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Analysis of the G93E mutant allele of KpsM, the membrane component of an ABC transporter involved in polysialic acid translocation in Escherichia coli K1
FEMS Microbiology Letters 156 (1997) 217^222
Analysis of the G93E mutant allele of KpsM, the membrane component of an ABC transporter involved in polysialic acid translocation in Escherichia coli K1 Ronald P. Pigeon, Richard P. Silver * Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Box 672, Rochester, NY 14642, USA
Received 2 July 1997; revised 5 September 1997; accepted 8 September 1997
Abstract
KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1. The allele is a point mutation in the first cytoplasmic loop (CI) of KpsM which partially disrupts translocation of the capsule. While producing polymer of wild-type length, strains harboring the G93E allele exhibit a decreased production of capsular polymer and a reduced rate of polymer translocation to the cell surface. kpsMG93E
Keywords : Escherichia coli
; Capsule; K1 antigen ; Polysialic acid; ABC transporter; Polysaccharide transport
1. Introduction
Capsules have long been recognized as important virulence determinants of extraintestinal pathogenic Escherichia coli. The K1 capsular polysaccharide, an K(2,8)-linked polymer of sialic acid, is the most common capsular antigen isolated from neonates with meningitis caused by E. coli. The 17 kb kps gene cluster of E. coli K1 encodes the information required for synthesis and expression of the polysialic acid (polySia) capsule [1^4]. The cluster is divided into three functional regions [1^4]. A central biosynthetic cassette, region 2, contains the genes for sialic acid synthesis, activation and polymerization while the £anking regions 1 and 3 encode proteins that function in polySia transport to the bacterial surface * Corresponding author. Tel.: +1 (716) 275-0680; Fax: +1 (716) 473-9573; E-mail: [email protected]
[1^4]. Region 3 contains two genes essential for the export of polySia across the cytoplasmic membrane: kpsM, encoding a 29.8 kDa hydrophobic protein, and kpsT, which encodes a 25 kDa hydrophilic protein with a consensus ATP-binding site [5]. KpsM and KpsT belong to the ATP-binding cassette (ABC) transporter superfamily [6]. The family contains a diverse group of transporters including the periplasmic permeases of enteric bacteria, the hemolysin exporter of E. coli, the eukaryotic cystic ¢brosis transductance regulator, and the P-glycoprotein which is associated with multiple drug resistance in mammalian tumor cells. ABC transporters share a common organizational motif consisting of four domains: two hydrophobic membrane components and two hydrophilic ATP-binding components [6]. KpsM and KpsT are postulated to function as homodimers promoting polySia transport across the cytoplasmic membrane utilizing energy from ATP hydrolysis [1].
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It is our view that the products of kps gene cluster form a hetero-oligomeric complex responsible for synthesis and concurrent translocation of polySia to the bacterial cell surface. We view the KpsMT transporter to be central to the functioning of the complex, and have presented a model in which the KpsT protein forms a ternary complex with polySia and KpsM [1]. According to our model, ATP binding results in a conformational change in KpsT and insertion of the domain associated with polySia into the membrane. We envision that subsequent ATP hydrolysis returns KpsT to its native conformation and releases polySia [1,7]. KpsM is proposed to have six membrane spanning domains, with three periplasmic loops (PI, PII, PIII), two cytoplasmic loops (CI and CII), the N and C termini facing the cytoplasm, and a short domain within the third periplasmic loop, the SV^SVI linker, localizing in the membrane [8]. Linker insertion and site-directed mutagenesis de¢ned the N terminus, CI loop, and the SV^SVI linker as regions important for the function of KpsM. In addition, we detected limited homology in the CI loop to a short motif composed of glutamate, alanine, alanine, and followed by an invariant glycine, which is conserved among the membrane components of some ABC transporters [9]. This domain, referred to as the EAA motif, is thought to interact with the ATP-binding components and to transmit conformational changes to the membrane components [9]. Although the analysis of Saurin et al. [9] indicated that ABC transporters, other than the bacterial binding protein-dependent permeases, lacked this conserved region, we considered the region a candidate as an interaction site between KpsM and KpsT. We recently described the construction of a mutant plasmid harboring a glycine 93 to glutamic acid change in the CI loop (G93E) of KpsM [8]. The introduction of this plasmid into the kpsM deletion strain RS2604 yielded an intermediate transport phenotype characterized by smaller immunoprecipitin halos when plated on antiserum agar and an altered plaque morphology when infected with K1 speci¢c bacteriophage [8]. We postulated that the G93E mutation disrupts the interaction of KpsM and KpsT, which causes a decrease in capsular polysaccharide export. In this study, we report further studies of the phenotype associated with the G93E allele. Cells harboring the
G93E mutant allele showed a decrease in polymer production, no di¡erence in polymer chain length, and an altered e¤ciency of polymer export. 2. Materials and methods
2.1. Bacterial strains
The E. coli K12-K1 hybrid strains used in this work were EV36 (galP23 rpsL9 kps) [10] and EV138 (nanA4 neuB25 zgj-791 ::Tn10)[11]. RS2699 and RS2700 are derivatives of EV36 and EV138, respectively, that carry the G93E allele [8]. They were constructed utilizing the suicide vector system described by Donnenburg and Kaper [12] as previously described [8]. Details of constructions can be obtained from the corresponding authors. The presence of the G93E allele was con¢rmed by restriction endonuclease analysis of PCR ampli¢ed kpsM from RS2699 which revealed the loss of an AlwNI site, indicative of the G93E mutation. RS218 is a naturally occurring K1 isolate [13]. Bacterial cultures were routinely grown in L-broth or L-agar at 37³C. Horse 46 (H.46) antiserum agar plates were used to detect capsule expression as described previously [5]. Bacteriophage K1F is speci¢c for encapsulated E. coli K1 [11]. 2.2. Characterization of capsule polymer
Polyacrylamide gel electrophoresis (PAGE) of puri¢ed polysaccharide was done as described by Pelkonen et al. [14]. Polysaccharide for PAGE was isolated by the method of Vann and Freese [15]. Rocket immunoelectrophoresis (RIE) was done according to the method described by Stevens et al. [16] using puri¢ed K1 antisera and standard concentrations of polysialic acid (polySia). The polySia standards were hydrolyzed into monomers by acid treatment (0.05 N H2 SO4 at 80³C for 1 h) and quantitated by the thiobarbituric acid assay [17]. Samples for RIE were prepared essentially as described by Vermeulen et al. [18]. Cultures were grown at 37³C for 3.5 h in BHI medium supplemented with 5% fetal calf serum and 0.7% glucose to an OD600 = 1.40 yielding 2.8U109 CFU/ml and harvested by centrifugation (10000Ug for 10 min). Culture supernates containing free cap-
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and RS2700 were diluted about 1:20 to an OD600 = 0.06 in fresh M63 minimal medium supplemented with glycerol (0.4% w/v), thiamine (50 mg ml31 ) and amino acids (40 Wg ml31 ), then grown at 37³C for 5 h to an OD600 = 0.55. Alternatively, cultures of RS2700 were harvested by centrifugation (10 000Ug for 10 min), then concentrated by resuspending the cells in one half the original volume of medium to an OD600 = 0.95. To initiate polymer synthesis, exogenous sialic acid (0.32 mM ¢nal concentration) was added to these cultures 1 min prior to phage addition. K1F phage were added to the cultures at an initial concentration (Po) of 3U107 PFU per ml at time zero. Samples of unabsorbed phage (P) were removed from the cultures at various time points, treated with 0.85% NaCl saturated with chloroform, and titered using EV36 cells previously grown to stationary phase [11]. Data were expressed as the fraction of unabsorbed K1F phage (P/Po) per min. 3. Results and discussion
Fig. 1. Characteristics of plaques produced by K1F bacteriophage. Note di¡erences in plaque morphology with wild-type EV36 (A) and the mutant RS2699 (B) harboring the G93E allele. Although the bacteriophage form plaques with equal e¤ciency on both strains, the plaques are more turbid and slightly smaller in RS2699 infected with K1F phage in comparison to EV36.
sular polymer were aliquoted and stored at 320³C. Pelleted cells harboring cell associated polymer were washed and then resuspended in phosphate bu¡ered saline. The cell suspensions were sonicated using a Branson Soni¢er 250 at 50% power for 4 min and then stored at 320³C. 2.3. Phage adsorption assay
Phage adsorption assays were done essentially as described by Vimr [11]. Overnight cultures of EV138
In this study, we constructed the strain RS2699 to examine the e¡ect of a chromosomally encoded G93E allele on capsular polymer production. RS2699 is a derivative of EV36, a K1/K12 hybrid strain which contains the entire kps gene cluster [10]. RS2699, generated by allelic exchange of the G93E allele with the wild-type chromosomal kpsM gene in EV36, displayed the distinct phenotypes which are characteristic of the G93E mutant allele, i.e. smaller precipitin halos [8] and smaller plaques when infected with K1 phage (Fig. 1). To characterize the G93E allele, capsule polymer Table 1 K1 polysaccharide content Cell associated polymerb Strain Free polymer in culture supernatanta EV36 4.2 þ 0.4 U 4.5 þ 0.2 U RS2699 0.8 þ 0.3 U 2.2 þ 0.3 U U = Wg polySia/109 CFU, n = 3 þstandard deviation. a Polymer content in the supernatants of late stationary cultures at the same optical density and cell number. b Cell associated polymer includes both the intra- and extracellular capsule polymer which is cell associated.
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lated about 80% less polymer in the supernates and produced approximately 50% less cell associated polymer than wild-type EV36. RS2699 produced smaller precipitin halos on antiserum agar than EV36. Although this observation could be explained by defective translocation in RS2699, we postulated that the smaller halos might also re£ect a qualitative di¡erence in the capsule polymer. To determine if RS2699 synthesizes polymer altered in length, polysialic acid was isolated from RS2699 and compared by polyacrylamide gel electrophoresis to polymer puri¢ed from EV36 as well as from a clinical isolate of E. coli K1, RS218 [13]. The migration pattern of the polysaccharides obtained from EV36 (data not shown) was similar to the pattern found with RS218 (Fig. 2). Colominic acid (Sigma), a polymer of sialic acid that is about 30 residues in length (lane 1), migrates faster than full
Fig. 2. Gel electrophoresis of polysialic acid. PolySia puri¢ed from RS218 and RS2699 were subjected to gel electrophoresis in 10% polyacrylamide gels (1.5 mm thick, 32 cm long) at 600 V for 3 h at 4³C, and the gels were stained with alcian blue followed by silver. Lanes: 1, colominic acid (Sigma) (10 Wg); 2, RS218 polySia (1 Wg); 3, RS218 polySia (10 Wg); 4, RS2699 polySia (1 Wg); 5, RS2699 polySia (10 Wg).
production was compared in cultures of RS2699 and EV36 using RIE. The free polymer in culture supernates and cell associated polymer were quantitated by comparison with polySia standards and the results are summarized in Table 1. The G93E allele led to a reduction in the amount of polymer in culture supernates as well as cell-associated polysaccharide. RS2699 harboring the G93E allele accumu-
Fig. 3. K1F phage adsorption kinetics of EV138 and RS2700. Cultures of EV138 and RS2700 were exposed to 0.32 mM sialic acid 1 min prior to phage infection at time zero. The titer of unabsorbed phage was determined after dilution into chloroform saturated saline. The average cell numbers (n) of the cultures were expressed in CFU/ml: EV138, OD600 = 0.550, n = 4.4U108 ; RS2700,OD600 = 0.550, n = 3.4U108 ; RS2700, OD600 = 0.950, n = 7U108 . The adsorption curve data are the means þ standard deviation for each experiment (n = 3).
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length capsule polymer (lane 2) which consists of approximately 200 sialic acid residues [3]. The higher molecular mass polysaccharides from RS218 (lanes 2 and 3) and RS2699 (lanes 4 and 5) migrated at the same rate, indicating that the polymers were about the same length. Therefore, the smaller halo size associated with RS2699 harboring the G93E allele re£ected decreased polymer production and not any major di¡erences in polymer length. The rate of polySia expression at the cell surface was examined by a K1 speci¢c phage adsorption assay developed by Vimr [11] using strain EV138. This strain, which provides an e¤cient system for investigating polySia transport in vivo, has two mutations leading to defects in sialic synthesis (neuB24) and degradation (nanA4) [11]. NeuB is required for sialic acid synthesis while NanA is an aldolase that degrades sialic acid to N-acetyl-mannosamine and pyruvate [19]. In this strain, the neuB mutation enables capsule synthesis to be controlled experimentally by the exogenous addition of sialic acid to the growth medium [11]. After addition, sialic acid is imported into the cell, activated to CMP-sialic acid, polymerized to polySia and exported [11]. Once transported to the cell surface, polySia serves as the bacterial receptor for binding of phage K1F. Using this assay, polySia translocation was detected within 2 min of sialic acid addition [11]. The G93E mutation was transferred to EV138 by allelic exchange methodology resulting in the strain RS2700. To examine the e¡ect of the G93E mutant allele on the rate of capsule polymer transport, the K1F adsorption kinetics of RS2700 were compared to that of EV138 (Fig. 3). The rate of phage adsorption is inversely proportional to the slope of the curve. It is apparent that following the addition of sialic acid, K1 speci¢c phage remained in the culture supernates of RS2700 signi¢cantly longer than EV138. After 15 min of exposure to K1F phage, RS2700 adsorbed only 30% of the phage while EV138 cultures bound 90% of the phage over the same time period. RS2700 cultures adsorbed a signi¢cant fraction of phage only after 20 min, and there appeared to be about a 10 min di¡erential in phage binding when compared to EV138. These experiments were performed with cultures of RS2700 and EV138 at the same optical density, however viable counts indicated slightly fewer RS2700 cells were
221
present. To ensure that the di¡erence in phage adsorption did not occur because of the reduced number of RS2700 cells, additional phage adsorption assays were performed using RS2700 at a greater cell number when compared to EV138. The results in Fig. 3 indicate that the slower rate of phage adsorption for RS2700 was independent of cell concentration, consistent with a lack of phage receptors on the cell surface. Thus, the G93E mutation caused a reduction in the rate of polymer expression on the surface of RS2700 in comparison to EV138. In EV138 cultures treated with K1F phage and exogenous sialic acid, the rate of phage adsorption parallels the rate of polySia expression on the cell surface [11]. As shown by the K1F phage adsorption kinetics, RS2700 harboring the G93E allele adsorbed phage less e¤ciently than EV138 which indicated a decrease in the rate of polySia expression on the cell surface. In cultures of EV138 and RS2700, polySia expression depends on the separate rates of sialic acid import, activation, polymerization and polySia export [11]. The strains EV138 and RS2700 are isogenic except for the G93E allele in KpsM, which is directly involved in capsule polymer export across the cytoplasmic membrane. Thus, it is likely that the G93E allele caused a reduction in the rate of polymer export which resulted in the observed decrease in the rate of polySia expression in RS2700. We favor a hypothesis that the G93E mutation partially disrupted an intermolecular interaction between KpsM and KpsT required for polymer transport. Consequently, the rate of polymer transport is slowed, causing a reduction in the overall rate of polymer expression. Alternatively, the mutation might modify an interaction between KpsM and the transported substrate, polysialic acid. We cannot rule out the possibility, however, that a disruption in polymer export also e¡ects sialic acid polymerization. A fundamental theme that has emerged from the study of acapsular mutants in the kps gene cluster is that polymer export and polymer synthesis are linked. Cells with mutations in regions 1 and 3 of the kps gene cluster involved in polySia transport accumulate polymer intracellularly and show a decrease in the endogenous sialyltransferase activity in vitro [5,10]. In E. coli K1, the sialyltransferase enzyme, encoded by the region 2 neuS gene, is capable of transferring activated sugar (CMP
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sialic acid) onto acceptor molecules [4]. An endogenous sialyltransferase assay measures the transfer of labeled sugar onto pre-existing receptors within the membranes while an exogenous assay measures the transfer of labeled sugar onto exogenously added polysialic acid. The association of polySia transport mutations with lowered endogenous sialyltransferase activity suggested that defects in transport a¡ect polymer synthesis [10]. Thus, it is possible that the
[5] Pavelka, Jr., M.S., Hayes, S.F. and Silver, R.P. (1994) Characterization of KpsT, the ATP-binding component of the ABC-transporter involved with the export of capsular polysialic acid in
Escherichia coli
K1. J. Biol. Chem. 269, 20149^
20158. [6] Higgins, C.F. (1992) ABC transporters : from microorganisms to man. Annu. Rev. Cell Biol. 8, 67^113. [7] Bliss, J.M., Garon, C.F.and Silver, R.P. (1996) Polysialic acid export in
Escherichia coli
K1 : the role of KpsT, the ATP-
binding component of an ABC transporter, in chain translocation. Glycobiology 6, 445^452.
G93E allele has a dual e¡ect on polymer synthesis
[8] Pigeon, R.P. and Silver, R.P. (1994) Topological and muta-
and translocation which led to a decreased rate of
tional analysis of KpsM, the hydrophobic component of the
polySia expression on the surface of RS2700. This decrease in polySia expression ultimately resulted in
ABC-transporter involved in the export of polysialic acid in
Escherichia coli
K1. Mol. Microbiol. 14, 871^881.
[9] Saurin, W., Koster, W. and Dassa, E. (1994) Bacterial bind-
smaller halos when G93E mutants are plated on
ing-protein dependent permeases : characterization of distinct
antiserum agar and reduced rates of K1F phage ad-
signatures for functionally related integral cytoplasmic mem-
sorption. In any event, these studies of the G93E allele underscore the signi¢cance of the CI loop of KpsM for polymer export and are consistent with the postulated role of this region as an interaction site within the KpsMT transporter of
E. coli
K1.
brane proteins. Mol. Microbiol. 12, 993^1004. [10] Vimr, E. R., Aaronson, W. and Silver, R.P. (1989) Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of
Escherichia coli
K1. J. Bacteriol. 171, 1106^1117.
[11] Vimr, E.R. (1992) Selective synthesis and labeling of the poly-
Escherichia coli K1 neuB. J. Bacteriol. 174,
sialic acid capsule in tions in
nanA
and
strains with muta6191^6197.
[12] Donnenberg, M.S. and Kaper, J.B. (1991) Construction of an
eae
Acknowledgments
Escherichia coli
deletion mutant of enteropathogenic
by
using a positive-selection vector. Infect. Immun. 59, 4310^ 4317.
We are grateful to Willie Vann for providing H46
[13] Silver, R.P., Aaronson, W., Sutton, A. and Schneerson, R.
immune globin, puri¢ed polysialic acid from RS218,
(1980) Comparative analysis of plasmids and some metabolic
and technical advice for the TBA assay. We would also like to thank Eric Vimr for providing EV138,
characteristics
of
Escherichia coli
K1
amide gel electrophoresis of the
electrophoresis, and Joe Bliss for critically reviewing
bacteria. J. Bacteriol. 170, 2646^2653.
Grants AI3915 and AI26655 from the National Institutes of Health.
diseased
and
[14] Pelkonen, S., Hayrinen, J. and Finne, J. (1988) Polyacryl-
Marilyn Loeb for technical advice concerning rocket
the manuscript. This work was supported in part by
from
healthy individuals. Infect. Immun. 29, 200^206.
Escherichia coli
K1 and other
[15] Vann, W.F. and Freese, S.J. (1994) Puri¢cation of
coli
Escherichia
K antigens. Methods Enzymol. 235, 304^311.
[16] Stevens, P., Chu, C.L. and Young, L.S. (1980) K1 antigen content and the presence of an additional sialic acid containing antigen among bacteremic K1
Escherichia coli :
correlation
with susceptibility to opsonophagocytosis. Infect. Immun. 29,
References
1055^1061. [17] Vann, W.F., Silver, R.P., Abeijon, C., Chang, K., Aaronson,
[1] Bliss, J.M. and Silver, R.P. (1996) Coating the surface : a model for expression of capsular polysialic acid in
coli
Escherichia
K1. Mol. Microbiol. 21, 221^231.
[2] Roberts, I.S. (1996) The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu. Rev. Microbiol.
synthetase. J. Biol. Chem. 262, 17556^17562. [18] Vermeulen, C., Cross, A., Byrne, W.R. and Zollinger, W. Quantitative
relationship
and killing of K1 encapsulated
between
capsular
Escherichia coli.
content
Infect. Im-
munity. 56, 2723^2730.
Glycobiology 2, 5^23. [4] Vimr, E., Steenbergen, S. and Cieslewicz, M. (1995) Biosynthesis of the polysialic acid capsule in
Escherichia coli cytidine 5P-monophosphate N-acetylneuraminic acid (1987) Puri¢cation, properties, and genetic location of
(1988)
50, 285^315. [3] Troy, F.A. II (1992) Polysialylation : from bacteria to brains.
J. Ind. Microbiol. 15, 352^360.
W., Sutton, A., Finn, C.W., Lindner, W. and Kotsatos, M.
Escherichia coli
K1.
[19] Vimr, E.R., and Troy, F.A. (1985) Regulation of sialic acid metabolism in
Escherichia coli :
role of N-acylneuraminate
pyruvate-lyase. J. Bacteriol. 164, 845^853.
FEMSLE 7855 10-11-97
Analysis of the G93E mutant allele of KpsM, the membrane component of an ABC transporter involved in polysialic acid translocation in Escherichia coli K1 Ronald P. Pigeon, Richard P. Silver * Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Box 672, Rochester, NY 14642, USA
Received 2 July 1997; revised 5 September 1997; accepted 8 September 1997
Abstract
KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1. The allele is a point mutation in the first cytoplasmic loop (CI) of KpsM which partially disrupts translocation of the capsule. While producing polymer of wild-type length, strains harboring the G93E allele exhibit a decreased production of capsular polymer and a reduced rate of polymer translocation to the cell surface. kpsMG93E
Keywords : Escherichia coli
; Capsule; K1 antigen ; Polysialic acid; ABC transporter; Polysaccharide transport
1. Introduction
Capsules have long been recognized as important virulence determinants of extraintestinal pathogenic Escherichia coli. The K1 capsular polysaccharide, an K(2,8)-linked polymer of sialic acid, is the most common capsular antigen isolated from neonates with meningitis caused by E. coli. The 17 kb kps gene cluster of E. coli K1 encodes the information required for synthesis and expression of the polysialic acid (polySia) capsule [1^4]. The cluster is divided into three functional regions [1^4]. A central biosynthetic cassette, region 2, contains the genes for sialic acid synthesis, activation and polymerization while the £anking regions 1 and 3 encode proteins that function in polySia transport to the bacterial surface * Corresponding author. Tel.: +1 (716) 275-0680; Fax: +1 (716) 473-9573; E-mail: [email protected]
[1^4]. Region 3 contains two genes essential for the export of polySia across the cytoplasmic membrane: kpsM, encoding a 29.8 kDa hydrophobic protein, and kpsT, which encodes a 25 kDa hydrophilic protein with a consensus ATP-binding site [5]. KpsM and KpsT belong to the ATP-binding cassette (ABC) transporter superfamily [6]. The family contains a diverse group of transporters including the periplasmic permeases of enteric bacteria, the hemolysin exporter of E. coli, the eukaryotic cystic ¢brosis transductance regulator, and the P-glycoprotein which is associated with multiple drug resistance in mammalian tumor cells. ABC transporters share a common organizational motif consisting of four domains: two hydrophobic membrane components and two hydrophilic ATP-binding components [6]. KpsM and KpsT are postulated to function as homodimers promoting polySia transport across the cytoplasmic membrane utilizing energy from ATP hydrolysis [1].
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It is our view that the products of kps gene cluster form a hetero-oligomeric complex responsible for synthesis and concurrent translocation of polySia to the bacterial cell surface. We view the KpsMT transporter to be central to the functioning of the complex, and have presented a model in which the KpsT protein forms a ternary complex with polySia and KpsM [1]. According to our model, ATP binding results in a conformational change in KpsT and insertion of the domain associated with polySia into the membrane. We envision that subsequent ATP hydrolysis returns KpsT to its native conformation and releases polySia [1,7]. KpsM is proposed to have six membrane spanning domains, with three periplasmic loops (PI, PII, PIII), two cytoplasmic loops (CI and CII), the N and C termini facing the cytoplasm, and a short domain within the third periplasmic loop, the SV^SVI linker, localizing in the membrane [8]. Linker insertion and site-directed mutagenesis de¢ned the N terminus, CI loop, and the SV^SVI linker as regions important for the function of KpsM. In addition, we detected limited homology in the CI loop to a short motif composed of glutamate, alanine, alanine, and followed by an invariant glycine, which is conserved among the membrane components of some ABC transporters [9]. This domain, referred to as the EAA motif, is thought to interact with the ATP-binding components and to transmit conformational changes to the membrane components [9]. Although the analysis of Saurin et al. [9] indicated that ABC transporters, other than the bacterial binding protein-dependent permeases, lacked this conserved region, we considered the region a candidate as an interaction site between KpsM and KpsT. We recently described the construction of a mutant plasmid harboring a glycine 93 to glutamic acid change in the CI loop (G93E) of KpsM [8]. The introduction of this plasmid into the kpsM deletion strain RS2604 yielded an intermediate transport phenotype characterized by smaller immunoprecipitin halos when plated on antiserum agar and an altered plaque morphology when infected with K1 speci¢c bacteriophage [8]. We postulated that the G93E mutation disrupts the interaction of KpsM and KpsT, which causes a decrease in capsular polysaccharide export. In this study, we report further studies of the phenotype associated with the G93E allele. Cells harboring the
G93E mutant allele showed a decrease in polymer production, no di¡erence in polymer chain length, and an altered e¤ciency of polymer export. 2. Materials and methods
2.1. Bacterial strains
The E. coli K12-K1 hybrid strains used in this work were EV36 (galP23 rpsL9 kps) [10] and EV138 (nanA4 neuB25 zgj-791 ::Tn10)[11]. RS2699 and RS2700 are derivatives of EV36 and EV138, respectively, that carry the G93E allele [8]. They were constructed utilizing the suicide vector system described by Donnenburg and Kaper [12] as previously described [8]. Details of constructions can be obtained from the corresponding authors. The presence of the G93E allele was con¢rmed by restriction endonuclease analysis of PCR ampli¢ed kpsM from RS2699 which revealed the loss of an AlwNI site, indicative of the G93E mutation. RS218 is a naturally occurring K1 isolate [13]. Bacterial cultures were routinely grown in L-broth or L-agar at 37³C. Horse 46 (H.46) antiserum agar plates were used to detect capsule expression as described previously [5]. Bacteriophage K1F is speci¢c for encapsulated E. coli K1 [11]. 2.2. Characterization of capsule polymer
Polyacrylamide gel electrophoresis (PAGE) of puri¢ed polysaccharide was done as described by Pelkonen et al. [14]. Polysaccharide for PAGE was isolated by the method of Vann and Freese [15]. Rocket immunoelectrophoresis (RIE) was done according to the method described by Stevens et al. [16] using puri¢ed K1 antisera and standard concentrations of polysialic acid (polySia). The polySia standards were hydrolyzed into monomers by acid treatment (0.05 N H2 SO4 at 80³C for 1 h) and quantitated by the thiobarbituric acid assay [17]. Samples for RIE were prepared essentially as described by Vermeulen et al. [18]. Cultures were grown at 37³C for 3.5 h in BHI medium supplemented with 5% fetal calf serum and 0.7% glucose to an OD600 = 1.40 yielding 2.8U109 CFU/ml and harvested by centrifugation (10000Ug for 10 min). Culture supernates containing free cap-
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and RS2700 were diluted about 1:20 to an OD600 = 0.06 in fresh M63 minimal medium supplemented with glycerol (0.4% w/v), thiamine (50 mg ml31 ) and amino acids (40 Wg ml31 ), then grown at 37³C for 5 h to an OD600 = 0.55. Alternatively, cultures of RS2700 were harvested by centrifugation (10 000Ug for 10 min), then concentrated by resuspending the cells in one half the original volume of medium to an OD600 = 0.95. To initiate polymer synthesis, exogenous sialic acid (0.32 mM ¢nal concentration) was added to these cultures 1 min prior to phage addition. K1F phage were added to the cultures at an initial concentration (Po) of 3U107 PFU per ml at time zero. Samples of unabsorbed phage (P) were removed from the cultures at various time points, treated with 0.85% NaCl saturated with chloroform, and titered using EV36 cells previously grown to stationary phase [11]. Data were expressed as the fraction of unabsorbed K1F phage (P/Po) per min. 3. Results and discussion
Fig. 1. Characteristics of plaques produced by K1F bacteriophage. Note di¡erences in plaque morphology with wild-type EV36 (A) and the mutant RS2699 (B) harboring the G93E allele. Although the bacteriophage form plaques with equal e¤ciency on both strains, the plaques are more turbid and slightly smaller in RS2699 infected with K1F phage in comparison to EV36.
sular polymer were aliquoted and stored at 320³C. Pelleted cells harboring cell associated polymer were washed and then resuspended in phosphate bu¡ered saline. The cell suspensions were sonicated using a Branson Soni¢er 250 at 50% power for 4 min and then stored at 320³C. 2.3. Phage adsorption assay
Phage adsorption assays were done essentially as described by Vimr [11]. Overnight cultures of EV138
In this study, we constructed the strain RS2699 to examine the e¡ect of a chromosomally encoded G93E allele on capsular polymer production. RS2699 is a derivative of EV36, a K1/K12 hybrid strain which contains the entire kps gene cluster [10]. RS2699, generated by allelic exchange of the G93E allele with the wild-type chromosomal kpsM gene in EV36, displayed the distinct phenotypes which are characteristic of the G93E mutant allele, i.e. smaller precipitin halos [8] and smaller plaques when infected with K1 phage (Fig. 1). To characterize the G93E allele, capsule polymer Table 1 K1 polysaccharide content Cell associated polymerb Strain Free polymer in culture supernatanta EV36 4.2 þ 0.4 U 4.5 þ 0.2 U RS2699 0.8 þ 0.3 U 2.2 þ 0.3 U U = Wg polySia/109 CFU, n = 3 þstandard deviation. a Polymer content in the supernatants of late stationary cultures at the same optical density and cell number. b Cell associated polymer includes both the intra- and extracellular capsule polymer which is cell associated.
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lated about 80% less polymer in the supernates and produced approximately 50% less cell associated polymer than wild-type EV36. RS2699 produced smaller precipitin halos on antiserum agar than EV36. Although this observation could be explained by defective translocation in RS2699, we postulated that the smaller halos might also re£ect a qualitative di¡erence in the capsule polymer. To determine if RS2699 synthesizes polymer altered in length, polysialic acid was isolated from RS2699 and compared by polyacrylamide gel electrophoresis to polymer puri¢ed from EV36 as well as from a clinical isolate of E. coli K1, RS218 [13]. The migration pattern of the polysaccharides obtained from EV36 (data not shown) was similar to the pattern found with RS218 (Fig. 2). Colominic acid (Sigma), a polymer of sialic acid that is about 30 residues in length (lane 1), migrates faster than full
Fig. 2. Gel electrophoresis of polysialic acid. PolySia puri¢ed from RS218 and RS2699 were subjected to gel electrophoresis in 10% polyacrylamide gels (1.5 mm thick, 32 cm long) at 600 V for 3 h at 4³C, and the gels were stained with alcian blue followed by silver. Lanes: 1, colominic acid (Sigma) (10 Wg); 2, RS218 polySia (1 Wg); 3, RS218 polySia (10 Wg); 4, RS2699 polySia (1 Wg); 5, RS2699 polySia (10 Wg).
production was compared in cultures of RS2699 and EV36 using RIE. The free polymer in culture supernates and cell associated polymer were quantitated by comparison with polySia standards and the results are summarized in Table 1. The G93E allele led to a reduction in the amount of polymer in culture supernates as well as cell-associated polysaccharide. RS2699 harboring the G93E allele accumu-
Fig. 3. K1F phage adsorption kinetics of EV138 and RS2700. Cultures of EV138 and RS2700 were exposed to 0.32 mM sialic acid 1 min prior to phage infection at time zero. The titer of unabsorbed phage was determined after dilution into chloroform saturated saline. The average cell numbers (n) of the cultures were expressed in CFU/ml: EV138, OD600 = 0.550, n = 4.4U108 ; RS2700,OD600 = 0.550, n = 3.4U108 ; RS2700, OD600 = 0.950, n = 7U108 . The adsorption curve data are the means þ standard deviation for each experiment (n = 3).
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length capsule polymer (lane 2) which consists of approximately 200 sialic acid residues [3]. The higher molecular mass polysaccharides from RS218 (lanes 2 and 3) and RS2699 (lanes 4 and 5) migrated at the same rate, indicating that the polymers were about the same length. Therefore, the smaller halo size associated with RS2699 harboring the G93E allele re£ected decreased polymer production and not any major di¡erences in polymer length. The rate of polySia expression at the cell surface was examined by a K1 speci¢c phage adsorption assay developed by Vimr [11] using strain EV138. This strain, which provides an e¤cient system for investigating polySia transport in vivo, has two mutations leading to defects in sialic synthesis (neuB24) and degradation (nanA4) [11]. NeuB is required for sialic acid synthesis while NanA is an aldolase that degrades sialic acid to N-acetyl-mannosamine and pyruvate [19]. In this strain, the neuB mutation enables capsule synthesis to be controlled experimentally by the exogenous addition of sialic acid to the growth medium [11]. After addition, sialic acid is imported into the cell, activated to CMP-sialic acid, polymerized to polySia and exported [11]. Once transported to the cell surface, polySia serves as the bacterial receptor for binding of phage K1F. Using this assay, polySia translocation was detected within 2 min of sialic acid addition [11]. The G93E mutation was transferred to EV138 by allelic exchange methodology resulting in the strain RS2700. To examine the e¡ect of the G93E mutant allele on the rate of capsule polymer transport, the K1F adsorption kinetics of RS2700 were compared to that of EV138 (Fig. 3). The rate of phage adsorption is inversely proportional to the slope of the curve. It is apparent that following the addition of sialic acid, K1 speci¢c phage remained in the culture supernates of RS2700 signi¢cantly longer than EV138. After 15 min of exposure to K1F phage, RS2700 adsorbed only 30% of the phage while EV138 cultures bound 90% of the phage over the same time period. RS2700 cultures adsorbed a signi¢cant fraction of phage only after 20 min, and there appeared to be about a 10 min di¡erential in phage binding when compared to EV138. These experiments were performed with cultures of RS2700 and EV138 at the same optical density, however viable counts indicated slightly fewer RS2700 cells were
221
present. To ensure that the di¡erence in phage adsorption did not occur because of the reduced number of RS2700 cells, additional phage adsorption assays were performed using RS2700 at a greater cell number when compared to EV138. The results in Fig. 3 indicate that the slower rate of phage adsorption for RS2700 was independent of cell concentration, consistent with a lack of phage receptors on the cell surface. Thus, the G93E mutation caused a reduction in the rate of polymer expression on the surface of RS2700 in comparison to EV138. In EV138 cultures treated with K1F phage and exogenous sialic acid, the rate of phage adsorption parallels the rate of polySia expression on the cell surface [11]. As shown by the K1F phage adsorption kinetics, RS2700 harboring the G93E allele adsorbed phage less e¤ciently than EV138 which indicated a decrease in the rate of polySia expression on the cell surface. In cultures of EV138 and RS2700, polySia expression depends on the separate rates of sialic acid import, activation, polymerization and polySia export [11]. The strains EV138 and RS2700 are isogenic except for the G93E allele in KpsM, which is directly involved in capsule polymer export across the cytoplasmic membrane. Thus, it is likely that the G93E allele caused a reduction in the rate of polymer export which resulted in the observed decrease in the rate of polySia expression in RS2700. We favor a hypothesis that the G93E mutation partially disrupted an intermolecular interaction between KpsM and KpsT required for polymer transport. Consequently, the rate of polymer transport is slowed, causing a reduction in the overall rate of polymer expression. Alternatively, the mutation might modify an interaction between KpsM and the transported substrate, polysialic acid. We cannot rule out the possibility, however, that a disruption in polymer export also e¡ects sialic acid polymerization. A fundamental theme that has emerged from the study of acapsular mutants in the kps gene cluster is that polymer export and polymer synthesis are linked. Cells with mutations in regions 1 and 3 of the kps gene cluster involved in polySia transport accumulate polymer intracellularly and show a decrease in the endogenous sialyltransferase activity in vitro [5,10]. In E. coli K1, the sialyltransferase enzyme, encoded by the region 2 neuS gene, is capable of transferring activated sugar (CMP
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222
sialic acid) onto acceptor molecules [4]. An endogenous sialyltransferase assay measures the transfer of labeled sugar onto pre-existing receptors within the membranes while an exogenous assay measures the transfer of labeled sugar onto exogenously added polysialic acid. The association of polySia transport mutations with lowered endogenous sialyltransferase activity suggested that defects in transport a¡ect polymer synthesis [10]. Thus, it is possible that the
[5] Pavelka, Jr., M.S., Hayes, S.F. and Silver, R.P. (1994) Characterization of KpsT, the ATP-binding component of the ABC-transporter involved with the export of capsular polysialic acid in
Escherichia coli
K1. J. Biol. Chem. 269, 20149^
20158. [6] Higgins, C.F. (1992) ABC transporters : from microorganisms to man. Annu. Rev. Cell Biol. 8, 67^113. [7] Bliss, J.M., Garon, C.F.and Silver, R.P. (1996) Polysialic acid export in
Escherichia coli
K1 : the role of KpsT, the ATP-
binding component of an ABC transporter, in chain translocation. Glycobiology 6, 445^452.
G93E allele has a dual e¡ect on polymer synthesis
[8] Pigeon, R.P. and Silver, R.P. (1994) Topological and muta-
and translocation which led to a decreased rate of
tional analysis of KpsM, the hydrophobic component of the
polySia expression on the surface of RS2700. This decrease in polySia expression ultimately resulted in
ABC-transporter involved in the export of polysialic acid in
Escherichia coli
K1. Mol. Microbiol. 14, 871^881.
[9] Saurin, W., Koster, W. and Dassa, E. (1994) Bacterial bind-
smaller halos when G93E mutants are plated on
ing-protein dependent permeases : characterization of distinct
antiserum agar and reduced rates of K1F phage ad-
signatures for functionally related integral cytoplasmic mem-
sorption. In any event, these studies of the G93E allele underscore the signi¢cance of the CI loop of KpsM for polymer export and are consistent with the postulated role of this region as an interaction site within the KpsMT transporter of
E. coli
K1.
brane proteins. Mol. Microbiol. 12, 993^1004. [10] Vimr, E. R., Aaronson, W. and Silver, R.P. (1989) Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of
Escherichia coli
K1. J. Bacteriol. 171, 1106^1117.
[11] Vimr, E.R. (1992) Selective synthesis and labeling of the poly-
Escherichia coli K1 neuB. J. Bacteriol. 174,
sialic acid capsule in tions in
nanA
and
strains with muta6191^6197.
[12] Donnenberg, M.S. and Kaper, J.B. (1991) Construction of an
eae
Acknowledgments
Escherichia coli
deletion mutant of enteropathogenic
by
using a positive-selection vector. Infect. Immun. 59, 4310^ 4317.
We are grateful to Willie Vann for providing H46
[13] Silver, R.P., Aaronson, W., Sutton, A. and Schneerson, R.
immune globin, puri¢ed polysialic acid from RS218,
(1980) Comparative analysis of plasmids and some metabolic
and technical advice for the TBA assay. We would also like to thank Eric Vimr for providing EV138,
characteristics
of
Escherichia coli
K1
amide gel electrophoresis of the
electrophoresis, and Joe Bliss for critically reviewing
bacteria. J. Bacteriol. 170, 2646^2653.
Grants AI3915 and AI26655 from the National Institutes of Health.
diseased
and
[14] Pelkonen, S., Hayrinen, J. and Finne, J. (1988) Polyacryl-
Marilyn Loeb for technical advice concerning rocket
the manuscript. This work was supported in part by
from
healthy individuals. Infect. Immun. 29, 200^206.
Escherichia coli
K1 and other
[15] Vann, W.F. and Freese, S.J. (1994) Puri¢cation of
coli
Escherichia
K antigens. Methods Enzymol. 235, 304^311.
[16] Stevens, P., Chu, C.L. and Young, L.S. (1980) K1 antigen content and the presence of an additional sialic acid containing antigen among bacteremic K1
Escherichia coli :
correlation
with susceptibility to opsonophagocytosis. Infect. Immun. 29,
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