Marine Photosynthetic Bacterium Rhodopseudomonas sp. - Journal of

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Seventy-six strains of marine photosynthetic bacteria were analyzed by agarose gel electrophoresis for plasmid DNA content. Among these strains, 12 carried ...
JOURNAL OF BACTERIOLOGY, OCt. 1986, p. 460-463 0021-9193/86/100460-04$02.00/0 Copyright © 1986, American Society for Microbiology

Vol. 168, No. 1

Development of a Gene Cloning System for the Hydrogen-Producing Marine Photosynthetic Bacterium Rhodopseudomonas sp. TADASHI MATSUNAGA,1* NAOKI MATSUNAGA,1 KAZUFUMI TSUBAKI,1 AND TERUO TANAKA2 Department of Applied Chemistry for Resources, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, and Laboratory of Biochemical Reactions and Biocatalysis, Mitsubishikasei Institute of Life Sciences, Machida, Tokyo 194,2 Japan Received 24 March 1986/Accepted 22 July 1986

Seventy-six strains of marine photosynthetic bacteria were analyzed by agarose gel electrophoresis for plasmid DNA content. Among these strains, 12 carried two to four different plasmids with sizes ranging from 3.1 to 11.0 megadaltons. The marine photosynthetic bacterium Rhodopseudomonas sp. NKPBOO2106 had two plasmids, pRD06S and pRD06L. The smafler plasmid, pRD06S, had a molecular weight of 3.8 megadaltons and was cut at a single site by restriction endonucleases Sall, SmaI, PstI, XhoI, and Bgl. Moreover, the marine photosynthetic bacterium Rhodopseudomonas sp. NKPB002106 containing plasmid pRD06 had a satisfactory growth rate (doubling time, 7.5 h), a hydrogen-producing rate of 0.96 ,umol/mg (dry weight) of cells per h, and nitrogen fixation capability. Plasmid pRD06S, however, had neither drug resistance nor heavy-metal resistance, and its copy number was less than 10. Therefore, a recombinant plasmid consisting of pRD06S and Escherichia coli cloning vector pUC13 was constructed and cloned in E. coli. The recombinant plasmid was transformed into Rhodopseudomonas sp. NKPBOO2106. As a result, Rhodopseudomonas sp. NKPBOO2106 developed ampicillin resistance. Thus, a shuttle vector for gene transfer was constructed for marine photosynthetic bacteria.

Recently, considerable interest has arisen worldwide in the development of new energy production systems. Hydrogen production utilizing marine microorganisms and solar energy is one approach that has been investigated. Several strains of marine photosynthetic bacteria exhibit high hydrogen production rates (8). Moreover, organic wastes can be utilized as electron donors by photosynthetic bacteria. Both hydrogen production and wastewater treatment are simultaneously carried out by photosynthetic bacteria (9). However, increased hydrogen-producing capability is still required for the application of marine photosynthetic bacteria to energy production. For wastewater treatment, cellulose degradation capability is also necessary in the marine photosynthetic bacteria. Recently, techniques of genetic recombination have progressed rapidly and have been applied in many fields. It may be possible to introduce nitrogenase and cellulase genes into marine photosynthetic bacteria by genetic recombination techniques. For this purpose, a hostvector system for marine photosynthetic bacteria is necessary. Cloning requires a suitable host and an appropriately marked vector that can be propagated in the host. Suyama and Gibson (14) were the first to find plasmid DNA in photosynthetic bacteria. Plasmid DNA was detected in Rhodopseudomonas sphaeroides and Chromatium sp. Gibson and Niederman (4) isolated two plasmids having masses approximating 75 megadaltons (MDa) from R. sphaeroides, whereas Saunders et al. (13) detected three plasmids in R. sphaeroides and estimated their masses to be 28, 66, and 75 MDa. Hu and Marrs (5) showed that R. capsulata has two plasmids of 74 and 94 MDa. A single plasmid DNA of 36.3 MDa was also found in Rhodospirillum rubrum (7). However, the structural and functional properties of these plasmids remain unknown, although Saunders et al. (13), Fomari et al. (3), and Nano and Kaplan (10) observed a correlation between a photosynthesis-minus phe*

notype and rearranged plasmid sequences. Therefore, at least one marker had to be introduced to develop a cloning system of photosynthetic bacteria. Moreover, plasmid DNA has not been isolated from marine photosynthetic bacteria. This paper describes the isolation and partial characterization of plasmid DNAs from the marine photosynthetic bacterium Rhodopseudomonas sp. Furthermore, a recombinant plasmid consisting of a plasmid isolated from this bacterium and of M13-derived cloning vector pUC13 was constructed and introduced in E. coli. It was observed that the recombinant plasmid was capable of replicating in the Rhodopseudomonas cells. Hydrogen production capability and substrate specificity were also studied for marine photosynthetic bacteria containing plasmid DNAs. Marine photosynthetic bacteria were isolated in our laboratory at the Tokyo University of Agriculture and Technology. Modified RCVB medium was made by adding 3% NaCl to the RCVB medium described by Ormerod et al. (11). Isolated marine photosynthetic bacteria were designated by genera according to the classification of Imhoff and Truper

(6).

The isolation of plasmid DNA from marine photosynthetic bacteria on a preparative scale was carried out by the modified procedure of Van den Hondel et al. (16). The isolation of plasmid DNA from E. coli was done by the alkaline extraction method (1), followed by CsCl-ethidium bromide density gradient centrifugation. Digestion with restriction endonucleases was carried out under the conditions described by Thomas and Davis (15). Construction of a hybrid plasmid was performed with a plasmid obtained from marine photosynthetic bacteria and the cloning vector pUC13 (1.8 MDa) (12). pUC13 and plasmids pRD06L and pRD06S obtained from Rhodopseudomonas sp. NKPBOO2106 were linearized by Sail, BamHI, SmaI, or EcoRI, and plasmids were ligated with T4 ligase. The ligation mixtures were transformed into E. coli JM103 by the standard Ca2+ treatment procedure similar to

Corresponding author. 460

NOTES

VOL. 168, 1986

461

TABLE 1. Plasmids in marine photosynthetic bacteria No. of

Species and strain

plasmids

Rhodopseudomonas sp. NKPBOO2106

2

Plasmid size (MDa)

NKPBOO2114 NKPBOO2130 NKPBO11707 NKPBO31703 NKPB031725

3 4 4 4 4

3.8 4.6 3.5 4.55 3.9 6.4 3.9 4.9 3.5 5.0 3.6, 3.9, 4.65 3.9, 4.9, 8.8, 11.0 3.1, 4.2, 6.6, 9.3 3.6, 3.9, 4.9, 9.5 3.1, 4.15, 7.6, 9.9

Rhodospirillum sp. NKPB030637 NKPB032739

4 6

3.6, 4.9, 7.4, 9.9 3.6, 4.9, 7.4, 7.8, 8.55, 9.9

NKPBO11714

NKPB011731

NKPB031702 NKPB033718

2 2

2 2

that described by Cohen and Chang (2). Selection was made for ampicillin resistance, and the isolates were screened for inactivation of the P-galactosidase gene. NKPB002106 cells were transformed by the same procedure as that used for E. coli. Occurrence of plasmid DNA in marine photosynthetic bacteria and restriction enzyme analysis of plasmid DNA. We have collected and isolated 300 strains of marine photosynthetic bacteria in Japan (T. Matsunaga, unpublished work). Among these strains, 76 were tested for plasmid occurrence by agarose gel electrophoresis. Molecular weights of plasmid DNAs were estimated with the aid of marker covalently closed circular DNAs, pBR322 (2.8 MDa), the replicative form of M13mp8 (4.6 MDa), pET272 (12.6 MDa) (T. Tanaka, unpublished work), pTM3 (9.3 MDa) (Tanaka, unpublished work), and pUC13 (1.8 MDa). Twelve strains of marine photosynthetic bacteria carried two to six different plasmids with molecular weights ranging from 3.1 to 11.0 MDa (Table 1). Rhodospirillum spp. NKPB030637 and NKPB032739 showed plasmids, four of which were of identical size (Table 1). Plasmids of 3.6, 3.9, 4.9, 7.4, and 9.9 MDa were found in several strains. However, 20 other plasmids were different in size. Five strains containing only two kinds of plasmids were employed for further restriction enzyme analysis. Ten plasmids were cut with EcoRI, BamHI, SalI, SmaI, and PstI. The 3.9-MDa plasmids of NKPBO11731 and NKPBO31702 seemed to be identical. The 3.5-MDa plasmids of NKPB011714 and NKPB033718 had the same restriction pattern. The NKPBOO2106 4.6-MDa plasmid has the same restriction sites as the 3.9-MDa plasmid. Strain NKPB0O 2106 has two plasmids, pRD06S (3.8 MDa) and pRD06L (4.6 MDa). The smaller plasmid, pRD06S, had a single cleavage site by restiction endonucleases SalI, SmaI, and PstI, but no sites for EcoRI, HindIlI, or BamHI. The larger plasmid, pRD06L, had single restriction sites for EcoRI and BamHI, no restriction site for Sall or SmaI, and two sites for PstI. Therefore, either plasmid is suitable for construction of a shuttle vector. Strain NKPB002106, containing plasmids pRD06S and pRD06L, produced hydrogen at rates as high as 0.96 ,mol/mg (dry weight) of cells per h. This strain utilized

No. of restriction Sall sites for:Smal

EcoRI

BamHI

0 1 0

0 1 0 0 1

Many 1 2 1 3 0

Many

Many 1

Many 0

Many

1 0

Many Many 0 0 0 0

Many Many

1 0 3 0 0 0 0 2 3 0

Pstl 1 2

Many Many 2

Many 2

Many Many 0

acetate, propionate, butyrate, fumarate, lactate, malate, pyruvate, sucrose, xylan, galactose, fructose, maltose, mannitol, and xylose. However, it did not produce hydrogen from and grow on cellulose (Matsunaga and Matsunaga, unpublished work). Therefore, for this strain to produce hydrogen from cellulose, it is necessary to introduce complete cellulose degradative genes including cellobiase into the marine photosynthetic bacterium by using genetic recombination techniques. Plasmids pRD06S and pRD06L of strain NKPBOO2106 were cured with ethidium bromide, and the colonies were picked up randomly. The absence of plasmids was confirmed by agarose electrophoresis in some presumptive cured strains. The hydrogen production rate of a cured strain was similar to that of NKPB002106 containing the plasmids. Utilization by the cured strain of the various substrates described above was also the same as that of the parental strain containing the plasmids. These results show that the genes involved in hydrogen production are not located on pRD06S and pRD06L. The parental and the plasmid-cured strains were sensitive to penicillin, ampicillin,

tetracycline, streptomycin, chloramphenicol, erythromycin, gentamicin, and neomycin, indicating that those plasmids do not specify resistance to these antibiotics. Therefore, selectable markers such as antibiotic resistance have to be introduced into pRD06S or pRD06L to construct a cloning system in strain NKPB0O2106. Construction of a shuttle vector between marine photosynthetic bacterium Rhodopseudomonas sp. and E. coli. Further restriction enzyme analysis of pRD06S and pRD06L was carried out with BglII, PvuII, XhoI, BamHI, and KpnI. Plasmid pRD06S was cut at a single site by Sall, SmaI, PstI, and XhoI. Plasmid pRD06L was cut at a single site by EcoRI and BamHI and at two sites by PstI. Digestion of the plasmid mixture from NKPB002106 with Sail, SmaI, and PstI resulted in linearization of pRD06S. pRD06L was linearized with EcoRI and BamHI or was cut twice with PstI. Plasmids linearized by the appropriate restriction enzymes were ligated with T4 ligase to the corresponding restriction enzyme digests of the E. coli vector pUC13 and were introduced in E. coli JM103 by transformation. Plasmids were reisolated from the ampicillin-resistant white colonies. As a result,

NOTES

462

J. BACTERIOL.

Eco RI

A

Sal I Bgl

PRD 06S

3 3.8 Md

1

2

Sma

I

Xho I Pst I B

Sal I

(pUG 13 1. 8 Md

Sal I

RDI

~~~~SmaI

ampicillin resistance gene, which is a useful marker for cloning in a marine photosynthetic bacterium such as Rhodopseudomonas sp. NKPBOO2106. Strain NKPBOO2106, containing plasmids pRD06S and pRD06L, shows sufficient hydrogen production capability. Recently, Kuhl et al. suggested that a plasmid of 55 kilobases might be involved in photosynthesis in Rhodospirillum rubrum (7). On the other hand, Hu and Marrs have demonstrated that all photosynthesis genes are coded by chromosomal DNA in Rhodopseudomonas capsulata (5). Rhodopseudomonas sp. NKPBOO2106 is ovoid (2 to 2.5 ,m wide by 2 to 3.5 ,um long), is motile by means of polar flagella, and multiplies by binary fission. The bacterium was dark brown. Therefore, Rhodopseudomonas sp. NKPBOO2106 is tentatively assigned to R. sphaeroides (T. Matsunaga and K. Tsubaki, unpublished work). Plasmids pRD06S and pRD06L were small plasmids (3.8 and 4.6 MDa, respectively; that is, 5.8 and 7 kilobases) compared with plasmid DNAs reported earlier. Plasmids pRD06S and PRD06L do not appear to be involved in photosynthesis. The function of these plasmids is not yet known. The hybrid plasmid pURDA might be a useful vector for gene cloning in hydrogen-producing marine photosynthetic bacterium Rhodopseudomonas sp. NKPBOO2106. Further studies are in progress in our laboratory for the cloning of cellulase gene in this bacterium. This work was partially supported by grant-in-aid for scientific research 59850134 from the Ministry of Science and Culture of Japan.

Sal I Sma I FIG. 1. (A) Restriction

maps of plasmids pRD06S and pRD06L from Rhodopseudomonas sp. NKPBOO2106. The map shows the restriction sites for Sall, EcoRI, SmaI, BamHI, PstI, and XhoI. (B) Diagrammatic representation of the construction of the hybrid plasmid pURDA. A Sall digest of each plasmid was used for ligation with T4 ligase. pURDA has ampicillin resistance and an inactivated 3-galactosidase gene.

hybrid plasmids (5.6 MDa) between pRD06S and pUC13 obtained when these plasmids were digested with SalI and SmaI. The recombinant plasmids pURDA and pURDB obtained by SalI and SmaI digestion, respectively, were tested for their ability to transform strain NKPBOO2106 to ampicillin resistance. Transformation to ampicillin resistance occurred at a frequency of 2.7 x 103 colonies per ,ug of DNA, and the recombinant plasmid pURDA was not lost in the host NKPBOO2106 through repeated subculturing in the medium containing ampicillin. No spontaneous loss of the ampicillin resistance marker was observed. The copy number of pURDA was six in the marine photosynthetic bacterium Rhodopseudomonas sp., which was similar to the copy numbers of pRD06S. The physical map of recombinant plasmid pURDA is shown in Fig. 1. This plasmid was cut at two sites by Sall. There were unique restriction enzyme sites for HindIII, PstI, Sall, AccI, Hindll, XbaI, BamHI, SmaI, XmaI, SstI, and EcoRI, which are derived from pUC13 polylinker. The recombinant plasmid also has an were

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70:1293-1297. 3. Fornari, C. S., M. Watkins, and S. Kaplan. 1984. Plasmid distribution and analysis in Rhodopseudomonas sphaeroides. Plasmid 11:39-47. 4. Gibson, K. D., and R. A. Niederman. 1970. Characterization of two circular satellite species of deoxyribonucleic acid in Rhodopseudomonas sphaeroides. Arch. Biochem. Biophys.

141:694-704. 5. Hu, N. T., and B. L. Marrs. 1979. Characterization of the plasmid DNAs of Rhodopseudomonas capsulata. Arch. Microbiol. 121:61-69. 6. Imhoff, J. F., and H. G. Truper. 1982. General classification of photosynthetic organisms, p. 513-522. In A. Mitsui and C. C. Black (ed.), Handbook of biosolar resources, vol. 1. Basic principles. CRC Press, Inc., Boca Raton, Fla. 7. Kuhl, S. A., D. W. Nix, and D. C. Yoch. 1983. Characterization of a Rhodospirillum rubrum plasmid: loss of photosynthetic growth in plasmidless strains. J. Bacteriol. 156:737-742. 8. Matsunaga, T., and A. Mitsui. 1982. Seawater-based production of hydrogen by immobilized marine photosynthetic bacteria. Biotechnol. Bioeng. Symp. 12:441-450. 9. Mitsui, A., T. Matsunaga, H. Ikemoto, and B. R. Renuka. 1985. Organic and inorganic waste treatment and simultaneous

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VOL. 168, 1986 nitrogen metabolism. Arch. Biochem. Biophys. 94:449-463. 12. Ruther, U. 1980. Construction and properties of new cloning vehicle, allowing direct screening for recombinant plasmids. Mol. Gen. Genet. 178:475-477. 13. Saunders, V. A., J. R. Saunders, and P. M. Bennet. 1976. Extrachromosomal deoxyribonucleic acid in wild-type and photosynthetically incompetent strains of Rhodopseudomonas sphaeroides. J. Bacteriol. 125:1180-1187.

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14. Suyama, Y., and J. Gibson. 1966. Satellite DNA in photosynthetic bacteria. Biochem. Biophys. Res. Commun. 24:549-553. 15. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. 16. Van den Hondel, C. A. M. J. J., W. Keegstra, W. E. Borrias, and G. A. van Arkel. 1979. Homology of plasmids in strains of unicellular cyanobacteria. Plasmid 2:323-333.