Figure 1.
Chloropyridinylmethyl (CPM) neonitotinoids.
Four commercial chloropyridinylmethyl (CPM) neonitotinoids (A) and their degradation to 6-chloronicotinic acid via methylene hydroxylation (B).
Figure 2.
Growth and resting cells assays of SG-6C.
Degradation kinetics of NA, 6-CNA, and 6-HNA as sole carbon sources in growth and resting cell studies by strain SG-6C. Values are the means of three replicates with standard deviations, if visible.
Figure 3.
Appearance of 6-HNA as a metabolic intermediate of 6-CNA and NA in resting cells of strain SG-6C.
A) LC-MS TOF Total Ion Chromatogram (TIC) showing appearance of 6-HNA in the supernatant of resting cells cultures supplemented with 6-CNA. Mass spectra of the two compounds are shown underneath the TIC. B) LC-MS TOF TIC showing appearance of 6-HNA in the supernatant of resting cells cultures supplemented with NA. Mass spectra the two compounds are shown underneath the TIC.
Figure 4.
Nicotinic acid and 6-chloronicotinic acid degradation in SG-6C.
A) Nicotinate degradation cluster in Azorhizobium caulinodans and selected Bradyrhizobiaceae strains. Genes were identified by Blast searches using the nicotinate degradation cluster of Eubacterium barkeri [23]. Genome accession numbers and locus tags for the displayed genes are as follows: Azorhizobium caulinodans ORS571 – NC_009937 (AZC_2804 to AZC_2790), Bradyrhizobium japonicum USDA110 – NC004463 (blr3818 to blr3830), Rhodopseudomonas palustris HaA2 – NC007778 (RPB_1671 to RPB_1658), Bradyrhizobiaceae bacterium SG-6C – AFOF01000023 (CSIRO_2076 to CSIRO_2090). Genes not conserved in the nicotinate degradation cluster are shown in white. B) Pathway for nicotinate and 6-chloronicotinate degradation in SG-6C. Nicotinate is metabolised via THON by the nicotinate degradation cluster shown above. 6-CNA is also metabolised via THON, after being converted to 6-hydroxynicotinate by Cch2. Pathways for nicotinate degradation in other organisms (not observed in strain SG-6C) are shown in grey.
Figure 5.
Identifying an ICE containing cch2.
(A) Comparison of the Mesorhizobium loti symbiosis island (ICEMlSymR7A) and SG-6C. Blast matches for genes involved in the excision, conjugative transfer and integration of the symbiosis island are all found in SG-6C (indicated by grey shading). The location of cch2 in the accessory gene region of the SG-6C ICE is indicated; (B) The two potential forms of the SG-6C ICE, integrated and excised. Binding sites of primers used to confirm the presence of both forms are indicated by half arrows; (C) PCR of strain SG-6C genomic DNA using primer combinations as indicated. L: 1kb+ ladder, 1: CHR1 & ICE1, 2: ICE2 & CHR2, 3: CHR1 & CHR2, 4: ICE1 & ICE2, 5: negative control.