nicC expression is Na-responsive and BpsR-regulated

The same beneficial Na utilization effect should occur if the Nic pathway itself were inactivated. To test this hypothesis, a B. bronchiseptica ΔnicA mutant (RBB33) was constructed; the mutant should be blocked at the first reaction in the degradation pathway. The WT parent and ΔnicA mutant strains carrying the plasmid vector control, and the complemented ΔnicA mutant, were grown in SS medium with low concentrations of Na as the sole pyridine NAD precursor (Fig. 5). The mutant exhibited exalted growth at all of the Na concentrations compared with the WT strain. For unknown reasons, complementation of the ΔnicA mutant strongly decreased growth yields at all Na concentrations, compared with the mutant and WT strains. In sum, these results suggest that inactivating the Nic pathway allows B. bronchiseptica to use its limiting Na for NAD biosynthesis that is essential for growth. Additionally, it is possible that inactivation of the Nic pathway, by mutation or excessive repression, influences NAD homeostasis, promoting efficient recycling of pyridines and NAD breakdown products.

 The initial reactions of the Na catabolic pathway would need to be carefully controlled to avoid pathway activation when NAD precursor abundance is limiting. Therefore, transcriptional responsiveness to the Na substrate or to the 6-HNa product of the first dedicated step in the pathway would serve as an effective means of control. In P. putida, the nicAB operon, encoding products catalyzing the first step in Na degradation, is transcriptionally repressed by NicS, the activity of which is inhibited by binding either Na or 6-HNa, resulting in nicAB derepression (Jiménez et al., 2011). In Bordetella, nicB2 is in a separate operon from nicA and nicB1. The nicA coding sequences begin 109 bp downstream of nicC, suggesting that they are transcribed independently, which Cummings et al. also predicted based on gene expression patterns observed by cDNA microarray (Cummings et al., 2006). However, other in silico analysis using Microbes Online (Dehal et al., 2010) indicated that they are likely to be operonic. Using a DNA fragment encompassing the predicted nicC-nicA intergenic region, a nicA-lacZ transcriptional fusion was constructed, but showed no appreciable LacZ activity under any growth medium condition tested (data not shown). Since nicC appeared to be the promoter-proximal gene in a nicCAB1 operon involved in the early steps of Na catabolism, a nicC-lacZ transcriptional fusion plasmid was constructed to assess the influence of various pyridines and BpsR on nicC promoter activity.

WT strain RB50 carrying nicC-lacZ plasmid pMP/nicC was cultured in SS medium supplemented with Na, Nm, or Qa as the sole pyridine source, at either 30 μM or 2 mM concentration, and LacZ activities were measured after 24 h. In this strain, nicC transcriptional activity was strongly upregulated in the presence of 2 mM Na, but not by Na at 30-μM concentration or by Nm or Qa at either concentration (Fig. 6A). Comparison of nicC-lacZ expression in strain RB50 versus RBB27 (ΔbpsR) (Fig. 6B) determined that nicC-lacZ expression was Na-inducible in the WT, but constitutive in the ΔbpsR mutant.

To further characterize nicC-lacZ Na-responsiveness, expression levels were measured after 24 h culture in SS supplemented with Na as the sole pyridine source at concentrations ranging from 30 μM to 4 mM (Fig. 6C). In WT strain RB50, nicC expression levels varied directly with the concentration of Na in the culture medium, exhibiting maximal expression levels at 2 mM. The ΔbpsR strain was virtually blind to Na, with nicC expression being uniformly elevated. Trans-complementation of the ΔbpsR mutant using a bpsR+ plasmid restored some nicC transcriptional responsiveness to Na, as evidenced by elevated nicC-lacZ expression levels associated with 1, 2, and 4 mM Na concentrations, compared with expression levels at 30 μM Na (P ≤ 0.05). However, overall nicC-lacZ expression levels were markedly lower in the complemented mutant versus the WT or mutant strains (P ≤ 0.05 for all Na concentrations tested), presumably because BpsR overproduction in the complemented strain resulted in enhanced repression. Overall, these results indicate that in B. bronchiseptica, BpsR represses nicC transcription, and that Na supplied at high concentration relieves that repression to promote expression of early Nic pathway genes. Sublingual

6-HNa is the inducer of nicC expression. The P. putida KT2440 nic gene cluster is comprised of three Na-inducible operons: nicAB, encoding enzymes that convert Na to 6-HNa, and the two divergently transcribed nicCDEFTP and nicXR operons, the products of which catabolize 6-HNa to fumarate or regulate transcription. The P. putida nicAB genes are repressed by NicS, which responds to the Na or 6-HNa inducers, resulting in derepression. P. putida NicR exerts negative control at the nicC-nicX divergent control region and is responsive only to 6-HNa (Jiménez et al., 2011).

Since Na relieved BpsR-mediated repression of nicC-lacZ expression in B. bronchiseptica RB50, we tested the hypothesis that, as in P. putida KT2440, Na was being converted to 6-HNa, and that 6-HNa was the actual inducer. nicC-lacZ expression was assessed in the ΔnicA strain RBB33, predicted to be unable to produce 6-HNa from Na. Because 6-HNa does not satisfy the pyridine requirement of B. bronchiseptica for NAD cofactor production (data not shown), all cultures were supplemented with 30 μM Nm, and induction by Na and 6-HNa was assessed (Fig. 7). In the WT strain, nicC-lacZ expression was strongly induced by 2 mM Na, but not by 30 μM Nm, as predicted. However, expression was also strongly induced by 2 mM 6-HNa. In the ΔnicA strain, nicC-lacZ expression was no longer responsive to Na, but responded strongly to 6-HNa. Complementation of the ΔnicA mutation restored induction by Na. Similar results were achieved using 1 mM 6-HNa (data not shown). Since transcription of nicC in the ΔnicA mutant only occurred when 6-HNa, the product of the first pathway reaction, was supplied, but not by Na itself, these results indicate that the BpsR repressor binds the 6- HNa inducer, thus inhibiting its function and allowing nicC expression.


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