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Pneumococci are described as alpha-haemolytic but under certain circumstances they produce zones of beta-haemolysis on blood-containing medium. This observation was investigated using wild type strains and a genetically-modified strain unable to produce the haemolytic toxin, pneumolysin. beta-haemolysis was produced by all pneumococci tested. It was not inhibited by anti-pneumolysin antibody but could be inactivated by cholesterol. These data confirm that pneumococci elaborate a second haemolysin, distinct from pneumolysin.

Mice were infected intranasally with a serotype 2 pneumococcus, a pneumolysin-negative derivative (PLN-A), or an autolysin-negative derivative (AL-2). Numbers of wild type pneumococci were seen in the lung from approximately 12 h after infection and were first detected in the blood around this time. Immunofluorescent staining of lung sections showed that pneumolysin was produced in vivo. Pneumococcal infection resulted in alteration of the composition of the blood but not the bone marrow. Some of the hematologic changes did not occur after PLN-A. PLN-A had a slower growth rate in the lung and bacteremia was delayed. AL-2 was rapidly cleared from the lungs and was not detected in the blood. These events paralleled the pattern of histology in the lung, with the severity of inflammation reduced with PLN-A and no inflammation or hematologic changes with AL-2.

Our previous work has demonstrated the importance of pneumolysin in the virulence of S. pneumoniae in a rabbit intracorneal model. This was accomplished by showing that deletion of the gene encoding pneumolysin resulted in reduced virulence, whereas restoration of the wild-type gene resulted in restoration of the virulent phenotype. To assess the importance of a particular domain in the pneumolysin molecule, we have now constructed a strain which produces a pneumolysin molecule which is hemolytic but which bears a site-specific mutation in the domain known to be associated with the complement-activating properties of this molecule. Comparison of the virulence of this strain with that of a strain bearing the wild-type gene showed statistically significantly lower total slit lamp examination (SLE) scores at 12, 18, 24, and 36 h (particularly with respect to fibrin formation), but no difference at 48 h. Determination of colony forming units (CFU) in eyes infected with the two strains showed approximately 10(6) bacteria per cornea until 36 h. Between 36 and 48 h, the bacteria were almost completely cleared with very few bacteria recoverable at the later time point. The loss of virulence observed with this mutation in the complement-activation domain of pneumolysin, though less than that observed with the gene deletion mutant, suggests that complement activation by pneumolysin has a significant role in the pathology observed in this model of corneal infection.

A gene from Streptococcus pneumoniae (nanA), with features entirely consistent with a neuraminidase gene, has been sequenced. High levels of neuraminidase activity were obtained after cloning of this gene, without flanking sequences, into a high-expression vector. RNA hybridization studies have shown that the gene is transcribed by a virulent pneumococcus strain. The predicted molecular weight of the protein and certain amino acid sequences are typical of other neuraminidases. NanA contains the four copies of the sequence SXDXGXTW that is present in all the bacterial neuraminidases previously described. Kyte and Doolittle analysis showed that NanA is a hydrophilic protein with hydrophobic domains at the N terminus and the C terminus. A putative signal peptide was found in the N terminus of this protein, indicating that the protein is exported from the pneumococcus. The C terminus has the features of the anchor motif found in other surface proteins from gram-positive bacteria. Electron microscopy studies showed the presence of neuraminidase associated with the cell surface of the pneumococcus.

Cell binding of pneumolysin to target cells is an important step in the lysis of cells by this toxin. We sought to locate the cell-binding region of pneumolysin. Deletion of the six C-terminal amino acids decreased cell-binding activity by 98%. Furthermore, mutagenesis of an amino acid near the C-terminus decreased the cell-binding activity of full-length pneumolysin by 90%. The C-terminus of pneumolysin has an important role in cell-binding activity.

Aims: To develop a system of species specific polymerase chain reaction (PCR) and DNA hybridisation based on 16s ribosomal RNA sequences for the identification of Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia in sputum from children with cystic fibrosis., Methods: Most of the 16s rRNA sequences from strains of Ps aeruginosa, Ps (Burkholderia) cepacia, and Ps putida were determined. PCR primers and DNA probes were synthesised from suitable sequences and then evaluated on bacterial cultures and sputum samples., Results: About 1000 bases of sequence was obtained from strains of Ps aeruginosa, Ps (Burkholderia) cepacia, and Ps putida. PCR of bacterial cultures was species specific, but PCR on sputum resulted in some non-specific amplification products. The subsequent hybridisation reaction was species specific., Conclusion: A species specific system of PCR and DNA hybridisation based on 16s rRNA sequences is applicable in clinical practice, and may aid the early diagnosis of respiratory tract infection with small numbers of Ps aeruginosa and Ps (Burkholderia) cepacia in patients with cystic fibrosis.

The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters.

The gene cluster of the capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three regions responsible for polymerization and surface expression have been defined (I.S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1330, 1988). Region 1 has now been sequenced, and five open reading frames (kpsEDUCS) have been defined (C. Pazzani, C. Rosenow, G. J. Boulnois, D. Bronner, K. Jann, and I. S. Roberts, J. Bacteriol. 175:5978-5983, 1993). In this study, we characterized region 1 mutants by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of their K5 polysaccharides. Certain mutations within region 1 not only effected polysaccharide transport (lack of region 1 gene products) but also impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain length. KDO and phosphatidic acid (phosphatidyl-KDO) substitution was found with extracellular and periplasmic polysaccharide and not with cytoplasmic polysaccharide. This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO synthetase) showed that CMP-KDO is engaged not in initiation of polymerization but in translocation of the polysaccharide.

Two families of Escherichia coli capsules, termed groups I and II, have been defined previously on the basis of a number of biochemical and genetic criteria. Recently, a third group of capsules, termed I/II has been suggested on the basis of chemical structure and mode of expression. In this paper, we show that group I capsule-producing strains lack the serA-linked group II capsule genes. In addition, group I/II capsule-producing strains lack the group II capsule genes despite the former genes also mapping near to serA. Therefore, the genetic data presented in this paper support the existence of three groups of capsule gene clusters, two of which are linked to serA. Sequences flanking the K4 capsule genes were found in the chromosome of all E. coli strains examined and were sometimes present in multiple copies at different loci, indicating that this chromosomal region is highly polymorphic.

The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.

The cytolytic toxin, pneumolysin, from the gram positive bacterium, Streptococcus pneumoniae, when perfused through the scala tympani of the guinea pig cochlea reduced the amplitude of both the compound action potential and the cochlear microphonic potential. When the surface of the organ of Corti was examined by scanning electron microscopy, both inner and outer hair cells and supporting cells were found to be damaged. Inner hair cells and outer hair cells of row 3 were the most susceptible to damage by pneumolysin, followed by row 2 and then by row 1 of the outer hair cells. Damage to hair cells included disruption and splaying of stereocilia, loss of stereocilia and complete dissolution of hair bundles. Apical surfaces of hair cells and supporting cells were torn, pitted and cratered with shrinkage and tearing of cell boundaries. Within the dose range perfused (0.05-1 micrograms/microliters in a 10 microliters aliquot), the magnitude of the physiological and anatomical lesions was concentration dependent. The cytotoxic effects of pneumolysin reported here may be clinically significant factors in deafness caused by meningitis and otitis media in humans.

For many years the virulence of Streptococcus pneumoniae has largely been attributed to its antiphagocytic polysaccharide capsule. Recent evidence, however, indicates that certain pneumococcal proteins play an important part in the pathogenesis of disease, either as mediators of inflammation or by directly attacking host tissues. Pneumococci carrying defined mutations in the genes encoding any one of at least three pneumococcal proteins (the toxin pneumolysin, the major pneumococcal autolysin, and pneumococcal surface protein A) have significantly reduced virulence. Pneumococcal hydrolytic enzymes, such as neuraminidase, hyaluronidase, and IgA1 protease may also contribute to colonization and/or invasion of the host. Several of these proteins (or their detoxified derivatives) are protective immunogens in animal models and therefore warrant consideration for inclusion in human antipneumococcal vaccine formulations.

Immunization with Freund's adjuvant and pneumolysin and stimulation with Freund's adjuvant alone both reduced the severity of the pneumonia caused by injections of bacteria into the apical lobe bronchi of rats. Neither protocol influenced the incidence of pneumococcal bacteremia. Illness sufficiently severe to require sacrifice was delayed from 2.8 days in nonimmunized animals to 5.7 days in those immunized with Freund's adjuvant and pneumolysin (P < 0.05) and 4.5 days in those stimulated with Freund's adjuvant alone (P, not significant).

Pneumolysin is a thiol-activated, membrane-damaging, multifunctional toxin and a known virulence factor of Streptococcus pneumoniae. The toxin can interfere with the functioning of both cellular and soluble components of the human immune system which protects against pneumococcal infection. Different amino acids within the toxin which are important in promoting oligomerization of the toxin in membranes and for the generation of functional lesions have been identified by site-directed mutagenesis. Pneumolysin can also activate the classical pathway of complement, and this appears to involve antibody binding (via Fc) by a region of the toxin homologous to C-reactive protein, a human acute-phase protein also capable of classical pathway activation and implicated in host defence against pneumococcal infection.

Streptococcus pneumoniae infections are common, but how they cause host tissue injury and death is incompletely understood. Immunization with pneumolysin, a thiol-activated toxin produced by the pneumococcus, partially protects animals during subsequent infection. The mechanism by which pneumolysin contributes to disease is not known. The aim of the present investigation was to determine the histologic changes induced by recombinant pneumolysin in the rat lung and to compare them with the changes induced by live organisms. Injection of either toxin (200 or 800 ng) or bacteria into the apical lobe bronchus was associated with the development of a severe lobar pneumonia restricted to the apical lobe. The changes induced by the toxin were greater at the higher concentration, and changes were most severe in those animals in which there was partial ligation of the apical lobe bronchus. The pneumonitis was less severe following injection of a modified toxin with decreased hemolytic activity, generated by site-directed mutagenesis of the cloned pneumolysin gene, indicating that this property of the toxin was important in generating pulmonary inflammation. There was still considerable pneumonitis after injection of a modified toxin with decreased capacity to activate complement.

A gene from Streptococcus pneumoniae was cloned in lambda EMBL301 and then expressed in Escherichia coli, which cleaved the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid. The cloned gene therefore encodes an enzyme with neuraminidase activity. On the basis of restriction mapping and DNA hybridization studies, this gene could be distinguished from another pneumococcal neuraminidase gene cloned previously (A. M. Berry, J. C. Paton, E. M. Glare, D. Hansman, and D. E. A. Catcheside, Gene 71:299-305, 1988). Both neuraminidase genes were found in each of five isolates, covering at least three serotypes, of pneumococci tested.

Pneumolysin, a membrane-damaging toxin, is known to activate the classical complement pathway. We have shown that 1 microgram ml-1 of pneumolysin can activate complement, which is a much lower level than observed previously. We have identified two distinct regions of pneumolysin which show homology with a contiguous sequence within acute-phase proteins, including human C-reactive protein (CRP). Site-directed mutagenesis of the pneumolysin gene was used to change residues common to pneumolysin and CRP. Some of the modified toxins had a reduced ability both to activate complement and bind antibody. We suggest that the ability of pneumolysin to activate complement is related to its ability to bind the Fc portion of immunoglobulin G.

As part of an ongoing study concerned with improving human vaccines against Streptococcus pneumoniae, the genes for two defined pneumolysin (PL) toxoids (pneumolysoids), Pd-A (PL with a Cys----Gly substitution at amino acid 428) and Pd-B (PL with a Trp----Phe substitution at position 433), were inserted into the high-expression vector pKK233-2 in Escherichia coli and the pneumolysoids were purified. Groups of mice which had been immunized with either Pd-A, Pd-B, or native PL purified from S. pneumoniae were then challenged either intranasally or intraperitoneally with virulent pneumococci. Mice in all immunized groups survived significantly longer than sham-immunized controls. Both pneumolysoids were more effective than PL as protective immunogens. Pneumolysoid Pd-B was conjugated covalently with pneumococcal type 19F capsular polysaccharide (19F PS), and the immunogenicities of both the protein and the PS moieties of the conjugate in mice were determined. Significant anti-PL titers were obtained, and the immunogenicity of the 19F PS moiety was markedly enhanced compared with that of unconjugated PS. Conjugation also appears to have converted the 19F PS into an antigen capable of inducing a booster effect. These results support the notion that the efficacy of human, PS-based antipneumococcal vaccines might be improved by supplementation with pneumolysoid in the form of a covalent pneumolysoid-PS conjugate.

A 1.5 kb EcoRI-BamHI restriction fragment from Mycobacterium tuberculosis was found to hybridize specifically with genomic DNA from M. tuberculosis-complex organisms. Primers were designed from the terminal sequences of this fragment and used to amplify uniquely M. tuberculosis-group DNA in a polymerase chain reaction. It is suggested that a combination of these primers and probe will prove a useful tool for the early diagnosis of tuberculous infections.

The gene encoding the Candida albicans aspartate proteinase that is secreted by cells grown in protein-containing media was cloned from a C. albicans genomic bank. The base sequence of the insert shows a 1173 bp open-reading frame and indicates an amino acid sequence typical of aspartate proteinases, with amino acid sequence homology to other enzymes of this class and a putative signal peptide consisting of 50 amino acids upstream of the active enzyme.

The complete nucleotide sequence has been determined of a region of the Escherichia coli K5 antigen gene cluster postulated to encode functions for the translocation of capsular polysaccharide across the inner membrane. This revealed two genes, designated kpsM and kpsT, organized in a single transcriptional unit. Analysis of the predicted amino acid sequence of the KpsM and KpsT proteins indicates that they may function as dual components in a polysaccharide export system analogous to the periplasmic binding protein-dependent transport systems of Gram-negative bacteria. We propose that the KpsT protein acts as an energizer, coupling ATP hydrolysis to the transport process mediated by the KpsM protein. Extensive sequence homology between the KpsM and KpsT proteins and the products of the bexB and bexA genes present in the capsulation (cap) locus of Haemophilus influenzae, indicates that a common mechanism for the export of polysaccharide across the inner membrane may exist in these two micro-organisms.

Streptococcus pneumoniae culture filtrates and pneumolysin both slow human ciliary beating and damage respiratory epithelium in vitro. A polyclonal pneumolysin antibody bound to sepharose beads removed pneumolysin from culture filtrates and showed that pneumolysin alone was responsible for the effects on epithelium. In a 48-h organ culture pneumolysin caused ciliary slowing and epithelial disruption in a dose-dependent manner down to 5 ng/ml. Comparison of the ciliary slowing activity and pneumolysin concentration in filtrates in a continuous broth culture showed a maximal effect at 16 h (pneumolysin 7.5 micrograms/ml). Later the activity decreased while the pneumolysin concentration increased (8.8 micrograms/ml). This loss of activity was prevented by neutralisation of the acid pH of the culture medium. Eight different culture filtrates produced significant (P less than 0.05) ciliary slowing which correlated (r = 0.95) with simultaneously measured haemolytic (pneumolysin) activity. Substitution of tryptophan (position 433) by phenylalanine reduced the haemolytic and ciliary slowing activity of pneumolysin, but did not affect its ability to activate complement. There was no correlation between the ciliary slowing produced by the culture filtrate and that produced by the autolysate of a particular strain, nor between ciliary slowing and the extent of autolysis or the serotype of the strain.

Many Escherichia coli K12 strains undergo switching between two forms which differ in a number of surface properties including colony morphology and the ability to auto-aggregate. This paper describes the identification of a gene which appears to play a part in controlling this switching phenomenon. This gene has been designated mor and is located at 89 minutes on the E. coli chromosome map between the argECBH operon and the trmA gene. By manipulation of this gene it is possible to overcome the switching of surface properties and fix a strain in one form or the other. The mor gene has been cloned and its DNA sequence determined. The putative protein sequence shows a high level of homology with four regulatory genes, the ilvY, cysB and lysR genes from E. coli and the metR gene from Salmonella typhimurium. It has also been shown that the mor gene is autoregulated at the transcriptional level.

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.

The sog gene of the IncI alpha group plasmid ColIb is known to encode a DNA primase that can substitute for defective host primase in dnaG mutants of Escherichia coli during discontinuous DNA replication. The biological significance of this enzyme was investigated by using sog mutants, constructed from a derivative of ColIb by in vivo recombination of previously defined mutations in a cloned sog gene. The resultant Sog- plasmids failed to specify detectable primase activity and were unable to suppress a dnaG lesion. These mutants were maintained stably in E. coli, implying that the enzyme is not involved in vegetative replication of ColIb. However, the Sog- plasmids were partially transfer deficient in E. coli and Salmonella typhimurium matings, consistent with the hypothesis that the normal physiological role of this enzyme is in conjugation. This was confirmed by measurements of conjugal DNA synthesis. Studies of recipient cells have indicated that plasmid primase is required to initiate efficient synthesis of DNA complementary to the transferred strand, with the protein being supplied by the donor parent and probably transmitted between the mating cells. Primase specified by the dnaG gene of the recipient can substitute partially for the mutant enzyme, thus providing an explanation for the partial transfer proficiency of the mutant plasmids. Conjugal DNA synthesis in dnaB donor cells was deficient in the absence of plasmid primase, implying that the enzyme also initiates synthesis of DNA to replace the transferred material.

The complete nucleotide sequence of a cloned streptolysin O (SLO) gene and the amino acid sequence of SLO, predicted from the DNA sequence, are reported. SLO contains a single cysteine residue located close to the C terminus of the molecule and shares extensive structural homologies with other thiol-activated toxins, which allow us to predict functionally important features.

A DNA primase encoded by an IncI alpha plasmid promotes efficient DNA replication in a primase-defective mutant of Escherichia coli. This finding implies that the plasmid enzyme can prime discontinuous DNA synthesis of the bacterial chromosome. The plasmid gene encodes two large, antigenically related proteins which differ from E. coli primase.

Plasmid ColIb-P9 of the I alpha incompatibility group is known to encode a DNA primase that acts in the conjugal transfer of the plasmid and can substitute for mutant dnaG gene product in vegetative replication of the Escherichia coli chromosome. The relevant genetic determinant (sog) has previously been cloned into a small multicopy vector plasmid. Prototype IncB plasmid R16 also suppresses host dnaG mutations. The equivalent gene(s) (pri) of R16 was cloned into plasmid pBR325 and shown by filter hybridization studies to be different from the ColI primase gene(s). The cloned fragment carrying the pri determinant encoded an enzyme which could initiate DNA synthesis in vitro on single-stranded phage M13 DNA template, but which was antigenically distinct from ColI primase. We used the cloned primase genes as probes in colony hybridization screening of strains carrying plasmids of the IncI complex and IncB group. Plasmids R64, R144 (I alpha), R621a (I gamma), RIP72, and R864a (B) contained nucleotide sequences homologous with the cloned ColI sog genes. Plasmids R805 (I xi), R724, (I delta), TP125, and PLG101 (B) showed sequence homology with the R16 pri determinant.

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.

Genes coding for production of the K1 polysaccharide capsule of Escherichia coli have been cloned. Complementation, insertion, and deletion analyses were used to localize the K1 genes and demonstrated that a minimum of 9 kilobases of DNA split into at least two gene blocks is involved in synthesis and assembly of the capsule. One of the gene blocks is responsible for biosynthesis of the polysaccharide, and the other is responsible for extracellular appearance of capsular material. Using cloned K1 genes as probes in Southern blot experiments, we detected homology to DNA from strains of E. coli capsular types K92, K7, and K100. In contrast, no homology was apparent between K1 genes and DNA from meningococcus group B, although the K1 and group B capsules are chemically and immunologically identical.

DNA restriction enzyme fragments have been used to programme the synthesis of polypeptides in an in vitro system without apparent loss in fidelity compared with supercoiled templates. The system is extremely sensitive, less than 1 microgram of DNA can be used to direct the synthesis of 35S-labelled polypeptides of sufficiently high specific activity such that products can be identified by SDS-PAGE after a few hours autoradiography. The ability to analyse fragments can be used to readily assign specific proteins to small regions of the coding template, to identify cloned gene products distinct from those of the vector, and to identify cloned genes expressed from their own promoters. The in vitro system can be used successfully with bacterial DNA from other species and efficient extracts can be prepared from any E. coli K-12 strain, which should greatly facilitate the purification of factors controlling the expression of specific genes by complementation assay.

A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.

The amount of ColI DNA transferred between mating cells of Escherichia coli K-12 increased about fourfold when rifampin-resistant donors were mated with sensitive recipients in the presence of the drug. Conjugational synthesis of ColI in dnaB recipients, shown primarily to reflect conversion of the transferred DNA into double-stranded material, was also enhanced when the recipients were treated with either rifampin or streptomycin. It is suggested that the amount of ColI transfer is normally limited by the synthesis of one or more proteins in the newly infected recipients. The protein is thought to be plasmid-specified because rifampin also quadrupled transfer to UV-irradiated recipients which were deficient in the transcription of the resident DNA. Successive strands of ColI appear to be transferred discontinuously, because the transferred DNA accumulated in normal and rifampin-treated recipients in the form of circular and linear monomeric units. Although rifampin treatment of recipients also increased transfer of a second Ialpha plasmid, R144drd-3, by about four times, the drug failed to cause a substantial increase of Flac transfer in comparable matings.

The gene for pneumolysin, the thiol-activated toxin from Streptococcus pneumoniae, has been expressed in Escherichia coli. The recombinant protein has been purified using a rapid, high yield, purification procedure and has been shown to be identical with respect to N-terminal amino-acid sequence, specific activity, effect on human polymorphonuclear phagocytes and effect on human complement to the native toxin purified from the pneumococcus. This provides a large enough source of material to begin investigation of pneumolysin as a candidate for inclusion in a pneumococcal vaccine.

A system for the production of monoclonal antibodies, particularly of the IgG type, against weakly immunogenic bacterial polysaccharide antigens is described. This system, which is based on the autoimmune NZB mouse strain, has been used to produce a monoclonal IgG2a antibody against the meningococcus group B and Escherichia coli K1 polysaccharides, identical homopolymers of alpha (2----8)-linked units of N-acetylneuraminic acid that are extremely poor immunogens. Comparison of the humoral immune responses of normal BALB/c mice and autoimmune NZB mice to hyperimmunization with group A, B, and C meningococci showed that, although both strains mounted a weak meningococcal B polysaccharide-specific IgM response, only the NZB strain mounted an IgG response. Similarly, NZB mice mounted a stronger IgG response to the more immunogenic group C meningococcal polysaccharide than did BALB/c mice, although this difference was less pronounced than that observed with meningococcal B polysaccharide. No difference between the two strains of mice was demonstrable with the strongly antigenic group A meningococcal polysaccharide. These results indicate that the NZB system may be generally useful for the production of monoclonal antibodies against weakly antigenic bacterial determinants.

The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.

The effects of rifampin and chloramphenicol on the transfer of ColIdrd-1 have been examined to determined whether transfer requires the synthesis of an untranslated species of ribonucleic acid (RNA), as proposed in models for the transfer of another IncIalpha plasmid, R64drd-11. When RNA synthesis was inhibited throughout mating by rifampin, ColI transfer between dna+ bacteria occurred at the normal rate for about 10 min and then stopped abruptly. Conjugational deoxyribonucleic acid (DNA) synthesis in dnaB mutants indicates that plasmid DNA was made in the rifampin-treated donors to replace the transferred material but the DNA tended to be unstable. In the presence of chloramphenicol, transfer of ColI gradually diminished over a longer period. Rifampin, but not chloramphenicol, was found to have unpredicted effects on chromosomal DNA metabolism in unmated dna+ and dnaB bacteria when they harbor any of three IncIalpha plasmids (ColIdrd-1, R144drd-3, and R64drd-11). Replication of the bacterial chromosome in such cells stopped abruptly about 15 min after the addition of rifampin, and at 41 degrees C, but not 37 degrees C, this was followed by extensive DNA breakdown. These findings suggest that curtailment of IncIalpha plasmid transfer by the drug results from a general disruption of DNA metabolism rather than from inhibition of a species of RNA essential for transfer.

The complete nucleotide sequence of a 2,971 base pair EcoRI fragment carrying the structural gene for colicin Ib has been determined. The length of the gene is 1,881 nucleotides which is predicted to produce a protein of 626 amino acids and of molecular weight 71,364. The structural gene is flanked by likely promoter and terminator signals and in between the promoter and the ribosome binding site is an inverted repeat sequence which resembles other sequences known to bind the LexA protein. Further analysis of the 5' flanking sequences revealed a second region which may act either as a second LexA binding site and/or in the binding of cyclic AMP receptor protein. Comparison of the predicted amino acid sequence of colicin Ib with that of colicins A and E1 reveals localised homology. The implications of these similarities in the proteins and of regulation of the colicin Ib structural gene are discussed.

A 2.2 kilobase (kb) fragment of ColIdrd-1 cloned in pBR325 causes phage T5 and BF23 infections of Escherichia coli K-12 to abort. This abortive infection is associated with leakage of beta-galactosidase from the cell. A recombinant plasmid containing a 2.8 kb fragment of ColIdrd-1 encodes colicin Ib but fails to cause abortive infection. Tn5 and Tn10 insertions into ColIdrd-1 that abolish colicin Ib production have no effect on the abortive infection phenotype. These findings are inconsistent with a previously proposed role for colicin Ib in causing phage infections to abort.

Synthesis of DNA complementary to the transferred strand of an IncI alpha plasmid has been shown previously to require DNA polymerase III. The possible involvement of the two defined priming proteins of Escherichia coli K12, RNA polymerase and primase, in initiating this conjugal DNA synthesis had been examined. Primase was inactivated using temperature-sensitive dnaG3 mutants and RNA polymerase was inhibited using rifampicin. When these two proteins were simultaneously inactivated in both parental strains, the average recipient synthesised at least one single-stranded equivalent of R144drd-3 before the rifampicin-treated donors lost the ability to transmit DNA. It is proposed that the product of a plasmid transfer gene is responsible for initiating this DNA synthesis in recipients. The results imply that this protein is supplied by the donors.

The role of the single cysteine residue in the activity of the thiol-activated toxin pneumolysin was investigated using oligonucleotide-mediated, site-directed mutagenesis. Three modified toxins in which the cysteine residue was changed to an alanine, a serine, or a glycine residue were purified to homogeneity and examined for activity. The Cys-428----Ala modified toxin was indistinguishable from the wild-type recombinant toxin in terms of hemolytic activity and lytic and inhibitory effects on human polymorphonuclear leukocytes (PMN), indicating that the cysteine residue is not essential for toxin activity. The Cys-428----Ser and Cys-429----Gly modified toxins had reduced activity on erythrocytes and polymorphonuclear leukocytes, being 6 and 20 times less active than the wild type, respectively. However, all the modified toxins formed oligomers in erythrocyte membranes to the same extent as the wild-type recombinant toxin. This suggests that the cysteine residue at position 428 is involved in neither the binding of toxin to membranes nor its insertion into the membrane, and also that the formation of oligomers is not by itself sufficient for toxin activity.

The sog gene of the large plasmid ColIdrd-1 has previously been shown to encode a DNA primase and a smaller antigenically related polypeptide. Genesis of these two products has been examined using Sog+ recombinant plasmids. Effects of amber mutations, isolated after in vitro mutagenesis, and deletions into or within sog suggest that the smaller polypeptide is a separate translation product which is encoded by DNA specifying the C-terminal region of the larger protein. Under control of the lac promotor, synthesis of both polypeptides is reduced when transcription is repressed. These findings imply that transcription of sog yields a single transcript which is translated from two initiation sites.