## 3 Results
### Molecular Detection of Nitrate-Reducing Functional Genes napA and narG  
For all functional genes, DNA sequencing of the PCR product was performed and matched against sequences in GenBank in order to confirm that the sequence of the PCR product matched that of the expected gene for its full length. Many strains that were shown to reduce nitrate and/or nitrite were not amplified by the primers for functional genes. In some cases, PCR products were produced but failed due to reading frame shifts that produced stop codons and/or contained regions that had little to no similarity to the expected gene. Gene sequences of control strains, by contrast, did not have these problems, nor did the 16S rRNA sequences of any strain. Supplemental Table S3 lists for each strain both any detected PCR products (\"+\") and those verified as the correct sequence (\"++\"). Only the verified sequences are used for the following analyses. Primers designed to detect _napA_ and _narG_ nitrate reductase genes were used to screen for the presence of nitrate-reducing genes in all 75 strains. The _napA_ nucleotide sequence was obtained from seven strains out of the 75 screened (Tables 2 and 3). The seven strains include \(\alpha\)-Proteobacteria (three strains: two _Bosea_ and one _Ensifer_), \(\beta\)-Proteobacteria (one strain: _Achromobacter_), and \(\gamma\)-Proteobacteria (three strains: _Pseudomonas_). The match similarity between the _napA_ sequences of these seven strains (e.g., OR416962-OR416968) and _napA_ sequences of other strains from GenBank ranged from 91% to 98% (Supplemental Table S4). Gene sequences are highly similar (99.61%) to that coding for membrane-bound nitrate reductase (_narG_) of _Gentorophomonas geniculata_ strain BR23 (CP134450) were confirmed in two _Stenotrophomonas_ strains (e.g., strains I-39, OR416969 and I-42, OR416970) (Supplemental Table S4). \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Strain \# (Genus)** & **Soil Type** & _napA_ & _narG_ & _nirS_ & _cnorB_ & _nosZ_ & **NR or DN \({}^{*}\)** \\ \hline
1 (_Pseudomonas_) & Brine/Oil & Yes & Yes & & DN \\ \hline
2 (_Pseudomonas_) & Brine/Oil & & & Yes & & DN \\ \hline
4 (_Ensifer_) & Brine/Oil & & & & Yes & DN \\ \hline
7 (_Bosea_) & Brine/Oil & Yes & & & & None \\ \hline
8 (_Bosea_) & Brine/Oil & Yes & & & & None \\ \hline
39 (_Stenotrophomonas_) & Prairie & & Yes & & & NR \\ \hline
42 (_Stenotrophomonas_) & Prairie & & Yes & & & NR \\ \hline
49 (_Achromobacter_) & Prairie & Yes & & & & NR \\ \hline
65 (_Pseudomonas_) & Prairie & & & Yes & Yes & DN \\ \hline
67 (_Pseudomonas_) & Prairie & Yes & & & NR \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Strains in which gene sequences for nitrate reduction or denitrification were detected. Molecular Detection of Penitrifying Functional Genes \(nirS\), \(nirK\), \(cnorB\), \(qnorB\), and \(nosZ\)  
All 75 strains were screened for the presence of nitrite reductase gene(s) using primers designed to detect the \(nirS\) nitrite reductase gene and \(nirK\) nitrite reductase gene. In addition, screening with \(cnorB\), \(qnorB\), and \(nosZ\) primers, as another indication of denitrification genes, was applied to a subset of the chosen 33 strains that showed the ability to reduce nitrate/nitrite in the microtiter plate assay and that represented the phylogenetic diversity of the strains. The selected 33 strains are indicated in bold letters in Supplemental Table S3 and summarized in Table 2.  
A total of four strains were found, where one or more denitrification functional genes were detected by PCR amplification and confirmed by sequencing to have high similarity to the denitrification genes (Table 3). The gene sequence coding for nitrite reductase (\(nirS\)) was detected in two _Pseudomonas_ strains (I-1 and I-2, Supplemental Table S4), isolated from oil/brine-contaminated soil, but not in a third _Pseudomonas_ strain classified as DN. _Pseudomonas_ _frederikskergensis_ strain AS1 provided the best match to the \(nirS\) of I-1 and I-2, as well as to I-1 \(napA\).