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In this study, the composition of the metabolically active fraction of the microbial community occurring in Gulf of Mexico marine sediments (water depth, 550 to 575 m) with overlying filamentous bacterial mats was determined. The mats were mainly composed of either orange- or white-pigmented Beggiatoa spp. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from three different sediment depths (0 to 2, 6 to 8, and 10 to 12 cm) that had been subjected to reverse transcription-PCR amplification. Domain-specific 16S PCR primers were used to construct 12 different 16S crDNA libraries containing 333 Archaea and 329 Bacteria clones. Analysis of the Archaea clones indicated that all sediment depths associated with overlying orange- and white-pigmented microbial mats were almost exclusively dominated by ANME-2 (95% of total Archaea clones), a lineage related to the methanogenic order Methanosarcinales . In contrast, bacterial diversity was considerably higher, with the dominant phylotype varying by sediment depth. An equivalent number of clones detected at 0 to 2 cm, representing a total of 93%, were related to the γ and δ classes of Proteobacteria , whereas clones related to δ- Proteobacteria dominated the metabolically active fraction of the bacterial community occurring at 6 to 8 cm (79%) and 10 to 12 cm (85%). This is the first phylogenetics-based evaluation of the presumptive metabolically active fraction of the Bacteria and Archaea community structure investigated along a sediment depth profile in the northern Gulf of Mexico, a hydrocarbon-rich cold-seep region.

A bifunctional oligonucleotide integrates in situ synthesis of a fluorogenic silver cluster with recognition of a target DNA sequence. With the template C(3)AC(3)AC(3)GC(3)A, a complex forms with 10 silver atoms that possesses electronic transitions in the near-infrared and that is detected at nanomolar concentrations using diode laser excitation. Pendant to this cluster encoding region, the recognition component binds a target DNA strand through hybridization, and decoupling of these two regions of the composite sensor renders a modular sensor for specific oligonucleotides. A target is detected using a quencher strand that bridges the cluster template and recognition components and disturbs cluster binding, as indicated by static quenching. Competitive displacement of the quencher by the target strand restores the favored cluster environment, and our key finding is that this exchange enhances emission through a proportional increase in the number of emissive clusters. DNA detection is also accomplished in serum-containing buffers by taking advantage of the high brightness of this fluorophore and the inherently low endogenous background in the near-infrared spectral region. Cluster stability in this biological environment is enhanced by supplementing the solutions with Ag(+).

Few-atom silver clusters harbored by DNA are promising fluorophores due to their high molecular brightness along with their long- and short-term photostability. Furthermore, their emission rate can be enhanced when co-illuminated with low-energy light that optically depopulates the fluorescence-limiting dark state. The photophysical basis for this effect is evaluated for two near-infrared-emitting clusters. Clusters emitting at ∼800 nm form with C(3)AC(3)AC(3)TC(3)A and C(3)AC(3)AC(3)GC(3)A, and both exhibit a trap state with λ(max) ∼ 840 nm and an absorption cross section of (5-6) × 10(-16) cm(2)/molecule that can be optically depopulated. Transient absorption spectra, complemented by fluorescence correlation spectroscopy studies, show that the dark state has an inherent lifetime of 3-4 μs and that absorption from this state is accompanied by photoinduced crossover back to the emissive manifold of states with an action cross section of ∼2 × 10(-18) cm(2)/molecule. Relative to C(3)AC(3)AC(3)TC(3)A, C(3)AC(3)AC(3)GC(3)A produces a longer-lived trap state and permits more facile passage back to the emissive manifold. With the C(3)AC(3)AC(3)AC(3)G template, a spectrally distinct cluster forms having emission at ∼900 nm, and its trap state has a ∼4-fold shorter lifetime. These studies of optically gated fluorescence bolster the critical role of the nucleobases in both the formation and excited state dynamics of these highly emissive metallic clusters.

In this study, ribosomes and genomic DNA were extracted from three sediment depths (0-2, 6-8 and 10-12 cm) to determine the vertical changes in the microbial community composition and identify metabolically active microbial populations in sediments obtained from an active seafloor mud volcano site in the northern Gulf of Mexico. Domain-specific Bacteria and Archaea 16S polymerase chain reaction primers were used to amplify 16S rDNA gene sequences from extracted DNA. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from each sediment depth that had been subjected to reverse transcription polymerase chain reaction amplification. Twelve different 16S clone libraries, representing the three sediment depths, were constructed and a total of 154 rDNA (DNA-derived) and 142 crDNA (RNA-derived) Bacteria clones and 134 rDNA and 146 crDNA Archaea clones obtained. Analyses of the 576 clones revealed distinct differences in the composition and patterns of metabolically active microbial phylotypes relative to sediment depth. For example, epsilon-Proteobacteria rDNA clones dominated the 0-2 cm clone library whereas gamma-Proteobacteria dominated the 0-2 cm crDNA library suggesting gamma to be among the most active in situ populations detected at 0-2 cm. Some microbial lineages, although detected at a frequency as high as 9% or greater in the total DNA library (i.e. Actinobacteria, alpha-Proteobacteria), were markedly absent from the RNA-derived libraries suggesting a lack of in situ activity at any depth in the mud volcano sediments. This study is one of the first to report the composition of the microbial assemblages and physiologically active members of archaeal and bacterial populations extant in a Gulf of Mexico submarine mud volcano.