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Vol. 173, No. 3
Transcriptional Analysis, Translational Analysis, and Sequence
ofthe kilA-Tellurite Resistance Region of Plasmid RK2Ter
EMILY G. WALTER,1 CHRISTOPHER M. THOMAS,2 JOHN P. IBBOTSON,2 AND
DIANE E. TAYLOR'*Department of Medical Microbiology and Infectious
Diseases, University of Alberta, Edmonton,
Alberta, Canada T6G 2H7,1 and School of Biological Sciences,
University ofBirmingham,Birmingham, United Kingdom B15 2TT2
Received 20 August 1990/Accepted 4 December 1990
The tellurite resistance (Ter) determinant of the IncPa plasmid
RK2Ter, a variant of RK2 (also called RP4),is located between the
kilA and korA genes involved in plasmid replication control.
Transcriptional andtranslational fusions were constructed between
the gene for I-galactosidase and the kilA and Ter genes by usingthe
transpositional phage mini-Mu. These fusions indicated that the Ter
genes are transcribed in the samedirection as kilA and that
transcription and translation of the cloned kilA gene are occurring
and may not belethal to the bacterial cell even in the absence of
korA. The nucleotide sequence of this region was determined,and
three open reading frames (ORFs) were identified. The first ORF
codes for KilA, a 28-kDa hydrophilicprotein. The second ORF, teUl,
codes for a hydrophilic protein of 42 kDa. The third ORF, teiB,
codes for ahydrophobic protein of 32 kDa. This protein appears to
be located in the inner membrane of the bacterial cell,since
fusions of TelB to alkaline phosphatase were obtained by using
TnphoA. All three proteins were detectedby sodium dodecyl
sulfate-polyacrylamide gel electrophoresis after overproduction
using the T7 RNApolymerase/promoter system. The same three proteins
were produced when Tes and Ter derivatives of RP4were expressed in
an in vitro transcription-translation system. A single Ser-to-Cys
missense mutation in telBwas found to be responsible for mutation
of RK2 to Ter.
The 60-kb plasmid RK2 is a member of the Pot incompat-ibility
group of plasmids, which have the ability to replicateand be stably
maintained in a wide variety of gram-negativebacteria (for reviews,
see references 45 and 48). PlasmidRK2 appears to be identical to
plasmids named RP4 and RP1(7, 13). A number of determinants
involved in plasmidstability and host range have been identified on
RK2. Theseinclude kil genes, which are potentially lethal to the
Esche-richia coli host bacterium, and kor or kil-override
genes,which inhibit the effect of kil genes on the cell (15).
Tran-scription of the kilA gene is negatively regulated by the
korA(also called trfB or korD) and korB gene products (52,
53).Their structural genes have been located to the 0- to 2.3-
and56- to 60-kb regions, respectively, on the map of RK2 (2,
40,52).A cryptic tellurite resistance (Ter) determinant has
been
located between the kilA and korA genes (5, 42).
Bacteriacarrying RP4 do not express this resistance unless
Tervariants are selected on plates containing tellurite,
followingwhich Ter is stable and constitutive (44, 50). This
Terdeterminant is carried on a transposon which appears toinclude
the kilA and korA genes (5, 6). The Ter determinantof RK2 was
recently cloned and expressed in pUC8 (50). TheTer genes were found
to be transcribed in the same directionas the kilA, korA, and korB
genes, and a single Ter protein of40 kDa was detected by using in
vitro transcription-transla-tion. No homology with the Ter
determinant of the IncHIIplasmid pHH1508a could be found by DNA-DNA
hybridiza-tion, and unlike the Ter determinant of the IncHI-2
plasmidpMER610, the IncPac Ter determinant was
constitutivelyexpressed (20, 50).
In this study, the kilA and tellurite resistance region
fromRK2Ter was sequenced. The transcription and translation of
* Corresponding author.
these genes were studied by using 3-galactosidase andalkaline
phosphatase fusions and overexpression in a T7RNA
polymerase/promoter system.
(This work has been presented by E.G.W. to the Facultyof
Graduate Studies and Research at the University ofAlberta in
partial fulfillment of the requirements for thePh.D. degree.)
MATERIALS AND METHODS
Bacterial strains, plasmids, and phages. Bacterial strainsand
plasmids carrying all or part of the tellurite resistanceregion of
RK2 or RP4 are described in Tables 1 and 2,respectively. Plasmid
pDT1555 was constructed by insertingthe BamHI-HindIII restriction
endonuclease fragment ofpDT1366 (50) carrying the Ter and kilA
genes of RK2 into thecorresponding sites of pACYC184 (9), thus
interrupting thetetracycline resistance determinant (Fig. 1).
PlasmidpMS202 consists of pBR325 with a single RP4 SphI
fragment(coordinates 58.8 to 7.7) inserted (23). The
derivativespMS202-2 and pMS202-4 were obtained as spontaneous
Termutants by growth on 5 p.g of potassium tellurite per ml.
Bacterial plasmids were isolated by the method of Birn-boim and
Doly (3), followed by cesium chloride-ethidiumbromide density
gradient centrifugation. Replicative formsof the bacteriophages
M13mpl8 and M13mpl9 (51) wereobtained from Boehringer Mannheim
Biochemicals, Mon-treal, Quebec, Canada.
Transposition of TnlOOO and TnphoA. TnIOOO insertionmutants of
pDT1558 were prepared by using F'::TnJOOO asdescribed previously
(50). TnphoA insertions into pDT1558were prepared by using ATnphoA
as described by Gutierrezet al. (16). Expression of alkaline
phosphatase was detectedby the formation of blue colonies on Luria
broth platescontaining 5-bromo-4-chloro-3-indolylphosphate
(XP).
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1112 WALTER ET AL.
TABLE 1. Bacterial strains
E. coli Genotype Referencestrain
JM83 ara A(lac-pro) rpsL thi 48odlacZAM15 49JM107 endAI gyrA96
thi hsdR17 supE44 51
relAl X- A(lac-proAB) (F' traD36proAB laclqZAM15)
K38 HfrC (X) 35CC118 araDI39 A(ara 1eu)7697 AIacX74 25
phoAA20 galE galK thi rpsE rpoBargE(Am) recAl
PoI1681TR F- MudI1681 (Kmi) ara::(Mu cts)3 A
8(proAB-argF-1acIPOZYA)XIII rpsLrecA56 srl::TnlO (Tc)
PoII1681TR F- rpsL MudII11681 ara::(Mu cts)3 8araD leu+ lac+
pro' recAS6srl::TnlO (Tcr)
M8820Mu F- araD139 Aara-leu)7697 A(proAB- 8argF-lacIPOZYA)XIII
rpsL Mu
Mini-Mu transposition. Plasmid pDT1555 was transformedinto the
mini-Mu-containing strains PoI1681TR andPoII1681TR as described by
Cohen et al. (12) except that theheat pulse was at 30°C for 2 min
rather than at 42°C. Mini-Muplasmid transduction was carried out as
described byCastilho et al. (8). Mini-Mu lysogens were constructed
in E.coli M8820Mu by plating transducing lysates of M3320Mu
onMacConkey agar containing 50 p.g of chloramphenicol per mlto
select for the drug resistance marker on pACYC184;plates were then
incubated at 30°C for 48 h. Colonies
TABLE 2. Bacterial plasmids derived from RK2 or RP4
Plasmid Resistance Source Referencedesignation marker(s)a
RK2 Ap Km Tc Laboratory stock 29RK2Ter Ap Km Tc Te Ter variant
of RK2 (labo- 5
ratory stock)RP4 Ap Km Tc Laboratory stock 17, 18, 48pDT1366 Ap
Te pUC8 clone derived from 50
RK2TerpDTI555 Cm Te pACYC184 clone derived This study
from pDT1366pDT1558 Ap Te pUC8 clone derived from 50
pDT1366pDT1558-1, Ap TnlO00 insertion mutants 50pDT1558-2 of
pDT1558
pDT1558-4 Ap Teb TnJO00 insertion mutant 50of pDT1558
pDT1558-5 Ap TnlO00 insertion mutant This studyof pDT1558
pDT1969 Ap Te pT7-5 clone derived from This studypDT1558c
pDT2031 Ap pT7-5 clone derived from This studypDT1558c
pDT2034 Ap pT7-6 clone derived from This studypDT1558c
pMS202 Ap Cm pBR325 clone derived 46from RP4
pMS202-2 Ap Cm Te Ter variant of pMS202 This studypMS202-4 Ap Cm
Te Ter variant of pMS202 This study
a Ap, Ampicillin; Cm, chloramphenicol; Km, kanamycin; Tc,
tetracycline;Te, tellurite.
b Low-level resistance of 8 pjg/ml, compared with 0.25 to 1
,ug/ml forplasmidless E. coli and 256 jjg/ml for E. coli(pDT1558)
(50).
c Regions contained in pT7 recombinant plasmids are illustrated
in Fig. 5a.
expressing the Lac+ phenotype were tested for resistance
tokanamycin (16 p,g/ml), the marker present on both themini-MudI
(transcriptional fusion) and mini-Mudll (transla-tional fusion)
phages, as well as for resistance to potassiumtellurite (50
,ug/ml).A series of strains containing mini-MudI and mini-Mudll
insertions into pDT1555, which were unable to grow on 50p.g of
potassium tellurite per ml, were thus identified. Simi-larly, two
tellurite-sensitive (Tes) derivatives of pMS202were obtained by
insertion of mini-MudI. Positions ofmini-Mu insertions were
identified by restriction endonucle-ase analysis with selected
enzymes.DNA sequencing. The nucleotide sequence was deter-
mined by a combination of Maxam-Gilbert chemical modifi-cation
(28) and Sanger dideoxy (36) methods. Endpoints forMaxam-Gilbert
sequencing were obtained by using restric-tion sites and BAL 31
deletion analysis. Dideoxy DNAsequencing reactions were performed
by using the Seque-nase kit obtained from United States
Biochemicals. Single-stranded DNA sequencing by the chain
termination method(36) was carried out by using the bacteriophages
M13mpl8and M13mpl9 (51). For double-stranded sequencing, theDNA was
mixed with primer and chemically denatured in asolution of 0.2 M
sodium hydroxide for 5 min, neutralizedwith 0.2 volumes of 3 M
sodium acetate (pH 4.5), and thenprecipitated twice with ethanol. A
second amount of primerwas added, and hybridization was carried out
at 37°C for 15min. Oligonucleotide primers were kindly synthesized
byK. L. Roy at the University of Alberta. Sequencing reac-tions
were subjected to electrophoresis in 6% polyacryl-amide gels for 2
to 10 h at a constant power to maintain atemperature of 50°C.
Analyses of nucleotide sequences andderived protein sequences were
carried out on an IBMPC-XT computer using the Beckman Microgenie
program(33) or on a Macintosh SE computer using DNA Strider
(26).Overproduction of proteins by using the T7 RNA polymer-
ase/promoter system. Restriction fragments from the Terregion of
RK2Ter were inserted into the multiple cloning sitebehind the T7
promoter in plasmid pT7-5 or pT7-6 (41a). Therecombinant plasmids
were transformed into E. coli JM83,with selection for ampicillin
resistance. After confirmation oftheir restriction maps, the
recombinant plasmids were trans-formed into E. coli K38 carrying
plasmid pGP1-2, withselection for resistance to both ampicillin and
kanamycin.Proteins were overproduced by incubation of the
bacterialculture at 42°C as described by Tabor and Richardson
(41).[35S]methionine was obtained from DuPont, Canada. Methi-onine
assay medium was obtained from Difco. The cell pelletwas
resuspended in loading buffer and boiled, and aliquotswere
subjected to electrophoresis in 12% acrylamide gelscontaining 0.1%
sodium dodecyl sulfate (SDS) (22). Gelswere dried and exposed to
Kodak XAR-5 film. Standardproteins were obtained from Bio-Rad
Laboratories (Canada)Ltd., Missauga, Ontario, Canada.
In vitro transcription-translation analysis. The polypep-tides
encoded by various plasmids (Table 2) were identifiedin an E.
coli-derived in vitro transcription-translation systemas described
previously (14). The [35S]methionine-labeledpolypeptides were
separated on 15% polyacrylamide gelscontaining 0.1% SDS as
described by Laemmli (22).
Nucleotide sequence accession number. The nucleotidesequence
data reported in this paper have been submitted toGenBank and
assigned accession number M38697.
J. BACTERIOL.
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kilA-Ter REGION OF RK2Ter 1113
Ba A/Sa E A H B E X Sm A/B * **TTI I T TT I, T
II4 1 It2 3 4 5
i1t 1 1t6 6.85
tet cat telB telA kilAFIG. 1. Mini-Mu insertions into pDT1555
that eliminate or reduce resistance to potassium tellurite. Plasmid
pDT1555 contains the tellurite
resistance region of RK2Ter inserted into pACYC184 (solid line).
The direction of transcription of the chloramphenicol
transacetylase geneis indicated by a horizontal arrow under the map
of pACYC184. Lower symbols ( denote lac+ transcriptional fusions in
which amini-Mudllac phage was inserted to inactivate Ter. Upper
symbols denote lac" translational fusions in which a mini-MudIIlac
phage wasinserted either to inactivate Ter (MIC c 1 p.g/ml) (t) or
to reduce the level of resistance (*) to a Te MIC of 16 ,ug/ml. In
each case, the directionof insertion of mini-Mu indicated the
direction of transcription and translation of the genes kilA, telA,
and telB, as indicated by arrows underthe map. Restriction
endonuclease sites: AccI, A; BamHl, Ba; BssHII, B; EcoRI, E; Sall,
Sa; SmaI, Sm; XhoI, X.
RESULTS
Transcriptional and translational fusions with I-galactosi-dase.
Thirteen Tes mutants of pDT1555 that expressed,-galactosidase were
isolated by insertion of the transcrip-tional fusion phage
mini-MudI. These were mapped to 10different positions within the
3-kb insert of pDT1555 (Fig. 1).The orientation of insertion of
mini-MudI in each mutantindicated that transcription was occurring
from right to lefton the map of pDT1555. All transcriptional
fusions resultedin the inactivation of the Ter determinant
[potassium telluriteMIC of s1 ,ug/ml, versus 256 ,ug/ml for E.
coli(pDT1555)]. Atotal of seven Tes mutations mapped within the
kilA gene.
Similar results were obtained with the translational fusionsto
3-galactosidase using mini-MudlI: of 18 mutants isolated,11
insertions were mapped in the kilA gene. Again, thedirection of
insertion indicated translation in only one direc-tion, from right
to left on the map of pDT1555 (Fig. 1).However, not all of the
mini-MudlI inserts showed a com-plete loss of Ter. Three mutants,
in which the inserts were ator close to the beginning of the kilA
gene, specified low-levelresistance to Te (MIC = 16 jxg/ml). Since
insertion ofmini-Mu into kilA eliminated or reduced the level of
telluriteresistance, it appeared that the promoter for the Ter
genesmay be close to or identical to the kilA promoter.
Alterna-tively, the kilA gene product could be involved in
telluriteresistance. The isolation of a large number of
0-galactosi-dase transcriptional and translational fusions
throughoutkilA indicated that transcription and translation of the
clonedkilA gene were occurring.DNA sequence analysis of the kilA
and Ter genes. The
nucleotide sequence of the Ter region from RK2Ter wasdetermined
by subcloning fragments of DNA from pDT1558or its TnJOOO insertion
mutants into the bacteriophageM13mpl8 or M13mpl9. Portions of the
sequence weredetermined by double-stranded sequencing ofpDT1558 or
itsinsertion mutants, using specially designed
oligonucleotideprimers.
Potential open reading frames (ORFs) were identified
bylocalization of probable initiation codons (ATG) and termi-nation
codons (TAA, TAG, and TGA) (Fig. 2). Three largeORFs were
identified (Fig. 3). The first ORF, extending fromnucleotide (nt)
coordinates 75 to 845, could encode a proteinof 257 amino acids in
length with a predicted molecularweight of 28,391. This ORF
corresponds to the kilA gene,which has been localized to this
region (15). An insertion ofTn1000 into this region was isolated
which resulted in loss ofresistance to tellurite (pDT1558-5). The
point of insertionwas determined by DNA sequencing and found to be
nearthe beginning of the kilA gene (Fig. 2). This result again
suggested that kilA may be required for expression oftellurite
resistance or that the Ter genes are transcribed froma promoter
near or identical to the kilA promoter.The promoter and operator
sequences for kilA have al-
ready been identified (52). The -35 region present matchesthe
consensus E. coli -35 hexanucleotide (TTGACA; 34) infive of six
positions. An A+T-rich hexamer corresponding tothe -10 portion of
the promoter is located 18 nucleotidesdownstream. The sequence CAT,
commonly found at thetranscription initiation site, is found 6 nt
downstream of theproposed -10 region (nt 37 to 39 in Fig. 2). RNA
primerextension analysis of plasmids pDT1558, RP4, and RP4Terhave
been used to show that transcription initiation occurs atnt 39
(49a). The sequence GAGG found within the E. coliribosome-binding
sequence identified by Shine and Dalgarno(38) is located 9 nt
upstream of the proposed translationalinitiation codon.A second
ORF, extending from coordinates 866 to 1999,
can encode a protein of 378 amino acids with a
predictedmolecular weight of 42,130. This gene has been
designatedtelA, since insertions of Tn1000 or mini-Mu into this
regioneliminate Ter (50). This ORF follows only 20 nt after the
endof kilA. The sequence GGAG found within the Shine-Dalgarno
ribosome-binding sequence is located 6 basesupstream of the
proposed start codon. The site of insertionof Tnl000 in plasmid
pDT1558-4 (50) was located to a site 20nt before the proposed start
codon for telA by DNA se-quencing. This plasmid confers an
intermediate level oftellurite resistance upon its host (MIC = 8
pg/ml), probablyas a result of expression of the telA and telB
genes from apromoter within TnJ000.A third ORF, extending from
coordinates 1999 to 2949,
was identified and named telB, since transposon insertions
inthis region also result in loss of resistance to tellurite.
Thisreading frame can code for a polypeptide 316 amino acids
inlength with a molecular weight of 32,375. Because of theabsence
of sequences resembling a promoter before telB, itappears that telA
and telB are transcribed as a single unit.One nucleotide of the
proposed start codon for telB overlapswith the last codon of telA;
therefore, it is predicted that thisORF would be translated
immediately after translation oftelA is completed, without
dissociation of the ribosome.However, there is a potential
ribosome-binding sequence,AGGAG, 5 nt upstream of the proposed
start codon.
Shortly after the end of telB is an inverted repeat, nt 2963to
2974 and 2978 to 2989, in which 11 of 12 residues in eachhalf
match. A long T-rich region (nt 2984 to 2995) overlapsthe second
half of the inverted repeat. These featuresindicate the presence of
a rho-independent terminator of
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1114 WALTER ET AL. J. BACTERIOL.
mRNA-35 -10 S
GTTGACGAGGGATAGAAAGTTTAGCTAAACTTCTTCCATC
GAAAAGCAATTAACCCACCGCGAGGGTGTATCGAATGGAA 80M E (kilA)
GAACAAAGCGTGAACATGGCGCGATTGAAGGGGGAGGTTT
TGCCCGCCCTCTTCGCGTCGCCGGCGACGATTGGCGAGTAE Q S V N M A R L K G E V L
P A L F A S P A T I G E Y
I TnlOOO (1558-5)CGGGGCCGGCATCGACGGGGCGGATTCCCTCAACGAGCTG
TCGAATCTGAT1IGAGCACGGCGCAGTTGCCGCGCTGGCCG
G A G I D G A D S L N E L S N L M E H G A V A A L A
D K I S Q I V A K L A D A D P R K I A E K P T W F E K
ATGCTTGGCCGTGAGGTTGAACGCCAGGTGAGGTATCAGG
TCGCCCGCAAGACGCTCGACCAGTTGCTGGACGAAGCCGAM L G R E V E R Q V R Y Q V
A R K T L D Q L L D E A E
_Bs AGGGCGTAGCGCAGCGCGTGCGGGACACGTTGCGCGCCTTG
GATGACATGCTCAATACGCATGAGGCCGAGGTAGACCGGC
G V A Q R V R D T L R A L D D M L N T H E A E V D R
TCAGAGCCTACATTCAAGCCGGGCGCGAGTTCCTGGACGA
GAACCCCGAGGCCGGCGCGGCCAAGGCCGGCGTGATCGAGL R A Y I Q A G R E F L D E
N P E A G A A K A G V I E
BsTTCGACAAGCCGCGCGAACGCTTCGCGCGCAAGCTCGCCe
ACCTGGCAACCCTCATGGCGTCCCATGAAATGAGCGTCACF D K P R E R F A R K L A N
L A T L M A S H E M S V T
TCAGATGAAGCTCACGCGGGCGCAGGCCGTGGACATGCTG
GACCGCTTCTCTGAAACGGCATCCGTCCTGGTGCCCGTCTQ M K L T R A Q A V D M L D
R F S E T A S V L V P V
GGCGTCAGCACACCCTCGCGCTCATCACCACCAAGAACAT
GAATCCGGCAATGGTCGCCGAGGCGGCCAAAGCTCACCAGW R Q H T L A L I T T K N M
N P A M V A E A A K A H Q
TnlOOO(pDT1558-4)| DGCGCTCATGCGGAGCCTTTCGCAGAGCCTGGAAGGCATCA
ACCAAprAACACGGCGGGAGAACCCTAIGAACGCACTGAAAA L M R S L S Q S L E G I
N Q * M N A L K
160
240
320
400
480
560
640
720
800
880(telA)
960T T H D A K A P I V A F D M T P A T L R E L G L Q E S
CGACGTGCCGGAAGTCCATGCGGTCGCGCAGCGGATCGAG
GTCGGCAGTCCGCAGACCGTTGCCGAGTTCGGCCGCGACG 1040D V P E V H A V A Q R
I E V G S P Q T V A E F G R D
TGGCCGAGCACACGTCCCGCTACGCCGATAGCCTGCTGGA
CCAGGTGCGCAACAGCGACCTGGACGAAGCAGGCGAGAAA 1120V A E H T S R Y A D S
L L D Q V R N S D L D E A G E K
CTGACCCAGGTTGTCGCCAAGGCCCGTTCCCTGAACGTCG
GCCCTTTGTCCGACAACCGTTCCCGCCTGCCCCTGATTGGL T Q V V A K A R S L N V G
P L S D N R S R L P L I G
Bs
1200
CCCGCTGATCGACCGCTTCCGCGTCCGTTCGACGGGCTTC
ATGGCGCGCTTCGACACGACCCGCGAGCAGATCGAACACC 1280P L I D R F R V R S T
G F M A R F D T T R E Q I E H
TGGTCAGCGAAGTGCAGACCACCCAGCAAGGCATCGCGCA
GCGCAATGCCTCGCTCGACGAAATGTTCGCAGCCGTGCGC 1360L V S E V Q T T Q Q G
I A Q R N A S L D E M F A A V R
GAGGAACACCGCCTTCTTGGCGTCCACATCGCGGCCGGCA
AGGTCCGCCTTGCCGAGCTGCGCGAGCAGGCCGAGGGTCT 1440E NH R L L G V H I A A
G K V R L A E L R E Q A E G L
SmGCGCGGCAATGTCGGGAACGACCCGGGCCGCGTGCAGGAG
CTGGCCGACCTCGATGCGATGGTTGCCAACCTGGACAAGC 1520
R G N V G N D P G R V Q E L A D L D A M V A N L D K
FIG. 2. Nucleotide and predicted amino acid sequences of the
tellurite resistance region of RK2Ter. The nucleotide sequence of
the cbdingstrand of the kilA-tellurite resistance region of RK2TeF
is shown. The deduced amino acid sequences of the three largest
ORFs are shownunderneath in the standard ohe-letter code. The ORFs
have been named kilA, telA, and telB. ATG start codons have been
underlined.Hexanucleotides corresponding to the probable promoter
are indicated by -35 and -10; the mRNA start site is marked mRNA;
possibleribosome-binding (Shine-Dalgarno) sequences are marked SD.
The single nucleotide difference in RK2 is indicated by a small
letter above thesequence at nt 2371. An inverted repeat after telB
is marked by lines above the sequence (nt 2963 to 2989). A T-rich
region from nt 2984 to2995 is marked by a line under the sequence.
Points of insertion of TnlO00 and TnphoA ih various insertion
mutants are indicated by verticallines. Restriction endonuclease
sites are indicated above the sequence as follows: AccI, A; BssHII,
Bs; EcoRl, E; SmaI, Sm; XhoI, Xh.
transcription (34). This corresponds to the terminator
previ-ously identified upstream of the korA/trfB/korD
promoter(47).The G+C content of this region is 64%, which is close
to
that of other genes on plasmid RP4 (60%) and in thePseudomonas
chromosome (66%) (17, 27), and there is acorresponding preference
for G or C in the third position ofthe codon.The nucleotide
sequence of the kilA-Ter region of RK2Ter
was compared with that of the Tes plasmid RK2 (14a). Overthe
3-kb region compared, only a single difference wasfound: a missense
mutation at nt 2371 (Fig. 2; A in RK2 andT in RK2Ter) resulting in
a Ser (RK2)-to-Cys (RK2Te)transition in telB. An identical mutation
in this residue wasfound in the sequence of an isolate of the Te'
plasmid RP4(45a).The DNA sequences and derived amino acid sequences
of
the Ter genes of RK2 were compared with those of the
IhcHI-2 plasmid pMER6iO (20) and the IncHII plasmidpHH1508a
(50a); however, no similarities could be detected.This lack of
similarity confirms earlier dot blot studies (50)and suggests that
the three Ter determinants are unrelated.In addition, our sequence
was used to search recent DNAand protein data banks, but no
significant homologies couldbe found.
Alkaline phosphatase fusion proteins. Insertions of trans-poson
TnphoA were constructed to determine whether anyof the proteins
encoded by this region were membraneassociated. Three independent
insertions were obtainedwhich eliminated the expression of
tellurite resistance andallowed the expression of active alkaline
phosphatase, asdetected by the formation of blue colonies on media
contain-ing XP. Each of these TnphoA insertions was mapped to
thesame position within the telB gene. The junction point of oneof
these insertions was sequenced and located immediately
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VOL. 173, 1991 kilA-Ter REGION OF RK2Ter 1115
GCATCGGCGACCTGATCGCCTTGCAACATTCGGCCATGCA
GAGCCTGCCGACCATCCGCATGATCCAGGCCAACAACCAG 1600R I G D L I A L Q H S
A M Q S L P T I R M I Q A N N Q
ATGCTGGTCGATAAATTCCACACCATCCGCGAAATCACCG
TGCCGGCGTGGAAGCGGCAATTCATGCTGGCCTTGAGCCT 1680M L V D K F H T I R E
I T V P A W K R Q F M L A L S L
CAACGAGCAGAAGAACGCCGTCGAACTGGCCACGGCCATC
GACGACACCACCAACGACCTGATGAAGCGCAATGCGGCCC 1760N E Q K N A V E L A T
A I D D T T N D L M K R N A A
TGCTGCATCGCACGTCCGTCGAGACGGCGAAGGAGAACCA
ACGCCTGGTGATCGACGTGGACACGCTCAAGCAGGTTCAG 1840L L H R T S V E T A K
E N Q R L V I D V D T L K Q V Q
TnlOOO (pDT1558-2)ACGACGCTCATCAAGACCGTCGAGGACIlrTATTCGCATCC
AGCAGGAAGGCGTGCAGAAGCGCAAGGATGCCGAGAAGCA 1920T T L I K T V E D V I
R I Q Q E G V Q K R K D A E K Q
S3DGATCGCCGCAATGCGTGGCGATCTTCAAGCCAAGCTGACC
CGCCAGCCCGTGCGCGAGCTGGCCCAACAGGAGTCCGTAI 2000
I A A M R G D L Q A K L T R Q P V R E L A Q Q E S VXh M
(teIB)
(AATGCCACAAACACCGATGTTTTCGCCCAGGTAGGCGGC
CTCGAGGCCCGAGGCGCGAAGATGAAGAAGCGGGGCACCC 2080N A T N T D V F A Q V
G G L E A R G A K M K K R G T
GCTTCCTCATCGCGGCGCTGGCAGTCCTTGCCATTGCCGG
GATCGGGGCAGTAACGGGATGGGCGATCAGCCCGAGCGCG 2160R F L I A A L A V L A
I A G I G A V T G W A I S P S A
ACGCCCGGAAGCATTGACGTGCCGCAGGTGCTGGCATCGA
CATTCAGCGACCAGGTGCCGGGCAGTGAGGGCGGCGGCCT 224CT P G S I D V P Q V L
A S T F S D Q V P G S E G G G L
GGGTGGCGGCCTGCCCTTCACTTCGGCCGTCGGGGCATTC
ACGGACTTCATGGCGGGGCCGGCAATTTTTACCTTGGGCA 2312G G G L P F T S A V G
A F T D F M A G P A I F T L G
E aTTCTTGGCATAGTGGTCGCGGGTGCCGTGCTCGTGTTCGG
GGGTGAATTCTGCGGGTTCGTGCGATCCGTCTGCATGATG 2400I L G I V V A G A V L
V F G G E F C G F V R S V C M M
GTGATAGCCGTCAGCATGATTTTCGTGTCGTCGAACTTGG
TGAAGGGCATTCTCGGCGGCGATCACGACGCCGGCCCTGC 2480V I A V S M I F V S S
N L V K G I L G G D H D A G P A
GGAGCCTTCGCCGCGTGCGCGATTCATGGCGGCCGTGGAG
GCCAAGGATTTCGCGCGAGTGCAAGAGCTGATCGAGGCGC 2560E P S P R A R F M A A
V E A K D F A R V Q E L I E A
BsGTGGAGCCAAGTCGGCGGCTGATTATGTCCTTGCGCAGCT
CGCCGTGGCCGAAGGTCTGGACCGCAAGCCTGGTGCGCGC 2640R G A K S A A D Y V L
A Q L A V A E G L D R K P G A R
ITnphoA TnlOOO
(pDT1558-1)GTCGTGGTCGGGAAAGCGGCGGGCAGCATGGCAATGCCGC
ICTGCGGCGCTGGGTTTTACGCCAAGGGGAGAAGCGGCATAI 2720V V V G K A A G S M
A M P P A A L G F T P R G E A A Y
CGCCATCGAGCGGTCAGCCTATGGTGAGCCGAGGTCCAGC ATTGCA I E R S A Y G E
P R S S I A
CGACCTGGTGGGCGATGGCCGGTGTGGCCGGCATCATCGG CGCGAA T W W A M A G V
A G I I G A
GCAGTGTCGATCCGCAACCGAGTGAAGCGCGTGCGCGACC TGTTGA V S I R N R V K
R V R D L L
AGACGAAAGCCCGGTTTCCGGGCTTTTGTTTTGTTACGCC 3000
after nt 2680; it was found to be in the correct reading
framefor fusion with the TelB protein (Fig. 2; 25).
Hydrophobicity plots. The amino acid sequences of KilA,TelA, and
TelB were analyzed by using the algorithms ofKyte and Doolittle
(21) (Fig. 4). Both KilA and TelA appearpredominantly hydrophilic.
In contrast, a number of hydro-phobic domains of about 20 amino
acid residues in length are
1000
3
2
1
-1
-2
-3
'GAAGCAGTACCAGCAGGAATGGAACCGGAAGGCGG 2800K Q Y Q Q E W N R K
A
TCCTGGCGGCGGCGGCAACCGGCTTTGTTGGGCTG 2880I L A A A A T G F V G
L
;GTGATGGAGCCGGGTGCAGAGCCATAAGCGGCAAG 2960V M E P G A E P
evident in the hydrophobicity plot of TelB. These domainsmay
anchor the TelB in the bacterial cell membrane. Amembrane location
for TelB is supported by the constructionof alkaline phosphatase
fusions with this protein, using thetransposon TnphoA. The point of
insertion of TnphoA wasdetermined by DNA sequencing and located
within aminoacid residue 228 (Fig. 2). This part of the protein is
predicted
2000 3000
3
2
1
-1
-2
-3
1000 2000 3000FIG. 3. ORFs encoded by the kilA-tellurite
resistance region of RK2Ter. The positions in the DNA sequence
(Fig. 2) are indicated above
and below in thousands of nucleotides. Possible translation
initiation codons (ATG) and termination codons (TAA, TAG, and TGA)
areindicated by short and full vertical lines, respectively, in the
three reading frames on both the coding strand (1, 2, and 3) and
the oppositestrand (-1. -2, and -3). The three largest ORFs on the
coding strand are shaded and marked kilA, telA, and telB.
I1':'''''llTite^,.lll lllll...
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1116 WALTER ET AL.
(a)
P -uAconas8
tolB ts';A i -
to A A:; t
100
.D 0|
>- -200
300-lTnphodiAs200%
-100-0100
-200
-300I 100 200 300 400
Residue NumberFIG. 4. Hydrophobicity plot analysis of the KilA,
TelA, and
TelB proteins. The deduced amino acid sequences of kilA (a),
telA(b), and telB (c) shown in Fig. 2 were used to predict the
hydropho-bicity plots by the algorithms of Kyte and Doolittle (21),
using awindow of 11 amino acids. The positive and negative values
indicatehydrophobicity and hydrophilicity, respectively. The point
of inser-tion of TnphoA in TelB is shown by an arrow in panel
c.
(b)A B C D
97-
66-
45-
31 -
* _
....-.sk
21-
to be located in the periplasm, since alkaline phosphatase
isactive only if it is exposed to the periplasmic
environment(25).Overproduction of the KilA, TeIA, and TelB
proteins.
Plasmids were constructed in which all (pDT1969) or part(pDT2031
and pDT2034) of the Ter region of RK2Ter wasplaced under the
control of the T7 promoter found inplasmids pT7-5 and pT7-6 (Fig.
5a). pDT1969 was con-structed by ligating the BamHI-HindIII
fragment ofpDT1555 (Fig. 1) into the corresponding sites within
themultiple cloning region of pT7-5. pDT2031 carrying the kilAand
telA genes of RK2Ter was constructed by ligating theXhoI-BamHI
fragment of pDT1555 into SalI-BamHI-di-gested pT7-5. pDT2034
carrying the kilA gene and part of thetelA gene was constructed by
ligating the BamHI-SmaIfragment of pDT1555 into similarly digested
pT7-6.
After induction at 42°C and labeling with [35S]methionine,cell
lysates containing the encoded proteins were subjectedto
SDS-polyacrylamide gel electrophoresis (PAGE). Theautoradiogram
showed that three proteins were produced bycells carrying pDT1969,
which contains the entire kilA-telABregion (Fig 5b). These proteins
had apparent molecularmasses of 48, 31, and 30 kDa. Cells carrying
pDT2031, whichencodes only the kilA and telA genes, did not produce
the30-kDa protein, indicating that this is the TelB protein.
Cellscarrying pDT2034, containing all of the kilA gene and part
ofthe telA gene, did not produce the 48-kDa protein, indicatingthat
this is TelA. The 31-kDa protein was still produced andappeared to
be KilA, and a new band of 28 kDa was seenwhich appeared to be a
truncated form of the TelA protein.
14-
FIG. 5. Overproduction of KilA, TelA, and TelB proteins in theT7
RNA polymerase/promoter system. (a) The indicated regions ofthe Ter
region of pDT1558 were put under the control of the T7promoter,
using plasmid pT7-5 or pT7-6. The start and stop codonsof the kilA,
telA, and telB genes are indicated by short vertical lines.(b)
Autoradiogram showing the proteins expressed in cells carryingeach
recombinant plasmid after overexpression and subjection toSDS-PAGE
as described in Materials and Methods. Lanes containcell lysates of
E. coli K38(pGP1-2) carrying the following plasmids:pT7-5 (lane A),
pDT1969 (lane B), pDT2031 (lane C), and pDT2034(lane D). The
protein TelA (48 kDa) and the closely spaced proteinsKilA (31 kDa)
and TelB (30 kDa) are indicated by closed arrow-heads; a truncated
derivative of TelA is indicated by an openarrowhead. The molecular
masses and positions of the markerproteins are indicated on the
left in kilodaltons (phosphorylase b97.4; bovine serum albumin,
66.2; ovalbumin, 45; carbonic anhy-drase, 31; soybean trypsin
inhibitor, 21.5; lysozyme, 14.4).
The sizes of the proteins overproduced in this system areclose
to those predicted from the nucleotide sequence.
Analysis of proteins encoded by Tes and Ter plasmids.Plasmid
pMS202 carries a portion of plasmid RP4, includingthat
corresponding to the Ter region of RK2Ter. Bacteriacarrying pMS202
are not resistant to tellurite; however,upon plating on media
containing tellurite, variants ofpMS202 that expressed tellurite
resistance (pMS202-2 andpMS202-4) could be obtained. The proteins
encoded by
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kilA-Ter REGION OF RK2Ter 1117
A B C D E F G
-92
-69
TelA
_ |* - s~KiIA
w *"TeIBGo~~
5_ 4
FIG. 6. Analysis of KiIA, TelA, and TelB products in an
E.coli-derived in vitro transcription-translation system. The
autoradio-gram shows the SDS-PAGE profiles of polypeptides encoded
bypMS202 and its derivatives. Lanes contain no DNA control
(A),pBR325 (B), pMS202 Te' (C), pMS202-2 Ter (D), pMS202-4 Ter
(E),and pMS202-4 derivatives into which mini-MudI was inserted
toinactivate the Ter determinant (F and G). The TelA (45 kDa),
KilA(31 kDa), and TelB (28 kDa) polypeptides are indicated by
arrows,and truncated derivatives of TelB are marked by an
arrowhead.Molecular masses of radiolabeled standard proteins are
marked inkilodaltons (phosphorylase b, 92; bovine serum albumin,
69; oval-bumin, 46; carbonic anhydrase, 30; lysozyme, 14).
these Tes and Ter plasmids were identified by in
vitrotranscription-translation followed by separation by SDS-PAGE
(Fig. 6). All three plasmids specified production of thesame three
polypeptides, TelA (45 kDa), KilA (31 kDa), andTelB (28 kDa). This
result showed that mutation to Ter didnot result in the production
of additional polypeptides.Furthermore, it supports the suggestion
that the mutationresponsible for the difference in expression of
Ter may be amissense mutation rather than a frameshift mutation or
amutation in the promoter for the Ter genes.
Mutation of pMS202-4 to tellurite susceptibility by inser-tion
of the mini-MudI phage (Fig. 6, lanes F and G) resultedin the loss
of the TelB polypeptide and the appearance of atruncated
polypeptide of approximately 23 kDa. All plas-mids, which were
derivatives of pBR325, specified chloram-phenicol acetyltransferase
of 24 kDa which was consistentlyvisible on the polyacrylamide
gels.
DISCUSSION
In this study, the molecular biology of the cryptic
telluriteresistance determinant from plasmid RK2 and its
relation-ship to the neighboring gene kilA was examined. By
usingtranscriptional and translation fusions to the lacZ gene,
thedirection of transcription and translation of the Ter geneswas
shown to be in the same direction as that of kilA. DNAsequencing of
this region revealed three ORFs, kilA, telA,and telB. The product
of kilA, predicted to be a 28,391-Dahydrophilic protein, was
observed after SDS-PAGE to have
an apparent molecular mass of 31 kDa. This protein isprobably
located in the cytoplasm, since a large number offusions were
obtained with ,3-galactosidase (LacZ), which isactive only in the
cytoplasm (39).The product of telA, predicted to be a 42,130-Da
protein,
has been detected by in vitro transcription-translation, inwhich
it had an apparent molecular mass of 40 to 45 kDa (50;this study).
The in vivo expression system used here indi-cated a molecular mass
of 48 kDa. Purification of TelA iscurrently being attempted to
determine the reason for thisvariation in size. The isolation of
several translational fu-sions of lacZ and the absence of any long
stretches ofhydrophobic residues in its sequence suggest that TelA
is acytoplasmic protein.The telB gene, however, appears to code for
a membrane-
spanning polypeptide, as judged from the hydrophobicityprofile.
The predicted molecular mass of 32,375 Da is closeto the 30-kDa
size seen in the T7 overproduction system.The slightly smaller size
seen with SDS-PAGE may be dueto this protein's hydrophobic nature,
causing it to bind lessSDS than other proteins of its size.
Hydrophobic membraneproteins often show abnormally fast migration
on SDS-PAGE gels (11).
Isolation of fusions of TelB to alkaline phosphatase
andP-galactosidase suggests that this protein is located in
theinner membrane, with parts extending into the periplasm
andcytoplasm. Gene fusions to both P-galactosidase and alka-line
phosphatase have been used extensively to study pro-tein location
and topology (24). However, cell fractionationexperiments are
currently under way to confirm the pro-posed locations of these
proteins.The mechanism by which TelA and TelB confer resistance
to tellurite is not clear. In contrast to the results
obtainedwith the mercury resistance determinant of plasmid
R100(30), no hypersensitive mutants were obtained during isola-tion
of insertions of Tn1000, TnphoA, or mini-Mu. Thissuggests that the
IncPa Ter determinant does not encode atellurite uptake system
despite its membrane location. TheTelA and TelB proteins may
directly reduce toxic potassiumtellurite to metallic tellurium,
which has been shown to bedeposited near the membrane (43).
Alternatively, since bothTer and Tes E. coli are capable of
tellurite reduction (49a),these proteins may simply increase the
rate of reduction byhost cell proteins. A tellurite reductase has
recently beenpartially purified from Thermus thermophilus (10). The
pos-sibility that TelA and TelB form a tellurite-efflux system
isalso under investigation.No similarity could be found between the
sequence of this
TeT determinant and those of the IncHI-2 plasmid pMER610(20) or
the IncHII plasmid pHH1508a (50a). However, eachTer determinant
appears to encode a membrane protein ofsimilar size which could
have some functional similarity.The presence of at least three
different plasmid-encoded Terdeterminants with no sequence homology
suggests that theevolution of Ter may occur quite readily and that
thisresistance is important to bacteria either in the environmentor
in colonization of the human body.The relationship of tellurite
resistance to the plasmid
replication control genes on RK2 is still unclear. The kilAgene
does not appear to be essential for expression oftellurite
resistance, since some mutants with insertions in themiddle of kilA
still express an intermediate level of resis-tance. However, many
other insertions in kilA eliminateexpression of Ter. This effect
may be due to interruption oftranscription of the Ter genes from
the kilA promoter.The isolation of transcriptional and
translational fusions in
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1118 WALTER ET AL.
the kilA gene indicated that the promoter for this gene
wasfuinctional despite interruption of the -35 region during
thecloning of this region (50). Even in the absence of korA,
theexpression of cloned kilA was not lethal to the host bacte-rium.
Its previously observed lethal effect may require otherregions on
RK2, or the host cell may have developed someresistance to its
function which is as yet unknown (15, 52).Our overexpression of
kilA will enable further studies on itspossible roles in either RK2
plasmid host range or plasmidreplication control (1, 37, 48).The
difference between RK2 and its tellurite-resistant
variant RK2Ter was investigated by comparing their DNAsequences.
The only difference found was a missense muta-tion in telB which
results in a Ser (RK2)-to-Cys (RK2Ter)change at amino acid residue
125. This finding suggests thatthis Cys residue is essential to the
expression of telluriteresistance by RK2Ter and that a mutation in
this residue isresponsible for lack of expression of Ter by RK2.
AnotherCys residue is found close by in the TelB amino
acidsequence. Two closely spaced Cys residues are also found
inmercuric reductase, MerA; its periplasmic mercury-bindingprotein,
MerP; the cadmium resistance protein, CadA (com-pared in reference
31); and glutathionine reductase (32). Theimportance of the Cys
residues in TelB in, for example, thebinding of metal ions,
cofactors, or reducing equivalentsrequires further
investigation.
Stable Ter variants of RP4 are readily generated uponplating on
tellurite media (4). This characteristic is mostconsistent with a
single mutation. Plasmids RK2, RP4, RP1,and R68 were all isolated
from the same hospital in Birming-ham in 1969 and are identical by
restriction endonucleaseanalyses and heteroduplex analysis (7, 13,
17, 18). Loss ofexpression of Ter by these plasmids may have
occurredeither before isolation of the plasmid or during
subsequentlaboratory subculturing. Further studies are required
todetermine whether all isolates of these plasmids have iden-tical
mutations in their Ter determinants.
ACKNOWLEDGMENTS
This work was supported by grants from the Medical
ResearchCouncil of Canada (to D.E.T.) and the U.K. Medical
ResearchCouncil (to C.M.T.). E.G.W. is the recipient of a
studentship fromthe Medical Research Council of Canada and a
research allowancefrom the Alberta Heritage Foundation for Medical
Research. D.E.T.is a Heritage Medical Scholar. J.P.I. was supported
by MRC grantG8511172CB awarded to C.M.T.We thank M. Casadaban for
mini-Mu strains, D. H. Figurski for
communication of DNA sequences prior to publication, E.
K.Manavathu for performing the in vitro
transcription-translationexperiments, S. Tabor for T7 plasmids and
overexpression system,and J. Weiner for the preparation of
hydrophobicity plots andcritical reading of the manuscript.
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