Effect of the Na degradation pathway on Bvg phenotypic phase modulation.

In standard Bordetella laboratory SS culture, Na is used at micromolar concentrations required for sufficient NAD biosynthesis; however, phenotypic modulation of Bvg-regulated genes occurs at millimolar Na concentrations that would also potentially be sufficient to serve as a carbon source. To determine whether Na catabolic pathway activity influences modulation, expression of a low-copy transcriptional lacZ fusion to the Bvg-activated adenylate cyclase toxin gene promoter (cyaA-lacZ) (Brickman et al., 2017) was used to monitor the response (Fig. 8). As expected, typical Bvg phenotypic phase modulation using 2 mM Na resulted in maximally decreased cyaA transcriptional activity compared to bacteria grown on 30 μM Na. The B. bronchiseptica ΔbpsR mutant was modestly more resistant to phenotypic modulation than the WT parent strain at the intermediate Na concentrations. This effect was consistently observed in multiple experiments. Conversely, the ΔnicA mutant was remarkably more sensitive to phenotypic modulation than the WT strain. Its cyaA-lacZ expression levels in 30 μM Nm were similar to those of the WT and the ΔbpsR mutant. However, compared with those strains, ΔnicA mutant expression of cyaA was significantly lower in response to all Na concentrations, with full modulation occurring at 1 mM. These results support the concept that the bacteria can sense and respond to fluctuations in intracellular Na pools. Loss of BpsR-mediated repression would be predicted to enhance Na flux through the Nic catabolic pathway, making Na less abundant in the cytoplasm, thereby dampening the modulation response. Conversely, blockage of the pathway via nicA mutation would make Na more abundant in the cytoplasm, triggering modulation at concentrations that are insufficient to significantly affect Bvg-dependent gene expression in WT strains.

DISCUSSION

A diversity of bacterial species can degrade and use aromatic compounds as nutrients. Regulation of these pathways often involves a transcriptional regulator, the function of which is either activated or inhibited by binding the aromatic substrate (Parke and Ornston, 2003; Tropel and van der Meer, 2004; Hiromoto et al., 2006; Wang et al., 2014). The Diaz group characterized the aerobic Na catabolic pathway of the γ proteobacterium P. putida and searched for orthologous systems (Jiménez et al., 2008). Rather than identifying other taxonomically related Pseudomonas or γ proteobacterium species, the strongest hits corresponded to members of the Burkholderiales of the β proteobacteria. These included the predicted nic gene products of Bordetella species, which also had a highly similar gene organization. Bordetella locus 1 nic genes that differ from those of P. putida include those encoding the tripartite Na hydroxylase enzymes (NicA, NicB1, NicB2) that carry out the first step of the pathway, contrasting with P. putida NicA and NicB. Whereas the P. putida nic cluster has genes predicted to encode a porin protein and a major facilitator superfamily transporter, presumably for Na uptake, Bordetella locus 1 contains genes encoding a predicted ATP binding cassette transporter. We were surprised to find locus 2, which P. putida lacks, with its duplicated nicEDXF genes. Since the locus 2 promoter-proximal gene encodes a predicted 2,3-dihydroxybenzoate decarboxylase, these genes likely function in other aromatic catabolism that uses the NicEDXF enzymes. Under the tongue

Recent studies produced recombinant B. bronchiseptica NicF and NicC in Escherichia coli, confirmed and characterized their maleamate amidohydrolase and 6-hydroxynicotinate 3-monooxygenase activities, respectively, and determined the NicC crystal structure (Kincaid et al., 2012; Hicks et al., 2016). These findings indicate that the B. bronchiseptica nicF and nicC products have the same activities as the P. putida enzymes. Therefore, one might predict Bordetella growth stimulation via catabolism of Na to ammonium and products including fumarate that can enter the TCA cycle. Since relatively little is known of Bordetella metabolism, a systematic analysis of the effects of combinations of growth medium components will be needed to establish whether these organisms can use Na as a primary carbon and/or nitrogen source.

For pyridine auxotrophs like Bordetella spp., utilization of Na as a carbon or nitrogen source would need to be carefully controlled, especially if Na were the only available NAD precursor. P. putida NicR and Bordetella BpsR are members of the MarR family of regulators that typically bind effectors that modify their activity. Based on the present studies and those of P. putida (Jiménez et al., 2011), BpsR, like NicR, represses transcription by binding to upstream control regions of target nic genes. When BpsR binds the 6-HNa inducer, it loses its DNA-binding activity and the nic genes are expressed (derepressed). By responding to the 6-HNa product of the first step in the Nic pathway, flux of Na into that pathway can be monitored and controlled. Derepression in the presence of 6-HNa allows the pathway to function in catabolism for the cell only when sufficient substrate concentrations are available. In our studies, a nicA mutant strain with an inactivated Nic pathway showed enhanced growth in 30-μM Na medium compared with growth of WT cells. These findings suggest that even at concentrations of exogenously added Na that are insufficient for maximal induction of nic gene expression in WT cells, some portion of that Na may be diverted from NAD biosynthesis to the Na catabolic pathway. Conversely, constitutive expression of the pathway in a bpsR mutant was associated with significantly reduced growth in the same 30-μM Na medium. Growth promotion of a bpsR mutant by a low concentration of Nm but not by Na suggests that the cell utilizes transported pools of Na differently than Na that is produced from Nm via PncA, which could be rapidly bound by PncB (Fig. 1). Since ectopic expression of de novo NAD biosynthesis genes rescued growth of the bpsR mutant, together our findings reveal significant interaction between NAD homeostatic mechanisms and the Na catabolic pathway. Furthermore, fluctuations in Nic pathway activity appear to alter intracellular Na concentrations, impacting Bordetella virulence gene expression.