Molecular Microbiology (1990) 4(9), 1567-1574

Mutagenesis, cloning and complementation analysis of C4-dicarboxylate transport genes from Rhodobacter capsulatus M. J. Hamblin, J. G. Shaw, J. P. Curson and D. J. Kelly* Robert Hill Institute. Department of Molecular Biology and Biotechnology. University of Sheffield. Western Bank, Sheffield SW 2TN. UK. Summary Transposon mutagenesis was used to isolate insertion mutants of the photosynthetic bacterium Rhodobacter capsulatus which were unable to grow under aerobic conditions in the dark on malate, succinate or fumarate as sole carbon sources. Of five mutants isolated, all were deficient in C4-dicarboxylate transport. However, these mutants were still capable of photoheterotrophic growth, although at a slower rate than the wild type, on malate and succinate (but not fumarate). The mutated locus (designated dct) was complemented in trans using a cosmid gene bank. Subcloning and complementation analysis indicated that at least three closely linked genes essential for aerobic dicarboxylate transport were contained within an 8.3kb region of the Rhodobacter capsulatus chromosome.

Introduction Purple photosynthetic bacteria of the genus Rhodobacter are nutritionally versatile prokaryotes (Madigan and Gest. 1979). They are capable of rapidly adapting their metabolism from an aerobic to an anaerobic mode and can utilize a wide range of organic carbon sources to support growth. One group of carbon sources, the Ca-dicarboxylic acids, has long been knov*(n to be particularly effective in promoting fast growth rates and producing high ceil yields of purple bacteria under both photo- and chemoheterotrophic growth conditions (Van Niel, 1944; Stahl and Sojka, 1973). Indeed, most media formulations for these bacteria contain either malate or succinate as the sole or major carbon source.

Received 19 January. 1990; revised 22 May, 1990. "For correspondence. Tel. (0742) 768555, ext. 4414; Fax (0742) 728697.

In view of the key role of these substrates, it is surprising that virtually nothing is known about the transport systems mediating C4-dicarboxylate uptake in photosynthetic bacteria or the nature of the genes and gene products essential for this process. Although the work of Gibson (1975) showed that Rhodobacter sphaeroides possessed an inducible transport system for malate, succinate and fumarate, the only other detailed study to date concerning C4-dicarboxylate transport in any photosynthetic bacterium was carried out with Ectothiorhodospira shaposhnikovii (Karzanov and Ivanovsky, 1980). for which evidence was obtained for the operation of a sodiumdependent dicarboxylate symport. In both enterobacteria and rhizobia. the uptake of C4-dicarboxylates is mediated by a common system that transports malate. succinate and fumarate (Kay and Kornberg, 1969: Finan et al., 1981). However, the transport mechanism may differ in the two bactenal groups. In Rhizobium leguminosarum. the product of the dctA gene is apparently the only protein necessary for the actual transport process (Ronson ef ai, 1984). while in Esoherichia coli the products of at least three genes have been implicated (Lo and Sanwal. 1975), including one thought to encode a periplasmic binding protein. Cloning and sequencing of the dct regulon from R. leguminosarum (Ronson ef ai, 1984) has shown that the expression of dotA is under the control of two linked regulatory genes {dctB and dctD) which share strong sequence homology with the Klebsiella pneumoniae ntrB and ntrC genes, respectively, and which are members of a two-component signal-transducing and response system (Ronson ef ai. 1987a). The DctB protein may detect the presence of C4-dicarboxylates in the environment, while the DctD protein is thought to be a positive transcriptional activator of the structural gene. dctA (Ronson et ai. 1987b). This process additionally requires an RNA polymerase containing the ntrA{rpoN) sigma factor (Ronson et al.. 1987c), In order to study the molecular details of carbon-substrate transport in photosynthetic bacteria, we have investigated the uptake of C4-dicarboxylates by cells of R. capsulatus. In this paper, we describe the isolation, by transposon mutagenesis. of mutants that are impaired in dicarboxylate uptake and we report the cloning of the corresponding wild-type genes by complementation.

1568

M. J. Hamblin, J. G. Shaiv, J. P. Curson and D. J. Kelly phenotype was simply due to a defect in the aerobic metabolism of C4-dicarboxylates. measurements were made of the specific activities of the enzymes of the dicarboxylic acid branch of the citric acid cycle in three of the mutants isolated (data not shown). Malate dehydrogenase, succinate dehydrogenase and fumarase were present at similar specific activities in both mutant and wild-type strains grown aerobically in the dark. In addition, the three Ce branch enzymes (citrate synthase. aconitase and isocitrate dehydrogenase) were also present at wildtype levels in one mutant. MJH28. which was chosen as a representative strain for more detailed characterization.

Results Isolation, growth characteristics and enzyme profiles of Ca-dicarboxylate transport mutants Nine thousand kanamycin-resistant transconjugants resulting from tive independent matings between Escherichia co/(S17-1(pSUP2021) and R. oapsulatus37b4 were selected under aerobic conditions in the dark on minimal pyruvate plates. Each transconjugant was then screened for the inability to grow on minimal-malate plates under the sanne incubation conditions. It was reasoned that this screening procedure would select against the isolation of citric acid cycle mutants, as both malate and pyruvate are known to be metabolized through this pathway in aerobically grown R. capsulatus (Willison. 1988). Five mutants which showed the desired phenotype were isolated. In addition to a lack of aerobic growth on minimal-malate plates, none of these mutants grew on media containing either succinate or fumarate as sole carbon source. Plate tests on glucose or acetate, however, showed that all of the mutants could utilize these substrates.

Transport of ['''CJ-L-malate into wild-type and mutant cells Cells of the wild type and MJH28 grown under aerobic conditions in the dark on pyruvate medium were harvested, incubated for two hours under the same conditions in malate medium and assayed for their ability to transport radiolabelled malate (Fig. la). In contrast to the wild type, in which malate transport was easily demonstrated, the mutant cells showed no malate uptake. Simitar results were also obtained for the other four mutants isolated. This confirms that the lesion in these mutants is at the level of transport rather than metabolism and the designation dct has therefore been used to denote the mutated locus.

Table 1 summarizes the growth rates of wild-type and mutant strains on pyruvate and C4-dicarboxylates during cultivation under chemoheterotrophic or photoheterotrophic conditions. The doubling times of all the mutants on pyruvate were similar to the wild type under both sets of growth conditions. None of the mutants grew on malate, succinate or fumarate under aerobic conditions in the dark, confirming the results of the plate tests described above. Unexpectedly, however, grovrth did occur under anaerobic conditions in the light when either malate or succinate, but not fumarate. was the carbon source, although the growth rates were significantly slower on succinate compared with malate under these conditions (Table 1). The final optical densities reached by such cultures were similar to those of wild-type cells, and it was found to be possible to repeatedly subculture the mutants under phototrophic conditions on o.L-malate. However, no growth occurred upon subsequent transfer to malate minimal medium under aerobic conditions in the dark.

Transport assays were also carried out with both wild-type and MJH28 cells grown photoheterotrophically on malate. In these experiments (Fig. 1b). the bacteria were maintained initially under anaerobic conditions in the dark by passing a slow stream of Oj-free nitrogen gas over the surface of the suspension contained within the oxygen electrode. No uptake of ('"CJ-L-malate was observed in either wild-type or MJH28 cells under such conditions. Upon illumination, however, uptake proceeded at a linear rate in the wild-type suspension after a reproducible short lag period (Fig. 1b). Surprisingly, significant light-dependent malate transport could not be detected using MJH28 cells, even at higher cell densities and with longer incubation times than used for the wild type (Fig. 1 b). Similarly,

In order to rule out the possibility that the mutant Table 1. Growth rales ot wild-type and mutant strains on pyruvate and Cj-dicarboxylates.

Culture Doubling Ti Chemoheterotrophic growth MJH Cartjon source Pyruvate D,L-malate Succinate Fumarate

Wild type 4 5 5 5

Photoheterotrophic groswth MJH

20

22

25

2B

40

Wild type

20

22

25

26

40

5 —

5 — —

5 — —

5 — —









5 — — —

4 5 5 5

5 14 25 —

5 15 25 —

4 12 25 —

5 15 18 —

4 12 25 —

b

a. Measured in hours by the increase in optical density at 580nm. b. —, no growth detectable.

Cloning ofdd genes from Rhodobacter capsulatus 1569 isolated. Revertants of all the mutants were selected on malate medium and arose at a frequency of between 10"^ and 10"'", The majority of these proved to be kanamycinsensitive and to have simultaneously regained the ability to grow on succinate and fumarate as well as malate. These data are consistent with reversion occurring by the precise excision of a single copy of Tn5 in each mutant. In order to establish unequivocally that a single Tn5

(0)

18

o

60

20

Time (s)

MJH

(b)

a.

X ?7bi. 20

.2 9 •

22

25 28

Kb

Kb 2*1 — 91.— 66-

u

UCHT CN

0 0

/

100

(a) Tn5 probe EcgR]

( . • ! . -

AT

iO 20 Time (s,Al 8



16

60 24

Time (min,»l

0-56—

Fig. 1. a. Uptake o( L malate into chemoheterotrophic ally grown cells of R. capsu/afus Strains 37b4 (wild type, A — A ) , MJH28 (•—•) and MJH28 complemented with pDCT205 {O—O). The cells were incubaled under aerobic conditions in a darkened oxygen electrode and uptake Initiated by the addition of |'*C|-L malate |6(i.M|. b. Uptake of L-malate into photoneterotrophically grown cells of R. capsulatus strains 37b4 {wi(d type. A — A ) and MJH28 ! • — • ) . The cells were incubated under anaerobic conditions in a darkened oxygen electrode and ("CJ-L-malate added at time zero. Uptake was initiated by Illuminating the cells.

no uptake was observed with these cells if the assay conditions were changed to aerobiosis in the dark. Thus, although MJH28 is capable of phototrophic but not aerobic growth on malate as sole added carbon source. this difference was not reflected in transport assays with radiolabelled malate. The affinity of intact cells of MJH28 for malate was also measured. Cultures were set up with different starting concentrations of L- or D-malate as sole added carbon source and incubated under anaerobic conditions in the light. The exponential growth rate achieved was plotted as a function of the initial malate concentration using a double-reciprocal plot, so that the x-axis intercept could be used to calculate an approximate Kg value (Pirt. 1975). The data conformed to Michaelis-Menten kinetics and gave a K^ value of 4,5 mM for L-malate and 6.6 mM for D-malate, Reversion analysis. Southern hybridization and mapping of the Tn5 insertion sites A reversion analysis established that the Tn5 insertions were genetically linked to the phenotype of the mutants

-2 6

79—

[bl Tn5 probe

(c) pSUP202 probe EC12R1

Fig, 2. Southern hybridization analysis of dicarboxylate transport mutants. Chromosomal DNA trom the wild type (strain 37b4) and each mutant was completely digested with either EcoRI or Psd and 2|ig run on 0.5% agarose gels. After blotting onto nitrocellulose, a digoxeninlabelled Tn5 probe (pRZ104: ColE::Tn5) or pSUP202 probe was hybridized to the blots at 68"C in 5 K SSC. After 16h. ihe blots were incubated with anti-digoxenin followed by colour development with X-phosphate and nitro-blue tetrazolium. a. The Tn5 probe was hybridized to £coRI-cut DNA. b The Tn5 probe was hybridized to Psd-cut DNA. THe three sizelabelled bands refer to internal Tn5 fragments. c. The PSUP202 probe was hybridized to EcoRI-cut DNA. pSUP202 DNA was run on the gel as a positive control. In the case of (a) and (b), the sizes and positions of HirjdIII-digested lambda standards are shown.

1570

M. J. Hamblin. J. G. Shaw. J. P. Curson and D. J. Kelly

insertion was responsible for the unexpected difference in growth of the mutants under photo- and chemoheterotrophic conditions, a series of Southern hybridizations was performed using both a digoxenin-labelled Tn5 probe (pRZ104; ColE1::Tn5) and a pSUP202 probe (to rule out the possibility that 'suicide vector' sequences were also present in the mutant genomes). The results (Fig. 2a) clearly showed that all five mutants contained a single band of about 10kb which hybridized to the Tn5 probe in an EcoRI digest of chromosomal DNA and that no pSUP202 sequences were present (Fig. 2c). As EcoRI does not cut within Tn5, these results indicate that each mutant contains only a single copy of the transposon. In addition, the size of the chromosomal EcoRI fragment containing the insertions is about 4 kb (assuming Tn5to be 5.8kb (Jorgansen etai, 1979) and that each insertion has occurred in the same fragment). Hybridization of the Tn5 probe to Psfl digests of chromosomal DNA revealed clear differences in the sites of the insertions (Fig. 2b). The precise locations of the Tn5 insertions were determined using these data and additional information from Southern blots probed with a part of the 4kb fcoRI fragment cloned from MJH28. The results are shown in Fig. 3b and confirm that all the insertions are in the same EcoRI fragment.

Cloning and complementation analysis of C4dicarboxylate transport genes A wild-type cosmid gene bank (Colbeau ef al., 1986) consisting of a collection of recombinant pLAFRI clones constructed from R. capsulatus strain BIO DNA was transferred to MJH28 at a frequency of approximately 10"^ tetracycline-resistant colonies per recipient in a triparental mating with pRK2013 as helper plasmid. pLAFRI clones able to complement the lesion in M JH28 in trans were selected aerobically on minimal-malate plates containing both kanamycin (to maintain selection for Tn5) and tetracycline (to select pLAFRI). The frequency of such mar Km" Tc"^ colonies was approximately 10"^ per pLAFRI-containing recipient selected on pyruvate. Twelve colonies were purified and their cosmids examined by complete EcoRI digestion. Three distinct types of cosmid could be distinguished from the restriction pattern. One of each type — designated pDCTIOO. pDCT200 and pDCT300 — was subjected to more detailed restriction mapping to order the EcoR) fragments wtfhin the inserts (Fig. 3a). The three cosmids were found to contain overlapping insert DNA with an 8.3 kb region in common, consisting of two EcoRI fragments of 4.0 and 4,3 kb. pDCT300 has since proved to be unstable and to undergo deletions of the insert DNA; it has not been analysed further. All of the EcoRI fragments from pDCT200 were subcloned individually into pRK415.

but none of the resulting plasmids was able to complement MJH28. However, a subclone which contained the entire 8.3kb common region (pDCT205) was able to restore MJH28 to aerobic growth on malate plates. pDCT205 was subsequently found to complement the other four dot mutants isolated in this study. The transport properties and growth rates of the complemented mutants were examined. The rate of aerobic malate uptake in MJH28 complemented with pDCT205 was similar to that of the wild type, whereas no uptake was observed in the mutant in the absence of this plasmid (Fig. la). The doubling times of the wild type and MJH28{pDCT205) under aerobic conditions in the dark and under anaerobic conditions in the light with malate as carbon source were found to be the same (4,2h), A comparison of the restriction maps of the inserts in pDKlOO and pDCT205 Is shown Jn Fig. 3b. along with the positions of the corresponding chromosomal Tn5 insertions in each of the mutants. It is evident that there is some restriction-site polymorphism in the DNA derived from strains MJH28(pDK100) and B10{pDCT205), However, the alignment shown is based on the positions of nine restnction enzyme sites which were identical in the two DNAs over the 4.0 kb region. Moreover, Southern blots of EcoRI-cut pDCT205 showed that the 4.0kb fragment in this plasmid hybridized specifically with that in pDKlOO (not shown), whereas there was no hybridization between pDKlOO and the contiguous 4.3 kb fragment in pDCT205. Subclones of pDCT205 were constructed in pRK415 (Fig, 3b), with all of the inserts being cloned in the same orientation. From the complementation patterns obtained in trans after SI 7-1-mediated conjugation, the mutants could be divided into three distinct groups: MJH28 defined one group; MJH20. 22 and 25 defined a second group; and MJH40 defined the third (Fig. 3b).

Discussion Transposon mutagenesis combined with a simple screening procedure for pyruvate-positive, D,L-malate-negative grovrth under aerobic conditions has led to the isolation of mutants of R. capsulatus deficient in their ability to transport C4-dicarboxylates, The five mutants isolated in this study appear to have an identical phenotype. and each contains a single Tn5 insertion as judged both by Southern blotting and reversion analysis. Evidence from both genetic and physiological experiments suggests that the locus inactivated encodes a transport system that recognizes malate. succinate and fumarate. We have cloned this locus and designated it dct by analogy with similar systems identified in both E. coli (Kay and Kornberg. 1969) and Rhizobium {Ronson etaL. 1981), The dc( genes identified in this study are located within an 8.3 kb region of the R. capsulatus chromosome.

Cloning o f dct genes from Rhodobacter capsulatus 1571

MJH 28 mQl»-ma['*"

pDCTlOO;



pDCT200. E E INSERT SIZE 67

pDCT201. 202 203

43 3-5 6-3

205

b) (.0

22ffi 20 26

i.0

22/2S 20 2S

TTTT

2Kb

pDCT 205 pDCT 207 pDCT20B pDK 101 pDCT 209 pDCT212

Although the Tn5 insertions in the five mutants isolated were all located on a single 4.0kb fcoRI fragment, it is clear from the complementation pattern obtained with MJH28 that the dct gene region extends into the contiguous 4,3kb fragment to a point beyond the unique SamHI site. The complementation pattern obtained with the other mutants was more complex. A second complementation group was defined by the insertions in MJH20, 22 and 25. based on the pattern of complementation by PDCT208, 209 and pDKIOI. However, MJH40 (and no other mutant) was complemented by pDCT209, thus separating it into a third complementation group. pDCT209 does not contain a 0,8kb region of DNA between the extreme left-hand fcoRI and Psfl sites of the pDCT205 insert which is present in the other constructs used. One possible explanation for the observed complementation pattern is that this region may encode an additional gene product essential for growth on C4-dicarboxylafes. If the Tn5 insertions in MJH20, 22 and 25 were polar on the expression of this product, then pDCT209 would be unable to complement these mutants. The complementation observed when pDCT209 was placed in

Fig, 3. a. Physical maps of cosmid inserts and plasmid subclones thereof which are able lo complement MJH28 to aerobic growth on malate. The cosmida IpDCTI 00, 200 and 300| were selected from the pLAFRI gene bank as described m the Experimentai procedures and mapped using fcoRI. H/ndlll, BamHI and Psfl digestions to establish the order of the fragments and to confirm the area of overlap. Only the fcoRI map of the inserts is shown tor clarity. Subclones of pDCT2CI0 were made in pRK415 and complementation was checked by mobilization of plasmids from E. cod S17-1 into MJH28. b. Mapping of the positions of Tn5 insertions in ihe dct mutants, alignment of the cloned wildtype DNA, and complementation analyses of pDCT205 and pDKlOO. The filled triangles represent the positions of chromosomal Tn5 insertions, the numbers above them refernng to the corresponding MJH mutant. The surrounding restriction map of the 4.0Kb EcoRI fragment was determined from the insert in pDKlOO, which was cloned from MJH28. The alignment of this fragment with the insert in pOCT205 (denved (rom the strain BIO gene bank) is based on the restnction-site identities shown and the results of Southern hybridization (see text). The subclones indicated were transferred to each dct mutant by E. co//S17-i-mediated conjugation. Complementation to aerobic growth on malate is indicated by ' * ' and was distinguished (rom repair of the lesions through recombination {which occurs at low frequency) by checking that Ihe number of malate-positive colonies obtained was similar to the number of colonies obtained on pyruvate plates containing kanamycin and tetracycline. M.C.S.: multiple cloning site of the pRK415 vector.

an MJH40 background, however, suggests that the product of this putative additional gene is being expressed from the chromosome in this mutant. This would only be possible if the insertion in MJH40 is non-polar. Downstream activation by some Tn5 insertions is a well-known phenomenon, and it has been reported that up to a third of Tn5 insertions may exhibit this property (Berg et ai, 1980). This explanation could be tested by the isolation of chromosomal transposon or interposon insertions in the 0.8kb EcoRI-PsM fragment, in order to determine whether yet another complementation group is present. Our data do, however, indicate the existence of at least three genes in this region, and we are now sequencing the 4.0kb fcoRI fragment to determine their location and identity. The R. capsulatus dct system has a high affinity for L-malate. with a K, value of about 3 |i.M (J, G. Shaw, M. J. Hamblin and D. J. Kelly, unpublished) and it is clearly essential for chemoheterotrophic growth on C4-dicarboxyiates. However, all of the mutants isolated in this study were still able to grow slowly under photohelerotrophic conditions on malate and succinate. The possibility

1572

M. J. Hamblin. J. G. Shaw, J. P. Curson and D. J. Kelly

that simple diffusion is responsible for the observed phototrophic growth of the mutants seems unlikely for several reasons: (i) at physiological pH values, malate is almost completely dissociated (pKI = 3.40. pK2 = 5,11) and the anionic forms cannot be transported by diffusion or uniport in the presence of an opposing membrane potential, inside negative; (ii) no growth of the mutants was observed aerobically on malate or succinate. yet diffusion would occur irrespective of growth conditions; and (iii) fumarate, which has very similar dissociation characteristics to malate, did not support photoheterotrophic growth of the mutants. These observations strongly suggest that a specific transporter is involved. However, it is clear from the Kg values determined for the phototrophic

growth of MJH28 on malate that such an additional transport system would have a very low affinity for C4-dicarboxylates. This may be the reason why we were unable to detect any appreciable malate uptake in MJH28 cells grown on this substrate photoheterotrophically, as the concentrations used in the transport assays were in the micromolar range and thus well below the Ks of the cells for malate. Confirmation of the presence of such a transport system will clearly require the development of suitable assays, and it will be important to determine if it is distinct from the dct system or related to it in some way. We are currently constructing mutants that are completely unable to grow on malate under photo- or chemoheterotrophic conditions

Table 2. Strains and plasmids used in this study. strain or plasmid

Genotype/ phenotype

Construction

Source or reference

Rhodobacter capsulatus 37b4 MJH20 MJH22 MJH25 MJH28 MJH40

Wild type, Sm" dcf20::Tn5, Km"

G. Drews This study This study This study This study This study

dct22:Jr\5. Km" dct25.Tn5. Km" dcf28::Tn5, Km" C/cM0::Tn5. Km"

Escherictiia coli lRP4-2(Tc::Mu) (Km::Tn?ll in chromosome

Simon efa/. (1983)

S17-1

hsdR. pro. recA. Tp", Sp"

HB101

r m , recA^2. ara-\4. proA2.lacY-\.galK2. rpsL20. xyl-b, mf(-1 supE4&. \ , F

F.C. H. Franklin

DH1

r m *, recA 1, endA 1.

A. Motr

A , r

Cosmids and plasmids pLAFRI pRZ104 pBR322 pSUP202 PSUP2021 PRK2013 pRK415 pDCTlOO PDCT200 PDCT201 pDCT202 pDCT203 PDCT204 pDCT205 pDCT207 pDCT20e pDCT209 PDCT212 PDCT300 pDKlOO pDKlOl

IncP, Tc" Km" Ap", Tc" Mob'. Ap", Tc", Cm" Mob', Ap", Km", Cm" Mob', Tra •, Km" IncP, Tc" Tc" Tc" Tc" Tc"* TC" Tc" Tc" Tc" Tc" Tc" Ic" Tc^ Ap", Tc", Km" Ap", Tc"

pRK290::cos ColEl;:Tn5 pBR325::mod pBR325:;mob::Tn6 Helper plasmid pRK404 + pUC19 polylinker 30kb insert of R. capsutatus DNA in pLAFRI 18.5kb insert of R. capsulatus DNA in pLAFRI 6.7kb EcoRI subclone in pRK415 4.0kb EcoRI subclone in pRK415 4.3kb EcoRI subclone in pRK415 3.5kb EcoRI subclone in pRK415 8.3kO EcoRI subclone in pRK415 5.5kb H/ndlll subclone in pRK415 4.5kb EcoRI-BamHI subclone in pRK415 2.9kb Psfl subclone in pRK415 2.8kb EraRI-Hiodlll subclone in pRK415 20kb insert of R. capsulatus DNA in pLAFRl 10kb insert of MJH28 DNA in pBR322 EcoRI-Sstl subclone ot pDKlOO (3.4 kb insert) in pRK415

Fnedman efa'. (1982) Jorgensen e/af (1979) Bolivar el a/. (1977) Simon efa/. (1983) Simon etal. (1983) Ditta efa/. (1980} Keen efa/. (1988) This study This study This study This study This study This study This sludy This study This study This study This study This study This study This study

Cloning of6cX genes from Rhodobacter capsulatus and which should help in identifying the function and characteristics of any additional dicarboxylate uptake routes in this bacterium. Experimental procedures Bacterial strains, plasmids and growth conditions The strains and plasmids used in this study are listed in Table 2. R. capsulatus was grown routinely in RCV minimal medium (Weaver e( ai. 1975; Hillmer and Gest, 1977) at SO'C With D,L-malate or sodium pyruvafe as carbon source. Photoheterolrophic growth curves of wild-type and mutant strains were obtained in completely filled test tubes capped with suba-seals, which were designed to fit into the sample compartment of a Corning type 252 photometer equipped with a 580 nm filter. The tubes were illuminated by a 100W tungsten bulb at a distance of 30cm. Chemoheterotrophic growth curves were obtained using 25 ml of medium in 250ml shake flasks with side-arms tor optical density measurement as above. The flasks were shaken al 250 r.p.m. in Ihe dark. In all cases, inocula were 0.4% (v/v) and carbon sources 0.4% (w/v). All E. coli strains were grown al 37^0 in nutrient broth under aerobic conditions. Antibiotics were used at the fallowing concentrations: tetracycline, 10 ^.g ml ' (for E. coli) or 1 [ig ml ' (for R. capsulatusY. kanamycin, 25 \i.g ml ' for both species.

Transposon mutagenesis Conjugations between E. co//S17-1 (pSUP2021) and R. capsulatus 37b4 were performed aerobically on membrane filters placed on fhe surface of RCV-malate plates containing 0.05% [w/v} yeast extract (Kelly ef al., 1988). After 5-6 h at 30X, serial dilutions were plated on RCV-pyruvate minimal medium containing kanamycin to select for Tn5. The donor was counterselected by auxotrophy.

Enzyme assays Cell-free extracts were prepared from 250ml cultures grown aerobically in the dark on RCV-pyruvate n'ledium by resuspension in 1 ml 50mM Tris-HCI buffer (pH 8.0) followed by sonicalion and microfugation (10 min) to remove debris. Citrate synthase and malate dehydrogenase were assayed according to Kelly (1988), aconilase and fumarase at 240nm according to Reeves ef al. (1971), isocitrate dehydrogenase according to Leyland et al. (1989), and succinate dehydrogenase according to Morgan ef aA (1986). Protein was determined by the Lowry method.

1573

in the assay mixture. When required, the electrode could be illuminated via a fibre-optiC light guide connected to a Schott KL-1500T 150W light source. Cells were added to a final concentration of 0.1-0.2 mg cell protein ml ' (wild type) or 3-4 mg cell protein ml ' (mutant cells) and allowed to equilibrate for 5 mm before addition of 6jj.M (22.2kBq) ["*C]-L-malate (uniformly labelled; specific activity 1.85-2.2 MBq nmol '). This concentration was found to give near-maximal uptake rates with wild-lype cells. Samples (lOOjil) were taken at intervals, added to 5 ml of stop buffer (lOmM Na D,L-malate, 0.2mM Na fluoracetate in lOmM phosphate buffer, pH 7.0) and rapidly filtered onto nitrocellulose filter discs which were then washed with 10ml of stop buffer, dried, and scinlillalion-counted in 3ml of Cocktail T fluid (Beckman).

Cloning of transport genes A gene bank constructed by A. Colbeau, P. Godfroy and P. Vignais (Colbeau ef ai. 1986) was generously donated by H. Hudig (University of Freiburg, FRG). It consists of eight pools of 200 randomly picked colonies of E. coli HB101, each containing a size-fraciionated partial fcoRI fragment ot chromosomal DNA from R. capsulatus strain BIO, cloned into the broad host-range cosmid vector, pLAFRl (Fnedman ef ai. 1982). The average insert size is approximately 20kb. Each pool was conjugated with R. capsulatus MJH28 in a separate triparental filter mating with pRK2013 as helper plasmid. Transconjugants were selected aerobically in the dark on RCV-malate plates containing kanamycin and letracycline. The efficiency of transfer was determined on RCV-pyruvate plates with the same antibiotics.

DNA isolation and manipulation Small-scale isolation of cosmids and plasmids from both R. capsulatus and E. coli was done using the rapid boiling method (Holmes and Quigley, 1981). Large-scale preparations were obtained after lysozyme/sodium dodecyl sulphate treatment followed by PEG precipitation and caesium chloride isopycnic cenlrifugation. Restriction mapping, agarose gel electrophoresis and ligation were performed according to standard techniques [Maniatis ef al., 1982). Bacterial transformation was performed according to Hanahan (1985). For Southern blotting, DNA fragments were separated on 0.5% agarose gels, denatured, and transferred to nitrocellulose. Filters were hybridized lo digoxeninlabelled probes made using a non-radioactive DNA labelling and detection kit (Boehringer Mannheim) under the conditions recommended by the manufacturer.

Acknowledgements Transport assays After han/esting by centrifugation. cells were washed once in RCV medium lacking any carbon source (RCV-C) and resuspended in the same medium at a concentration of 30-40mg cell protein ml '. The cells were stored in the dark on ice and used within 6 h. A Clarke-type oxygen electrode maintained at 3O'C was filled with 2 ml of RCV-C and sparged continuously either with compressed air in the dark for aerobic assays, or with a slow stream of oxygen-free nitrogen gas passed over the surface for anaerobic assays. In the latter case, an oxygen-scavenging system (0.3% glucose, 2,6 U glucose oxidase and 0.02 U catalase) was included

We would like lo Ihank the Science and Engineenng Research Council for a research grant to D.J.K. and a studentship to J.G.S. We also thank the Society for General Microbiology for generous financial support in the form of a consumables and equipment grant to D.J.K.

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Mutagenesis, cloning and complementation analysis of C4 -dicarboxylate transport genes from Rhodobacter capsulatus.

Transposon mutagenesis was used to isolate insertion mutants of the photosynthetic bacterium Rhodobacter capsulatus which were unable to grow under ae...
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