Identification of the gene lon (capR) product as a 94-kilodalton polypeptide by cloning and deletion analysis.

A mutation in the lon (capR) gene of Escherichia coli K-12 effects several phenotypic alterations in the mutant cell, such as overproduction of capsular polysaccharide and sensitivity to ultraviolet or ionizing radiation. A previously cloned 9.2-megadalton (Md) EcoRI fragment contained the capR+ gene and specified two polypeptides, 94 kilodaltons (K) and 67K. To provide evidence that the 94K polypeptide is the capR+ gene product, we constructed a capR+ plasmid pJMC40, having a 2.0-Md EcoRI-PstI fragment which codes only for the 94K polypeptide. Plasmids pJMC22 and pJMC30, having deletions of 0.7 and 0.8 Md, respectively, from one end of the 2.0-Md fragment, were also constructed. Each codes for a shortened stable polypeptide (from the 94K). Neither plasmid can confer the capR+ phenotype to capR mutants, confirming that the unaltered 94K polypeptide is the capR+ gene product. Plasmids pJMC51 and pJMC52 each have a deletion of 0.7 Md from the other end of the 2.0-Md fragment, differing only in the orientation of the remaining 1.3-Md fragment with respect to the cloning vehicle. They are nonfunctional with respect to capR+ and do not code for a common polypeptide from the 1.3-Md fragment. These data indicate that the fragments in pJMC22 and pJMC30, which both code for shortened 94K polypeptides, contain the promoter-operator region of the capR gene. The deletion plasmids were also used to map chromosomal capR mutations.     

Molecular mechanism for dominance of a mutant allele of an ATP-dependent protease

We have demonstrated that the lon⁺ (capR⁺) ATP-hydrolysis-dependent protease is inhibited by the addition of an enzymatically inactive mutant form of the protein (capR9 protein). The enzymatic activity of the capR⁺-capR9 protein mixture is also more stable to heat than the capR⁺ protein alone indicating that hybrid molecules are formed. Independent measurements demonstrated that the lon⁺ ATP-hydrolysis-dependent protease is a tetramer of identical 94 × 10₃ molecular weight monomers. The pattern of defective subunit (capR9) inhibition indicates only tetramers containing three capR9 monomers and one capR⁺ monomer are inactive, i.e. two or more capR⁺ monomers are required for a tetramer to act as an ATP-hydrolysis-dependent protease. Two molecular mechanisms to explain the dominance of a mutant allele, termed anticomplementation, are considered.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Bacterial Cell Division Regulation: Physiological Effects of Crystal Violet on Escherichia coli lon+ and lon− Strains

The Escherichia coli lon⁻ mutants apparently are defective in the ability to recommence cell division after temporary periods of deoxyribonucleic acid (DNA) synthesis inhibition. They are also more susceptible to cell division inhibition by the basic dye, crystal violet (CV), than are lon⁺ strains. In enriched broth, the lon⁺ strain continued to grow and divide in the presence of CV, but lon⁻ cell division was inhibited and filamentous growth resulted. In a supplemented minimal medium containing CV, lon⁻ cell division was only temporarily inhibited. There was no detectable specific effect on DNA synthesis, although CV slowed the rate of mass increase in both media. Trichloroacetic acid-insoluble lipid synthesis was preferentially inhibited in both lon⁺ and lon⁻ strains. In CV-containing enriched broth, diaminopimelic acid incorporation into trichloroacetic acid-insoluble compounds occurred at a rate greater than the rate of mass increase in both lon⁺ and lon⁻ strains. In a CV-containing supplemented minimal medium, diaminopimelic acid was incorporated to a greater extent by lon⁻ cells than by lon⁺ cells.     

Bacterial Cell Division Regulation: Lysogenization of Conditional Cell Division lon− Mutants of Escherichia coli by Bacteriophage Lambda

The lon⁻ mutants of Escherichia coli grow apparently normally except that, after temporary periods of inhibition of deoxyribonucleic acid synthesis, septum formation is specifically inhibited. Under these conditions, long, multinucleate, nonseptate filaments result. The lon⁻ mutation also creates a defect such that wild-type bacteriophage λ fails to lysogenize lon⁻ mutants efficiently and consequently forms clear plaques on a lon⁻ host. Two lines of evidence suggest that this failure probably results from interference with expression of the λcI gene, which codes for repressor, or with repressor action:-(i) when a lon⁻ mutant was infected with a λcII, cIII, or c Y mutant, there was an additive effect between the lon⁻ mutation and the λc mutations upon reduction of lysogenization frequency; and (ii) lon⁻ mutants permitted the growth of the λcro⁻ mutant under conditions in which the repressor was active. The isolation of λ mutants (λtp) which gained the ability to form turbid plaques on lon⁻ cells is also reported.     

Involvement of Recombination Genes in Growth and Viability of Escherichia coli K-12

We have studied the growth properties of 17 isogenic strains of Escherichia coli K-12 differing only in the recA, recB, recC, and sbcA alleles. We have observed the following. (i) All recombination deficient strains have decreased growth rates and decreased viabilities compared with recombination proficient strains. The large populations of nonviable cells in Rec⁻ cultures may arise by spontaneous lethal sectoring (9). (ii) A recA mutant strain which is entirely recombination deficient and which shows high ultraviolet sensitivity and “reckless” deoxyribonucleic acid (DNA) breakdown has approximately the same growth rate and twice the viability as recB and recC mutant strains which have residual recombination proficiency, moderate ultraviolet sensitivity, and “cautious” DNA breakdown. (iii) Indirectly suppressed (sbcA⁻) recombination proficient (Rec⁺) revertants of recB and recC mutant strains have approximately normal growth rates and are three times as viable as their Rec⁻ ancestors (but not as viable as rec⁺ cells). We suggest the following hypothesis to account for the low viability of Rec⁻E. coli. Single-strand breaks in the DNA duplex, necessary for normal bacterial growth, may be repaired in a Rec⁺ cell. Failure of Rec⁻ cells to repair this normal DNA damage may lead to the observed loss of viability.     

Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)⁺ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)⁺ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 10⁶ and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10⁶. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.     

GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.     

Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis

Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry⁻) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized (González and Carlton, 1980). The plasmid patterns of both Cry⁻ and Cry⁺ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry⁻ mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry⁻ mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (˜130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.     

The Genetic Basis for Mucoidy and Radiation Sensitivity in capR (lon) Mutants of E. coli K-12

CapR mutants of E. coli K-12 overproduce capsular polysaccharide (mucoid phenotype) and enzymes involved in capsular polysaccharide synthesis, and they are sensitive to radiation. It has been uncertain whether both properties are mediated by damage to a single cistron or by a polar effect on a second cistron in the same operon. Introduction of a polarity suppressor caused no change in the overproduction of polysaccharide, in the enzymes of polysaccharide synthesis or in radiation sensitivity of the capR mutant. Thus mucoidy and radiation sensitivity resulting from capR (lon) mutations are both the consequences of impairment of the same cistron. The experiments demonstrate the advantage of the use of polarity suppressors (over conventional nonsense suppressors) in determining whether pleiotropic effects of a mutation are the result of polarity.     

Delayed lysis with a mutant of salmonella bacteriophage p22

A mutant of bacteriophage P22 (Lys⁻) was isolated which shows a plaque morphology on mixed plates comparable to the r⁺ plaques of the T-even phages. When Lys⁻ and normal Lys⁺ plaques are juxtaposed on a petri dish, the Lys⁺ plaque exhibits a flat side adjacent to the Lys⁻ plaque. The mutant is identical to P22 under an electron microscope, is inactivated at the same rate by antiserum and heat, and has the same kinetics of attachment. It does not plate on Salmonella lysogenic for phage P22 nor on strain St/22. In liquid culture, the lysis of mutant infections in M9CAA medium is delayed between 20 and 40 min. Cells mixedly infected in M9CAA with Lys⁻ and Lys⁺ phage lyse later than Lys⁺-infected cells and even later than Lys⁻-infected cells. In unsupplemented M9 medium, however, mixedly infected cells again lyse later than Lys⁺-infected cells, but Lys⁻-infected cells require more than 3 hr to lyse. In supplemented and unsupplemented M9 media, intracellular phage development and endolysin synthesis proceed in Lys⁻ infections at least as rapidly as in Lys⁺-infected cells. In diluted infections, the latent and eclipse periods of Lys⁻ and Lys⁺ infections are indistinguishable. The possible mechanisms involved in the control and timing of lysis are discussed.     

Rhomboids of Mycobacteria: Characterization Using an aarA Mutant of Providencia stuartii and Gene Deletion in Mycobacterium smegmatis

Background

Rhomboids are ubiquitous proteins with unknown roles in mycobacteria. However, bioinformatics suggested putative roles in DNA replication pathways and metabolite transport. Here, mycobacterial rhomboid-encoding genes were characterized; first, using the Providencia stuartii null-rhomboid mutant and then deleted from Mycobacterium smegmatis for additional insight in mycobacteria.

Methodology/Principal Findings

Using in silico analysis we identified in M. tuberculosis genome the genes encoding two putative rhomboid proteins; Rv0110 (referred to as “rhomboid protease 1”) and Rv1337 (“rhomboid protease 2”). Genes encoding orthologs of these proteins are widely represented in all mycobacterial species. When transformed into P. stuartii null-rhomboid mutant (ΔaarA), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of P. stuartii). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in M. smegmatis, the ΔMSMEG_4904 single mutant (which lost the gene encoding MSMEG_4904, orthologous to Rv1337, “rhomboid protease 2”) formed the least biofilms and was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. However, the ΔMSMEG_5036 single mutant (which lost the gene encoding MSMEG_5036, orthologous to Rv0110, “rhomboid protease 1”) was not as susceptible. Surprisingly, the double rhomboid mutant ΔMSMEG_4904–ΔMSMEG_5036 (which lost genes encoding both homologs) was also not as susceptible suggesting compensatory effects following deletion of both rhomboid-encoding genes. Indeed, transforming the double mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ΔMSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin).

Conclusions/Significance

Mycobacterial rhomboid-encoding genes exhibit differences in complementing aarA whereby it''s only genes encoding “rhomboid protease 2” orthologs that fully restore AarA activity. Additionally, gene deletion data suggests inhibition of DNA gyrase by MSMEG_4904; however, the ameliorated effect in the double mutant suggests occurrence of compensatory mechanisms following deletion of genes encoding both rhomboids.     

Glyceraldehyde-3-Phosphate Dehydrogenase (NADP) from Sinapis alba L: Reversible Association of the Enzyme with a Protein Factor as Controlled by Pyridine Nucleotides in Vitro

Aggregation of glyceraldehyde-3-P dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings during gel filtration on Sepharose 6B is dependent on the presence of a fraction (“binding fraction”) which can be separated from the enzyme by precipitation with 55% ammonium sulfate. Association of the enzyme with this binding fraction is NAD-dependent whereas NADP⁺ causes release. Dithioerythritol (2 mM) has no influence on these reversible processes.

Binding fractions, partially purified by ammonium sulfate and acetone fractionation, were submitted to dodecylsulfate-polyacrylamide gel electrophoresis. They always contain one or two dominant polypeptides with apparent molecular weights 42,000 and 58,000. The 42,000 polypeptide comigrates during dodecylsulfate electrophoresis with the corresponding subunit of the enzyme. It comprises up to 70% of the total protein in partially purified binding fractions and can be regarded as a major protein in seedling extracts.

The differential transport behavior of glyceraldehyde-3-P dehydrogenase (NADP) on Sephadex G-200 in the presence of NAD⁺ and NADP⁺ can be used as a simple and effective purification procedure. The enzyme isolated in this way has an isoelectric point of about 4.5 and maintains under all tested conditions a heterogeneous subunit composition of at least three different polypeptide chains (apparent molecular weights, 39,000, 42,000, 43,000).

The present data suggest that NAD(P)-controlled aggregation of glyceraldehyde-3-P dehydrogenase (NADP) from Sinapis alba L. is due primarily to enzyme association with a separate binding fraction rather than to enzyme polymerization. It is possible that a major component of this binding fraction, the 42,000 polypeptide, consists of “surplus” nonactive enzyme subunits, which self-associate and interact with the NAD-conformer of the enzyme.

     

Oxidation of Trichloroethylene, 1,1-Dichloroethylene, and Chloroform by Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1

Toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, which oxidizes toluene and o-xylene, was examined for its ability to degrade the environmental pollutants trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-DCE, trans-1,2-DCE, chloroform, dichloromethane, phenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, and 2,3,4,5,6-pentachlorophenol. Escherichia coli JM109 that expressed ToMO from genes on plasmid pBZ1260 under control of the lac promoter degraded TCE (3.3 μM), 1,1-DCE (1.25 μM), and chloroform (6.3 μM) at initial rates of 3.1, 3.6, and 1.6 nmol/(min · mg of protein), respectively. Stoichiometric amounts of chloride release were seen, indicating mineralization (2.6, 1.5, and 2.3 Cl⁻ atoms per molecule of TCE, 1,1-DCE, and chloroform, respectively). Thus, the substrate range of ToMO is extended to include aliphatic chlorinated compounds.     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

Conjugal Transfer of Bacteriophage Resistance Determinants on pTR2030 into Streptococcus cremoris Strains

Agar surface conjugal matings were used to introduce heat-sensitive phage resistance (Hsp⁺) determinants carried on the conjugal plasmid pTR2030 into Streptococcus cremoris KH, HP, 924, and TDM1. Lactose-fermenting (Lac⁺) transconjugants were selected from matings of Lac⁻ variants of S. cremoris KH, HP, 924, and TDM1 with Streptococcus lactis ME2 or a high-frequency donor, S. lactis T-EK1 (pTR1040, Lac⁺; pTR2030, Hsp⁺). For all of the S. cremoris strains examined, select Lac⁺ transconjugants were completely resistant to plaquing by their homologous lytic phages. In all cases the plaquing efficiencies were less than 10⁻⁹. Acquisition of a 30-megadalton plasmid (pTR2030) in the S. cremoris phage-resistant transconjugants was demonstrated by direct plasmid analysis, by hybridization with ³²P-labeled probes, or by conjugal transfer of pTR2030 out of the phage-resistant transconjugants into a plasmid-cured recipient, S. lactis LM2302. Acid production, coagulation ability, and proteolytic activity of phage-resistant transconjugants in milk were comparable to those of their phage-sensitive parents. Further, S. cremoris phage-resistant transconjugants were not attacked by phage in starter culture activity tests, which included a 40°C incubation period. The results demonstrated that phage resistance determinants on pTR2030 could be conjugally transferred to a variety of S. cremoris strains and confer resistance to phage under conditions encountered during cheese manufacture. Phage-resistant transconjugants of S. cremoris M43 and HP were also constructed without the use of antiblotic markers to select conjugal recipients from mating mixtures.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ^? cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon.     

Nasal Colonisation by Staphylococcus aureus Depends upon Clumping Factor B Binding to the Squamous Epithelial Cell Envelope Protein Loricrin

Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the “dock, lock and latch” mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB⁺ S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor^−/−) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB⁻ mutant colonised wild-type mice less efficiently than the parental ClfB⁺ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB⁻ mutant in the Lor^−/− mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor^−/− mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by S. aureus.     

The acetylcholine receptor: Isolation of a brain nicotinic receptor and its preliminary characterization in lipid bilayer membranes

The technique of affinity chromatography with the curarizing neurotoxins of Naja naja venom has been employed to extract nicotinic acetylcholine receptors from the brain tissues of mouse and hog. Both carbochol and hexamethonium were used as linear or step gradients to elute the receptor and its properties were investigated in lipid bilayer membranes. Of particular interest is the observation that discrete quanta of conductance could be observed across an NaCl gradient of 1.0:0.1 M. By switching the voltage-clamp across the bilayer between a positive and negative 80 mV, the separate Na⁺ and Cl⁻ conductances of these quanta could be estimated and the following conductances of the smallest discrete quanta were observed: 3.7 · 10⁻¹¹ Ω⁻¹ (Na⁺) and 5.9 · 10⁻¹¹ Ω⁻¹ (Cl⁻) for mouse brain receptors; 3.8 · 10⁻¹¹ Ω⁻¹ (Na⁺) and 4.7 · 10⁻¹¹ Ω⁻¹ (Cl⁻) for hog brain receptors. Large aggregates of receptors appeared to activate and deactivate as multiples of a basic conductance size, although there is evidence that they may not represent the actual gating of ion channels. A “background noise” that is not within the temporal capability of the recording system is also present at an intensity that seems to parallel the number of activated receptors, and in view of recent electrophysiological evidence that the relaxation lifetime of the open channel state is of a millisecond duration, it may be that this “noise” actually represent the channel gating.