ARCHIVES

OF BIOCHEMISTRY

Vol. 292, No. 2, February

Cytochrome

AND

BIOPHYSICS

1, pp. 419-426, 1992

c2 Mutants of Rhodobacter capsulatus

Michael Caffrey,*,’ Edgar Davidson,?

Michael

Cusanovich,*

and Fevzi Daldal$

*Department of Biochemistcg, University of Arizona, Tucson, Arizona 85721; tlnstitute for Structural and Functional 3401 Mark Street, Philadelphia, Pennsylvania 19104; and $Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Studies,

Received June 17, 1991, and in revised form September 24,1991

Although structurally related to other members of the class I c-type cytochromes, the cytochromes cg have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have develalped a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome cz-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement 4or photosynthetic growth in R. we describe the generation, sphaeroides. Further, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overprodu.ced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These flndings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants. 0 1992 Academic Press, Inc.

With their well-established chemical and structural properties, the cytochromes c are among the best characterized proteins (1). In general, they are small, watersoluble molecules which a.re structurally, and often functionally, related to each other. A subclass of the class I c-type cytochromes, the cytochromes c2 from photosynthetic bacteria, functions as soluble electron carriers between membrane-bound redox centers. They transfer 1 To whom correspondence should be addressed at present address: Centre d’Etudes Nucleaires, Laboratoire de Resonance Magnetique, 85X38041 Grenoble Cedex, France. 0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

electrons from the cytochrome bcr complex to the photosynthetic reaction center under photosynthetic conditions (2) and to cytochrome oxidase under aerobic conditions (3). Cytochromes c2 have been purified from a large number of phototrophic bacteria, and the high-resolution rubrum (4,5) and structures of those from Rhodospirillum Rhodobacter capsulatus (6) have been determined. Although having substantial structural homology with the other class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology (less than 40%) and exhibit distinct properties. For example, the redox potentials of the cytochromes c2 range from 250 to 470 mV and their isoelectric points range from 4.5 to greater than 9. This is in sharp contrast to the eukaryotic cytochromes c which have redox potentials of 260 * 5 mV and are strongly basic (PI = 10.5). The development of a genetic system for the study of R. capsulatus cytochrome c2 by site-directed mutagenesis is of particular interest for a number of reasons. With respect to the eukaryotic cytochromes c, R. capsulatus cytochrome c2 has a relatively high redox potential of 367 mV at pH 7.0 and it is therefore interesting to examine the basis for this difference in redox potential (7). In addition, the isoelectric point of R. capsulatus cytochrome c2 is near neutrality (PI = 7.1) which makes it amenable to studies characterizing electrostatic and steric interactions with other electron donors or acceptors (1). In contrast, the protein-protein interactions of the eukaryotic cytochromes c are dominated by a strong positive electrostatic field which complicates the interpretation of the electrostatic and steric effects of individual residues. Finally, the availability of the R. capsulatus cytochrome c2 three-dimensional structure provides the foundation for choosing residues to be mutated and aids in the interpretation of the resulting mutant properties (6). Importantly, the gene encoding R. capsulatus cytochrome c2 (cycA) has been cloned, and a cytochrome c2minus strain of R. capsulatus prepared (8) which can serve as a host for the overexpression of mutants. It is well established that elimination of cytochrome c2 does not 419

Inc. reserved.

CAFFREY

420

prevent aerobic or photosynthetic growth in R. capsulatus, indicating that alternative pathways of electron transfer bypassing this soluble electron carrier must exist in this species (8, 9). This situation is in sharp contrast to that of R. sphaeroides which requires a functional cytochrome c2 for photosynthetic but not aerobic growth (10). Thus, if R. capsulatus cytochrome c2 could overcome the photosynthetic growth defect of the R. sphaeroides cytochrome cz-minus strain, the in vivo functionality of R. capsulatus mutants in R. sphaeroides could be tested by simple growth tests. In what follows, we report the expression of R. capsulutus cytochrome c2 in cytochrome cz-minus strains of R. capsulatus and R. sphaeroides. In addition, the expression and in vivo functionality properties of nine R. capsulatus cytochrome c2 site-directed mutants will be described. MATERIALS

AND

METHODS

Bacterial strains, plasmids, media, growth conditions, and genetic procedures. Escherichia coli, R. cap&a&s, and R. sphaeroides strains used and derivatives of the plasmids pBR322 and pRK2 are listed in Table I. E. coli strains were grown on LB or YT media supplemented with appropriate concentrations of antibiotics when required (8). The strain 71-18 was used as a recipient to detect a-complementation for pUC19 derivatives on LB plates supplemented with 80 PM IPTG and 32 pg/ml X-GAL.’ Photosynthetic bacteria were grown by respiration (aerobic, dark) or by photosynthesis (anaerobic, light provided by 40-W tungsten lamps at a distance of 15 cm) on MYPE rich or RCV minimal media (22) for R. cupsulatus species and on Medium A of Sistrom for R. sphaeroides species (23). Cultures were supplemented when necessary with tetracycline (0.5-2.5 pg/ml) or spectinomycin (10 pg/ml). The broad host-range plasmid pRK404 derivatives carrying the wild-type and mutant cytochromes c2 were transferred into R. capsulatus and R. sphaeroides strains via triparental crosses using the helper plasmid pRK2013 and selecting for tetracycline resistance (20). and site-directed mutagenesis. Recombinant DNA techniques Recombinant DNA techniques have been described previously (11,24). The sequencing primers and the mutagenic primers were derived from the cycA gene (8) and are listed in Table II. For site-directed mutagenesis, the phage Ml3 derivative cz-mplSB/S, carrying the previously sequenced 1.25-kb SalI-EglII fragment containing the cycA gene, was used as a template (8). Mutagenesis reactions were carried out as in Nisbet and Beilharz (25) with uracylated templates (26). Mutagenized phages were screened by dideoxy DNA sequencing (27). Following mutagenesis, the 1.25-kb HindIII-KpnI fragment of the replicative form of the Ml3 cpmplSB/S mutant derivatives was transferred into the plasmid pUC19 or pUC119, selecting for ampicillin resistance (AmpR) and screening for loss of ol-complementation (Fig. 1). All pUC derivatives thus obtained were cloned into the unique Hind111 restriction site of pRK404 by selecting for tetracycline resistance (TetR) and ampicillin resistance. The composite plasmids containing both RK2 and ColEl origins of repli-

’ Abbreviations used: cyt, cytochrome; wt, wild-type; K12D, lysine 12 substituted by aspartate; K14E, lysine 14 substituted by glutamate; K32E, lysine 32 substituted by glutamate; K14E/K32E, lysines 14 and 32 substituted by glutamates; P35A, proline 35 substituted by alanine; W67Y, tryptophan 67 substituted by tyrosine; Y75C, Y75F, and Y75S, tyrosine 75 substituted by cysteine, phenylalanine, and serine, respectively; IPTG, isopropyl B-D-thiogalactoside; R, resistance; BCA, bicinchoninic acid, X-GAL, 5.bromo-4-chlro-3-indolyl$-D-galactoside; RCV, R. capsulates minimal media with vitamins; MYPE, minimal peptone media with yeast extract.

ET AL. cations were found to be stable in the E. coli and R. capsulates strains used. andpED8 carrying the R. capsulates Construction of theplasmidspED5 ana’ R. sphaeroides cycA genes and isolation of a R. sphaeroides cytochrome The plasmid pED5 was constructed in two steps. c2-minus mutant. First the 1.25-kb fragment of pBc, (8) was cloned into the SalI-PstI sites of the plasmid pUC8 after appropriate modifications of the BglII and PstI sites using T4 DNA polymerase. The plasmid obtained, pSH3, was cloned into the unique Hind111 site of the broad host-range plasmid pRK404 (20), selecting for Amps and TetR, and yielded pED5. The plasmid pED8 was obtained by cloning the entire plasmid pC2P2-71 (18), kindly provided by Dr. T. J. Donohue, into the unique EcoRI site of the broad host-range plasmid pRK291 (20). A cytochrome c,-minus mutant of R. sphaeroides strain Ga (obtained from Dr. W. Sistrom) was constructed in a way similar to that reported in (8) with the exception that the inactivating interposon employed here conferred resistance to spectinomycin (SpeR) and contained transcription and translation terminators in both orientations (19). The R. sphueroides cytochrome c,-minus mutant, GadCP, thus obtained was also unable to grow photoheterotrophically either on minimal or on rich medium (Table III), although its respiratory growth appeared unaffected. Further, as shown in Fig. 2 ascorbate-reduced minus ferricyanide-oxidized optical difference spectra of cell-free extracts obtained from GadC2 had much less 550.nm-absorbing material (attributed to soluble c-type cytochromes) than the parental strain Ga. These phenotypes of GadC2 were similar to that of the mutant CYC65 reported earlier by Donohue et al. (10). Finally, the Tets merodiploid pED8/GadCP was competent for photosynthetic growth (Table III) and overproduced cytochrome c1 (Fig. 2), as was reported earlier (28). Cell extract preparation and spectroscopic analysis. Cell extracts were prepared by resuspending bacteria in 1:4 ratio in 100 mM potassium phosphate buffer (pH 7.4) and sonicating 3 X 20 s (Branson 200 sonicator with a microtip probe at a power setting of 30%) at 0°C. Cellular debris was removed by centrifugation (45,000 rpm for 2 h in a Beckman 50Ti rotor) at 5°C. Total protein concentrations of cell-free extracts were determined either by the BCA method (29) or according to Lowry et al., (30). Cell-free extracts were oxidized with potassium ferricyanide and reduced with sodium ascorbate. Cytochrome c2 concentrations were estimated from analysis of the ascorbate-reduced minus ferricyanide-oxidized difference spectra with respect to the difference spectra obtained using the cytochrome c,-minus mutant MT-G4/S4 grown under the appropriate growth conditions. All absorbance changes were normalized to a total protein concentration of 1 mg/ml. The percentage of cytochrome cz present with respect to total protein concentration was estimated by using a difference extinction coefficient of 20 mM~‘cm~’ and a molecular weight of 13 kDa (31). Absorption spectra were taken using either Cary 15 or Hitachi U3210 spectrophotometers.

RESULTS

AND

DISCUSSION

Expression of the R. capsulatus cycA gene and overproduction of R. capsulatus cytochrome cp in R. capsulatus. The structural gene of R. capsulatus cytochrome c2 (cycA) is confined to the 1.25-kb SalI-BglII fragment present on plasmid pBcz (8). The plasmid pED5, constructed as described under Materials and Methods, was conjugated into the R. capsulatus strain MT-G4/S4 to determine whether this fragment can direct the in vivo synthesis of cytochrome cg. Ascorbate-reduced minus ferricyanide-oxidized optical difference spectra of cell-free extracts from photosynthetically grown R. capsulatus strains MT1131, MT-G4/S4, and pEDS/MT-G4/S4 are shown in Fig. 3a. These data indicated that the merodiploid cells containing pED5 overproduced cytochrome c2

CYTOCHROME

C2 MUTANTS

OF Rhodobacter

TABLE

421

capsulatus

I

E. coli, R. capsulatus, and R. sphaeroides Strains and Plasmids Used Strains and plasmids E. coli HBlOl JL1245 JMlOl JM103 71-18 R. capsulatus MT1131 MT-G4/S4 R. sphaeroides Ga GadC2 Plasmids pBR322 pUC8 puc19 puc119 PBC~ pC2P2-71 pHP45w pRK404 pRK291 pSUP202 pSH3 pED5 pED6 pED7 pED8 pMCl0 pMCl1 pMC12 pMC13 pMC14 pMCl5 pMC16 pMC17 pMC18 pMC19

Description

F- proA leu hsdS20(ri mg) recA ara lacy1 dut ung thil relA spoT1 zdd279::TnlO/F’ [ly.sA] A(lac pro) thi supE/F’ [traD36 proAB la0 ZaM15] A(lac pro) thi strA supE endA sbcB h.sdR-/F [traD36 proAB lacZq ZdMlB] A(& pro) /F’ [proAB la0 ZAM15] crtD121 RifR crtD121 Al(cycA: :kan) Rifa

Source

(11) J. Little

(12) (12) (13) (14)

(8)

crt

(15) This work

crt Al(cycA::spe) Ampa, TetR Ampa AmpR AmpR derivative of pUC19 carrying Ml3 OR1 AmpR (Rc. cyt c2) AmpR (Rs. cyt cg) Ampa, Spea TetR TetR AmpR, CamR AmpR derivative of pUC8 carrying Rc. cyt cp AmpR, TetR derivative of pRK404 carrying Rc. cyt c2 AmpR, speR derivative of pC2P2-71 SpeR derivative of pSUP202 AmpR, TetR derivative of pRK291 (Rs. cyt c2) AmpR derivative of pUC19 carrying K12D AmpR derivative of pUC19 carrying K14E AmpR derivative of pUC19 carrying K32E AmpR derivative of pUC19 carrying K14E/K32E AmpR derivative of pUC19 carrying P35A AmpR derivative of pUC19 carrying W67Y Ampa derivative of pUC19 carrying Y75F AmpR derivative of pUC119 carrying Y75C AmpR derivative of pUC119 carrying Y75S AmpR derivative of pUC19 carrying Y75S

(11) (16) (17) J. Messing

(8) (18) (19) (20) (20) (21) This This This This This This This This This This This This This This This

work work work work work work work work work work work work work work work

Note. AmpR, CamR, KanR, RifR, TetR and SpeR indicate resistance to ampicillin, chloramphenicol, kanamycin, rifampicin, tetracycline, and spectinomycin, respectively. Rc. and Rs.‘indicate R. capsulates and R. sphaeroides, respectively. Plasmids pMCl0 to pMC19 were cloned into the plasmid pRK404 using their unique Hind111 sites to yield the plasmids pMClO-404 to pMC19-404, respectively.

(compare pED5/MT-G4/S4 to MT-G4/S4 and MT1131). Amounts of cytochrome c2 similar to those detected in pED5/MT-G4/S4 were also found in MT-G4/S4 strains harboring several related plasmids containing the cycA gene in all four possible orientations with respect to pUC8 and pRK404 plasmids (d.ata not shown). Therefore, the synthesis of cytochrome c2 on pED5 must be directed by the 365-bp-long region located 5’ upstream to the coding part of cycA (8). That this region carries at least part of the elements directing the expression of cycA was further confirmed using transcriptional fusions between the cycA and the E. coli la& genes (to be reported). Complementation of the photosynthetic growth defect of a R. sphaeroides cytochrome c2-minus mutant by R. cap-

sulatus cytochrome c2. Cytochrome c,-minus mutants of R. capsulates are photosynthesis-proficient (8) while those of R. sphaeroides are photosynthesis-deficient (10). Since these bacteria are closely related (32), we reasoned that R. capsulatus cytochrome c2 may complement a R. sphaeroides cytochrome c2-minus mutant for photosynthetic growth and that this feature could be used to detect mutant R. capsulatus cytochromes c2 that are either nonfunctional or poorly functional in Go. The plasmid pED5, expressing the cytochrome c2 of R. capsulatus, was conjugated into the R. sphaeroides cytochrome c2-minus mutant, GadC2. The TetR merodiploid pED5/GadC2 obtained grew photosynthetically on minimal and on rich medium (Table III). This indicated that R. capsulatus cy-

422

CAFFREY

S

ET AL.

-

KD

D mutagenesis UW

g

c2 mplBB/S

c 2 mp18B ‘S

3

MCS

E

pRK404

R cap

-

puc19

K

pUC19del

c;

(MT-G4/S4) . overproduction

R sph. c?(Gad C2) l m v/v0 functtonaltty

pRK404der

FIG. 1. Construction of R. capsnlatus mutant cytochromes cp. C, corresponds to the structural gene for R. capsulatus cytochrome c2 (cycA), with the arrow indicating the direction of transcription. D, S, and K correspond to the restriction enzyme sites HindIII, SalI, and KpnI, respectively, and the hybrid BamHI/BglII site is indicated as B*. amp and tet correspond to the structural genes for p-lactamase and tetracycline resistance. MCS denotes the multiple cloning site: U and (A) are for the uracylated template and the mutagenic primers, respectively.

tochrome c2 complemented the photosynthetic growth defect of R. sphaeroides strain GadC2. Furthermore, ascorbate-reduced minus ferricyanide-oxidized optical difference spectra of cell-free extracts of pED5/GadC2 confirmed that the cytochrome cp from R. capsulatus was overproduced in R. sphaeroides as well as in R. capsulatus (Fig. 2). In an earlier study, Zilsel et al. (33) have shown that the puf operon encoding the light harvesting I complex subunits and the reaction center L and M subunits of R. sphaeroides could complement for photosynthetic growth a R. capsulatus strain devoid of reaction center. Similarly, Davidson et al. (34) have demonstrated that the fbc (pet) operon of R. sphaeroides could overcome the photosynthetic defect of a R. capsulatus cytochrome bq-minus mutant. These in uiuo results are also supported by the work of Gabellini et al., (35) who have shown that the gene products of the fbc (pet) operon of R. capsulatus [although reported mistakenly as being from R. sphaeroides (36)] were synthesized in vitro using a coupled transcription/translation system from R. sphaeroides (37). It is therefore clear that all three important components of a

bacterial photosynthetic apparatus (i.e., the membranebound reaction center and oxidoreductase as well as the soluble cytochrome CJ are functionally interchangeable between the R. capsulatus and R. sphaeroides species. Isolation of R. capsulatus cytochrome c2 site-directed mutants and their growth properties. Using the scheme described in Fig. 1, nine site-directed mutants of R. capsulatus cytochrome ca were obtained. In general, mutated residues were highly conserved (Fig. 4) and designed to test the importance of a residue to a specific property of c-type cytochromes. For example, the role of electrostatics in the electron transfer reactions of cytochromes c2 was tested by replacing lysines 12, 14, and 32 with aspartate or glutamate residues [mutants K12D (pMClO), K14E (pMCll), and K32E (pMC12) and the double mutant K14E/K32E (pMC13)]. To test the importance of the hydroxyl and aromatic moieties at position 75 to redox potential and stability, tyrosine 75 was replaced with cysteine, phenylalanine, and serine [mutants Y75C (pMC17), Y75F (pMC16), and Y75S (pMC18)]. In addition, proline 35 was replaced by alanine to ascertain the importance of structural constraint at this position to redox potential

CYTOCHROME TABLE

423

capsulatus GadC2

pEDB/GcdCP

pED5/GadCZ

and

Sequence (5’ to 3’)

Sequencing SPl SP2 U17” Mutagenesis 12A 14A

-TGTGGTAAAGCGTGGA-AGCAAACAGACGAAGG-GTTTTCCCAGTCACGAC-AAAGAATTCAACGA/CG/TTGCAAGACC-

520

-CGAAGACCGGCGG/CGAACCTCT-

67A

-GGGCTTC!GCCTA/TTACCGAGGA-

75A 75B

-ATCGCGACCTC/G/TTGTGAA-ATCGCGACCTTTGTGAAl7-mer from New IEngland Biolabs.

and stability [mutant P35A (pMC14)]. Finally, to test the importance of tryptophan 167as a hydrogen bonding partner to the rear heme propionate, tryptophan 67 was replaced by tyrosine [mutant W67Y (pMC15)]. To test the growth properties of the mutant R. capsulatus cytochromes cz in R. capsulatus and R. sphaeroides

TABLE

520

560

520

“In

560

520

nm

560 “IT3

FIG. 2. Normalized ascorbate-reduced minus ferricyanide-oxidized optical difference spectra of cell-free extracts of R. sphaeroides strains Ga and GadC2 (cytochrome c,-minus) producing R. sphaeroides (pEDS/ GadC2) and R. capsulatus (pED5/GadC2) cytochromes c2 under photosynthetic growth conditions.

-GTGAAAGGCGCGGA/CG/TACCGGCCCG-

35A

560 nm

-CAACAAGTGCC/GAGACCTG-

32A

a Universal

OF Rhodobacter

II

Oligonucleotides Used for Sequencing Site-Directed Mutagenesis Oligonucleotide

Cz MUTANTS

cytochrome cz-minus mutants, the plasmids pMClO-404, pMCll-404, pMC12-404, pMC13-404, pMC14-404, pMC15-404, pMC16-404, pMC17-404, and pMC18-404 (Table I) were conjugated into the strains MT-G4/S4 and GadC2, selecting for TetR under respiratory growth conditions (Fig. 1). The growth ability of the merodiploids obtained were estimated on rich and minimal medium under various conditions by measuring their colony size (Table III). Except for strains pMC17-404/MT-G4/S4 (Y75C) and pMC18-404/MT-G4/S4 (Y75S), the growth ability of all R. capsulatus merodiploids producing various

III

Complementation of the Photosynthetic Growth Defect of GadC2, a Cytochrome c,-Minus Mutant of R. sphaeroides with Wild-Type and Mutant Derivatives of R. capsulatus Cytochromes c2 Photosynthetic Strain Ga GadC2 pED5/GadC2 pED8/GadC2 pMC!lO-404/GadC2 pMCll-404/GadC2 pMC12-404/GadC2 pMC13-404/GadC2 pMC14-404/GadC2 pMC15-404/GadC2 pMC16-404/GadC2 pMC17-404/GadC2 pMC18-404/GadC2

Med A

MYPE

cyt c2 Rs. cyt c2 No cyt c2 Rc. cyt cg Rs. cyt cg K12D K14E K32E K14E/K32E P35A W67Y Y75F Y75C Y75S

growth”

UT1131A

I * 520

+ + +

(1.0) (