The Bordetella bronchiseptica nic locus encodes a nicotinic acid degradation pathway

Description and organization of Bordetella nic genes. Jimenez and coworkers reported that Na could serve as the sole carbon and energy source for P. putida KT2440, identifying and characterizing the nic gene locus responsible for aerobic Na catabolism (Jiménez et al., 2008). They determined that aside from P. putida, the only other organisms with orthologous nic gene clusters were β- proteobacteria of the Burkholderiales order. The study showed that in the Burkholderiales, the chromosomal organization of the nic genes, as well as their predicted protein products, are remarkably similar to those in P. putida. The predicted Bordetella Nic pathway enzymes and NicR regulator proteins have 41 – 80 % amino acid sequence identity with those of P. putida (Table 1). The prototypic nic gene cluster of P. putida contains genes encoding enzymes for Na degradation, the NicR MarR-family transcriptional regulator, and a putative Na transport system.

Our in silico analyses confirmed that this orthologous nic gene cluster (also denoted as locus 1) is present in B. bronchiseptica (Fig. 1B), B. pertussis and B. parapertussis. Comparison of nic gene cluster sequences of representative classical species strains found that, with the exception of the annotated pseudogenes, the nic gene orthologs of B. pertussis Tohama I and B. parapertussis 12822 share over 98% nucleotide sequence identity with those of B. bronchiseptica RB50. Bordetella avium and Bordetella ansorpii also have this nic locus. Bordetella species predicted to lack nic locus 1 include B. petrii, B. hinzii, B. pseudohinzii, B. trematum, B. holmesii, B. bronchialis and B. flabilis. The nicR regulator gene described by Jimenez et al. (Jiménez et al., 2008) as associated with the Bordetella nic cluster was then later described as bpsR (Conover et al., 2012), encoding a transcriptional repressor of the bpsABCD genes involved in production of extracellular polysaccharide during biofilm formation. At present, in this study we use the bpsR gene designation. The bpsR (nicR) gene of B. bronchiseptica is separated from bpsABCD by genes encoding a putative membrane protein (BB1770) and a tRNAArg (BBt23). As with the nic loci of P. putida and other Burkholderiales species (Jiménez et al., 2008), there are predicted transport genes in Bordetella nic locus 1, located downstream of bpsR. Fifty bp downstream of Bordetella nicB2 is a gene encoding a putative flavocytochrome (BB1784), orthologs of which are not associated with the nic loci of P. putida or other species within the Burkholderiales. Only classical Bordetella species appear to have this flavocytochrome gene.

A separate gene locus (locus 2: BB0271-BB0275) was identified, encoding paralogs of the locus 1 nic cluster nicEDXF genes in the classical Bordetella species, B. avium and B. ansorpii. In B. bronchiseptica RB50, this locus differs in DNA sequence from the paralogous core nic cluster DNA region by only 10 nucleotides, and encodes proteins with primary amino acid sequences that are identical to those of the locus 1 nicEDXF gene products. The locus 2 nicEDXF paralogs are predicted to be operonic with a promoter-proximal gene encoding a putative 2,3-dihydroxybenzoate decarboxylase (BB0271) predicted to be involved in catabolism of aromatic compounds. In silico analysis indicates that P. putida KT2440 does not have an orthologous locus 2. B. holmesii, B. bronchialis and B. flabilis lack locus 1 but appear to have locus 2 or a derivative of it. B. petrii strain J51 has a prototypic nic locus 2 but strains DSM12804 and J49 do not. NMN

In B. pertussis strains Tohama I, UT25, and CS, the nic genes are adjacent to the bpsR and bpsABCD genes, as they are in B. bronchiseptica RB50 and other B. bronchiseptica strains (Fig. 1B). However, in some other B. pertussis strains, there are genome rearrangements affecting the region, such that nic cluster genes are not proximal to the bpsR or bpsABCD genes. Although some B. pertussis strains lack nic locus 1, they all have an intact locus 2. For B. pertussis strains that have nic locus 1, the nicB1 (BP1952), nicD (BP1956) and nicB2 (BP1960) genes have been annotated as pseudogenes. It is interesting to note that the locus 1 nicD pseudogene is an exact duplicate of its paralog (BP0580) in B. pertussis locus 2 (BP0578-BP0582). All of the B. pertussis strains examined have locus 2 genes, with the same nicD pseudogene. Analysis of the few B. parapertussis strains for which sequences were available indicates two nic pseudogenes that vary, depending on the strain.

Based on studies of the P. putida nicotinate degradation pathway, the following model for Na degradation in Bordetella species is proposed (Fig. 1A, Table 1). NicAB is a multisubunit Na hydroxylase that catalyzes the production of 6-HNa from the substrate, Na. As with P. putida NicA (hydroxylase small subunit), Bordetella NicA shares motifs likely involved in binding two [2Fe-2S] clusters. In P. putida, the NicB large subunit is comprised of two domains: an N-terminal domain with three molybdopterin cytosine dinucleotide (MCD) cofactor binding motifs, and a C-terminal domain with three cytochrome c heme binding motifs. In Bordetella, these NicB domains appear to reside on two separate polypeptides, dubbed NicB2 (BB1783) and NicB1 (BB1776). As proposed previously (Jiménez et al., 2008), Bordetella NicB2, corresponding to the N-terminal domain of the P. putida NicB protein, binds the substrate and MCD cofactor, and NicB1, with both MCD and heme binding motifs, would mediate electron transfer to cytochrome c oxidase. NicC catalyzes the oxidative decarboxylation of 6-HNa to produce 2,5-dihydroxypyridine. NicX then opens the dihydroxypyridine ring between ring carbon positions 5 and 6 by insertion of two oxygen atoms, yielding N- formylmaleamic acid, which is then deformylated by NicD to generate formic acid and maleamic acid. NicF converts the maleamic acid to maleic acid and ammonia, and in the final step, NicE catalyzes the conversion of maleic acid to the TCA cycle intermediate, fumaric acid.