Date Published: June 29, 2017
Publisher: Public Library of Science
Author(s): Yilei Liu, Martina Lardi, Alessandro Pedrioli, Leo Eberl, Gabriella Pessi, Eric Cascales.
Burkholderia cenocepacia is a versatile opportunistic pathogen that survives in a wide variety of environments, which can be limited in nutrients such as nitrogen. We have previously shown that the sigma factor σ54 is involved in the control of nitrogen assimilation and virulence in B. cenocepacia H111. In this work, we investigated the role of the σ54 enhancer binding protein NtrC in response to nitrogen limitation and in the pathogenicity of H111. Of 95 alternative nitrogen sources tested the ntrC showed defects in the utilisation of nitrate, urea, L-citrulline, acetamide, DL-lactamide, allantoin and parabanic acid. RNA-Seq and phenotypic analyses of an ntrC mutant strain showed that NtrC positively regulates two important phenotypic traits: exopolysaccharide (EPS) production and motility. However, the ntrC mutant was not attenuated in C. elegans virulence.
The betaproteobacterium Burkholderia cenocepacia is an opportunistic pathogen that thrives in different environments, which can be limited in essential elements such as nitrogen [1, 2]. Bacterial adaptations to changes in nitrogen availability have been shown to be stringently regulated [3–7]. Enterobacteria respond to nitrogen starvation by activating the nitrogen regulatory response (Ntr) to facilitate N scavenging from alternative nitrogen sources. The Ntr system monitors the intracellular ratio of glutamine to α-ketoglutarate. Under nitrogen limiting conditions, the PII signal transduction proteins encoded by glnB and glnK are uridylylated and, by controlling the kinase and phosphatase activities of the regulator NtrB, regulate the transcription of nitrogen-regulated target genes [3, 6, 8, 9]. NtrB is a sensor kinase, and is part of the NtrBC two-component regulatory system. Under nitrogen limiting conditions, NtrB phosphorylates the response regulator NtrC, which then binds to DNA sequences in the promoter and together with the alternative sigma factor σ54 (or RpoN) activates transcription [10–12]. The sigma factor σ54 reversibly associates with the core RNA polymerase and recognizes its cognate promoter sequences via defined consensus sequences at positions –12 and –24 bp (relative to the transcription start site) . The initiation of σ54-dependent transcription usually requires such an interaction with an enhancer binding protein (EBP). The specific protein involved varies depending on the respective environmental signals. NtrC is the EBP in the case of nitrogen starvation conditions [14, 15]. EBPs share a conserved modular structure, which consists of three domains: i) an amino terminal regulatory domain, ii) a central catalytic domain that belongs to the AAA+ superfamily of ATPases and is required for interaction with σ54, iii) a carboxy-terminal DNA-binding domain with a helix–turn–helix motif that is required for recognition of upstream activator sequences . The EBP catalyzes ATP hydrolysis and thereby promotes conversion of the closed promoter into an open promoter complex from which transcription can proceed [16–20]. In enterobacteria, hexamers of phosphorylated NtrC bind to an upstream activator sequence (UAS), which is usually located 100–150 nucleotides upstream of the transcriptional start site [21, 22].
We previously identified the B. cenocepacia genes responding to nitrogen starvation conditions and found that the alternative sigma factor σ54 (or RpoN) plays a major role in the control of nitrogen metabolism. That study furthermore uncovered that σ54 controls other important cellular processes such as EPS production, biofilm formation and C. elegans virulence . Sigma 54 is an alternative sigma factor since it binds to a characteristic -24/-12 binding sequence (GGcacg-N4-ttGC) in the promoter of target genes and requires an additional ATP-dependent activation event to initiate transcription. This step is provided by transcriptional activators with an AAA (ATPase Associated with various cellular Activities) protein domain, which bind as inactive dimers to a consensus sequence upstream of the promoter, assemble as hexameric rings (in their active form) and interact through DNA looping with the σ54 promoter complex to activate transcription . This requirement for an activator protein (or EBP) allows σ54 to tightly and rapidly control gene expression in response to cellular and extracellular signals that regulate the activity of a specific AAA-domain containing protein. Bacteria usually encode several AAA-family activator proteins and each one is needed for a specific and precise response to an environmental change. By looking for all B. cenocepacia proteins containing a σ54 activation AAA domain (PFAM family: PF00158), we were able to identify 22 proteins that could potentially serve as σ54 activator proteins (S4 Table). Among them we identified the regulatory protein NtrC, which is known to be the master regulator of nitrogen control in other bacteria. The ntrC gene had previously been shown to have increased expression under nitrogen limiting conditions . The ntrC gene is usually located downstream of ntrB, which encodes a sensor kinase that phosphorylates the response regulator NtrC in nitrogen limited environments. In this study, we first performed a comprehensive growth analysis of an ntrC mutant on 95 different nitrogen sources including all common amino acids, the nitrogenous bases, several di-peptides and other compounds (Biolog Phenotype MicroArray). The ntrC mutant was revealed to be affected in the utilisation of 7 N sources i. e. nitrate, urea, L-citrulline, acetamide, DL-lactamide, allantoin and parabanic acid suggesting that utilisation of these sources in B. cenocepacia is dependent on the presence of a functional NtrC. We next used RNA-Seq analysis to elucidate the role of NtrC in the control of transcription during nitrogen starvation conditions. The results clearly show that NtrC is a major regulator under nitrogen limiting conditions and that the large majority of the NtrC-regulated genes are co-regulated by σ54. At the phenotypic level, the ntrC mutant strain behaved like a σ54 mutant strain for the utilization of alternative nitrogen sources such as nitrate and urea. Both urea and nitrate were not utilised as nitrogen sources by the mutant strain (Fig 1 and S2 Table). Accordingly, the transcription of genes involved in urea and nitrate assimilation (urease and nitrate/nitrite reductase, respectively) was significantly down-regulated in the ntrC mutant strain (Table 1). In addition to, and in line with, a poor utilization of allantoin in the ntrC mutant (S2 Fig and S2 Table), two genes coding for allantoicases (I35_0495 and I35_1962, Table 2 and S3 Table) showed significantly decreased expression in the ntrC mutant. Inspection of the NtrC regulon revealed two clusters of genes (bceI and bceII) involved in the production of cepacian, the main EPS in B. cenocepacia [29, 45–47]. Accordingly, EPS production was clearly reduced in an ntrC mutant and this defect could be rescued by genetic complementation. Control of EPS synthesis by NtrC has been demonstrated in several bacteria including the human pathogen Vibrio vulnificus , Agrobacterium sp. ATCC 31749  and Sinorhizobium meliloti . However, and in contrast to our previous results obtained with the σ54 mutant , biofilm formation was only slightly reduced in the ntrC mutant, suggesting that σ54 is using another EBP for controlling biofilm formation. This result also suggested that the reduced transcription of the bceI and bceII clusters does not drastically affect biofilm formation in microtiter plates.