Molecular analyses of the diversity in marine bacterioplankton assemblages along the coastline of the northeastern Gulf of Mexico

Bacterial community diversity in marine bacterioplankton assemblages were examined in 3 coastal locations along the northeastern Gulf of Mexico (GOM) using 16S rRNA gene libraries and fluorescence in situ hybridization approaches. The majority of the sequences (30%-60%) were similar to the 16S rRNA gene sequences of unknown bacteria; however, the operational taxonomic units from members of the Cyanobacteria, Proteobacteria, and Bacteroidetes were also present at the 3 GOM sites. Overall, sequence diversity was more similar between the Gulf sites of Carrabelle and Ochlockonee than between either of the Gulf sites and Apalachicola Bay. Fluorescence in situ hybridization analyses revealed the quantitative predominance of members of the Alphaproteobacteria subclass and the Cytophaga-Flavobacterium cluster within the bacterioplankton assemblages. In general, the study further reveals the presence of many bacterial taxa that have been previously found to be dominant in coastal marine environments. Differences observed in the representation of the various bacterial phylogenetic groups among the GOM coastal sites could be partly attributed to dynamic variations in several site-specific conditions, including intermittent tidal events, nutrient availability, and anthropogenic influences.     

Resistance of marine bacterioneuston to solar radiation

A total of 90 bacterial strains were isolated from the sea surface microlayer (i.e., bacterioneuston) and underlying waters (i.e., bacterioplankton) from two sites of the northwestern Mediterranean Sea. The strains were identified by sequence analysis, and growth recovery was investigated after exposure to simulated solar radiation. Bacterioneuston and bacterioplankton isolates were subjected to six different exposure times, ranging from 0.5 to 7 h of simulated noontime solar radiation. Following exposure, the growth of each isolate was monitored, and different classes of resistance were determined according to the growth pattern. Large interspecific differences among the 90 marine isolates were observed. Medium and highly resistant strains accounted for 41% and 22% of the isolates, respectively, and only 16% were sensitive strains. Resistance to solar radiation was equally distributed within the bacterioneuston and bacterioplankton. Relative contributions to the highly resistant class were 43% for gamma-proteobacteria and 14% and 8% for alpha-proteobacteria and the Cytophaga/Flavobacterium/Bacteroides (CFB) group, respectively. Within the gamma-proteobacteria, the Pseudoalteromonas and Alteromonas genera appeared to be highly resistant to solar radiation. The majority of the CFB group (76%) had medium resistance. Our study further provides evidence that pigmented bacteria are not more resistant to solar radiation than nonpigmented bacteria.     

Diversity and metabolism of marine bacteria cultivated on dissolved DNA

Dissolved DNA (dDNA) is a potentially important source of energy and nutrients in aquatic ecosystems. However, little is known about the identity, metabolism, and interactions of the microorganisms capable of consuming dDNA. Bacteria from Eel Pond (Woods Hole, MA) were cultivated on low-molecular-weight (LMW) or high-molecular-weight (HMW) dDNA, which served as the primary source of carbon, nitrogen, and phosphorus. Cloning and sequencing of 16S rRNA genes revealed that distinct bacterial assemblages with comparable levels of taxon richness developed on LMW and HMW dDNA. Since the LMW and HMW dDNA used in this study were stoichiometrically identical, the results confirm that the size alone of dissolved organic matter can influence bacterial community composition. Variation in the growth and metabolism of isolates provided insight into mechanisms that may have generated differences in bacterial community composition. For example, bacteria from LMW dDNA enrichments generally grew better on LMW dDNA than on HMW dDNA. In contrast, bacteria isolated from HMW dDNA enrichments were more effective at degrading HMW dDNA than bacteria isolated from LMW dDNA enrichments. Thus, marine bacteria may experience a trade-off between their ability to compete for LMW dDNA and their ability to access HMW dDNA via extracellular nuclease production. Together, the results of this study suggest that dDNA turnover in marine ecosystems may involve a succession of microbial assemblages with specialized ecological strategies.     

Diversity and Metabolism of Marine Bacteria Cultivated on Dissolved DNA

Dissolved DNA (dDNA) is a potentially important source of energy and nutrients in aquatic ecosystems. However, little is known about the identity, metabolism, and interactions of the microorganisms capable of consuming dDNA. Bacteria from Eel Pond (Woods Hole, MA) were cultivated on low-molecular-weight (LMW) or high-molecular-weight (HMW) dDNA, which served as the primary source of carbon, nitrogen, and phosphorus. Cloning and sequencing of 16S rRNA genes revealed that distinct bacterial assemblages with comparable levels of taxon richness developed on LMW and HMW dDNA. Since the LMW and HMW dDNA used in this study were stoichiometrically identical, the results confirm that the size alone of dissolved organic matter can influence bacterial community composition. Variation in the growth and metabolism of isolates provided insight into mechanisms that may have generated differences in bacterial community composition. For example, bacteria from LMW dDNA enrichments generally grew better on LMW dDNA than on HMW dDNA. In contrast, bacteria isolated from HMW dDNA enrichments were more effective at degrading HMW dDNA than bacteria isolated from LMW dDNA enrichments. Thus, marine bacteria may experience a trade-off between their ability to compete for LMW dDNA and their ability to access HMW dDNA via extracellular nuclease production. Together, the results of this study suggest that dDNA turnover in marine ecosystems may involve a succession of microbial assemblages with specialized ecological strategies.     

Patterns of marine bacterioplankton biodiversity in the surface waters of the Scotia Arc, Southern Ocean

Spatial patchiness in marine surface bacterioplankton populations was investigated in the Southern Ocean, where the Antarctic Circumpolar Current meets the islands of the Scotia Arc and is subjected to terrestrial input, upwelling of nutrients and seasonal phytoplankton blooms. Total bacterioplankton population density, group-specific taxonomic distribution and six of eight dominant members of the bacterioplankton community were found to be consistent across 18 nearshore sites at eight locations around the Scotia Arc. Results from seven independent 16S rRNA gene clone libraries (1223 sequences in total) and fluorescent in situ hybridization suggested that microbial assemblages were predominantly homogeneous between Scotia Arc sites, where the Alphaproteobacteria, Gammaproteobacteria and the Cytophaga-Flavobacterium-Bacteroidetes cluster were the dominant bacterial groups. Of the 1223 useable sequences generated, 1087 (89%) shared ≥?97% similarity with marine microorganisms and 331 (27%) matched published sequences previously detected in permanently cold Arctic and Antarctic marine environments. Taken together, results suggest that the dominant bacterioplankton groups are consistent between locations, but significant differences may be detected across the rare biodiversity.     

Diversity of bacterioplankton in coastal seawaters of Fildes Peninsula,King George Island,Antarctica

The bacterioplankton not only serves critical functions in marine nutrient cycles, but can also serve as indicators of the marine environment. The compositions of bacterial communities in the surface seawater of Ardley Cove and Great Wall Cove were analyzed using a 16S rRNA multiplex 454 pyrosequencing approach. Similar patterns of bacterial composition were found between the two coves, in which Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria were the dominant members of the bacterioplankton communities. In addition, a large fraction of the bacterial sequence reads (on average 5.3 % per station) could not be assigned below the domain level. Compared with Ardley Cove, Great Wall Cove showed higher chlorophyll and particulate organic carbon concentrations and exhibited relatively lower bacterial richness and diversity. Inferred metabolisms of summer bacterioplankton in the two coves were characterized by chemoheterotrophy and photoheterotrophy. Results suggest that some cosmopolitan species (e.g., Polaribacter and Sulfitobacter) belonging to a few bacterial groups that usually dominate in marine bacterioplankton communities may have similar ecological functions in similar marine environments but at different geographic locations.     

Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates

A total of 319 bacterial strains isolated from the surfaces of seaweeds and invertebrates were tested for their effects on settlement of Ulvalactuca spores and Hydroidesezoensis larvae in laboratory bioassays. Of the 192 bacterial strains isolated from the surfaces of seaweeds 63 isolates were shown to be inhibitory against the settlement of algal spores and 62 isolates were inhibitory against larval settlement. Thirty-seven percent of the 127 bacterial strains isolated from the surfaces of marine invertebrates were shown to be inhibitory against algal spores and larval settlement. The strain CI4 isolated from an adult Cionaintestinalis showed the strongest inhibitory effect and was identified as Pseudoalteromonas sp. via 16S rRNA gene sequencing. The high proportions of host associated bacteria producing antifouling compounds suggest that these bacteria may help the host organism in the defense against fouling.     

Growth patterns of two marine isolates: adaptations to substrate patchiness?

During bottle incubations of heterotrophic marine picoplankton, some bacterial groups are conspicuously favored. In an earlier investigation bacteria of the genus Pseudoalteromonas rapidly multiplied in substrate-amended North Sea water, whereas the densities of Oceanospirillum changed little (H. Eilers, J. Pernthaler, and R. Amann, Appl. Environ. Microbiol. 66:4634-4640, 2000). We therefore studied the growth patterns of two isolates affiliating with Pseudoalteromonas and Oceanospirillum in batch culture. Upon substrate resupply, Oceanospirillum lagged threefold longer than Pseudoalteromonas but reached more than fivefold-higher final cell density and biomass. A second, mobile morphotype was present in the starved Oceanospirillum populations with distinctly greater cell size, DNA and protein content, and 16S rRNA concentration. Contrasting cellular ribosome concentrations during stationary phase suggested basic differences in the growth responses of the two strains to a patchy environment. Therefore, we exposed the strains to different modes of substrate addition. During cocultivation on a single batch of substrates, the final cell densities of Oceanospirillum were reduced three times as much as those Pseudoalteromonas, compared to growth yields in pure cultures. In contrast, the gradual addition of substrates to stationary-phase cocultures was clearly disadvantageous for the Pseudoalteromonas population. Different growth responses to substrate gradients could thus be another facet affecting the competition between marine bacteria and may help to explain community shifts observed during enrichments.     

Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates

A total of 319 bacterial strains isolated from the surfaces of seaweeds and invertebrates were tested for their effects on settlement of Ulvalactuca spores and Hydroidesezoensis larvae in laboratory bioassays. Of the 192 bacterial strains isolated from the surfaces of seaweeds 63 isolates were shown to be inhibitory against the settlement of algal spores and 62 isolates were inhibitory against larval settlement. Thirty-seven percent of the 127 bacterial strains isolated from the surfaces of marine invertebrates were shown to be inhibitory against algal spores and larval settlement. The strain CI4 isolated from an adult Cionaintestinalis showed the strongest inhibitory effect and was identified as Pseudoalteromonas sp. via 16S rRNA gene sequencing. The high proportions of host associated bacteria producing antifouling compounds suggest that these bacteria may help the host organism in the defense against fouling.     

Low Intraspecific Diversity in a Polynucleobacter Subcluster Population Numerically Dominating Bacterioplankton of a Freshwater Pond

Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a Polynucleobacter subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the Polynucleobacter cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a Polynucleobacter population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.     

Culturability and In situ abundance of pelagic bacteria from the North Sea

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.     

Low intraspecific diversity in a polynucleobacter subcluster population numerically dominating bacterioplankton of a freshwater pond

Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a Polynucleobacter subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the Polynucleobacter cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a Polynucleobacter population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.     

Specific detection, isolation, and characterization of selected, previously uncultured members of the freshwater bacterioplankton community

High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the alpha-Proteobacteria, beta-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental alpha-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.     

渤海沉积物产脂肪酶细菌的筛选及其多样性分析

【目的】从渤海沉积物中分离筛选产脂肪酶细菌,分析其物种多样性,增加人们对渤海生态系统中产脂肪酶菌多样性的认识,获取高效产脂肪酶菌株,为海洋产脂肪酶微生物的挖掘提供菌群资源。【方法】分别将8个渤海沉积物样品梯度稀释涂布至吐温-80筛选平板和三丁酸甘油酯筛选平板,选择性分离产脂肪酶细菌;分析基于16SrRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;利用对硝基苯酚法测定胞外脂肪酶活性,筛选出高效产脂肪酶菌株。【结果】从8个渤海沉积物样品中分离获得51株产脂肪酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria和Firmicutes三个门的8个属,其中Pseudoalteromonas(35.2%)、Marinobacter(23.5%)和Sulfitobacter(17.6%)是优势菌群;脂肪酶酶活性实验表明所有测定菌株都能够分泌脂肪酶,菌株70623分泌的脂肪酶酶活最高,为42.4 U/m L。【结论】渤海沉积物中可培养产脂肪酶细菌类群较为丰富,Pseudoalteromonas、Marinobacter和Sulfitobacter菌株是优势菌群,测定菌株所产胞外脂肪酶能力不同,获得了一株高效产脂肪酶菌株Marinobacter sp.70623。     

Screening of marine bacteria for fucoidan hydrolases

Twenty-five strains of epiphytic marine bacteria isolated from the brown algae Fucus evanescens and Chorda filum and fifty-three bacteria isolated from the sea cucumber Apostichopus japonicus were screened for fucoidanases using fucoidans prepared from the brown algae F. evanescens, Laminaria cichorioides, and L. japonica. Eighteen bacterial epiphytes and thirty-eight bacterial isolates from the sea cucumber were found to contain fucoidanases, which were able to hydrolyze either all of the fucoidans studied or some of them. Bacteria of the genera Cytophaga and Alteromonas/Pseudoalteromonas exhibited the highest fucoidanase activities, which, however, did not exceed the activity of fucoidanases from the already known sources.     

Marine bacteria from Danish coastal waters show antifouling activity against the marine fouling bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis independent of bacteriocidal activity

The aims of this study were to determine if marine bacteria from Danish coastal waters produce antifouling compounds and if antifouling bacteria could be ascribed to specific niches or seasons. We further assess if antibacterial effect is a good proxy for antifouling activity. We isolated 110 bacteria with anti-Vibrio activity from different sample types and locations during a 1-year sampling from Danish coastal waters. The strains were identified as Pseudoalteromonas, Phaeobacter, and Vibrionaceae based on phenotypic tests and partial 16S rRNA gene sequence similarity. The numbers of bioactive bacteria were significantly higher in warmer than in colder months. While some species were isolated at all sampling locations, others were niche specific. We repeatedly isolated Phaeobacter gallaeciensis at surfaces from one site and Pseudoalteromonas tunicata at two others. Twenty-two strains, representing the major taxonomic groups, different seasons, and isolation strategies, were tested for antiadhesive effect against the marine biofilm-forming bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis. The antiadhesive effects were assessed by quantifying the number of strain S91 or Ulva spores attaching to a preformed biofilm of each of the 22 strains. The strongest antifouling activity was found in Pseudoalteromonas strains. Biofilms of Pseudoalteromonas piscicida, Pseudoalteromonas tunicata, and Pseudoalteromonas ulvae prevented Pseudoalteromonas S91 from attaching to steel surfaces. P. piscicida killed S91 bacteria in the suspension cultures, whereas P. tunicata and P. ulvae did not; however, they did prevent adhesion by nonbactericidal mechanism(s). Seven Pseudoalteromonas species, including P. piscicida and P. tunicata, reduced the number of settling Ulva zoospores to less than 10% of the number settling on control surfaces. The antifouling alpP gene was detected only in P. tunicata strains (with purple and yellow pigmentation), so other compounds/mechanisms must be present in the other Pseudoalteromonas strains with antifouling activity.     

Spatial differences in bacterioplankton composition along the Catalan coast (NW Mediterranean) assessed by molecular fingerprinting

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments was used to compare surface bacterioplankton assemblages along the Catalan coast (NW Mediterranean). Samples from three coastal stations were compared with samples taken inside the Barcelona harbour and open sea samples taken during a cruise. The bacterial assemblage of each sample showed a characteristic and reproducible DGGE fingerprint. Between 17 and 35 bands were detected in each sample, and about 40% of the bands accounted for more than 80% of the band intensity in each sample. The presence of bands as well as their relative intensity was used to compare bacterial assemblages. Clear differences between the harbour samples and the coastal samples were evident during all periods. Marked temporal changes in the bacterial assemblages were detectable for the coastal sites, suggesting seasonal succession of coastal bacterioplankton. During each season, two stations presented a very similar bacterial composition (Barcelona and Masnou) whereas bacterial assemblages in Blanes were slightly different. These differences were consistent with the different hydrography of the area. Diversity indices calculated from DGGE fingerprints were relatively similar for all samples analysed, even though harbour samples were expected to present lower diversity values.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.     

Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis.

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1, 632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.