A Metagenomic and Amplicon Sequencing Combined Approach Reveals the Best Primers to Study Marine Aerobic Anoxygenic Phototrophs.

Studies based on protein-coding genes are essential to describe the diversity within bacterial functional groups. In the case of aerobic anoxygenic phototrophic (AAP) bacteria, the pufM gene has been established as the genetic marker for this particular functional group, although available primers are known to have amplification biases. We review here the existing primers for pufM gene amplification, design new ones, and evaluate their phylogenetic coverage. We then use samples from contrasting marine environments to evaluate their performance. By comparing the taxonomic composition of communities retrieved with metagenomics and with different amplicon approaches, we show that the commonly used PCR primers are biased towards the Gammaproteobacteria phylum and some Alphaproteobacteria clades. The metagenomic approach, as well as the use of other combinations of the existing and newly designed primers, show that these groups are in fact less abundant than previously observed, and that a great proportion of pufM sequences are affiliated to uncultured representatives, particularly in the open ocean. Altogether, the framework developed here becomes a better alternative for future studies based on the pufM gene and, additionally, serves as a reference for primer evaluation of other functional genes.

Taxonomic differences shape the responses of freshwater aerobic anoxygenic phototrophic bacterial communities to light and predation.

Aerobic anoxygenic phototrophic (AAP) bacteria are a phylogenetically diverse and ubiquitous group of prokaryotes that use organic matter but can harvest light using bacteriochlorophyll a. Although the factors regulating AAP ecology have long been investigated through field surveys, the few available experimental studies have considered AAPs as a group, thus disregarding the potential differential responses between taxonomically distinct AAP assemblages. Here, we used sequencing of the pufM gene to describe the diversity of AAPs in 10 environmentally distinct temperate lakes, and to investigate the taxonomic responses of AAP communities in these lakes when subjected to similar experimental manipulations of light and predator removal. The studied communities were clearly dominated by Limnohabitans AAP but presented a clear taxonomic segregation between lakes presumably driven by local conditions, which was maintained after experimental manipulations. Predation reduction (but not light exposure) caused significant compositional shifts across most assemblages, but the magnitude of these changes could not be clearly related to changes in bulk AAP abundances or taxonomic richness of AAP assemblages during experiments. Only a few operational taxonomic units, which differed taxonomically between lakes, were found to respond positively during experimental treatments. Our results highlight that different freshwater AAP communities respond differently to similar control mechanisms, highlighting that in-depth knowledge on AAP diversity is essential to understand the ecology and potential role of these photoheterotrophs.

Phenology and ecological role of aerobic anoxygenic phototrophs in freshwaters.

BACKGROUND: Aerobic anoxygenic phototrophic (AAP) bacteria are heterotrophic bacteria that supply their metabolism with light energy harvested by bacteriochlorophyll-a-containing reaction centers. Despite their substantial contribution to bacterial biomass, microbial food webs, and carbon cycle, their phenology in freshwater lakes remains unknown. Hence, we investigated seasonal variations of AAP abundance and community composition biweekly across 3 years in a temperate, meso-oligotrophic freshwater lake. RESULTS: AAP bacteria displayed a clear seasonal trend with a spring maximum following the bloom of phytoplankton and a secondary maximum in autumn. As the AAP bacteria represent a highly diverse assemblage of species, we followed their seasonal succession using the amplicon sequencing of the pufM marker gene. To enhance the accuracy of the taxonomic assignment, we developed new pufM primers that generate longer amplicons and compiled the currently largest database of pufM genes, comprising 3633 reference sequences spanning all phyla known to contain AAP species. With this novel resource, we demonstrated that the majority of the species appeared during specific phases of the seasonal cycle, with less than 2% of AAP species detected during the whole year. AAP community presented an indigenous freshwater nature characterized by high resilience and heterogenic adaptations to varying conditions of the freshwater environment. CONCLUSIONS: Our findings highlight the substantial contribution of AAP bacteria to the carbon flow and ecological dynamics of lakes and unveil a recurrent and dynamic seasonal succession of the AAP community. By integrating this information with the indicator of primary production (Chlorophyll-a) and existing ecological models, we show that AAP bacteria play a pivotal role in the recycling of dissolved organic matter released during spring phytoplankton bloom. We suggest a potential role of AAP bacteria within the context of the PEG model and their consideration in further ecological models.

Mixed-culture aerobic anoxygenic photosynthetic bacterial consortia reduce nitrate: Core species dynamics, co-interactions and assessment in raw water of reservoirs.

Three consortia of mixed-culture Aerobic Anoxygenic Photosynthetic Bacteria (AAPB) with excellent aerobic denitrifying ability were isolated from drinking water source reservoirs. The results showed that the removal of dissolved organic carbon (DOC) and nitrate nitrogen (NO3--N) by mixed-culture AAPB were higher than 90% and 99%, respectively. The Illumina MiSeq sequencing of pufM gene revealed that the dominant genera and their relative abundance changed over the culture periods. Sphingomonas sanxanigenens was the most dominant species observed at 9 h, whereas at 48 h, the most abundant species was Rhodobacter blasticus. A network analysis demonstrated that the co-interactions among the different genera were complex and variable. Mixed-culture AAPB removed more than 30% of NO3--N and 25% of DOC from the source water and this study suggests that mixed-culture AAPB can be regarded as a latent denitrifying microbial inoculum in the reservoir raw water treatment.

Distribution of Phototrophic Purple Nonsulfur Bacteria in Massive Blooms in Coastal and Wastewater Ditch Environments.

The biodiversity of phototrophic purple nonsulfur bacteria (PNSB) in comparison with purple sulfur bacteria (PSB) in colored blooms and microbial mats that developed in coastal mudflats and pools and wastewater ditches was investigated. For this, a combination of photopigment and quinone profiling, pufM gene-targeted quantitative PCR, and pufM gene clone library analysis was used in addition to conventional microscopic and cultivation methods. Red and pink blooms in the coastal environments contained PSB as the major populations, and smaller but significant densities of PNSB, with members of Rhodovulum predominating. On the other hand, red-pink blooms and mats in the wastewater ditches exclusively yielded PNSB, with Rhodobacter, Rhodopseudomonas, and/or Pararhodospirillum as the major constituents. The important environmental factors affecting PNSB populations were organic matter and sulfide concentrations and oxidation‒reduction potential (ORP). Namely, light-exposed, sulfide-deficient water bodies with high-strength organic matter and in a limited range of ORP provide favorable conditions for the massive growth of PNSB over co-existing PSB. We also report high-quality genome sequences of Rhodovulum sp. strain MB263, previously isolated from a pink mudflat, and Rhodovulum sulfidophilum DSM 1374T, which would enhance our understanding of how PNSB respond to various environmental factors in the natural ecosystem.

[Spatial and Temporal Succession Characteristics of Aerobic Anoxygenic Photosynthesis Bacteria in a Stratified Reservoir].

Aerobic anoxygenic photosynthesis bacteria (AAPB) play a significant role in the material circulation of the hydrosphere, with diverse community structure and unique metabolic functions. To investigate the spatial and temporal succession characteristics of AAPB abundance and community structure in Jinpen Reservoir, a quantitative real-time polymerase chain reaction and Illumina MiSeq high-throughput sequencing technique targeting the pufM gene were applied. Furthermore, redundancy analysis was used to determine the influence of environmental factors on their community structure. The results showed that the AAPB abundance ranged from (6.70±0.43)×103 to (2.69±0.15)×104 copies·mL-1, with the maximum value appearing in October, and decreased with an increase in water depth. Samples were mainly classified into 19 genera (except for the unclassified genus); the most abundant AAPB genera were Bradyrhizobium sp. and Methylobacterium sp., which were affiliated to the α-Proteobacteria, and the proportion of the Bradyrhizobium sp. was highest in November, accounting for more than 60% (except 10 m). Furthermore, Rubrivivax sp., belonging to ß-Proteobacteria, was found to have a low proportion. There was a strong interaction relationship between AAPB genera. For example, Rhodobacter sp. was positively correlated with Rhodovulum sp., while Hydrogenophaga sp. was negatively correlated with Bradyrhizobium sp.. The community structure composition and distribution of AAPB were significantly different, mainly affected by temperature (T), total nitrogen (TN), NO3--N, and light intensity and comprehensively regulated by environmental factors. For instance, T, TN, and total phosphorus had a significant impact on the AAPB community structure of water samples at 0, 5, and 15 m in October, whereas light intensity, pH, DO, and chlorophyll-a were major structuring factors in the AAPB assemblages of water samples at 5 m in December. The results have guiding significance for parsing the spatial and temporal variability of AAPB abundance and diversity in stratified reservoirs, and simultaneously provide a theoretical basis for exploring the driving factors of AAPB population structure.

Diversity and Distribution of Freshwater Aerobic Anoxygenic Phototrophic Bacteria across a Wide Latitudinal Gradient.

Aerobic anoxygenic phototrophs (AAPs) have been shown to exist in numerous marine and brackish environments where they are hypothesized to play important ecological roles. Despite their potential significance, the study of freshwater AAPs is in its infancy and limited to local investigations. Here, we explore the occurrence, diversity and distribution of AAPs in lakes covering a wide latitudinal gradient: Mongolian and German lakes located in temperate regions of Eurasia, tropical Great East African lakes, and polar permanently ice-covered Antarctic lakes. Our results show a widespread distribution of AAPs in lakes with contrasting environmental conditions and confirm that this group is composed of different members of the Alpha- and Betaproteobacteria. While latitude does not seem to strongly influence AAP abundance, clear patterns of community structure and composition along geographic regions were observed as indicated by a strong macro-geographical signal in the taxonomical composition of AAPs. Overall, our results suggest that the distribution patterns of freshwater AAPs are likely driven by a combination of small-scale environmental conditions (specific of each lake and region) and large-scale geographic factors (climatic regions across a latitudinal gradient).

Diversity of purple nonsulfur bacteria in shrimp ponds with varying mercury levels.

This research aimed to study the diversity of purple nonsulfur bacteria (PNSB) and to investigate the effect of Hg concentrations in shrimp ponds on PNSB diversity. Amplification of the pufM gene was detected in 13 and 10 samples of water and sediment collected from 16 shrimp ponds in Southern Thailand. In addition to PNSB, other anoxygenic phototrophic bacteria (APB) were also observed; purple sulfur bacteria (PSB) and aerobic anoxygenic phototrophic bacteria (AAPB) although most of them could not be identified. Among identified groups; AAPB, PSB and PNSB in the samples of water and sediment were 25.71, 11.43 and 8.57%; and 27.78, 11.11 and 22.22%, respectively. In both sample types, Roseobacter denitrificans (AAPB) was the most dominant species followed by Halorhodospira halophila (PSB). In addition two genera, observed most frequently in the sediment samples were a group of PNSB (Rhodovulum kholense, Rhodospirillum centenum and Rhodobium marinum). The UPGMA dendrograms showed 7 and 6 clustered groups in the water and sediment samples, respectively. There was no relationship between the clustered groups and the total Hg (HgT) concentrations in the water and sediment samples used (<0.002-0.03 µg/L and 35.40-391.60 µg/kg dry weight) for studying the biodiversity. It can be concluded that there was no effect of the various Hg levels on the diversity of detected APB species; particularly the PNSB in the shrimp ponds.

The hydrological context determines the beta-diversity of aerobic anoxygenic phototrophic bacteria in European Arctic seas but does not favor endemism.

Despite an increasing number of studies over the last 15 years, aerobic anoxygenic photoheterotrophic (AAP) bacteria remain a puzzling functional group in terms of physiology, metabolism, and ecology. To contribute to a better knowledge of their environmental distribution, the present study aims at analyzing their diversity and structure at the boundary between the Norwegian, Greenland, and Barents Seas. The polymorphism of a marker gene encoding a sub-unit of the photosynthetic apparatus (pufM gene) was analyzed and attempted to be related to environmental parameters. The Atlantic or Arctic origin of water masses had a strong impact on the AAP bacterial community structure whose populations mostly belonged to the Alpha- and Gammaproteobacteria. A majority (>60%) of pufM sequences were affiliated to the Gammaproteobacteria reasserting that this class often represents the major component of the AAP bacterial community in oceanic regions. Two alphaproteobacterial groups dominate locally suggesting that they can constitute key players in this marine system transiently. We found that temperature is a major determinant of alpha diversity of AAP bacteria in this marine biome with specific clades emerging locally according to the partitioning of water masses. Whereas we expected specific AAP bacterial populations in this peculiar and newly explored ecosystem, most pufM sequences were highly related to sequences retrieved elsewhere. This observation highlights that the studied area does not favor AAP bacteria endemism but also opens new questions about the truthfulness of biogeographical patterns and on the extent of AAP bacterial diversity.

Summer community structure of aerobic anoxygenic phototrophic bacteria in the western Arctic Ocean.

Aerobic anoxygenic phototrophic (AAP) bacteria are found in a range of aquatic and terrestrial environments, potentially playing unique roles in biogeochemical cycles. Although known to occur in the Arctic Ocean, their ecology and the factors that govern their community structure and distribution in this extreme environment are poorly understood. Here, we examined summer AAP abundance and diversity in the North East Pacific and the Arctic Ocean with emphasis on the southern Beaufort Sea. AAP bacteria comprised up to 10 and 14% of the prokaryotic community in the bottom nepheloid layer and surface waters of the Mackenzie plume, respectively. However, relative AAP abundances were low in offshore waters. Environmental pufM clone libraries revealed that AAP bacteria in the Alphaproteobacteria and Betaproteobacteria classes dominated in offshore and in river-influenced surface waters, respectively. The most frequent AAP group was a new uncultivated betaproteobacterial clade whose abundance decreased along the salinity gradient of the Mackenzie plume even though its photosynthetic genes were actively expressed in offshore waters. Our data indicate that AAP bacterial assemblages represented a mixture of freshwater and marine taxa mostly restricted to the Arctic Ocean and highlight the substantial influence of riverine inputs on their distribution in coastal environments.