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Bacteriochlorophyll Biosynthesis in Rhodopseudomonas Capsulata in Response to Oxygen

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Bacteriochlorophyll Biosynthesis in Rhodopseudomonas Capsulata in Response to Oxygen JOURNAL OF BACTERIOLOGY, Nov. 1983, p. 686-694 Vol. 156, No. 2 0021-9193/83/110686-09$02.00/0 Copyright © 1983, American Society for Microbiology Transcriptional Regulation of Several Genes for Bacteriochlorophyll Biosynthesis in Rhodopseudomonas capsulata in Response to Oxygen ALAN J. BIELt AND BARRY L. MARRSt* E. A. Doisy Department ofBiochemistry, Saint Louis University School of Medicine, St. Louis, Missouri 63104 Received 27 June 1983/Accepted 23 August 1983 Although it has been shown that bacteriochlorophyll synthesis in Rhodopseudo- monas capsulata is repressed by oxygen and high light intensity, few details of regulation by these environmental factors are known, primarily owing to a lack of assays for the biosynthetic enzymes. We have examined regulation at the transcriptional level by isolating and studying fusions between the Mu dl(Apr lac) phage and various bch genes. In these strains, the lacZ gene of the phage is under the control of bch gene promoters. We have found that atmospheric oxygen tension (20% 02) reduces the expression of these fusions at least twofold compared with low oxygen tension (2% 02). Therefore, transcription of the bchA, bchB, bchC, bchG, and bchH genes is regulated in response to oxygen. In Rhodopseudomonas capsulata, the biosyn- by whole-cell suspensions of Rhodopseudo- thesis of protoheme and bacteriochlorophyll fol- monas spheroides. This conversion only oc- lows a common pathway from the condensation curred when the cells were grown under low of succinyl coenzyme A with glycine to the oxygen tension. However, further work was formation of protoporphyrin IX. Biosynthesis of hampered by the inability of cell extracts to protoheme and bacteriochlorophyll proceeds catalyze the chelation of magnesium. from protoporphyrin IX by the chelation of iron To elucidate the mechanism by which bacte- and magnesium, respectively. The pathway riochlorophyll synthesis is regulated in response from magnesium chelation to formation of bacte- to oxygen, it is necessary to be able to measure riochlorophyll is depicted in Fig. 1. The work of the activity of each of the genes coding for Cohen-Bazire et al. (6) was instrumental in dem- bacteriochlorophyll biosynthetic enzymes. Be- onstrating that oxygen represses bacteriochloro- cause of the lack of assays for these enzymes, phyll synthesis in purple nonsulfur photosyn- we have used an alternative method for measur- thetic bacteria. However, study of the details of ing the transcription of the biosynthetic genes. regulation by oxygen has proven difficult. One This report details the use of the Mu dl(Apr lac) of the main reasons for this is the lack of assays phage developed by Casadaban and Cohen (5) to for most of the enzymes involved in the conver- create fusions between the lacZ (f3-galacto- sion of protoporphyrin IX to bacteriochloro- sidase) gene and various bch genes. By placing phyll. One enzyme of the common pathway that the lacZ gene under the control of various bch has been studied in some detail is 8-aminolevu- gene promoters, the activity of these promoters linate synthase. This enzyme appears to be under different culture conditions can be deter- repressed in highly aerated cultures (10). The mined by measuring the amount of P-galacto- only other enzyme whose activity can be direct- sidase formed. Using these fusions, the regula- ly determined is magnesium protoporphyrin tion of many of the bch gene promoters in methyl transferase. Synthesis of this enzyme response to oxygen and light has been deter- also appears to be repressed by oxygen (10). mined. We have found that growth in a low- Gorchein (7, 8) was able to demonstrate the oxygen environment increased the expression conversion of exogenous protoporphyrin IX to from the bch gene promoters two- to fourfold. magnesium protoporphyrin monomethyl ester MATERIALS AND METHODS t Present address: Department of Biology, Tulane Universi- Bacterial strains and plasmids. Bacterial strains used ty, New Orleans, LA 70118. in this work are described in Table 1. pRPS404 is a t Present address: Corporate Research Science Labora- derivative of RP1 containing all of the known bch and tories, Exxon Research and Engineering Co., Annandale, NJ crt genes. The plasmid carries a gene for kanamycin 08801. resistance and the crtD223 allele. The genes for ampi- 686 VOL. 156, 1983 REGULATION OF R. CAPSULATA bch GENES BY OXYGEN 687 Mg2+, SAM Mg-2,4-divinyl Protoporphyrin IX - * - Mg Protoporphyrdn - pheoporphyrdn as 2+ monomethyl ester bchE monomethyl ester + Fe bchD, bchH (P590) (P631) Protohem bchB + 2H bchF 2-Desecetyl.2-vinyl bchA + H 0 bacteriochlorophyllide a*> +2H +2H ' eP730) Chlorophyllide a *- - Mg2vnyl,4-ethyl 2.Descetyl-2-hydroxyethyl + H20 (P665) pheoporphyrin a,, bactertochlorophyllde a b20/ monomethyl ester (P720) ' +2H bchF N 2-Devinyl,2-hydroxyethyl -2H bchC bchA chlorophyllide a (P662) + phytolp Bacterlochlorophylllde a Becteriochlorophyll (P770) bchG FIG. 1. Bacteriochlorophyll biosynthetic pathway (after references 15 and 16). The order of reactions has been inferred from consideration of the structures of accumulated intermediates. The red-most spectral peaks of the intermediates are indicated in parentheses. Genes have been positioned along the pathway on the basis of the bacteriochlorophyll intermediates accumulated in strains with lesions in those genes, but unambiguous assignments await further enzymological and genetic studies. SAM, S-Adenosylmethionine. cillin and tetracycline resistance have been inactivated extracted with acetone-methanol (7:2) as described by amber mutations (11, 13). previously by Cohen-Bazire et al. (6). The rest of the Media. R. capsulata was routinely grown either in a culture was harvested and suspended in 4 ml of Z- malate-minimal salts medium (RCV) (19) or in 0.3% buffer (12). The cells, at 0°C, were disrupted by two Difco Bacto-Peptone-0.3% Difco yeast extract (PYE). 20-s bursts of sonication in a model 140 Branson Escherichia coli was grown in minimal salts medium Sonifier. The suspension was centrifuged for 20 min at Al (12) modified by the omission of sodium citrate and 27,000 x g. Absorbances of the acetone-methanol thiamine. The carbon source included in this medium extract, the sonic extract, and the culture fluid were was either 0.2% glucose or 0.2% sorbitol. L-broth (2) recorded from 300 to 900 nm with a Beckman DU7 modified by omitting glucose and decreasing the sodi- spectrophotometer and compared with spectra ob- um chloride to 0.5% was used as a rich medium. tained from strains carrying known bch lesions. Antibiotics, when used, were added to yield the fol- Identification of porphyrins accumulated by bchD, lowing final concentrations (,ug/ml): ampicillin, 25; bchE, and bchH strains. Strains containing mutations kanamycin, 10; streptomycin, 75; rifampin, 75. When in bchD, bchE, and bchH were grown in 200 ml of solid media were employed, 1.5% agar (Difco) was RCV+ for 2 to 3 days without shaking and extracted added to the above media. Soft agar overlays con- with 10 ml of acetone-methanol as described above. tained 0.7% agar. 5-Bromo-4-chloro-3-indoyl-,-D-ga- The emission spectra of these extracts, along with lactopyranoside (X-gal), used to determine the pres- protoporphyrin IX, magnesium protoporphyrin IX, ence of P-galactosidase activity in colonies, was in- and protoporphyrin monomethyl ester standards (Por- cluded in agar-containing media at a concentration of phyrin Products), were recorded by a Perkin-Elmer 40 ,ug/ml. Oxygen tensions, where indicated, were 512 fluorescence spectrophotometer with the excita- measured polarimetrically in the liquid phase of each tion wavelength set at 402 nm. The extracted porphy- culture with a Clark-type oxygen electrode (YSI model rins and standards were also characterized by chroma- 53). tography on Silica Gel G TLC plates (Fisher). The Mating procedures. Matings between E. coli donors plates were developed in benzene-ethyl acetate-etha- and R. capsulata recipients were as described previ- nol (4:1:1) and viewed under long-wave UV light while ously by Marrs (11). Matings between E. coli donors still wet. and recipients were accomplished by the liquid mating Determination of j-galactosidase activity in bch::Mu technique of Barth (1). d insertion strains. Overnight PYE broth cultures of Induction of Mu dl(Apr lac) phage. Mu dl(Apr lac) the strains to be assayed were used to inoculate 10 ml lysates were obtained by thermal induction of strain of PYE in culture tubes (16 by 150 mm) to a Klett (red MAL103 as described previously by Casadaban and filter) value of 25. The tubes were fitted with rubber Cohen (5). septa, each containing a glass tube as a gas inlet and a Identification of precursors accumulated by Mu d syringe needle as a gas outlet. The cultures were insertion strains. Mu d insertion strains were grown for incubated with appropriate lighting and sparged with 2 to 3 days at 30°C in the dark without shaking in 30 ml different gases as described below. The incubation was of RCV containing 0.6% glucose, 0.5% pyruvate, and stopped when the cultures reached a density of ca. 100 0.5 M dimethyl sulfoxide (RCV+) to obtain low oxy- Klett units, and the dissolved oxygen concentration of gen tensions. Cultures were grown under high oxygen each culture was determined with a YSI model 53 tension by inoculating 30 ml of RCV+ in a 1-liter flask dissolved oxygen monitor. Eight milliliters of culture with the appropriate strain and incubating it for 1 day were harvested by centrifugation at 27,000 x g for 10 with vigorous
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