Genetic analysis of the histidine utilization (hut) genes in Pseudomonas fluorescens SBW25

Abstract

The histidine utilization (hut) locus of Pseudomonas fluorescens SBW25 confers the ability to utilize histidine as a sole carbon and nitrogen source. Genetic analysis using a combination of site-directed mutagenesis and chromosomally integrated lacZ fusions showed the hut locus to be composed of 13 genes organized in 3 transcriptional units: hutF, hutCD, and 10 genes from hutU to hutG (which includes 2 copies of hutH, 1 of which is nonfunctional). Inactivation of hutF eliminated the ability to grow on histidine, indicating that SBW25 degrades histidine by the five-step enzymatic pathway. The 3 hut operons are negatively regulated by the HutC repressor with urocanate (the first intermediate of the histidine degradation pathway) as the physiological inducer. 5'-RACE analysis of transcriptional start sites revealed involvement of both sigma(54) (for the hutU-G operon) and sigma(70) (for hutF); the involvement of sigma(54) was experimentally demonstrated. CbrB (an enhancer binding protein for sigma(54) recruitment) was required for bacterial growth on histidine, indicating positive control of hut gene expression by CbrB. Recognition that a gene (named hutD) encoding a widely distributed conserved hypothetical protein is transcribed along with hutC led to analysis of its role. Mutational and gene fusion studies showed that HutD functions independently of HutC. Growth and fitness assays in laboratory media and on sugar beet seedlings suggest that HutD acts as a governor that sets an upper bound to the level of hut activity.

Figure 1.—

The histidine degradation pathways (A) and structure of the P. fluorescens SBW25 hut locus (B) with details of the promoter regions of P_hutF and P_hutU (C). (A) The five-step histidine degradation pathway of Pseudomonas (Hu et al. 1987) is shown by solid arrow with gene product involved in each reaction: HutH, histidine ammonia-lyase or histidase; HutU, urocanase; HutI, imidazolone propionate (IPA) amidohydrolase; HutF, formiminoglutamate (FIGLU) iminohydrolase; HutG, formylglutamate (FG) amidohydrolase. The divergent final step of the four-enzyme pathway (Magasanik 1978) is indicated by a dashed arrow and it is catalyzed by FIGLU formiminohydrolase. The gene encoding this enzyme has been referred to as hutG; however, it shares little in sequence identity with the hutG from Pseudomonas and thus it is not shown in the diagram to avoid confusion. (B) The metabolic hut genes and the hut regulators are shown by open and solid arrows, respectively. Crosshatched arrows represent putative transporters. ORF numbers are derived from the current SBW25 genome annotation (http://www.sanger.ac.uk/Projects/P_fluorescens). Insertion sites of the Ω-Sp cassette are indicated by open triangles. Location and orientation of the three histidine-induced promoters are indicated by bent arrows. Positions of the lacZ fusions are shown by open circles with attached arrow. (C) The putative HutC-binding sites are marked with underlined letters in boldface type. They were identified by their sequence similarities with the known HutC-binding site (CTTGTACATACAAG) from P. putida (Hu et al. 1989). Transcriptional start site determined by 5′-RACE experiments is indicated by +1.

Figure 2.—

Expression of hutU in wild-type SBW25 and the hutCD mutants. β-Galactosidase activities (aM 4MU min⁻¹ cell⁻¹) were measured in hutU-‘lacZ fusion strains PBR813 (wild type), PBR823 (ΔhutC), PBR824 (ΔhutD), and PBR825 (ΔhutCD). Bacteria were grown in M9 salts plus glucose and ammonia (open bars), M9 salts plus urocanate (stippled bars), and M9 salts plus histidine (solid bars). Values are means and standard errors of three independent cultures. Bars that are not connected by the same letter (shown above each) are significantly different (P < 0.05) by Tukey’s HSD.

Figure 3.—

Growth dynamics of the hutCD deletion mutants. Growth was measured for wild-type SBW25 (open circles) and mutants PBR805 (ΔhutC, solid triangles), PBR806 (ΔhutD, open triangles), and PBR807 (ΔhutCD, open squares) in M9 (M9 salts plus glucose and ammonia, A), M9 salts plus urocanate (B) and M9 salts plus histidine (C). Results are means of eight independent cultures. Data were collected at 5-min intervals, but two hourly time points are shown for clarity. Standard errors are within the symbols and thus not visible.

Figure 4.—

Fitness of the hutCD deletion mutants relative to lacZ-marked P. fluorescens SBW25 in laboratory media. (A) Fitness of SBW25 (wild type), PBR805 (ΔhutC), PBR806 (ΔhutD), and PBR807 (ΔhutCD) relative to SBW25-lacZ grown in M9 medium, or M9 salts medium with histidine or urocanate as sole carbon and nitrogen sources. Data are means and standard errors of 10 independent cultures. A fitness of zero indicates that the fitness of the mutant is identical to wild type (a negative value indicates a reduction in fitness relative to wild type). (B) Fitness of PBR805 (ΔhutC, solid triangles), PBR806 (ΔhutD, open triangles), and PBR807 (ΔhutCD, open squares) relative to SBW25-lacZ grown on M9 salts medium supplemented with 5 mm glutamate and varying concentrations of histidine. Data are means and standard errors of six independent cultures.

Figure 5.—

Fitness of the hutCD deletion mutants relative to lacZ-marked P. fluorescens SBW25 on sugar beet seedlings. Data are shown in an order of strains: ancestral SBW25 (wild type), PBR805 (ΔhutC), PBR806 (ΔhutD), and PBR807 (ΔhutCD) and PBR815 (hutU∷Ω). Fitness was determined after 2 weeks of competitive colonization. Results are means and standard errors of data collected from the shoot and rhizosphere of eight plants, which were inoculated by independent cultures. A fitness of zero indicates that the fitness of the mutant is identical to wild type (a negative value indicates a reduction in fitness relative to wild type).