construction tnphoagene fusions in rhodobacter isolation … · construction of tnphoa gene fusions...

JOURNAL OF BACTERIOLOGY, Aug. 1989, p. 4385-4394 Vol. 171, No. 80021-9193/89/084385-10$02.00/0Copyright © 1989, American Society for Microbiology

Construction of TnphoA Gene Fusions in Rhodobacter sphaeroides:Isolation and Characterization of a Respiratory Mutant Unable ToUtilize Dimethyl Sulfoxide as a Terminal Electron Acceptor during

Anaerobic Growth in the Dark on GlucoseMARK D. MOORE AND SAMUEL KAPLAN*

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received 2 February 1989/Accepted 15 May 1989

We have constructed a suicide vector, pUI800, containing the transposable element TnphoA (TnSISSOL::phoA), for the purpose of producing protein fusions in vivo between the Escherichia coli alkalinephosphatase (APase) and proteins of the facultative photoheterotroph, Rhodobacter sphaeroides. We introducedTnphoA into the genome of R. sphaeroides at a coupled conjugation-transposition frequency of approximately1 x 10-6. Fusions giving rise to APase expression, as judged by blue-colony pigmentation when exconjugantswere plated on growth medium containing the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate,were observed in about 1% of the exconjugants. Numerous, distinguishable mutant phenotypes have beengenerated by this method, including those which lack the ability to use dimethyl sulfoxide as a terminal electronacceptor during anaerobic respiration, as well as those which are photosynthetically incompetent or altered inpigment synthesis, and others that express resistance to chlorate. The growth and spectral characteristics ofseveral of these mutants, as well as the localization and quantitation of subcellular APase activity underdifferent physiological conditions, have been examined. The presence of TnphoA in the host genome has beenconfirmed for each mutant analyzed, and specifically tagged DNA fragments containing TnphoA have beenidentified and localized; cosmids containing R. sphaeroides genomic DNA capable of complementing individualmutants have also been isolated. The usefulness of this approach in studying gene activity in R. sphaeroides isdiscussed.

Rhodobacter sphaeroides is a purple, nonsulfur, faculta-tive photoheterotrophic bacterium which is useful, as amodel system, for the study of membrane biogenesis, bio-energetics, and photosynthesis. This organism is capable ofchemoheterotrophic growth under aerobic conditions byusing a variety of organic substrates; under anaerobic con-ditions in the light, cyclic electron flow provides the ener-getic capability for either photoautotrophic or photohetero-trophic growth (9, 21).

R. sphaeroides is also capable of anaerobic growth onglucose in the absence of light, provided that an alternateelectron acceptor, such as trimethylamine N-oxide (TMAO)or dimethyl sulfoxide (DMSO), is present in the growthmedium (12). The ability to use DMSO and TMAO asterminal electron acceptors has been observed in the relatedorganism Rhodobacter capsulatus (22, 44), from whichMcEwan and co-workers have identified a single periplasmi-cally localized oxidoreductase as the terminal component ofthis anaerobic respiratory pathway (29). Recent studies haveconfirmed the presence of a periplasmic enzyme reducingDMSO in R. sphaeroides forma sp. denitrificans (31), whileseparate reductases utilizing either DMSO or TMAO havebeen isolated in Escherichia coli (34, 42), Salmonella typhi-murium (19), and Proteius vulgaris (38). Despite the exist-ence of structural information concerning the DMSO reduc-tase from R. capsiulatius and R. sphaeroides forma sp.denitrificans, very little is known about the gene(s) whichencodes the enzyme system, how DMSO reductase activityis regulated, or what factors govern its expression in vivo.

* Corresponding author.

Only recently has a gene encoding DMSO reductase beenidentified in E. coli (5) and a mutant in the DMSO-TMAOreductase of R. capsulatius been isolated by Tn5 mutagenesis(17).To address these and other questions in R. sphaeroides, as

well as to extend the utility of the TnphoA (TnS IS50L::phoA) gene fusion system, we have used protein fusions tothe E. coli alkaline phosphatase (APase) (EC 3.1.3.1) as amethod of generating and localizing mutants in compartmen-talized proteins in R. sphaeroides. Developed by Hoffmanand Wright (15), fusions to APase were first used to study themechanisms of protein secretion. By this method, they wereable to generate APase fusions to a variety of cloned genesby in vitro manipulation.More recently, Manoil and Beckwith (24) extended the use

of APase to the isolation of protein fusions in vivo bydevelopment of a transposon-mediated fusion system. Theyconstructed a hybrid transposon, TnphoA, containing the E.coli phoA gene (minus its Shine-Dalgarno sequence, as wellas the coding region for the signal peptide and five additionalamino-terminal amino acid residues) ('phoA) cloned into theleft insertion sequence of TnS. By using this system, theywere able to identify APase-active fusions to both periplasm-and membrane-localized proteins in E. coli.The development of TnphoA led to the possibility that we

could exploit APase fusions to generate mutants in R.sphaeroides which would permit us to examine the complex-ities of the induction, regulation, and enzymology, as well asprotein structure in pathways such as anaerobic respirationand photosynthesis. We report here the first evidence thatTnphoA is useful for generating mutants in key compartmen-talized proteins of R. sphaeroides and present data which

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4386 MOORE AND KAPLAN

TABLE 1. Bacterial strains and plasmid vectors

Strain or plasmid Relevant genotype-phenotype" Source Reference

StrainE. coliTB1 hsdR hsdM+ derivative of JM83 T. Baldwin 3S17-1 C600::RP-4 2-Tc::Mu-Km::Tn7 hsdR hsdM+ recA thi pro A. Puhler 35CC118 A(ara leu)7697 AIacXJ74 phoAA20 galE galK thi argE(Am) araDI39 rpoB recAl J. Beckwith 24

rpsECC202 CC118(F42lacI3 zzf-2::TnphoA) J. Beckwith 24

R. sphaeroides 2.4.1 Wild type W. Sistrom 40

PlasmidspSUP203 Apr Tcr Cmr mob A. Puhler 35pUI800 15.6-kb Tcr Cmr Kmr pSUP203 derivative containing TnphoA in-frame in bla gene This studypUI8738 Tcr pLA2917 containing 23-kb insert of R. sphaeroides DNA complementing S. Dryden This study

MM1004

" Apr, Cmr, Kmr, and Tcr denote resistance to ampicillin, chloramphenicol, kanamycin, and tetracycline, respectively; mob, mobilization genes from RP4.

support its application to the study of gene activity in vivo inthis organism.(A preliminary report of this work has been presented

[M. D. Moore and S. Kaplan, Abstr. Annu. Meet. Am. Soc.Microbiol. 1988, H-33, p. 150].)

MATERIALS AND METHODS

Bacterial strains, plasmid vectors, growth conditions, andmedia. Bacterial strains and plasmid vectors are described inTable 1. Cultures of R. sphaeroides 2.4.1 were grown inSistrom minimal medium A (SMM) containing succinate, aspreviously described (9, 14); strains of E. coli were grownaerobically in either glucose-M9 minimal medium or LBbroth (23). In the case of strain CC202, LB broth wassupplemented with 0.2% lactose (wt/vol); low-phosphateSMM was prepared as previously described (9). Whennecessary, antibiotics were added to the following finalconcentrations: ampicillin, 25 ,ug/ml; tetracycline, 20 jig/mlin E. coli and 1 pLg/ml in R. sphaeroides; and kanamycin, 25,ug/ml.

Conditions for anaerobic growth in the dark of R.sphaeroides on SMM containing glucose and DMSO, pho-toheterotrophic light intensities, and culture conditions havebeen reported by Donohue et al. (11). Anaerobic growth onSMM containing glucose and TMAO was accomplished bythe substitution of 30 mM TMAO for DMSO in this medium(11). Resistance to chlorate (Chl') was assessed by theaddition of 100 mM KCl03 to SMM.

Conjugal matings. Conjugal matings between E. coli andR. sphaeroides were performed essentially as described byDavis et al. (8), with the following modifications: E. coli andR. sphaeroides were used at cell densities of 6.5 x 108 cellsper ml and 1.4 x 109 cells per ml, respectively, at thedonor-to-recipient ratio of 1:20. Conjugation plates wereincubated at 32°C for 6 h to facilitate mating, with filtersbeing kept moist by the addition of 5 to 15 ,ul of sterile SMMat 1- to 2-h intervals. Exconjugants were spread onto SMMagar plates containing kanamycin and were incubated aero-bically for 2 to 5 days at 32°C.

Screening of exconjugants for APase activity and sensitivityto RS1 bacteriophage. APase activity in vivo was detected byusing the p-toluidine salt of the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate (X-P), which was pre-pared as a 40-mg/ml stock in dimethylformamide and addedto agar medium to a final concentration of 40 jig/ml (24). R.sphaeroides-specific RS1 phage was prepared essentially by

the method of Abeliovich and Kaplan (1) and was used toscreen exconjugants for sensitivity by the method of Dono-hue et al. (10).Complementation of mutants by using an R. sphaeroides

genomic cosmid library. A genomic DNA library of R.sphaeroides 2.4.1 (S. C. Dryden and S. Kaplan, unpublishedresults), with the cosmid vector pLA2917 (2), was screenedfor trans complementation of the TnphoA-derived mutants.Strains of E. coli S17-1 containing individual cosmids werecultured in LB containing tetracycline as 150-pAl samples in96-well microdilution dishes (Falcon 3070 MicroTest IIIplates; Becton Dickinson Labware, Oxnard, Calif.) to ap-proximately 6.0 x 108 cells per ml and were then inoculatedonto freshly prepared LB agar plates. Recipient cultures ofR. sphaeroides were cultured similarly and, at a cell densityof approximately 5 x 108 cells per ml, were inoculated ontothe agar plates previously inoculated with donor cells. Cellswere mixed, and plates were incubated at 32°C for 6 h. Afterconjugation, cells were transferred to a selective mediumcontaining tetracycline and kanamycin. Individual cosmidclones in E. coli S17-1 were stored at -72°C in LB contain-ing sterile glycerol (20%, vol/vol).

Isolation of nucleic acids and recombinant DNA techniques.Highly purified plasmid DNA was isolated from chloram-phenicol-amplified cultures of E. coli TB1 grown in glucose-M9 minimal medium containing 0.2% (wt/vol) CasaminoAcids (Difco Laboratories, Detroit, Mich.). Triton X-100lysates were prepared essentially as described previously(25); CsCI-ethidium bromide gradients were centrifuged byusing Quick-Seal polyallomer centrifuge tubes (BeckmanInstruments, Inc., Palo Alto, Calif.) at 20°C in a Beckman70.1Ti rotor with a Sorvall RC80 ultracentrifuge (DuPontCompany Biotechnology Systems, Wilmington, Del.) in stepmode (16 h; 220,000 x g) and then by relaxation of thegradient (1 h; 110,000 x g). Covalently closed circular DNAwas collected from the gradient, extracted with isopentylalcohol, and dialyzed as previously described (23). Amplifi-cation of plasmid DNA in chloramphenicol-resistant strainswas accomplished by the addition of spectinomycin (300pg/ml). Small-scale plasmid preparation was performed aspreviously described (6).DNA restriction fragments were separated by gel electro-

phoresis (30, 37) and were sized relative to restrictionenzyme digests of bacteriophage X cI ts857 on agarose gelsor pBR322 on polyacrylamide gels (37). E. coli strains weretransformed with plasmid DNA by the CaCl2 heat shock

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CONSTRUCTION OF TnphoA GENE FUSIONS IN R. SPHAEROIDES 4387

method (7). Competent cells were stored in 0.1 M CaCl2 insterile glycerol (20%, vol/vol) at -72°C for at least 6 monthswithout significant loss of viability or competence. Restric-tion fragments were purified directly from agarose gel slicesby centrifugation (10 min; 12,000 x g) through a 0.22-p.mcellulose acetate Spin-X membrane filtration unit (Costar,Cambridge, Mass.). Radioactively labeled restriction frag-ment probes were prepared by using [_x-32P]dCTP and were

used in Southern hybridization analyses as described previ-ously (11).

Southern hybridization analysis of genomic DNA. Intactchromosomal DNA from R. sphaeroides was isolated as

described previously (33) and was analyzed by transverse,alternation field electrophoresis (TAFE) on 1.0% agarose

gels at 12°C by using a GeneLine Electrophoresis System(Beckman Instruments, Inc.) as described elsewhere (A.Suwanto and S. Kaplan, manuscript submitted). Concate-meric A cI ts857 DNA, used as a molecular weight ladder,was prepared as described previously (41), except that thefinal buffer used for reconstitution of the DNA contained0.30 M NaCl and 0.03 M sodium citrate (2x SSC [lx SSC is0.15 M NaCl plus 0.015 M sodium citrate]) (Suwanto andKaplan, manuscript submitted).

Protein purification. Anion-exchange column chromato-graphic separation of periplasmic proteins with DEAE-Sepharose CL-6B (Pharmacia LKB Biotechnology, Inc.,Piscataway, N.J.) was performed at 4°C by the method ofMcEwan et al. (29). Conductivity in each fraction was

measured with a Radiometer-Copenhagen conductivitymeter, model CDM-2E. The protease inhibitors N-cL-p-tosyl-L-lysine chloromethyl ketone and N-tosyl-L-phenylalaninechloromethyl ketone, freshly prepared in dimethylforma-mide, were added to cell extracts to final concentrations of1.0 mM each prior to chromatography.

Cell fractionation and enzyme assays. Subcellular fractionswere prepared by the method of Weiss (43), as used by Taiand Kaplan (39). The protease inhibitor phenylmethylsulfo-nyl fluoride, freshly prepared in absolute isopropanol, was

added to cell extracts at a final concentration of 0.5 mM.Succinate dehydrogenase (EC 1.3.99.1) and malate dehydro-genase (EC 1.1.1.37) were used as membrane and cytoplas-mic markers, respectively, and were assayed according tothe methods of Markwell and Lascelles (26); their methodwas also used to assay APase. Cytochrome c2 was assayedas previously described (11). DMSO reductase was assayedby the reduced methyl viologen (MVH) method described byMcEwan et al. (28). Protein content was determined by theMarkwell sodium dodecyl sulfate modification of the Lowrymethod (20, 27), with bovine serum albumin as the standard.Western immunoblot analysis. Protein fractions were elec-

trophoresed on 10% sodium dodecyl sulfate-polyacrylamidegels and were transferred to nitrocellulose filters (pore size,0.20 ,um) at 4°C by electrophoresis (10 V, 12 h) (18).Immunoblotting was accomplished with a ProtoBlot AP kit(Promega Biotec, Madison, Wis.), with rabbit anti-E. coliAPase as the primary antibody and goat anti-rabbit immu-noglobulin G conjugated to bovine APase as the secondaryantibody. An APase-active column chromatographic frac-tion of E. coli periplasmic proteins was obtained from SigmaChemical Co. (St. Louis, Mo.).

Materials. Restriction endonucleases and nucleic acid-modifying enzymes were purchased from Bethesda Re-search Laboratories Life Technologies, Inc. (Gaithersburg,Md.) or New England BioLabs, Inc. (Beverly, Mass.).DNase, the Klenow fragment of DNA polymerase, oxalac-etate, and RNase A were the products of Boehringer-

Mannheim Biochemicals (Indianapolis, Ind.). [ox-32P]dCTP(800 Ci/mmol) was obtained from Amersham Corporation(Arlington Heights, Ill.). Nitrocellulose (0.45 urm and 0.20p.m) used for Southern hybridization and Western immuno-blot analyses, respectively, was from Schleicher & Schuell,Inc. (Keene, N.H.). Calbiochem bovine APase-conjugated,goat anti-rabbit immunoglobulin G was obtained from Be-hring Diagnostics (La Jolla, Calif.). All antibiotics, 2,6-dichlorobenzenone-indophenol, methyl viologen, NADH,Nitro Blue Tetrazolium, p-nitrophenyl phosphate, phenazinemethosulfate, phenol red, phenylmethylsulfonyl fluoride,N-co-p-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-pheny-lalanine chloromethyl ketone, and X-P were obtained fromSigma Chemical Co. With the exception of phenol (J. T.Baker Chemical Co., Phillipsburg, N.J.), which was distilledbefore use, all other chemicals were of reagent grade purityand were used without further purification.

RESULTS

Construction of pSUP203::TnphoA. To introduce TnphloAinto R. sphaeroides, we used the methods of Manoil andBeckwith (24) to generate transpositions of TnphoA onto amulticopy suicide vector, pSUP203 (35). TnphoA was intro-duced into S17-1(pSUP203) (35) via a diparental mating withE. coli CC202 (24). Exconjugants were selected by platingonto glucose-M9 minimal medium containing thiamine, pro-line, trimethoprim (50 ,ug/ml), tetracycline, and kanamycin(300 Rg/ml), with the high concentration of kanamycin usedto enrich for transpositions of TnphoA onto the multicopyvector, pSUP203. Exconjugants were replica plated ontoampicillin-containing medium to test for inactivation of thebla gene. Colonies failing to grow on ampicillin-containingmedium were cultured in LB broth, and small-scale plasmidpreparations were performed. One pSUP203 derivative con-taining an in-frame bla-'phoA fusion was designated pUI800.Comparison to the published restriction maps of TnS (16)and pSUP203 (35) and detailed restriction endonucleasemapping of pUI800 confirmed its size and the location ofTnphoA in the bla gene (Fig. 1).pUI800 was able to confer APase activity on E. coli CC118

(24) after transformation and plating on agar medium con-taining the chromogenic indicator X-P. Transformants weresubsequently shown to possess periplasmically localizedAPase activity in vitro (data not shown).

Demonstration of endogenous phosphatase activity in R.sphaeroides. Although no APase activity was detected in R.sphaeroides in situ, under the conditions of in vitro APaseassay, a slight, but reproducible, endogenous activity wasdetected in the wild-type strain. This activity was primarilylocalized to the periplasmic fraction (0.89 U of protein permg), although APase activity was also observed in themembrane fraction as well (0.73 U of protein per mg). Thelevel of phosphatase activity was similar for cells grownphotoheterotrophically at 10 W/m2 (data not shown). For thedetermination of TnphoA-specific APase activity, the endog-enous activity was subtracted from the corresponding sub-cellular fraction for each of the mutants.

Introduction of TnphoA into R. sphaeroides 2.4.1 by conju-gal mating with E. coli S17-1(pUI800). After the mating of E.coli S17-1(pUI800) and R. sphaeroides 2.4.1, we recovered atotal of 3,112 Kmr exconjugants, at a coupled conjugation-transposition frequency of 1.1 x 10-6 (Table 2). Of these, 31(approximately 1%) showed visible X-P hydrolysis. Thisfrequency is similar to results obtained for TnphoA muta-genesis in E. coli (J. Beckwith, personal communication). A

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FIG. 1. Restriction map of the 15.6-kb suicide vector pUI800containing a single transposition of TnphoA into the Apr gene ofpSUP203, which resulted in an in-frame bla-'phoA fusion. Therestriction sites of the chimeric plasmid were identified in previousmaps of pSUP203, TnphoA, and Tn5 (16, 24, 35). They wereconfirmed by detailed restriction endonuclease mapping of pUI800and were drawn to scale.

total of 66 additional exconjugants, although showing nohydrolysis of X-P, were altered in their pigmentation, sug-gesting an alteration in either carotenoid or bacteriochloro-phyll biosynthesis. Both APase-active and -inactive colonieswere screened for sensitivity to tetracycline and the R.sphaeroides species-specific bacteriophage RS1. A total of 4APase-active and 14 APase-inactive colonies were found tobe RS1Y Tcr and were thus discarded. The 27 APase-activestrains and 5 of the remaining 52 APase-inactive, Kmr TcsRS1s strains (Table 2) were then subjected to biochemical

TABLE 2. Introduction of TnphoA into R. sphaeroides 2.4.1 viaE. coli S17-1(pUI800)a

TotalKMr APase inactiveb

exconjugants (altered APase activeExpt exvpigmentation)

No. i' No. i No. s'

1,554 1.3 x 10-6172 2.8 x 10-7

1,386 1.8 x 10-6

38 3.4 x 10-85 8.1 x 10-8

23 2.9 x 10-8

20 1.5 x io-83 4.9 x 10-98 1.1 x io-8

Summary 3,112 1.1 x 10-6 66 4.8 x 10-8 31 3.1 x 10-8Final" 52 4.0 x 10-8 27 2.6 x 10-8

" Conjugations were performed as described in Materials and Methods.Frequencies are expressed relative to the total number of viable R. sphaelroi-des recipient cells after conjugation.

6 APase active and APase inactive refer to the visually detectable in vivohydrolysis of X-P.

' Represents the final number of APase-active and APase-inactive excon-

jugants which were subsequently shown to be sensitive to RS1 infection andtetracycline.

and physiological characterization, with only a subset ofthese being described in this report.

Identification of genomic transpositions of TnphoA bySouthern hybridization. To characterize the transposition ofTnphoA in the R. sphaeroides mutants, we sought to confirm(i) the presence of the transposon, (ii) the absence of thedelivery vector, and (iii) the uniqueness of each genomictransposition. Southern hybridization of Asel-digested ge-nomic DNA resolved by TAFE (Suwanto and Kaplan,manuscripts submitted) with a TnphoA-specific probe re-vealed transpositions to a number of unique genomic frag-ments, suggesting that TnphoA transposition in R. sphaer-oides was random, consistent with previous observations inE. coli (23). Shown (Fig. 2) is a composite photograph of theTAFE analysis in which 1% agarose gels containing AseI-digested total genomic DNA from seven TnphoA-containingstrains were used. MM1003 contained a single copy ofTnphoA in a 244-kilobase (kb) AseI fragment (Fig. 2A, lane4), while MM1004 contained a transposition of TnphoA to a410-kb AseI fragment (Fig. 2A, lane 5). Transposition ofTnphoA onto the 73-kb AseI fragment was observed forMM1006 (Fig. 2A, lane 6). In a separate analysis, the largestof the two AseI fragments were separated and probed withTnphoA, which resided in the 1,105-kb fragment in MM1005(Fig 2B, lane 1) and in the 910-kb AseI fragment in MM1001(Fig. 2B, lane 2). The absence of a hybridization signal inlanes 3 and 7 suggested that in strains MM1002 and MM1007,TnphoA might reside within one of two endogenous plasmidswhich are not cleaved by AseI (A. Suwanto and S. Kaplan,manuscript submitted). In data not shown, we confirmed thetransposition of TnphoA to separate endogenous plasmids instrains MM1002 and MM1007. A vector-specific 1.5-kbEcoRI-SalI fragment from pUI800 (Fig. 1) detected nohybridization to any of the DNA preparations shown in Fig.2 or to the wild-type strain (data not shown).

Characterization of TnphoA mutants. We examined thegrowth characteristics under both photoheterotrophic andchemoheterotrophic conditions, including generation timeand in situ expression of APase for each of the TnphoA-generated mutants. These results (Table 3) identified threepigment mutants, MM1003, MM1005, and MM1007, amongthe TnphoA-containing strains. Each of these strains showedan increased generation time with respect to the wild-typestrains under both aerobic and photosynthetic growth con-ditions. At limiting-incident light intensity under photosyn-thetic growth conditions, MM1003 exhibited twofold-slowergrowth than 2.4.1 did. While all three of these strains weredefective in normal pigment synthesis, the nature of eachmutation was unique. Although MM1003 and MM1005 ap-peared normal under aerobic growth conditions, they ex-pressed altered pigmentation under photosynthetic growthconditions: the former appeared dark green, and the latterappeared very pale brown. Conversely, while MM1007appeared wild type in its photosynthetic pigmentation, aer-obic colonies of this strain were a very pale pink.

Also identified among the pigment mutants was a photo-synthetically incompetent strain, designated MM1006,which formed pale orange colonies under aerobic growthconditions and had a threefold-longer doubling time than thewild-type strain did. No APase activity was detected by insitu hydrolysis of X-P in this strain (Table 3).

In addition to the recovery of photosynthetically incom-petent and pigment mutants, we identified three strainswhich possessed defects in anaerobic respiratory functions.These strains, described in detail below, hydrolyzed X-Pprimarily during anaerobic growth, in which the colonies

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CONSTRUCTION OF TnphioA GENE FUSIONS IN R. SPHAEROIDES 4389

a:ICac£

A.

rN K It too o O oD__0 0 0E

Z-F 2 E n- M

B.

k b!

~- 41

2J

- 244

l2 3 4 i. 7

FIG. 2. Southern hybridization analysis of chromosomal DNA isolated from the wild-type strain and seven TnphoA-induced mutants. (A)TAFE analysis of genomic DNA digested with Asel by using an 18-h, 23-s pulse separation. Lane 1, Wild-type DNA digested with Asel; lane2, X cI ts857 concatemeric molecular weight ladder. Shown below is the composite autoradiograph of these gels, with a 3.0-kb HindlIl DNAfragment from TnphoA (Fig. 1) as a probe. The corresponding molecular sizes of the positively hybridizing AseI fragments are shown to theright. (B) TAFE analysis by 18-h, 55-s pulse separation to resolve the high-molecular-weight doublet.

appeared intensely dark blue, although pale blue colonieswere also observed during aerobic growth (Table 3). Theminimal expression of APase under aerobic conditions onagar medium could be due to the slightly anaerobic nature ofcells within each colony.

Intracellular localization and quantitation of enzymaticactivity. APase activity in five X-P-hydrolyzing strains wasquantitated and localized (Tables 3 and 4). As controls, thewild-type strains and two of the non-X-P-hydrolyzing mu-tants were also assayed. Subcellular fractionation was mon-

TABLE 3. Characterization of TnphoA-generated mutants and quantitation of APase activity

'phoA-derived APase activityGeneration time (h)

In situ X-P hydrolysis" In vitro total units of APasedStrain Phenotype"

Photosynthetic' Photosynthetic PhotosyntheticAerobic Aerobic Aerobic

100 W/m2 10 W/m2 3 W/m2 10 W/m2 3 W/m2 10 W/m2 3 W/m2

2.4.1 Wild type 3.1 3.0 3.0 12.4 - - - 0 0 0MM1001 Chlr 4.4 3.2 2.9 12.0 + ++++ + + + + 110 890 1,180MM1002 Chlr 5.6 3.1 3.0 11.8 + + + + + + + 470 330 520MM1003 Dark green 4.4 3.4 4.9 22.0 + + + + + 370 190 27MM1004 DMSO- 5.0 4.0 4.0 15.0 + + + + + + + + + 840 4,200 990MM1005 Pale brown 9.0 3.3 3.1 10.5 +++ ++ + 950 500 310MM1006 PS- 9.0 NGe NG NG - NA' NA 11 NA NAMM1007 Light pink 6.0 4.4 4.8 10.5 - - - 2 <2 3

" Colonies of MM1003 and MM1005 appeared dark green and pale brown under photoheterotrophic growth, respectively. MM1007 appeared pale pink underaerobic growth.

" Generation times, except for those of the wild type, were determined in SMM containing succinate and 25 ,ug of kanamycin per ml. Photosynthetic growthwas measured at incident light intensities of 100, 10, and 3 W/m2.

- and +, Relative extent of X-P hydrolysis in situ on the basis of the intensity of the blue-colony phenotype."One unit is the amount required for the hydrolysis of 1 nmol of p-nitrophenyl phosphate per min at 20°C. 'phoA-derived APase activity was calculated by

subtracting wild-type phosphatase activity from the total APase activity measured." NG, No growth.- NA, Not applicable.

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TABLE 4. Subcellular localization of 'PhoA-derived APase activity

Sp act" [U/mg of protein (%)J

Periplasm Membrane' CytoplasmStrain

Photosynthetic Photosynthetic PhotosyntheticAerobic Aerobic Aerobic

10 W/m2 3 W/m2 10 W/m2 3 W/m2 10 W/m2 3 W/m2

MM1001 NDC (<0.1) 58.0 (62.5) 85.0 (80.1) 6.6 (99.9) 5.4 (11.6) 10.0 (12.9) ND (<0.1) 14.0 (25.9) 24.0 (6.9)MM1002 ND (<0.1) 26.4 (94.9) 92.1 (96.3) 17.7 (91.6) 0.21 (4.7) ND (<0.1) 3.1 (8.3) 0.11 (0.3) 4.0 (3.6)MM1003 12.2 (14.0) 3.9 (16.7) ND (<0.1) 35.0 (85.1) 6.7 (79.9) 1.9 (91.7) 0.23 (0.8) 0.31 (3.3) 0.51 (8.2)MM1004 255.0 (99.3) 69.1 (86.3) 128.0 (88.5) 1.6 (0.7) 11.0 (13.4) 7.8 (8.5) ND (<0.1) 0.15 (0.3) 2.0 (2.9)MM1005 89.2 (97.1) 132.2 (94.9) 28.8 (78.8) ND (<0.1) 1.2 (1.2) 3.8 (18.6) 1.8 (2.9) 5.5 (4.0) 5.0 (2.5)MM1006 ND (<0.1) NGd NG ND (<0.1) NG NG 11.0 (99.9) NG NGMM1007 ND (<0.1) ND (<0.1) 0.03 (1.1) 0.13 (99.9) 0.4 (99.9) 0.04 (96.8) ND (<0.1) ND (<0.1) 0.02 (2.0)

"Expressed as units per milligram of total protein, with the level of lysozyme removed from the calculations. Values in parentheses represent percent of totalactivity. Unit definition given in footnote d of Table 3.

b Includes total residual membrane after spheroplast lysis.'ND, None detected.d NG, No growth.

itored by use of malate dehydrogenase, succinate dehydro-genase, and cytochrome c2, which are localized in thecytoplasm, the cytoplasmic membrane, and the periplasm,respectively, as previously described (11, 26). We routinelyachieved greater than 95% localization of each marker to itsrespective subcellular compartment (data not shown). Thedata in Table 3 are expressed in total units of APase for eachstrain, while the data in Table 4 represent specific activity ofAPase in each subcellular fraction. Values in both tablesrepresent only 'phoA-specific APase activity after subtrac-tion of endogenous phosphatase activity.Both MM1001 and MM1002 contained significant APase

activity, the former having almost 2.5-fold-higher totalAPase activity than the latter, on the basis of in vitrohydrolysis of p-nitrophenyl phosphate (Table 3). In bothstrains, APase activity was associated with the membraneunder aerobic conditions and with the periplasmic fractionunder photoheterotrophic conditions (Table 4). Moreover,we observed a twofold increase in specific activity in low-light-grown cells when compared with 10-W/m2-grown cellsin both of these strains (Table 4).MM1005 contained a high level-APase activity; 950 U of

total APase activity was detected under aerobic growthconditions, while 500 and 310 U were detected in photohet-erotrophically grown cells at 10- and 3-W/m2 incident lightintensity, respectively (Table 3). The majority of this activitywas confined to the periplasmic fraction (Table 4), with lessthan 50 U of APase activity being observed in the membranefraction of MM1005 under any growth condition (data notshown). Unlike MM1001 and MM1002, the highest APasespecific activity in MM1005 was obtained under 10-W/m2photosynthetic growth. This represented a fivefold increasein specific activity over that seen in cultures of MM1005grown at 3 W/m2 (Table 4).MM1003 possessed membrane-associated APase activity

and, like MM1005, was most active under aerobic conditions(370 U of total APase). Only 27 U of total APase activity wasobserved under conditions of 3-W/m2 photoheterotrophicgrowth, representing a 15-fold decrease in activity whencompared with expression during aerobic growth (Table 3).The most active APase fusion protein observed was in the

DMSO- mutant MM1004. Over 4,200 U of total APaseactivity was detected in 10-W/m2 photosynthetically growncells; nearly 1,000 U was observed in photosynthetic cellsgrown at 3 W/m2. Subcellular fractionation demonstratedthat greater than 85% of the APase activity in MM1004 was

localized to the periplasmic fraction under all growth condi-tions examined (Table 4).As anticipated, we detected no significant APase activity

in the pigment mutant MM1007, which failed to hydrolyzeX-P in situ (Tables 3 and 4). In contrast was the photosyn-thetically incompetent (PS-) mutant, MM1006, which alsofailed to hydrolyze X-P in situ. By enzymatic analysis wedetected 11 U of APase activity above endogenous phos-phatase level in aerobically grown cells (Table 3). Thisactivity was >99% localized to the cytoplasmic fraction(Table 4).Western immunoblot analysis. To demonstrate the pres-

ence of an APase fusion protein in the TnphoA-derivedmutants, periplasmic (for MM1003, total membrane) pro-teins were separated by sodium dodecyl sulfate-polyacryla-mide gel electrophoresis, transferred to nitrocellulose, andreacted with E. coli APase-specific antibodies, as describedin Materials and Methods. These data (Fig. 3) confirmed thepresence of APase fusion proteins in all of those strainsexhibiting APase activity. Strain MM1001 showed at least

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FIG. 3. Western immunoblot analysis of periplasmic fractions ofthe TnphoA-induced mutants with antibodies raised against E. coliAPase. Goat anti-rabbit immunoglobulin G conjugated to bovineAPase was used for detection. Protein (80 jig) was loaded in eachlane. Lane 8 contains 0.10 ,ug of an APase-active column chromato-graphic fraction of E. coli periplasmic proteins used as a positivecontrol, with the major band at approximately 43 kDa being matureE. coli APase.

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three species of APase fusion proteins present in the peri-plasmic fraction, the largest of which had a molecular massof approximately 85 kilodaltons (kDa) (lane 1). The minorcross-reacting species at 45 kDa and the major species at 43kDa were attributed to degraded forms of the fused proteinsequences, since bands of the same electrophoretic mobilitywere observed in several of the other strains examined (lanes3 through 6), including the periplasmic fraction of E. coliused as a positive control (lane 8). This conclusion was

supported by our observation that an increase in denatur-ation temperature of the protein samples prior to electropho-resis led to the accumulation of the 43-kDa polypeptide anda disappearance of the higher-molecular-weight immunore-active species (unpublished observations).MM1003 contained a membrane-localized APase fusion

protein at 43.4 kDa (Fig. 3, lane 3). An APase fusion proteinwas also observed in the periplasmic fraction of both theDMSO- mutant, MM1004, and the cosmid-complementedstrain, MM1004(plUI8738), at approximately 43.5 kDa (lanes4 and 6). MM1005 contained at least four immunoreactivepolypeptides; the largest was observed at 110 kDa, withthree visible degradation products at 60, 45, and 43 kDa alsovisible (lane 5). MM1007 appeared to contain a single APasefusion protein at 98 kDa (lane 7). No significant cross-

reacting protein species were seen in the periplasm ofwild-type R. sphaeroides (lane 2). E. coli APase was ob-served as having a molecular mass of approximately 43 kDa(lane 8).

In each of the lanes containing R. sphaeroides periplasmicfractions, a very minor cross-reacting protein species wasobserved at a molecular mass of approximately 37 kDa,although the identity of this species is not yet known.

Identification of two mutants expressing ChIr. R. sphaer-oides is unable to grow in a medium containing C103-,presumably due to its reduction to the toxic ion C102- via amechanism as yet unidentified in this organism (A. G.McEwan and S. Kaplan, unpublished results). By plating theAPase-active exconjugants onto SMM containing C103, weidentified two strains, designated MM1001 and MM1002,that expressed Chl' during both aerobic and photohetero-trophic growth (Table 3); both strains were able to grow inSMM containing glucose and DMSO (data not shown). Wedetermined that MM1001 and MM1002 contained mem-brane-localized APase activity under aerobic growth andperiplasmically localized APase activity under anaerobicgrowth (Table 4); the strains also possessed normal levels ofperiplasmically localized DMSO reductase activity (Table5). Southern hybridization analysis, as shown previously,confirmed that these are unique mutations (Fig. 2A, lane 3;Fig. 2B, lane 2). The fact that MM1002 contained a copy ofTnphoA on an endogenous plasmid is discussed later.

Identification and characterization of a DMSO- mutant,MM1004. We screened the 27 APase-active exconjugants byplating onto SMM containing glucose, 0.2% yeast extract,and 30 mM TMAO with 0.1% phenol red added as a pHindicator. TMAO is reduced by the DMSO reductase in R.sphaeroides to yield the basic product trimethylamine. OneAPase-active strain, MM1004, produced acidic by-productsonly; a yellow zone evident around those colonies suggesteda lack of trimethylamine production. As discussed above, byspectrophotometric analysis we demonstrated the presenceof substantial periplasmically localized APase activity in thisstrain (Tables 3 and 4).MM1004 did not grow in SMM containing glucose and

DMSO under anaerobic growth conditions in the dark andshowed negligible growth under similar conditions with

TABLE 5. Subcellular localization of DMSO reductase activity

Sp actb [U/mg of protein (%)J

Strain Subcellular Photosynthetic'fraction" Aerobic10 W/m2 3 W/m2

2.4.1 Periplasm 2.99 (98.3) 10.56 (96.9) 8.65 (96.8)Membrane 0.40 (1.1) 2.11 (2.2) 1.21 (2.9)Cytoplasm 0.21 (0.5) 0.51 (0.8) 0.31 (0.2)

MM1001 Periplasm 2.12 (97.7) 9.20 (94.9) 6.63 (95.7)Membrane 0.81 (2.2) 3.84 (4.2) 2.32 (3.9)Cytoplasm ND' (<0.1) 0.31 (0.8) 0.21 (0.3)

MM1002 Periplasm 3.57 (97.7) 13.87 (97.8) 7.71 (96.6)Membrane 0.81 (2.1) 1.51 (1.5) 1.22 (3.2)Cytoplasm ND (<0.1) 0.11 (0.6) ND (<0.1)

MM1004 Periplasm 0.40 (98.7) 1.20 (98.5) 0.71 (97.2)Membrane 0.31 (1.1) 0.21 (0.4) 0.22 (1.9)Cytoplasm ND (<0.1) 0.10 (1.0) 0.30 (0.8)

"Membrane fraction includes total residual membrane after spheroplastlysis.

/' One unit of activity is the amount required for the oxidation of 1 ,umol ofMVH per min at 20°C. The amount of lysozyme used was subtracted from thecalculations. Values in parentheses represent the percent of total activity ineach fraction.

Values refer to cells grown at either 10 or 3 W/m2 incident light intensity.d ND, None detected.

TMAO (data not shown). Protein fractions from both aerobicand photosynthetic cultures failed to produce significantDMSO-dependent MVH oxidation, although we localizedDMSO reductase activity to the periplasmic fraction of thewild-type strain (Table 5). In the wild-type strain, there wasa three- to fourfold increase in DMSO reductase activity inphotoheterotrophically grown cells when compared withaerobically grown cells, consistent with previous observa-tions in this laboratory (L. A. Cohen, A. G. McEwan, and S.Kaplan, unpublished results).To confirm the absence of DMSO reductase in MM1004,

periplasmic proteins of both mutant and wild-type strainswere analyzed by ion-exchange column chromatography(Fig. 4). Approximately 85% of the periplasmic proteinsbound to the column and less than 8% of the total DMSOreductase activity from the wild-type strain eluted in theflow-through. Fractionation was accomplished with a linear(80 to 400 mM) NaCl gradient (29). In the wild-type strain, asingle peak of DMSO reductase activity was observed elut-ing at a conductivity of approximately 10 mS; no peak ofactivity was identified in the mutant. Likewise, while a singlepeak of APase activity was observed in the mutant (whicheluted at approximately 4 mS), no 'phoA-derived APaseactivity was detected in the wild-type strain.By using a cosmid library of R. sphaeroides 2.4.1 genomic

DNA (S. C. Dryden and S. Kaplan, unpublished results), weisolated a cosmid, designated pUI8738, which restored toMM1004 both anaerobic growth in the dark in SMM con-taining glucose and DMSO and a red zone around colonieson TMAO-phenol red agar medium. Fractionation of theperiplasmic proteins of the complemented strain confirmednot only the presence of an APase-active fusion but also aDMSO-dependent MVH-oxidizing component which elutedat essentially the same conductivity as that for DMSOreductase in the wild-type strain (Fig. 4). Thus, the restora-tion of anaerobic growth in the dark on SMM containingglucose and DMSO in MM1004 by pUI8738 was most likely

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2a0 8800.4e2.4- -~~~NCi.4

1~.5 6 v- 0Q3 )

1s042B 02

0.5-2 -

0 O0C) 00 8 16 24 32 40 48 5

Fraction NumberFIG. 4. Elution profile of the DEAE-Sepharose CL-6B3 ion-exchange chromatographic separation of periplasmic proteins from R.

sphaeroides 2.4.1. Bound proteins were eluted with an 80 to 400 mM linear NaCi gradient (shown as a plot of conductivity [in inS]).Superimposed over the wild-type elution profile are the activities and relative elution peaks for DMSO reductase and APase in the DMSO-mutant MM1004 and the cosmid-complemented strain MM1004(pUI8738) grown under identical conditions. The break in conductivity is dueto titration of EDTA in the sample and the large protein fraction which elutes immediately upon addition of NaCl.

due to the restoration of DMSO reductase activity in theperiplasmic fraction of the complemented strain.As discussed above, we confirmed by Western analysis

(Fig. 3) the presence of an approximately 43.5-kDa APasefusion protein in both MM1004 and the complementedstrain. A fusion protein of that size is consistent withtransposition of TnphoA into the coding region immediatelyfollowing the signal peptide sequence of the R. sphaeroidestarget gene.

DISCUSSION

Protein fusions have played a significant role in the mo-lecular genetic analysis of gene expression and regulation inbacteria (4). Recently, their utility was extended to the studyof the mechanisms of protein compartmentalization andexport by using fusions to the E. coli APase (15). Thedevelopment of TnphoA by Manoil and Beckwith (24) com-bined the facility of TnS mutagenesis with the selectivity ofAPase as a reporter protein to allow the ready isolation ofprotein fusions in vivo in E. coli.By using a suicide delivery vehicle with broad host range

capability, we have further extended the use of TnphoA toproduce protein fusions in vivo to compartmentalized pro-teins in the facultative photoheterotroph R. sphaeroides.Because all major protein components of anaerobic respira-tion and photosynthetic electron transport are either mem-brane or periplasmically localized, the use of TnphoA as atool for gene fusion analysis is both attractive and highlyselective. The transposition of TnphoA in R. sphaeroideswas apparently random, as judged by insertions into bothchromosomal DNA and at least two of the five endogenousplasmids (13, 30).

Colonies giving rise to the blue-colony phenotype wereshown to contain APase activity localized either to themembrane or to the periplasmic fraction. The extent of X-P

hydrolysis in situ (as monitored by the intensity of theblue-colony phenotype on agar medium) correlated well withthe level of APase activity measured by enzymatic assay(Tables 3 and 4): highly active strains such as MM1002 andMM1003 were intensely blue on agar medium containingX-P, while MM1001, which possessed significantly lessAPase activity in vitro, appeared pale blue under the sameconditions. A photosynthetically incompetent mutant,MM1006, which failed to hydrolyze X-P in situ, also lackedsignificant APase activity in vitro (Tables 3 and 4). Thesedata support the earlier hypothesis of Manoil and Beckwith(24) that the extent of X-P hydrolysis in situ is proportionalto the level of APase activity detected in vitro.Our observations that the PS- strain, MM1006, failed to

hydrolyze X-P in situ and possessed negligible APase activ-ity in vitro (Table 3) strengthen the argument of Hoffman andWright that APase is not active unless it is exported (15).However, we cannot unambiguously conclude that cytoplas-mically localized 'phoA fusion proteins, such as those seenin MM1006, are not active in vivo, since it has not beenconclusively shown that X-P can cross the cytoplasmicmembrane.We have recovered APase-active fusion proteins in at

least three distinct categories on the basis of subcellularlocalization: (i) soluble periplasmic fusions (MM1004 andMM1005); (ii) integral membrane fusions (MM1003); and (iii)periplasmic fusions which appear to be associated with thecytoplasmic membrane (MM1001 and MM1002). This reportalso presents the first evidence demonstrating the utility ofTnphoA as a means of monitoring the regulation of compart-mentalized proteins in R. sphaeroides by light or oxygen orboth.Western immunoblot analysis confirmed the presence of

APase fusion proteins in each of the APase-active strainsexamined. In most cases, several different-sized fusion prod-ucts were detected in the strains possessing fusion proteins

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by using antibody against APase. This observation is prob-ably explained by the degradation of the heterologous fusedproteins, in vivo or during sample preparation, giving rise tosmaller immunoreactive polypeptides. This conclusion wassupported by the presence of significant amounts of cross-reacting protein migrating at an electrophoretic mobilityidentical to that of the native E. coli APase. The observationof in vivo degradation of TnphoA-derived protein fusions hasbeen made previously in E. coli (24), and we have observedthat extensive denaturation of APase fusion protein samplesprior to electrophoresis resulted in the accumulation of asingle immunoreactive polypeptide at a molecular massequal to that of native E. coli APase (unpublished observa-tions).Although no APase activity was detected in situ or in vitro

in MM1007 (Tables 3 and 4), a low level of cross-reactingprotein was observed by Western analysis of the periplasmicproteins from this strain (Fig. 3, lane 7). This suggested thatthe 'phoA gene fusion in MM1007 gives rise to an immuno-logically reactive, yet enzymatically inactive, APase fusion.This observation could be explained by the inability of theprotein fusion in that strain to correctly dimerize in situ, acondition prerequisite for enzymatic activity of APase (15,24).We have demonstrated the utility of APase fusions as a

tool for monitoring gene expression and regulation in R.sphaeroides by the recovery of mutants such as MM1002,which expressed APase activity both in situ and in vitrodifferentially, dependent upon the incident light intensityunder which cells were grown. Likewise, expression of theperiplasmic fusion in MM1005 and the membrane-localizedAPase fusion in MM1003 was shown to be regulated not onlyby photoheterotrophic growth conditions but also by oxy-gen. Not surprising was the observation that APase fusionsof components involved in anaerobic respiration, such asMM1001 and MM1004, also contained APase fusion proteinswhich were regulated by the presence of oxygen or incidentlight intensity.Because the genomic locations of TnphoA in the two Chl'

strains were different, these must represent unique muta-tions in at least two separate components of the pathwayleading to C103 reduction in vivo. The nearly normal levelsof DMSO reductase activity in these two mutants supportthe conclusion that Chlr in R. sphaeroides may involve othercomponents of anaerobic respiration besides the DMSOreductase. An earlier report by McEwan et al. (29) suggestedthat DMSO-TMAO reductase may be involved in Chlr in R.capsulatuis; however, a recent report identifying a transpo-son-induced DMSO-TMAO reductase mutant in that organ-ism (17) did not indicate a concomitant Chlr phenotype.TnphoA present in MM1006 was localized to a 73-kb AseI

chromosomal DNA restriction fragment (Fig. 2), whichencompasses the majority of the photosynthetic gene cluster(A. Suwanto and S. Kaplan, manuscript submitted). Com-plementation analyses have identified two cosmids whichmap to this region and which restore photosynthetic compe-tence to MM1006 (unpublished results). Complementationwith the R' plasmid, pWS2, derived from R. sphaeroidesWS108 (36), also resulted in the restoration of photosyn-thetic competence to MM1006 (unpublished observations).MM1007 contained a TnphoA transposition on a plasmid

of R. sphaeroides. While previous studies have been unableto define specific functions for the five plasmids in 2.4.1 (13,30), Saunders and co-workers ascribed a PS- phenotype tothe insertional inactivation of an unknown (but essential)locus on the 42-kb plasmid (32). However, Nano and Kaplan

have reported a strain of R. sphaeroides, CU1022, whichlacks this plasmid but is still photosynthetically competent,although showing an altered ratio of spectral complexes (30).Their work implicated rearrangement between the 42- and99-kb plasmids in the formation of spontaneous PS- andpigment mutants. The recovery of a Chlr mutant (MM1002)containing a transposition of TnphoA onto one of theseendogenous plasmids suggests that one or more componentsof anaerobic respiration may be encoded by loci on theseplasmids.We have identified, and partially purified by anion-ex-

change chromatography, a DMSO-dependent MVH-oxidizing protein component in the periplasmic fraction of R.sphaeroides which eluted at a conductivity similar to thatreported for R. capsulatius DMSO-TMAO reductase (29).The fact that a TMAO-dependent oxidation of MVH by thiscomponent has been observed (M. D. Moore and S. Kaplan,unpublished results) agrees with earlier studies in E. coli(42), R. capsulatus (17, 28), and P. vulgaris (38) whichsuggest that a single enzyme is responsible for both DMSOand TMAO reduction. Expression of DMSO reductase in R.sphaeroides appears to be repressible-derepressible, sinceanaerobically grown cells possess three- to fourfold-higherDMSO reductase activity than aerobically grown cells. Thisis consistent with the expression of the TMAO reductase ofS. typhimrnriiirn (19), the DMSO-TMAO reductase of R.capsiulatus (29), and the TMAO reductase of E. coli (42) butcontrasts with the constitutive expression observed for theDMSO reductases of E. coli (42) and P. vulgaris (38).Localization of DMSO reductase activity to the periplasmicfraction in R. sphaeroides agrees with the localization ofDMSO-TMAO reductase in R. capsiulatus (28), althoughWeiner and co-workers have shown the DMSO reductase inE. coli (42) is an intrinsic cytoplasmic membrane proteinreleased from the membrane only after detergent solubiliza-tion.

Additional work in our laboratory (A. R. Varga and S.Kaplan, manuscript submitted) has demonstrated that theconstruction of specific cycA:phoA fusions leads to exten-sive accumulation of APase in the periplasm of R. sphaer-oides. In summary, the results presented here have demon-strated the activity, specificity, and utility of using TnphoAin R. sphaeroides.

ACKNOWLEDGMENTS

We thank J. Beckwith for his gift of E. coli CC118 and CC202,S. C. Dryden for construction of the R. sphaeroides genomiccosmid library, A. R. Varga for preparation of rabbit anti-E. coliAPase for Western immunoblot analysis, J. K. Wright for his advicein preparation for column chromatography, and A. Suwanto for hisinvaluable assistance with TAFE analysis.

This work was supported by Public Health Service grantsGM15590 and GM31667 from the National Institutes of Health toS.K. M.D.M. was a Predoctoral Fellow supported by Public HealthService grant GM07283 from the National Institutes of Health.

LITERATURE CITED1. Abeliovich, A., and S. Kaplan. 1974. Bacteriophages of Rhodo-

psecudol,nonas sphaeroides: isolation and characterization of aRhodopseiudonmonas sphaelroides bacteriophage. J. Virol. 13:1392-1399.

2. Allen, L. N., and R. S. Hanson. 1985. Construction of broad-host-range cosmid cloning vectors: identification of genes nec-essary for growth of Methylohacteri,mn organophiliun on meth-anol. J. Bacteriol. 161:955-962.

3. Baldwin, T. O., T. Berends, T. A. Bunch, T. F. Holzman, S. K.Rausch, L. Shamansky, M. L. Treat, and M. M. Ziegler. 1984.

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Cloning of the luciferase structural genes from Vibrio harveyiand expression of bioluminescence in Escherichia coli. Bio-chemistry 23:3663-3667.

4. Bassford, P. J., T. J. Silhavy, and J. R. Beckwith. 1979. Use ofgene fusions to study secretion of maltose-binding protein toEscherichia coli periplasm. J. Bacteriol. 139:19-31.

5. Bilous, P. T., and J. H. Weiner. 1988. Molecular cloning andexpression of the Escherichia coli dimethyl sulfoxide reductaseoperon. J. Bacteriol. 170:1511-1518.

6. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

7. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromo-somal antibiotic resistance in bacteria: genetic transformation ofE. coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69:2110-2114.

8. Davis, J., T. J. Donohue, and S. Kaplan. 1988. Construction,characterization, and complementation of a Puf- mutant ofRhodobacter sphaeroides. J. Bacteriol. 170:320-329.

9. Donohue, T. J., B. D. Cain, and S. Kaplan. 1982. Alterations inthe phospholipid composition of Rhodopseudomonas sphaer-oides and other bacteria induced by Tris. J. Bacteriol. 152:595406.

10. Donohue, T. J., J. Chory, T. E. Goldsand, S. P. Lynn, and S.Kaplan. 1985. Structure and physical map of Rhodopseudomo-nas sphaeroides bacteriophage RS1 DNA. J. Virol. 55:147-157.

11. Donohue, T. J., A. G. McEwan, and S. Kaplan. 1986. Cloning,DNA sequence, and expression of the Rhodobacter sphaeroidescytochrome c2 gene. J. Bacteriol. 168:962-972.

12. Ferguson, S. J., J. B. Jackson, and A. G. McEwan. 1987.Anaerobic respiration in the Rhodospirillaceae: characterisa-tion of pathways and evaluation of roles in redox balancingduring photosynthesis. FEMS Microbiol. Rev. 46:117-143.

13. Fornari, C. S., M. Watkins, and S. Kaplan. 1984. Plasmiddistribution and analyses in Rhodopseudomonas sphaeroides.Plasmid 11:39-47.

14. Fraley, R. T., D. R. Leuking, and S. Kaplan. 1979. The relation-ship of intracytoplasmic membrane assembly to the cell divisioncycle in Rhodopseudomonas sphaeroides. J. Biol. Chem. 254:1980-1986.

15. Hoffman, C. S., and A. Wright. 1985. Fusions of secretedproteins to alkaline phosphatase: an approach for studyingprotein secretion. Proc. Natl. Acad. Sci. USA 82:5107-5111.

16. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. Arestriction enzyme cleavage map of Tn5 and location of a regionencoding neomycin resistance. Mol. Gen. Genet. 177:65-72.

17. Kelly, D. J., D. J. Richardson, S. J. Ferguson, and J. B. Jackson.1988. Isolation of transposon Tn5 insertion mutants ofRhodobacter capsulatus unable to reduce trimethylamine-N-oxide and dimethyl sulphoxide. Arch. Microbiol. 150:138-144.

18. Kiley, P. J., A. Varga, and S. Kaplan. 1988. Physiological andstructural analysis of light-harvesting mutants of Rhodobactersphaeroides. J. Bacteriol. 170:1103-1115.

19. Kwan, H. S., and E. L. Barrett. 1983. Purification and propertiesof trimethylamine oxide reductase from Salmonella typhimu-rium. J. Bacteriol. 155:1455-1458.

20. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

21. Lueking, D. R., R. T. Fraley, and S. Kaplan. 1978. Intracyto-plasmic membrane synthesis in synchronous cell populations ofRhodopseudomonas sphaeroides. J. Biol. Chem. 253:451-457.

22. Madigan, M. T., and H. Gest. 1978. Growth of a photosyntheticbacterium anaerobically in darkness supported by "oxidantdependent" sugar fermentation. Arch. Microbiol. 117:119-122.

23. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

24. Manoil, C., and J. Beckwith. 1985. TnphoA: a transposon probefor protein export signals. Proc. NatI. Acad. Sci. USA 82:

8129-8133.25. Maquat, L. E., and W. S. Reznikoff. 1978. In vitro analysis of

the Escherichia coli RNA polymerase interaction with wild-typeand mutant lactose promoters. J. Mol. Biol. 125:467-490.

26. Markwell, J. P., and J. Lascelles. 1978. Membrane-bound,pyridine nucleotide-independent L-lactate dehydrogenase ofRhodopseudomonas sphaeroides. J. Bacteriol. 133:5934600.

27. Markwell, M. A., S. M. Haas, L. L. Bieber, and N. E. Tolbert.1978. A modification of the Lowry procedure to simplify proteindetermination in membrane and lipoprotein samples. Anal.Biochem. 87:206.

28. McEwan, A. G., H. G. Wetzstein, S. J. Ferguson, and J. B.Jackson. 1985. Periplasmic location of the terminal reductase intrimethylamine N-oxide and dimethylsulphoxide respiration inthe photosynthetic bacterium Rhodopseudomonas capsulata.Biochim. Biophys. Acta 806:410-417.

29. McEwan, A. G., H. G. Wetzstein, 0. Meyer, J. B. Backson, andS. J. Ferguson. 1987. The periplasmic nitrate reductase ofRhodobacter capsulatus; purification, characterisation and dis-tinction from a single reductase for trimethylamine N-oxide,dimethylsulfoxide, and chlorate. Arch. Microbiol. 147:340-345.

30. Nano, F. E., and S. Kaplan. 1984. Plasmid rearrangements in thephotosynthetic bacterium Rhodopseudomonas sphaeroides. J.Bacteriol. 158:1094-1103.

31. Satoh, T., and F. N. Kurihara. 1987. Purification and propertiesof dimethylsulfoxide reductase containing a molybdenum cofac-tor from a photodenitrifier, Rhodopseudomonas sphaeroidesf.s. denitrificans. J. Biochem. 102:191-197.

32. Saunders, V. A., J. R. Saunders, and P. M. Bennett. 1976.Extrachromosomal deoxyribonucleic acid in wild type andphotosynthetically incompetent strains of Rhodopseudomonassphaeroides. J. Bacteriol. 125:1180-1187.

33. Schwartz, D. C., and C. R. Cantor. 1984. Separation of yeastchromosome-sized DNAs by pulsed-field gradient gel electro-phoresis. Cell 37:67-75.

34. Silvestro, A., J. Pommier, and G. Giordano. 1988. The inducibletrimethylamine-N-oxide reductase of Escherichia coli K12: bio-chemical and immunological studies. Biochim. Biophys. Acta954:1-13.

35. Simon, R., U. Priefer, and A. PiihIer. 1983. A broad host rangemobilization system for in vivo genetic engineering: transposonmutagenesis in Gram-negative bacteria. Bio/Technology 1:37-45.

36. Sistrom, W. R., A. Macaluso, and R. Pledger. 1984. Mutants ofRhodopseudomonas sphaeroides useful in genetic analysis.Arch. Microbiol. 138:161-165.

37. Southern, E. 1979. Gel electrophoresis of restriction fragments.Methods Enzymol. 68:152-176.

38. Styrvold, 0. B., and A. R. Str0m. 1984. Dimethylsulphoxide andtrimethylamine oxide respiration of Proteus vulgaris. Evidencefor a common terminal reductase system. Arch. Microbiol.140:74-78.

39. Tai, S. P., and S. Kaplan. 1985. Intracellular localization ofphospholipid transfer activity in Rhodopseudomonas sphaer-oides and a possible role in membrane biogenesis. J. Bacteriol.164:181-186.

40. Van Niel, C. B. 1944. The culture, general physiology, andclassification of the non-sulfur purple and brown bacteria.Bacteriol. Rev. 8:1-118.

41. Waterbury, P. G., and M. J. Lane. 1987. Generation of lambdaphage concatemers for use as pulsed field electrophoresis sizemarkers. Nucleic Acids Res. 15:3930.

42. Weiner, J. H., D. P. MacIsaac, R. E. Bishop, and P. T. Bilous.1988. Purification and properties of Escherichia coli dimethylsulfoxide reductase, an iron-sulfur molybdoenzyme with broadsubstrate specificity. J. Bacteriol. 170:1505-1510.

43. Weiss, R. L. 1976. Protoplast formation in Escherichia coli. J.Bacteriol. 128:6684670.

44. Yen, H.-C., and B. Marrs. 1977. Growth of Rhodopseudomonascapsulata under anaerobic dark conditions with dimethylsul-phoxide. Arch. Biochem. Biophys. 181:411-418.

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