Mar 13, 1990 - kDa polypeptides in mba region of the virulence plasmid,. pKDSC50, of Salmonella choleraesuis. Hidenori Matsui, Kazuyoshi Kawahara, ...
Nucleic Acids Research, Vol. 18, No. 8 2181
1990 Oxford University Press
Nucleotide sequences of genes encoding 32 kDa and 70 kDa polypeptides in mba region of the virulence plasmid, pKDSC50, of Salmonella choleraesuis Hidenori Matsui, Kazuyoshi Kawahara, Nobuyuki Terakadol and Hirofumi Danbara* Department of Bacteriology, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108 and 1Department of Bacteriology, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305, Japan EMBL accession
Submitted March 13, 1990 We previously reported the 50 kilobase (kb) plasmid, pKDSC5O, of Salmonella choleraesuis associated with virulence of this organism (1). Virulence mapping demonstrated that 6.2 kb 1 GAATTCCTGA AATGGCACCA CGCCAGCGGT 61 AGCCCTCAAC GTTGTTTATT CCTGAGTTAA 121 CGCGATTCCG CACACCAGAA AAATAGCACA 181 CTGGCAATAC AAAATAATTC CCCTGCACAC
241 ATTTTCAGG
CACGTTCTTC CCGGCCTCAC CCGACGCCGG TTTGTTGTGC CATCTTTGCA CACCGGCAAC
no.
X52035
region, mba region, was essential for causing bacteremia in mice, and polypeptides were identified in E. coli minicells (2). Recently the 4.1 kb EcoRI fragment within the mba region was subcloned 1621 GCCCCGCACG GAGAGCAGTT TTTATCGCCT P R T E S S F Y R L
TAAATAAACT CAATATAACC CACTCATTTT 1681
ATTATCAGTC TTCAGGATTT CATTCTGTTT
GGAGTACTCG GTGCGCAACA GCAACGGCGA E Y W N S N G D c V
TCATTTCTGG TTACTGCATG ACAGTAACGG CATCCTGCAC CTGCTGGGGA AAACCGCCGC I D F W L L H D S N G L II L L G K T A A
20 ~~32 k CTCATCATTA TTTATGAATA TCAATCAGAC CACCACTCCG GCACTTTCAC 1741 AGCACGCCTC AGCGATCCCC AGGCCGCCTC TCATACGGCG CAATGGCTGG TTGAGGACTC M N H N Q T T S P
A L S Q A R L S D P Q A A S 301 AGGTCGAAAC CGCCATCCGG GTCCCGGCAG GCAATTTTGC AAAATATAAT TATTATTCCG 1801 GGTGACCCCT GCCGGCGAGC ATATCTATTA V E T A I R V P A N F A G K N Y s v V T r A G E If I Y Y 361 TGTTTGATAT TGTCCGTCAG ACCCGTAAAC AGTTTATTAA CGCCAATATG TCATGGCCGG 1861 GGACCTCAAT GGGAACGAGC CCGGACGCGA F D I V R Q T R K Q F I N A N M S W P G G N E A D L N G R D 421 GCTCCCGCGG AGGTAAACCC TGCGACCTGG CGATGGGCCA CGCGCAGTAT ATCCGCTGCA 1921 ACAGTATGGC AACGCCACCC CCGCCGCCCA S R G G K A W D L A M G Q A Q Y I R C M Q Y G N A T P A A D 481 TGTTCCGAGA AAATCAATTG ACCCGCAGAG TTCGGGGGAC CTTGCAGCAG ACACCGGACA 1981 GGTACAGTGG CTGTTCACCC TAGTGTTTGA F R E N Q L T R R V R G T L Q Q T P D N V Q W L F T L V F D 541 ATGGCACCAA CCTGACCAGT TCCGCTGTCG GCCGTATTCA GGCACAGGCA GAGCGTCCGC 2041 ACCGCCTGCA TTCACTGCTC AGAACAGCTG T C G N L S S S A V G I Q G Q A E R R P A F T A Q P P N S W 601 CGGACCTGGC CACCCTGATG GTGGTTAATG ATGCCATTAA CCAGCAAATA CCGACCCTGC 2101 TAACTACGGC TTTGAGATCC GCCTCCATCG D L A T L M V V N D A I N Q Q I P T L L N Y G F E I R L It R 661 TGCCGTATCA TTTTCCACAC GACCAGGTGG AGTTATCTCT GCTGAATACC GATGTGTCGC 2161 CTTTCCTGAT GAACTGGGTG AAGCCGATAC P Y H F P H D Q V E L S L L N T D V S L E L G E F P D A D T 721 TGGAAGATAT TATCAGCGAG AGCAGCATTG ACTGGCCGTG GTTCCTGAGC AACTCGCTGA 2221 CGAAAATCCG ATACTGACAC AGCTTTCCGC E D I I S E S S I D W P W F L S N S L T E N P I L T Q L C A 781 CCGGCGATAA CAGTAACTAT GCCATGGAGC TCGCCAGCCG GCTGTCACCA GAGCAGCAGA 2281 TTATAGAAGA GCTCCTGTCA ACAATATAAT G D N S N Y A M E L A S R L S P E Q Q T R R Y A P V N N I M 841 CACTCCCGAC CGAGCCGGAC AACAGTACCG CCACTGACCT GACCTCTTTT TACCAGACCA 2341 AGGTAATTCA TCTCGACCAA AATCAAAATG L P T E P D N S T A G N S T D L T S F Y Q T S R P K N x w S
H
T
A
Q
W
L
V
E
E
S
CTCCTACTTG GCGGACAACC GTGACAATGT S
Y
L
A
E
N
G
D
N
V
TCGCAGCGCC ATGCGCTATC TCAGCAAGGT R
S
A
M
R
Y
L
S
K
V
TCTGTACCTC TGGACTAGCG CCACACCCCC L
Y
L
W
T
S
A
T
P
A
CTACGGCGAA CGTGGTGTAG ATCCACAGGT Y
G
E
R
G
V
D
P
Q
V
GCTCGCCCGC CAGGATCCCT TCTCCCTGTA L
A
R
Q
D
P
F
S
L
Y
CCTGTCCCGC CAAGTCCTCA TGTTCCACCA L
C
R
Q
V
L
M
F
If
H
GCTGGTTTCC CGTCTGCTGC TGGAGTATGA L
V
S
R
L
L
L
E
Y
D
TGCTCGGACG CTGGCCTATG AAGGCGACGG A
R
T
L
A
Y
E
G
D
G
CCCACCGCCA CCGCCTCCTC CGATGATGGG P
P
P
P
P
P
P
M
M
G
GGCGATTGTA GAGGAATCAA AGCAGATTCA A I V E E S K Q I Q
901 ATCTCGGGCT GAAAACCGCC GACTATACGC CATTTGAAGC ACTGAATACC TTTGCCCGAC L G L K T A D Y T P F E A L N T F A R Q
2401 AGCTCTG ACG TACTATTCAC GTCAACCCTA CACTGTCATT AATAAATATT TACCTCGCGA A L R Y Y S R I Q C Y S V N K Y L R G D
961 AGTTAGCGAT TACCGTTCCC CCAGGTGGAA CAGTTGATTG CGGGTACTCT GCGTGCCAGC L A I T V P P G G T V D C A C Q P G Y S
2461 TGATTATCCT GAAACACAGG CAAAAGAAAC TCTGCTCTCC AGAGACTATC TTTCCACAAA D Y P E T Q A K E T L L S R D Y L T N S
1021 CGGCAGTTTA GCTTCCCGCG CTACCAGAGT AGTGAGCAGC AGACCATTCT GCAGAATCTG A V *
2521 TGAACCCAGT GATGAGGAGT TTAAAAATGC CATGTCAGTT TATATAAATG ATATTGCCGA E P S D E E F K N A M S Y I N D V I A E
1081 ATGCGACGTC ATTGTTCAGG TGCATTCTAC CGCGCTGTAC GGCGGCAGCA CTTTTCAAC
2581 GGGATTAAGT TCACTTCCCG AAACAGATCA CAGAGTCGTA TACCGGGGCC TGAAGCTTGA G L S S L P E T D H R V V Y R G L I L D
-IN
1141 GGCCGTAGAG
CAGACGCCtr' AAGCAGAAAA TATACCTGGC CATCGTCAGA CGGCCAGTTT k
121 1201 CAGGATAG TGTATGTTGA TACTAAATGG TTTTTCATCT GCCACTTTAG CGCTGATCAC M L I L N G F S S A T L A L I T 1261 TCCCCCTTCC CTGCCAAAAG GGGGCAAGGC GCTGAGTCAG TCAGGCCCTG ACGGCCTAGC P P S L P K G G A L S Q S G P D G L A
1321 CAGTATAACG CTGCCTCTGC CCATCAGCGC CGAACGCGGC TTTGCGCCTG CGCTGGCGCT S I T L P L P I S A F A P A L A L E R G 1381 GCACTACAGC AGCGGTCGCG CCAATGCCCC CTTCGGCCTG CGCTGGTCCT GCGCGACAAT H Y S S G G G N G P F G V A T M G W S C 1441 GAGCATTGCC CGCAGCACCA GCCATGGCGT GCCGCAGTAT AACGACAGCG ATGAGTTTCT S I A R S T S P Q Y I G V N D S D E F L 1501 GGGGCCGGAC GGAGAACTGC TGGTTCAAAC GCTCAGCACC GGTGATGCCC CCAATCCCGT V Q T G P D G E V L L S T N P V G D A P
1561 CACCTCGTTC GCGTACGGTG ACGTATCGTT CCCGCAAAGC TACACGGTGA CCCGCTATCA T S F V S F A Y G D Y T V T P Q S R Y Q
*
To whom correspondence should be addressed
2641 TAAGCCCCCA TTATCGGATG TGCTGAAGGA ATACACTACT ATACGTAATA TAATAATAGA K P A L S D V E I I D L T T I C N I Y 2701 TAAAGCTTTT ATGAGTACAT CGCCAGATAA GGCATGGATA AATGACACTA TTCTCAACAT K A F H S T S P D A W I N D T I L N I 2761 ATACCTAGAA AAAGGACATA AAGGTAGAAT ACTCGGAGAT GTTGCACATT TTAAGGGAGA Y L E K G H K L G D G R I V A H F K G E
2821 GGCAGAGATG CTTTTCCCTC CAAATACTAA ACTCAAAATC GAAAGCATTG TAAATTGTGG A E M L F P P N T K L K I N C G E S I V 2881 ATCCCAAGAC TTTGCAAGCC AGCTTAGTAA GCTGAGATTA AGTGATGATG CAACTGCTGA S Q D F A S Q L S K S D D A L R L T A D 2941 CACAAACAGG ATAAAAAGAA TAATAAACAT GAGGGTACTC AACTCATAGA TACTAAGAAT T N R I K R I I N M N S * R V L 3001 3061 3121 3181
CTATTCCAGA AGTCCTATCA GCGGCCTACC TCTATAAGGG GTTATACTCC GGAACCCCAC ATTTTTCCGT CACCCTAGCC CCGCAAAGTA GTGCATCTAA ACTTTTGCCA TTACCCTTCT TTAACTTTCT GCTCGGAACG GACCGAAATA TCATTTTTTC GCCTGATAAA AAATGAGGTT TTCTGGATAA CTAATCGTTT TATTAA
2182 Nucleic Acids Research, Vol. 18, No. 8 into pUCi 18, and proteins expressed by genes on this fragment were analyzed after isopropylthiogalactoside induction. SDS polyacrylamide gel electrophoresis analysis followed by Coomassie brilliant blue staining indicated the presence of at least three proteins with apparent molecular weights of 70 kilodaltons (kDa), 32 kDa and 29 kDa (data will be published elsewhere). Sequence analysis showed three open reading frames corresponding to these polypeptides in the 4.1 kb fragment. The DNA sequence for the 29 kDa protein has been previously reported (3). In the present paper we described the nucleotide and the deduced amino acid sequences of the 70 kDa and 32 kDa proteins, and the putative sequences for promoter, Shine-Dalgano (SD) and transcription stop with potential dyad symmetry are indicated in the Figure. The deduced molecular weight was 65,340 (591 amino acids) for 70 kDa protein, and 28,184 (255 amino acids) for 32 kDa protein. The amino acid sequences of the 70 kDa and 32 kDa proteins are significantly homologous to the gene products of mkaA (4) and mkaB (5), respectively, of the virulence plasmid of S. typhimurium.
ACKNOWLEDGEMENTS This work was partly supported by Grant-in-Aid provided by the Ministry of Education, Science and Culture, the Japanese Government (No. 62304036 and No. 63570205).
REFERENCES 1. Kawahara,K. et al. (1988) Microbial Pathogenesis 4, 155-163. Kawahara,K. et al. (1990) Microbial Pathogenesis 8, in press. Matsui,H. et al. (1990) Nucd. Acids Res. 18, in press. Taira,S. and Rhen,M. (1989) Microbial Pathogenesis 7, 165-173. Taira,S and Rhen,M. (1989) FEBS Lett. 257, 274-278.
2. 3. 4. 5.
