PCR amplification of the <i>alkB</i> gene from alkane-degrading bacterial strains using different <i>alkB</i>-targeting primers.

a<p>The codes of <i>alkB</i>-targeting primers are those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone-0066565-t001" target="_blank">Table 1</a>.</p>b<p>Phylogenetic position of isolated alkane-degrading bacterial strains; α, Alphaproteobacteria; β, Betaproteobacteria; γ, Gammaproteobacteria.</p><p>HEP – all strains were able to grow with heptadecane as the sole carbon source.</p

The Use of a Combination of <i>alkB</i> Primers to Better Characterize the Distribution of Alkane-Degrading Bacteria

<div><p>The alkane monooxygenase AlkB, which is encoded by the <i>alkB</i> gene, is a key enzyme involved in bacterial alkane degradation. To study the <i>alkB</i> gene within bacterial communities, researchers need to be aware of the variations in <i>alkB</i> nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three <i>alkB</i>-targeting primers to enhance the detection of the <i>alkB</i> gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, <i>alkB</i>-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study <i>alkB</i> richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of <i>alkB</i>-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 <i>alkB</i> phylotypes were detected. Venn diagram analyses revealed that only low numbers of <i>alkB</i> phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three <i>alkB</i>-targeting primers enhanced the richness of <i>alkB</i> phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single <i>alkB</i>-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the <i>alkB</i> composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of <i>alkB</i> primers was an efficient strategy for enhancing the detection of the <i>alkB</i> gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments.</p></div

Phylogenetic tree of the <i>alkB</i> sequences obtained from sI, sR, sY, s3 and sC soil libraries and from the closely related <i>alkB</i> genes from the GenBank database (A).

<p>The tree was constructed using the neighbor-joining (NJ) method and MEGA 5 software. The corresponding colors for the different sampling sites are: red (sI), blue (sR), yellow (sY), black (s3) and purple (sC). (B) The same phylogenetic tree showing the distribution of the <i>alkB</i> phylotypes resulting from the amplification with the different targeting primers. The colors corresponding to the primers used are: yellow - (d), blue - (e) and red - (f). The color white was used for phylotypes that originated from the PCR amplification with more than one primer.</p

Data obtained from the statistical analyses of clone libraries.

a<p>The clone libraries are denoted as: letters d, e and f (representing the <i>alkB</i>-targeting primers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone-0066565-t001" target="_blank">Table 1</a>), followed by the sampling site codes sI, sR, sY (three uncontaminated (pristine) soil samples) and s3 (one diesel contaminated soil sample) from King George Island, Antarctica (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565.s001" target="_blank">Fig. S1</a>) and sC (oil-contaminated soil) from Carmópolis.</p>b<p>Species richness <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565-Chao1" target="_blank">[28]</a>;</p>c<p>Confidence intervals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565-Chao2" target="_blank">[46]</a>;</p>d<p>Species richness <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565-Chao3" target="_blank">[47]</a>;</p>e<p>Shannon’s diversity index (H’) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565-Shannon1" target="_blank">[29]</a>;</p>f<p>Boneh estimator <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565-Boneh1" target="_blank">[31]</a>.</p

Dendrogram describing the dissimilarity (1-similarity) among the sampling sites (A).

<p>The groups were clustered using the UPGMA algorithm and the Jaccard similarity coefficient based on the observed richness. The clone libraries are denoted as follows: the letters (d), (e) and (f) correspond to the <i>alkB</i>-targeting primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone-0066565-t002" target="_blank">Table 2</a>, and the sampling sites (sI, sR, sY, s3 and sC) are described in the Materials and Methods. (B) and (C) Venn diagrams of all <i>alkB</i> phylotypes detected in each sampling site using a distance level of 97% similarity.</p

List of <i>alkB</i>-targeting primers used in this study.

a<p>Primer code used throughout the results section and figures.</p>b<p>Reference position of the amplified fragment based on complete <i>alkB</i> gene sequence of <i>Pseudomonas putida</i> Gp01.</p>c<p>nt = nucleotide.</p

A Venn diagram of <i>alkB</i> clone libraries using a distance level of 97% similarity.

<p>The numbers represent the richness and the shared richness of each library using the Chao richness index <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565-Chao1" target="_blank">[28]</a>. The Venn diagram groups are denoted as follows: sI, sR, sY, s3 and sC represent the sampling sites, and the letters (d), (e) and (f) correspond to the <i>alkB</i>-targeting primers used in the PCR amplification of the <i>alkB</i> gene, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone-0066565-t002" target="_blank">Table 2</a>.</p

Rarefaction analysis of <i>alkB</i> clone libraries using a distance level of 97% similarity.

<p>The clone libraries are denoted as follows: the letters (d), (e) and (f) correspond to the <i>alkB</i>-targeting primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone-0066565-t001" target="_blank">Table 1</a>, and sI, sR, sY, s3 and sC are the sampling site codes (described in Materials and Methods).</p

Chemical and physicochemical properties of the soils from Carmópolis, Brazil and King George Island in Maritime Antarctica^a.

a<p>data from Jurelevicius et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565-Jurelevicius2" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066565#pone.0066565-Jurelevicius3" target="_blank">[23]</a> and this study.</p>b<p>not determined.</p>c<p>under the detection limit of the method used.</p