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The ability of bacteria to detoxify mercurial compounds by reduction and volatilization is conferred by mer genes, which are usually plasmid located. The narrow spectrum (Hg2+ detoxifying) Tn501 and R100 determinants have been subjected to molecular genetic and DNA sequence analysis. Biochemical studies on the flavoprotein mercuric reductase have elucidated the mechanism of reduction of Hg2+ to Hg0. The mer genes have been mapped and sequenced and their protein products studied in minicells. Based on the deduced amino acid sequences, these proteins have been assigned a role in a mechanistic scheme for mercury flux in resistant bacteria. The mer genes are inducible, with regulatory control being exerted at the transcriptional level both positively and negatively. Attention is now focusing on broad-spectrum resistance involving detoxification of organomercurials by an additional enzyme, organomercurial lyase. Lyase genes have recently been cloned and sequencing studies are in progress.

Summary: The mechanism of resistance to chloramphenicol specified by 18 plasmids from Gram-negative bacteria representing different incompatibility groups was investigated. Most determined the drug-inactivating enzyme chloramphenicol acetyltransferase. The enzymes were purified and their properties were compared with those of the previously characterized enzyme types specified by R429 (type I), s-a (type II) and R387 (type III). The type I enzyme was determined by plasmids representing incompatibility groups FII, C, S, I, H, L, O and Com9. Plasmids from incompatibility groups K, Iγ and the A-C complex specified the type III enzyme, while elements representing incompatibility groups V and W determined the type II variant.

The gene coding for epidermolytic toxin serotype B (etb) was cloned in a plasmid expression vector in Escherichia coli. Its expression was dependent on the vector plasmid's trc promoter. A polypeptide immunochemically indistinguishable from the purified staphylococcal toxin and with the same molecular weight was predominantly localized in the periplasm of E. coli. The etb gene resides on a 1.7 kb HindIII fragment of the 42 kb plasmid pTC142 present in the parental Staphylococcus aureus strain.

Spontaneous tetracycline-sensitive, transfer-deficient mutants of R100-1 were selected and analysed by genetic complementation tests and with the restriction endonuclease EcoR1. While some of the Tets Tra- mutants were caused by a single deletion event which removed the Tetr genes and extended into the neighbouring transfer genes, other mutants were the result of the deletion of the Tetr genes within Tn10 which was accompanied by an inversion of adjacent DNA sequences. A clustering of deletion and inversion endpoints occurred in the traA gene. Some of the transfer genes of R100-1 were assigned to EcoR1 fragments.

Transcription of the mer genes of plasmid R100 is regulated by the product of the merR gene. The merR gene negatively regulates its own expression and also controls the transcription of the merTCA operon both negatively (in the absence of inducer) and positively (in the presence of inducer). We used transcriptional mer-lac fusions of R100-1 in complementation tests to measure the ability of the merR products of different mercury-resistant transposons and plasmids to functionally interact with R100-1. Plasmids from incompatibility groups C, B, S, L, and P, as well as the Pseudomonas transposons Tn501 and Tn3401, regulated the expression of the R100 mer genes in a similar fashion to the R100-1 merR product itself, suggesting that these elements are closely related. Only plasmid R391 (IncJ) did not complement.

Methicillin- and gentamicin-resistant strains of Staphylococcus aureus isolated in Dublin hospitals have been classified into groups I, II, and III based on resistance to antimicrobial agents and plasmid profiles. Each group expresses a characteristic level of resistance to gentamicin, tobramycin, and sisomicin. Enzyme assays showed that resistant strains expressed 2"-aminoglycoside phosphotransferase-6'-aminoglycoside transferase activity by a determinant which is known to be chromosomally located. The gentamicin resistance (Gmr) determinants were transferred from group I, II, or III strains by transduction into a laboratory strain where each expressed the same low level of resistance. This finding suggests that high-level resistance in some clinical strains is due to a second, unlinked resistance mechanism. No evidence was obtained by hybridization experiments that clinical isolates or spontaneous mutants expressing high-level Gmr carried more than one copy of the Gmr determinant, thus eliminating the possibility that a gene dosage effect was responsible for high-level resistance. Hybridization experiments with transductants and wild strains suggested that the Gmr determinant was located at homologous sites in wild strains from different groups, although restriction site differences were observed in flanking sequences. Electron microscope analysis of a cloned Gmr determinant and genetic evidence suggested that a Dublin clinical isolate harbored a transposon very similar to Tn4001.

Chloramphenicol acetyltransferases specified by fi − (F fertility-noninhibiting) R factors were compared with those specified by the fi + R factors R1, 222, and R6-S. Three classes of enzyme were distinguished: class I was determined by group N fi − R factors and was indistinguishable from the enzyme determined by fi + R factors, class II was correlated with group W fi − R factors, and class III was determined by R387, an unclassified fi − R factor.

Staphylococcus aureus is a premier human pathogen and the most common cause of osteoarticular, wound, and implanted device infections. We recently demonstrated S. aureus efficiently binds the classical complement regulator C4b-binding protein (C4BP) inhibiting antibody-initiated complement-mediated opsonization. Here we identify S. aureus surface protein SdrE as a C4BP-binding protein. Recombinant SdrE and recombinant bone sialoprotein-binding protein (Bbp), an allelic variant of SdrE, both efficiently bound to C4BP in heat-inactivated human serum. We previously described SdrE as binding alternative pathway regulator factor H. Recombinant SdrE and Bbp efficiently bound C4BP and factor H in serum without apparent interference. Gain of function studies utilizing Lactococcus lactis clones expressing SdrE or Bbp increased serum C4BP and factor H binding, compared with empty-vector control (WT) approximately 2-fold. Correspondingly, classical pathway-mediated C3-fragment opsonization and bacterial killing by human neutrophils decreased by half for L. lactis clones expressing SdrE or Bbp compared with WT. In summary, we identify SdrE and allelic variant Bbp as S. aureus surface proteins that bind the complement regulator C4BP inhibiting classical pathway-mediated bacterial opsonization and killing.

Colonization of human skin with Staphylococcus aureus is a common feature in a variety of dermatologic diseases. In order to reproducibly investigate the adherence of Staphylococcus aureus to human epidermal cells, an in vitro assay was established using the biotin/streptavidine labeling system and the HaCaT cell line. This assay was used to define the role of several Staphylococcus aureus surface proteins with regard to their function in the staphylococcal adhesion process. Our studies included the standard laboratory strain Newman as well as its genetically constructed mutants DU5873, DU5852, DU5854, and DU5886 generated by allele replacement or transposon mutagenesis, which are deficient in the elaboration of staphylococcal protein A (spa), clumping factor (clfA), coagulase (coa), and the fibronectin-binding proteins A and B (fnbA/B), respectively. In comparison with strain Newman all mutants showed remarkably reduced adherence to the HaCaT keratinocyte cell line in our assay, yielding only between 43% and 60% of the adherence capacity of strain Newman after 60 min. Bacterial adherence could be re-established by introducing the cloned wild-type genes for the surface proteins on shuttle plasmids into the chromosomally defective mutants, thus suggesting a pathogenetic role of these proteins in the attachment of Staphylococcus aureus to human keratinocytes. Bacterial adherence was additionally enhanced by alkaline pH-values that are characteristic for skin conditions with epidermal barrier dysfunction. The use of Staphylococcus aureus mutant strains, deficient in the elaboration of defined proteins, allows specific investigation of colonization and virulence factors of this dermatologic relevant microorganism. Key word: surface proteins. J Invest Dermatol 111:452–456 1998

Epizootiological studies on Aeromonas salmonicida are important in view of its role as the causative agent of furunculosis. The use of DNA probes to detect restriction fragment length variations promised to provide a new way to distinguish between different strains of the organism. We used four different DNA probes in combination with seven restriction enzymes to compare seven strains of A. salmonicida. Despite the diversity of the geographical origins of the organisms no differences in the patterns of the hybridizing bands were found. This suggests that the DNA sequences of A. salmonicida are very strongly conserved and that the potential for DNA probes to identify different strains may be limited.

Plasmids R26 (Inc P) and R55-1 (Inc C) specify inducible nonenzymatic resistance to chloramphenicol. Escherichia coli K-12 strains harboring these plasmids encoded low-level resistance to thiamphenicol analogs Sch 25298 and Sch 25393 but failed to specify resistance to the fluorinated chloramphenicol analog Sch 24893. The analogs were efficient inducers of high-level chloramphenicol resistance.

Prior to 1976 methicillin-resistant Staphylococcus aureus (MRSA) \TNas associated with sporadic infection in the eight hospitals studied in the Dublin area (total of 1850 beds). These organisms were gentamicin-sensitive and although sporadic infection was seen there was no bacteraemia. 1

A series of 23 transposon 801(Tn801)-induced mutations of plasmid R100-1 from mercuric salts resistance to sensitivity was studied. Although Tn801 transposed frequently into the mer region of the plasmid, fine structural analysis showed that the site of insertion within mer varied. About one-half of the Tn801 insertion events also caused a deletion of greater than 1 megadalton. Genetic and restriction endonuclease EcoRI and BamHI analysis of the mutant plasmid deoxyribonucleic acid elucidated the organization of the mer operon and suggested the existence of a trans-acting regulatory factor governing resistance to mercuric salts. Tn801 insertions leading to mercuric sensitivity occurred in the restriction endonuclease fragments EcoRI-H and EcoRI-I. Regulatory mutations leading to a 50-fold-reduced synthesis of mercuric reductase enzyme occurred in two complementation classes thought to represent the gene for a trans-acting inducer molecule and a cis-acting operator-promoter sequence. Mutations leading to total loss of the enzyme mercuric reductase occurred on both the EcoRI-H and EcoRI-I fragments, showing that the structural gene for this enzyme (merA) bridges the EcoRI cleavage site separating the segments. Hypersensitivity to mercuric salts resulted when Tn801 insertion occurred in the reductase gene in the operatordistal portion of the operon. Hypersensitive cells inducibly bound three to five times more Hg2+ at low concentrations than did sensitive (plasmidless) cells. This finding led to the proposal that another gene (merT) controls uptake of Hg2+ by the cells. Transcription of the operon was deduced to start in the EcoRI-H fragment and to move into the EcoRI-I fragment of the plasmid genome.

S. aureus strain 8325-4 was shown to produce alpha-, beta-, delta- and gamma-haemolysins by haemolytic assays and immunoblotting. Hybridization experiments indicated that a single copy of the alpha-haemolysin gene (hla) resides in the chromosome. Site-directed mutagenesis was used to inactivate the hla gene. This gene, which had previously been cloned in E. coli, was inactivated in vitro by inserting a fragment carrying an erythromycin resistance marker. Shuttle plasmids were constructed and transformed into 8325-4 and non-haemolytic recombinants enriched by a plasmid incompatibility technique. A previously isolated Tn551 insertion defective in alpha-haemolysin was not located in hla. It had pleiotropic defects in expression of alpha-, beta- and delta-haemolysins. Expression of alpha-haemolysin from a plasmid-located hla gene was very low. In contrast, hla-erm mutants were deficient only in alpha-haemolysin and allowed high level expression of the plasmid-borne hla gene. The Tn551 insertion is probably located in a gene encoding a positive regulatory element required for expression of several exoproteins. An hla-erm mutant was less virulent than the otherwise isogenic 8325-4 hla+ strain in a mouse peritonitis model, confirming that alpha-haemolysin is an important virulence factor.

SUMMARY: The isolation of Escherichia coli chromosomal mutants that increased, the level of resistance of a partially tetracycline-sensitive mutant of R100-1 is described. Plasmid-less derivatives of these moderately resistant mutants were phenotypically similar to the cmlB mutants described by Reeve (1966, 1968), and also mapped in the same region. The level of intrinsic resistance to both chloramphenicol and tetracycline was increased about twofold. Also, the levels of R factor-determined resistance to these drugs were increased by this host mutation and tetracycline resistance was expressed constitutively. A cmlB mutant accumulated tetracycline at a threefold] lower rate than the wild-type strain, and it is proposed that the mutants have an altered permeability to the drugs and that this acts syner-gistically with the products of the R factor chloramphenicol and tetracycline resistance genes.

The majority of TN10 insertions in the lacZ gene of Escherichia coli occurred in a small region of the promoter distal part of the gene. The resulting mutations were polar on lacY and reverted to Lac+ at a frequency of 10(-8). None of the revertants were Tcr. Furthermore Lac+ Tcr revertants could not be selected directly. Relief of polarity revertants of the lacZ::Tn10 mutants were formed at a frequency of 10(-5) - 10(-4). Most resulted from a deletion event internal to the transposon which removed the Tcr genes and the putative transcription terminator. It is postulated that a fragment of Tn10 remains at the original insertion point to cause a revertible Lac- mutation.

The tet genes of transposon Tn10 have been mapped in a 2,200 bp DNA sequence by analysing deletion and Tn5 insertion mutations. When the tet genes were present on multi-copy plasmids the level of resistance expressed was about ten-fold lower than that determined by a single copy of Tn10 in the E. coli chromosome. The 36K tet protein known to be encoded by R100 in E. coli minicells was not detected when they harboured a multicopy tet plasmid. However, normal high levels of resistance were expressed when the tet genes were recombined into the host chromosome as part of a lambda lysogen, showing that the multicopy effect was phenotypic. Most of the Tn5 insertions and deletions in tet which caused Tcs mutations also prevented expression of high level Tcr from a chromosomal Tn10 element present in the same cell. Only those insertions in the promoter-proximal 90–130 bp of a 1,275 bp HindII fragment known to carry the gene encoding the 36K tet protein did not reduce the single copy Tn10 resistance level. A gene fusion system that results in the constitutive synthesis of β-galactosidase from a tet promoter has been used to assay tet repressor activity. The basal (uninduced) β-galactosidase level in cells carrying multicopy tet plasmids was 10–20 fold lower than those carrying a single copy. The tet:: Tn5 mutants defective in the trans-dominant multicopy effect still made normal amounts of tet repressor showing that repressor overproduction was not responsible for this effect. In addition a repressor-defective constitutive mutant did not exhibit a higher resistance level when located on a multicopy plasmid vector. We postulate that a regulatory mechanism recognises the amino-terminus of the tet structural gene product when attempts are being made to overproduce the protein and prevents further translation.

Transposon Tn10 is 9300 bp in length, with 1400 bp inverted repeats at its ends. The inverted repeats are structurally intact IS-like sequences (Ross et al., 1979). Analysis of deletion mutants and structural variants of Tn10, reported below, shows that the two IS10 segments contain all of the Tn10-encoded genetic determinants, both sites and functions, that are required for transposition. Furthermore, the two repeats (IS10-Right and IS10-Left) are not functionally equivalent: IS10-Right is fully functional and is capable by itself of promoting normal levels of Tn10 transposition; IS10-Left functions only poorly by itself, promoting transposition at a very low level when IS10-Right is inactivated. Complementation analysis shows that IS10-Right encodes at least one function, required for Tn10 transposition, which can act in trans and which works at the ends of the element. Also, all of the sites specifically required for normal Tn10 transposition have been localized to the outermost 70 bp at each end of the element; there is no evidence that specific sites internal to the element play an essential role. Finally, Tn10 modulates its own transposition in such a way that transposition-defective point mutants, unlike deletion mutants, are not complemented by functions provided in trans; and wild-type Tn10, unlike deletion mutants, is not affected by functions provided in trans from a "high hopper" Tn10 element.

Publisher Summary Transposable elements are discrete sequences of DNA that can move from one genetic locus to another on the same or on a different replicon by a process of recombination called “transposition or translocation.” They are ubiquitous among bacteria and many different ones have been discovered. The size of these elements can vary markedly from one another and many different properties can be transposable. When a transposable element contains an accessory gene, encoding some marker, it is called a “transposon:” the terms “transposable element” and “transposon” are often used interchangeably. The transposable markers may be antibiotic resistance determinants, toxins and other virulence factors, or metabolic functions. The perceived importance of drug resistance transposons best reflects the human interest in this class of transposon and the ease with which they can be detected and studied. Much more investigation is needed before a decision on this point can be made. However, the point is not trivial, because transposons give to bacterial DNA and, in particular, to bacterial plasmids an enormous genetic flexibility that is reflected in the adaptability displayed by bacteria to the alterations in their environment.

Forty-one isolates of multiply resistant gram-negative bacteria causing infection in a urological unit of a Dublin hospital were collected during a 6-month period. Twenty-one isolates transferred multiple resistance to an Escherichia coli K-12 recipient in liquid matings. Serratia marcescens, Proteus morganii, Proteus vulgaris, and E. coli isolates harbored similar 120-megadalton IncC plasmids, whereas Enterobacter cloacae strains transferred a 160-megadalton plasmid of a different Inc group. Southern hybridization experiments were performed with purified fragments cloned from one IncC plasmid as probes. They were hybridized to plasmid sequences in total cellular DNA extracts, showing that the IncC plasmids were very closely related. This suggests that the same plasmid has transferred to different bacterial species in the hospital environment.

Toxic shock syndrome (TSS) toxin 1 (TSST1) is produced by strains of Staphylococcus aureus associated with TSS. Purified TSST1 induces in rabbits a shock-like illness with many features similar to TSS in humans. These symptoms were also induced by TSST1-producing bacteria in diffusion chambers implanted in the rabbit uterus. Naturally occurring TSST1+ strains and a TSST1- strain harboring a pE194-derived plasmid carrying the cloned TSST1 determinant tst gave the same symptoms. TSST1- strains and a TSST1- strain carrying a pE194-tst plasmid with a deletion of the tst gene had no effect in rabbits. The results with the plasmid-carrying TSST1+ and TSST1- strains, which were isogenic apart from tst, show that the toxin is responsible for the illness in rabbits and suggest that it is a major factor in the pathogenesis of TSS.

Mutants of Staphylococcus aureus which fail to express alpha-toxin (Hly), beta-toxin (Hlb), or both have been constructed by site-specific mutagenesis. The virulence of the mutants was compared with that of wild-type toxigenic strains by intramammary inoculation of lactating mice. A bovine strain, M60, and a laboratory strain, 8325-4, caused acute mastitis and death within 48 h for 60% of the mice inoculated. Animals inoculated with Hly mutants also developed acute mastitis, but no deaths occurred. Comparisons of Hly- or Hlb-positive strains with the double mutation Hly Hlb showed that both toxins led to a significantly higher recovery of S. aureus from the gland 48 h postinfection. Histopathological examination of mammary glands showed that phagocytosis of bacteria occurred irrespective of toxigenicity, but toxigenic strains, particularly those which were Hly+, continued to multiply, invaded the interalveolar tissues, and produced severe lesions. Stimulation of an inflammatory response by inoculation of the mammary gland with endotoxin prior to challenge with S. aureus reduced recovery of the bacteria 10- to 100-fold and, under these conditions, neither alpha-toxin nor beta-toxin contributed significantly to growth and survival.

The cml gene of plasmid R26 is carried on a 1.9-kb HindIII fragment and specifies low-level, inducible resistance to chloramphenicol (Cm). In this paper we report the identification of its product as an approx. 31 kDa protein in minicell experiments, and the determination of the nucleotide sequence of cml, which indicates that the gene product is a relatively hydrophobic protein of Mr 33,800. The protein has no detectable homology to other characterised chloramphenicol-resistance (CmR) proteins, nor any to the membrane-associated tetracycline-resistance (TcR) proteins. The presumptive ribosome-binding site (RBS) of cml mRNA is within a region showing potential for secondary structure.

Transfer-deficient derivatives of pDU202 (a Tcs deletion mutant of R100-1) caused by the insertion of Tn10 into the R factor's transfer genes have been described previously. Tetracyline-sensitive mutants of four of these were selected. In the majority of cases the Tcs mutation was caused by a deletion of the Tcr genes which was often accompanied either by a deletion of some of the flanking transfer genes or by a secondary mutation which was probably an inversion. A number of preferred end points for the deletions and inversions occur in the transfer operon of pDU202. Analysis of the mutants by complementation tests with Flac tra elements confirmed that the order of genes in the promoter distal part of the tra region of pDU202 is traKBCFHGSD and traI.

The mercuric resistance (mer) genes of plasmid R100 were cloned into plasmid pBR322. A series of transposon Tn5 insertion mutations in the mer genes were isolated and mapped. The mutants were characterized phenotypically by their sensitivity to Hg2+ and by binding and volatilization of 203Hg2+. Dominance and complementation tests were also performed. Mutations affecting the previously described mer genes merR (regulation), merT (transport), and merA (reductase) were characterized. Evidence was obtained for two new mer genes, which have been called merC and merD. A restriction enzyme map of the mer region was drawn with the gene order merRTCAD. Transcriptional merR-lac and merA-lac fusions were generated by insertion of phage Mu d amp lac into plasmid R100-1. These were used to study regulation of mer gene expression. The merR gene product appears to regulate negatively its own expression as well as acting as both a negative and a positive regulator of the merTCA genes.

SUMMARY: The gene coding for protein A (spa) has been mapped close to nov on the genetic map of the chromosome of Staphylococcus aureus 8325-4. A rapid mapping procedure has been developed which first allowed the region of the chromosome carrying the spa gene to be identified by blot hybridization of large DNA fragments which had been separated by pulsed-field gel electrophoresis. Restriction endonuclease Smal fragment G was shown to carry the spa gene. An insertion mutation in spa was constructed by in vitro insertion of a fragment of DNA expressing resistance to kanamycin and neomycin. A spa::KanrNeor mutation was isolated in S. aureus 8325-4 by allele replacement. This provided a selectable marker which allowed the spa gene to be mapped by transformation analysis.

SUMMARY: The extent of the deletions of five Tets Tra- mutants of R100–1 was determined by complementation experiments with wild-type and tra mutants of Flac. The presence or absence of the origin of transfer on the mutants was also investigated. Using the results, a tentative map of this region of the R factor was drawn: it was essentially similar to the analogous region of the E. coli K12 F factor, except that tet was located between traJ and traA. Some of the deletions had removed the promoter for the transfer operon. This allowed detection of the transcription of traC and distal genes from a weak, traJ-independent promoter. This is probably the Is2 promoter, since R100–1 carries an Is2 insertion sequence located immediately to the left of traC in the correct orientation. Since neither the transfer operon promoter nor the Is2 promoter seemed to be required for transcription of traI, it was concluded that, unlike the F factor, this was located in a separate operon.

Publisher Summary Transposons are discrete sequences of ONA that can move from one replicon to another by a process of recombination called “transposition” or “translocation.” Many types of transposons are found in nature. The size of the elements can vary markedly from one to another and many different properties can be transposable. The transposable markers may be antibiotic resistance determinants, toxins and other virulence factors, or metabolic properties. Transposons have been discovered in many species of both gram-negative and gram-positive bacteria. This chapter describes the better-characterized transposons of gram-negative bacteria. It highlights that it is now possible to perform genetic manipulations in organisms where no genetic system was previously available. These include the isolation of auxotrophic mutations, provision of portable regions of homology for chromosome transfer, and the generation of deletions.

We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.

The nucleotide sequence of the eta gene, which codes for the epidermolytic toxin serotype A of Staphylococcus aureus TC16, is reported. The coding sequence of 840 nucleotides specifies a protein which, when secreted, has a predicted molecular weight of 26,950. The sequence of eta and the deduced amino acid sequence of the toxin have been compared with those of epidermolytic toxin serotype B. The coding sequences have 52% identical residues, and the polypeptides have 40% identical residues. Amino acid residues have been conserved in the areas of the proteins which correspond to major hydrophobic domains, whereas the regions likely to specify antigenic determinants occur in hydrophilic sequences that have diverged. The level of expression of epidermolytic toxin A in S. aureus 8325-4 was shown to be dependent on the integrity of a regulatory gene called agr.

Transcriptional (operon) and translational (gene) fusions between the R100 merR gene and lacZ were constructed in vitro in a pBR322 plasmid carrying the mer genes derived from plasmid R100. The translational fusions were oriented in the opposite direction to and divergently from the merTCAD genes. This shows that the reading frame previously thought to be merR was incorrect. Expression of the gene fusion was repressed in trans by a compatible plasmid carrying the R100 merR+ gene, as was a similarly oriented transcriptional fusion. In contrast, expression of beta-galactosidase by the lac fragment located at the same site but in the opposite orientation was at a lower level and was not repressed by merR+.

The gene coding for protein A (spa) of Staphylococcus aureus 8325-4 has been inactivated by substituting part of the spa coding sequence for a DNA fragment specifying resistance to ethidium bromide. The in vitro-constructed spa::EtBrr substitution mutation was introduced into the S. aureus chromosome by recombinational allele replacement. Southern blot hybridization showed that the in vitro-constructed mutation was present in the chromosomal spa locus. We have previously reported the inactivation of the alpha-toxin gene (hly) by allele replacement with an in vitro-constructed hly::Emr (erythromycin resistance) mutation (M. O'Reilly, J.C.S. de Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathogen. 1:125-138, 1986). A double Spa- Hly- mutant was constructed by transduction. The virulence of Spa- and Hly- mutants was tested by experimental infection of mice. When subcutaneous injections were given, Hly- mutants formed a flat, darkened lesion, whereas Hly+ strains caused a raised, cream lesion. Alpha-toxin was shown to be a major factor in forming subcutaneous lesions and in causing the death of mice injected intraperitoneally. Spa- mutants were slightly less virulent than their Spa+ counterparts, which suggests that protein A is also a virulence factor of S. aureus.

The gene coding for serotype A of epidermolytic (exfoliative) toxin has been cloned from Staphylococcus aureus in Escherichia coli phage λ and plasmid vectors. The coding sequence for eta was localised by subcloning and transposon Tn 5 mutagenesis experiments. The eta gene was probably expressed from its natural promoter in E. coli . The protein synthesised in E. coli was located predominantly in the periplasm. It was immunochemically indistinguishable from the toxin purified from S. aureus culture supernatants and had the same molecular weight. Furthermore, subcutaneous injection of this material caused epidermal splitting (the Nikolsky reaction) showing that it was biologically active. An eta shuttle plasmid was transformed into protoplasts of S. aureus . The level of expression of toxin in strain 8325-4 was shown to be dependent on the integrity of the agr gene which is known to be required for the expression of several exoproteins.

We have localized the sites and functions encoded by transposon Tn10 which are relevant for Tn10 transposition. Analysis of deletion mutants and structural variants shows that the outer-most 70 basepairs at each end of the element encode all of the sites essential for normal transposition, and that at least one function which recognizes these ends is encoded by the 1400-base-pair inverted repeat segments which form the ends of Tn10. Genetic analysis has revealed a functional asymmetry between the left and right sides of Tn10 and a number of complexities in the functioning of the two IS10 elements as they occur in the wild type element. In addition, complementation analysis has revealed the unexpected fact that most transposition-defective mutants of Tn10 are not complementable in trans , although other data indicate that transposition functions are in fact freely diffusible. Finally, analysis of Tn10 mutants for their effect on precise excision and nearly-precise excision of Tn10 makes it clear that both events can occur by pathways which are independent of transposition functions, and that the inverted repeats of the element play a structural role in excision by these pathways.

SUMMARY: Thirty-four epidermolytic toxin-producing strains of Staphylococcus aureus obtained from a variety of sources were screened for the presence of plasmid DNA. All serotype ii toxin producers harboured a large 42 kilobase pairs (kb) plasmid. Elimination of these plasmids resulted in the simultaneous loss of a bacteriocin determinant (Bac+) and type ii toxin production (Tox+ ii). Some strains producing serotype i toxin (Tox+ i) contained similar 42 kb plasmids. Elimination of these plasmids resulted in the loss of only bacteriocin production. Strains producing both toxin serotypes readily lost Tox+ ii and Bac+, which were carried on the same plasmid, but Tox+ i could not be eliminated. Thus Tox+ i was probably chromosomally determined, while Tox+ ii was a plasmid-encoded marker. In some strains cadmium resistance was also linked to the 42 kb plasmid. The 42 kb plasmids from seven strains with different phenotypes were analysed with restriction endonucleases EcoRI and HindIII. The plasmids shared 19 of 22 HindIII fragments indicating that they are closely related to each other.

Recombination between single-site and multisite chloramphenicol-sensitive mutants of the F-like R factors R1 and R100-1 indicates that the chloramphenicol resistance region is a single structural gene coding for the 20,000-molecular weight subunit of chloramphenicol acetyltransferase.

Bacterial colonisation of prosthetic material can lead to clinical infection or implant failure, or both, often requiring removal of the device. Adherence of Staphylococcus aureus to bioprosthetic materials is mediated by adhesins belonging to the MSCRAMM (microbial surface components recognising adhesive matrix molecules) family of microbial cell surface proteins. The objective of this study was to compare the virulence of a mutant strain of S. aureus Newman that possesses all three fibrinogen-, fibronectin- and collagen-binding MSCRAMMs (MSCRAMM-positive strain) with that of a mutant strain that lacks all three types of MSCRAMMs (MSCRAMM-negative strain) in a rabbit model of orthopaedic device-related infection. After a hole was drilled into the knee joint of each animal, a group of 10 rabbits was inoculated with the MSCRAMM-positive strain and another group of 10 rabbits received the MSCRAMM-negative strain. A stainless steel screw was then placed into the drilled hole. Two weeks later, the rabbits were killed and serum samples, bone tissue and implants were harvested for bacteriological and histopathological evaluation. No significant difference in infection rates was demonstrated between the two groups. The ability to delineate the role of S. aureus surface adhesins in causing orthopaedic device-related infection could be model-dependent.

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.

C-reactive protein (CRP) is a well established marker for inflammation, and a good predictor of coronary heart disease. It is also known to interact with the platelet FcγRIIa and to enhance the inhibition of platelet aggregation by aspirin by an unknown mechanism. CRP has also recently been demonstrated to compete for PAC-1 binding in collagen stimulated platelets, suggesting that CRP interacts with GPIIb/IIIa. In order to study the mechanism of interaction with platelets directly, we carried out platelet adhesion assays. We coated plates with recombinant CRP which was in the native pentameric form as confirmed by size exclusion chromatography. Platelets adhered to immobilized CRP and to immobilized fibrinogen to a similar extent. Adhesion to CRP and fibrinogen was inhibited by GPIIb/IIIa antagonists but not by antibody to the FcγRIIa (IV.3). Platelet adhesion to CRP was increased 5-fold when platelets were treated with 1 mM MnCl2. Adhesion of MnCl2 treated platelets was also inhibited by GPIIb/IIIa antagonists. When viewed by confocal microscopy, the adherent platelets displayed pseudopodia, but did not spread fully. Treatment of platelets with MnCl2 stimulated lamellipodia formation and full platelet spreading on CRP. Analysis of the exposed residues on the surface of the CRP crystal structure identified two RGD-like sequences, RQD and DGK. The peptides KRQDN and VDGKP derived from CRP inhibited platelet adhesion to fibrinogen suggesting that these sequences could be responsible for the interaction with GPIIb/IIIa. Our data demonstrates that CRP in the native pentameric form interacts with GPIIb/IIIa and that it has an increased affinity for the activated conformation of GPIIb/IIIa. This interaction is likely to be due to an interaction with the surface exposed KRQDN and VDGKP CRP sequences. We have previously demonstrated that CRP acts as a GPIIb/IIIa antagonist when in solution. Furthermore, pentameric CRP cannot support platelet adhesion under shear conditions. Thus it is likely that elevated circulating levels of soluble CRP reduces the overall thrombotic potential.

In this chapter we introduce the concepts of upcycling and valorisation of food waste by highlighting some work at the University of Nottingham looking at valorisation of feed-stocks from brewers’ spent grain, Citrus waste, Pea Haulm and Bran, producing value-added products spanning macronutrients: fibre and protein, and lipids (chloroplast-rich fractions and waxes), as well as identifying carry over of micronutrients, as a result of careful and sustainable processing. The functionalised materials provide novel structuring capability and also nutritional benefits. Finally, a case study is presented turning food by-products into functionalised food ingredients that have been used to produce safe ingredients and final products with higher dietary fibre content, where products were well accepted in initial sensory tests, providing waste reduction and lower carbon emissions across the supply chain.

We used an ex vivo canine arteriovenous shunt model, previously developed to study plasma protein adsorption and thrombogenesis on polymeric biomaterials, to define the role of host proteins in promoting adhesion of Staphylococcus aureus. Either polyethylene or polyvinyl chloride tubings were exposed to canine blood for 5, 15, or 60 min at a flow rate of 300 ml/min and then were flushed in phosphate-buffered saline (PBS), cut into 1.5-cm segments, and stored at -70 degrees C. After thawing, each segment was preincubated in 0.5% albumin in PBS to prevent nonspecific staphylococcal attachment to surfaces that were not exposed to blood. Each segment was then incubated with 4 x 10(6) CFU of [3H]thymidine-labelled S. aureus per ml for 60 min at 37 degrees C in an in vitro adhesion assay. Two site-specific mutants of S. aureus were tested: one specifically defective in adhesion to surface-bound fibronectin (FnAd-def) and the other defective in adhesion to fibrinogen (FgAD-def) [corrected]. Compared with their respective parental strains, the FgAd-def, but not the FnAd-def, mutant of S. aureus showed a strong (> 80%) decrease in attachment to ex vivo tubings. The adhesion of each strain of S. aureus onto polyethylene was consistently more than twofold higher than the adhesion onto polyvinyl chloride segments exposed to flowing blood for 5 or 15 min, but adhesion became similar to that on polyvinyl chloride after 60 min of exposure. In conclusion, the specific adhesion-defective mutants of S. aureus suggested that fibrinogen was the most active adhesion-promoting protein in a short-term blood-material interaction. The experimental approach described in this study should prove useful for screening materials thought to be resistant to protein-mediated staphylococcal adhesion and colonization.

Block copolymers are being introduced in food research as promising stabilisers able to control the digestibility of lipid-based systems such as oil-in-water emulsions. Lipid digestion takes place mainly in the duodenum and involves the action of different agents competing for the oil-water interface of emulsified lipids; bile salts secreted by the gallbladder play a crucial role preparing this interface for the adsorption of the enzyme pancreatic lipase. The aim of this work was to analyse the bulk interactions between triblock copolymers of the family of Pluronics (F127 or F68) and a bile salt (sodium taurodeoxycholate, NaTDC) in Pluronic-stabilised emulsions. These new findings can be exploited in tailoring novel food and pharmacological matrices with improved functional properties, while increasing the scope for identifying functionalities of other potential (bio)polymers.

Background Fibronectin binding proteins A and B (FnBPA and FnBPB) mediate adhesion of S. aureus to fibrinogen, elastin and fibronectin. We previously identified seven different isotypes of FnBPA based on divergence in the fibrinogen- and elastin-binding A domains. The variation created differences in antigenicity while ligand binding functions were retained. Here, FnBPB variation was examined in both human and bovine isolates and compared to that of FnBPA. Results Seven different fnbB allelic variants were identified. Some strains that cluster by phylogenetic analysis contain different fnbB variants, whereas more divergent strains contain the same fnb B variant. The phylogeny of fnb B alleles does not match the phylogeny of fnbA alleles. Some FnBPA and FnBPB isotypes that are specified by human S. aureus strains are also found in bovine strains. The seven fnb B allelic variants encode seven distinct isotypes of the FnBPB A domain that are 61 to 85% identical in amino acid sequence. Variant amino acid residues were mapped on a three-dimensional model of the FnBPB A domain and were predicted to be surface-exposed. They are responsible for the antigenic diversity detected with polyclonal antibody and a monoclonal antibody raised against isotype I. Ligand binding by recombinant FnBPB N23 isotypes was compared by ELISA-based solid phase assays and surface plasmon resonance. Each bound to immobilized fibrinogen, elastin and fibronectin dose-dependently and saturably with similar affinities. Binding to fibronectin was surprising because the A domains do not contain any known motifs that mediate binding to fibronectin. This raises the possibility that the A domain of FnBPB contains a novel fibronectin binding motif that binds fibronectin by a novel mechanism. Conclusions Seven different isoforms of FnBPB A domain retain ligand-binding functions but are antigenically distinct. The variation in FnBPA and FnBPB occurs in human and bovine S. aureus strains and may act as an immune evasion mechanism. All seven isotypes of FnBPB are capable of binding fibronectin though none contain any known fibronectin-binding motifs. These results have implications for the development of vaccines or immunotherapeutics that target FnBPB