Amplified expression and large-scale purification of protein L'.

The gene fragment (PPL') encoding the functional unit of peptostreptococcus protein L was isolated using PCR and expressed in E. coli. As the gene fragment lacked its own promoter, the 5' PCR primer was designed to incorporate an Nde1 restriction site (CATATG) into the gene. This enabled the gene to be cloned in frame into an Nde1 restriction site immediately downstream of a trp promoter. To prevent read through, a stop codon was introduced into the 3' primer. Expression of PPL' was up to 27% total cell protein which compares favourably to the 0.1% total soluble cell protein obtained from the original clone of peptostreptococcus. Following a heat step homogeneous PPL' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in yields of 90%.

The functional units of a peptostreptococcal protein L.

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.

Nucleotide sequence of the gene for peptostreptococcal protein L.

A gene bank of Peptostreptococcus magnus DNA was established using an E. coli host-vector system. Western blot analysis identified a clone expressing protein L which bound to the light chain of human immunoglobulins. DNA sequence determination and analysis revealed an open reading frame of 992 amino acids, giving a theoretical secreted protein of 106 kD with a pl of 4.67.

Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR.

Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers. The patterns were compared with the patterns generated from strains of closely related species belonging to the "Bacillus cereus group" of bacteria, including B. cereus, Bacillus thuringiensis, and Bacillus mycoides. All B. anthracis profiles were identical for each of 18 restriction enzymes, each of 10 arbitrary PCR primers, and a repetitive extragenic palindrome-specific PCR primer. The PCR profiles generated with a coliphage M13-based primer exhibited slight pattern variation in a 400- to 500-bp band region. The B. anthracis profiles were unique compared with the profiles of the other species examined. In these other species, strain-to-strain variations were observed. Our results showed that isolates of B. anthracis are almost completely homogeneous, indicating a clonal lineage, and are distinct from other members of the B. cereus group and that B. anthracis, as a species in its own right, may have evolved only relatively recently.

The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis.

Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.

Identification of Bordetella avium using the polymerase chain reaction.

A DNA fragment from Bordetella pertussis, encoding the fim2 fimbrial subunit gene with adjacent sequences, was used as a probe for the detection of homologous sequences in chromosomal DNA of Bordetella avium. A 1.8 kb Sa1I-PstI fragment from the genome of B. avium, which hybridized with the probe, was isolated and sequenced. No fimbrial subunit gene was located on the B. avium DNA fragment. Two regions could be distinguished in the sequence of the fragment. Region 1, which was 80% identical to the sequence upstream of the fim2 gene of B. pertussis and region 2, which had no identity with any known sequence. A 491 bp EagI DNA fragment (probe A) within region 1 and a 650 bp EagI DNA fragment (probe B) within region 2 were used as DNA probes on restriction endonuclease digests of chromosomal DNA from various bacterial species. This hybridization experiment showed that the region 2 sequence was specific for B. avium. A polymerase chain reaction (PCR) with specific primers within region 2 resulted in the amplification of a 500 bp DNA fragment with B. avium DNA only. This PCR is a useful method for the rapid detection of B. avium and appeared useful to discriminate B. avium from other Bordetella species and also from Alcaligenes faecalis.

Chromosomal DNA from both flagellate and non-flagellate Bordetella species contains sequences homologous to the Salmonella H1 flagellin gene.

The genus Bordetella contains four species: two are non-motile, the human pathogens B. pertussis and B. parapertussis; and two are motile, the broad host-range mammalian pathogen B. bronchiseptica, and the avian pathogen B. avium. The motility of the latter two species is due to peritrichous flagella. Here we show that strains of all four species contain DNA sequences homologous to flagellin genes. Two types of gene probe were hybridised to Bordetella chromosomal DNa in Southern blots: the structural gene for H1 flagellin of Salmonella typhimurium and an oligonucleotide derived from the conserved N-terminal amino acid sequences of various flagellin proteins. ClaI-digested DNa from all four Bordetella species hybridised with both probes in Southern blots, although each species gave a characteristic pattern of hybridisation. This indicates that the non-motile B. pertussis and B. parapertussis species contain non-expressed flagellin genes.

High frequency of independent IS50 transposition during Tn5 mutagenesis of Bordetella pertussis virulence-associated genes.

Transposon Tn5-generated mutants of Bordetella pertussis were selected on the basis of their inability to bind the dye Congo red (Crb-). No mutants which were solely Crb- were found. Ten mutants were phenotypically equivalent to previously described strains with mutations in the virulence regulatory (bvg) locus and failed to express a range of virulence-associated factors. Two of these mutants were shown to have Tn5 insertions within the bvg locus, while another two mutants showed deletions in this regulatory region. Complementation studies indicated that the other six mutants had spontaneous mutations in the bvg locus, but with Tn5 inserted elsewhere in the chromosome. Several of the mutants, besides having a single Tn5 insertion, also showed additional IS50 insertions, indicating that the IS50 element contained within Tn5 had transposed independently. Such additional insertion events, which themselves would have the potential to cause mutation, could complicate the interpretation of mutant phenotypes which could thus arise from the insertional inactivation of more than one gene.

Bacillus anthracis but not always anthrax.

Gram-positive bacilli isolated during epidemiological investigations which, on the basis of conventional tests, resemble Bacillus anthracis but which fail to produce the capsule or to induce anthrax in test animals have long been dismissed in clinical and veterinary laboratories as B. cereus or simply as unidentified Bacillus spp. and thereupon discarded as inconsequential. In this study, the application of newly available DNA probe, polymerase chain reaction and specific toxin antigen detection technology has revealed that a proportion of such strains are B. anthracis which lack the plasmid carrying the capsule gene (pXO2). While these techniques cannot, of course, be used to confirm the identities of strains resembling B. anthracis but which also lack the plasmid carrying the toxin genes (pXO1), the likelihood that these also are bona fide B. anthracis becomes more acceptable. (As yet no naturally occurring pXO1-/2+ strains have been found.) At this point, the significance of the presence of such avirulent forms of B. anthracis in specimens can only be a subject for speculation, but the possibility that they may be indicators of virulent parents somewhere in the system being examined must be considered.

Serotype variation in Bordetella pertussis is governed by cis-acting elements.

Bordetella pertussis contains two genes encoding the serospecific fimbrial subunit proteins 2 and 3 which are assembled into completed fimbriae, which elicit the formation of agglutinating antibodies. Expression of these agglutinogens can vary independently of each other. A gene library from a B. pertussis strain (fimbrial serotype 0.3) was probed with an oligonucleotide probe specific for fimbrial subunit genes. Three homologous genetic loci were identified; an active fim 3 gene, an inactive fim 2 gene and an unknown fim-homologous region. The fim 3 gene carried on a cosmid produced agglutinating fimbrial structures in B. parapertussis and in variants of B. pertussis which had lost the capacity to produce the agglutinogen. This indicated that cis-acting factors are associated with serotype variation in B. pertussis rather than the production of trans-acting repressor molecules.

Evidence for a DNA inversion system in Bordetella pertussis.

The expression of virulence-associated genes in Bordetella pertussis can be lost in three ways: phase variation, antigenic modulation, or serotype conversion. The mechanism(s) of these alterations in gene expression is unclear. B. pertussis chromosomal DNA was probed with cloned pin genes from Escherichia coli and cloned hin genes from Salmonella typhimurium. DNA duplex melting temperature experiments indicated significant homology between B. Pertussis chromosomal DNA and both DNA inversion genes. Southern blots using the hin gene probe showed homology with a 15 kb EcoRI fragment of B. pertussis chromosomal DNA. We postulate here that B. pertussis contains a DNA inversion system which may be responsible for serotype conversion or virulence phase change in this organism.

Cloning and nucleotide sequence analysis of the serotype 2 fimbrial subunit gene of Bordetella pertussis.

An oligonucleotide probe complementary to the beginning of the gene encoding the serotype 2(ST2) fimbrial subunit of Bordetella pertussis was synthesized and a cloned DNA fragment hybridizing with the probe identified and sequenced. Several lines of evidence indicate that an open reading frame with coding information for a polypeptide of 207 amino acids, including a 26-amino-acid signal sequence, is the ST2 gene. The protein deduced from the nucleotide sequence shows good agreement with the NH2-terminal amino acid sequence, amino acid composition and molecular weight of the purified fimbrial subunit. In addition, the proposed ST2 subunit is shown to have homology with other fimbrial subunits.

Nucleotide sequence analysis of the gene for protein A from Staphylococcus aureus Cowan 1 (NCTC8530) and its enhanced expression in Escherichia coli.

Nucleotide sequence analysis of the gene (spa) for staphylococcal protein A (SPA) from Staphylococcus aureus strain Cowan 1 (NCTC8530) shows that the sequence differs from previously reported SPA nucleotide sequences, especially in the number of repeat units in the cell-wall-binding region of the gene. Dot matrix comparison with streptococcal protein G and the macrophage receptor for the constant fragment (Fc) of immunoglobulins shows a limited but significant homology. The homology to the latter probably identifies the Fc-binding region in the immunoglobulin-binding domains of SPA. Enhanced production of SPA in Escherichia coli was achieved using the lac promoter immediately upstream from the spa gene.

Is there evidence for a common amino acid sequence in proteins with membrane attaching ability?

A comparison between the primary sequence of staphylococcal protein A (SpA) and a wide range of published protein sequences revealed a limited, but striking homology in approximately two-thirds of them. The region in the SpA sequence with the common homology was identified as the octapeptide repeats comprising the cell wall peptidoglycan-binding domain. Available structural information and the known location of the proteins within their host cells suggests that this common octapeptide may be important in interaction of the protein with the cell surface (either membrane or wall).

Cloning and expression of the Staphylococcus aureus protein A gene in Escherichia coli.

A gene bank of Staphylococcus aureus strain Cowan I was established using an E. coli HB101/pBR327 host-vector system. Recombinants expressing staphylococcal protein A (SPA) were detected using an IgG-binding assay. A 3.2 Kb DNA fragment directing the synthesis of SPA in E. coli was identified. SPA produced by E. coli was characterised in minicells and by Western blotting and double diffusion experiments.

Use of recombinant plasmids to investigate the structure of the human cytomegalovirus genome.

Human cytomegalovirus (HCMV) DNA was digested with restriction endonucleases and the fragments characterized with respect to molecular weight and relative mole proportions. The terminal fragments were identified by digesting HCMV DNA with exonucleases before restriction endonuclease treatment and subsequent gel analysis. The HindIII fragments of HCMV DNA were cloned in Escherichia coli and recombinant plasmids were characterized by digestion with restriction endonucleases and by molecular hybridization with HindIII, Bg/II and XbaI fragments of the virus genome. Data from these experiments were used to construct physical maps of HCMV DNA for the HindIII, Bg/II and XbaI restriction endonucleases. The terminal regions of the genome and the region containing fragment HindIII M were shown to be heterogeneous.

An endonuclease cleavage map of the plasmid pWWO-8, a derivative of the TOL plasmid of Pseudomonas putida mt-2.

Cleavage sites on the pWWO-8 plasmid were determined for the restriction endonucleases HindIII and XhoI. Terminal labelling using DNA polymerase I was particularly useful both for the characterisation of the smaller cleavage products and for confirmation of the order of fragments in the intact plasmid.

Molecular relationships of degradative plasmids determined by in situ hybridisation of their endonuclease-generated fragments.

Plasmid inter-relationships were studied by hybridisation of a radioactively labelled DNA probe to endonuclease-derived fragmentation patterns of plasmids bound to a nitrocellulose filter. The degradative plasmids SAL and NAH were found to be very closely related, but probably one did not give rise to the other by just a single deletion or insertion. Relationships between SAL and other degradative plasmids are complex; substantial homology was found with TOL and other plasmids encoding toluate dissimilation and significant homology was found with OCT.

Two modes of loss of the Tol function from Pseudomonas putida mt-2.

Some of a set of independently arising Tol- (non toluate-utilising) derivatives of Pseudomonas putida mt-2 have lost the unique plasmid present in the parent strain. In others this plasmid has suffered a deletion of a specific region of about 27 Md.