Journal of General Microbiology (1992), 138, 1365-1 370. Printed in Great Britain
1365
Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis secY SHIN-KWON KANG, TOSHIAKIKUDO^,^* and KOKIHORIKOSHI' Laboratory of Microbiology' and Biodesign Research Group2,Riken Institute and JRDC, Wako, Saitama 351-01, Japan (Received 4 February 1992; revised 25 March 1992; accepted 2 April 1992)
A 1.8 kb Hind111 DNA fragment containing the secY gene of alkalophilic BaciZZus sp. C125 has been cloned into plasmid pUC119 using the B. subtiZis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequencesof the B. subtilis proteins indicated that they were the genes for ribosomal protein L1 5-SecY-adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M, value has been calculated to be 47 100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic BaciZZus sp. C125 secY homologue showed 69.7% homology with that of B. subtiZissec Y. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmicdomains (73% identity) with less homologous regions present in extracellular domains (43% identity).
Introduction Genetic studies suggest that a secretion machinery exists for the export of protein to the cell envelope (Michaelis & Beckwith, 1982). Theprl series of genes, prlA (secY) (It0 et al., 1983), prlB, prlC and prlD (Emr et al., 1981; Bankatis & Bassford, 1985) identified by their ability to suppress mutations of an export-defective signal peptide, are believed to be involved in the secretion machinery of Escherichia coli. One of these genes, sec Y (prlA), encodes an integral membrane protein of 10 transmembrane segments (Akiyama & Ito, 1987) which is essential for protein export from the cytosol to the periplasm or outer membrane. Bacillus sp. strains have a remarkable ability to secrete many proteins, such as amylases, cellulases or proteases, into the culture medium. Recently several groups isolated and analysed the secY gene from B. subtilis (Yoshikawa & Doi, 1990; Nakamura et al., 1990). However, the protein export pathway or other protein components related to the protein secretion process in Bacillus remain unclear. Our group has a specific interest in alkalophilic
* Author for correspondence. Tel. 484 62 111 1; fax 484 66 5209. The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number D 10360. Abbreviation: Adk, adenylate kinase.
0001-7380 O 1992 SGM
bacteria (Horikoshi, 1991) and have recently selected alkalophilic Bacillus sp. C125 as a model alkalophile for molecular biological study (Kudo et al., 1990). In alkalophiles a cell-membrane-maintained pH difference between the internal (pH 8-8.6) and the external (pH 1011) environments exists. A membrane protein is likely to show special adaptations to alkaline conditions. In order to clarify the protein secretion system in alkalophilic Bacillus and to study the secY protein as a model membrane protein, the sec Y gene of alkalophilic Bacillus sp. C125 was cloned using the B. subtilis secY gene as a probe, and the cloned gene was characterized.
Methods Bacterial strains and plasmid. Alkalophilic Bacillus sp. C 125 (TrpUra- Cms) (Kudo et al., 1990) was used as the DNA source for Southern hybridization analysis and cloning of the secY gene. E. coli JM109 [recAI endAI gyrA96 thi-1 hsdRI7 supE44 relAl A- A(1acproAB) (F' proAB lacPZAMI5 traD36)I was used as the host (Nakamura et al., 1990). Plasmid pSY912 containing the B. subrilis secY gene was kindly provided by Dr H. Yoshikawa (Tokyo University, Tokyo, Japan) and used as a probe (Yoshikawa et al., 1990). Media and maintenance of strains. E. coli strain JM 109 was grown and maintained on LB medium (Maniatis et al., 1982). When necessary, ampicillin was added to the medium (1OOpg ml-I). Alkalophilic Bacillus sp. C125 was grown and maintained on Horikoshi I1 medium (Kudo et al., 1990).
I366
S . - K . Kang, T. Kudo and K. Horikoshi
General D N A techniques. DNA manipulations and transformation of E . coli were accomplished by the methods outlined by Maniatis et al. ( 1 982). Southern hybridization. Alkalophilic Bacillus sp. C 125 chromosomal DNA was digested with restriction endonucleases, electrophoresed on a l o < ( w j v ) agarose gel and then blotted on to a nylon membrane (Amersham) by the method of Southern (Southern, 1975). The membrane was prehybridized and hybridized in a 2004 (v/v) formamide hybridization solution at 42 "C. Subsequent washing and DNA detection were carried out according to the protocol provided with the digoxigenin DNA labelling and detection kit (Boehringer Mannheim). Sequence analysis. Nucleotide sequencing was carried out by the method of Sanger et al. (1977) on an Applied Biosystems DNA sequencer (373A) using single-stranded DNA prepared by the method of Vieira & Messing (1987) and an Applied Biosystems sequencing kit. Exonuclease I11 deletions were carried out by the method of Henikoff (1984).The DNA sequences were analysed using Macmolly Software.
Fig. 1. Southern hybridization analysis of alkalophilic bluc~llussp. C125 chromosomal DNA. Lanes: 1, Size marker (HindIII-digested 1 DNA and HaeIII-digested 4x174 DNA mixture); 2, EcoRI-digested C125 D N A ; 3, HindIII-digested C125 DNA. The plasmid pSY912 containing the B. subtilis secY gene was used as a probe.
Results and Discussion
L15
Cloning of' see Y D N A
0
To verify the existence of the secY gene in alkalophilic Bacillus sp. C125 chromosomal DNA, the DNA was digested with HindIII or EcoRI and then analysed by Southern hybridization using the B. subtilis secY gene as a probe (Fig. 1). A 1-8kb HindIII fragment and two EcoRI fragments ( 5 and 0.5 kb) were detected. The 1.8 kb HindIII fragment was chosen for cloning since it gave a strong signal and the size was such that it was likely to contain the complete secY gene. Fragments of HindIII-digested Bacillus sp. (2125 DNA of 1-8kb were purified from a 1% (w/v) agarose gel by electroelution, cloned into the HindIII site of pUC119 and transformed into E. coli. Two-thousand Ampr transformants were subjected to colony hybridization analysis and two positive colonies identified. Southern analysis of purified plasmid DNA from these two positive colonies showed positive bands. Restriction mapping of the two plasmids revealed that they contained the same 1.8 kb fragment in the same orientation. The plasmid was designated as pASY3. The physical map of the 1.8 kb HindIII fragment contained in pASY3 is presented in Fig. 2. D N A sequence analysis of the secY gene of alkalophilic Bacilius sp. Ci25
The nucleotide sequence of the entire 1.8 kb fragment contained in pASY3 was determined. The nucleotide and deduced amino acid sequences are presented in Fig. 3. Three ORFs, all in the same orientation, were identified. The first ORF lacking the N-terminal region consisted of 66 amino acids. This ORF shared 79.1% amino acid identity (51/66) with the ribosomal protein
SecY
.4d k I , ,
1 8 kb
1
EcoRI
\
BuniH I
C'/u I
Fig. 2. Physical map and alignment of the three ORFs in the cloned 1.8 kb HindIII fragment from alkalophilic Bacillus sp. C 115 chromosomal DNA.
L15 of B. subtilis (Yoshikawa & Doi, 1990; Nakamura et al., 1990). The second ORF, beginning with an ATG codon at position 202, consisted of 431 amino acids and showed 69.7% amino acid identity with the B. subtifis secY gene product (Yoshikawa & Doi, 1990; Nakamura et al., 1990). The third ORF, beginning with a TTG codon at position 1570, consisted of 107 amino acids and was terminated by the HindIII site. This ORF had 78.5% amino acid identity with the B. subtilis adk gene product (Yoshikawa & Doi, 1990; Nakamura et al., 1990). Typical sequences for ribosomal binding, upstream of the 2nd and 3rd ORFs were observed. These results indicated that the gene order around secY, given by the deduced gene product sequences was L 15-SecY-adenylate kinase (Adk) and identical to that observed in B. subtilis. The predicted amino acid sequence of the secY gene product of alkalophilic Bacillus sp. C125 was compared with that of the B. subtilis secY gene product (Fig. 4) (Yoshikawa & Doi, 1990). The number of amino acid residues of both gene products was the same (431 amino acids) and alkalophilic Bacillus sp. C125 SecY shared 69.7% amino acid identity with B. subtilis SecY. The hydrophobic profiles in the amino acid sequences were analysed by the procedure of Kyte & Doolittle (1982).
Alkalophilic Bacillus sp. C125 secY gene L15
Lye L e u A e n Arg P h e G l u A s p G l y T h r G l u V a l S e r P r o G l u L e u Leu L e u G l u BAG c?T AAT CGT TIT GAA GAT GGA ACA GAG GTT TCA CCA GAA CI" rlTG CIT GAA
54
Thr G l y V a l V a l Ser A s n A l a L y s Asp G l y I l e L y s I l e L e u G l y A s n G l y L y s L e u Glu ACA GGT G" GTI' AGC AAC GCA AAG GAC GGC AT" AAG ATC CTC GGT AAT GGA AAG CTA GAG
114
Hind I I I
Lye L y s Leu Thr V a l L y a A l a A a n L y e GTG AAA GCC AAC AAA Hpa I A l a G l y G l y L y S T h r G l u V a l I l e TER GCC GGC GGG AAA ACT W T G ATC TAA SD G l y A s p Leu A r g A r g LYE V a l I l e P h e AAG
P h e Ser A l a Ser A l a V a l G l u A l a I l e G l u A l a GAA GCT ATI' GAA GCT
TI'C TCT GCT T G T -
SecY
Pst I
174
M e t P h e Arg Thr Ile S e r A a n Ile Phe Arg V a l ATG "I'C CGA ACG ATC TCC AAC ATI' TIT CGA GTG
2 34
Thr L e u L e u M e t L e u I l e V a l P h e A r g I l e G l y GGT GAT TI'G CGC CGT AAG GTC ATT TTC ACC cIy3 CTC ATG CTC ATC GTT TIT CGG ATC GGA
294
Ser P h e I l e P r o V a l Pro G l y Thr A s n Arg G l u V a l L e u A e p P h e V a l A s p G l n A l a A s n AGT TIT ATC CCC GTI' CCA GGT ACA AAC CGA GAA GTG CTA GAT l?IT GTC GAT CAG GCA AAT
354
A l a Phe G l y P h e L e u A s n Thr P h e G l y G l y G l y A l a L e u G l y A m P h e Ser I l e P h e A l a GCA TTC GGT TI" TTA AAT ACG ??T GGA GGA GGG GCA Cl" GGG AAT TTC TCC ATC TIT GCG
4 14
M e t G l y I l e M e t P r o Tyr I l e Thr Ala Ser I l e V a l M e t G l n L e u L e u G l n M e t A e p V a l ATG GGG ATC ATG CCA TAC ATT ACA GCA TCC ATT GTC ATG CAA TTA TI'G CAG ATG GAT GTC
47 4
V a l P r o L y s Phe A l a G l u Trp A l a L y e G l u G l y G l u A l a G l y Arg Arg Lye Leu A l a G l n G a CAA GTT CCG AAA TI" GCT GAG TGG GCG AAA GAG GGC GAA GCA GGG CGT CGT
534
NheI
P h e Thr Arg Tyr G l y Thr I l e V a l V a l Leu G l y Phe I l e G l n A l a L e u G l y M e t Ser V a l
?TT ACC CGC TAT GGA ACG ATT GTT G'IT TTA GGG TIT ATT CAG GCA CTC GGG ATG TCG GTT
594
G l y Phe A s n A s n P h e Phe P r o G l y Leu I l e Pro A m Pro Ser V a l Ser V a l Tyr L e u Phe GGT TIT AAC AAC TTC TI'C CCA GGA 'ITA ATT CCT AAT CCA AGC GTG TCG GTG TAT CI'C TIT
6 54
I l e A l a Leu V a l Leu Thr Ala G l y Thr A l a Phe L e u M e t T r p L e u G l y G l u G l n I l e Thr ATC GCA CIT GTC Cl" KT GCLGGA ACG GCT W C TI'A ATG TGG CTC GGG GAG CAG ATT ACA
714
A l a L y e G l y V a l G l y A s n G l y I l e Ser I l e I l e I l e P h e A l a G l y I l e A l a A l a G l y I l e GCT AAG GGA GTC GGA AAC GGA ATC TCC ATT ATC ATC 'MT GCA GGG ATT GCT GCT
77 4
Pst I
EcoRI
Pro A m G l y L e u A e n Leu I l e T y r Ser T h r Arg I l e G l n A s p A l a G l y G l u G l n Leu P h e CCA AAT =GG TTA AAT CTC ATT TAC TCA ACA cr;C ATC CAA GAC GCA GGG GAG CAA TIy3 TTC
834
BumHI
L e u A m I l e V a l V a l I l e Leu Leu Leu A l a L e u A l a I l e L e u A l a I l e I l e V a l G l y V a l ! I " AAC ATI' GTG GTG ATC 'ITG ! I " CI" GCT -3" GCG ATT CXT GCC ATC ATI' G'FS GGC GTI'
894
I l e Phe V a l G l n G l n Ala Leu Arg L y s I l e Pro V a l G l n Tyr A l a L y s Arg Leu V a l G l y CGT AAA ATC CCT GTI' CAG TAT GCC AAG CGT CTA GTA GGG ATT TTC d C CAA CAA GCG
9 54
Arg A e n Pro V a l G l y G l y G l n Ser Thr H i s Leu P r o L e u L y e V a l A s n A l a A l a G l y V a l AGA AAC CCT G" GGA GGT CAG TCG ACC CAT CTI? CCG TTA AAA GTG AAT GCC GCA GGC GTT
1014
Sal I
I l e Pro V a l I l e Phe Ala L e u S e r Leu L e u I l e Phe P r o P r o Thr V a l A l a G l y L e u P h e ATP CCG GFC AT" ' I " GCG TI'A TCG "'G CTC ATT ' I " CCA CCA ACE GTT GCT GGG CTG TIT
1074
G l y Ser A8p H i e Pro V a l Ala A l a V a l I l e G l u Thr Phe A s p Tyr Thr H i e Leu I l e GGC AGT GAT CAT CCG W C GCI' GCA TGG Gn; ATI' GAG ACA TIT GAC TAT ACC CAC TIY; ATI'
1134
G l y M e t Ala V a l T y r Ala Leu Arg I l e I l e G l y Phe Thr Tyr P h e Tyr Ala Phe I l e G l n GGG ATG GCG GTA TAT GCC T I y 3 CGC ATC ATC GGA TIT ACG TAT "C TAT GCG TIT ATC CAA
1194
V a l A m Pro G l u Arg M e t A l a G l u A m L e u L y e Lye G l n G l y G l y T y r Ile P r o G l y I l e GTC AAC CCA GAG CGA AT0 GCG GAG AAC "G AAA AAA CAG GGT GGC TAT ATI' CCT
1254
EcoRI Arg Pro G l y Lye Ala Thr G l n Thr Tyr I l e Thr Pro I l e Leu Tyr Arg Leu Thr Phe V a l G T CCA GGG AAA GC?i ACG CAG ACG TAC ATC ACG CCT ATC TIG A--T & T . ACG TIT GTC ClUI Fig. 3.
1314
1367
1368
S.-K. Kang, T. Kudo and K . Horikoshi G l y Ser L e u Phe Leu Ala V a l V a l Ala I l e Leu P r o V a l Phe Phe I l e L y e P h e A l a A s p GGA TCA c?T TI'C CTC GCA GTG GTG GCG ATC "G CCG GTA TTC TIT ATT AAG TIT GCC GAC
1374
TTG CCA CAG GCG ATT
Leu P r o G l n A l a I l e G l n I l e G l y G l y Thr G l y Leu L e u I l e V a l V a l G l y V a l A l a L e u CAA ATT GGT GGT ACG GGC 'ITS CTC ATC GTC GTC GGG GTT GCC CTT
1434
A e p Thr M e t L y e G l n I l e G l u A l a G l n Leu I l e L y e Arg Ser Tp-L y e G l y Phe I l e L y e GAT ACG ATG AAG CAA ATT GAA GCA CAG "G ATC AAA CGT TCT TAT AAA GGG ' I " ATX' AAG
1494
TER TAG GGG GAG CAG GGA AGC GTG GTG TCT TCC CGT TCG CCT TCT GTT CIT AAA TTA TTA GAC
Adk Met A e n Leu A e n Leu M e t G l y Leu Pro G l y A l a G l y L y e G l y Thr
-G
1554
ATA GAC "G AAT CTG AAC CIT ATG GGA CTT CCT GGT GCT GGT AAA GGT ACA
1614
G l n A l a G l u Lye I l e I l e G l u L y s Tyr G l y I l e Pro His I l e S e r T h r G l y A s p M e t P h e CAG GCA GAA AAG ATC A'IT GAG AAG TAC GGC ATC CCA CAC ATT TCA ACA GGT GAC ATG TIT
1674
SD
Nsp I Arg Ala Ala M e t L y e A m G l u Thr G l u L e u G l y L e u L y e Ala L y e Ser Tyr M e t A s p A l a CGT GCT GCG ATG AAG AAC GAG ACC GAG C I T GGA TTA AAA GCA AAA TCG TAC ATG GAT GCA
1734
G l y G l u Leu V a l P r o A e p G l u V a l Thr I l e G l y I l e V a l Arg A e p Arg L e u S e r G l n A a p GGG GAA CI'G GTT CCT GAT GAA GTA ACG ATI' GGT ATC GTT CGG GAT CXT CTC AGT CAA GAC
1794
A e p Cye G l n A e n G l y Phe L e u L e u A s p G l y Phe P r o Arg Thr V a l A l a G l n A l a G l u A l a GAT TGC CAA AAT GGC TI" TIy3 CIT GAC GGG TI" CCA CGG ACT GTC GCT CAA GCA GAA GCG
1854
L e u G l u A e p I l e L e u A l a S e r L e u A e p LYE L y s Leu TTA GAA GAT ATT "G GCG TCA C l T GAT AAA BAG CIT
189 0
HindIII Fig. 3. Nucleotide and deduced amino acid sequences of the 1.8 kb HindIII fragment. Nucleotides are numbered from the 5'-end. The possible ribosome binding sites (SD) preceding each of the ORFs are underlined.
Hydrophobic 3.0
-1
-
I
.
1365
Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis secY SHIN-KWON KANG, TOSHIAKIKUDO^,^* and KOKIHORIKOSHI' Laboratory of Microbiology' and Biodesign Research Group2,Riken Institute and JRDC, Wako, Saitama 351-01, Japan (Received 4 February 1992; revised 25 March 1992; accepted 2 April 1992)
A 1.8 kb Hind111 DNA fragment containing the secY gene of alkalophilic BaciZZus sp. C125 has been cloned into plasmid pUC119 using the B. subtiZis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequencesof the B. subtilis proteins indicated that they were the genes for ribosomal protein L1 5-SecY-adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M, value has been calculated to be 47 100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic BaciZZus sp. C125 secY homologue showed 69.7% homology with that of B. subtiZissec Y. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmicdomains (73% identity) with less homologous regions present in extracellular domains (43% identity).
Introduction Genetic studies suggest that a secretion machinery exists for the export of protein to the cell envelope (Michaelis & Beckwith, 1982). Theprl series of genes, prlA (secY) (It0 et al., 1983), prlB, prlC and prlD (Emr et al., 1981; Bankatis & Bassford, 1985) identified by their ability to suppress mutations of an export-defective signal peptide, are believed to be involved in the secretion machinery of Escherichia coli. One of these genes, sec Y (prlA), encodes an integral membrane protein of 10 transmembrane segments (Akiyama & Ito, 1987) which is essential for protein export from the cytosol to the periplasm or outer membrane. Bacillus sp. strains have a remarkable ability to secrete many proteins, such as amylases, cellulases or proteases, into the culture medium. Recently several groups isolated and analysed the secY gene from B. subtilis (Yoshikawa & Doi, 1990; Nakamura et al., 1990). However, the protein export pathway or other protein components related to the protein secretion process in Bacillus remain unclear. Our group has a specific interest in alkalophilic
* Author for correspondence. Tel. 484 62 111 1; fax 484 66 5209. The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number D 10360. Abbreviation: Adk, adenylate kinase.
0001-7380 O 1992 SGM
bacteria (Horikoshi, 1991) and have recently selected alkalophilic Bacillus sp. C125 as a model alkalophile for molecular biological study (Kudo et al., 1990). In alkalophiles a cell-membrane-maintained pH difference between the internal (pH 8-8.6) and the external (pH 1011) environments exists. A membrane protein is likely to show special adaptations to alkaline conditions. In order to clarify the protein secretion system in alkalophilic Bacillus and to study the secY protein as a model membrane protein, the sec Y gene of alkalophilic Bacillus sp. C125 was cloned using the B. subtilis secY gene as a probe, and the cloned gene was characterized.
Methods Bacterial strains and plasmid. Alkalophilic Bacillus sp. C 125 (TrpUra- Cms) (Kudo et al., 1990) was used as the DNA source for Southern hybridization analysis and cloning of the secY gene. E. coli JM109 [recAI endAI gyrA96 thi-1 hsdRI7 supE44 relAl A- A(1acproAB) (F' proAB lacPZAMI5 traD36)I was used as the host (Nakamura et al., 1990). Plasmid pSY912 containing the B. subrilis secY gene was kindly provided by Dr H. Yoshikawa (Tokyo University, Tokyo, Japan) and used as a probe (Yoshikawa et al., 1990). Media and maintenance of strains. E. coli strain JM 109 was grown and maintained on LB medium (Maniatis et al., 1982). When necessary, ampicillin was added to the medium (1OOpg ml-I). Alkalophilic Bacillus sp. C125 was grown and maintained on Horikoshi I1 medium (Kudo et al., 1990).
I366
S . - K . Kang, T. Kudo and K. Horikoshi
General D N A techniques. DNA manipulations and transformation of E . coli were accomplished by the methods outlined by Maniatis et al. ( 1 982). Southern hybridization. Alkalophilic Bacillus sp. C 125 chromosomal DNA was digested with restriction endonucleases, electrophoresed on a l o < ( w j v ) agarose gel and then blotted on to a nylon membrane (Amersham) by the method of Southern (Southern, 1975). The membrane was prehybridized and hybridized in a 2004 (v/v) formamide hybridization solution at 42 "C. Subsequent washing and DNA detection were carried out according to the protocol provided with the digoxigenin DNA labelling and detection kit (Boehringer Mannheim). Sequence analysis. Nucleotide sequencing was carried out by the method of Sanger et al. (1977) on an Applied Biosystems DNA sequencer (373A) using single-stranded DNA prepared by the method of Vieira & Messing (1987) and an Applied Biosystems sequencing kit. Exonuclease I11 deletions were carried out by the method of Henikoff (1984).The DNA sequences were analysed using Macmolly Software.
Fig. 1. Southern hybridization analysis of alkalophilic bluc~llussp. C125 chromosomal DNA. Lanes: 1, Size marker (HindIII-digested 1 DNA and HaeIII-digested 4x174 DNA mixture); 2, EcoRI-digested C125 D N A ; 3, HindIII-digested C125 DNA. The plasmid pSY912 containing the B. subtilis secY gene was used as a probe.
Results and Discussion
L15
Cloning of' see Y D N A
0
To verify the existence of the secY gene in alkalophilic Bacillus sp. C125 chromosomal DNA, the DNA was digested with HindIII or EcoRI and then analysed by Southern hybridization using the B. subtilis secY gene as a probe (Fig. 1). A 1-8kb HindIII fragment and two EcoRI fragments ( 5 and 0.5 kb) were detected. The 1.8 kb HindIII fragment was chosen for cloning since it gave a strong signal and the size was such that it was likely to contain the complete secY gene. Fragments of HindIII-digested Bacillus sp. (2125 DNA of 1-8kb were purified from a 1% (w/v) agarose gel by electroelution, cloned into the HindIII site of pUC119 and transformed into E. coli. Two-thousand Ampr transformants were subjected to colony hybridization analysis and two positive colonies identified. Southern analysis of purified plasmid DNA from these two positive colonies showed positive bands. Restriction mapping of the two plasmids revealed that they contained the same 1.8 kb fragment in the same orientation. The plasmid was designated as pASY3. The physical map of the 1.8 kb HindIII fragment contained in pASY3 is presented in Fig. 2. D N A sequence analysis of the secY gene of alkalophilic Bacilius sp. Ci25
The nucleotide sequence of the entire 1.8 kb fragment contained in pASY3 was determined. The nucleotide and deduced amino acid sequences are presented in Fig. 3. Three ORFs, all in the same orientation, were identified. The first ORF lacking the N-terminal region consisted of 66 amino acids. This ORF shared 79.1% amino acid identity (51/66) with the ribosomal protein
SecY
.4d k I , ,
1 8 kb
1
EcoRI
\
BuniH I
C'/u I
Fig. 2. Physical map and alignment of the three ORFs in the cloned 1.8 kb HindIII fragment from alkalophilic Bacillus sp. C 115 chromosomal DNA.
L15 of B. subtilis (Yoshikawa & Doi, 1990; Nakamura et al., 1990). The second ORF, beginning with an ATG codon at position 202, consisted of 431 amino acids and showed 69.7% amino acid identity with the B. subtifis secY gene product (Yoshikawa & Doi, 1990; Nakamura et al., 1990). The third ORF, beginning with a TTG codon at position 1570, consisted of 107 amino acids and was terminated by the HindIII site. This ORF had 78.5% amino acid identity with the B. subtilis adk gene product (Yoshikawa & Doi, 1990; Nakamura et al., 1990). Typical sequences for ribosomal binding, upstream of the 2nd and 3rd ORFs were observed. These results indicated that the gene order around secY, given by the deduced gene product sequences was L 15-SecY-adenylate kinase (Adk) and identical to that observed in B. subtilis. The predicted amino acid sequence of the secY gene product of alkalophilic Bacillus sp. C125 was compared with that of the B. subtilis secY gene product (Fig. 4) (Yoshikawa & Doi, 1990). The number of amino acid residues of both gene products was the same (431 amino acids) and alkalophilic Bacillus sp. C125 SecY shared 69.7% amino acid identity with B. subtilis SecY. The hydrophobic profiles in the amino acid sequences were analysed by the procedure of Kyte & Doolittle (1982).
Alkalophilic Bacillus sp. C125 secY gene L15
Lye L e u A e n Arg P h e G l u A s p G l y T h r G l u V a l S e r P r o G l u L e u Leu L e u G l u BAG c?T AAT CGT TIT GAA GAT GGA ACA GAG GTT TCA CCA GAA CI" rlTG CIT GAA
54
Thr G l y V a l V a l Ser A s n A l a L y s Asp G l y I l e L y s I l e L e u G l y A s n G l y L y s L e u Glu ACA GGT G" GTI' AGC AAC GCA AAG GAC GGC AT" AAG ATC CTC GGT AAT GGA AAG CTA GAG
114
Hind I I I
Lye L y s Leu Thr V a l L y a A l a A a n L y e GTG AAA GCC AAC AAA Hpa I A l a G l y G l y L y S T h r G l u V a l I l e TER GCC GGC GGG AAA ACT W T G ATC TAA SD G l y A s p Leu A r g A r g LYE V a l I l e P h e AAG
P h e Ser A l a Ser A l a V a l G l u A l a I l e G l u A l a GAA GCT ATI' GAA GCT
TI'C TCT GCT T G T -
SecY
Pst I
174
M e t P h e Arg Thr Ile S e r A a n Ile Phe Arg V a l ATG "I'C CGA ACG ATC TCC AAC ATI' TIT CGA GTG
2 34
Thr L e u L e u M e t L e u I l e V a l P h e A r g I l e G l y GGT GAT TI'G CGC CGT AAG GTC ATT TTC ACC cIy3 CTC ATG CTC ATC GTT TIT CGG ATC GGA
294
Ser P h e I l e P r o V a l Pro G l y Thr A s n Arg G l u V a l L e u A e p P h e V a l A s p G l n A l a A s n AGT TIT ATC CCC GTI' CCA GGT ACA AAC CGA GAA GTG CTA GAT l?IT GTC GAT CAG GCA AAT
354
A l a Phe G l y P h e L e u A s n Thr P h e G l y G l y G l y A l a L e u G l y A m P h e Ser I l e P h e A l a GCA TTC GGT TI" TTA AAT ACG ??T GGA GGA GGG GCA Cl" GGG AAT TTC TCC ATC TIT GCG
4 14
M e t G l y I l e M e t P r o Tyr I l e Thr Ala Ser I l e V a l M e t G l n L e u L e u G l n M e t A e p V a l ATG GGG ATC ATG CCA TAC ATT ACA GCA TCC ATT GTC ATG CAA TTA TI'G CAG ATG GAT GTC
47 4
V a l P r o L y s Phe A l a G l u Trp A l a L y e G l u G l y G l u A l a G l y Arg Arg Lye Leu A l a G l n G a CAA GTT CCG AAA TI" GCT GAG TGG GCG AAA GAG GGC GAA GCA GGG CGT CGT
534
NheI
P h e Thr Arg Tyr G l y Thr I l e V a l V a l Leu G l y Phe I l e G l n A l a L e u G l y M e t Ser V a l
?TT ACC CGC TAT GGA ACG ATT GTT G'IT TTA GGG TIT ATT CAG GCA CTC GGG ATG TCG GTT
594
G l y Phe A s n A s n P h e Phe P r o G l y Leu I l e Pro A m Pro Ser V a l Ser V a l Tyr L e u Phe GGT TIT AAC AAC TTC TI'C CCA GGA 'ITA ATT CCT AAT CCA AGC GTG TCG GTG TAT CI'C TIT
6 54
I l e A l a Leu V a l Leu Thr Ala G l y Thr A l a Phe L e u M e t T r p L e u G l y G l u G l n I l e Thr ATC GCA CIT GTC Cl" KT GCLGGA ACG GCT W C TI'A ATG TGG CTC GGG GAG CAG ATT ACA
714
A l a L y e G l y V a l G l y A s n G l y I l e Ser I l e I l e I l e P h e A l a G l y I l e A l a A l a G l y I l e GCT AAG GGA GTC GGA AAC GGA ATC TCC ATT ATC ATC 'MT GCA GGG ATT GCT GCT
77 4
Pst I
EcoRI
Pro A m G l y L e u A e n Leu I l e T y r Ser T h r Arg I l e G l n A s p A l a G l y G l u G l n Leu P h e CCA AAT =GG TTA AAT CTC ATT TAC TCA ACA cr;C ATC CAA GAC GCA GGG GAG CAA TIy3 TTC
834
BumHI
L e u A m I l e V a l V a l I l e Leu Leu Leu A l a L e u A l a I l e L e u A l a I l e I l e V a l G l y V a l ! I " AAC ATI' GTG GTG ATC 'ITG ! I " CI" GCT -3" GCG ATT CXT GCC ATC ATI' G'FS GGC GTI'
894
I l e Phe V a l G l n G l n Ala Leu Arg L y s I l e Pro V a l G l n Tyr A l a L y s Arg Leu V a l G l y CGT AAA ATC CCT GTI' CAG TAT GCC AAG CGT CTA GTA GGG ATT TTC d C CAA CAA GCG
9 54
Arg A e n Pro V a l G l y G l y G l n Ser Thr H i s Leu P r o L e u L y e V a l A s n A l a A l a G l y V a l AGA AAC CCT G" GGA GGT CAG TCG ACC CAT CTI? CCG TTA AAA GTG AAT GCC GCA GGC GTT
1014
Sal I
I l e Pro V a l I l e Phe Ala L e u S e r Leu L e u I l e Phe P r o P r o Thr V a l A l a G l y L e u P h e ATP CCG GFC AT" ' I " GCG TI'A TCG "'G CTC ATT ' I " CCA CCA ACE GTT GCT GGG CTG TIT
1074
G l y Ser A8p H i e Pro V a l Ala A l a V a l I l e G l u Thr Phe A s p Tyr Thr H i e Leu I l e GGC AGT GAT CAT CCG W C GCI' GCA TGG Gn; ATI' GAG ACA TIT GAC TAT ACC CAC TIY; ATI'
1134
G l y M e t Ala V a l T y r Ala Leu Arg I l e I l e G l y Phe Thr Tyr P h e Tyr Ala Phe I l e G l n GGG ATG GCG GTA TAT GCC T I y 3 CGC ATC ATC GGA TIT ACG TAT "C TAT GCG TIT ATC CAA
1194
V a l A m Pro G l u Arg M e t A l a G l u A m L e u L y e Lye G l n G l y G l y T y r Ile P r o G l y I l e GTC AAC CCA GAG CGA AT0 GCG GAG AAC "G AAA AAA CAG GGT GGC TAT ATI' CCT
1254
EcoRI Arg Pro G l y Lye Ala Thr G l n Thr Tyr I l e Thr Pro I l e Leu Tyr Arg Leu Thr Phe V a l G T CCA GGG AAA GC?i ACG CAG ACG TAC ATC ACG CCT ATC TIG A--T & T . ACG TIT GTC ClUI Fig. 3.
1314
1367
1368
S.-K. Kang, T. Kudo and K . Horikoshi G l y Ser L e u Phe Leu Ala V a l V a l Ala I l e Leu P r o V a l Phe Phe I l e L y e P h e A l a A s p GGA TCA c?T TI'C CTC GCA GTG GTG GCG ATC "G CCG GTA TTC TIT ATT AAG TIT GCC GAC
1374
TTG CCA CAG GCG ATT
Leu P r o G l n A l a I l e G l n I l e G l y G l y Thr G l y Leu L e u I l e V a l V a l G l y V a l A l a L e u CAA ATT GGT GGT ACG GGC 'ITS CTC ATC GTC GTC GGG GTT GCC CTT
1434
A e p Thr M e t L y e G l n I l e G l u A l a G l n Leu I l e L y e Arg Ser Tp-L y e G l y Phe I l e L y e GAT ACG ATG AAG CAA ATT GAA GCA CAG "G ATC AAA CGT TCT TAT AAA GGG ' I " ATX' AAG
1494
TER TAG GGG GAG CAG GGA AGC GTG GTG TCT TCC CGT TCG CCT TCT GTT CIT AAA TTA TTA GAC
Adk Met A e n Leu A e n Leu M e t G l y Leu Pro G l y A l a G l y L y e G l y Thr
-G
1554
ATA GAC "G AAT CTG AAC CIT ATG GGA CTT CCT GGT GCT GGT AAA GGT ACA
1614
G l n A l a G l u Lye I l e I l e G l u L y s Tyr G l y I l e Pro His I l e S e r T h r G l y A s p M e t P h e CAG GCA GAA AAG ATC A'IT GAG AAG TAC GGC ATC CCA CAC ATT TCA ACA GGT GAC ATG TIT
1674
SD
Nsp I Arg Ala Ala M e t L y e A m G l u Thr G l u L e u G l y L e u L y e Ala L y e Ser Tyr M e t A s p A l a CGT GCT GCG ATG AAG AAC GAG ACC GAG C I T GGA TTA AAA GCA AAA TCG TAC ATG GAT GCA
1734
G l y G l u Leu V a l P r o A e p G l u V a l Thr I l e G l y I l e V a l Arg A e p Arg L e u S e r G l n A a p GGG GAA CI'G GTT CCT GAT GAA GTA ACG ATI' GGT ATC GTT CGG GAT CXT CTC AGT CAA GAC
1794
A e p Cye G l n A e n G l y Phe L e u L e u A s p G l y Phe P r o Arg Thr V a l A l a G l n A l a G l u A l a GAT TGC CAA AAT GGC TI" TIy3 CIT GAC GGG TI" CCA CGG ACT GTC GCT CAA GCA GAA GCG
1854
L e u G l u A e p I l e L e u A l a S e r L e u A e p LYE L y s Leu TTA GAA GAT ATT "G GCG TCA C l T GAT AAA BAG CIT
189 0
HindIII Fig. 3. Nucleotide and deduced amino acid sequences of the 1.8 kb HindIII fragment. Nucleotides are numbered from the 5'-end. The possible ribosome binding sites (SD) preceding each of the ORFs are underlined.
Hydrophobic 3.0
-1
-
I
.
