Article Figures & Data
Figures
- FIG. 1.
(A) Biphenyl catabolic pathway of Rhodococcus sp. strain RHA1. Compounds: I, biphenyl; II, benzoate; III, HPD; IV, 4-hydroxy-2-oxovalerate; V, acetaldehyde; VI, pyruvate; VII, acetyl-CoA. Enzymes: BphA, biphenyl dioxygenase complex; BphB, dihydrodiol dehydrogenase; BphC, 2,3-dihydroxybiphenyl dioxygenase; BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase; BphE1, HPDH; BphF1, HOVA; BphG, AADH. (B) Restriction map and subclones of the 5.7-kb ApaI fragment carrying the bphRGF1E1 genes. The arrows indicate the bphR, bphG, bphF1, and bphE1 ORFs. The segment included in each subclone plasmid is indicated by a horizontal bar. The BphE1 (HPDH), BphF1 (HOVA), and BphG (AADH) activities of subclones are presented on the right. +, present; −, absent; NT, not tested.
- FIG. 2.
Agarose gel electrophoresis of RT-PCR products across the boundaries of bphGF1E1 genes. RHA1 cells were grown on biphenyl (BP) or ethylbenzene (ETB) or in LB medium. Molecular size markers are in lanes M. The sizes of the marker fragments are presented on the left. The carbon sources used are shown above the lane numbers. The boundaries indicated on the top were amplified using the primer sequences described in Materials and Methods. No detectable products were obtained in control reactions with each pair of primers from which RT had been omitted (lanes 1, 3, 5, 7, 9, and 11) or in reactions performed with LB medium-grown cells (lanes 2 and 8).
- FIG. 3.
Identification of the 5′ end of the RHA1 bphGF1E1 transcript. (A) Nucleotide sequence obtained with cloned bphG and fluorescent bphG primer. (B) Primer extension product obtained by using RNA from biphenyl-grown RHA1 cells as a template and the bphG primer. The retention time of the product is indicated. (C) Nucleotide sequence of the upstream region of bphG. The vertical arrow indicates the transcription start point estimated from panels A and B. The horizontal arrow indicates the position of the bphG primer (boxed). The deduced ribosome-binding site (RBS) for bphG is enclosed in a box. The stop codon of bphR and the start codon of bphG are underlined.
- FIG. 4.
Growth of bphGF1E1 disruption mutants on ethylbenzene. The strains RDE1, RDF1, and RDG1, which are disruption mutants of bphE1 (A), bphF1 (B), and bphG (C), respectively, and their derivatives, RDE1/pK4Etsr, RDF1/pK4Ftsr, and RDG1/pK4Gtsr, which have plasmids containing the respective intact genes, together with RHA1 were grown in W minimal medium at 30°C on ethylbenzene.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s) Source or reference Strains Rhodococcus RHA1 Wild type; Bph+ 18 RDE1 Mutant derivative of RHA1; aphII gene insertion mutant of bphE1; Kmr This study RDF1 Mutant derivative of RHA1; aphII gene insertion mutant of bphF1; Kmr This study RDG1 Mutant derivative of RHA1; aphII gene insertion mutant of bphG; Kmr This study R. erythropolis IAM1399 Wild type; Bph− IAM1399 culture collection Plasmids pUC18, pUC19, pUC119 Cloning vector; Apr Takara Shuzo pBluescript II SK(+) Cloning vector, Apr Stratagene pBluescript II KS(+) Cloning vector, Apr Stratagene pMS21 pBluescript II KS(+) with 5.7-kb ApaI fragment carrying bphRGF1E1 This study pMS211 pBluescript II SK(+) with 4.2-kb PvuII-ApaI fragment carrying bphGF1E1 This study pT7-blue TA-cloning vector; Apr Novagen pK4 Rhodococcus-E. coli shuttle vector, Kmr 11 pK4HKcos pK4 containing cos region This study pK4E pK4 with 1.7-kb SphI-BamHI fragment of RHA1 carrying bphE1 This study pK4F pK4 with 1.8-kb EcoRI-SspI fragment of RHA1 carrying bphF1 This study pK4G pK4 with 1.4-kb PvuII-SacII fragment of RHA1 carrying bphG This study pK4GF pK4 with 2.5-kb PvuII-SspI fragment of RHA1 carrying bphGF1 This study pK4GFE pK4 with 4.2-kb PvuII-ApaI fragment of RHA1 carrying bphGF1E1 This study pK4RGFE pK4 with 5.7-kb ApaI fragment of RHA1 carrying bphRGF1E1 This study pUCKmD pUC19 with 790-bp aphII gene fragment; Kmr This study pDE1 pUCKmD containing a 430-bp BssHII bphE1 internal fragment This study pDF1 pUCKmD containing a 600-bp SacII bphF1 internal fragment This study pDG1 pUCKmD containing a 450-bp KpnI-EcoRI bphG internal fragment This study pKH402KF pUC119 with 1.1-kb KpnI fragment of KKS102 carrying bphE 10 pBsRG6 Cloning vector; Apr Tsr R. van der Geize pUJ1-tsr pUC18 with insertion of tsr gene from pBsRG6; Apr Tsr This study pK4Etsr pK4E with insertion of tsr gene from pUJI-tsr; complements the bphE1 mutant This study pK4Ftsr pK4F with insertion of tsr gene from pUJI-tsr; complements the bphF1 mutant This study pK4Gtsr pK4G with insertion of tsr gene from pUJI-tsr; complements the bphG mutant This study - TABLE 2.
Characteristics of RHA1 bph gene products
ORF Sizea Similar protein (% identity) Accession no. 1 228 P. pentosaceus negative transcriptional regulator PadR (27%) AJ276891 2 299 M. tuberculosis H37Rv hypothetical protein (61%) Z82098 Pseudomonas sp. strain CF600 AADH DmpF (53%) X60835 3 359 M. tuberculosis H37Rv hypothetical protein (50%) Z82098 Pseudomonas sp. strain CF600 4-hydroxy-2-oxovalerate aldolase DmpG (45%) X60835 4 268 M. tuberculosis H37Rv hypothetical protein (60%) Z82098 Pseudomonas sp. strain CF600 2-hydroxypenta-2,4-dienoate hydratase DmpE (37%) X60835 ↵ a Number of amino acid residues.
