pufM Gene Research Articles

Environmental controls and composition of anoxygenic photoheterotrophs in ultraoligotrophic high‐altitude lakes (Central Pyrenees)

The phylogenetic composition of freshwater anoxygenic photoheterotrophs (APs) has been poorly investigated as compared with their marine counterparts. In this study, we explored a set of ultraoligotrophic cold high mountain lakes (Central Pyrenees, Spain) by both pufM gene denaturing gradient gel electrophoresis fingerprinting, and cloning and sequencing of selected lakes samples. Different ranges of limnological and physico-chemical values were explored as environmental drivers of APs richness and composition. We did not observe significant relationships between richness/diversity of pufM and any of the limnological characteristics measured or trophic status, but a negative correlation with ammonia concentration. Conductivity, pH and nitrate concentration were significantly related to changes in APs community composition, whereas lake area, altitude, temperature and trophic status did not. Most of the sequences (> 85%) had the pufM sequences of Limnohabitans (Betaproteobacteria) as the closest relative in databases, whereas less abundant clones were more closely related to Rhodobacter, Sulfitobacter and Brevundimonas (Alphaproteobacteria), in agreement with 16S rRNA gene sequences previously found in the area. Congregibacter-like Gammaproteobacteria were not detected. Comparison with available studies in inland waters showed taxonomic partitioning along salinity gradients, and Congregibacter-like sequences restricted to high saline conditions in continental water bodies.

Marked seasonality of aerobic anoxygenic phototrophic bacteria in the coastal NW Mediterranean Sea as revealed by cell abundance, pigment concentration and pyrosequencing of pufM gene

The abundance and diversity of aerobic anoxygenic phototrophs (AAPs) were studied for a year cycle at the Blanes Bay Microbial Observatory (NW Mediterranean) and their potential links to an array of environmental variables were explored. Cell numbers were low in winter and peaked in summer, showing a marked seasonality that positively correlated with day length and light at the surface. Bacteriochlorophyll a concentration, their light-harvesting pigment, was only detected between April and October, and pigment cell quota showed large variations during this period. Pyrosequencing analysis of the pufM gene revealed that the most abundant operational taxonomic units (OTUs) were affiliated to phylogroup K (Gammaproteobacteria) and uncultured phylogroup C, although they were outnumbered by alphaproteobacterial OTUs in spring. Overall, richness was higher in winter than in summer, showing an opposite trend to abundance and day length. Clustering of samples by multivariate analyses showed a clear seasonality that suggests a succession of different AAP subpopulations over time. Temperature, chlorophyll a and day length were the environmental drivers that best explained the distribution of AAP assemblages. These results indicate that AAP bacteria are highly dynamic and undergo seasonal variations in diversity and abundance mostly dictated by environmental conditions as exemplified by light availability.

Anoxygenic phototrophic bacterial diversity within wastewater stabilization plant during ‘red water’ phenomenon

The molecular diversity of the purple photosynthetic bacteria was assessed during temporal pigmentation changes in four interconnected wastewater stabilization ponds treating domestic wastewater by denaturant gel gradient electrophoresis method applying pufM gene. Results revealed high phylogenetic diversity of the purple phototrophic anoxygenic bacteria community characterized by the presence of the purple non-sulfur, purple sulfur, and purple aerobic photosynthetic anoxygenic bacteria. This phototrophic bacterial assemblage was dominated by the purple non-sulfur bacteria group (59.3 %) with six different genera followed by the purple sulfur community (27.8 %) with four genera and finally 12.9 % of the pufM gene sequences were assigned throughout the aerobic anoxygenic phototrophic bacterial group. The purple phototrophic bacterial community was characterized by the presence of salt-dependant bacterial species belonging to the genus Marichromatium, Thiorhodococcus, Erythrobacter, and Roseobacter. The wastewater treatment plant performances were unsatisfactory, and the biological and chemical parameters suggested that the purple photosynthetic bloom was correlated with the eutrophic state.

Roseibacterium beibuensis sp. nov., a Novel Member of Roseobacter Clade Isolated from Beibu Gulf in the South China Sea

A novel aerobic, bacteriochlorophyll-containing bacteria strain JLT1202r(T) was isolated from Beibu Gulf in the South China Sea. Cells were gram-negative, non-motile, and short-ovoid to rod-shaped with two narrower poles. Strain JLT1202r(T) formed circular, opaque, wine-red colonies, and grew optimally at 3-4% NaCl, pH 7.5-8.0 and 28-30°C. The strain was catalase, oxidase, ONPG, gelatin, and Voges-Proskauer test positive. In vivo absorption spectrum of bacteriochlorophyll a presented two peaks at 800 and 877nm. The predominant cellular fatty acid was C(18:1) ω7c and significant amounts of C(16:0), C(18:0), C(10:0) 3-OH, C(16:0) 2-OH, and 11-methyl C(18:1) ω7c were present. Strain JLT1202r(T) contained Q-10 as the major respiratory quinone and the genomic DNA G+C content was 76.3mol%. Phylogenetic analysis based on 16S rRNA gene sequences of various species with validly published names showed that strain JLT1202r(T) fell within the genus Roseibacterium, family Rhodobacteraceae, sharing the highest similarity with Roseibacterium elongatum OCh 323(T) (97.9% similarity), followed by Dinoroseobacter shibae DFL 12(T) (95.4% similarity). The phylogenetic distance of pufM genes between strain JLT1202r(T) and R. elongatum OCh 323(T) was 9.4%, suggesting that strain JLT1202r(T) was distinct from the only strain of the genus Roseibacterium. Based on the variabilities of phylogenetic and phenotypic characteristics, strain JLT1202r(T) stands for a novel species of the genus Roseibacterium and the name R. beibuensis sp. nov. is proposed with JLT1202r(T) as the type strain (=JCM 18015(T)=CGMCC 1.10994(T)).

Sulfur bacteria in wastewater stabilization ponds periodically affected by the ‘red-water’ phenomenon

Several wastewater stabilization ponds (WSP) in Tunisia suffer periodically from the ‘red-water’ phenomenon due to blooming of purple sulfur bacteria, indicating that sulfur cycle is one of the main element cycles in these ponds. In this study, we investigated the microbial diversity of the El Menzeh WSP and focused in particular on the different functional groups of sulfur bacteria. For this purpose, we used denaturing gradient gel electrophoresis of PCR-amplified fragments of the 16S rRNA gene and of different functional genes involved in microbial sulfur metabolism (dsrB, aprA, and pufM). Analyses of the 16S rRNA revealed a relatively high microbial diversity where Proteobacteria, Chlorobi, Bacteroidetes, and Cyanobacteria constitute the major bacterial groups. The dsrB and aprA gene analysis revealed the presence of deltaproteobacterial sulfate-reducing bacteria (i.e., Desulfobacter and Desulfobulbus), while the analysis of 16S rRNA, aprA, and pufM genes assigned the sulfur-oxidizing bacteria community to the photosynthetic representatives belonging to the Chlorobi (green sulfur bacteria) and the Proteobacteria (purple sulfur and non sulfur bacteria) phyla. These results point on the diversity of the metabolic processes within this wastewater plant and/or the availability of sulfate and diverse electron donors.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-012-3931-5) contains supplementary material, which is available to authorized users.

Diversity of Purple Phototrophic Bacteria, Inferred from <i>pufM</i> Gene, within Epilithic Biofilm in Tama River, Japan

The diversity of purple phototrophic bacteria in algae-dominated biofilm of a streambed in Tama River, Japan was investigated. Clone library analysis of the pufM gene encoding a subunit of the photochemical reaction center of purple bacteria detected 18 operational taxonomic units (OTUs) in several classes of Proteobacteria. Most of the OTUs showed less than 85% identity to the PufM amino acid sequences of known phototrophic bacteria. These results suggest that phylogenetically divergent and unknown purple phototrophic bacteria are present in the epilithic biofilm of the river.

Rhodoferax‐related pufM gene cluster dominates the aerobic anoxygenic phototrophic communities in German freshwater lakes

The presence of aerobic anoxygenic phototrophs (AAPs) has been repeatedly reported from various marine environments, but their distribution in freshwater lakes was neglected until recently. We investigated the phylogenetic composition of AAP communities in 10 lakes in Northeastern Germany with different trophic status including oligotrophic Lake Stechlin and humic matter rich Lake Grosse Fuchskuhle. The AAP community was composed by members of Alpha- and Betaproteobacteria, but their contribution varied largely among the studied lakes. Our results show that AAP community composition in the studied lakes was affected mostly by pH and humic matter content. While alkaline lakes were mostly composed of Betaproteobacteria, the acidic and humic matter rich south-west (SW) basin of Lake Grosse Fuchskule was dominated (87%) by Alphaproteobacteria. The most frequent group within Betaproteobacteria was a cluster of pufM genes which was phylogenetically related to Rhodoferax representing 38.5% of all retrieved sequences. Alphaproteobacteria-related sequences had a broader phylogenetic diversity including six different taxa dominated by Sphingomonas- and Rhodobacter-like bacteria in lakes with alkaline to neutral pH. In the acidic and humic matter-rich SW basin of Lake Grosse Fuchskuhle, however, Methylobacterium-related sequences dominated the AAP community. We suggest that the variable AAP community structure might reflect the potential of these bacteria to cope with the contrasting conditions in freshwater environments.

Diversity of cultivated and metabolically active aerobic anoxygenic phototrophic bacteria along an oligotrophic gradient in the Mediterranean Sea

Abstract. Aerobic anoxygenic phototrophic (AAP) bacteria play significant roles in the bacterioplankton productivity and biogeochemical cycles of the surface ocean. In this study, we applied both cultivation and mRNA-based molecular methods to explore the diversity of AAP bacteria along an oligotrophic gradient in the Mediterranean Sea in early summer 2008. Colony-forming units obtained on three different agar media were screened for the production of bacteriochlorophyll-a (BChl-a), the light-harvesting pigment of AAP bacteria. BChl-a-containing colonies represented a low part of the cultivable fraction. In total, 54 AAP strains were isolated and the phylogenetic analyses based on their 16S rRNA and pufM genes showed that they were all affiliated to the Alphaproteobacteria. The most frequently isolated strains belonged to Citromicrobium bathyomarinum, and Erythrobacter and Roseovarius species. Most other isolates were related to species not reported to produce BChl-a and/or may represent novel taxa. Direct extraction of RNA from seawater samples enabled the analysis of the expression of pufM, the gene coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis. Clone libraries of pufM gene transcripts revealed that most phylotypes were highly similar to sequences previously recovered from the Mediterranean Sea and a large majority (~94 %) was affiliated to the Gammaproteobacteria. The most abundantly detected phylotypes occurred in the western and eastern Mediterranean basins. However, some were exclusively detected in the eastern basin, reflecting the highest diversity of pufM transcripts observed in this ultra-oligotrophic region. To our knowledge, this is the first study to document extensively the diversity of AAP isolates and to unveil the active AAP community in an oligotrophic marine environment. By pointing out the discrepancies between culture-based and molecular methods, this study highlights the existing gaps in the understanding of the AAP bacteria ecology, especially in the Mediterranean Sea and likely globally.

Elevated Ground-Level O3 Changes the Diversity of Anoxygenic Purple Phototrophic Bacteria in Paddy Field

The knowledge of the impact of elevated ground-level O(3) below ground the agro-ecosystem is limited. A field experiment in China Ozone Free-Air Concentration Enrichment (FACE-O(3)) facility on a rice-wheat rotation system was carried out to investigate responses of anoxygenic phototrophic purple bacteria (AnPPB) to elevated ground-level O(3). AnPPB community structures and sizes in paddy soil were monitored by molecular approaches including PCR-DGGE and real-time quantitative PCR based upon the pufM gene on three typical rice growth stages. Repetitive sequence-based PCR (rep-PCR) in combination with culture-reliant method was conducted to reveal changes in genotypic diversity. Elevated ground-level O(3) statistically reduce AnPPB abundance and percentage in total bacterial community in flooded rice soil via decreasing their genotypic diversity and metabolic versatility. Concomitantly, their community composition changed after rice anthesis stage under elevated ground-level O(3). Our results from AnPPB potential responses imply that continuously elevated ground-level O(3) in the future would eventually harm the health of paddy ecosystem through negative effect on soil microorganisms.

Soil purple phototrophic bacterial diversity under double cropping (rice-wheat) with free-air CO2 enrichment (FACE)

Soil purple phototrophic bacterial (PPB) communities and their responses to elevated atmospheric carbon dioxide (CO 2 ) concentration and nitrogen (N) fertilizer were investigated under a rotation of paddy rice (Oryza sativa L.) and winter wheat (Triticum aestivum L. cv. Yangmai 14) cultivation in a FACE (free-air CO 2 enrichment) system. Community structures and abundances of PPB were determined by denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR respectively, targeting the pufM gene, which encodes a protein in the light reaction centre of PPB. Soil PPB communities were more diverse and larger under rice than under wheat cultivation, which may be attributed to the flooding of the paddy field and soil moisture changes. Elevated atmospheric CO 2 concentration significantly increased the abundance and biodiversity of PPB in soils under rice cultivation, while N fertilizer application rate had less effect on the abundance and diversity. Phylogenetic analysis showed that two common dominant DGGE bands belonged to Bradyrhizobium- and Rhodopseudomonas palustris-like PPB in both rice and wheat soils. The results demonstrated a significant shift in soil PPB communities during the rice-wheat rotation, and a strong positive response of PPB communities to an elevated atmospheric CO 2 concentration. Our results also indicated that a diverse and abundant soil PPB community could occur in upland crop fields as well as in aquatic environments and paddy-rice fields. These findings extend our understanding of the ecological significance of PPB in terrestrial soil environments and their responses to future climate change.

Diversity and abundance of aerobic anoxygenic phototrophic bacteria in two cyanobacterial bloom-forming lakes in China

Aerobic anoxygenic phototrophic (AAP) bacteria are widely distributed in marine and freshwater ecosystems. The aims of this study were to investigate the diversity and abundance of AAP bacteria in cyano- bacterial bloom-forming eutrophic lakes and to study the association of AAP bacteria with the bloom- forming cyanobacteria. Analysis of pufM gene (the light-reaction center gene) clone libraries indicated that in eutrophic lakes (Lake Taihu and Lake Chaohu, China) with cyanobacterium Microcystis blooms, the AAP bacteria were related to members of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. In Lake Taihu and Lake Chaohu, Alphaproteobacteria accounted for 81.5% and 75.0% of Microcystis- associated AAP bacteria, respectively, and 84.6% and 72.5% of free-living AAP bacteria, respectively. The predominance of Alphaproteobacteria in the two lakes was different from the previously reported predominance of Betaproteobacteria in freshwater lakes. Quantitative real-time PCR analysis indicated that in Lake Taihu and Lake Chaohu, AAP bacteria represented an important part of the bacterial community associated with Microcystis, and the abundance of Microcystis-associated AAP bacteria (18.3% and 11.7%, respectively) was higher than that of free-living AAP bacteria (5.1% and 7.9%, respectively). The abundance of AAP bacteria in the two bloom-forming lakes was higher than the previously reported level in other eutrophic freshwater bodies.

Diversity of aerobic anoxygenic phototrophic bacteria in paddy soil and their response to elevated atmospheric CO₂.

SummaryAerobic anoxygenic phototrophic bacteria (AAnPB) are recognized as an important group driving the global carbon cycling. However, the diversity of AAnPB in terrestrial environment remains largely unknown as well as their responses to the elevated atmospheric CO2. By using culture‐independent techniques, the diversity of AAnPB in paddy soil and the changes in response to the rising atmospheric CO2 were investigated within China FACE (Free‐air CO2 enrichment) platform. There was a phylogenetically diverse AAnPB community with large population size residing in paddy soil. The community structure of AAnPB in bulk and rhizospheric soils stayed almost identical, while the population size was higher in rhizospheric [2.0–2.5 × 108 copy number of pufM genes g−1 dry weight soil (d.w.s.)] than that in bulk (0.7–0.8 × 108 g−1 d.w.s.) soils. Elevated atmospheric CO2 appeared to significantly stimulate AAnPB abundance (up to 1.4–1.5 × 108 g−1 d.w.s.) and result in a higher AAnPB percentage in total bacterial community (from 0.5% up to 1.5%) in bulk soil, whereas no significant effect was observed in rhizospheric soil. Our results would extend the functional ecotypes of AAnPB and indicate that environmental changes associated with the rising atmospheric CO2 might affect AAnPB community in paddy soil.

Distribution of Roseobacter RCA and SAR11 lineages in the North Sea and characteristics of an abundant RCA isolate

The Roseobacter group and SAR11 clade constitute high proportions of the marine bacterioplankton, but only scarce information exists on the abundance of distinct populations of either lineage. Therefore, we quantified the abundance of the largest cluster of the Roseobacter group, the RCA (Roseobacter clade affiliated) cluster together with the SAR11 clade by quantitative PCR in the southern and eastern North Sea. The RCA cluster constituted up to 15 and 21% of total bacterial 16S ribosomal RNA (rRNA) genes in September 2005 and May 2006, respectively. At a few stations, the RCA cluster exceeded the SAR11 clade, whereas at most stations, SAR11 constituted higher fractions with maxima of 37%. In most samples, only one RCA ribotype was detected. RCA abundance was positively correlated with phaeopigments, chlorophyll, dissolved and particulate organic carbon (POC), turnover rates of dissolved free amino acids (DFAAs), temperature, and negatively correlated with salinity. The SAR11 clade was only correlated with POC (negatively, May) and with DFAA turnover rates (positively, September). An abundant RCA strain, 'Candidatus Planktomarina temperata', was isolated from the southern North Sea. This strain has an identical 16S rRNA gene sequence to the dominant RCA ribotype. Detection of the pufM gene, coding for a subunit of the reaction center of bacteriochlorophyll a, indicates the potential of the isolate for aerobic anoxygenic photosynthesis. Our study shows that a distinct population of the RCA cluster constitutes an abundant bacterioplankton group in a neritic sea of the temperate zone and indicates that this population has an important role during decaying phytoplankton blooms.

Response of Aerobic Anoxygenic Phototrophic Bacterial Diversity to Environment Conditions in Saline Lakes and Daotang River on the Tibetan Plateau, NW China

Variations of the pufM gene [encoding the M subunit of the photosynthetic reaction center in aerobic anoxygenic phototrophic (AAnP) bacteria] diversity in response to environmental changes were investigated in waters of six aqueous regimes (including Daotang River and five saline/hypersaline lakes on the Tibetan Plateau) representing a full salinity gradient from freshwater to NaCl-saturation. AAnP bacterial community structures responded to salinity change: Gamma-like AAnP community was predominant in freshwater Daotang River (0.01% salinity). AAnP community structure shifted from Loktanella-like sequences of the Alphaproteobacteria in saline Qinghai Lake to Roseobacter-like sequences in hypersaline lakes (Gahai, Xiaochaidan and Charhan). In addition to salinity, other environmental variables (e.g. N and P availability, TOC and/or DOC, and HCO− 3/CO2− 3 ions) were also important in affecting the pufM gene diversity in hypersaline lakes. These data have important implications for our understanding of the response of AAnP bacterial community to environmental variables in high-altitude aquatic ecosystems.

Bacteriochlorophyll and community structure of aerobic anoxygenic phototrophic bacteria in a particle-rich estuary

Photoheterotrophic microbes use organic substrates and light energy to satisfy their demand for carbon and energy and seem to be well adapted to eutrophic estuarine and oligotrophic oceanic environments. One type of photoheterotroph, aerobic anoxygenic phototrophic (AAP) bacteria, is especially abundant in particle-rich, turbid estuaries. To explore questions regarding the controls of these photoheterotrophic bacteria, we examined their abundance by epifluorescence microscopy, concentrations of the light-harvesting pigment, bacteriochlorophyll a (BChl a) and the diversity of pufM and 16S ribosomal RNA (rRNA) genes in the Chesapeake Bay. Concentrations of BChl a varied substantially, much more so than AAP bacterial abundance, along the estuarine salinity gradient. The BChl a concentration was correlated with turbidity only when oceanic and estuarine waters were considered together. Concentrations of BChl a and BChl a quotas were higher in particle-associated than in free-living AAP bacterial communities and appear to reflect physiological adaptation, not different AAP bacterial communities; pufM genes did not differ between particle-associated and free-living communities. In contrast, particle-associated and free-living bacterial communities were significantly different, on the basis of the analysis of 16S rRNA genes. The BChl a quota of AAP bacteria was not correlated with turbidity, suggesting that pigment synthesis varies in direct response to particles, not light attenuation. The AAP bacteria seem to synthesize more BChl a when dissolved and particulate substrates are available than when only dissolved materials are accessible, which has implications for understanding the impact of substrates on the level of photoheterotrophy compared with heterotrophy in AAP bacteria.

Purple nonsulfur bacteria diversity in activated sludge and its potential phosphorus-accumulating ability under different cultivation conditions

This study investigates the diversity and the potential phosphorus-accumulating ability among the purple nonsulfur (PNS) bacteria. Traditional methods and molecular biotechniques were applied. Microscopic visualization using 4',6-diamidino-2-phenylindole staining as well as chemical analysis demonstrated that most of the isolated PNS bacteria presented different levels of phosphorus accumulation. Four of the pure cultures, denoted as Rhodopseudomonas palustris CC1, CC7, G11, and GE1, based on their differences in the PNS's pufM gene, exhibited higher internal phosphorus content compared to other isolated strains in this study. In addition, substantial polyphosphate accumulation was observed after the bacteria entered their stationary growth phase. Among them, the isolated R. palustris G11 could accumulate internal phosphorus up to 13%-15% of its cell dry weight under anaerobic illuminated incubation conditions. When the incubation status was switched from anaerobic to aerobic, the bacterial phosphorus content had a tendency to decrease slightly or remain about the same throughout the whole aerobic stage. The growth rate and biomass were higher when the PNS bacteria grew under photoheterotrophic conditions rather than the chemoheterotrophic ones. Furthermore, the environmental pH value could affect the contents of internal bacterial phosphate. Results of this study demonstrated that PNS bacteria are a group of the polyphosphate-accumulating organisms, of which this ability had never been properly studied. The conditions that PNS bacteria accumulating polyphosphate presented from this study were unique and showed characteristics that were different from the well-known enhanced biological phosphorus removal model.

Diversity ofpufMgenes, involved in aerobic anoxygenic photosynthesis, in the bacterial communities associated with colonial ascidians

Ascidians are invertebrate filter feeders widely distributed in benthic marine environments. A total of 14 different ascidian species were collected from the Western Mediterranean and their bacterial communities were analyzed by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene. Results showed that ascidian tissues harbored Bacteria belonging to Gamma- and Alphaproteobacteria classes, some of them phylogenetically related to known aerobic anoxygenic phototrophs (AAPs), such as Roseobacter sp. In addition, hierarchical cluster analysis of DGGE patterns showed a large variability in the bacterial diversity among the different ascidians analyzed, which indicates that they would harbor different bacterial communities. Furthermore, pufM genes, involved in aerobic anoxygenic photosynthesis in marine and freshwater systems, were widely detected within the ascidians analyzed, because nine out of 14 species had pufM genes inside their tissues. The pufM gene was only detected in those specimens that inhabited shallow waters (<77 m of depth). Most pufM gene sequences were very closely related to that of uncultured marine bacteria. Thus, our results suggest that the association of ascidians with bacteria related to AAPs could be a general phenomenon and that ascidian-associated microbiota could use the light that penetrates through the tunic tissue as an energy source.

Free-air CO2 enrichment (FACE) enhances the biodiversity of purple phototrophic bacteria in flooded paddy soil

Purple phototrophic bacteria (PPB) are thought to be crucial in the nutrient cycling of rice field. However, it remains unclear how PPB would respond to the climate change associated with the projected atmospheric CO2 in the future. A factorial design of field experiments was set up with two levels of atmospheric CO2 concentration (350 and 550 μmol mol−1) and N application rate (150 and 250 kg N ha−1) to investigate the abundance and composition changes of PPB in rhizospheric and bulk soils in response to the rising atmospheric CO2 concentration. Based on denaturant gradient gel electrophoresis (DGGE) analysis of pufM gene encoding the M subunit of anoxygenic PPB light reaction center, elevated CO2 appeared to enhance the biodiversity of PPB in flooded paddy soils. This was further supported by canonical correspondence analysis (CCA) of DGGE fingerprinting pattern of pufM genes in paddy soils as well as Shannon diversity indices. Real-time quantitative PCR analysis of pufM gene further indicated that PPB abundance was stimulated by elevated CO2 in bulk soil, while the contrasting result was observed in rhizospheric soil. Our result for the first time demonstrated that elevated CO2 enhanced the biodiversity of PPB within α and β subdivisions of Proteobacteria.

Genetic diversity of aerobic anoxygenic photosynthetic bacteria in open ocean surface waters and upper twilight zones

Aerobic anoxygenic phototrophic bacteria (AAPB) represent a widespread mixotrophic bacterial group in marine ecosystems. Here we investigated AAPB genetic diversity in the surface waters and upper twilight zones of the central Pacific, Atlantic, and Indian oceans by amplifying an AAPB marker gene (pufM, encoding photosynthetic reaction center small subunit) directly from bacterioplankton community DNA. Phylogenetic and statistical analysis of 267 pufM partial sequences in six clone libraries revealed a high diversity pattern in open ocean AAPB communities. Various AAPB subgroups belonging to Alpha- and Gamma-proteobacteria were found in both surface and upper twilight zone waters. In most samples, subgroups in which no pure culture was isolated as yet were predominant. By sampling a wide size range of bacterioplankton (0.22–200 μm) and introducing nested PCR to amplification, we retrieved abundant pufM fragments (136 sequences in 37 OTUs) directly from upper twilight zone samples. AAPB populations in upper twilight zones covered major subgroups found in surface waters and had a slightly lower diversity, higher dominance, and lower GC and GC3 contents in pufM genes than those in surface AAPB populations. These diversity data combined with previous BChl.a data in upper twilight zones support the hypothesis that AAPB may be present below euphotic zones based on the speculation that AAPB can utilize the dim light in twilight zones as a supplement to energy supply in their heterotrophic lives.

The use of denaturing gradient gel electrophoresis with fully degenerate pufM primers to monitor aerobic anoxygenic phototrophic assemblages

Aerobic Anoxygenic Photosynthetic bacteria (AAnP) were recently proposed to be significant contributors to the global ocean carbon and energy cycles, but relatively little is known about the spatial and temporal distribution of AAnP populations. Here, we report on the development of denaturing gradient gel electrophoresis (DGGE) analyses to monitor naturally occurring marine AAnP populations using the pufM gene. We developed an approach using fully degenerate universal pufM primers, and tested the effect of different primers and PCR conditions on DGGE band resolution and migration. PufM‐DGGE analyses of eastern Mediterranean environmental DNA samples show that AAnP composition was coherent among stations and seasons. Furthermore, samples from different depths of stratified water columns resulted in clearly different DGGE patterns, while very similar DGGE patterns at varying depths were observed under well‐mixed conditions. These results demonstrate the potential use of DGGE with fully degenerate primers as a tool for rapid analyses of natural marine AAnP communities.