See discussions, stats, and author profiles for this publication at:
`https://www.researchgate.net/publication/15281840
`
`Molecular Characterization of the Gene
`Operon of Heat Shock Proteins HSP60
`and HSP10 in Methicillin-Resistant
`Staphylococcus aureus
`
`ARTICLE in BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS · JULY 1993
`
`Impact Factor: 2.3 · DOI: 10.1006/bbrc.1993.1686 · Source: PubMed
`
`CITATIONS
`28
`
`READS
`7
`
`5 AUTHORS, INCLUDING:
`
`Makoto Kuroda
`National Institute of Infectious Diseas…
`
`111 PUBLICATIONS 4,318 CITATIONS
`
`SEE PROFILE
`
`Available from: Makoto Kuroda
`Retrieved on: 18 February 2016
`
`

`
`Vol. 193, No. 2, 1993
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`June 15 1993
`
`Page-5 730'737
`
`MOLECULAR CHARACTERIZATION OF THE GENE OPERON
`
`OF HEAT SHOCK PROTEINS HSP60 AND HSP10
`
`IN METHICILLIN-RESISTANT Staphylococcus aureus
`
`Toshiko Ohta, Kyoko Honda, Makoto Kuroda, Keiko Saito,
`and Hideo Hayashi
`
`Department of Microbiology, Institute of Basic Medical Sciences,
`University of Tsukuba,
`Tsukuba, Ibaraki, 305, JAPAN
`
`Received April 24, 1993
`
`SUMMARY: Methicillin-resistance of S.aureus (MRSA) was diminished or depressed at 44‘C. In
`order to investigate whether bacterial heat shock response is correlated with mathicillin resistance, we
`examined the inducibility of the heat shock proteins (HSPs) in MRSA, and cloned and sequenced of
`their genes. A temperature shift from 37‘C to 46‘C enhanced the production of at least 8 kinds of
`cytoplasmic proteins as measured with two-dimensional PAGE. The induced protein profile was
`almost the same as methicillin sensitive S.aurcus, and stress conditions due to ethanol, cadmium or
`low pH. Two of these proteins were HSP60 and HSP10. Their N—terminal amino acid sequences
`were 79%, and 83%, homologous with thermobacterium PS3, respectively. A positively hybridized
`4.2kbp DNA fragment encoding both proteins was isolated from the chromosomal DNA of MRSA.
`The resulting sequence revealed two reading frames and showed high homology to those of hsp60
`(groEL) and hspl0 (groES) of bacteria (E0011) and several other species. The genes of HSP60 and
`HSP10 in S.aureus comprised an operon as in E.coli. The relationship between those HSPS and
`PBP2' is currently under investigation.
`« 1993 A<:adem.t<: PM-.5,
`Inc.
`
`Heat shock proteins are temporarily expressed when cells are exposed to sublethal heat shock or
`
`the other stresses (1,2). These proteins are highly conserved from bacteria to animals (3). Some
`
`HSPs are chaperonins, which are involved in the folding, refolding, assembly, and disassembly of
`
`polypeptide chains during protein translocation processes (1,4,5). Homologous proteins to the
`
`GroEL and GroES of Escherichia coli chaperonins (3) have been found in other bacteria (6,7). The
`
`groE genes comprise an operon under positive transcriptional control promoted by heat shock sigma
`
`factor, 032 (8,9).
`
`Saphylococcus aureus, a pathogenic gram—positive bacteria, can grow under various stress
`
`conditions
`
`including high temperature, low pH, high pH, high osmotic pressure, or at lethal
`
`$4.00
`0006-291X/93
`Copyright © 1993 by Academic‘ Press, Inc.
`All rights of reproduction in any form reserved.
`
`730
`
`Page 2
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`Page 2
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`

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`Vol. ‘I93, No. 2, 1993
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`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`concentration of drugs. Methicil1in—resistant S.aurcus (MRSA), can resist most [5—lactam compounds.
`
`This drug-resistance was depressed with an exposure to high temperature at 44‘C (10), suggesting
`
`possible involvement of heat shock proteins in the resistance mechanism in S.aureus. In order to
`
`clarify whether the heat shock response correlates with expression of the resistance, we examined the
`
`inducibility of the HSPs in MRSA and sequenced their genes. Here we determined the structure of
`
`the gene operon encoding HSP60 and HSPIO.
`
`MATERIALS AND METHODS
`
`Bacterial strains, plasmids, and culture conditions: Clinically isolated and identified S.
`
`aureus 912 (MIC for methicillin = l00ug/ml) was used for the cloning experiments. Plasmid
`pUCl 19 was used as a vector for the construction of genomic libraries, E.coli JMIO9 was the host.
`T-vector, a DNA fragment derived from pCR1000 was the vector for polymerase chain reaction
`(PCR) products, purchased from Invitrogen Co.(U.S.A.). S. aureus was grown at 37‘C in a brain
`heart infusion broth (BHI broth). P1asmid—containing E. coli was routinely grown at 37‘C in Luria—
`
`Bertani medium (LB broth) supplemented with ampicillin (50|.tg/ml).
`Chemicals: Pharmalytes (pH3—7 and pH7-9) were obtained from Pharmacia LKB(Sweden).
`
`Adenosine [y—32P] triphosphate (6,000 Ci/ m mol) was obtained from Amersham (1 Ci=37 GBq).
`Lysostaphin, DNase I, RNase A, and proteinase K were purchased from Sigma Chemical
`Co.(St.Louis). Primers for DNA sequencing, DNA polymerase Klenow fragment, phosphokinase,
`and Ampli Taq-DNA polymerase were obtained from Takara Shuzo(Kyoto), and dideoxynucleoside
`triphosphates was from Boehringer Mannheim Inc.(Germany). Other chemicals used were of
`biochemical grade.
`Two-dimensional polyacrylamide gel electrophoresis: Two—dimensional gel electrophoresis
`was carried out as described by I-lirabayashi eta! .(l1).
`Amino acid sequencing analysis: N—terminal sequences of the heat—inducib1e proteins obtained
`from two—dimensional gel electrophoresis were analyzed by a gas—phase protein sequenator (Model
`470A connected to a Model 120A phenylthiohydantoin-amino acid analyzer, and a Model 900A
`Integrator, Applied Biosystems Inc.) after being transblotted onto a po1y(vinyI-diphenyl)floridone
`(PVDF) membrane. In this method, the Coomassie-Blue—stained spots corresponding to the heat-
`inducible proteins were cut out from the membrane and applied to the sequenator without Polybrene
`treatment, as described previously (12,13).
`Preparation of DNA: Chromosomal DNA was prepared from S. aureus in the late logarithmic
`phase in L—B broth by the method of Matsuhashi etal . (14) with some modifications. Large—scale or
`small~scale preparation of plasmid DNA was carried out by the alkaline-SDS method (15).
`Construction, screening of gene libraries and DNA sequencing: Gene libraries for
`cloning of the hsp60 were constructed with completely digested chromosomal DNA of S. aureus
`912. The positively hybridized DNA fragments were ligated to the appropriately digested and
`dephosphorylated vectors (pUCl19). Transformation in E. coli JMIO9 was carried out by the method
`of Norgard er al.(l6). Positive clones were checked by restriction endonuclease digestion and were
`sequenced by the method of Sanger et al .(l7).
`Southern blot hybridization analysis: Total chromosomal and plasmid DNA fragments were
`isolated on agarose gels. Southern blots on nylon membranes (Gene Screen Plus, Du Pont) were
`carried out by the blotter (STRATAGENE, Funakoshi Co., Tokyo) and hybridization was performed
`as described previously (18), according to the manufacturer's instructions. DNA fragment of 600 bp
`for the probe was amplified by the PCR using chromosomal DNA of S. aureus as template. The
`
`731
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`Page 3
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`Page 3
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`

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`Vol. 193, No. 2, 1993
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`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`oligonucleotide primers synthesized for the PCR were 5'-GG(AGCT)GA(CT)GG(AGCT)AC(AG
`CT)AC(AGCT)AC(AGCT)GC(AGCT)AC(AGCT)GT—3' corresponding to the codons down stream
`of the sequence Gly35 of Pseudomonus aeruginosa GroEL and the complementary primer, 5'~TC(A
`GCT)CC(AG)AA(AGCT)CC(AGCT)GG(CT)GC(AGCT)TT(AGCT)AC(AGCT)GC-3'
`
`corresponding to the codons upstream of Asp232 (19). PCR was performed for 30 cycles of 95‘C for
`lmin, 45‘C for 2min, and 72‘C for 2.5min after denaturation at 95‘C for 3min in the DNA thermal
`cycler (PJ2000, Perkin Elmer Cetus) employing the following conditions: 200mM dNTPs, 25mM
`TAPS (N—tris(hydroxymethyl)methyl—3-aminopropane—sulfonic acid), pH9.3, 50mM KC], 2mM
`MgCl2, 1mM 2—mercaptoethanol, 0.25 mg/ml activated salmon sperm DNA, chromosomal DNA
`
`(about 100 ng), the primers (100 pmoles), and Ampli Taq DNA polymerase (2.5 units). The
`amplified DNA fragments were cloned into pCR1000 using a TA Cloning kit (Invitrogen C0,,
`U.S.A.), and sequenced as described above. These DNA probes were labeled with digoxigenin using
`the DNA labeling kit (Boehringer Mannheim).
`
`RESULTS AND DISCUSSION
`
`At 46 ‘C, the growth of S.aureus was depressed to 50%, and the production of coagulase and
`
`methicillin resistance were diminished whereas DNase, enterotoxin, [Hactamase were intact (data not
`
`shown). Two dimensional PAGE (Fig.1) revealed that a temperature shift from 37‘C to 46C
`
`1. Two-dimensional gel electrophoresis of cytosolic proteins. Logarithmic-phase cells of
`Fig.
`MRSA were soaked at 37 T and 46°C for 25min and the cytosolic proteins from the cell lysate
`were prepared as in MATERIALS AND METHODS. The samples were subjected to isoelectric
`focusing using a pH range of 4-9 for the first dimension, and then to a SDS-PAGE with 10% gel
`for the second dimension. Numbers with an arrow indicate the positions of heat-inducible
`proteins.
`
`732
`
`Page 4
`
`Page 4
`
`

`
`Vol. 193, No. 2, 1993
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`enhanced the production of at least 8 kinds of proteins. The production of heat—inducible-proteins in
`
`MRSA, like the Gram—positive bacterium, thermobacterium PS3 (20), was much stronger than that of
`
`Gram-negative E.coli. The de novo synthesis of these proteins detected by pulse—labeling were
`
`significantly inducible (data not shown). These heat—inducibIe proteins were also enhanced under
`
`10% ethanol, ZOOLLM cadmium, and pH5. Two proteins numbered as 5 and 8 were identified to be
`
`HSP6O (60kDa) and HSP10 (l0kDa), respectively. The N-terminal amino acid sequences of
`
`VKQLKFSEDARQAMLRGVDQ(HSP60) and MLKPIGNRVHEKKEQEQFTKSGIVLTDSAKEKS
`
`(HSP10) were 79% and 83% homologous to those of therrnobacterium PS3 (20), respectively. An
`
`oligonucleotide primer set for HSP6O was synthesized and a DNA probe was then prepared by PCR
`
`(polymerase chain reaction) using chromosomal DNA of S.aureus as template shown in
`
`MATERIALS AND METHODS. The DNA sequence of the amplified DNA fragment revealed that it
`
`was highly homologous to HSP60 genes of thermobacterium PS3 (20) and E.coli (3). Hindlll and
`
`Pst I digested DNA fragment of 4.2kbp was found to be positive with the above probe in total
`
`Southern hybridization. The sequencing strategy of the isolated positive clone of HSP60 is outlined
`
`in Fig.2.
`
`Corresponding nucleotide sequences of the partial amino acid sequences of HSP10 and HSP60
`
`were found in the cloned DNA sequence (the broken lines in Fig.3), and the DNA fragment
`
`comprised two genes encoding HSP10 and HSP60 (Fig.3). The putative heat-shock—promoter
`
`sequences of -35 (TTGAAA) and -10 (TATTAT) were identified only upstream of the HSP10 gene,
`
`by their homology to the consensus sequences in several heat shock genes of other bacteria (7).
`
`_ pKHS-137
`
`Hind m
`
`'
`
`’
`
`' Hincll
`
`Pstl Hincll
`
`
`
`Fig. 2. Sequencing strategy of HSP10 and HSP60 genes. Two open boxes on the solid bar
`(named as pKHS-137 clone) indicate the open reading frames of HSP10 and HSP60,
`respectively. Selected Hind III, Hinc II, and Pst I sites are shown. The arrows below the
`restriction map indicate the length of insert to a vector plasmid (pUCl 19) and the extents of the
`sequenced fragments.
`
`733
`
`Page 5
`
`Page 5
`
`

`
`Vol. 193, No. 2, 1993
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`80
`70
`60
`50
`40
`30
`20
`10
`TGG A1 '1"’I' l"'l'AAT’l"1'1‘TAAATTAA’I‘TAAAA‘FAAACG’l‘CGTGGCGGCGA’I"[‘AAAAA’1"AGAACAAGTTGTATGTAAATGACTG
`90
`100
`110
`120
`130
`140
`150
`160
`C‘1"I"1‘AGTC AGTTCTATGCCACTAI1’A'I"1‘GTACAAA'I'GGT}\A'1'T’1‘TTTTACAATCACAAGCCCTAAAAAT T‘CACACCA'TA'1'A
`170
`180
`190
`200
`210
`220
`230
`240
`TAAATAATAACAGTTAGCAATGATGCCCATAATCTTGTCATAATTTTCCTCCAAATATTGTTTATAATTTATTTTATCGT
`250
`260
`270
`280
`290
`300
`310
`320
`AAATAACTTGAAGTTACAAAACTTAATTACAGAGGTTATGACTTGAAATTTTGACCAAATTTGATTATTATAAATCTATG
`-35
`-10
`400
`390
`380
`370
`360
`350
`340
`330
`TTAGCACTCTTTAATGTTAAGTGCTAAACTTTAGCTTTTTTAAGGAGGAACAATCATGCTAAAACCAATTGGAAATCGTG
`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
`M L
`K
`P
`I
`G N
`R V
`4IIIIIll
`IIIIIII»~
`460
`470
`480
`450
`440
`430
`420
`410
`TGATTATTGAGAAAAAAGAACAAGAACAAACAAC1AAAAGTGGTATTGTTTTAACTGATAGTGCTAAAGAAAAATCAAAC
`I
`I
`E
`K
`K
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`Q
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`T
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`K
`B
`K
`S
`N
`
`
`
`560
`550
`540
`530
`520
`510
`500
`490
`GAAGGCGTTATCGTTGCAGTAGGAACTGGACGTCTATTAAATGATGGTACAAGAGTGACTCCTGAAGTGAAAGAAGGGGA
`E
`G
`V
`I
`V A V G
`T
`G
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`L
`L
`N
`D
`G
`T
`R V T
`P
`E V K
`I
`G
`D
`
`640
`630
`620
`610
`600
`590
`580
`570
`CCGTGTCGTGTTCCAACAATATGCTGGTACAGAAGTTAAACGAGATAATCAAACATATCTAGTATTAAATGAAGAAGATA
`R V V
`P
`Q
`Q
`Y A G
`T
`E
`V
`K
`R
`D
`N
`E
`T
`Y
`L V
`L
`N
`E
`E
`D
`I
`
`720
`710
`700
`690
`680
`670
`660
`650
`TTTTAGCGCTAATTGAATAATATAAAATTAAATTCATAGATAAATTGTAAAGAACCAAAATGAAATATGACTAAACAAAT
`L A V
`I
`E
`,
`union}
`—-Qnlllluu
`800
`790
`780
`730
`/40
`750
`760
`770
`CCAGG11'["1‘ATCA'I"I"I'A1‘GC I"1"A}\ACAATTC.7\AA‘I‘TCTCTGAIXGATCCACGTC’AAGCAATCTTACGTGCTGTACACCAATT
`
`S/D
`
`Iyllllvlllllxllll Inan::5:u-Eu-§uIEI--?uIIhnu§Iu III%un[“-Iu!I"IIuRn|Ig-lxuunnllol I
`
`880
`8/0
`860
`850
`840
`830
`820
`810
`AGCAAATGCAGTTAAAGTAACGATTGGTCCTAAAGGACGTAATGTTC1ATTAGATAAAGAGTTTACAGCACCTTTAATTA
`A
`N A V
`K
`V
`T
`I
`G
`P
`K
`G
`R
`N
`V
`V
`L
`D
`K
`E
`F
`T A
`P
`L
`I
`T
`
`960
`950
`940
`930
`920
`910
`900
`890
`CGAACGATGGTGTAACGATTGCTAAAGAAATCGAATTAGAAGATCCATATGAAAATATGGGGGCTAAACTAGTTCAAGAA
`N
`D
`G
`V
`T
`I
`A K
`E
`I
`E
`L
`E
`D
`P
`Y
`E
`N M G A
`K
`L
`V
`O
`B
`
`1040
`1030
`1020
`1010
`1000
`990
`980
`970
`GTTGCAAATAAGACAAATGAAATTGCTGGTGACGGTACGACAACTGCAACAGTATTAGCTCAAGCAATGATTCAAGAAGG
`V
`A
`N
`K
`T
`N
`E
`I
`A G
`D
`G
`T
`T
`T A T
`V
`L A Q
`A H
`I
`Q
`B
`G
`
`1120
`1110
`1100
`1090
`1080
`1070
`1060
`1050
`CTTGAAAAATGTTACAAGTGGTGCGAACCCAGTTGGTTTACGTCAAGGTATCGACAAAGCAGTTAAAGTTGCTGTAGAAG
`L
`K
`N
`V
`T
`S
`G A
`N
`P V G L
`R
`Q
`G
`I
`D
`K
`A
`V
`K
`V
`A V E
`A
`
`1200
`1190
`1180
`1170
`1160
`1150
`1140
`1130
`CATTACA1CAAAATTCTCAAAAAGTTGAAAATAAAAATGAAATAGCGCAAGTAGGTGCGATTTCAGCAGCAGATGAAGAA
`L
`H
`E
`N
`S
`O
`K V E
`N
`K
`N
`E
`I
`A Q
`V
`G A
`I
`S A A
`D
`E
`E
`
`1280
`1270
`1260
`1250
`1240
`1230
`1220
`1210
`ATTGGACGTTATAWTTCTGAAGCTACGGAAAAAGTAGGTAACGATGGTGTCATTACAATTATTACAATTGAAGAATCAAA
`I
`G
`R
`Y
`I
`S
`E
`A
`T
`E
`K V G
`N
`D
`G
`V
`I
`T
`I
`I
`T
`I
`E
`E
`S
`N
`
`1360
`1350
`1340
`1330
`1320
`1310
`1300
`1290
`TCGACTAAACACTCAACTAGAATTGGGTATGCAATTTGATCGTGGTTATCAATCACCATATATGGTTACTGATTCAGATA
`R
`L
`N
`T
`E
`L
`E
`L G M Q
`P
`D
`R G Y
`Q S
`P
`Y M V
`T
`D
`S
`D
`K
`
`Fig. 3. Nucleotide sequence of the genes encoding HSP60 and HSPIO, and the predicted amino
`acid sequence of these in S. aureus. Translation stop signals are marked by asterisks below the
`respective codons. The putative ribosome-binding sites (Shine-Dalgano sequence) and heat
`shock promoter (-10 and -35 consensus sequence) are underlined. Broken lines with arrowheads
`show the stem-and-loop. Arrows below nucleotide sequence show the positions of primer. The
`amino acid sequences determined by N-terminal analysis are presented with broken lines.
`
`734
`
`Page 6
`
`Page 6
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`

`
`Vol. 193. No. 2, 1993
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`1440
`1430
`1420
`1410
`1400
`1390
`1380
`1370
`AAATGGTTGCTGAATTAGAACGCCCATACATTTTAGTAACAGATAAGAAAATCTCGTCTTTCCAAGATATCTTACCTTTA
`H V A
`E
`L
`B
`R
`P
`Y
`I
`L V T
`D
`K
`K
`I
`S
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`I
`L
`P
`L
`
`1520
`1510
`1500
`1490
`1480
`1470
`1460
`1450
`T1AGAACAAGTGGTTCAATCTAATCGTCCAATCTTAATTGTAGCTGATGAAGTTGAAGGCGATGCATTAACAAATATCGT
`L
`E
`Q
`V
`V Q
`S
`N
`R
`P
`I
`L
`I
`V A
`D
`E V
`B G D
`A L
`T
`N
`I
`V
`
`1600
`1590
`1580
`1570
`1560
`1550
`1540
`1530
`GCTAAACCGAATGCGTGGCACATTTACAGCTGTTGCAGTAAAGGCGCCTGGTTTCGGTGATCGTCGTAAAGCAATGT1‘AG
`LNRMRGTFTAVAVKAPGPGDRRRAMLE
`.&.
`1680
`1670
`1640
`1650
`1660
`1630
`1620
`1610
`AAGATTTAGCTATTTTAACTGCTGCGCAAGTGATTACTGATGATTTAGGCTTACATTTAAAAGATGCATCAATTGATATG
`D
`L A
`I
`L
`T
`G
`A O V
`I
`T
`D
`D
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`D
`L
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`D
`A
`S
`I
`D M
`
`1760
`1750
`1740
`1730
`1720
`1710
`1700
`1690
`T1AGGTACTGCAAGTAAAGTAGAAGTAACTAAAGATAATACCACTGTTGTTGATGGTGACGGTGACGAAAACAGCATTGA
`L
`G
`T A
`S
`K
`V E V T
`K
`D N
`T
`T V V D
`G
`D G
`D
`E N S
`I
`D
`
`1840
`1830
`1820
`1810
`1800
`1790
`1780
`1770
`TGCACGTGTAAGCCAATTGAAATCTCAAATTGAAGAAACTGAATCTGACTTTGATCGTGAAAAATTACAAGAGCGCTTAG
`A
`R V
`S
`Q
`L
`K
`S
`Q
`I
`E
`E
`T
`B
`S
`D
`P
`D
`R
`E
`K
`L
`Q
`E
`R
`L A
`
`1920
`1910
`1900
`1890
`1880
`1870
`1860
`1850
`CTAAATTAGCAGGTGGTGTTGCAGTTATTAAAGTAGGTGCAGCAAGTGAAACAGAGCTTAAAGAACGTAAATTACGTATT
`K
`L A G
`G V A
`V
`I
`K
`V
`G
`A A
`S
`E
`T
`E
`L
`K
`E
`R
`K
`L
`R
`I
`
`2000
`1990
`1980
`1970
`1960
`1950
`1940
`1930
`GAACATGCATTAAATTCTACACGTGCAGCAGTTGAAGAAGGTATTGTTGCAGGTGGTGGTACTGCATTAGTAAATGTTTA
`B
`D A
`L
`N
`S
`T
`R A A V
`E
`E
`G
`I
`V A G
`G
`G T A L V N V Y
`
`2080
`2070
`2060
`2050
`2040
`2030
`2020
`2010
`CCAAAAAGTAACTGAAAATGAAGCAGAAGGTGACATTGAAACAGGTGTAAATATTGTACTTAAAGCACTAACTGCACCAG
`Q
`K V
`S
`E
`N
`E A E
`G
`D
`I
`E
`T
`G
`V
`N
`I V L
`K A L
`T A P V
`
`2160
`2150
`2140
`2130
`2120
`2110
`2100
`2090
`1TCG1CAAATTGCTGAAAATGCAGGATTACAAGGTTCTGTTATTGTAGAACGTTTGAAAAACGCAGAGCCGGGTGTTGCT
`R
`Q
`I
`A
`E
`N
`A G
`L
`E
`G
`S
`V
`I
`V E
`R
`L
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`E
`P
`G V G
`
`2240
`2230
`2220
`2210
`2200
`2190
`2180
`2170
`T1TAACGGTGCTACAAACGAGTGGGTTAATATGT1AAGAAGAGGTATCGTTGATCCAACTAAAGTAACACGCTCAGCATT
`P
`N G A
`T
`N
`E W V N
`M
`L
`R
`R G
`I
`V D
`P
`T
`K V T
`R
`S
`A
`L
`
`2320
`2310
`2300
`2290
`2280
`2270
`2260
`2250
`ACAACATGC1GCAAGTGTTGCAGCAATGTTCTTAACGACTGAAGCGGTTGTAGCATCAATTCCAGAAAAAAATAATGACC
`O
`H A A
`S V A A M
`P
`L
`T
`T
`E
`A V V A
`S
`I
`P
`E
`K
`N
`N
`D
`Q
`
`2400
`2390
`2380
`2370
`2360
`2350
`2340
`2330
`AACCTAACATGGGTGGCATGCCGGGAATGATGTAAAACGACTGTTAAACGCTGATTTTATAAAGTTGTAATATTGGGTGG
`P
`N
`M
`G
`G M
`P
`G M H
`'
`2480
`2470
`2460
`2450
`2440
`2410
`2420
`2430
`CCGTAATTIGG1CATAGGAAATTTTAAAATAAATCYYTTGAGACGTTTTCCATGAGTTCACTAAACTTTTGGGAACCGTC
`2490
`2500
`2510
`2520
`2530
`2540
`2550
`2560
`1 'I"’I‘T’I TGTATGAGCCCGTAA'['C1"TAGCGTACA1G1'1‘CATAGTGG1‘G’I"l‘TATATCTTTGTGTCGCAAGCGTTCTTGTA1 'T‘I'
`25/0
`2580
`2590
`2600
`2610
`2620
`2630
`2640
`CCFYAATATGCACACCAGCCTCTATAAGTAACGCGCAATGAGTATGACGAAATCAATCAGTGCTTATTTGTTTATFAGTT
`2650
`2660
`2670
`2680
`2690
`2700
`2709
`ATGTTAGTCTTTTTAAGGTATAGCTTTTATCCATAACTGTAGTTTCTTAATTACGAGGGGATAGCCGTTGAC
`
`Fig.
`
`3
`
`— Continued
`
`Thus, the genes for two HSPs were found to be organized as an operon. Two putative stem-and-loop
`
`structures were identified in the upstream region of the HSPIO gene and in the junction between the
`
`two Coding frames. The coding frames had a G+C content of 35%. The two putative polypeptides
`
`735
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`Page 7
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`

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`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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`had 94 and 538 amino acids and a molecular mass of 10,416 Da and 57,855 Da, respectively. The
`
`HSPs of S. aureus also showed amino acid sequence homology with E. coli GroES and GroEL (3)
`
`by 44.7% and 56.2%, respectively, and with those of thennobacterium PS3 by 60.2% and 68.5% .
`
`Thus, in comparison with HSPs of the Gram-negative bacteria, E. coli, those of S.aureus have higher
`
`homology with therrnobacterium PS3 and Clostridium acetoburylicum (7), which are Gram-positive.
`
`The effector of methicillin resistance is believed to be penicillin-binding protein 2' (PBP2'), the
`
`product of mecA gene, the expression of which may be controled by femA and/or femC. At the
`
`moment, we have not observed a direct correlation between PBP2' production and HSP60 or HSP10
`
`expression, however, considering some reports concerning supression of protein synthesis by HSPs,
`
`we are now attempting to isolate HSP mutants in S.aureus to elucidate the role of HSPs in drug
`
`resistance. In fact, overexpression of the heat shock proteins GroES and GroEL in E. coli has been
`
`reported to supress some temperature sensitive mutations in vivo (21).
`
`ACKNOWLEDGMENTS
`
`We thank Prof. H.Hirata for the protein sequencing analysis and Dr. J.Miyazaki for kind
`
`technical support. This work was supported by a grant from the University of Tsukuba.
`
`REFERENCES
`
`.‘°?°>’?‘!J'.*‘
`
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