Article Figures & Data
Figures
- FIG. 1.
Pathways for the biosynthesis of AroAAs in M. maripaludis. The precursors for the de novo pathway in methanogens are not known, but homologs for genes at all the steps after 3-dehydroquinate are present in the genomic sequence (see the text). After chorismate, the pathway splits into three branches, one of which leads to tryptophan. At prephenate, the branches leading to phenylalanine and tyrosine form. The aryl acid pathway starts from phenylacetate, p-hydroxyphenylacetate, and indoleacetate, precursors for phenylalanine, tyrosine, and tryptophan, respectively. The enzymes shown are DHQ, CM, PDT, PDH, AroAT, and IOR. Possible transcriptional regulation of DHQ is indicated by double dashed lines. Inhibition (−) or activation (+) of the PDT and PDH enzyme activities are indicated by single dashed lines.
- FIG. 2.
Construction of the ΔaroD::pac mutation. (A) M. maripaludis aroD (MMP1394) gene region. The ORFs MMP1396, MMP1395 and MMP1391 were annotated as aminotransferase, DEAD/DEAH box helicase and aspartate-semialdehyde dehydrogenase, respectively. The ORFs MMP1393 and MMP1392 were annotated as hypothetical proteins (J. Leigh, personal communication). The locations of the primers U1, U2, D1, and D2 used to clone the upstream and downstream regions flanking MMP1394 are shown. (B) Confirmation of the genotypes of the wild-type S2 and mutants S83 and S87 by Southern hybridization. The genomic DNA (3.2 μg) was digested with BglII prior to hybridization with the probe indicated in panel A. Lanes 1, 2, and 3, genomic DNAs of S2, S83, and S87, respectively, digested with BglII. Lane 4, pJA03-aroD digested with PvuII and NheI as a positive control.
- FIG. 3.
Growth requirement of the auxotroph S87 for AroAAs. The McNA medium contained 1 mM AroAAs or aryl acids, except when otherwise indicated. (A) Growth in the presence of AroAAs or aryl acids. The inoculum was 105 cells. Shown are growth of the wild-type S2 with or without acids (◊) and growth of S87 with all three aryl acids (▴), with all three AroAAs (□), and without any addition (•). (B) Assimilation of phenylalanine and tyrosine limit the growth rate of the mutant S87. The inoculum was 107 cells washed in McNA medium. Shown are growth of S87 without any addition (•); with all three aryl acids (▴); with phenylalanine, indoleacetate, and p-hydroxyphenylacetate (▪); with tyrosine, indoleacetate, and phenylacetate (⧫); with indoleacetate and p-hydroxyphenylacetate alone (□); and with indoleacetate and phenylacetate alone (◊). (C) Growth yield of the mutant S87 with limiting concentrations of indoleacetate (▪), p-hydroxyphenyacetate (▴), and phenylacetate (•). The inoculum was 107 cells washed in McNA medium.
- FIG. 4.
NMR spectra of the mixture of amino acids produced by acid hydrolysis of labeled proteins. The amino acids (10 mg) were isolated by Robert's method from S87 cells grown in the presence of 0.1 mM [1-13C]phenylacetate. (A) 13C spectrum of the mixture of amino acids produced by acid hydrolysis of the labeled proteins. (B) Proton spectrum of the 13C-labeled amino acids demonstrating coupling to 13C.
- FIG. 5.
Phylogeny of the iorA genes that encode the α subunit of IOR. The phylogenetic tree was constructed by using the PHYLIP package based upon an alignment of conserved positions using the Fitch-Margoliash algorithm. Similar phylogenetic trees were generated by neighbor-joining and parsimony algorithms (data not shown). The bootstrap values for all three algorithms were very close and are labeled in the tree by the symbols at the branch points: •, values of >90%; ○, values of >60%; unlabeled, values of ≤60%. The scale bar is 0.5 expected amino acid substitutions per site. The six clades found are labeled A to F. The accession numbers for protein sequences from the National Center for Biotechnology Information database (from top to bottom in the tree) are: AAM05134 , AAM32330 , CAF29872.1 , AAR21228 , AAL80969 , NP_143041 , NP_126757 , C90374 , BAB65740 , BAB59718 , CAC12141 , Q9UZ57 , O58495 , AAL80657 , BAA20528 [formerly named Pyrococcus sp KOD1(43)], NP_615972 , NP_634117 , O28783 , AAB86318 , ZP_00000830 , ZP_00001160 , ZP_00080126 , NP_106112 , BAC48676 , CAD15531 , ZP_00056522 , ZP_00129724 , AAM32484 , AAM05385 , NP_661020 , AAO75537 , ZP_00054515 , ZP_00015617 , ZP_00010808 , ZP_00079304 , NP_623747 , NP_348620 , ZP_00059944 , AAR21230 , and CAF30269.1 .
- FIG. 6.
Construction of ΔiorA2::pac mutation. (A) M. maripaludis iorA2 (MMP0713) gene region. The ORFs MMP0712, MMP715, MMP0716, and MMP0717 were annotated as solute-binding protein/glutamate receptor, coenzyme F390 synthetase II, acetohydroxyacid synthase small-subunit related, and hypothetical protein, respectively (J. Leigh, personal communication). The primers U3, U4, D3 and D4 were used to clone the flanking regions for construction of pIJA03-iorA. The primers E1 and E2 were used for cloning iorA2 and iorB2 during the construction of pMEV2-iorAB2. (B) Confirmation of the genotype by Southern hybridization with the wild-type S2 and the iorA2 mutant S122. The genomic DNA (3.3 μg) was digested with EcoRV prior to hybridization to the probe indicated in panel A. Lanes 1 and 2, S122 and S2 genomic DNAs, respectively; lane 3, pJA03-iorA2 DNA digested with PvuII and NheI as a positive control.
- FIG. 7.
Effects of aryl acids on growth of the ΔiorA2::pac mutant S122. The McNA medium contained 1 mM aryl acids or AroAAs as indicated. The inoculum was ∼2 × 105 cells. (A) Inhibition of growth of strain S122 by phenylacetate or p-hydroxyphenylacetate. ◊, wild-type S2 without any addition; •, S122 without any addition; ▵, S122 with phenylacetate alone, p-hydroxyphenylacetate alone, both phenylacetate and p-hydroxyphenylacetate, or all three aryl acids. (B) Restoration of growth by AroAAs. Shown are S122 without any addition (•), with phenylacetate (▵), with phenylacetate and phenylalanine (▴), with p-hydroxyphenylacetate (□), and with p-hydroxyphenylacetate and tyrosine (▪). (C) Complementation of the ΔiorA2::pac mutant with pMEV2-iorAB2. Shown are growth of wild-type S2 without any addition (◊), the complemented strain S155 without any addition (•), S155 with phenylacetate (▵), S155 with p-hydroxyphenylacetate (□), S155 with phenylacetate and p-hydroxyphenylacetate (○), and S155 with the three aryl acids (▴).
- FIG. 8.
Effects of aryl acids on growth of wild type S2. The McNA medium contained 1 mM aryl acids where indicated. The inoculum was ∼2 × 105 cells. Shown is the S2 strain growing without any addition (◊), with phenylacetate alone (•), with indoleacetate alone (▪), with p-hydroxyphenylacetate alone (▴), and with the three aryl acids (▵). The inset shows the growth lag of strain S2 during growth with increasing concentrations of phenylacetate (•), indoleacetate (▪), and p-hydroxyphenylacetate (▴).
Tables
- TABLE 1.
Bacterial strains and plasmids
Bacterial strain or plasmid Genotype or description Source or reference M. maripaludis S2 Wild type 65 S83 ΔaroD::pac This work S87 ΔaroD::pac This work S122 ΔiorA2::pac This work S151 ΔiorA2::pac/pMEV2-iorAB2 This work S153 ΔiorA2::pac/pMEV2-iorAB2 This work S155 ΔiorA2::pac/pMEV2-iorAB2 This work E. coli DH5α F− (φ80dlacZΔM15) recA1 endA1 gyrA96 thi-1 hsdR17 (rK − mK +) supE44 Δ(lacZYA-argF) U169 19 Plasmids pIJA03 Purr methanogen integration vector 54 pIJA03-aroD pIJA03 with the upstream and downstream regions of the aroD gene This work pIJA03-iorA2 pIJA03 with the upstream and downstream regions of the iorA2 gene pMEV2 Neomycin shuttle vector 32 pMEV2-iorAB2 pMEV2 with iorAB2 genes This work - TABLE 2.
Indolepyruvate oxidoreductase specific activities of the wild-type S2 and mutant S122 and S155 strainsa
Strain Genotype Sp act (mU mg−1) with: Phenylpyruvate p-Hydroxyphenylpyruvate Indole-3-pyruvate S2 Wild type 162 ± 33 101 ± 31 43 ± 12 S122 ΔiorA2::pac 39 ± 5 26 ± 7 25 ± 10 S155b ΔiorA2::pac/pMEV2-iorAB2 519 ± 44 381 ± 10 88 ± 2 ↵ a Except as noted, specific activities were determined in triplicate from four independent cultures grown in McNA medium. The concentrations of the substrates phenylpyruvate and p-hydroxyphenylpyruvate were 1 mM. The concentration of the substrate indolepyruvate was 0.5 mM. Means and one standard deviation are reported.
↵ b Specific activities were determined in duplicate. The two additional complementation strains S151 and S153 gave results similar to these with S155.
