The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export

The nucleotide sequence of a 5.1 kb region in the Haemophilus influenzae type b capsulation locus has been determined and found to contain four open reading frames: bexD, bexC, bexB, and bexA. Comparison of the deduced products of bexC, bexB, and bexA to known proteins, and TnphoA mutagenesis, suggests that they form components of an ATP-driven polysaccharide export apparatus. Furthermore, close sequence similarity between BexA and BexB and products of the kpsT and kpsM genes at the Escherichia coli K5 capsulation locus (Smith et al., 1990--accompanying paper) suggests that capsulation genes in these organisms may have a common ancestry.     

The Haemophilus influenzae capsulation gene cluster: a compound transposon

The population of capsulate Haemophilus influenzae is divided into two phylogenetic divisions. Here we show that in division I strains the capsulation (cap) gene cluster lies between direct repeats of a novel insertion sequence (IS)-like element, IS1016. cap has apparently been mobilized in the chromosome as a compound transposon by IS1016, and the repeats have provided a molecular substrate for reversible cap gene amplification, with augmentation of capsule production, through unequal homologous recombination. Such amplification has occurred in serotype b strains, but in these a large direct repeat of cap genes has become fixed in the population. We have found a 1.2 kb deletion at one end of this duplicated capb locus, removing most of one copy of the polysaccharide export gene bexA. We have shown that this makes capsulation dependent on preservation of the direct repeat structure in order to avoid recombination-mediated loss of the other copy of bexA. Type b strains with this cap configuration are disseminated worldwide and currently cause nearly all invasive Haemophilus infections, leading us to speculate that the 1.2 kb deletion occurred in an ancestral type b strain and conferred significant biological advantage.     

Functional organization of the gene cluster involved in the synthesis of the pneumococcal capsule.

Streptococcus pneumoniae is a major human pathogen and its capsular polysaccharide has been shown to be the main virulence factor. The molecular organization of the genes governing the formation of this capsule was not studied until the 1990s. The capsular clusters (cap) of eight of the 90 known pneumococcal types have now been studied. The cap operon, located between the dexB and aliA genes, is arranged as a central region comprising the genes coding for the specific-type polysaccharide, flanked by open reading frames that are mostly common to all of the serotypes. The biochemical functions of 24 genes required for capsular polysaccharide biosynthesis have been elucidated but the precise role of the flanking regions in capsular formation is unknown. The natural genetic transformation characteristic of pneumococci, the arrangement of the cap locus and the abundance of transposable elements at this locus favor the genetic variability of the capsule in this microorganism. These well-documented observations together with the finding that some genes located outside the cap cluster may also participate in capsule formation increase the complexity of pneumococcal infection control.     

Capsulation gene loss and 'rescue' mutations during the Cap+ to Cap- transition in Haemophilus influenzae type b.

Genes for Haemophilus influenzae type b capsule expression are duplicated to form a potentially unstable structure, cap, of directly-repeated chromosomal regions of approximately 17 kb. Capsule-deficient mutants arise in a two-stage process, initiated by rec-dependent reduction of this region from two copies to one. This recombinational event is usually lethal, only about 1/200 surviving to form slow-growing colonies of organisms that continue to synthesize polysaccharide but are defective in its export. A variety of secondary 'rescue' mutations within cap can occur to reduce polysaccharide synthesis and restore normal organism appearance and colony morphology.     

The Haemophilus influenzae Type b hcsA and hcsB gene products facilitate transport of capsular polysaccharide across the outer membrane and are essential for virulence

Haemophilus influenzae type b is a common cause of invasive bacterial disease, especially among children in underdeveloped countries. The type b polysaccharide capsule is a polymer of ribose and ribitol-5-phosphate and is a critical determinant of virulence. Expression of the type b capsule is dependent upon the cap b locus, which consists of three functionally distinct regions, designated regions 1 to 3. Region 3 contains the hcsA and hcsB genes, which share significant homology with genes that have been implicated in encapsulation in other pathogenic bacteria but have unclear functions. In this study, we inactivated hcsA alone, hcsB alone, and both hcsA and hcsB together and examined the effects of these mutations on polysaccharide transport and bacterial virulence properties. Inactivation of hcsA alone resulted in accumulation of polysaccharide in the periplasm and a partial decrease in surface-associated polysaccharide, whereas inactivation of hcsB alone or of both hcsA and hcsB together resulted in accumulation of polysaccharide in the periplasm and complete loss of surface-associated polysaccharide. All mutations eliminated serum resistance and abrogated bacteremia and mortality in neonatal rats. These results indicate that the hcsA and hcsB gene products have complementary functions involved in the transport of polysaccharide across the outer membrane and are essential for virulence.     

Common organization of chromosomal loci for production of different capsular polysaccharides in Haemophilus influenzae.

Cloned Haemophilus influenzae type b capsulation genes were used as hybridization probes to isolate DNA from the capsulation loci (cap) of other serotypes of H. influenzae. Mapping of the resulting clones and Southern hybridization analysis of chromosomal DNAs from type a, b, c, and d strains showed that in each strain cap was organized in the same way: a central DNA segment specific to each serotype flanked by DNA segments of common structure. We infer that enzymes necessary for the synthesis of specific capsular polysaccharide are encoded in the central segment of cap, while proteins involved in a more general way in the process of capsulation are encoded in the flanking segments. Studies of the function of the DNA in one of these non-serotype-specific flanking segments (J. S. Kroll, I. Hopkins, and E. R. Moxon, Cell 53:347-356, 1988) have previously identified a gene encoding a protein necessary for polysaccharide export, an event now deduced to proceed by a mechanism independent of the nature of the disaccharide subunit in the polysaccharide. The near-total duplication of cap that has been found in most type b strains was not found at the analogous locus in the other serotypes. This reinforces our previous hypothesis, based on study of type b strains alone, that while such a duplication is unnecessary for capsulation, it confers some unexplained survival advantage on the widely prevalent strains with this clinically important serotype.     

Translocation of capsular polysaccharides in pathogenic strains of Escherichia coli requires a 60-kilodalton periplasmic protein.

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.     

Vertebrate histone genes: nucleotide sequence of a chicken H2A gene and regulatory flanking sequences.

The DNA sequence of a chicken genomal fragment containing a histone H2A gene has been determined. It contains extensive 5' and 3' flanking regions and encodes a protein identical in sequence to the histone H2A protein isolated from chicken erythrocytes. In the 5' flanking region, a possible "TATA box" and three possible "cap sites" can be recognised upstream from the initiation codon. To the 5' side of the "TATA box" is found an unusual sequence of 21 A's interrupted by a central G residue. It occupies the same relative position as the P. miliaris H2A gene-specific 5' dyad symmetry sequence and the "CCAAT box" seen in other eukaryotic polymerase II genes but is clearly different from both. A significant feature of the 3' non-coding region is the presence of a 23 base-pair sequence that is nearly identical to a conserved region found in sea urchin histone genes. The coding region is extremely GC rich, with strong selection for these bases in the third position of codons. Not a single coding triplet ends in U. No intervening sequences were found in this gene.     

Comparative biochemistry of chondroitin sulphate–proteins of cartilage and notochord

Fragments that consisted mainly of two polysaccharide chains joined by a short polypeptide bridge (doublets) were prepared from chondroitin sulphate-proteins of lamprey, sturgeon, elasmobranch and ox connective tissues after hydrolysis with trypsin and chymotrypsin. Consideration of molecular parameters, compositions and behaviour on gel electrophoresis and density-gradient fractionation leads to a proposed parent structure for chondroitin sulphate-proteins. A single polypeptide chain of about 2000 amino acid residues contains alternating short and long repeating sequences. A short sequence consists of less than 10 amino acid residues with one N-terminal and one C-terminal serine residue, each of which carries a polysaccharide chain linked glycosidically to its hydroxyl group. This structure constitutes the doublet subunit. Some variation is introduced when the doublet subunit carries only a single polysaccharide chain. The long sequence contains about 35 amino acid residues and is subject to cleavage by trypsin and chymotrypsin. The main polypeptide is probably homologous in the vertebrate sub-phylum with strong conservation of structure suggested for the short sequence. However, polymorphism of polypeptide structures cannot be excluded.     

Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1.

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.     

Interstrain Variation of the Polysaccharide B Biosynthesis Locus of Bacteroides fragilis: Characterization of the Region from Strain 638R

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.