The capsule biosynthetic locus of Pasteurella multocida A:1

Pasteurella multocida is the aetiological agent of fowl cholera, bovine haemorrhagic septicaemia and atrophie rhinitis in pigs. Many strains of P. multocida express a capsule on their surface. However, nothing is known about the capsule biosynthetic locus in P. multocida although the capsule has been implicated as a virulence factor. The entire capsule locus of P. multocida A:1 was cloned and sequenced. The locus is divided into three regions. Region 1 comprises four ORFs which are involved in the transport of the capsule polysaccharide to the surface. Region 2 comprises five ORFs whose postulated protein products are involved in the biosynthesis of the polysaccharide capsule. Region 3 comprises two ORFs whose postulated products show similarity to proteins that are involved in the phospholipid substitution of the polysaccharide capsule.     

Genetic and molecular analyses of Escherichia coli K1 antigen genes

The plasmid pSR23, composed of a 34-kilobase E. coli chromosomal fragment inserted into the BamHI site of the pHC79 cosmid cloning vector, contains genes encoding biosynthesis of the K1 capsular polysaccharide. Deletions, subclones, and Tn5 insertion mutants were used to localize the K1 genes on pSR23. The only deletion derivative of pSR23 that retained the K1 phenotype lacked a 2.7-kilobase EcoRI fragment. Subclones containing HindIII and EcoRI fragments of pSR23 did not produce K1. Cells harboring pSR27, a subclone containing a 23-kilobase BamHI fragment, synthesized K1 that was not detectable extracellularly. Six acapsular Tn5 insertion mutants of three phenotypic classes were observed. Class I mutants synthesized K1 only when N-acetylneuraminic acid (NANA) was provided in the medium. Reduced amounts of K1 were detectable in cell extracts of class II mutants. Class III mutants did not produce detectable K1 in either extracts or when cells were provided exogenous NANA. All mutants had sialyltransferase activity. Analysis in the E. coli minicell system of proteins expressed by derivatives of pSR23 identified a minimum of 12 polypeptides, ranging in size from 18,000 to 80,000 daltons, involved in K1 biosynthesis. The 16-kilobase coding capacity required for the proteins was located in three gene clusters designated A, B, and C. We propose that the A cluster contains a NANA operon of two genes that code for proteins with apparent molecular weights of 45,000 and 50,000. The A region also includes a 2-kilobase segment involved in regulation of K1 synthesis. The B region encoding five protein species appears responsible for the translocation of the polymer from its site of synthesis on the cytoplasmic membrane to the cell surface. The C region encodes four protein species. Since the three gene clusters appear to be coordinately regulated. we propose that they constitute a kps regulon.     

Phospholipid substitution of capsular polysaccharides and mechanisms of capsule formation in Neisseria meningitidis

Within the capsule gene complex (cps) of Neisseria meningitidis two functional regions B and C are involved in surface translocation of the cytoplasmically synthesized capsular polysaccharide, which is a homopolymer of α-2,8 polyneuraminic acid. The region-C gene products share characteristics with transporter proteins of the ABC (ATP-binding cassette) superfamily of active transporters. For analysis of the role of region B in surface translocation of the capsular polysaccharide we purified the polysaccharides of region B- and region C-defective Escherichia coli clones by affinity chromatography. The molecular weights of the polysaccharides were determined by gel filtration and the polysaccharides were analysed for phospholipid substitution by polyacrylamide gel electrophoresis and immunoblotting. The results indicate that the full-size capsular polysaccharide with a phospholipid anchor is synthesized intracellularly and that lipid modification is a strong requirement for translocation of the poly saccharide to the cell surface. Proteins encoded by region B are involved in phospholipid substitution of the capsular polysaccharide. Nucleotide sequence analysis of region B revealed two open reading frames, which encode proteins with molecular masses of 45.1 and 48.7 kDa.     

A novel outer membrane protein, Wzi, is involved in surface assembly of the Escherichia coli K30 group 1 capsule

Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface. Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface. Group 1 capsule biosynthesis gene clusters (cps) in E. coli and Klebsiella pneumoniae include a conserved open reading frame, wzi. The wzi gene is the first of a block of four conserved genes (wzi-wza-wzb-wzc) found in all group 1 K-antigen serotypes. Unlike wza, wzb, and wzc homologs that are found in gene clusters responsible for production of exopolysaccharides (i.e., predominantly cell-free polymer) in a range of bacteria, wzi is found only in systems that assemble capsular polysaccharides. The predicted Wzi protein shows no similarity to any other known proteins in the databases, but computer analysis of Wzi predicted a cleavable signal sequence. Wzi was expressed with a C-terminal hexahistidine tag, purified, and used for the production of specific antibodies that facilitated localization of Wzi to the outer membrane. Circular dichroism spectroscopy indicates that Wzi consists primarily of a beta-barrel structure, and dynamic light scattering studies established that the protein behaves as a monomer in solution. A nonpolar wzi chromosomal mutant retained a mucoid phenotype and remained sensitive to lysis by a K30-specific bacteriophage. However, the mutant showed a significant reduction in cell-bound polymer, with a corresponding increase in cell-free material. Furthermore, examination of the mutant by electron microscopy showed that it lacked a coherent capsule structure. It is proposed that the Wzi protein plays a late role in capsule assembly, perhaps in the process that links high-molecular-weight capsule to the cell surface.     

The Escherichia coli K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm

The intracellular expression of the K5 lyase enzyme, which degrades the K5 polysaccharide, decreased cell surface expression of the Escherichia coli K5 capsule. This indicates that biosynthesis of K5 polysaccharide in the cytoplasm is accessible to the action of K5 lyase and is not synthesized within a protected cytoplasmic compartment.The polysaccharide capsules of bacteria have been studied in most detail in Escherichia coli (13). E. coli has over 80 chemically and serologically distinct polysaccharide capsules, which are designated K antigens and classified into four groups (13). Group 2 polysaccharide capsules, of which K1 and K5 have been most studied (12, 13), are commonly expressed in pathogenic extraintestinal E. coli (1, 3, 7) and closely resemble the capsules of Neisseria meningitidis and Haemophilus influenzae (13). Group 2 capsule gene clusters have a conserved genetic organization comprising three regions. Regions 1 and 3 are common to all group 2 capsule gene clusters and encode the Kps proteins involved in polysaccharide export across the inner membrane, periplasm, and outer membrane. Region 2, flanked by regions 1 and 3, contains the highly variable serotype-specific genes involved in the biosynthesis of the particular polysaccharide (12, 13). In the case of the K5 capsule gene cluster, this involves the kfiABCD genes (9).The biosynthesis of the K5 polysaccharide occurs through the sequential addition of GlcA and GlcNAc residues to the nonreducing end of the polysaccharide chain catalyzed by two glycosyltransferases, KfiA and KfiC (4, 6). Polysaccharide biosynthesis occurs at the cytoplasmic face of the inner membrane and involves a hetero-oligomeric complex, consisting of proteins involved in both biosynthesis and export, that is localized at the pole of the cell (8). Such a complex would facilitate a linkage between polysaccharide synthesis and export, although at this stage the mechanism by which synthesis and export are linked is unclear. A recent paper in which the K1-specific endosialidase was expressed in the cytoplasm of a K1-expressing strain indicated that K1 polysaccharide synthesis may occur within a protected cytoplasmic compartment that is inaccessible to endosialidase cleavage (10). To test whether this was also true for the synthesis of the K5 polysaccharide, we expressed the K5-specific lyase, an enzyme that specifically degrades K5 and is associated with the tail spike of K5-specific bacteriophage (2, 5), in the cytoplasm of a K5-encoding strain. In contrast to the situation with K1, we found that expression of the K5 lyase in the cytoplasm reduced the cell surface expression of K5 polysaccharide, suggesting that unlike K1 polysaccharide synthesis, K5 polysaccharide is not synthesized within a protected cytoplasmic compartment.     

Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1.

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.     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

Molecular cloning and expression of the genes encoding the Escherichia coli K4 capsular polysaccharide, a fructose-substituted chondroitin

The majority of capsular polysaccharides (K antigens) are linear molecules and their genes have a common functional organisation encoding common steps in capsule biogenesis. However, the K4 antigen is a substituted polymer composed of a chondroitin backbone with a fructose side chain. In order to determine whether K4 biosynthesis uses these common mechanisms the K4 antigen genes were cloned. DNA probes taken from the two conserved regions of the K1 genes were used to isolate one plasmid, pRD1, homologous to both probes. Immunological analysis was used to show that pRD1 directs the production of the substituted K4 antigen on the cell surface. Southern hybridisation was used to show that the cloned genes are organised in the same way as other K antigen gene clusters. We conclude that the branched K4 antigen is handled by the same post-polymerisation mechanisms as other linear K antigens.     

Selective synthesis and labeling of the polysialic acid capsule in Escherichia coli K1 strains with mutations in nanA and neuB.

The enzymes required for polysialic acid capsule synthesis in Escherichia coli K1 are encoded by region 2 neu genes of the multigenic kps cluster. To facilitate analysis of capsule synthesis and translocation, an E. coli K1 strain with mutations in nanA and neuB, affecting sialic acid degradation and synthesis, respectively, was constructed by transduction. The acapsular phenotype of the mutant was corrected in vivo by exogenous addition of sialic acid. By blocking sialic acid degradation, the nanA mutation allows intracellular metabolite accumulation, while the neuB mutation prevents dilution by the endogenous sialic acid pool and allows capsule synthesis to be controlled experimentally by the exogenous addition of sialic acid to the growth medium. Complementation was detected by bacteriophage K1F adsorption or infectivity assays. Polysialic acid translocation was observed within 2 min after addition of sialic acid to the growth medium, demonstrating the rapidity in vivo of sialic acid transport, activation, and polymerization and translocation of polysaccharide to the cell surface. Phage adsorption was not inhibited by chloramphenicol, demonstrating that de novo protein synthesis was not required for polysialic acid synthesis or translocation at 37 degrees C. Exogenous radiolabeled sialic acid was incorporated exclusively into capsular polysaccharide. The polymeric nature of the labeled capsular material was confirmed by gel permeation chromatography and susceptibility of sialyl polymers to K1F endo-N-acylneuraminidase. The ability to experimentally manipulate capsule expression provides new approaches for investigating polysialic acid synthesis and membrane translocation mechanisms.     

Identification of hairy root loci in the T-regions of Agrobacterium rhizogenes Ri plasmids

Summary Agrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the T_L-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the T_L-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the T_L-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the T_L-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the T_L-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114     

Influence of the culture conditions on extracellular lyase activity related to K5 polysaccharide

The K5 capsular polysaccharide antigen of some Escherichia coli strains is the non-sulphated precursor in heparin biosynthesis. It is composed by two components, 16000 and 1500 Da, whose ratio depends on the activity of the extracellular form of a lyase synthesized by the same K5 producer strain. The lyase activity on the K5 chain size was greatly influenced by the medium composition employed for the lyase production. The control of lyase activity results in defined ratios of the two components in the K5 polysaccharide that is suitable for the semisynthetic production of heparin-like molecules.     

Common organization of gene clusters for production of different capsular polysaccharides (K antigens) in Escherichia coli.

Southern blot analysis of cloned K5- and K7-antigen genes, using DNA fragments from cloned K1 genes as radiolabeled probes, demonstrated that each K-antigen gene cluster is organized in a manner similar to that shown for the K1 antigen. That is, a central DNA segment unique for a given antigen type is flanked by DNA sequences that encode common functions for the management of intracellular polymer. This has been confirmed by transposon and deletion mutagenesis of plasmids carrying the K5 and K7 genes. We also describe a series of complementation experiments in which transport or postpolymerizational modification functions for one K antigen are used to complement mutations in the corresponding regions of a different K-antigen gene cluster. Thus, postpolymerizational modification of polysaccharide and transport of mature polysaccharide from the periplasmic space are common mechanisms and are independent of polysaccharide structure.     

The capsule and slime polysaccharides of the wild type and a phage resistant mutant ofRhodopseudomonas capsulata St. Louis

A rhamnose, galactose and pyruvic acid containing polysaccharide (capsule) together with the peptidoglycan was isolated fromRhodopseudomonas capsulata St. Louis as the insoluble sediment after sodium dodecyl sulfate extraction of cell envelope fractions. Treatment with pronase E separated the soluble polysaccharide from the insoluble peptidoglycan. After lysozyme-digestion, both the capsule polysaccharide and peptidoglycan were soluble.The capsule was also accumulated in the combined interphase/phenol-phase of hot phenol-water extracts of whole cells. Again, the capsule and peptidoglycan were sedimented together as long as no pronase E-treatment was performed. With the phage-resistant mutant (R. capsulata St. Louis RC1^-), no capsule polysaccharide was obtained in the combined interphase/phenol phase.An acidic polysaccharide (slime) different from the capsule in composition and serology was obtained by Cetavlon fractionation of hot phenol/water extracts of cells of both the wild-type and the mutant cells. It was shown to consist mainly of rhamnose, glucosamine and galacturonic acid.The use of O/K-antisera and of capsule polysaccharideantisera allowed a separate visualization of the capsule and slime layers.This paper is dedicated to Professor Hans G. Schoegel on the occasion of his 60th birthday     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ Nucleotide Sequence Databanks and have been assigned the accession number X78413     

Functional analysis of combinations in astaxanthin biosynthesis genes from<Emphasis Type="Italic">Paracoccus haeundaensis</Emphasis>

Carotenoids are important natural pigments produced by many microorganisms and plants. We have previously reported the isolation of a new marine bacterium,Paracoccus haeundaensis, which produces carotenoids, mainly in the form of astaxanthin. The astaxanthin biosynthesis gene cluster, consisting of six carotenogenic genes, was cloned and characterized from this organism. Individual genes of the carotenoid biosynthesis gene cluster were functionally expressed inEscherichia coli and each gene product was purified to homogeneity. Their molecular characteristics, including enzymatic activities, were previously reported. Here, we report cloning the genes for crtE, crtEB, crtEBI, crtEBIY, crtEBIYZ, and crtEBI-YZW of theP. haeundaensis carotenoid biosynthesis genes inE. coli and verifying the production of the corresponding pathway intermediates. The carotenoids that accumulated in the transformed cells carrying these gene combinations were analyzed by chromatographic and spectroscopic methods.