characterization of kpst, the atp-binding component of the abc

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 31, Issue of August 5, pp. 20149-20158, 1994 Printed in U.S.A.

Characterization of KpsT, the ATP-binding Component of the ABC-Transporter Involved with the Export of Capsular Polysialic Acid in Escherichia coli K1*

(Received for publication, January 15, 1994, and in revised form, May 27, 1994)

Martin S. Pavelka, Jr.S§, Stanley F. Hayesn, and Richard P. SilverSll From the $Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642 and the Waboratoy of Vectors and Pathogens, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, Montana 59840

The 17-kilobase kps gene cluster of Escherichia coli K1 contains all the information necessary for the expression of capsular polysaccharide. Region 3 of the cluster encodes two genes, kpsM and kpsT, whose products belong to the ATP-Binding Cassette (ABC)-transporter protein family. The KpsMT system is involved with the export of capsular polysaccharide in E. coli. Earlier work indicated that interaction between KpsT and ATP is important for transport. In this study, we report that KpsT, a peripheral inner membrane protein, can be photola- beled by the ATP analog, 8-N,[y-s2PlATP. The derivatization of KpsT by this reagent is inhibited by cold ATP or ATPyS. Furthermore, the protein seems to require a membrane environment for efficient photolabeling, but does not require any other kps gene products. Results obtained from saturation mutagenesis of the ATP-binding consensus sequence of KpsT and the phenotypes of strains with defined mutations in the chromosomal gene, are consistent with the view that ATP-binding by KpsT is required for transport of polymer across the inner membrane. The structure of KpsT was compared to a model developed for other ABC-transport proteins, and important functional regions were determined. The results obtained from chemical mutagenesis of kpsT are consistent with the model and revealed characteristics particular to capsule transporters.

Extraintestinal infections caused by Escherichia coli include pyelonephritis, septicemia, and meningitis (Levine, 1984; Philip, 1985). E. coli K1 is the most prevalent capsular serotype in cases of neonatal meningitis caused by E. coli (Robbins et al., 1974). The K1 capsular polysaccharide, an a-2,8-linked polymer of N-acetylneuraminic acid (sialic acid), is an essential virulence determinant for this organism (Robbins et al., 1974; Silver and Vimr, 1990). Synthesis of capsular polysialic acid (polySia) in E. coli K1 is a complicated process, requiring the synthesis, activation, and polymerization of sialic acid and transport of the polymer to the cell surface (reviewed in Troy (1992)). These activities are chromosomally encoded by the 17-

* This work was supported in part by Grant AI26655 from the Na- tional Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Supported by a National Institutes of Health Predoctoral Training Grant in Microbial Pathogenesis 5T32-AI07362. Current address: Dept. of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461.

11 To whom correspondence and reprint requests should be addressed. Tel.: 716-275-0680; Fax: 716-473-9573. E-mail: [email protected] biophysics.rochester.edu.

kb’ kps gene cluster (Fig. 1) (Silver et al., 1984; Vimr et al., 1989; Boulnois and Roberts, 1990). In E. coli K1, sialic acid is synthesized within the cytoplasm in a condensation reaction between N-acetylmannosamine and phosphoenolpyruvate and activated to a nucleotide derivative, CMP-sialic acid, which is subsequently used as the sialic acid donor during polymerization (Troy, 1992). The mechanisms responsible for chain initia- tion, polymerization, termination, and export are not fully understood. The observation, however, that capsular polysaccharide synthesis occurs on the cytoplasmic surface of the inner membrane (Boulnois and Jann, 1989; Kroncke et al., 1990b; Troy, 1992) necessitates a mechanism for transporting polymer across two membranes prior to attachment to the cell surface. The aim of this study was to gain a better understanding of the export of polymer from the cytoplasm by investigating the structure-function aspects of KpsT, a protein involved with this process.

Mutational studies implicate the gene products in region 1 of the kps gene cluster (Fig. 1) in the transport of polymer across the outer membrane, as cells with region 1 mutations tend to accumulate polysaccharide within the periplasmic space (Boulnois and Roberts, 1990; Wunder et al., 1994). Conversely, cells with mutations within region 3 of the gene cluster (Fig. 1) accumulate polymer within the cytoplasm, suggesting that region 3 gene products are important for translocation of polysaccharide across the cytoplasmic membrane (Boulnois and Rob- erts, 1990; Kronke et al . , 1990a). We previously reported (Pavelka et al . , 1991) that region 3 of the K1 kps gene cluster contains two genes, designated kpsM and kpsT, whose products belong to the ATP-binding Cassette (ABC)-transporter superfamily (Higgins et al., 1988, 1990; Blight and Holland, 1990; Ames et al., 1992). This family, also known as the “traffic ATPases’’ (Ames and Joshi, 19901, includes both prokaryotic and eukaryotic proteins involved with a variety of ATP-dependent export and import processes. Within this family are the periplasmic binding protein-dependent permeases of enteric bacteria, such as Mal, His, and Opp (Higgins et al., 1986, 1990; Ames et al., 19921, the hemolysin exporter, HlyB, of E. coli (Blight and Holland, 19901, the cystic fibrosis transductance regulator of humans (Riordan et al., 19891, and the P-glycoprotein (MDR), responsible for multiple drug resistance in mammalian tumor cells (Gros et al., 1986). The members of the ABC-transporter superfamily share a common organizational motif consisting of a hydrophobic membrane component and a hydrophilic, ATP-binding component (Ames et al., 1992; Hig- gins, 1992). I t is believed that a functional transporter consists

The abbreviations used are: kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; 8-N,ATP, 8-azidoadenosine 5‘-triphosphate; ATPyS, adenosine 5’-0- (3-thiotriphosphate).

20149

20150 Characterization of KpsT from E. coli Kl

B C C H H E HE B w P B H H

1 1 1 I I1 I I I I 1 I I I , b 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 1 4 1 5 16 17

t u 1 7 I D 1 L) I A I c l l E l r , l r ) I c ,I,,, 0 , E *

FIG. 1. Genetic organization of the kps gene cluster of E. coli K1. Flanking regions 1 and 3 are conserved among different capsular serotypes of E. coli, while the central region 2 is unique for a given capsular polysaccharide. The designation neu is used for the region 2 genes of E. coli K1. Restriction endonuclease sites are shown, using the following abbreviations: B, BglII; C , ClaI; H, HindIII; E, EcoRI; BH, BamHI; P, PstI. The arrows below the map indicate the direction of transcription for each region.

of two of each type of component. Our model of the KpsMT transporter postulates a KpsM dimer within the cytoplasmic membrane and two KpsT proteins peripherally associated with the membrane, each one in contact with a KpsM protein. We and others have proposed that KpsM and KpsT constitute a system that transports capsular polysaccharide across the cytoplasmic membrane in E. coli, utilizing the energy from ATP hydrolysis (Smith et al., 1990; Pavelka et al., 1991). In addition, KpsM and KpsT are conserved among different capsular serotypes of E. coli, and homologous proteins are involved in capsular polysaccharide export in Haemophilus infZuenzae and Neisseria meningitidis (Kroll et al . , 1988; Frosch et al . , 1991; Pavelka et al., 1991).

We have shown that interaction between KpsT and ATP is important for export of capsular polysaccharide. A mutant al- lelle of kpsT (K44E), containing a mutation in a highly conserved lysine residue within the ATP-binding consensus sequence of the protein, cannot complement a strain with a chromosomal mutation in kpsT (Pavelka et al., 1991). In this study, we report further work on the interaction of KpsT with ATP, the subcellular localization of the protein, the construction and characterization of chromosomal kpsT mutants, and the determination of important functional regions of the protein by mutational analysis.

MATERIALS AND METHODS Bacteria, Plasmids, Bacteriophage, and Media-Table I contains de-

scriptions of the bacterial strains and plasmids used in this study. Bacterial cultures were grown in L-broth or on L-agar at 37 “C supple- mented with appropriate antibiotics. The use of Horse 46 (H.46) antiserum agar plates to assay K1 capsule precipitin halo formation has been described previously (Silver et al., 1984). Bacteriophage E is specific for encapsulated E. coli K1 (Gross et al., 1977).

Nucleic Acid Manipulation-Nucleic acid manipulations were done as previously described (Sambrook et al., 1989; Ausubel et al., 1987). Plasmid DNA was prepared, and DNA sequencing was done as previously described (Pavelka et al., 1991). Partial sequencing of some of the hydroxylamine mutants was accomplished using an Applied Biosystems 373A DNA Sequencer. Oligonucleotides used as primers for sequencing and the polymerase chain reaction (PCR) were produced by an Applied Biosystems 380B DNA Synthesizer. The University of Wisconsin Ge- netics Computer Group (UWGCG) software package was used for protein sequence analysis (Genetics Computer Group, 1991).

SDS-PAGE-Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was done according to Laemmli (1970). High range prestained molecular mass standards (Life Technologies, Inc.) were used: lysozyme, 14.3 kDa; @-lactoglobulin, 18.4 kDa; carbonic anhydrase, 29 kDa; ovalbumin, 43 kDa; bovine serum albumin, 68 kDa, phosphorylase b, 97.4 kDa; and myosin (heavy chain), 200 kDa.

Production ofKpsKpsTAntiserum-Overexpression of KpsT from a high copy number plasmid results in the production of insoluble inclusion bodies. We prepared inclusions (Harlow and Lane, 1988) from HBlOl containing pSR.340, solubilized them in Laemmli SDS-PAGE loading buffer, and separated the proteins via denaturing SDS-PAGE. The

area of the gel containing KpsT was excised (Harlow and Lane, 1988), and the slices were frozen a t -20 “C and used as a source of antigen for production of polyclonal antiserum in two New Zealand White rabbits at East Acres Biologicals (Southbridge, MA). The immune sera recognized KpsT in whole cell lysates prepared from both wild-type cells and cells overexpressing the protein, while the preimmune sera did not. The immune sera from both rabbits were pooled, and nonspecific antibodies were partially removed by batch absorption to acetone

JL3664. powders (Harlow and Lane, 1988) prepared from HBlOl/pKS+ and

Western Blotting-Immunoblotting was performed essentially as described (Ausubel et al., 1987). Proteins were electrophoretically transferred from polyacrylamide gels to nitrocellulose filters using a semidry transblotting apparatus (PolyBlot,American Bionetics, Emeryville, CA) at constant voltage as recommended by the manufacturer. Polyclonal rabbit KpsT antiserum was used as the primary antibody a t a typical dilution of 1:lOOO. Detection of primary antibody was either by lz5I conjugated to Staphylococcus aureus protein A (57.0 mCi/mg, 1 Ci = 37 GBq, ICN Biochemicals) followed by autoradiography or by a secondary goat anti-rabbit IgG conjugated to horseradish peroxidase (Boehringer Mannheim) used at a dilution of 15000, followed by incubation with the chromogenic substrate, 3,3‘,5,5’-tetramethylbenzidine (Kirkegaard and Perry Laboratories, Inc., Gaithersberg, MD) as recommended by the manufacturer.

Cell Fractionation-Fractionation of cells and separation of inner and outer membranes was done as previously described (Smit et al., 1975). Alternatively, inner and outer membrane proteins were separated using the detergent n-lauryl sarcosinate (Sarcosyl, Sigma), according to the procedure of Lambert, (1988). 50 pg of protein were loaded per well for analysis of the fractions by SDS-PAGE and immunoblotting.

Protein Quantitation-Protein concentration was determined by the Bradford assay (Bradford, 1976) using the Bio-Rad reagent kit (Bio- Rad). Bovine y-globulin dissolved in distilled water was used as the standard.

Immunodiffusion-Immunodiffusions, utilizing H.46 serum, were done on culture supernatants or cell lysates as previously described (Silver et al., 1984; Vimr et al., 1989).

Preparative and Diagnostic PCR Amplification-PCR amplification was done essentially as described (Pavelka et al., 19911, using specific primers flanking the kpsT gene, unless otherwise noted below. All nucleotide positions given for PCR oligomers used in this study corre- spond to the previously published DNA sequences of kpsM and kpsT from E. coli K1 (Pavelka et al., 1991), resident within GenBank under accession numbers M57382 and M57381, respectively. In all cases, amplification was accomplished using AmpliTaq polymerase and the GeneAmp DNA kit (Perkin Elmer) as suggested by the manufacturer. Reactions were run on a Perkin Elmer DNA Temperature Cycler using previously described parameters (Pavelka et al., 1991). The template material used for preparative PCR reactions was a gel-purified 1.25-kb ClaI DNA fragment containing kpsT (Fig. 1). For diagnostic amplification of chromosomal DNA, template DNA was prepared as described (Gussow and Clackson, 19891, and 10 pl of the preparation was used for each 100 p1 of PCR mixture.

PCR-directed Saturation Mutagenesis of kpsT-Site-directed mutagenesis by overlap extension (Ho et al., 1989) was done essentially as described (Ho et al., 1989; Pavelka et al., 19911, with the following changes to perform saturation mutagenesis. Mutagenesis primers “C” and “D” in our original procedure (Pavelka et al., 1991) were replaced with degenerate oligonucleotide primers spanning nucleotides 864 to 898 and 937 to 896, respectively. These new mutagenesis primers were synthesized using doped reaction mixtures in order to produce a small population of primers containing one point mutation at any position within each oligomer (Ner and Smith, 1989). The PCR reactions were carried out as described (Pavelka et al., 19911, and full-length DNA products were recovered, ligated into Bluescript pKS’, and introduced into EV95. We obtained 24 acapsular clones which produced KpsT at levels comparable to the wild-type plasmid pSR340. DNA sequence analysis of these 24 clones identified 5 with a single missense mutation in the kpsT gene. Transformation of RS2436 with these five plasmids resulted in an acapsular phenotype, confirming that the original phenotype was not due to mutations within the chromosomal kps gene cluster.

Chemical Mutagenesis-Hydroxylamine mutagenesis of plasmid DNA was done essentially with the method of Humphreys et al. (1976). The mutagenized DNA was recovered by absorption to glass powder (Geneclean, Bio 101) eluted into sterile distilled water and added to aliquots of competent RS2436 cells. Mutant transformants were se-

Characterization of KpsT from E. coli K1 20151 TABLE I

Bacterial strains and plasmids

Strain or plasmid Relevant genotype Source or reference

E. coli K-12 HBlOl

XL-1 Blue

SY327Apir SMlOhpir

JL3664

EV5 EV36 EV95 RS2400 RS2436

E. coli K1K-12

Plasmids PKS' vACYC184 pCVD442 pSR340

pSR346

pSR366

pSR385 pSR386

pSR387 pSR389

pSR390

pSR402

pSR415 pSR416

pSR417

hsds20 recAl3 ara-14 proA2 lacy1 galK2 rpsL20 xyl-5

recAl lac en&l gyrA96 thi hsdRl7 supE44 relAl

A(1ac pro) argE(Am) rif nalAreeA56 pirR6K thi thr leu ton4 lacy supE44recA::RP4-2-Tc::"

argA22 galP23 rha-200

EV1 neuAZ2 galP23 rpsL9 (arg*rha+kps+) EV36 kpsT32::TnZO EV36 kpsT10 K44E EV36 AkpsT

Bluescript cloning vector, 2.95-kb, Ap' Cloning vector, 4.0-kb, CmTc' Suicide vector, 6.2-kb, sacB oriR6K mobRP4, Ap' 700-bp EcoRI-BamHI PCR fragment containing wild-type

kpsT cloned into pKS+, downstream of lac promoter 700-bp EcoRI-BamHI PCR fragment containing kpsT

with the K44E mutation cloned into pKS', downstream

2.95-kb PuuII-ClaI fragment containing region 3 and part of lac promoter

pACYC184 minus both BglI sites, Cm' of region 2 cloned into pKS'

2.95-kb BamHI-Sal1 region 3 fragment from pSR366

2.0-kb BumHI-BglII deletion of pSR386 500-bp BglI-DraIII fragment from pSR346, containing the

kpsT K44E gene, cloned into pSR387 2.1-kb HzndIII-Sal1 fragment from pSR389 cloned into

2.1-kb SacI-Sal1 fragment from pSR390 cloned into

500-bp BglI-DraIII deletion of kpsT in pSR387 1.6-kb HindIII-Sal1 fragment from pSR415 cloned into

1.6-kb SacI-Sal1 fragment from pSR416 cloned into

mtl-1 supE44 leu

(F'proAE lacZAM15 TnlO)

Km'pirR6K

cloned into pSR385

P M '

pCVD442

PKS'

pCVD442

Laboratory collection

Laboratory collection

Miller and Mekalanos, 1988 Miller and Mekalanos, 1988

Vimr and Troy, 1985

Vimr et al., 1989 Vimr et al., 1989 Vimr et al., 1989 This study This study

Stratagene Chang and Cohen, 1978 Donnenberg and Kaper, 1991 Pavelka et al., 1991

Pavelka et al., 1991

This study

This study This study


This study

This study


This study

lected on the basis of ampicillin and bacteriophage E resistance, yielding 28 clones. Only six clones produced wild-type levels of KpsT and were confirmed plasmid mutations.

Construction of RS2436 by AZlelic Exchange-Deletion of the chromosomal kpsT gene was accomplished by allelic exchange, following homologous recombination, utilizing the suicide vector pCVD442 (Don- nenberg and Kaper, 1991) and the selection procedure of Blomfield et al. (1991). The suicide vector construct used (pSR417) is pCVD442 containing a 1.6-kb kps region 3 insert with a 500-bp Bgl I-Dra I11 internal deletion in kpsT (Table I). One acapsular clone (as assayed on antiserum agar plates) was confirmed by its resistance to lysis by bacteriophage E and designated RS2436. Confirmation of the chromosomal deletion of kpsT in RS2436 was accomplished by diagnostic PCR amplification of the region 3 DNA by primers flanking the 3' end of kpsT (Pavelka et al., 1991) and the BglII site upstream of the 5' end of kpsM (5' GACGGAAATAGATCTATTTATCCCCTGCGG 3') (Fig. 1). Absence of the KpsT protein was confirmed by immunoblotting.

Construction of RS2400-Return of the K44E allele to the chromosome of EV36 was done by the above method used to construct RS2436, resulting in the acapsular clone RS2400. For this experiment we used pSR402, a pCVD442 construct containing a 2.1-kb kps region 3 insert encoding the kpsT K44E allele (Table I). The presence of the K44E mutation in the chromosome of RS2400 was confirmed by diagnostic PCR amplification of the allele, utilizing primers flanking the kpsT gene (Pavelka et al., 1991), with subsequent screening by restriction endonuclease digestion. The K44E mutation creates a unique HphI restriction endonuclease site not found in the wild-type kpsT sequence (Pavelka et al., 1991).

Preparation of Bacteria for Electron Microscopy-Bacteria were grown to midlogarithmic phase, and cells from 3-ml aliquots were gently pelleted at 8,000 rpm in a Microfuge for 10 min at 4 "C, washed once with 0.1 rnl of L-broth, and processed as previously described (Wunder et al., 1994). Samples were examined by transmission electron microscopy at 75 kV with a Hitachi 11-E-1 electron microscope.

8;tlzidoATP Photolabeling Assays-Photolabeling experiments using 8-azidoATP analogs were based upon previously described methods (Potter and Haley, 1983; Hobson et al., 1984). When using 8-NJy- 32PlATP (18.0 Ciimmol, 1 Ci = 37 GBq, ICN Biochemical), the reaction mixtures contained protein at a concentration of 1 mg/ml, 32P-labeled analog at 0.2-0.5 p, in a total reaction volume of 75 to 200 pl in photolabeling buffer (10 m~ Tris-HC1, pH 7.5, 10 m~ MgCl,, and 5 mM CaCI,). Reactions were irradiated with a model UVGL-25 mineralight (UVP, Inc., San Gabriel, CA) on the short wave setting (254 nm), at a distance of 2.5-3.0 cm for 3 min. 50 pg of protein was loaded directly per well for separation by denaturing SDS-PAGE, followed by autoradiography. In experiments utilizing 8-N3[y-32PlATP, the amount of KpsT present in each sample tested was determined by immunoblotting. When using nonradioactive 8-N,ATP (Sigma), the assay conditions were almost the same as above, except the protein concentration was 0.5 mg/ml and the analog concentration 100 PM in a total reaction volume of 200 pl. Samples used for labeling were either total membrane preparations or whole cell lysates (equivalent results were obtained from either type of sample). Approximately 6 pg of protein was loaded per well for separation by denaturing SDS-PAGE, followed by immunoblotting with KpsT antiserum. Derivatization of KpsT by the analog results in an altered mobility of the protein in SDS-PAGE gels, with the modi- fied species migrating more slowly than the unmodified species (Silver et al., 1993). For inhibition experiments, ATP or ATPyS (Sigma) was added at concentrations described in the text, from stock solutions prepared in photolabeling buffer and stored at -20 "C.

Sialyltransferase Assays-Endogenous and exogenous sialyltransferase assays were done essentially as previously described (Troy et al., 1982; Vimr et al., 19891, using CMP-[l4C1sialic acid (11.2 GBq/mmol, DuPont NEN) and colominic acid (Sigma), with total membrane fractions freshly prepared as described above. Specific activity was calcu- lated as nanomoles of CMP-['4Clsialic acid transferred/h/mg of total membrane protein and expressed as a percentage of the wild-type control. Results shown are averaged from multiple assays.

20152 Characterization of KpsT from E. coli K l

TABLE I1 KpsT mutant data

Sialyltransferase Strain Relevant

genotype H.46” E phageh Immunodiffusion< activityd 8-AzidoATP

endogenous exogenous photolabelinge

Wild-type + S SUP 100 100 NDf EV36 RS2436 RS2436/pKS+ RS2436lpSR340 RS2436lpSR346 RS2400 RS2400lpSR340 Motif A

saturation mutantsg

Hydroxylamine mutantsh

EV36 k a s T - R LVS 18 AkpsTlpkS’ AkpsTlkpsP AkpsTlkpsT K44E’ EV36kpsT K44E EV36kpsT K44EIkpsT

G41E K44I K44N S45L T46P

G43D G155R G84D S126F C163Y H181Y

- R + S - R - R + S

ND 118 11 20

NDf

ND ND ND ND ND

ND ND 18 15 18 19

109 ND ND ND 109 +++ 92

109 +/- ND

ND ND

ND ND ND ND ND

ND ND ND +++ 105 +++ 123 84

113 ++ +++ +++

Precipitin halo formation on H.46 antiserum plates. Lysis by K1-specific bacteriophage E; S = sensitive, R = resistant. Detection of polySia in culture supernatants (Sup) or lysates (Lys) via immunodiffusion using H.46 antiserum. Sialyltransferase activities in total membranes, expressed as percentage of the wild-type, EV36.

e Results of photolabeling experiments done with 8-azidoATP as described under “Materials and Methods.” The amount of derivatization for each was estimated visually from the immunoblots, since densitometry scans lacked sufficient resolution. Generally, 40-50% of wild-type KpsT was derivatized in each exDeriment (+++). Mutants either had a similar (+++), slightly reduced (++), or poor (+I-) level of derivatization.

f ND = not determined. Motif A saturation mutagenesis clones in RS2436. Hydroxylamine mutagenesis clones in RS2436.

RESULTS AND DISCUSSION

Characterization of the kpsT Deletion Strain RS2436- Previous studies used strains with ill-defined mutations in region 3 of the kps gene cluster. For this study, a strain (RS2436), with a well-defined chromosomal deletion in the kpsT gene, was constructed by removing 500 bp of DNA internal to the gene in the chromosome of EV36. RS2436 was shown to be acapsular, as indicated by the lack of precipitin halo formation on antiserum agar and resistance to lysis by K1-specific bacteriophage. Furthermore, a-2,8 polysialic acid was detected by immunodiffusion in a cell lysate, but not in a culture superna- tant prepared from RS2436 (Table 11). These observations are consistent with the phenotype of region 3 mutations and the role of KpsT in the transport of polymer to the cell surface (Vimr et al., 1989; Boulnois and Roberts, 1990; Pavelka et al., 1991). To more accurately determine the location of polymer, electron microscopy was performed on ultrathin sections of fixed and embedded specimens of EV36 and RS2436. At low magnification, a striking difference between the two strains was clearly apparent (Fig. 2, panels A and B ) . RS2436 cells contained electron lucent zones within the cytoplasm, indica- tive of intracellular polysaccharide accumulation (Pelkonen, 1990; Bronner et al., 1993). Moreover, the accumulations were located a t discrete sites around the periphery of the cell (Fig. 2, panel C). Higher magnification revealed that the accumulated polysaccharide was located against the inner membrane (Fig. 2, panel D ) . These results are consistent with the observation that capsular polysaccharide remains attached to the inner surface of the cytoplasmic membrane during synthesis (Troy, 1992).

Clearly, cells lacking KpsT are capable of synthesizing polymer but unable to transport it across the inner membrane. Mutations within region 3 of the gene cluster have been shown

to not only block export of polymer, but to also affect sialyltransferase activity (Vimr et al., 1989). In E. coli K1, the sialyltransferase enzyme, encoded by the region 2 neuS gene (Weisgerber et al., 1991; Steenbergen et al., 19921, is capable of transferring activated sugar (CMP-sialic acid) onto acceptor molecules, but cannot initiate polymerization (Steenbergen et al., 1992). In vitro sialyltransferase activity is defined as the ability of isolated membranes to transfer chromatographically mobile CMP-[’4Clsialic acid onto acceptor molecules, yielding a chromatographically immobile product (Troy et al., 1982). An endogenous sialyltransferase assay measures the transfer of labeled sugar onto pre-existing acceptors within the membranes, while an exogenous assay measures the transfer of labeled sugar onto exogenously added colominic acid (a-2,8 polysialic acid oligomers approximately 30 residues in length). As shown in Table 11, the endogenous sialyltransferase activity of RS2436 was 18% that of wild type, while the exogenous activity was normal, consistent with the phenotype of other region 3 mutations (Vimr et al., 1989). The reason for this reduced activity is unclear, since the exogenous activity in these cells is normal, indicating that the sialyltransferase enzyme itself is not affected. It has been suggested that the gene products of the kps cluster form a multiprotein complex that synthesizes and polymerizes sialic acid and transports the polymer to the cell surface (Silver et al., 1993; Vimr and Steen- bergen, 1993). According to this view, the loss of any one component could prevent the proper assembly of such a complex, causing pleiotropic effects upon polysaccharide synthesis and export. However, a comparison between the phenotypes of RS2436 and other mutants, described below, shows that the decrease in endogenous sialyltransferase activity in RS2436 is not due to the lack of KpsT, but is related to the inability of the cells to transport polymer across the inner membrane. Synthe-

Characterization of KpsT from E. coli K l 20153

of ultrathin sections of EV36 (A), FIG. 2. Electron photomicrographs

RS24.76 (I& C, and D ) , and RS2400 ( E ) . The inner and outer memhranw are indi-

A, E , and C, while the hnr = 50 nm in D cnted hy the nrrotos. The hnr = 0.5 pm in

and E .

I

sis and export of capsular polysaccharide likely occurs at the same time, and, therefore, it may be possible that a block in export could feed back on the synthesis pathway, perhaps affecting the nature or number of available endogenous acceptors.

Regions 3 and 2 are transcribed in the same direction (Fig. 1); however, knowledge of the transcriptional organization of the hps gene cluster is incomplete. It is possible that deletion of hps?" could have polar effects upon downstream region 2 mRNA. However, slot blot analysis of total cellular RNA prepared from RS2436 and EV36, using DNA probes encompassing region 2 genes which are proximal and distal to region 3, revealed no differences in steady state mRNA levels (data not shown). Furthermore, introduction of hps?", carried on a multicopy plasmid (pSR340) restored RS2436 to wild-type (Table I1 ).

Strhcellular Localization of KpsT-The ATP-binding components of the ARC-transporters, whether domains of large proteins or separate polypeptides, always face the cytoplasm (Pugsley, 1991, Ames et al., 1992; Higgins, 1992). The recent observation that HisP, a KpsT homolog of the histidine permease, is also exposed to the periplasmic space (Raichwal et a/., 1993) indicates that the specific orientation of these proteins may vary from system to system. Our model of the KpsMT transporter posits KpsT on the cytoplasmic surface of the inner membrane. We immunoblotted various cell fractions from EV36 with KpsT antiserum to determine the subcellular loca-

tion of the protein. KpsT was present in the inner memhranc fraction and the soluble fraction of EV.36 (Fig. 3. pnnrl B , middle), consistent with the idea that KpsT is a peripheral inner membrane protein. This localization was independent of KpsM, as similar results werr obtained from immunoblots done using fractions prepared from a strain with a chromnsomal deletion in hpsM (data not shown). Unexpcctcdly. however. KpsT was also present in the outer mcmbrane fractions from both strains (Fig. 3 R , micfdlr). This was not due to the prcscncc of capsular polysaccharide, as immunoblots of fractions prepared from EVFi, a strain which cannot synthesize polysialic acid due to a defect in nruA, the gene cncoding CMP-sialic acid synthetase (Zapata rt al., 1989: Vann rt nl., 19931, wcre similar to wild type (Fig. 3, panel R , hottom ). As an altcrnativr mctbotl for membrane separation, we utilized srlcctiw solubilization of the inner membrane by the detergent n-lauryl sarcosinate fsar- cosyl) (Filip et nl., 1973,. Extraction of a whole cell lysatc of EV36 with 2c'r sarcosyl resulted in a similar separation of the inner and outer membranes comparrd to the sucrosc prnce- dure, as judged by Coomassie staining (Fig. 4.pc1nrIA. compare to Fig. 3, p n n d A ). Furthermore, immunoblotting determinrd that KpsT was present in both memhranc fractions (Fig. 4. panel B , hottom ), comparable to the results ohtainrd with thc sucrose gradient-separated memhrancs. \Vhy KpsT appears in the outer membrane fraction from both these procedures is not clear. KpsT, a very basic protein with a calculntcd pl of 10.W (Isoelectricpoint, Genetics Computer Group, 1991 I. may h a w

Characterization of KpsT from E. coli KI 20154

A - s

68-

43-

0 1 B - w s I O -

29-

FIG. 3. Location of KpsT in membranes separated by sucrose gradient centrifugation. A is a Coomassie-stained 12‘7 polyacrylamide gel containing the following fractions from EV36: S, soluhle; I , inner membrane; and 0, outer memhrane. Molecular mass s tandards are indicated in kDa. R contains anti-KpsT immunohlots of RS24.76 ( t o p ) , EV.76 ( m i d d ! ~ ) , and EV6 (hottom 1. Key: W , wholr cell lysate; S . soluble fraction; I , inner membrane; 0, outer membrane. KpsT is indicatrd by the nrrotus.

A. BS BI SS SI

68-

43-

29-

18.4-

FIG. 4. Location of KpsT in membranes separated by sarcosyl extraction. A is :I Coomassie-stained 12f> polyacnlamide gel contnin- ing buffer (control) and sarcosyl fractions from EV36. Fractions: R S , buffer-soluble (soluble protrins); R I , huffrr-insoluhle (total mrm- branes); S S , sarcosyl-soluble tinnrr membrane proteins); and SI, sarcosyl-insoluble (outer membrane proteins). Molecular mass s tandards are indicated in kDa. R contains anti-KpsT immunoblots of the same fractions from RS24.76 ( l o p ) and EV36 (hottom I. KpsT is indicated by the arrow.

an intrinsic ability to associate with a membrane. Interestingly, KpsT is fully removed from outer membranes and partially removed from inner membranes in the presence of 1.0 M NaCl (data not shown), suggesting that the presence of KpsT in the outer membrane fraction might be due to nonspecific electro- static interactions with outer membrane components during the separation procedure.

8-AzidoATP Photoaffinit.v Labeling of KpsT-Preliminary work in our laboratory has demonstrated that ATP interaction is important for the function of KpsT in capsule expression (Pavelka et al., 1991; Silver et al., 1993). Using the photoafin- ity ATP analog, 8-N3[y-’”P1ATP, we found that KpsT could be

derivatized in memhranes prepared from RS24.76 cells ovrrcx- pressing the protein from pSR.740 ( see Fig. 5. pnnrl A , lnnrs 1 and 2 ). The laheling of KpsT is due to thr hinding of the analog and suhsequent UVinduced derivatization. not to a phospho- rylation event utilizing the y - : T Iahrl. since wc did not scc radiolaheled KpsT in the ahsrnce of C?’ irradiation (data not shown). The photolaheling of KpsT from RS24.7WpSR.740 mcm- branes could he inhihited with the addition of a large excess of cold ATP to the reaction (Fig. 6, Innrs 3-6 I. Hnwrvcr, wr found that the nonhydrolyzahle ATP analog. ATPyS. inhihitrd photolabeling more eficiently (Fig. 6, lnnrs 7-10 I . Thr hrttcr inhihition observed with ATPyS is prohahlv due to the insensitivity of this analog to the high ATPase activities associated with total membrane preparations. In addition to laheling KpsT within the membranes from crlls overproducing the protrin. \vc w w r also ahle to derivatize KpsT in memhranrs prrparrd from wild- type cells (see Fig. 5 , pnnrl C, Innrs 1 and 2 ).

For other ABC-transporter systems. it has hrcn noted that the ATP-binding components may require contact with other components of their respective systems in order to hind ATP efficiently (Ames et nl., 1992; P a n a ~ o t i d i s rt nl., 199.7). Inter- estingly, KpsT present in a soluhle fraction prepared from RS24WpSR340 did not label as eficiently with R-N,[y-”PIATP as KpsT present within the memhrane fraction (compare Fig. 5. panrl R, lnnr 4 topanr l A, lane. 2 ). The amount of KpsT prrscnt in the soluble fraction was shown, by immunohlotting, to he nearly equivalent to the amount present in the memhrane fraction. Addition of a total memhrane preparation lacking KpsT to a KpsT-containing soluhle fraction did not improvr phntolahel- ing of the protein (data not shown). \71ile soluhlr KpsT could be derivatized to some extent hv the azido analog, the reaction was much more eficient within the memhranr preparation. However, derivatization of KpsT does not require a n y other k p . ~ gene products, as we were ahle to eficiently photolnhel thr protein in membranes from HRlOl carrying pSR.740 (Fig. 5, panrl A, lane 4 ) . Furthermore, photolaheling of KpsT in the soluble fraction from HR101ipSR.740 was similar to that scrn in the soluble fraction from RS24.76/pSR.740 ccomparr Fig. 5, panrl R, lane 6 to Innr 4 ). In hoth strains. the amount of KpsT present in each fraction was equivalent.

Characterization of RS2400-we have cstahlishcd that KpsT does hind ATP and that this is important for the function of the protein in transporting capsular polysaccharide across the inner membrane, since the nonfunctional K44E mutant protc4n does not hind ATP as eficiently as the wild type protein (Silver et 01. (199.7) and Tahle 11). We examined this mutant allcle mow closely by constructing a strain with the K44E gene in placc of the wild-type chromosomal kpsT gene in E\.%. The resultant strain, RS2400. was acapsular and accumulated intracellular polysialic acid (Tahle I1 ). Immunohlotting of cell fractions prepared from RS2400 demonstrated that the K44E protein \vas expressed at a level similar to wild tvpr and localizrd to thr inner and outer membrane fractions in a similar fashion (data not shown). Furthermore. consistent with rqiion 3 mutations, the endogenous sialyltransferase activity in mrmhrancs prc- pared from RS2400 was 2OPi that of wild-type, while the cxog- enous activity was normal. This phenotype was also s r rn in RS2436 cells carrying the K44E gene on the plasmid pSR.746 (Table 11). As expected, RS2400 was complemented hack to wild-type by introduction of pSR.740 (Table 11). >lost importantly, when ultrathin sections of RS2400 cells were examined under the electron microscope, we saw the same rlectron lucrnt zones that were present in the kpsT delrtion strain RS24.76 (Fig. 2, panel R 1. This provides further evidence for the importance of ATP binding hy KpsT in the transport of capsular polysaccharide across the inner memhrane.

Characterization. of KpsT from E. coli KI 20 15.5

RS2436 HBlOl 0

1 2 3 4

RS2436 HBlOl UY RS2436 B.

(? Q- N

UY n

VI a 3. w

pKS+ w - + - + - +

pSR340 pSR340

C . 1 2 3 4 5 6 1 2

m x a

0 Tp M pl4 ATP

% ..

a 1 5 0 100 250 500 I 5 10 25 50 ...

,:.; ...... ........ . , _ . . . . ,

4 . .~ .... > . . ... "... ... ,:,,,,. i. ...... .:..;. ......... -=. n..? . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -

1 2 3 4 5 6 7 8 9 1 0 FIG. 6. Autoradiogram of the inhihition of A-NJy-"PIATP pho-

tolaheling of KpsT in RS243WpSIU40 total memhranes hy ATP and ATPyS. I , o r t , s 1, pl<S' vvcttw c~)nt r (~I ; lnrrr~ 2, pSI{:btO, no inhihltor: lnws ;<-(;, inrrcmsing cnncrnt rat ions ofcn1t1 ATP. 1,nrrv.y 7-10. incrcvsing concrntrations of cold ATPyS. I'hotolahrld ICpsT is indicatrd.

Saturntion Mutngrnrsis of Motif A of thr ATP-binding Dn- moin of KpsT-Mutation of the high1.v conserved lysine residue a t position 44 within the ATP-hinding consensus sequence of KpsT had a marked eFFcct on the protein's function. We further investigated this domain by mutagenizing the glycine-rich area (GFX,G;YGXCKSTX,;GX,;CI) encompassing motif A of the consensus sequence (Walkrr et 01.. 1982,. This was accomplished using a comhination of PCR site-directed mutagenesis using overlap extension and randomly doped oligonucleotides. U'r selected a 30-amino acid residue stretch (from position 31 to 60, inclusive), as the region to target for mutagenesis, and isolated five mutant genes that did not complement a chromosomal kpsT mutation (Table 11). Each gene has a single mutation changing a highly conserved residue within motifA of the ATP- binding consensus sequence (shown in itnlics in Fig. 7 ) . The importance of the lvsine at position 44 was confirmed, as two of the saturation mutants had changes at this position (K44N, K44I ).

The fact that mutations in the consensus ATP-binding sequence of KpsT result in nonfunctional proteins underscores the importance of ATP in this transport process. The memhers ofthe ARC-transporter family are believed to utilize the energy from ATP hydrolysis to transport their respective suhstrates (Ames and Joshi, 1990). While the drug-stimulated ATPase activity of the eukaryotic P-glycoprotein has heen demonstrated (Amhudkarct nl., 19921. showing ATP hydrolysis hy the ATP-hinding components of prokaryotic systems, has been dif- ficult (Ames c t 01. . 1992). However, the cytoplasmic, ATP-hinding portion of the HlyR protein of the hemolysin exporter in E. roli has recently heen purified in a soluhle form and shown to have an intrinsic ATPase activity (Koronakisct nl., 1993). Likr- wise, successfully renatured, purified MalK protein from the maltose permease o f S. t,vphimurirrrn has an intrinsic ATPase activity independent of maltose and maltose hinding protein (Walter t>t al., 1992). However, when the maltose permease is reconstituted into transport-competent proteoliposomes, the

ATPase activity of the complex is dcprndcnt upon thc prrsrncr o f maltose and the maltosc hinding protcin ~1)nvidson P! nl., 1992,. These ohsewations suggest that thr ATI'asc activity of the ATP-hinding components in thrsr systrms may hv tlr~prnd- en t upon their context. Thr inahility of soluhlr KpsT to rffi- ciently hind ATP may reflect a requirc.mc,nt fnr :I mc.mhranc. environment, regardless of the prrsrncr or ahscncc. of ot hcr I:ps gene products. Alternatively. the soluhlr KpsT sprciw may h c .

irreversihly denaturcd during thc fractionation procdurc. . Our attempts to demonstratr ATI' hydrolysis via KpsT by assaying '"P release from [y- 'TIATP in wtrncts from cc~l ls nvc.rprodueing the protein or hy staining for ATPasc activity in nativc l':\(;la: gels containing similar cclll rxtracts. hnvr. hcvn unsucrc~ssful.

Strrrrtnrnl ,Wor/r~l of KpsT-To formul:ltc~ :I mcschanism for the coupling of ATP hinding and. prrsumahly. hydrolysis hy KpsT to the export of polymrr. additional functional rcyions of the protein nred to he dctcrmincd. This \voulrl h c , fnri1it:ltc.d hy the knowledge of the structurr of thc. KpsT protcxin. L-nfortu- nately, none of the ATP-hinding componc,nts within thv A R C ' - transportw family has hecn cnstallizcvl. so thrrc. is a p:lurityof structural data. FIowrvrr. thrsc proteins sham considt~rahlr sequencr homology outsidr of thr cnnsrnsus ATP-hinding sr- quencr, allowing thr dcvrlopmr.nt of srconday antl trrtiary structural models (Hydc. vt c r / . , 1990; 5Iimur:I vt ol. , 1 9 9 1 I . The models are based upon computrr prrtlirtions of thr srcondan. st.ructure ofthese proteins. compartd with known crystal struc- tures of proteins containing slmilar ATP-hintling scqurncrs,

20156 Characterization of KpsT from E. coli K1

FIG. 8. Topological diagram of the structural model for KpsT, inferred from the model proposed by Mimura et uZ. (1991). a helices are shown as cylinders, while 0 strands are represented as arrows. Designations are as described in the text and in Fig. 7.

such as adenylate cyclase, p21““, and elongation factor EF-Tu. Chemical Mutagenesis of kpsT-We investigated the func- The models’main conclusion is that the ABC-transport proteins tional role of the domains predicted from the Mimura model by are modular in their design, consisting of a conserved ATP- using hydroxylamine to randomly mutagenize plasmid DNA binding domain and a variable domain. By using the Pepplot containing kpsT. DNA sequence analysis determined that the program of the UWGCG software package to predict the sec- six resulting mutant clones had a single mutation within the ondary structure of KpsT followed by manual comparison to the kpsT gene (shown in bold type in Fig. 7). As noted in Table 11, proposed models (Hyde et al., 1990; Mimura et al., 1991), we RS2436 cells containing the HA mutations on plasmids accu- found that KpsT fit best with the Mimura structural model. mulated polymer intracellularly and had endogenous sialyl- The resulting structural motif assignments for KpsT are shown transferase activities that were approximately 15-19% of wild in Fig. 7, while a topological diagram of KpsT inferred from the type, while exogenous sialyltransferase activities were normal. Mimura model is shown in Fig. 8. According to the general Interestingly, as noted in Fig. 7, mutation G43D is located in a Mimura model, the ATP-binding domain, comprised of a five- conserved residue in motif A of the ATP-binding sequence, con- stranded p sheet with intervening a! helices (a Rossman mono- firming the importance of this region to the protein’s function. nucleotide binding fold), is interrupted by a variable “helical domain,” which appears to be important for the specific transport function of a particular system (Schneider and Walter, 1991; Ames et al., 1992). A short, conserved “linker peptide,” a flexible region thought to be important for intramolecular signalling, resides within the helical domain (Ames et al., 1992). For the ABC-transporter systems, it is believed that the helical domain is the region that interacts with the membrane-bound component while the linker peptide acts as a signalling path between the helical domain and the ATP-binding domain. In this scenario, conformational changes induced in the ATP-binding domain by ATP could be transmitted by the linker peptide to the helical domain, altering its conformation which, in turn, would affect the membrane component, allowing transport to occur (Ames et al., 1992; Higgins, 1992). Most of the proteins used in the construction of the model are predicted to contain four a! helices in the helical domain, the largest being helix three (H3). On the basis of primary sequence comparisons to some of these proteins, KpsT appears to have the H1, H2, and H4 helices, but lacks the large H3 helix. However, the second-

Additionally, we isolated a mutant with a change near motif B of the ATP-binding consensus sequence, in which a conserved glycine at position 155 was changed to an arginine, without much effect on the protein’s ATP-binding ability (Table 11). The ABC-transporters have a highly conserved histidine residue, situated toward the carboxyl terminus of the ATP-binding fold. This histidine is thought to be important for conformational events occurring after ATP binding, since mutations in this residue result in mutant proteins able to bind ATP, but unable to transport substrate (Shayamala et al., 1991). This residue in KpsT was changed to a tyrosine residue in the H181Y mutant (Fig. 7) and did not drastically affect the ATP-binding ability of the protein (Table 11). Most interestingly, two of the mutants had changes within residues of the proposed helical domain. G84D is within helix 1 (Hl), while S126F resides in the linker peptide. Consistent with the observation that mutations within the helical domain of the ABC-transporters do not affect ATP- binding, (Shayamala et al., 1991), the KpsT G84D and S126F mutants could still be derivatized by 8-NdTP (Table 11). It is interesting to note that a nonfunctional HisP mutant with a

ary structure predictions suggest that KpsT has two small a! Ser to Phe mutation and nonfunctional cystic fibrosis transduc- helices in the H3-H4 region. Therefore, we have designated this tance regulator proteins with Ser to Asn, Lys, and Ile mutations area within the helical domain as helix H3/H4. Interestingly, in the conserved Ser residue of the linker peptide have been the H3 helix region shows the highest degree of variation reported (Shayamala et al., 1991). In the context of the Mimura within the ABC-transporters (Mimura et al., 1991; Ames et al., model, the fact that these KpsT mutants are still able to bind 19921, suggesting that this particular region of KpsT may con- ATP suggests that the defect in these mutants may be due to tain residues that specifically interact with KpsM. some alteration of intra- or intermolecular signalling. The

Characterization of KpsT from E. coli Kl 20157

K ~ ~ T K 1 1 MIKIENLTKSYRTPTGRHYKNLNIIFP~GYNIALIGQNGAGKSTLLR~ 50

KpST K 5 1 MIKIENLTKSYRTPTGRHYVFKDLNIEIPSGKSVAFIGRNGAGKSTLLRM 50 I I I I I I I I I I I I I I I I I I I I I I : I I I : I . I . : l : l i . i i l l i l i l l l :

51 IGGIDRPDSGNIITEHKISWPVGLAGGFQGSLTGRENVKNARLYAKRDE 100

51 IGGIDI(PDSGKIITNKTISWPVGI-4GGFQGSLTGRENVKFVAFGYAKQEE 100 l / I I I I I l l I . I I l : . . l l l l l l l l l l l l i / l / l / / I l I l l l l l / l / . : I

101 LNERVDFVEEFSELGKYFDMPKTYSSGMRSRLAFGLSMAFKFDYYLIDE 150 i . I : : : I i I I I . i I l l i I i I i I I i i I I I I I I i i : I I I I I I I I I I I I : : I l

101 LKEKIEFVEEFAELGKYFDMIIKTYSSGMRSRLGFGLSWKFDYYIVDE 150

201 QGKFYKNVTEAIADYK. .KDL. . . 219

201 IIGYYENVQSGIDEYKMYQDLDIE 224 : I . I i . : I . : ( ( . I I

FIG. 9. Alignment of the KpsT proteins from E. coli K1 (Pavelka et al., 1991) and KS (Smith et al., 1990), using the Gap program of the UWGCG software package (Genetics Computer Group, 1991). The conserved His-181, Cys-163, Cys-190, and the areas of conserved identity within the carboxyl termini are indicated by asterisks and bores, respectively.

C163Y mutation is very interesting, for KpsT has 2 cysteine residues, 1 at position 163 and the other at position 190 (Fig. 7). These cysteines are not found in most of the ABC-transporter proteins, only within the capsular transport proteins within the family, namely, BexA and CtrD, of H. influenzae and N. men- iaggitidis, respectively (Kroll et al., 1988; Frosch et al., 1991, Pavelka et al., 1991). The cysteine residues are located in the ATP-binding fold, in a helices 3 and 4, which are situated on the same side of the p-sheet of the protein's ATP-binding domain (see Fig. 7). Analysis of these helices using the Helicalwheel program (Genetics Computer Group, 1991) indicates that both are somewhat asymmetric, consisting of hydrophobic residues on one side of the helix and charged or uncharged polar residues on the other. Each cysteine is situated within the middle of its respective helix, on the hydrophobic side. Taken together, these observations suggest that these 2 cysteines of KpsT are in proximity, allowing interaction with each other or some other lzps component, and this interaction is required for polymer export. This is supported by the observation that the C163Y mutant can still bind ATP, indicating some other functional defect in this protein. We compared the amino acid sequences of KpsT from E. coli K1 and the functionally identical KpsT protein from the K5 serotype (Smith et al., 1990) and found that the two proteins are virtually identical at their amino termini (Fig. 9). The identity decreases, however, at the carboxyl termini. In fact, the K5 protein is 5 amino acids longer at the carboxyl terminus than the K1 protein. In both cases, the 3' ends of the genes are located at the junction between region 3 and region 2 of the gene cluster, an area that appears to have been the site of DNA recombination (Roberts et al., 1988; Boulnois and Jann, 1989). We believe the proximity of KpsT to the junction explains the divergence in the C termini of the proteins. Most importantly, the 2 cysteine residues, as well as the conserved His-181 residue, are located within small conserved areas in the divergent part of the proteins, suggesting that changes in these areas are not tolerated (Fig. 9). These observations suggest that the conservation of these cysteines has some functional relevance to capsular polysaccharide ex- porters and is not a consequence of homology between closely related proteins which have not yet diverged.

The results presented here further our understanding of the role of the KpsMT transporter in capsular polysaccharide export by delineating specific regions of the KpsT protein important to its function. We speculate that the conserved cysteines of KpsT may be involved with sensing substrate availability. We propose that KpsT and ATP are required to open a KpsM pore or channel in the inner membrane. Isolation of extragenic suppressor mutations in the strains described in this study will allow us to explore these ideas.

Acknowledgments-We gratefully thank M. Donnenberg for the suicide vector pCVD442, P. Annunziato and W. Vann for critically review- ing the manuscript, and C. Garon and P. Small for their help while M. P. was a guest scientist at the Rocky Mountain Laboratories.

REFERENCES

Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M., and

Ames, G. F.-L., and Joshi, A. K. (1990) J. Bacteriol. 172,4133-4137 Pastan, I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8472-8476

Ames, G. F.-L., Mimura, C. S., Holbrook, S. R., and Shayamala, V. (1992) Adu. Emymol. Relat. Areas Mol. Biol. 66, 1-47

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A,, and Struhl, K. (1987) Current Protocols in Molecular Biology, Greene Pub-

Baichwal, V., Liu, D., and Ames, G. F.-L. (1993) Proc. Natl. Acad. Sei. U. s. A. 90, lishing Associates and Wiley-Intersciences, New York

Blight, M. A,, and Holland, I. B. (1990) Mol. Microbiol. 4, 873-880 620-624

Blomfield, I. C., Vaughn, V., Rest, R. F., and Eisenstein, B. I. (1991) Mol. Microbiol.

Boulnois, G. J., and Jann, K. (1989) Mol. Microbiol. 3, 1819-1823 Boulnois, G. J., and Roberts, I. S. (1990) Cur,: Topics Microbiol. Immunol. 160,

Bradford, M. (1976) Anal. Biochem. 72,248-254 Bronner, D., Sieberth, V., Pazzani, C., Roberts, I. S., Boulnois, G. J. , Jann, B., and

Jann, K. (1993) J. Bacteriol. 176, 5984-6992 Chang, A. C. Y., and Cohen, S. N. (1978) J. Bacteriol. 134, 1141-1156 Davidson, A. L., Shuman, H. A., and Nikaido, H. (1992) Proc. Natl. Acad. Sci.

Donnenberg, M. S., and Kaper, J. B. (1991) Infect. Immun. 69, 4310-4317 Filip, C., Fletcher, G., Wulff, J., andEarhart, C. F. (1973)J. Bacteriol. 116,717-722 Frosch, M., Edwards, U., Bousset, K., Kraupe, B., and Weisgerber, C. (1991) Mol.

6, 1447-1457

1-18

U. S. A. 89,2360-2364

Microbiol. 6, 1251-1263

7. A ~ r i l 1991. Genetics ComDuter GrouD. Madison. WI Genetics Computer Group (1991) Program Manual for the GCG Package, Version

Gros, P., Croop, J . , &id Housmen, D. (1986) Cell 47,371-380 I .~ ~

Gross, R. J., Cheasty, T., and Rowe, B. (1977) J. Clin. Microbiol. 6,54%550 Gussow, D., and Clackson, T. (1989) Nucleic Acids Res. 17,4000 Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring

Higgins, C. F., Hiles, I. D., Salmond, G. P. C., Gill, D. R., Downie, J. A., Evans, I. Higgins, C. F. (1992) Annu. Rev. Cell. Biol. E, 67-113

J. , Holland, I. B., Gray, L., Buckel, S. D., Bell, A. W., and Hermodson, M. A. (1986) Nature 323, 448-450

Higgins, C. F., Gallagher, M. P., Mimmack, M. L., and Pearce, S. R. (1988)Bioessays 8, 111-116

Higgins, C. F., Gallagher, M. P., Hyde, S. C., Mimmack, M. L., and Pearce, S. R. (1990) Philos. Pans . R . Soc. Lond. B Biol. Sci. 326,353365

Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, S. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59

Hobson, A. C., Weatherwax, R., and Ames, G. F.-L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,7333-7337

Humphreys, G. O., Willshaw, G. A,, Smith, H. R., and Anderson, E. S. (1976) Mol. & Gen. Genet. 146, 101-108

Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gill, D. R., Hubbard, R. E., and Higgins, C. F. (1990) Nature 346,362-368

Koronakis, V., Hughes, C., and Koronakis, E. (1993) Mol. Microbiol. 8, 1163-1175 Kroll, J. S., Hopkins, I., and Moxon, E. R. (1988) Cell 63, 347-356 Kroncke, K. D., Boulnois, G., Roberts, I., Bitter-Suermann, D., Golecki, J. R., Jann,

Kroncke, K. D., Golecki, J. R., and Jann, K. (1990b) J. Bacteriol. 172,3469-3472 Laemmli, U. K. (1970) Nature 227, 680485 Lambert, P. A. (1988) in Bacterial Cell Surface Techniques (Hancock, I. C., and

Levine, M. M. (1984) in Bacterial Vaccines (Germainer, R., ed) pp. 187-235, Aca-

Miller, V. L., and Mekalauos, J. J. (1988) J. Bacteriol. 170,2575-2583 Mimura, C. S., Holbrook, S. R., and Ames, G. F.-L. (1991) Proc. Natl. Acad. Sci.

Ner, S. S., and Smith, M. (1989) Mol. Biol. Rep. 7, 1 3 Panagiotidis, C. H., Reyes, M., Sievertsen, A., Boos, W., and Shuman, H. A. (1993)

Pavelka, M. S., Jr., Wright, L. F., and Silver, R. P. (1991) J. Bacteriol. 173, 4603-

Pelkonen, S. (1990) Cur% Microbiol. 21, 23-28 Philip, A. G. S. (1985) Neonatal Sepsis and Meningitis, Hall Medical Publishers,

Potter, R. L., and Haley, B. E. (1483) Methods Enzymol. 91,613-633 Pugsley, A. P (1991) in Prokaryotic Structure and Function: A New Perspective

(Mohan. S., Dow, C., and Cole, J. A., ed) pp. 223-248, Cambridge University

Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Press, New York

Zielenski, J., Lok, S., Plavsic, N., Chou, J., Drumm, M. L., Annuzzi, M. C., Collins, F. S., and Tsui, L. (1989) Science 246, 106G1073

Robbins, J. B., McCraken, Jr., G. H., Gotschlich, E. C., Orskov, F., 0rskov, I., and Hansson, L. A. (1974) N. Engl. J. Med. 290, 1216-1220

Roberts, I., Mountford, R., Hodge, R., Jann, K. B., and Boulnois, G. (1988) J. Bacteriol. 170, 1305-1310

Sambrook, J., Fritsch, E. F., and Maniatis, T (1989) Molecular Cloning: A Labo- ratory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Harbor Laboratory, Cold Spring Harbor, NY

B., and Jann, K. (1990a) J. Bacteriol. 172, 1085-1091

Poxton, I. R., ed) pp. 113-114, John Wiley & Sons, New York

demic Press, Orlando, FL

U. S. A. 88,8688

J. Biol. Chem. 268,23685-23696

4610

Boston, MA

Schneider, E., and Walter, C. (1991) Mol. Microbiol. 6, 1375-1383

20158 Characterization of KpsT from E. coli Kl Shayamala, V., Baichwal, V, Beall, E., and Ames, G. F.-L. (1991) J. Bid. Chem. Polysialic Acid: From Microbes to Man (Roth, J., Rutishauser, U., and Troy, F.

266, 18714-18719 Silver, R. P., and Vimr, E. R. (1990) in The Bacteria. Vol. 11: Molecular Basis of Vimr, E. R., and &enbergen, s. M. (1993) in polysialic Acid: From Microbes to

Bacterid Pathogenesis (IdewSki, B., and Clark, v, eds) PP. 39-60, Academic Man (Roth, J., Rutishauser, U., and Troy, F, A, eds) pp. 73-92, Birkhauser Press, New York

A., eds) pp. 125-136, Birkhauser Verlag, Basel

Silver, R. P., Vann, W. E , and Aaronson, W. (1984) J. Bacteriol. 157, 568-575 Vimr, E. R,, and Troy, F. A. (1985) J. ~ ~ ~ ~ ~ ~ ~ ~ l , 845-853 Verlag. Basel

Silver, R. P., Annunziato, P. W., Pavelka, M. S., Pigeon, R. P., Wright, L. F., and vimr, E. R., A ~ ~ ~ ~ ~ ~ ~ , w., and silver, R. p, (1989) J , ~ ~ ~ ~ ~ ~ i ~ l , 171, 11og1117 Wunder, D. E. (1993) in Polysialic Acid: From Microbes to Man (Roth, J., Rut- ishauser, U., and Troy, F. A., eds) pp. 59-71, Birkhauser Verlag, Basel Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 8,

Smit, J., Kamio, Y, and Nikaido, H. (1975) J. Bacteriol. 124, 942-958 Smith, A. N., Boulnois, G. J., and Roberts, I. S. (1990) Mol. Microbiol. 4,1863-1869 C.9 Honer zu Bentrup, R, and Schneider, E. (lgg2) J . 267’ Steenbergen, 9. M., Wrona, T. J., and Vimr E. R. (1992) J. Bacteriol. 174, 109% 8863-8869

Troy, F. A. (1992) Glycobiology 2, 5-23 Wunder, D. E., Aaronson, W., Hayes, S. F., Bliss, J. M., and Silver, R. P. (1994) J. Troy, F. A., Vijay, I. K., McCloskey, M. A,, and Rohr, T. E. (1982) Methods Enzymol. Bacteriol. 176,4025-4033 83, 540-548 Zapata, G., Vann, W. F., Aaronson, W., Lewis, M. S., and Moos, M. (1989) J. Biol.

Vann, W. F., Zapata, G., Roberts, I. S., Boulnois, G. J., and Silver, R. P. (1993) in Chem. 264, 14769-14774

945-951

1108 Weisgerber, C., Hansen, A,, and Frosch, M. (1991) Glycobiology 1, 357-365

characterization of kpst, the atp-binding component of the abc

Documents