Assessment of Bacterial <i>bph</i> Gene in Amazonian Dark Earth and Their Adjacent Soils

<div><p>Amazonian Anthrosols are known to harbour distinct and highly diverse microbial communities. As most of the current assessments of these communities are based on taxonomic profiles, the functional gene structure of these communities, such as those responsible for key steps in the carbon cycle, mostly remain elusive. To gain insights into the diversity of catabolic genes involved in the degradation of hydrocarbons in anthropogenic horizons, we analysed the bacterial <i>bph</i> gene community structure, composition and abundance using T-RFLP, 454-pyrosequencing and quantitative PCR essays, respectively. Soil samples were collected in two Brazilian Amazon Dark Earth (ADE) sites and at their corresponding non-anthropogenic adjacent soils (ADJ), under two different land use systems, secondary forest (SF) and manioc cultivation (M). Redundancy analysis of T-RFLP data revealed differences in <i>bph</i> gene structure according to both soil type and land use. Chemical properties of ADE soils, such as high organic carbon and organic matter, as well as effective cation exchange capacity and pH, were significantly correlated with the structure of <i>bph</i> communities. Also, the taxonomic affiliation of <i>bph</i> gene sequences revealed the segregation of community composition according to the soil type. Sequences at ADE sites were mostly affiliated to aromatic hydrocarbon degraders belonging to the genera <i>Streptomyces</i>, <i>Sphingomonas</i>, <i>Rhodococcus</i>, <i>Mycobacterium</i>, <i>Conexibacter</i> and <i>Burkholderia</i>. In both land use sites, shannon's diversity indices based on the <i>bph</i> gene data were higher in ADE than ADJ soils. Collectively, our findings provide evidence that specific properties in ADE soils shape the structure and composition of <i>bph</i> communities. These results provide a basis for further investigations focusing on the bio-exploration of novel enzymes with potential use in the biotechnology/biodegradation industry.</p></div

Venn's diagram of <i>bph</i> data belonging to operational protein families (OPFs) for different soil types under different land uses.

<p>ADE  =  Amazon Dark Earth; ADJ  =  Adjacent soils; SF  =  Secondary Forest; M =  Manioc cultivation. Sequences were grouped into OPFs based on sequence identity of 94%.</p

Bar charts representing the taxonomic affiliation of <i>bph</i> gene sequences.

<p>Sequences were affiliated using the TBlastX tool available in the GenBank database. ADE  =  Amazon Dark Earth; ADJ  =  Adjacent soils; SF  =  Secondary Forest; M =  Manioc cultivation.</p

Principal Coordinate Analysis (PCoA) performed with the sequences obtained for the <i>bph</i> gene (based on Unweighted Unifrac distance).

<p>ADE  =  Amazon Dark Earth; ADJ  =  Adjacent soils; SF  =  Secondary Forest; M =  Manioc cultivation.</p

Redundancy analysis (RDA) based on T-RFLP data obtained for the <i>bph</i> gene, and soil properties, at the four studied sites.

<p>Arrows indicate correlation between the chemical parameters and community structure of samples. The significance of correlations was evaluated via Monte Carlo permutation test and it is indicated as follows: * <i>p</i><0.05. ADE  =  Amazon Dark Earth; ADJ  =  Adjacent soils; SF  =  Secondary Forest; M =  Manioc cultivation.</p

Log gene copy number (<i>y</i> axis) of (a) 16S rRNA and (b) <i>bph</i> genes across the studied sites.

<p>Sites are indicated in the <i>x</i> axis. Error bars represent the standard deviation of three independent replicates. ADE  =  Amazon Dark Earth; ADJ  =  Adjacent soils; SF  =  Secondary Forest; M =  Manioc cultivation. Different upper case letters refer to differences for the 16S rRNA across sites; while different lower case letters refer to differences for the <i>bph</i> gene (<i>P</i><0.01, Tukey test).</p

Clustering analysis of T-RFLP data based on Bray-Curtis similarity for the <i>bph</i> gene.

<p>‘a’ and ‘b’ indicate the segregation patterns according to soil type. ADE  =  Amazon Dark Earth; ADJ  =  Adjacent soils; SF  =  Secondary Forest; M =  Manioc cultivation.</p

ANOSIM test for the bacterial 16S rRNA and <i>bph</i> gene based on T-RFLP data of the Amazonian Dark Earth (ADE) and Adjacent soil (ADJ) under secondary forest (SF) and under manioc cultivation (M).

a<p>Samples were compared using T-RF peak height as a measure of abundance.</p

Comparison of diversity indices and richness estimators for the <i>bph</i> gene.

a<p>The operational protein family (OPFs), richness estimators (ACE, Chao1 and Jackknife), diversity indices (Shannon and Simpson) and ^bestimated sample coverage were calculated at a cutoff value of 94% of sequence identity.</p><p>ADE  =  Amazon Dark Earth soils; ADJ  =  Adjacent soils; SF  =  Secondary forest; M =  Manioc cultivation.</p

Patterns of expression of differentially expressed genes in fungus-inoculated tissue (p≤0.05).

<p>Expression of these genes in response to several other stresses was assessed through the publicly available microarray database, TAIR. Putative Arabidopsis orthologs of those bean genes were used to create a heat map obtained with the clustering tool of Genevestigator. Gene numbers in green and red colors indicate their down- or up-regulation in our study, respectively.</p