Role of a local transcription factor in governing cellular carbon/nitrogen homeostasis inPseudomonas fluorescens

Abstract

Autoactivation of two-component systems (TCSs) can increase the sensitivity to signals but inherently cause a delayed response. Here, we describe a unique negative feedback mechanism enabling the global NtrB/NtrC regulator to rapidly respond to nitrogen starvation over the course of histidine utilization (hut) in Pseudomonas fluorescens. NtrBC directly activates transcription of hut genes, but overexpression will produce excess ammonium leading to NtrBC inactivation. To prevent this from occurring, the histidine-responsive repressor HutC fine-tunes ntrBC autoactivation: HutC and NtrC bind to the same operator site in the ntrBC promoter. This newly discovered low-affinity binding site shows little sequence similarity with the consensus sequence that HutC recognizes for substrate-specific induction of hut operons. A combination of genetic and transcriptomic analysis indicated that both ntrBC and hut promoter activities cannot be stably maintained in the ΔhutC background when histidine fluctuates at high concentrations. Moreover, the global carbon regulator CbrA/CbrB is involved in directly activating hut transcription while de-repressing hut translation via the CbrAB-CrcYZ-Crc/Hfq regulatory cascade. Together, our data reveal that the local transcription factor HutC plays a crucial role in governing NtrBC to maintain carbon/nitrogen homeostasis through the complex interactions between two TCSs (NtrBC and CbrAB) at the hut promoter.

NtrBC Selectively Regulates Host-Pathogen Interactions, Virulence, and Ciprofloxacin Susceptibility of Pseudomonas aeruginosa

“…NtrBC is a two-component system of P. aeruginosa that is essential for sensing environmental nitrogen levels and responding to nitrogen starvation through initiation of nitrate assimilation ( Luque-Almagro et al., 2011 ). NtrC is known to activate a variety of other physiological processes, in part by increasing the binding affinity of the alternative sigma factor RpoN/σ 54 to RNA polymerase, including histidine utilization ( Naren and Zhang, 2021 ) and the stringent stress response ( Brown et al., 2014 ). We previously showed that NtrBC impacts on P. aeruginosa adaptive phenotypes including biofilm formation and swarming motility as well as invasiveness and virulence in vivo ( Alford et al., 2020 ), and that the effects of each of NtrB and NtrC appear to be additive for selected phenotypes.…”

The Regulatory Hierarchy Following Signal Integration by the CbrAB Two-Component System: Diversity of Responses and Functions

“…Apart from the previously described targets for CbrB in P. putida the small RNAs CrcZ and CrcY [ 74 ], the ChIP-seq analysis led to the identification of several undescribed targets including the putative efflux pump encoded by the operon PP2810 - PP2813 , the porin OprD, PP3074 encoding a putative permease or the histidine kinase PP3420 [ 31 ]. Other targets that have also been positively controlled by CbrB are CrcZ in P. aeruginosa and A. vinelandii [ 75 , 76 ], CrcZ and CrcX in P. syringae [ 30 ], the lipA (lipase) gene in P. alcaligenes [ 87 , 88 ], and the hutU (histidine utilization) operon of P. aeruginosa and P. fluorescens SBW25 [ 35 , 89 , 90 ].…”

“…The substrate-specific induction of the h istidine ut ilization hut genes in Pseudomonas is negatively regulated by the HutC repressor that binds to the hutC promoter in the absence of urocanate [ 84 , 85 ]. In contrast to the hut promoters of P. putida in the analogous operon in P. fluorescens , strain SBW25 is more complex, since it contains an insertion of five genes between hut U and hut H encoding a high-affinity transporter for urocanate ( hutT u ), an additional high-affinity ABC-type transporter ( hutXWV ) and a gene coding for an histidase ( hutH1 ), which, however, is not required for growth on histidine [ 35 , 84 ]. This last module is also present in the genome of P. aeruginosa , which bears an additional ABC transporter system encoded by orfXYZ , and an orfT encoding another histidine transporter ( Figure 4 B).…”

“…These non-coding RNAs regulate the activity of the Crc/Hfq complex by direct binding, thereby interfering with the translation repression of the corresponding mRNA targets in the process of catabolite repression in Pseudomonas and Azotobacter , corresponding to a post-transcriptional regulation mechanism. In addition, cross-talk with NtrBC regulatory systems for the assimilation of certain amino acids as a carbon or nitrogen source has been reported [ 32 , 33 , 34 , 35 ], as well as possible interactions with extracytoplasmic function (ECF) sigma factors [ 36 ].…”

The Regulatory Functions of σ54 Factor in Phytopathogenic Bacteria

“…It is also required to accumulate the sigma factor RpoS and core metabolites of aromatic and sugar metabolism [ 85 , 86 ]. Importantly, CbrAB and the master nitrogen regulator NtrBC directly control C/N homeostasis by regulating the transcription of histidine utilization genes ( hut ) [ 87 ]. When histidine is the sole source of N, the CbrAB-mediated promoter activity is weak, and NtrBC plays the dominant role in activating hut transcription.…”