Metagenomic Characterization of Soil Microbial Communities in the Luquillo Experimental Forest (Puerto Rico) and Implications for Nitrogen Cycling.

The phylogenetic and functional diversities of microbial communities in tropical rainforests and how these differ from those of temperate communities remain poorly described but are directly related to the increased fluxes of greenhouse gases such as nitrous oxide (N2O) from the tropics. Toward closing these knowledge gaps, we analyzed replicated shotgun metagenomes representing distinct life zones and an elevation gradient from four locations in the Luquillo Experimental Forest (LEF), Puerto Rico. These soils had a distinct microbial community composition and lower species diversity compared to those of temperate grasslands or agricultural soils. In contrast to the overall distinct community composition, the relative abundances and nucleotide sequences of N2O reductases (nosZ) were highly similar between tropical forest and temperate soils. However, respiratory NO reductase (norB) was 2-fold more abundant in the tropical soils, which might be relatable to their greater N2O emissions. Nitrogen fixation (nifH) also showed higher relative abundance in rainforest than in temperate soils, i.e., 20% versus 0.1 to 0.3% of bacterial genomes in each soil type harbored the gene, respectively. Finally, unlike temperate soils, LEF soils showed little stratification with depth in the first 0 to 30 cm, with ∼45% of community composition differences explained solely by location. Collectively, these results advance our understanding of spatial diversity and metabolic repertoire of tropical rainforest soil communities and should facilitate future ecological studies of these ecosystems. IMPORTANCE Tropical rainforests are the largest terrestrial sinks of atmospheric CO2 and the largest natural source of N2O emissions, two greenhouse gases that are critical for the climate. The microbial communities of rainforest soils that directly or indirectly, through affecting plant growth, contribute to these fluxes remain poorly described by cultured-independent methods. To close this knowledge gap, the present study applied shotgun metagenomics to samples selected from three distinct life zones within the Puerto Rico rainforest. The results advance our understanding of microbial community diversity in rainforest soils and should facilitate future studies of natural or manipulated perturbations of these critical ecosystems.

pH selects for distinct N2O-reducing microbiomes in tropical soil microcosms.

Nitrous oxide (N2O), a greenhouse gas with ozone destruction potential, is mitigated by the microbial reduction to dinitrogen catalyzed by N2O reductase (NosZ). Bacteria with NosZ activity have been studied at circumneutral pH but the microbiology of low pH N2O reduction has remained elusive. Acidic (pH < 5) tropical forest soils were collected in the Luquillo Experimental Forest in Puerto Rico, and microcosms maintained with low (0.02 mM) and high (2 mM) N2O assessed N2O reduction at pH 4.5 and 7.3. All microcosms consumed N2O, with lag times of up to 7 months observed in microcosms with 2 mM N2O. Comparative metagenome analysis revealed that Rhodocyclaceae dominated in circumneutral microcosms under both N2O feeding regimes. At pH 4.5, Peptococcaceae dominated in high-N2O, and Hyphomicrobiaceae in low-N2O microcosms. Seventeen high-quality metagenome-assembled genomes (MAGs) recovered from the N2O-reducing microcosms harbored nos operons, with all eight MAGs derived from acidic microcosms carrying the Clade II type nosZ and lacking nitrite reductase genes (nirS/K). Five of the eight MAGs recovered from pH 4.5 microcosms represent novel taxa indicating an unexplored N2O-reducing diversity exists in acidic tropical soils. A survey of pH 3.5-5.7 soil metagenome datasets revealed that nosZ genes commonly occur, suggesting broad distribution of N2O reduction potential in acidic soils.

Psychrobacter sanguinis sp. nov., recovered from four clinical specimens over a 4-year period.

An analysis of 16S rRNA gene sequences from archived clinical reference specimens identified a novel species of the genus Psychrobacter, of which four strains have been independently isolated from human blood. On the basis of 16S rRNA gene sequence similarity, the closest relatives with validly published names were Psychrobacter arenosus R7(T) (98.7%), P. pulmonis CECT 5989(T) (97.7%), P. faecalis Iso-46(T) (97.6%) and P. lutiphocae IMMIB L-1110(T) (97.2%). Maximum-likelihood phylogenetic analysis of 16S rRNA gene sequences showed that the isolates belonged to the genus Psychrobacter and were members of a cluster associated with Psychrobacter sp. PRwf-1, isolated from a silk snapper fish. DNA-DNA relatedness and partial 23S rRNA gene sequences also supported the finding that the isolates belonged to a species distinct from its closest phylogenetic neighbours. The predominant cellular fatty acids were C(18:1)ω9c, C(16:0), summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH), summed feature 5 (C(18:2)ω6,9c and/or anteiso-C(18:0)) and C(18:0). Biochemical and morphological analysis further supported the assignment of the four isolates to a novel species. The name Psychrobacter sanguinis sp. nov. is proposed. The type strain is 13983(T) (=DSM 23635(T)=CCUG 59771(T)).

The genome sequence of Psychrobacter arcticus 273-4, a psychroactive Siberian permafrost bacterium, reveals mechanisms for adaptation to low-temperature growth.

Psychrobacter arcticus strain 273-4, which grows at temperatures as low as -10 degrees C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (-10 degrees C to -12 degrees C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.

Biogeography of two cold-adapted genera: Psychrobacter and Exiguobacterium.

The genera Exiguobacterium and Psychrobacter have been frequently detected in and isolated from polar permafrost and ice. These two genera have members that can grow at temperatures as low as -5 and -10 degrees C, respectively. We used quantitative PCR (Q-PCR) to quantify members of these genera in 54 soil or sediment samples from polar, temperate and tropical environments to determine to what extent they are selected by cold environments. These results were further analyzed by multiple linear regression to identify the most relevant environmental factors corresponding to their distribution. Exiguobacterium was detected in all three climatic zones at similar densities, but was patchier in the temperate and tropical samples. Psychrobacter was present in almost all polar samples, was at highest densities in Antarctica sediment samples, but was in very low densities and infrequently detected in temperate and tropical soils. Clone libraries, specific for the 16S rRNA gene for each genus, were constructed from a sample from each climatic region. The clone libraries were analyzed for alpha and beta diversities, as well as for variation in population structure by using analysis of molecular variance. Results confirm that both genera were found in all three climatic zones; however, Psychrobacter populations seemed to be much more diverse than Exiguobacterium in all three climatic zones. Furthermore, Psychrobacter populations from Antarctica are different from those in Michigan and Puerto Rico, which are similar to each other.

Psychrobacter cryohalolentis sp. nov. and Psychrobacter arcticus sp. nov., isolated from Siberian permafrost.

Three Gram-negative, non-motile, non-pigmented, oxidase-positive coccobacilli capable of growth at temperatures from -10 to 30 degrees C and salinities of 0 to 1.7 M NaCl were isolated from Siberian permafrost and characterized. Both 16S rRNA and gyrB gene sequencing studies placed the isolates in the Gammaproteobacteria within the genus Psychrobacter. However, with higher bootstrap values and reproducible tree topologies, gyrB represented a more reliable phylogenetic marker for the taxonomy of Psychrobacter species. DNA-DNA hybridization data supported gyrB tree topologies and established two relatedness groups within the three isolates; neither of these groups was related at the species level to any previously described Psychrobacter species. The two groups of isolates could be differentiated phenotypically from 13 previously described Psychrobacter species using API strips. These results support the existence of two novel species of Psychrobacter, for which we propose the names Psychrobacter cryohalolentis sp. nov. (type strain K5(T) = DSM 17306(T) = VKM B-2378(T)) and Psychrobacter arcticus sp. nov. (type strain 273-4(T) = DSM 17307(T) = VKM B-2377(T)).

Correspondence between community structure and function during succession in phenol- and phenol-plus-trichloroethene-fed sequencing batch reactors.

The effects of more than 2 years of trichloroethene (TCE) application on community succession and function were studied in two aerobic sequencing batch reactors. One reactor was fed phenol, and the second reactor was fed both phenol and TCE in sequence twice per day. After initiation of TCE loading in the second reactor, the TCE transformation rates initially decreased, but they stabilized with an average second-order rate coefficient of 0.044 liter mg(-1) day(-1) for 2 years. In contrast, the phenol-fed reactor showed higher and unstable TCE transformation rates, with an average rate coefficient of 0.093 liter mg(-1) day(-1). Community analysis by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes showed that the phenol-plus-TCE-fed reactor had marked changes in community structure during the first 100 days and remained relatively stable afterwards, corresponding to the period of stable function. In contrast, the community structure of the phenol-fed reactor changed periodically, and the changes coincided with the periodicity observed in the TCE transformation rates. Correspondence analysis of each reactor community showed that different community structures corresponded with function (TCE degradation rate). Furthermore, the phenol hydroxylase genotypes, as determined by restriction fragment length polymorphism analysis, corresponded to community structure patterns identified by T-RFLP analysis and to periods when the TCE transformation rates were high. Long-term TCE stress appeared to select for a different and stable community structure, with lower but stable TCE degradation rates. In contrast, the community under no stress exhibited a dynamic structure and dynamic function.

Microbial dehalorespiration with 1,1,1-trichloroethane.

1,1,1-Trichloroethane (TCA) is a ubiquitous environmental pollutant because of its widespread use as an industrial solvent, its improper disposal, and its substantial emission to the atmosphere. We report the isolation of an anaerobic bacterium, strain TCA1, that reductively dechlorinates TCA to 1,1-dichloroethane and chloroethane. Strain TCA1 required H2 as an electron donor and TCA as an electron acceptor for growth, indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that strain TCA1 is related to gram-positive bacteria with low DNA G+C content and that its closest relative is Dehalobacter restrictus, an obligate H2-oxidizing, chloroethene-respiring bacterium.