4 Total organic carbon. 5 Total nitrogen. 6 Sulphur. CbbL clone libraries (Form IC & IA) CbbL clone sequences were grouped into OTUs based on a cut-off of 95% sequence similarity. Totals of 141, 99 and 103
form IC cbbL clone sequences were obtained from agricultural (AS) and two saline (SS1 & SS2) soils and termed BS, HS, and RS respectively. Overall, the red like clone sequences yielded 58, 32 and 40 unique phylotypes for AS, SS1 & SS2 clone libraries respectively. Heatmap (Additional file 1: Figure S1) generated by Mothur program depicts the relative abundance of these phylotypes within respective clone libraries. In spite of repeated attempts to amplify and clone PCR products, only 28 partial form IA clone sequences were obtained from the saline soil (SS2), termed “RG clones”, and could be grouped into 8 OTUs (Figure 1). Comparisons AZD6738 order with the NCBI database by BLAST searches revealed that these OTUs were only distantly related to the known Selleck AZD4547 green-like cbbL sequences (Figure 1). Figure 1 Phylogenetic analysis of green like cbbL clones. Neighbour-joining tree (Jukes–Cantor correction) was constructed from saline soil (SS2) clone library partial cbbL (form IA) nucleic acid sequences (phylotypes) with closely related
cbbL-gene sequences from known organisms and environmental clones. Clone sequences of form IA cbbL sequence are coded as ‘RG’. One representative phylotype is shown followed by phylotype number and the number of clones within each phylotype is shown at the Ixazomib chemical structure end. One thousand bootstrap analyses were performed and percentages are shown at
nodes. The scale bar indicates 0.05 substitutions per site. The red-like cbbL sequence of Xanthobacter autotrophicus was used as outgroup for tree calculations. Phylogenetic affiliation of RuBisCO genes The phylogenetic trees were constructed by neighbour joining method using Jukes-Cantor correction. A composite phylogenetic tree was generated from selected nucleotide sequences of form IC cbbL genes from all three soil samples and bacterial isolates (Figure 2). Separate trees for AS and SS1 & SS2 were also generated from aligned nucleotide sequences of form IC cbbL genes (Additional file 2: Figure S2a and Additional file 3: Figure S2b). In the composite tree, majority of the phylotypes (60%) from different soil types did not cluster close to the cbbL sequences of known Akt inhibitor autotrophs. The sequences of cluster 2 (4 OTUs), cluster 6 (12 OTUs), cluster 7 (5 OTUs, 7 cultured isolates), cluster 8 (6 OTUs), cluster 13 (8 OTUs) and cluster 14 (4 OTUs) formed novel monophyletic groups not affiliated to known cbbL gene containing bacteria. Some of the clone sequences clustered with cbbL sequences from known lithotrophs. OTUs from AS soil were grouped into one site specific cluster (cluster 8). The phylotypes from saline soils were closely clustered within cluster 3, cluster 6, cluster 7, cluster 14 and cluster 15.