Figure 1.
Locations of acuI relative to various ddd and dmdA genes in different bacteria.
Locations of the acuI genes relative to those that encode DMSP demethylase (dmdA – yellow fill) or the ddd genes that encode the DddD, DddL, DddP and DddY DMSP lyases (arrows filled with various colors) are shown. Grey-filled arrows signify genes with other, known roles in DMSP catabolism. The fccA and fccB genes in Shewanella sp. MR4 encode a flavocytochrome c and a tetraheme cytochrome c respectively. Gene tags from left to right as shown above are: Halomonas HTNK1: ACV84065 to ACV84073 inclusive Rhodobacter sphaeroides 2.4.1: RSP_1433 to RSP_1435 inclusive Canididatus Puniceispirillum marinum IMCC1322: SAR116_1428, SAR116_1427 Alcaligenes faecalis M3A: ADT64689 to ADT64696 inclusive Arcobacter nitrofigilis DSM 7299: Arnit_0113, Arnit_0112 Shewanella sp. MR4: Shewmr4_2154 to Shewmr4_2151 inclusive Ruegeria pomeroyi DSS-3: SPO1913, SPO1914 Also shown are the dimensions of the Ruegeria pomeroyi DSS-3 dmdA-lacZ and acuI-lacZ fusion plasmids (pBIO2020 and pBIO2021 respectively) in which the reporter lacZ gene in pBIO1878. The cloned R. pomeroyi DNA (shown as blue lines) was cloned into pBIO1878 to form pBIO2022 (acuI+dmdA) and pBIO2024 (dmdA alone) for the complementation tests.
Figure 2.
Molecular phylogenetic analysis of selected AcuI polypeptides.
Strains, clades, and protein identification numbers of a selection of the AcuI polypeptides in are shown. Strains in which MDR012-type polypeptides are encoded by acuI genes that are close to dmdA or to the various ddd genes are highlighted in yellow and the two MDR028 gene products near dddQ in green. Those cases where the cloned acuI genes were shown experimentally to correct the acrylate sensitivity of the E. coli YhdH− mutant are underlined. Other examples illustrate the wide taxonomic range of bacteria that harbour AcuI homologues, several of which are closely related to those encoded by genes linked to ddd or dmdA, and include some genera (Xanthomonas, Streptomyces, Geobacter) in which only some strains contain AcuI homologues. (C./V. = Chlamydiae/Verrucomicrobia; α-, β-, δ-, γ-, ε- refer to the corresponding sub-phylum of Proteobacteria). Three strains have two separate AcuI homologues, as indicated for Dechloromonas aromatica, Alkalilimnicola ehrlichii and Psychrobacter cryohalolentis. The tree with the highest log likelihoood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically using maximum parsimony method or by the BIONJ method with MCL distance matrix. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
Figure 3.
Effects of different acuI genes on the inhibitory effects of acrylate on the growth of Escherichia coli.
Overnight cultures of wild type E. coli strain BW25113, its YhdH− mutant derivative JW3222-1 and derivatives of JW3222-1 containing the cloned yhdH gene of E. coli (E. col.) or the acuI genes of Rhodobacter sphaeroides (Rh. sph.), Ruegeria pomeroyi DSS-3 (Ru. pom.), Halomonas HTNK1 (Halo), Alcaligenes faecalis M3A (Alc. fae.), Rhizobium leguminosarum 3841 (Rhi. leg.) or Burkholderia ambifaria AMMD (Bur. amb.) were spotted (10 µl) onto M9 minimal medium agar, with glycerol as the carbon source, plus acrylate at concentrations shown. Plates were incubated at 37°C for 20 hours.
Figure 4.
Chemical formulae of agents tested for their effects on the growth of Escherichia coli.
Values below each chemical indicate the highest concentrations tested at which the E. coli wild type strain would grow. Where there was a difference between the wild type and the YhdH− mutant strain, the maximum concentration at which the mutant would grow is shown in brackets.
Figure 5.
AcuI-mediated conversion of acryloyl-CoA to propionyl-CoA.
The pathway from 3-hydroxypropionate to propionyl-CoA is adapted from Schneider et al. [13]. Also shown is how DMSP lyases can generate acrylate, which is postulated to be converted to acryolyl-CoA by a CoA-ligase, as yet unidentified. The exact identity of the DMSP lyase is strain-dependent; e.g., Rhodobacter sphaeroides has the DddL lyase, and in Ruegeria pomeroyi, the acrylate can be generated from DMSP by DddP, DddQ and/or DddW.