Gene, 144 (1994) 139-140 0 1994 Elsevier Science B.V. All rights reserved. 0378-1119/94/$07.00

GENE 07961

Cloning and characterisation of the gene encoding P~~~d~~on~~ stutzeri

(Nucleotide sequencing; Gram-negative bacteria; di-heme; recombinant DNA)

H.E.M. Christensen

Institute ofMolecular and Cell Biology, National UniuersifJ? ofSingapore, Singupore 0511, Singapore

139

cytochrome c4 from

Received by J.A. Engler: 7 February 1994; Accepted: 21 February 1994; Received at publishers: 14 March 1994

SUMMARY

By use of a degenerate oligodeoxyribonucleotide probe corresponding to the N-terminal amino acid (aa) sequence and Southern blot analysis, the gene encoding the di-heme cytochrome cq (CC4) from Pseu~o~o~ffs stutzeri has been cloned. The aa sequence deduced from the nucleotide sequence shows a 20-aa signal peptide and a 190-aa mature protein with 79% identity to the Azotobacter vinelandii CC4 sequence, which had earlier been determined by aa sequencing.

CC4 is a di-heme single polypeptide periplasmic lo- cated protein found in Gram- bacteria. Its function is virtually unknown but it is believed to be an immediate electron donor to terminal oxidation systems (Pettigrew and Brown, 1988). The primary structure of genes encod- ing five other c-type heme proteins from Pseudomonas stutzeri has been reported (Jungst et al., 1991). Multi- heme cytochromes is expected to play an important role in future analysis of electron transport networks in multi- centre metalloproteins. In this respect CC4, with two centres is one of the simplest of the multi-centre metallo- proteins. In order to further elucidate its structure and function the CC4 from Pseudomonas stutzeri has been cloned and the first nt sequence for CC4 is reported here. Based on the N-terminal protein sequence available (Ambler and Murray, 1973) a mixture of degenerate 53-nt oligomers was synthesized and end-labelled with ‘*P. Chromosomal DNA of P. stutzeri (A.T.C.C. 11607) was

digested with SacI, NotI, EcoRI and XhoI and used in a Southern blot analysis shown in Fig. 1. The 4.5kb Sac1 band was cloned into hZAPI1 (Stratagene, La Jolla, CA, USA) and the clone of interest was then obtained by screening the &ZAP11 clones using the same probe as for the southern blot analysis. It was then converted into the SK-phagemid and sequenced. The nt sequence of the CC4 is shown in Fig. 2 together with the deduced aa sequence. It revealed several interesting features. It has a putative 20-aa signal peptide with a cleavage site (AlaXaaAlaAla) that is typical of outer membrane proteins (Lee et al., 1991; Jungst et al., 1991). The C+G content of the ORF is high at 61.8%, which is characteristic of P. stutzeri ORF’s. The mature protein has the same size, 190 aa, as the Azotobacter u~~e~andii CC4 (Ambler et al., 1984). The aa identity is 79% and with conservative substitutions included the similarity is 88%.

Correspondence to: Dr. H.E.M. Christensen, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore. Tel. (65) 7723035; Fax (65) 7591117.

Abbreviations: aa, amino acid(s) A.T.C.C., American Type Culture Collection; bp, base pair(s); CC4, cytochrome c,; CC4, gene encoding CC4; kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame; P., Pseudomonas; Xaa, any aa.

SSDI 0378-I 119(94)00172-0

140

A

kb

12.0

6.0

4.0

B

S E S

I i I I

0 2 3

kb

Fig. 1. Southern blot analysis of genomic DNA of P. stutzeri (A.T.T.C.

11607) (A) and restriction endonuclease map of the cloned DNA frag-

ment (B). The open arrow marks the position of the CC4 and the

direction of transcription. E, EcoRI; S, SacI.

REFERENCES

Ambler, R.P. and Murray, S.: N-terminal amino acid sequences of cyto-

chromes cg from Azotobacter ~~nela~dii and denitrif~ng pseudomon-

ades. Biochem. Sot. Trans. 1 (1973) 162-164.

Ambler, R.P., Daniel, M., Melis, K. and Stout, C.D.: The amino acid

sequence of the dihaem cytochrome cd from the bacterium

Azotobacter uinelandii. Biochem. J. 222 (1984) 217-227.

Jungst, A., Wakabayashi, S., Matsubara, H. and Zumft, W.G.: The

nirSTBM region coding for cytochrome cd, -dependent nitrite respi-

ration of pseudomonas stutzeri consists of a cluster of mono-, di-,

and tetraheme proteins. FEBS Lett. 279 (1991) 205-209.

Lee, H.S., Abdelal, A.H.T., Clark, M.A. and Ingraham, J.L.: Molecular

characterization of nosA, a Pseudomonas stutzeri gene encoding an

outer membrane protein required to make copper-containing N,O

reductase. J. Bacterial. 173 (1991) 5406-5413.

Pettigrew, G.W. and Brown. K.R.: Free and membrane-bound forms

of bacterial cytochrome c.,. Biochem. J. 252 (1988) 427-435.

GAATTCCTTTCAAACCCTTAGCCGTAGTTGGBTTAGCTGACTCGTGAGT 60 MNKVLVS 7

CTGCTGTTGACCCTTGGCATCACCGGTATGOCCCACGCGGT 120 L L L T L G I TG M A H AIA G DA E AG 27

CA~~~G~CAGM~~TGfGTGCCRCGGTGTCGATGCG 180 QGKVAVCGACHGVDGNSPAP 47

AATTTCCCGkAACTGGCTGGCCAGGGTGAGCGTTACeTGCGTTACCTGCTC~GC~C~AGGACATC 240 NFPKLAGQGERYLLKQLQDI 67

AAGGCTGGCAGCACACCGGGTGCCCCGGAAGGCGTAGGTCTGACC 300 KAGSTPGAPEGVGRKVLEMT 87

GGCATGCTGGACCCGfTGAGTGATCAGGATCTGGAAGACAC 360 GMLDPLSDQDLEDIAAYFSS 107

CAG~GGG~G~T~~ACGCCGATCC~TC~GC~~A~GAG~~~T~ 420 QKGSVGYADPALAKQGEKLF 127

CGTGGTGGCAAGCTCGATCAGGGCATGCCGGCGTGCACCG 480 RGGKLDQGMPACTGCHAPNG 147

GTCGGTAACGACCTGGCTGGCTTCCCCAAGCTGGGTGGGCCGCC 540 “GNDLAGFPKLGGQHAAYTA 167

AAGCAGCTGACCGACTTCCGCGRAGGCAATCGCACCAACG 600 KQLTDFREGNRTNDGDTMIN 287

CGTGGCGTCGCCGCCRAGeTGAGCAACAAGGACATCG~GffiCTGTCCAGCTACA~CAG 660 RGVAAKLSNKDIEALSSYIQ 207

GGCCTGCACTGATRACAGCGGCAGGTTGAACAGAAGGGCGCGGC 720 G L H * 210

GCCCTTTTGCGTTTTCACGCCTGGCTACAC~TCTTCGTCACGGCTTTACACGCGCGGAT 780

GRARRRCGCTGGCGAGTCCnACGTTTTGCTGCAGCCTCT 840

~CAG~GTATTT~A~CGC~~~TA~~AC~CCG 879

Fig. 2. The nt sequence of the CC4 from P. stutzeri and the deduced

aa sequence (GenBank accession No. U05988). The aa are numbered

starting with the first in-frame methionine as 1. A, N-terminal end of

mature CC4. The translation termination codon is designated by *. The

putative ribosome-binding site is underlined.

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