The histidine utilization (hut) genes of Pseudomonas fluorescens SBW25 are active on plant surfaces, but are not required for competitive colonization of sugar beet seedlings

Abstract

The histidine utilization (hut) genes of Pseudomonas fluorescens SBW25 are active on plant surfaces, but are not required for competitive colonization of sugar beet seedlings The ability to monitor the spatial and temporal distribution of signals in complex environments is necessary for an understanding of the function of bacteria in the wild. To this end, an existing recombinase-based transcriptional reporter strategy (recombinase-based in vivo expression technology, RIVET) has been extended and applied to the plant-colonizing bacterium Pseudomonas fluorescens SBW25. Central to the project was a rhizosphere-inducible locus, rhi14, which functional analyses show is hutT, a histidine-inducible gene that is required for histidine utilization. A transcriptional fusion between hutT and a promoterless site-specific recombinase (tnpR mut168 ) results in excision of a chromosomally integrated tetracycline-resistance cassette in a histidine-dependent manner. The dose-and time-responsiveness of the promoterless recombinase to histidine closely mirrored the histidine responsiveness of an identical hutT fusion to promoterless lacZ. To demonstrate the effectiveness of the strategy, the activity of hutT was monitored on sugar beet seedlings. Low levels of transcriptional activity were detected in the phyllosphere, rhizosphere and in plant extract, but not in vermiculite devoid of seedlings. The histidine concentration in the rhizosphere was estimated to be 0?6 mg ml "1 . The ecological significance of the hut locus was examined by competing a hutT deletion mutant against the wild-type during colonization of sugar beet seedlings. No impact on competitive fitness was detected, suggesting that the ability to utilize plant-derived histidine is not essential for bacterial colonization.

Genetic Analysis of the Histidine Utilization (hut) Genes in Pseudomonas fluorescens SBW25

“…Drawing upon the data shown in Figure 4 (and its interpretation) the data of fitness in the plant environment indicate that histidine is scarce in this environment-a finding consistent with previous measures that showed histidine to be present in the rhizosphere at $3 mm (Zhang et al 2006). The fitness of PBR815 (hutUTV) was indistinguishable from wild type (Figure 5), which indicates that neither histidine nor urocanate are important sources of carbon or nitrogen in the plant environment.…”

“…In addition to the core degradative enzymes (HutHUIG), the SBW25 hut operon harbors a second copy of the histidase gene (hutH) along with five open reading frames (ORFs) that are predicted to play a role in uptake: plfu0363-0365 are likely to encode an ABC-type transport system and pflu0362 and pflu0368 encode predicted permeases ½pflu0368 is required for histidine utilization (Zhang et al 2006). a The hut-'lacZ fusion strain was constructed by cloning the DNA fragment into the delivery vector pUIC3 followed by integration into the hut locus by insertion-duplication.…”

“…Of value has been the bringing together of various aspects of hut genetics within a single study to lay the foundations for further work. Indeed, our original goal in focusing attention on hut had been to study the function of the five predicted uptake components (Zhang et al 2006), but this proved impossible without the genetics of hut first achieving a contemporary position.…”

“…The histidine uptake and utilization pathway (the hut locus) confers on SBW25 the ability to utilize histidine as a sole source of carbon, nitrogen, and energy (Zhang et al 2006).…”

“…Relative fitness was expressed in terms of the selection rate constant (SRC) (Lenski 1991). A plant competitive colonization assay was performed as previously described (Zhang et al 2006).…”