RESUMEN
In this review, we discuss the kps cluster of Escherichia coli as the paradigm for the ABC capsular polysaccharide exporter (CPSE) family. Components of the cluster form a multimeric protein complex consisting of both biosynthetic and export machinery. We compare the Kps exporter with capsule export systems from other members of the CPSE family.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Bacterias Gramnegativas/metabolismo , Proteínas Periplasmáticas , Polisacáridos Bacterianos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/patogenicidad , Bacterias Gramnegativas/genética , Proteínas de Transporte de Membrana , Polisacáridos Bacterianos/genéticaRESUMEN
Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Cápsulas Bacterianas/fisiología , Campylobacter jejuni/patogenicidad , Proteínas de Escherichia coli , Proteínas de Transporte de Membrana , Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Clonación Molecular , Escherichia coli/genética , Prueba de Complementación Genética , Metabolismo de los Lípidos , Mutagénesis , Polisacáridos/metabolismo , Análisis de Secuencia de ADN , VirulenciaRESUMEN
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, Neu(III)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 neu(III)D homologue from GBS shares a similar enzymatic function.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205-212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background. However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame. Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in E. coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Oxo-Ácido-Liasas/metabolismo , Serina Endopeptidasas/metabolismo , Ácidos Siálicos/biosíntesis , Acetiltransferasas/genética , Proteínas Bacterianas/genética , Dimerización , Escherichia coli/genética , Vectores Genéticos , Sustancias Macromoleculares , Oxo-Ácido-Liasas/genética , Plásmidos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Serina Endopeptidasas/genéticaRESUMEN
Escherichia coli K1 produces a capsular polysaccharide of alpha(2-8) poly-N-acetylneuraminic acid. This polysaccharide is an essential virulence factor of these neuropathogenic bacteria. The genes necessary for the synthesis of neuNAc were localized to a plasmid containing the neuBAC genes of the K1 gene cluster. Cells harboring the neuB+ allele in an aldolase (nanA-) negative background produce neuNAc in vivo. Enzymatic synthesis of neuNAc could be demonstrated in extracts of cells harboring an expression plasmid (pNEUB) containing the neuB gene alone. NeuNAc synthetase was purified to homogeneity from extracts of cells harboring pNEUB. The molecular weight of the purified enzyme is 40 kDa, similar to that predicted by the nucleotide sequence of the neuB gene. The amino terminal sequence of the purified protein matches that predicted by the nucleotide sequence of the neuB gene. NeuNAc synthetase catalyzes the formation of neuNAc as indicated by its coupling to the CMP-neuNAc synthetase reaction. The enzyme condenses manNAc and PEP with the release of phosphate. The E. coli neuNAc synthetase is specific for manNAc and PEP, unlike rat liver enzyme that utilizes N-acetylmannosamine-6-phosphate to form neuNAc-9-PO4. This represents the first report of a purification of a sialic acid synthetase from either a eukaryotic or prokaryotic source to homogeneity. These experiments clearly demonstrate an aldolase-independent sialic acid synthetase activity in E. coli K1.
Asunto(s)
Escherichia coli/enzimología , Oxo-Ácido-Liasas/aislamiento & purificación , Oxo-Ácido-Liasas/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Durapatita , Escherichia coli/genética , Genes Bacterianos , Cinética , Hígado/enzimología , Peso Molecular , Familia de Multigenes , Oxo-Ácido-Liasas/química , Fragmentos de Péptidos/química , Ratas , Especificidad por Sustrato , TermodinámicaRESUMEN
KpsT utilizes ATP to effect translocation of the polysialic acid capsule of Escherichia coli K1. We have previously proposed a mechanistic model for the action of this protein. Here, we provide evidence to support two predictions of the model: that KpsT associates with polymer and that KpsT is accessible from the periplasmic surface of the inner membrane.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Ácidos Siálicos/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico Activo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Pruebas de Precipitina , Tripsina/metabolismoRESUMEN
KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1. The kpsMG93E allele is a point mutation in the first cytoplasmic loop (Cl) 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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Antígenos Bacterianos , Escherichia coli/genética , Polisacáridos Bacterianos/genética , Ácidos Siálicos/metabolismo , Alelos , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Transporte Biológico/genética , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Mutación , Polisacáridos/metabolismo , Polisacáridos Bacterianos/metabolismoRESUMEN
Capsules are well-studied components of the bacterial surface that modulate interactions between the cell and its environment. Generally composed of polysaccharide, they are key virulence determinants in invasive infections in humans and other animals. Genetic determinants involved in capsule expression have been isolated from a number of organisms, but perhaps the best characterized is the kps cluster of Escherichia coli K1. In this review, the current understanding of the functions of the kps gene products is summarized. Further, a proposed mechanistic model for capsule expression is presented and discussed. The model is based on the premise that the numerous components of the kps cluster form a hetero-oligomeric complex responsible for synthesis and concurrent translocation of the capsular polysialic acid through sites of inner and outer membrane fusion. We view the ATP-binding cassette (ABC) transporter, KpsMT, to be central to the functioning of the complex, interacting with the biosynthetic apparatus as well as the extracytoplasmic components of the cluster to co-ordinate synthesis and translocation. The model provides the basis for additional experimentation and reflects emerging similarities among systems responsible for macromolecular export in Gram-negative bacteria.
Asunto(s)
Escherichia coli/metabolismo , Modelos Biológicos , Polisacáridos Bacterianos/biosíntesis , Ácidos Siálicos/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Escherichia coli/genética , Escherichia coli/patogenicidad , Expresión Génica , Genes Bacterianos , Humanos , Sustancias Macromoleculares , Microscopía Electrónica , Familia de Multigenes , Polisacáridos Bacterianos/genética , Ácidos Siálicos/genética , VirulenciaRESUMEN
The polysialic acid (polySia) capsule of Escherichia coli K1 is a key virulence determinant of the organism, allowing it to evade host defenses. The proteins necessary for expression of the capsule are encoded by the 17 kb kps gene cluster. This cluster contains two genes, kpsM and kpsT, that are required for polySia transport across the cytoplasmic membrane. KpsM is a hydrophobic integral inner membrane protein, while KpsT is a peripheral inner membrane protein that binds ATP. They belong to the ATP-binding cassette (ABC) superfamily of transporters. To study the role of KpsT in polySia translocation, we used PCR mutagenesis to isolate dominant negative mutations of plasmid-encoded kpsT. All mutations mapped to the same glutamic acid residue at position 150, adjacent to Walker motif B of KpsT. Wild-type (kps+) cells harboring one such allele, E150G, did not transport polySia to the cell surface but accumulated intracellular polysaccharide and produced small colonies containing cells that grew as long filaments. The E150G protein still bound ATP as shown by 8-azidoATP photolabeling assays. We combined the E150G allele with each of five mutations isolated previously in kpsT. Mutations that disrupt ATP-binding (K44E) or alter regions of the protein thought to interact with KpsM (G84D, S126F) suppressed the dominant negative phenotype while mutations in the C-terminal portion of the protein (C163Y, H181Y) did not suppress. These studies have allowed the development of a working model for the role of KpsT in polySia chain translocation.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Ácidos Siálicos/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Alelos , Proteínas Bacterianas/genética , Transporte Biológico , Microscopía Electrónica de Rastreo , Mutagénesis , FenotipoRESUMEN
The K1 capsular polysaccharide, a polymer of sialic acid, is an important virulence determinant of extraintestinal pathogenic Escherichia coli. The genes responsible for the synthesis and expression of the polysialic acid capsule of E. coli K1 are located on the 17-kb kps gene cluster, which is functionally divided into three regions. Central region 2 encodes proteins necessary for the synthesis, activation, and polymerization of sialic acid, while flanking regions 1 and 3 are involved in polymer transport to the cell surface. In this study, we identified two genes at the proximal end of region 2, neuD and neuB, which encode proteins with predicted sizes of 22.7 and 38.7 kDa, respectively. Several observations suggest that the neuB gene encodes sialic acid synthase. EV24, a neuB chromosomal mutant that expresses a capsule when provided exogenous sialic acid, could be complemented in trans by the cloned neuB gene. In addition, NeuB has significant sequence similarity to the product of the cpsB gene of Neisseria meningitidis group B, which is postulated to encode sialic acid synthase. We also present data indicating that neuD has an essential role in K1 polymer production. Cells harboring pSR426, which contains all of region 2 but lacks region 1 and 3 genes, produce an intracellular polymer. In contrast, no polymer accumulated in cells carrying a derivative of pSR426 lacking a functional neuD gene. Unlike strains with mutations in neuB, however, neuD mutants are not complemented by exogenous sialic acid, suggesting that NeuD is not involved in sialic acid synthesis. Additionally, cells harboring a mutation in neuD accumulated sialic acid and CMP-sialic acid. We also found no significant differences between the endogenous and exogenous sialyltransferase activities of a neuD mutant and the wild-type organism. NeuD shows significant similarity to a family of bacterial acetyltransferases, leading to the theory that NeuD is an acetyltransferase which may exert its influences through modification of other region 2 proteins.
Asunto(s)
Acetiltransferasas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Familia de Multigenes , Oxo-Ácido-Liasas/genética , Ácidos Siálicos/biosíntesis , Secuencia de Aminoácidos , Cápsulas Bacterianas/metabolismo , Secuencia de Bases , Escherichia coli/enzimología , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de AminoácidoRESUMEN
The 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E. coli K1, is divided into three functional regions. Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP-binding site. KpsM and KpsT belong to the ATP-binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using beta-lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane-spanning regions, with the N- and C-terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV-SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space. In vivo cross-linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker-insertion and site-directed mutagenesis define the N-terminus, the first cytoplasmic loop, and the SV-SVI linker as regions that are important for the function of KpsM in K1 polymer transport.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana , Ácidos Siálicos/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Transporte Biológico Activo , Reactivos de Enlaces Cruzados , ADN Complementario/genética , Endopeptidasas/metabolismo , Escherichia coli/química , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
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 photolabeled by the ATP analog, 8-N3[gamma-32P]ATP. The derivatization of KpsT by this reagent is inhibited by cold ATP or ATP gamma S. 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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Ácidos Siálicos/metabolismo , Adenosina Trifosfato/análogos & derivados , Marcadores de Afinidad , Secuencia de Aminoácidos , Azidas , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Cartilla de ADN , Escherichia coli/genética , Escherichia coli/ultraestructura , Datos de Secuencia Molecular , Familia de Multigenes , Conformación Proteica , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The 17-kb kps gene cluster encodes proteins necessary for the synthesis, assembly, and translocation of the polysialic acid capsule of Escherichia coli K1. We previously reported that one of these genes, kpsD, encodes a 60-kDa periplasmic protein that is involved in the translocation of the polymer to the cell surface. The nucleotide sequence of the 2.4-kb BamHI-PstI fragment accommodating the kpsD gene was determined. Sequence analysis showed an open reading frame for a 558-amino-acid protein with a typical N-terminal prokaryotic signal sequence corresponding to the first 20 amino acids. KpsD was overexpressed, partially purified, and used to prepare polyclonal antiserum. A chromosomal insertion mutation was generated in the kpsD gene and results in loss of surface expression of the polysialic acid capsule. Immunodiffusion analysis and electron microscopy indicated that polysaccharide accumulates in the periplasmic space of mutant cells. A wild-type copy of kpsD supplied in trans complemented the chromosomal mutation, restoring extracellular expression of the K1 capsule. However, a kpsD deletion derivative (kpsD delta C11), which results in production of a truncated KpsD protein lacking its 11 C-terminal amino acids, was nonfunctional. Western blot (immunoblot) data from cell fractions expressing KpsD delta C11 suggest that the truncated protein was inefficiently exported into the periplasm and localized primarily to the cytoplasmic membrane.
Asunto(s)
Antígenos Bacterianos , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos/genética , Proteínas Periplasmáticas , Ácidos Siálicos/metabolismo , Secuencia de Aminoácidos , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/ultraestructura , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Transporte Biológico/genética , Análisis Mutacional de ADN , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , Polisacáridos Bacterianos/metabolismo , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
The polysialic acid capsule of Escherichia coli K1, a causative agent of neonatal septicemia and meningitis, is an essential virulence determinant. The 17-kb kps gene cluster, which is divided into three functionally distinct regions, encodes proteins necessary for polymer synthesis and expression at the cell surface. The central region, 2, encodes products required for synthesis, activation, and polymerization of sialic acid, while flanking regions, 1 and 3, are thought to be involved in polymer assembly and transport. In this study, we identified two genes in region 3, kpsM and kpsT, which encode proteins with predicted sizes of 29.6 and 24.9 kDa, respectively. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein, while KpsT contains a consensus ATP-binding domain. KpsM and KpsT belong to a family of prokaryotic and eukaryotic proteins involved with a variety of biological processes, including membrane transport. A previously described kpsT chromosomal mutant that accumulates intracellular polysialic acid was characterized and could be complemented in trans. Results of site-directed mutagenesis of the putative ATP-binding domain of KpsT are consistent with the view that KpsT is a nucleotide-binding protein. KpsM and KpsT have significant similarity to BexB and BexA, two proteins that are essential for polysaccharide capsule expression in Haemophilus influenzae type b. We propose that KpsM and KpsT constitute a system for transport of polysialic acid across the cytoplasmic membrane.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos , Familia de Multigenes , Polisacáridos Bacterianos/genética , Ácidos Siálicos/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al ADN/genética , Haemophilus influenzae/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Polisacáridos Bacterianos/biosíntesis , Homología de Secuencia de Ácido NucleicoRESUMEN
The kps gene cluster of Escherichia coli K1 encodes functions for sialic acid synthesis, activation, polymerization, and possibly translocation of polymer to the cell surface. The size and complexity of this membrane polysaccharide biosynthetic cluster have hindered genetic mapping and functional descriptions of the kps genes. To begin a detailed investigation of the polysialic acid synthetic mechanism, acapsular mutants were characterized to determine their probable defects in polymer synthesis. The mutants were tested for complementation with kps fragments subcloned from two separately isolated, functionally intact kps gene clusters. Complementation was assayed by immunological and biochemical methods and by sensitivity to the K1-specific bacteriophage K1F. The kps cluster consisted of a central 5.8-kilobase region that contained at least two genes coding for sialic acid synthetic enzymes, a gene encoding the sialic acid-activating enzyme, and a gene encoding the sialic acid polymerase. This biosynthetic region is flanked on one side by an approximately 2.8-kilobase region that contains a potential regulatory locus and at least one structural gene for a polypeptide that appears to function in polysialic acid assembly. Flanking the biosynthetic region on the opposite side is a 6- to 8.4-kilobase region that codes for at least three proteins which may also function in polymer assembly and possibly in translocating polymer to the outer cell surface. Results of transduction crosses supported these conclusions and indicated that some of the kps genes flanking the central biosynthetic region may not function directly in transporting polymer to the cell surface. The results also demonstrate that the map position and probable function of most of the kps cluster genes have been identified.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos , Familia de Multigenes , Mutación , Polisacáridos Bacterianos/genética , Ácidos Siálicos/genética , Clonación Molecular , Escherichia coli/ultraestructura , Prueba de Complementación Genética , Genotipo , Plásmidos , Mapeo RestrictivoRESUMEN
Epidemiologic, immunologic, and genetic evidence indicate that the K1 capsular polysaccharide confers invasiveness to Escherichia coli. The capsule, an alpha-2----8-linked homopolymer of sialic acid (NeuNAc), provides the bacterium with a physical antiphagocytic barrier. Structural similarities between K1 and human tissue components suggest that immune tolerance may also be a factor in pathogenesis of K1 disease. The molecular and genetic events involved in the synthesis and export of the K1 polysaccharide were examined. The cloned K1 genes encode at least 12 proteins involved in capsule biosynthesis. These genes appear to be coordinately regulated and functionally clustered. One cluster is associated with the synthesis and activation of NeuNAc and includes the gene encoding CMP-NeuNAc synthetase. This enzyme catalyzes the activation of NeuNAc to CMP-NeuNAc. A second region, encoding five proteins, is associated with translocation of polysaccharide to the bacterial surface. The K1 polysaccharide is a poor immunogen in humans, and an understanding of the key reactions involved in K1 synthesis may help in providing an alternative to anticapsular immunity.
Asunto(s)
Antígenos Bacterianos/genética , Escherichia coli/patogenicidad , Polisacáridos Bacterianos/fisiología , Animales , Antígenos Bacterianos/análisis , Antígenos Bacterianos/inmunología , Cápsulas Bacterianas , Escherichia coli/genética , Escherichia coli/inmunología , Humanos , Polisacáridos Bacterianos/análisis , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/inmunologíaRESUMEN
N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid. We have purified CMP-NeuAc synthetase from an Escherichia coli O18:K1 cytoplasmic fraction to apparent homogeneity by ion exchange chromatography and affinity chromatography on CDP-ethanolamine linked to agarose. The enzyme has a specific activity of 2.1 mumol/mg/min and migrates as a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis. The enzyme has a requirement for Mg2+ or Mn2+ and exhibits optimal activity between pH 9.0 and 10. The apparent Michaelis constants for the CTP and NeuAc are 0.31 and 4 mM, respectively. The CTP analogues 5-mercuri-CTP and CTP-2',3'-dialdehyde are inhibitors. The purified CMP-N-acetylneuraminic acid synthetase has a molecular weight of approximately 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding CMP-N-acetylneuraminic acid synthetase is located on a 3.3-kilobase HindIII fragment. The purified enzyme appears to be identical to the 50,000 Mr polypeptide encoded by this gene based on insertion mutations that result in the loss of detectable enzymatic activity. The amino-terminal sequence of the purified protein was used to locate the start codon for the CMP-NeuAc synthetase gene. Both the enzyme and the 50,000 Mr polypeptide have the same NH2-terminal amino acid sequence. Antibodies prepared to a peptide derived from the NH2-terminal amino acid sequence bind to purified CMP-NeuAc synthetase.
Asunto(s)
Escherichia coli/enzimología , N-Acilneuraminato Citidililtransferasa/aislamiento & purificación , Nucleotidiltransferasas/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Secuencia de Bases , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Magnesio/metabolismo , Manganeso/metabolismo , Metionina/metabolismo , N-Acilneuraminato Citidililtransferasa/genética , PlásmidosRESUMEN
An 11.6-kilobase (kb) region of a 34-kb fragment of Escherichia coli DNA that encodes the K1 capsular polysaccharide genes is necessary for translocation of the K1 polysaccharide to the bacterial cell surface. This 11.6-kb region contains a gene, kpsD, encoding a 60-kilodalton protein. The kpsD gene was localized to a 2.4-kb PstI-BamHI fragment. Cells harboring a Tn1000 insertion in kpsD did not synthesize the 60-kilodalton protein and did not express polysaccharide on the cell surface. Immunodiffusion and rocket immunoelectrophoresis of cell extracts, however, demonstrated that K1 polysaccharide was synthesized by these cells. We present evidence that the kpsD gene product is synthesized as a precursor and that the processed form is located in the periplasmic space. Analysis of alkaline phosphatase activity of a kpsD-phoA fusion demonstrated that kpsD expression was under positive regulation. A 260-base-pair AluI fragment located within the kpsD coding sequence was used as a probe and was found to hybridize to chromosomal DNA from E. coli that synthesizes the K2, K5, K7, K12, and K13 capsular polysaccharides but not K3 and K100. These results suggest that the kpsD gene product may be required for export not only of K1 but for other K antigens as well.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/metabolismo , Genes Bacterianos , Polisacáridos Bacterianos/metabolismo , Fosfatasa Alcalina/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/patogenicidad , Inmunodifusión , Inmunoelectroforesis , Peso Molecular , Mutación , Hibridación de Ácido Nucleico , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/genética , Precursores de Proteínas/biosíntesis , Señales de Clasificación de Proteína/metabolismo , Transcripción GenéticaRESUMEN
The incidence of the aerobactin system and the genetic location of aerobactin genes were investigated in Escherichia coli K1 neonatal isolates belonging to different clonal groups. A functional aerobactin system was found in all members of the O7 MP3, O1 MP5, O1 MP9, and O18 MP9 clonal groups examined and also in K1 strains having O6, O16, and O75 lipopolysaccharide types, which are less frequently associated with neonatal infections. In contrast, the aerobactin system was not detected in strains from the O18 MP6 clone. The combined results of plasmid and colony hybridization experiments showed that the aerobactin genes were located on the chromosome in the majority (75%) of the aerobactin-producing K1 isolates, the genetic location of the aerobactin genes was closely correlated with the outer membrane protein profile rather than the O lipopolysaccharide type, the K1 strains harboring a chromosome-mediated aerobactin system did not possess colicin V genes, and five of six K1 isolates possessing a plasmid-borne aerobactin system contained colicin V genes which were located on the same plasmids carrying the aerobactin genes. The comparison of hemolysin production with possession of the aerobactin system in virulent clones of E. coli K1 strains showed that all of the aerobactin-producing strains from the O18 MP9 and O7 MP3 clonal groups did not synthesize hemolysin, whereas 11 of 12 aerobactin-nonproducing O18 MP6 isolates were hemolytic. Of the K1 strains examined, 92.5% possessed either the aerobactin system or the ability to produce hemolysin or both.
Asunto(s)
Escherichia coli/patogenicidad , Genes Bacterianos , Proteínas Hemolisinas/genética , Ácidos Hidroxámicos/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Mapeo Cromosómico , Cromosomas Bacterianos/fisiología , Colicinas/genética , ADN Bacteriano/genética , Electroforesis , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Hierro/metabolismo , PlásmidosRESUMEN
Encapsulated Haemophilus influenzae type b produce nonencapsulated variants at high frequency (0.1-0.3%). Cosmid cloning was used to investigate the genetic mechanism responsible for this instability. Analysis of three independently derived cosmid clones showed that the b+ parental strain contains an 18-kilobase tandem duplication of genes involved in type b capsule expression. Loss of one complete copy of the 18-kilobase tandem duplication occurred following transformation of the cosmid clones into Rec+, but not Rec-, Escherichia coli, and in H. influenzae strains that had spontaneously lost capsule expression. These results suggest that high-frequency loss of type b capsule expression is due to rec-dependent recombination between the two copies of the 18-kilobase tandem repeat. This is further supported by our finding that introduction of the H. influenzae rec-1 mutation stabilized type b capsule expression.