Cloning and molecular characterization of Cu,Zn superoxide dismutase from Actinobacillus pleuropneumoniae.

Copper-zinc superoxide dismutases (Cu,Zn SODs), until recently considered very unusual in bacteria, are now being found in a wide range of gram-negative bacterial species. Here we report the cloning and characterization of sodC, encoding Cu,Zn SOD in Actinobacillus pleuropneumoniae, a major pathogen of pigs and the causative organism of porcine pleuropneumonia. sodC was shown to lie on a monocistronic operon, at the chromosomal locus between the genes asd (encoding aspartate semialdehyde dehydrogenase) and recF. The primary gene product was shown to have an N-terminal peptide extension functioning as a leader peptide, so that the mature Actinobacillus enzyme, like other bacterial examples, is directed to the periplasm, where it is appropriately located to dismutate exogenously generated superoxide. While the role of these secreted bacterial SODs is unknown, we speculate that in A. pleuropneumoniae the enzyme may confer survival advantage by accelerating dismutation of superoxide derived from neutrophils, a central host defense response in the course of porcine infection.

Region II of the Haemophilus influenzae type be capsulation locus is involved in serotype-specific polysaccharide synthesis.

The central (serotype-specific) Region II of the Haemophilus influenzae Type b capsulation locus cap is 8.3 kb long and contains a cluster of four genes. We show that these genes, designated orf1 to orf4, are involved in the biosynthetic steps required for the formation of the Type b capsular polysaccharide and that orf1 probably encodes a CDP-ribitolpyrophosphorylase. We present evidence that growth of polysaccharide chains takes place through the alternating addition of single sugar nucleotides.

Natural genetic transfer of a putative virulence-enhancing mutation to Haemophilus influenzae type a.

Haemophilus influenzae strains of serotype a very rarely cause life-threatening infections. Examination of strains from the Gambia, West Africa, that caused septicemia, meningitis, or both revealed that a clone has emerged that carries a DNA deletion previously identified only in type b strains that is hypothesized to contribute to the special virulence of that serotype. This clone appears to have arisen by transfer of DNA between type a and type b strains, a transformation event that has happened more than once, as shown by the discovery Kenya, East Africa, of a clonally distinct type a strain bearing the identical deletion. The implications for the emergence of clinically important non-type b strains of H. influenzae are obvious.

Molecular and genetic characterization of superoxide dismutase in Haemophilus influenzae type b.

Oxygen free radicals present a serious potential threat to microbial survival, through their ability to inflict indiscriminate damage on proteins and DNA. Superoxide dismutase (SOD, EC 1.15.1.1), among other oxygen-metabolizing enzymes, is essential to prevent these toxic molecules from accumulating in the bacterial cytosol during aerobic metabolism. The gene sodA, encoding manganese-containing SOD ([Mn]-SOD), has been cloned from a virulent strain of Haemophilus influenzae type b using degenerate oligonucleotides encoding regions of the gene conserved across different bacterial species. The gene product has been identified as [MN]-SOD by its similarity at key amino acid residues to known examples of the enzyme, by expression of enzymatically active protein from cloned DNA expressed in Escherichia coli, and by demonstration that an in-frame deletion in the gene abolishes this activity. In contrast to the situation in E. coli, this [Mn]-SOD is the only active SOD detected in H. influenzae. In further contrast to E. coli, [Mn]-SOD gene expression in H. influenzae has been found to be only partially repressed under anaerobic conditions. When expressed in E. coli the gene is regulated by Fur and Fnr, and the promoter region, identified experimentally, has been found to contain nucleotide sequence motifs similar to the Fur- and Fnr-binding sequences of E. coli, suggesting the involvement of analogues of these aerobiosis-responsive activators in H. influenzae gene expression.

An ancestral mutation enhancing the fitness and increasing the virulence of Haemophilus influenzae type b.

Capsulate Haemophilus influenzae is a major cause of septicemia and meningitis in children. Virtually all invasive strains have a type b polysaccharide capsule and belong to division I of the two phylogenetic divisions into which the H. influenzae population segregates. In this study 18 isolates, collected from all over the world and representative of the whole population of division I type b strains, have been shown to be the progeny of a common ancestor in which a founder mutation occurred, the deletion of one of two copies of the gene bexA. BexA is essential for exporting capsular polysaccharide to the bacterial surface, and a single copy of its gene lies at the center of an otherwise duplicated capsulation locus. Deletion of the other copy has had the paradoxical effect of enhancing pathogenicity, through increasing the potential for amplification of capsule biosynthetic genes and capsule production.

Palindromic Haemophilus DNA uptake sequences in presumed transcriptional terminators from H. influenzae and H. parainfluenzae.

We have found palindromic pairs of near matches to the 11-bp Haemophilus DNA uptake motif shortly after the stop codons of three Haemophilus genes. Short runs of thymidylate residues follow the stem-loop structures thus defined. This organization suggests that, in H. influenzae, the uptake motif may be preferentially incorporated into gene termination signals, as has been proposed for Neisseria gonorrhoeae.

Copper-zinc superoxide dismutase in Haemophilus species.

Copper-zinc superoxide dismutases ([Cu,Zn]-SODs) are ubiquitous in eukaryotes but have rarely been found in prokaryotes. A gene for [Cu,Zn]-SOD (sodC) has recently been cloned from Haemophilus influenzae type b and H. parainfluenzae, so other Haemophilus and related species were screened for the presence of [Cu,Zn]-SODs by visualization of bands of SOD activity in non-denaturing polyacrylamide gels and by gene probing. Strains of H. aphrophilus, H. paraphrophilus, H. haemolyticus, H. paraphrohaemolyticus, some non-typable H. influenzae, H. haemoglobinophilus (canis) and H. parasuis were all found to have [Cu,Zn]-SOD activity (inhibited by 2 mM-cyanide) in polyacrylamide gels. In a Southern blot analysis, DNA from H. aphrophilus, H. paraphrophilus, H. haemolyticus and [Cu,Zn]-SOD-containing non-typable H. influenzae--but not the other species--hybridized to a 360 nucleotide DNA probe containing the 5'-part of sodC cloned from H. influenzae type b. Bacterial [Cu,Zn]-SODs are more prevalent than has previously been recognized.

Copper-zinc superoxide dismutase of Haemophilus influenzae and H. parainfluenzae.

Copper-zinc superoxide dismutase ([Cu,Zn]-SOD) is widely found in eukaryotes but has only rarely been identified in bacteria. Here we describe sodC, encoding [Cu,Zn]-SOD in Haemophilus influenzae and H. parainfluenzae, frequent colonists and pathogens of the human respiratory tract. In capsulate H. influenzae, sodC was found in only one division of the bacterial population, and although the protein it encoded was clearly [Cu,Zn]-SOD from its deduced sequence, it lacked enzymatic activity. In H. parainfluenzae, in contrast, active enzyme was synthesized which appeared to be secreted beyond the cytoplasm when the gene was expressed in Escherichia coli minicells. The origin of gene transcription differed between the Haemophilus species, but protein synthesis from cloned genes in vitro was comparable. A C-T transition was found in the H. influenzae sequence compared with the H. parainfluenzae sequence, leading to a histidine, known to be crucial in eukaryotic [Cu,Zn]-SOD for copper ion coordination and so for enzymatic activity, to be changed to tyrosine. This is speculated to be the cause of inactivity of the H. influenzae enzyme. Secreted SODs have only been described in a few bacterial species, and this is the first identification of [Cu,Zn]-SOD in a common human upper respiratory tract colonist. The role of secreted bacterial SODs is unknown, and we speculate that in Haemophilus species the enzyme may confer survival advantage by accelerating dismutation of superoxide of environmental origin to hydrogen peroxide, disruptive to the normal mucociliary clearance process in the host.

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.

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.

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.