks of exposure, biofilms' parameters (dry-weight, chlorophyll a concentration, heterotrophic activity) were analyzed and tolerance acquisition of the heterotrophic communities was assessed using the toxicity test. Modifications of bacterial and eukaryotic community structure were assessed with Automated Ribosomal Intergenic Spacer Analysis (ARISA). Effects of metal exposure were observed on biofilms parameters in the Cd and Zn experiments. Tolerance levels increased for both Cd-exposed biofilms, and for the high metal treatment biofilms in the Ni and Zn experiments. Analysis of the ARISA profiles showed that metal exposure affected the structure of both bacterial and eukaryotic communities. Moreover, Cd tolerance of the Zn-exposed heterotrophic communities was evaluated, which showed that the Zn-tolerant community (high metal treatment in the Zn experiment) also became tolerant to Cd (co-tolerance). The study shows that tolerance acquisition can be detected after exposure to environmental metal concentrations using β-glucosidase activity as an endpoint in short-term toxicity tests.

Cyanobacterial mats from hot springs produce antimicrobial compounds and quorum-sensing inhibitors under natural conditions

Polar (water) and non-polar (ethyl acetate) extracts from the cyanobacterial layer (top 1–3 mm) of four hot spring microbial mats in the Sultanate of Oman were tested for their antibacterial, antidiatom and quorum-sensing inhibitory activities under natural conditions. The chemical composition of the active extracts was analysed using gas chromatography–mass spectrometry (GC-MS). Cyanobacteria within these mats were identified by direct microscopy while the total bacterial community composition was compared using automated ribosomal intergenic spacer analysis (ARISA). Only the extracts from Bowshar and Nakhl mats showed antibacterial properties against Bacillus sp., Micrococcus luteus, Shigella sonnei, Salmonella enterica and Klebsiella pneumoniae. All tested extracts inhibited the growth of the benthic diatom Amphora coffeaeformis. Extracts from Bowshar, Rustaq and Nakhl inhibited quorum-sensing of the reporter strains Chromobacterium violaceum CV017 and Agrobacterium tumefaciens NTL4. The highest bioactivity was recorded for ethyl acetate extracts from Nakhl mats, which had the lowest number of operational taxonomic units (OTUs). Using GC-MS, 74 chemical compounds were obtained, however with different distribution among the four mat extracts (similarity < 43%). Various cyanobacteria, belonging mainly to Chroococcus, Phormidium, Leptolyngbya, Spirulina and Lyngbya were detected in the different mats, and each mat had its unique bacterial community, as confirmed by ARISA profiles. We conclude that antimicrobial and quorum-sensing inhibitory compounds can be produced by hot spring mat microorganisms under natural conditions and the differences in these compounds could be attributed to the differences in the mats’ bacterial composition as well as the physical–chemical conditions of the springs.

Optimisation of automated ribosomal intergenic spacer analysis for the estimation of microbial diversity in fynbos soil

Automated ribosomal intergenic spacer analysis (ARISA) has become a commonly used molecular technique for the study of microbial populations in environmental samples. The reproducibility and accuracy of ARISA, with and without the polymerase chain reaction (PCR) are important aspects that influence the results and effectiveness of these techniques. We used the primer set ITS4/ITS5 for ARISA to assess the fungal community composition of two sites situated in the Sand Fynbos. The primer set proved to deliver reproducible ARISA profiles of the fungal community composition with little variation observed between ARISA-PCRs. Variation that occurred in a sample due to repeated DNA extraction is expected for ecological studies. This reproducibility made ARISA a useful tool for the assessment and comparison of diversity in ecological samples. In this paper, we also offered particular suggestions concerning the binning strategy for the analysis of ARISA profiles.

PH dynamics and bacterial community composition in the rumen of lactating dairy cows

The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminally cannulated Holstein cows fitted with indwelling electrodes that recorded pH at 10-min intervals over a 54-h period. Cows were fed a silage-based total mixed ration supplemented with monensin. Ruminal samples were collected each day just before feeding and at 3 and 6h after feeding. Solid and liquid phases were separated at collection, and extracted DNA was subjected to PCR amplification followed by automated ribosomal intergenic spacer analysis (ARISA). Although cows displayed widely different pH profiles (mean pH=6.11 to 6.51, diurnal pH range=0.45 to 1.39), correspondence analysis of the ARISA profiles revealed that 6 of the 8 cows showed very similar bacterial community compositions. The 2 cows having substantially different community compositions had intermediate mean pH values (6.30 and 6.33) and intermediate diurnal pH ranges (averaging 0.89 and 0.81 pH units). Fortuitously, these 2 cows alone also displayed milk fat depression, along with markedly higher ruminal populations of 1 bacterial operational taxonomic unit (OTU) and reduced populations of another ARISA amplicon. Cloning and sequencing of the elevated OTU revealed phylogenetic similarity to Megasphaera elsdenii, a species reportedly associated with milk fat depression. The higher populations of both M. elsdenii and OTU246 in these 2 cows were confirmed using quantitative real-time PCR (qPCR) with species-specific primers, and the fraction of total bacterial rDNA copies contributed by these 2 taxa were very highly correlated within individual cows. By contrast, the fraction of total bacterial rDNA copies contributed by Streptococcus bovis and genus Ruminococcus, 2 taxa expected to respond to ruminal pH, did not differ among cows (mean= <0.01 and 10.6%, respectively, of rRNA gene copies, determined by qPCR). The data indicate that cows with widely differing pH profiles can have similar ruminal bacterial community compositions, and that milk fat depression can occur at intermediate ruminal pH. The results support recent reports that milk fat depression is associated with shifts in bacterial community composition in rumine and is specifically related to the relative abundance of Megasphaera elsdenii.

NESTED AUTOMATED RIBOSOMAL INTERGENIC SPACER ANALYSIS: A RAPID AND ACCURATE METHOD FOR COMPARISON OF BACTERIAL COMMUNITY COMPOSITION

ABSTRACT Nested automated ribosomal intergenic spacer analysis (ARISA) was used to examine the community structure of epilithic biofilms in freshwater streams experiencing different levels of human impact. This molecular fingerprinting technique generated reproducible profiles of bacterial community structure that varied significantly between stream sites. Nested ARISA was determined to be a cost‐effective, high‐throughput approach to assess bacterial community composition from very small sample volumes, requiring little sampling effort and without the need for taxonomic identification of individual organisms. In combination with multidimensional scaling, nested ARISA provides a rapid and sensitive method to carry out complex analyses of bacterial community structure. PRACTICAL APPLICATIONSNested automated ribosomal intergenic spacer analysis (ARISA) provides a high‐throughput molecular method with which to screen large numbers of environmental samples for differences in microbial community structure. This sensitive approach benefits assessments from small sample volumes or environments exhibiting reduced microbial biomass (both aquatic and terrestrial). Differences in bacterial community structure (obtained from ARISA profiles) could be used to characterize the impact of anthropogenic disturbance on freshwater systems, analogous to the current use of macroinvertebrate indicators of freshwater ecological health.

Combined monitoring of changes in δ13CH4 and archaeal community structure during mesophilic methanization of municipal solid waste

Reconstituted municipal solid waste (MSW) with varying contents of putrescible and cellulosic waste was incubated anaerobically under mesophilic conditions. Standard physicochemical parameters were monitored, together with stable isotopic signatures of produced CH(4) and CO(2). delta(13)C values for CH(4) indicated a change of methanogenic metabolism with time. CH(4) was predominantly produced from H(2)/CO(2) at the beginning of the incubations. This period was associated with important shifts in archaeal communities monitored by automated ribosomal intergenic spacer analysis (ARISA) and FISH of oligonucleotidic probes targeting specifically 16S rRNA gene of various methanogenic groups. The onset of the active methane generation phase was characterized by an increase of CH(4)delta(13)C, indicating a progressive shift toward an aceticlastic metabolism. When the methane production levelled off, a decrease in the isotopic signature was observed toward values characteristics of hydrogenotrophic metabolism. ARISA profiles were, however, found to be stable from the beginning of the active methane generation phase until the end of the experiment. FISH observation indicated that members of the family Methanosarcinaceae were predominant in the archaeal community during this period, suggesting that these methanogens might exhibit a high metabolic versatility during methanization of waste.

Importance of DNA quality in comparative soil microbial community structure analyses

This study assessed how different in-situ lysis soil DNA extraction methods influence the DNA yield, quality and hence the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA). Of the methods tested in three soils, a modified hexadecyltrimethylammonium bromide-dithiotreitol (CTAB-DTT)-based method produced ⩾3 times more DNA of higher quality than the other methods (260/230nm ratios=1.64–1.82 and 260/280nm ratios=1.82–1.89 and extracts were less inhibitory of PCR). DNA extracted by this method also yielded more reproducible ARISA ribotypes (89−119 for bacteria and 48−88 for fungi; P<0.05) than DNA extracted by other methods, and consequently produced more reliable estimates of bacterial and fungal diversity in all three test soils. The significant correlations observed between the numbers of reproducible ribotypes and DNA extract 260/230nm ratios (r=0.88 and 0.72 for bacteria and fungi, respectively; P<0.001) reaffirmed the strong influence of DNA quality on the reliability of microbial diversity indices determined based on PCR-based DNA fingerprinting technique. Results of discriminant function analysis (DFA) and multivariate analysis of variances (MANOVA) performed on ARISA profiles (number and relative abundance of ribotype) revealed that the variability associated with DNA extraction methods did not exceed the biological variability among soil types; this supports the conclusion that high-quality DNA underpins DNA fingerprinting techniques.

Seasonal influences on fungal community structure in unimproved and improved upland grassland soils

Seasonal and management influences on the fungal community structure of two upland grassland soils were investigated. An upland site containing both unimproved floristically diverse (U4a) and improved mesotrophic (MG7b) grassland types was selected. Samples from both grassland types were taken at five times in one year. Soil fungal community structure was assessed using fungal automated ribosomal intergenic spacer analysis (ARISA), a DNA-profiling approach. A grassland management regime was found to strongly affect fungal community structure, with fungal ARISA profiles from unimproved and improved grassland soils differing significantly. The number of fungal ribotypes found was higher in unimproved than improved grassland soils, providing evidence that improvement may reduce the suitability of upland soil as a habitat for specific groups of fungi. Seasonal influences on fungal community structure were also noted, with samples taken in autumn (October) more correlated with change in ribotype profiles than samples from other seasons. However, seasonal variation did not obscure the measurement of differences in the fungal community structure that were due to agricultural improvement, with canonical correspondence analysis indicating grassland type had a stronger influence on fungal profiles than did season.

Microbial Succession in a Compost-packed Biofilter Treating Benzene-contaminated Air

Air artificially contaminated with increasing concentrations of benzene was treated in a laboratory scale compost-packed biofilter for 240 days with a removal efficiency of 81-100%. The bacterial community in the packing material (PM) at different heights of the biofilter was analysed every 60 days. Bacterial plate counts and ribosomal intergenic spacer analysis (RISA) of the isolated strains showed that the number of cultivable aerobic heterotrophic bacteria and the species diversity increased with benzene availability. Identification of the isolated species and the main bands in denaturing gradient gel electrophoresis (DGGE) profiles from total compost DNA during the treatment revealed that, at a relatively low volumetric benzene load (1.2< or =VBL< or =6.4 g m(-3) (PM) h(-1)), besides low G+C Gram positive bacteria, originally present in the packing compost, bacteroidetes and beta- and gamma-proteobacteria became detectable in the colonising population. At the VBL value (24.8 g m(-3) (PM) h(-1)) ensuring the maximum elimination capacity of the biofilter (20.1 g m(-3) (PM) h(-1)), strains affiliated to the genus Rhodococcus dominated the microflora, followed by beta-proteobacteria comprising the genera Bordetella and Neisseria. Under these conditions, more than 35% of the isolated strains were able to grow on benzene as the sole carbon source. Comparison of DGGE and automated RISA profiles of the total community and isolated strains showed that a complex bacterial succession occurred in the reactor in response to the increasing concentrations of the pollutant and that cultivable bacteria played a major role in benzene degradation under the adopted conditions.

Coupling 16S‐ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series

We outline an approach to simultaneously assess multilevel microbial diversity patterns utilizing 16S-ITS rDNA clone libraries coupled with automated ribosomal intergenic spacer analysis (ARISA). Sequence data from 512 clones allowed estimation of ARISA fragment lengths associated with bacteria in a coastal marine environment. We matched 92% of ARISA peaks (each comprising >1% total amplified product) with corresponding lengths from clone libraries. These peaks with putative identification accounted for an average of 83% of total amplified community DNA. At 16S rDNA similarities <98%, most taxa displayed differences in ARISA fragment lengths >10 bp, readily detectable and suggesting ARISA resolution is near the 'species' level. Prochlorococcus abundance profiles from ARISA were strongly correlated (r2=0.86) to Prochlorococcus cell counts, indicating ARISA data are roughly proportional to actual cell abundance within a defined taxon. Analysis of ARISA profiles for 42 months elucidated patterns of microbial presence and abundance providing insights into community shifts and ecological niches for specific organisms, including a coupling of ecological patterns for taxa within the Prochlorococcus, the Gamma Proteobacteria and Actinobacteria. Clade-specific ARISA protocols were developed for the SAR11 and marine cyanobacteria to resolve ambiguous identifications and to perform focused studies. 16S-ITS data allowed high-resolution identification of organisms by ITS sequence analysis, and examination of microdiversity.

Impact of light on marine bacterioplankton community structure

Reports of widespread proteorhodopsin and bacteriochlorophyll a genes suggest that light may facilitate the growth of many marine bacteria. We tested this hypothesis by conducting 3 light manipulation experiments with bacterial communities obtained off the Southern California coast. In each experiment, duplicate 20 l mesocosms were subjected to either 14:10 h light:dark or continual darkness for 5 to 10 d. Automated Ribosomal Intergenic Spacer Analysis (ARISA), a whole- community fingerprinting technique, was used to determine how light affects bacterial community structure. Light removal resulted in only minor changes in ARISA profiles (mean decrease in similar- ity between treatments: 6 ± 1% via Sorenson's index (only considers presence/absence of taxa), 15 ± 7% via Pearson's coefficient (considers relative abundance of taxa)) at the conclusion of the experi- ments. Oligotrophic communities responded nearly twice as strongly to light removal compared to mesotrophic communities. Phototrophs such as cyanobacteria exhibited consistent, sharp declines in dark treatments, indicating whatever heterotrophic abilities they possess were not sufficient to sus- tain them when faced with natural removal processes such as grazing or viral lysis. Members of the broad SAR86, SAR11, SAR116, CFB and Roseobacter phylogenetic groups exhibited minor, mixed responses to light removal, suggesting that only a few select members may rely on phototrophy to a measurable extent. Our results indicate that the majority of non-cyanobacterial bacteria in the ocean do not depend heavily on light to maintain themselves over 5 or 10 d periods in the presence of nat- ural removal processes, and hint that proteorhodopsin and bacteriochlorophyll a genes may have alternative, undetermined ecological benefits besides phototrophy.

Annual Patterns in Bacterioplankton Community Variability in a Humic Lake

Bacterioplankton community composition (BCC) was monitored in a shallow humic lake in northern Wisconsin, USA, over 3 years using automated ribosomal intergenic spacer analysis (ARISA). Comparison of ARISA profiles of bacterial communities over time indicated that BCC was highly variable on a seasonal and annual scale. Nonmetric multidimensional scaling (MDS) analysis indicated little similarity in BCC from year to year. Nevertheless, annual patterns in bacterioplankton community diversity were observed. Trends in bacterioplankton community diversity were correlated to annual patterns in community succession observed for phytoplankton and zooplankton populations, consistent with the notion that food web interactions affect bacterioplankton community structure in this humic lake. Bacterioplankton communities experience a dramatic drop in richness and abundance each year in early summer, concurrent with an increase in the abundance of both mixotrophic and heterotrophic flagellates. A second drop in richness, but not abundance, is observed each year in late summer, coinciding with an intense bloom of the nonphagotrophic dinoflagellate Peridinium limbatum. A relationship between bacterial community composition, size, and abundance and the population dynamics of Daphnia was also observed. The noted synchrony between these major population and species shifts suggests that linkages across trophic levels play a role in determining the annual time course of events for the microbial and metazoan components of the plankton.