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Nic pathway effects on the growth of wild-type and mutant B. bronchiseptica strains

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Analysis of a dataset from a previous cDNA microarray study comparing the Bvg regulons of B. pertussis Tohama I and B. bronchiseptica RB50 revealed that nic gene expression was strongly upregulated when 20 mM Na was used for Bvg phenotypic phase modulation (Cummings et al., 2006). For example, nicC transcript abundance was increased an average of 3.97-fold in response to high Na concentration. Expression of the bpsR regulator gene was not significantly altered (averaging a 0.85-fold increase in transcript abundance) and bpsA expression was increased 1.65-fold under those same conditions. However, in that study neither the nic loci nor the bps genes were found to be Bvg-regulated. Since the phenotypic phase modulation response requires millimolar concentrations of Na, and Bordetellae have putative Na catabolic pathway nic genes that are upregulated in response to Na, it was hypothesized that these organisms, like P. putida, may use this aromatic compound as a nutrient for growth.

The classical Bordetella species are typically grown in standard Stainer Scholte (SS) defined medium formulated with 32 μM Na as the required cofactor for NAD biosynthesis (Stainer and Scholte, 1970). Since Nm at the same concentration promotes better growth of B. pertussis than Na (Parker, 1976), the SS medium we routinely use in our studies has this modification. In SS medium, glutamate and proline serve as the main Bordetella carbon sources, which would presumably obviate the need to catabolize Na via the Nic pathway. Using medium modifications that included substitution of the glutamate and proline carbon sources for Na (2 – 5 mM), and altering the nitrogen source and inoculum density, growth stimulation was not observed (data not shown). To date, we have not been successful in formulating a defined medium that could demonstrate that B. bronchiseptica grows using Na as the sole carbon source (as does P. putida).

We observed that compared with wild-type (WT) B. bronchiseptica, in-frame ΔbpsR mutant strain RBB27 had dramatically reduced growth yields in SS formulated with 30 μM Na. Conover et al. also noted poor growth of this mutant in the standard SS medium (Conover et al., 2012). To further examine this growth defect, WT B. bronchiseptica RB50 and the isogenic ΔbpsR mutant RBB27, each carrying the plasmid vector control, and RBB27 complemented with a bpsR+ plasmid, were grown in SS medium containing low or high concentrations of Na, Nm or Qa as sole NAD precursors (Fig. 2). The WT strain showed typical good growth on each of the three pyridines at 30-μM concentration. In contrast, the ΔbpsR mutant showed poor growth on 30 μM Na, but had significantly higher growth yields with 30 μM Nm or Qa. Complementation of the mutant restored growth on 30 μM Na to levels equivalent to, or higher than, WT levels. All of the strains exhibited abundant growth on 1 mM concentrations of Na, Nm and Qa. These results demonstrate that the bpsR regulator mutant is specifically defective for growth on low concentrations of Na, strongly suggesting that, as predicted, the NicR-type BpsR regulator influences utilization of Na. Nicotinamide Mononucleotide

B. bronchiseptica lacks the nadA and nadB genes necessary for de novo synthesis of NAD (Parkhill et al., 2003). One possible explanation for the bpsR mutant growth defect on Na was that Nic pathway deregulation by loss of BpsR-mediated repression caused excessive Na degradation, depleting precursor pools needed for NAD synthesis (e.g., Fig. 1A). If true, then ectopic induction of de novo NAD biosynthesis was predicted to relieve this growth yield defect. Previous studies demonstrated that the Paraburkholderia phytofirmans nadAB de novo NAD biosynthesis genes (nadAB+Pphy) are expressed in B. bronchiseptica and allow it to grow in the absence of NAD precursor supplements (Brickman et al., 2017). B. bronchiseptica RB50 (WT) and RBB27 (ΔbpsR) carrying the plasmid vector control (nadAB-) or pBBR/nadAB+Pphy (nadAB+) were assessed for growth in the absence or presence of Na or Nm (Fig. 3). The WT strain carrying the control plasmid vector failed to grow in the absence of pyridines, but NAD prototrophy conferred by nadAB+Pphy rescued its growth, as previously observed (Brickman et al., 2017). ΔbpsR mutant RBB27 (vector control) similarly did not grow in the absence of pyridines, and also showed its typical poor growth on 30 μM Na. Expression of nadAB+Pphy in the ΔbpsR mutant not only promoted growth in the absence of NAD precursors, but also importantly, relieved the mutant growth defect on 30 μM Na as the sole pyridine.

It was noted that genetic complementation of the ΔbpsR mutant using a bpsR+ multicopy plasmid often restored growth on 30 μM Na to levels that were higher than those observed in the WT strain, suggesting that BpsR overproduction might enhance repression of the nic degradation pathway genes, thus sparing Na for NAD production. To determine whether bpsR overexpression could promote improved growth on limiting Na concentrations, we examined WT(vector) and the WT(pBpsR) strains for their growth on very low concentrations of Na (Fig. 4). The BpsR- overproducing strain consistently yielded higher levels of growth compared with the same WT strain lacking the bpsR+ plasmid. Remarkably, even at the lowest Na concentration of 0.94 μM, the overproducing strain grew extremely well, compared with the control strain. These results are consistent with the hypothesis that super-repression of the nic genes associated with BpsR overproduction blocks the Na catabolic pathway, allowing any available Na to be used for NAD synthesis.