Pseudomonas putida F1 has multiple chemoreceptors with overlapping specificity for organic acids

Abstract

Previous studies have demonstrated that Pseudomonas putida strains are not only capable of growth on a wide range of organic substrates, but also chemotactic towards many of these compounds. However, in most cases the specific chemoreceptors that are involved have not been identified. The complete genome sequences of P. putida strains F1 and KT2440 revealed that each strain is predicted to encode 27 methyl-accepting chemotaxis proteins (MCPs) or MCP-like proteins, 25 of which are shared by both strains. It was expected that orthologous MCPs in closely related strains of the same species would be functionally equivalent. However, deletion of the gene encoding the P. putida F1 orthologue (locus tag Pput_4520, designated mcfS) of McpS, a known receptor for organic acids in P. putida KT2440, did not result in an obvious chemotaxis phenotype. Therefore, we constructed individual markerless MCP gene deletion mutants in P. putida F1 and screened for defective sensory responses to succinate, malate, fumarate and citrate. This screen resulted in the identification of a receptor, McfQ (locus tag Pput_4894), which responds to citrate and fumarate. An additional receptor, McfR (locus tag Pput_0339), which detects succinate, malate and fumarate, was found by individually expressing each of the 18 genes encoding canonical MCPs from strain F1 in a KT2440 mcpS-deletion mutant. Expression of mcfS in the same mcpS deletion mutant demonstrated that, like McfR, McfS responds to succinate, malate, citrate and fumarate. Therefore, at least three receptors, McfR, McfS, and McfQ, work in concert to detect organic acids in P. putida F1.

Chemotaxis by natural populations of coral reef bacteria

“…This suggests that a subset of the chemotactic community is made of 'generalist chemotaxers' that have the capacity to sense and direct movement in response to diverse chemicals compounds. Currently, little is known about the chemoreceptors used by marine bacteria (Stocker and Seymour, 2012), but the generalist chemotaxers identified here may either have multiple sets of chemoreceptors, which allow them to respond to a variety of compounds (Wadhams and Armitage, 2004;Parales et al, 2013), or single chemoreceptors that allow for binding of multiple compounds (Adler, 1969;Glekas et al, 2012). However, the bulk (76.4%) of chemotactic OTUs identified here can be classified as 'specialist chemotaxers', as they responded to only one compound.…”

Cytosine chemoreceptor McpC in Pseudomonas putida F1 also detects nicotinic acid

“…The aer2 gene was deleted from the DmcpC mutant XLF004 by homologous recombination using the aer2 deletion construct pXLF019 (Luu et al, 2013), generating strain XLF119. …”

“…The nicB gene in P. putida F1 (locus tag Pput_1889) was insertionally inactivated with the kanamycin resistance gene from pRK415Km (Luu et al, 2013) using an Infusion-HD cloning kit (Clontech), and the suicide vector pEX18, which carried a gentamicin resistance gene and the sacB gene, conferring sucrose sensitivity (White & Metcalf, 2004). The primers used were pEX18_1889delfor (59-CGACGGCCAGTGCCAA-GCTTCACTCACAACAGGTGCCCAG-39)/pEX18_1889delrev (59-GC-TATGACCATGATTACGAATTCCATCATTACGTCGATAGCTGGC-A-39), pput_1889_intfor (59-TTGAATGGGCCCTACATGGTGTGG-TCAGGTACGCAGAAC-39)/pput_1889intrev (59-GAGTTCGGTCC-GATCAAGGTACCTGACCACACGCGGAT-39) and pETKm_RsrI-for/pETKm_ApaI-rev (Luu et al, 2013). To generate the mutant construct, 1 kb regions at the beginning and centre of the nicB gene were amplified by PCR, and the resulting PCR fragments were fused to the amplified kanamycin resistance gene and pEX18 using the complementary overhanging ends.…”

“…Qualitative capillary assays were carried out as described previously (Grimm & Harwood, 1997;Parales et al, 2013). Briefly, bacterial cells in mid-exponential phase (OD 660 0.3-0.4) were harvested by centrifugation at 5000 x g for 5 min, washed once with chemotaxis buffer (CB; 50 mM potassium phosphate buffer, pH 7.0, 10 mM disodium EDTA and 0.05 % glycerol) (Parales et al, 2000) and gently resuspended in CB to OD 660~0 .10.…”

“…Recent analyses of bacterial and archaeal genomes have shown that the number of MCPs encoded in a given genome is generally related to the metabolic diversity of the organism rather than the size of the genome (Lacal et al, 2010). We have been using P. putida F1 as a model organism in which to study the range of attractants detected and the complexity of chemotactic signalling Liu et al, 2009;Luu et al, 2013;Parales et al, 2000Parales et al, , 2013. Previous work demonstrated that McpC, one of the 27 predicted MCPs/ MCP-like proteins in P. putida F1, mediated the chemotactic response to the N-heteroaromatic pyrimidine base cytosine .…”