Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria, including Pelagibacter ubique, a member of the ubiquitous SAR11 clade. The goals of this study were to explore the diversity of PR genes and to estimate their abundance in the North Atlantic Ocean using quantitative polymerase chain reaction (QPCR). We found that PR genes in the western portion of the Sargasso Sea could be grouped into 27 clusters, but five clades had the most sequences. Sets of specific QPCR primers were designed to examine the abundance of PR genes in the following four of the five clades: SAR11 (P. ubique and other SAR11 Alphaproteobacteria), BACRED17H8 (Alphaproteobacteria), HOT2C01 (Alphaproteobacteria) and an uncultured subgroup of the Flavobacteria. Two groups (SAR11 and HOT2C01) dominated PR gene abundance in oligotrophic waters, but were significantly less abundant in nutrient- and chlorophyll-rich waters. The other two groups (BACRED17H8 and Flavobacteria subgroup NASB) were less abundant in all waters. Together, these four PR gene types were found in 50% of all bacteria in the Sargasso Sea. We found a significant negative correlation between total PR gene abundance and nutrients and chlorophyll but no significant correlation with light intensity for three of the four PR types in the depth profiles north of the Sargasso Sea. Our data suggest that PR is common in the North Atlantic Ocean, especially in SAR11 bacteria and another marine alphaproteobacterial group (HOT2C01), and that these PR-bearing bacteria are most abundant in oligotrophic waters.

To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a large insert metagenomic library constructed with bacterial DNA from the Delaware River, a polymerase chain reaction (PCR) library of 16S rRNA genes, and community structure determined by fluorescence in situ hybridization (FISH). The composition of the libraries and community structure determined by FISH differed for the major bacterial groups in the river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library compared with the PCR library and FISH, while Cytophaga-like bacteria were more abundant in the metagenomic library than in the PCR library and in the actual community according to FISH. The Delaware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the ACK-m1 and STA2-30 clusters of Actinobacteria, and the PRD01a001B Cytophaga-like bacteria cluster. Coverage of bacteria with > 97% sequence identity was 65% and 50% for the metagenomic and PCR libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composition of the PCR library. This study suggests that although it may miss some bacterial groups, the metagenomic approach can sample other groups (e.g. Cytophaga-like bacteria) that are potentially underrepresented by other culture-independent approaches.

Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that, if abundant, may be biogeochemically important in the oceans. We used epifluorescence microscopy and quantitative PCR (qPCR) to examine the abundance of these bacteria by enumerating cells with bacteriochlorophyll a (bChl a) and the light-reaction center gene pufM, respectively. In the surface waters of the Delaware estuary, AAP bacteria were abundant, comprising up to 34% of prokaryotes, although the percentage varied greatly with location and season. On average, AAP bacteria made up 12% of the community as measured by microscopy and 17% by qPCR. In the surface waters of the Chesapeake, AAP bacteria were less abundant, averaging 6% of prokaryotes. AAP bacterial abundance was significantly correlated with light attenuation (r ‫؍‬ 0.50) and ammonium (r ‫؍‬ 0.42) and nitrate (r ‫؍‬ 0.71) concentrations. Often, bChl a-containing bacteria were mostly attached to particles (31 to 94% of total AAP bacteria), while usually 20% or less of total prokaryotes were associated with particles. Of the cells containing pufM, up to 87% were associated with particles, but the overall average of particleattached cells was 15%. These data suggest that AAP bacteria are particularly competitive in these two estuaries, in part due to attachment to particles.Recent studies indicate that photoheterotrophic bacteria, which are capable of both phototrophy and heterotrophy, may be abundant and important in biogeochemical cycles of the oceans (3,23,28). One type of photoheterotroph, aerobic anoxygenic phototrophic (AAP) bacteria, can harvest light by use of bacteriochlorophyll a (bChl a) for the production of ATP (45). Phototrophy explains the higher growth rates (46) and viability (40) of these bacteria grown in the light compared with the results seen with dark-grown cultures. Some AAP bacteria reduce the production of photosynthetic pigments in response to higher organic carbon concentrations (27,40). These data suggest that AAP bacteria would have competitive advantages over typical heterotrophs when organic carbon concentrations are low (23).This hypothesis was supported by the discovery of AAP bacteria in the open ocean (28), where AAP bacteria can make up from Ͻ1% to 10% of the prokaryotic community (13, 18, 37). However, emerging evidence indicates that these bacteria may be as abundant in eutrophic as in oligotrophic environments (13,37,39). A global survey of bacteria containing bChl a and the reaction center pufM gene found that AAP bacteria were more abundant in the Long Island Sound and Chesapeake Bay than in the open ocean (37). Additionally, these bacteria were more abundant in the North Atlantic Ocean, where chlorophyll concentrations were higher, than in the North Pacific, where AAP bacteria comprised less than 5% of prokaryotes (13). These wide ranges suggest that more data on the abundance of AAP bacteria are needed to determine the ecological controls of these bacteria.To explore what environmental factors control AAP bacteria, we enumerated cells...