Dual Involvement of CbrAB and NtrBC in the Regulation of Histidine Utilization inPseudomonas fluorescensSBW25

Abstract

Pseudomonas fluorescens SBW25 is capable of growing on histidine as a sole source of carbon and/or nitrogen. Previous work showed that the two-component regulatory system CbrAB is required for expression of the histidine utilization (hut) locus when histidine is the sole source of carbon and nitrogen. Here, using mutational analysis and transcriptional assays, we demonstrate involvement of a second twocomponent system, NtrBC. When histidine is the sole carbon source, transcription of the hutU operon is initiated from a s 54 -type promoter and requires CbrB (an enhancer binding protein for s 54 -recruitment). However, when histidine is the sole nitrogen source, the hutU operon is transcribed from a s 70 -type promoter and requires either CbrB or the nitrogen regulator, NtrC. No role was found for the SBW25 homolog of the nitrogen assimilation control protein (NAC). Biolog phenotypic microarray analysis of the ability of the three mutants (DcbrB, DntrC, and DcbrB DntrC) to utilize 190 carbon and 95 nitrogen substrates confirmed the central regulatory roles of CbrAB and NtrBC in cellular carbon and nitrogen catabolism: deletion of cbrB abolished growth on 20 carbon substrates; deletion of ntrC eliminated growth on 28 nitrogen substrates. A double cbrB-ntrC mutant was unable to utilize a further 14 nitrogen substrates (including histidine, proline, leucine, isoleucine, and valine). Our data show that CbrAB plays a role in regulation of both carbon and nitrogen catabolism and maintains activity of catabolic pathways under different C:N ratios.

Regulation of the Histidine Utilization (Hut) System in Bacteria

“…It is interesting that like the Hut systems from enteric bacteria (46,84), all of the hut operons of P. fluorescens (and perhaps other pseudomonads [52,64]) can be transcribed by the "housekeeping" RNA polymerase, which carries 70 as its promoter recognition subunit (175,176). However, the hutU-G operon of P. fluorescens can also be transcribed from a promoter that is recognized by the unusual RNA polymerase that carries 54 (also known as N ) as its promoter recognition subunit (175,176), as described in more detail in the section on regulation (see below).…”

“…Finally, there appear to be several, possibly independent mechanisms that operate to create a balance between carbon and energy metabolism and biosynthetic requirements (130). What is clear is that the two-component CbrAB system is important for activation of the hut genes from P. fluorescens and P. aeruginosa in response to carbon limitation (176). Mutants lacking CbrA or CbrB cannot grow with histidine (or several other amino acids) as the sole carbon source, but they grow normally with histidine as the sole nitrogen source when glucose or succinate is present as a carbon source (112,176).…”

“…What is clear is that the two-component CbrAB system is important for activation of the hut genes from P. fluorescens and P. aeruginosa in response to carbon limitation (176). Mutants lacking CbrA or CbrB cannot grow with histidine (or several other amino acids) as the sole carbon source, but they grow normally with histidine as the sole nitrogen source when glucose or succinate is present as a carbon source (112,176). When histidine is the sole carbon source, the hutU-G operon of P. fluorescens is transcribed from a 54 -dependent promoter and the hutF gene is transcribed from a 70 -dependent promoter (176).…”

“…Mutants lacking CbrA or CbrB cannot grow with histidine (or several other amino acids) as the sole carbon source, but they grow normally with histidine as the sole nitrogen source when glucose or succinate is present as a carbon source (112,176). When histidine is the sole carbon source, the hutU-G operon of P. fluorescens is transcribed from a 54 -dependent promoter and the hutF gene is transcribed from a 70 -dependent promoter (176). This suggests that CbrB may directly activate 54 -dependent transcription of hutU-G, but this has not been demonstrated.…”

“…This residual expression of hutU-G is entirely dependent on another two-component regulator, NtrBC. In the absence of both CbrB and NtrC, hutU-G expression is not activated and the cells cannot grow with histidine as the sole carbon source or sole nitrogen source (176). In other words, cells with an active CbrAB system can use histidine as the sole carbon or nitrogen source and do not require NtrBC under either condition.…”