- Email: [email protected]
NeuD plays a role in the synthesis of sialic acid in Escherichia coli K1
FEMS Microbiology Letters 189 (2000) 281^284
www.fems-microbiology.org
NeuD plays a role in the synthesis of sialic acid in Escherichia coli K1 Dayle A. Daines a
a;1
, Lori F. Wright a , Donald O. Cha¤n b , Craig E. Rubens b , Richard P. Silver a; *
Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Box 672, Rochester, NY 14642, USA b Department of Pediatrics, University of Washington, Children's Hospital and Regional Medical Center, Seattle, WA 98105, USA Received 21 March 2000 ; received in revised form 19 June 2000; accepted 21 June 2000
Abstract The polysialic acid capsule of Escherichia coli K1 is an essential virulence determinant. The kps gene cluster, which encodes the proteins necessary for polymer synthesis and transport, is divided into three functional regions. In this report, we present evidence that the neuD gene from region 2 is involved in sialic acid synthesis. A non-polar chromosomal deletion in neuD was constructed. The defect was complemented by neuD in trans or by the addition of exogenous sialic acid. A NeuD homologue, NeuIII D, from serotype III Streptococcus agalactiae (GBS) also restored capsule expression to the neuD deletion strain. These data confirm the role of neuD in E. coli sialic acid capsule synthesis and demonstrate that the neuIII D homologue from GBS shares a similar enzymatic function. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Escherichia coli; K1 capsule; NeuD; Sialic acid ; Group B Streptococcus
1. Introduction The polysaccharide capsule of the neurotropic pathogen Escherichia coli K1 consists of linear homopolymers of K(2,8)-linked sialic acid (NeuNAc) [1,2]. The genetic locus (kps) responsible for K1 capsule synthesis is organized into three functional regions [3^5]. Genes in regions 1 and 3 are involved in transport of the polysialic acid capsule to the bacterial cell surface and are conserved among E. coli synthesizing serologically distinct capsules [3^5]. In contrast, the genes in the central region 2 are unique for a given capsular antigen. Region 2 of the kps cluster of E. coli K1 encodes six proteins, NeuDBACES, required for synthesis, activation, and polymerization of NeuNAc (Fig. 1) [4]. NeuC is the UDP-GlcNAc 2-epimerase that converts UDP-GlcNAc to ManNAc (D. Daines et al., submitted). NeuB, the sialic acid synthase, condenses ManNAc and PEP to form NeuNAc while NeuA is the CMP-NeuNAc synthetase that activates the sugar prior to * Corresponding author. Tel. : +1 (716) 275-0680; Fax: +1 (716) 473-9573; E-mail: [email protected] 1
Present address: Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, Columbia, MO 65212, USA.
polymerization by NeuS, the polysialyltransferase [6^8]. Reactions 1^3 summarize the enzymatic reactions for sialic acid synthesis in E. coli K1. UDP ÿ GlcNAc ! ManNAc UDP NeuC
1
ManNAc PEP ! NeuNAc Pi NeuB
2
NeuNAc CTP ! CMP ÿ NeuNAc PPi NeuA
3
NeuD belongs to a family of acetyl- or acyltransferases [9,10]. In this study, we have constructed a non-polar deletion mutant of neuD and provide evidence that supports the role of NeuD in the synthesis of NeuNAc. 2. Materials and methods The E. coli K-12/K1 hybrid strains used in this study were EV36 (galP23 rpsL9 kps ) [11] and two derivatives of EV36, RS2876 (vneuD), and RS2887 (vneuD nanA4). RS2876 was constructed utilizing the suicide vector system described by Donnenberg and Kaper [12]. RS2887 was constructed from RS2876 by P1 transduction of the nanA4 allele [13]. The plasmid pSR699 carries the E. coli K1 neuD gene cloned in the pTrcHisA vector (Invitrogen).
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 0 1 - 3
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PSR399 carries the E. coli K1 neuBAC genes [14] and pSD100 contains the serotype III Streptococcus agalactiae (GBS) neuIII D homologue from the type III GBS strain, COH1 [15] cloned into pBluescript KS+ (Stratagene). Details of strain and plasmid constructions can be obtained from the corresponding author. Bacterial cultures were routinely grown in LB-broth or LB-agar at 37³C. Horse 46 (H.46) antiserum agar plates and bacteriophage K1F were used to detect capsule expression as described previously [11]. 3. Results and discussion 3.1. Sialic acid restores capsule expression to a neuD deletion strain The strain RS2876 was constructed by allelic exchange and carries a 415-bp deletion in the 621-bp neuD gene. It has an acapsular phenotype, as demonstrated by resistance to K1-speci¢c bacteriophage and the absence of precipitin halos surrounding bacterial colonies on antiserum agar plates. We used sensitivity to K1 speci¢c bacteriophage to monitor the ability of NeuNAc to complement the neuD deletion in RS2876. To ensure that the NeuNAc added to the media was targeted to capsule synthesis and not catabolized, we transduced the nanA4 allele [13], which encodes a mutant NeuNAc aldolase, into RS2876, generating RS2887. NeuNAc aldolase, the product of the nanA gene, cleaves NeuNAc to ManNAc and pyruvate. As shown in Fig. 2, the addition of NeuNAc to the medium restored capsule expression to RS2887. We previously reported [16] that NeuD was not involved in NeuNAc synthesis. This conclusion was based, in large part, on the inability of exogenously added NeuNAc to restore polymer synthesis to a strain harboring an IS5 element in the neuD gene [16]. However, reexamination of the phenotype of this strain, RS2444 (neuD16: :IS5nanA4), indicates that exogenously added NeuNAc was able to restore capsule synthesis, although not very e¤ciently. We hypothesize that the poor complementation observed may due to the presence of a functional nanA gene in RS2444 [16]. 3.2. NeuD homologue from group B Streptococcus complements the neuD deletion in RS2876 In addition to NeuNAc, introduction of pSR699, a plasmid carrying the neuD gene, restored capsule expression to RS2876 (Fig. 3). Additional evidence that NeuD is in-
Fig. 1. Genetic organization of region 2 of the kps gene cluster of E. coli K1. The arrow indicates the direction of transcription.
Fig. 2. Sialic acid complements RS2887. A 50-ml culture of RS2887 (vneuDnanA4) was grown in M63 minimal medium at 37³C with 0.4% glycerol as the sole carbon source. At mid-exponential phase, the culture was split and 100 Wg ml31 sialic acid was added to one. Ten minutes later (time = 0), capsule speci¢c K1F bacteriophage was added to both cultures at a multiplicity of infection of 0.5. The cultures were monitored at 600 nm for lysis, indicative of capsule synthesis (8, no sialic acid) (F, sialic acid added).
volved in sialic acid synthesis comes from the observation that pSD100, a plasmid carrying the neuIII D homologue from the capsule gene cluster GBS type III, also restored capsule expression to the E. coli neuD deletion strain (Fig. 4). The NeuD homologue from GBS was previously designated CpsE [15]. To be consistent with Yamamoto et al. [17], the GBS genes involved in sialic acid synthesis were renamed neu. NeuD and NeuIII D are 45% similar and 34% identical. The homology is distributed throughout the proteins. GBS type III synthesizes a branched-chain capsule, unrelated to the polysialic acid capsule of E. coli K1, consisting of a backbone trisaccharide of glucose, N-acetylglucosamine, and galactose with a side chain of a single galactose that terminates with a NeuNAc residue. The NeuNAc residue of the GBS capsule is an essential virulence determinant [18]. Since the only similarity between the group B streptococcal capsule and the K1 capsule is a single terminal NeuNAc residue, the complementation data supports the notion that neuD plays a similar role in both pathogens and con¢rms its role in the synthesis of NeuNAc in E. coli K1. The data also suggests that the pathway for NeuNAc synthesis is probably identical in these two pathogens. Indeed, a plasmid containing the four GBS neu genes involved in NeuNAc synthesis com-
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283
Fig. 3. Overexpression of neuBAC does not restore capsule expression to RS2876. Exponential-phase cultures of RS2876 (vneuD) harboring plasmids encoding NeuD (pSR699) or NeuBAC (pSR399) homologues were cross-streaked with a suspension of K1-speci¢c bacteriophage on a minimal agar plate supplemented with the appropriate antibiotics and incubated at 37³C for 6 h. Note that in cells expressing a polysialic acid capsule, growth stopped at the margin of the bacteriophage streak (left). Phage-sensitive cells also produced halos on antiserum (H.46) agar plates (right).
plemented strains with defects in either neuD, neuB, neuC, or neuA, the E. coli K1 region 2 NeuNAc synthesis genes (D. Daines, unpublished). 3.3. Overexpression of neuBAC was not su¤cient to restore capsule expression to RS2876 In this report, we present several lines of evidence supporting the view that NeuD is essential for NeuNAc synthesis in E. coli K1. This, however, presents us with a paradox as to the role of NeuD in NeuNAc synthesis. NeuD belongs to a family of acetyl- or acyltransferases [9,10]. Like other members of this family [9,10,19], NeuD contains a signature hexapeptide repeat referred to as an isoleucine patch, and forms trimers of identical subunits in vivo (Daines and Silver, unpublished). We have been unable, however, to demonstrate transfer of an acetyl group from acetylCoA to any sugar substrate using a¤nity puri¢ed NeuD (Daines and Silver, unpublished). For that matter, there does not appear to be a sugar acetyl- or acyltransferase reaction needed in the pathway for sialic acid synthesis. The enzymes encoded by neuC, neuB, and neuA are su¤cient to convert UDPGlcNAc into CMP-NeuNAc. The observation by Vann et al. [14] that E. coli K-12 cells harboring pSR399, a plasmid that contained only the neuBAC genes, synthesized NeuNAc and CMP-NeuNAc is consistent with this view. However, as shown in Fig. 3, pSR399 was unable to restore capsule synthesis to RS2876. The inability to detect polymer in cells expressing neuBAC on a multicopy plasmid can be explained if NeuD is necessary for the e¤cient synthesis of sialic acid. We postulate that if any NeuNAc is made in the absence of NeuD, it is unable to either initiate or support polysialic acid synthesis in E. coli K1. We favor the view that NeuD may act upon other proteins in the sialic acid pathway, rather than on a sugar substrate, and enhance enzymatic activity. The proteins involved in polymer synthesis and transport form a heterooligomeric membrane bound biosynthetic complex. Perhaps NeuD is necessary to properly localize Neu proteins
to the complex. Interestingly, we have recently shown, using a LexA-based genetic system that NeuD interacts with NeuB in vivo (Daines and Silver unpublished). In any event, our results indicate that NeuD is involved in sialic acid synthesis in both E. coli K1 and GBS type III.
Fig. 4. NeuD homologue from group B Streptococcus complements RS2876. Fifty-milliliter cultures of EV36 (K1 ) (R), RS2876 (vneuD) (F) and RS2876 (pSD100) (8) were grown in M63 minimal medium at 37³C with appropriate antibiotics and 0.4% glycerol as the sole carbon source. At mid-exponential phase (time = 0), capsule speci¢c K1F bacteriophage was added to each culture at a multiplicity of infection of 0.5. The cultures were monitored at 600 nm for lysis, indicative of capsule synthesis.
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Acknowledgements This work was supported by NIH Grants AI39615 to R.P.S., AI25152 and AI22498 to C.E.R. D.A.D. was supported by a Molecular Pathogenesis of Bacteria and Viruses Training Grant AI07362 from the Public Health Service. References [1] Silver, R.P. and Vimr, E.R. (1990) Polysialic acid capsule of Escherichia coli K1. In: Molecular Basis of Microbial Pathogenesis (Iglewski, B.H. and Clark, V.L., Eds.), Vol. XI, pp. 39^60. Academic Press, San Diego, CA. [2] Troy, F.A. (1992) Polysialylation: from bacteria to brains. Glycobiology 2, 5^23. [3] Vimr, E., Steenbergen, S. and Cieslewicz, M. (1995) Biosynthesis of the polysialic acid capsule in Escherichia coli K1. J. Ind. Microbiol. 15, 352^360. [4] Bliss, J.M. and Silver, R.P. (1996) Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1. Mol. Microbiol. 21, 221^231. [5] Whit¢eld, C. and Roberts, I.S. (1999) Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol. Microbiol. 31, 1307^1319. [6] Vann, W.F., Silver, R.P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C.W., Lindner, W. and Kotsatos, M. (1987) Puri¢cation, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase. J. Biol. Chem. 262, 17556^17562. [7] Steenbergen, S.M., Wrona, T.J. and Vimr, E.R. (1992) Functional analysis of the sialyltransferase complexes in Escherichia coli K1 and K92. J. Bacteriol. 174, 1099^1108. [8] Vann, W.F., Tavarez, J.J., Crowley, J., Vimr, E. and Silver, R.P. (1997) Puri¢cation and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase. Glycobiology 7, 697^701.
[9] Downie, J.A. (1989) The nodL gene from Rhizobium leguminosarum is homologous to the acetyltransferases encoded by lacA and cysE. Mol. Microbiol. 3, 1649^1651. [10] Vaara, M. (1992) Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a six-residue periodicity theme. FEMS Microbiol. Lett. 97, 249^254. [11] 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. 172, 1106^1117. [12] Donnenberg, M.S. and Kaper, J.B. (1991) Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun. 59, 4310^4317. [13] Vimr, E.R. and Troy, F.A. (1985) Identi¢cation of an inducible catabolic system for sialic acids (nan) in Escherichia coli. J. Bacteriol. 164, 845^853. [14] Vann, W.F., Zapata, G., Roberts, I., Boulnois, G. and Silver, R.P. (1993) Structure and function of enzymes in sialic metabolism in polysialic producing bacteria. In: Polysialic Acid: From Microbes to Man (Roth, J., Rutishauser, U., and Troy II, F.A., Eds.), pp. 125^136. Birkhauser, Basle. [15] Cha¤n, D.O. and Rubens, C.E. (1998) Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression. Gene 219, 91^99. [16] Annunziato, P.W., Wright, L.F., Vann, W.F. and Silver, R.P. (1995) Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1. J. Bacteriol. 177, 312^319. [17] Yamamoto, S., Miyake, K., Koike, Y., Watanabe, M., Machida, Y., Ohta, M. and Iijimma, S. (1999) Molecular characterization of typespeci¢c capsular polysaccharide biosynthesis genes of Streptococcus agalactiae type Ia. J. Bacteriol. 181, 5176^5184. [18] Wessels, M.R., Rubens, C.E., Benedi, V.-J. and Kasper, D.L. (1989) De¢nition of a bacterial virulence factor: sialylation of the group B streptococcal capsule. Proc. Natl. Acad. Sci. USA 86, 8983^8987. [19] Beaman, T.W., Sugantino, M. and Roderick, S.L. (1998) Structure of the hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa. Biochemistry 37, 6689^6696.
FEMSLE 9525 26-7-00
www.fems-microbiology.org
NeuD plays a role in the synthesis of sialic acid in Escherichia coli K1 Dayle A. Daines a
a;1
, Lori F. Wright a , Donald O. Cha¤n b , Craig E. Rubens b , Richard P. Silver a; *
Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Box 672, Rochester, NY 14642, USA b Department of Pediatrics, University of Washington, Children's Hospital and Regional Medical Center, Seattle, WA 98105, USA Received 21 March 2000 ; received in revised form 19 June 2000; accepted 21 June 2000
Abstract The polysialic acid capsule of Escherichia coli K1 is an essential virulence determinant. The kps gene cluster, which encodes the proteins necessary for polymer synthesis and transport, is divided into three functional regions. In this report, we present evidence that the neuD gene from region 2 is involved in sialic acid synthesis. A non-polar chromosomal deletion in neuD was constructed. The defect was complemented by neuD in trans or by the addition of exogenous sialic acid. A NeuD homologue, NeuIII D, from serotype III Streptococcus agalactiae (GBS) also restored capsule expression to the neuD deletion strain. These data confirm the role of neuD in E. coli sialic acid capsule synthesis and demonstrate that the neuIII D homologue from GBS shares a similar enzymatic function. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Escherichia coli; K1 capsule; NeuD; Sialic acid ; Group B Streptococcus
1. Introduction The polysaccharide capsule of the neurotropic pathogen Escherichia coli K1 consists of linear homopolymers of K(2,8)-linked sialic acid (NeuNAc) [1,2]. The genetic locus (kps) responsible for K1 capsule synthesis is organized into three functional regions [3^5]. Genes in regions 1 and 3 are involved in transport of the polysialic acid capsule to the bacterial cell surface and are conserved among E. coli synthesizing serologically distinct capsules [3^5]. In contrast, the genes in the central region 2 are unique for a given capsular antigen. Region 2 of the kps cluster of E. coli K1 encodes six proteins, NeuDBACES, required for synthesis, activation, and polymerization of NeuNAc (Fig. 1) [4]. NeuC is the UDP-GlcNAc 2-epimerase that converts UDP-GlcNAc to ManNAc (D. Daines et al., submitted). NeuB, the sialic acid synthase, condenses ManNAc and PEP to form NeuNAc while NeuA is the CMP-NeuNAc synthetase that activates the sugar prior to * Corresponding author. Tel. : +1 (716) 275-0680; Fax: +1 (716) 473-9573; E-mail: [email protected] 1
Present address: Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, Columbia, MO 65212, USA.
polymerization by NeuS, the polysialyltransferase [6^8]. Reactions 1^3 summarize the enzymatic reactions for sialic acid synthesis in E. coli K1. UDP ÿ GlcNAc ! ManNAc UDP NeuC
1
ManNAc PEP ! NeuNAc Pi NeuB
2
NeuNAc CTP ! CMP ÿ NeuNAc PPi NeuA
3
NeuD belongs to a family of acetyl- or acyltransferases [9,10]. In this study, we have constructed a non-polar deletion mutant of neuD and provide evidence that supports the role of NeuD in the synthesis of NeuNAc. 2. Materials and methods The E. coli K-12/K1 hybrid strains used in this study were EV36 (galP23 rpsL9 kps ) [11] and two derivatives of EV36, RS2876 (vneuD), and RS2887 (vneuD nanA4). RS2876 was constructed utilizing the suicide vector system described by Donnenberg and Kaper [12]. RS2887 was constructed from RS2876 by P1 transduction of the nanA4 allele [13]. The plasmid pSR699 carries the E. coli K1 neuD gene cloned in the pTrcHisA vector (Invitrogen).
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 0 1 - 3
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PSR399 carries the E. coli K1 neuBAC genes [14] and pSD100 contains the serotype III Streptococcus agalactiae (GBS) neuIII D homologue from the type III GBS strain, COH1 [15] cloned into pBluescript KS+ (Stratagene). Details of strain and plasmid constructions can be obtained from the corresponding author. Bacterial cultures were routinely grown in LB-broth or LB-agar at 37³C. Horse 46 (H.46) antiserum agar plates and bacteriophage K1F were used to detect capsule expression as described previously [11]. 3. Results and discussion 3.1. Sialic acid restores capsule expression to a neuD deletion strain The strain RS2876 was constructed by allelic exchange and carries a 415-bp deletion in the 621-bp neuD gene. It has an acapsular phenotype, as demonstrated by resistance to K1-speci¢c bacteriophage and the absence of precipitin halos surrounding bacterial colonies on antiserum agar plates. We used sensitivity to K1 speci¢c bacteriophage to monitor the ability of NeuNAc to complement the neuD deletion in RS2876. To ensure that the NeuNAc added to the media was targeted to capsule synthesis and not catabolized, we transduced the nanA4 allele [13], which encodes a mutant NeuNAc aldolase, into RS2876, generating RS2887. NeuNAc aldolase, the product of the nanA gene, cleaves NeuNAc to ManNAc and pyruvate. As shown in Fig. 2, the addition of NeuNAc to the medium restored capsule expression to RS2887. We previously reported [16] that NeuD was not involved in NeuNAc synthesis. This conclusion was based, in large part, on the inability of exogenously added NeuNAc to restore polymer synthesis to a strain harboring an IS5 element in the neuD gene [16]. However, reexamination of the phenotype of this strain, RS2444 (neuD16: :IS5nanA4), indicates that exogenously added NeuNAc was able to restore capsule synthesis, although not very e¤ciently. We hypothesize that the poor complementation observed may due to the presence of a functional nanA gene in RS2444 [16]. 3.2. NeuD homologue from group B Streptococcus complements the neuD deletion in RS2876 In addition to NeuNAc, introduction of pSR699, a plasmid carrying the neuD gene, restored capsule expression to RS2876 (Fig. 3). Additional evidence that NeuD is in-
Fig. 1. Genetic organization of region 2 of the kps gene cluster of E. coli K1. The arrow indicates the direction of transcription.
Fig. 2. Sialic acid complements RS2887. A 50-ml culture of RS2887 (vneuDnanA4) was grown in M63 minimal medium at 37³C with 0.4% glycerol as the sole carbon source. At mid-exponential phase, the culture was split and 100 Wg ml31 sialic acid was added to one. Ten minutes later (time = 0), capsule speci¢c K1F bacteriophage was added to both cultures at a multiplicity of infection of 0.5. The cultures were monitored at 600 nm for lysis, indicative of capsule synthesis (8, no sialic acid) (F, sialic acid added).
volved in sialic acid synthesis comes from the observation that pSD100, a plasmid carrying the neuIII D homologue from the capsule gene cluster GBS type III, also restored capsule expression to the E. coli neuD deletion strain (Fig. 4). The NeuD homologue from GBS was previously designated CpsE [15]. To be consistent with Yamamoto et al. [17], the GBS genes involved in sialic acid synthesis were renamed neu. NeuD and NeuIII D are 45% similar and 34% identical. The homology is distributed throughout the proteins. GBS type III synthesizes a branched-chain capsule, unrelated to the polysialic acid capsule of E. coli K1, consisting of a backbone trisaccharide of glucose, N-acetylglucosamine, and galactose with a side chain of a single galactose that terminates with a NeuNAc residue. The NeuNAc residue of the GBS capsule is an essential virulence determinant [18]. Since the only similarity between the group B streptococcal capsule and the K1 capsule is a single terminal NeuNAc residue, the complementation data supports the notion that neuD plays a similar role in both pathogens and con¢rms its role in the synthesis of NeuNAc in E. coli K1. The data also suggests that the pathway for NeuNAc synthesis is probably identical in these two pathogens. Indeed, a plasmid containing the four GBS neu genes involved in NeuNAc synthesis com-
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Fig. 3. Overexpression of neuBAC does not restore capsule expression to RS2876. Exponential-phase cultures of RS2876 (vneuD) harboring plasmids encoding NeuD (pSR699) or NeuBAC (pSR399) homologues were cross-streaked with a suspension of K1-speci¢c bacteriophage on a minimal agar plate supplemented with the appropriate antibiotics and incubated at 37³C for 6 h. Note that in cells expressing a polysialic acid capsule, growth stopped at the margin of the bacteriophage streak (left). Phage-sensitive cells also produced halos on antiserum (H.46) agar plates (right).
plemented strains with defects in either neuD, neuB, neuC, or neuA, the E. coli K1 region 2 NeuNAc synthesis genes (D. Daines, unpublished). 3.3. Overexpression of neuBAC was not su¤cient to restore capsule expression to RS2876 In this report, we present several lines of evidence supporting the view that NeuD is essential for NeuNAc synthesis in E. coli K1. This, however, presents us with a paradox as to the role of NeuD in NeuNAc synthesis. NeuD belongs to a family of acetyl- or acyltransferases [9,10]. Like other members of this family [9,10,19], NeuD contains a signature hexapeptide repeat referred to as an isoleucine patch, and forms trimers of identical subunits in vivo (Daines and Silver, unpublished). We have been unable, however, to demonstrate transfer of an acetyl group from acetylCoA to any sugar substrate using a¤nity puri¢ed NeuD (Daines and Silver, unpublished). For that matter, there does not appear to be a sugar acetyl- or acyltransferase reaction needed in the pathway for sialic acid synthesis. The enzymes encoded by neuC, neuB, and neuA are su¤cient to convert UDPGlcNAc into CMP-NeuNAc. The observation by Vann et al. [14] that E. coli K-12 cells harboring pSR399, a plasmid that contained only the neuBAC genes, synthesized NeuNAc and CMP-NeuNAc is consistent with this view. However, as shown in Fig. 3, pSR399 was unable to restore capsule synthesis to RS2876. The inability to detect polymer in cells expressing neuBAC on a multicopy plasmid can be explained if NeuD is necessary for the e¤cient synthesis of sialic acid. We postulate that if any NeuNAc is made in the absence of NeuD, it is unable to either initiate or support polysialic acid synthesis in E. coli K1. We favor the view that NeuD may act upon other proteins in the sialic acid pathway, rather than on a sugar substrate, and enhance enzymatic activity. The proteins involved in polymer synthesis and transport form a heterooligomeric membrane bound biosynthetic complex. Perhaps NeuD is necessary to properly localize Neu proteins
to the complex. Interestingly, we have recently shown, using a LexA-based genetic system that NeuD interacts with NeuB in vivo (Daines and Silver unpublished). In any event, our results indicate that NeuD is involved in sialic acid synthesis in both E. coli K1 and GBS type III.
Fig. 4. NeuD homologue from group B Streptococcus complements RS2876. Fifty-milliliter cultures of EV36 (K1 ) (R), RS2876 (vneuD) (F) and RS2876 (pSD100) (8) were grown in M63 minimal medium at 37³C with appropriate antibiotics and 0.4% glycerol as the sole carbon source. At mid-exponential phase (time = 0), capsule speci¢c K1F bacteriophage was added to each culture at a multiplicity of infection of 0.5. The cultures were monitored at 600 nm for lysis, indicative of capsule synthesis.
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Acknowledgements This work was supported by NIH Grants AI39615 to R.P.S., AI25152 and AI22498 to C.E.R. D.A.D. was supported by a Molecular Pathogenesis of Bacteria and Viruses Training Grant AI07362 from the Public Health Service. References [1] Silver, R.P. and Vimr, E.R. (1990) Polysialic acid capsule of Escherichia coli K1. In: Molecular Basis of Microbial Pathogenesis (Iglewski, B.H. and Clark, V.L., Eds.), Vol. XI, pp. 39^60. Academic Press, San Diego, CA. [2] Troy, F.A. (1992) Polysialylation: from bacteria to brains. Glycobiology 2, 5^23. [3] Vimr, E., Steenbergen, S. and Cieslewicz, M. (1995) Biosynthesis of the polysialic acid capsule in Escherichia coli K1. J. Ind. Microbiol. 15, 352^360. [4] Bliss, J.M. and Silver, R.P. (1996) Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1. Mol. Microbiol. 21, 221^231. [5] Whit¢eld, C. and Roberts, I.S. (1999) Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol. Microbiol. 31, 1307^1319. [6] Vann, W.F., Silver, R.P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C.W., Lindner, W. and Kotsatos, M. (1987) Puri¢cation, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase. J. Biol. Chem. 262, 17556^17562. [7] Steenbergen, S.M., Wrona, T.J. and Vimr, E.R. (1992) Functional analysis of the sialyltransferase complexes in Escherichia coli K1 and K92. J. Bacteriol. 174, 1099^1108. [8] Vann, W.F., Tavarez, J.J., Crowley, J., Vimr, E. and Silver, R.P. (1997) Puri¢cation and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase. Glycobiology 7, 697^701.
[9] Downie, J.A. (1989) The nodL gene from Rhizobium leguminosarum is homologous to the acetyltransferases encoded by lacA and cysE. Mol. Microbiol. 3, 1649^1651. [10] Vaara, M. (1992) Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a six-residue periodicity theme. FEMS Microbiol. Lett. 97, 249^254. [11] 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. 172, 1106^1117. [12] Donnenberg, M.S. and Kaper, J.B. (1991) Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun. 59, 4310^4317. [13] Vimr, E.R. and Troy, F.A. (1985) Identi¢cation of an inducible catabolic system for sialic acids (nan) in Escherichia coli. J. Bacteriol. 164, 845^853. [14] Vann, W.F., Zapata, G., Roberts, I., Boulnois, G. and Silver, R.P. (1993) Structure and function of enzymes in sialic metabolism in polysialic producing bacteria. In: Polysialic Acid: From Microbes to Man (Roth, J., Rutishauser, U., and Troy II, F.A., Eds.), pp. 125^136. Birkhauser, Basle. [15] Cha¤n, D.O. and Rubens, C.E. (1998) Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression. Gene 219, 91^99. [16] Annunziato, P.W., Wright, L.F., Vann, W.F. and Silver, R.P. (1995) Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1. J. Bacteriol. 177, 312^319. [17] Yamamoto, S., Miyake, K., Koike, Y., Watanabe, M., Machida, Y., Ohta, M. and Iijimma, S. (1999) Molecular characterization of typespeci¢c capsular polysaccharide biosynthesis genes of Streptococcus agalactiae type Ia. J. Bacteriol. 181, 5176^5184. [18] Wessels, M.R., Rubens, C.E., Benedi, V.-J. and Kasper, D.L. (1989) De¢nition of a bacterial virulence factor: sialylation of the group B streptococcal capsule. Proc. Natl. Acad. Sci. USA 86, 8983^8987. [19] Beaman, T.W., Sugantino, M. and Roderick, S.L. (1998) Structure of the hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa. Biochemistry 37, 6689^6696.
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