Pseudomonas aeruginosa protease IV produces corneal damage and contributes to bacterial virulence

PURPOSE: A Pseudomonas mutant deficient in protease IV has significantly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augment the virulence of protease-IV-deficient bacteria were analyzed. METHODS: The toxicity of purified protease IV was determined by intrastromally injecting the exoenzyme (20-200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient strain, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV or 200 ng heat-inactivated protease IV. Changes in ocular disease, determined by slit-lamp examination, were measured at 3, 16, 22, and 27 hours after infection. Colony-forming units per cornea were quantified at 27 hours after infection. RESULTS: Purified protease IV at doses from 50 to 200 ng induced epithelial defects within 3 hours of injection. Injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the protease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P < or = 0.0001). Neither active nor heat-inactivated protease IV altered the growth of bacteria in the cornea (6 log units; P = 0.81). CONCLUSIONS: The important role of protease IV in corneal virulence was demonstrated by direct toxicity and by its ability to significantly augment the virulence of protease-IV-deficient Pseudomonas.     

Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence

PURPOSE: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV. METHODS: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent. Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria. RESULTS: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate. Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02). CONCLUSIONS: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface. Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.     

Pyoverdin is essential for virulence of Pseudomonas aeruginosa

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.     

Ecomycins, unique antimycotics from Pseudomonas viridiflava

While insulin is known to promote vascular smooth muscle (VSM) relaxation, it also enhances endothelin-1 (ET-1) secretion and action in conditions such as NIDDM and hypertension. We examined the effect of insulin pretreatment on intracellular free calcium ([Ca2+]i) responses to ET-1 in cultured aortic smooth muscle cells (ASMCs) isolated from Sprague-Dawley (SD) rats and measured ET(A) receptor characteristics and ET-1-evoked tension responses in aorta obtained from insulin-resistant, hyperinsulinemic Zucker-obese (ZO) and control Zucker-lean (ZL) rats. Pretreatment of rat ASMCs with insulin (10 nmol/l for 24 h) failed to affect basal [Ca2+]i levels but led to a significant increase in peak [Ca2+]i response (1.7-fold; P < 0.01) to ET-1. The responses to IRL-1620 (an ET(B) selective agonist), ANG II, and vasopressin remained unaffected. ET-1-evoked peak [Ca2+]i responses were significantly attenuated by the inclusion of the ET(A) antagonist, BQ123, in both groups. The ET(B) antagonist, BQ788, abolished [Ca2+]i responses to IRL-1620 but failed to affect the exaggerated [Ca2+]i responses to ET-1. Saturation binding studies revealed a twofold increase (P < 0.01) in maximal number of binding sites labeled by 125I-labeled ET-1 in insulin-pretreated cells and no significant differences in sites labeled by 125I-labeled IRL-1620 between control and treatment groups. Northern blot analysis revealed an increase in ET(A) mRNA levels after insulin pretreatment for 20 h, an effect that was blocked by genistein, actinomycin D, and cycloheximide. Maximal tension development to ET-1 was significantly greater (P < 0.01), and microsomal binding studies using [3H]BQ-123 revealed a twofold higher number of ET(A) specific binding sites (P < 0.01) in aorta from ZO rats compared with that of ZL rats. These data suggest that insulin exaggerates ET-1-evoked peak [Ca2+]i responses via increased vascular ET(A) receptor expression, which may contribute to enhanced vasoconstriction observed in hyperinsulinemic states.     

Influence of growth media on potential virulence factors of Pseudomonas aeruginosa

Potential virulence factors of three Pseudomonas aeruginosa strains after growth in three complex media (CM) and in one mineral medium (MM) were evaluated. Cell surface hydrophobicity demonstrated by adherence of bacteria to xylene as well as enzymatic activity (elastase, protease, lipase) of the strains grown in CM varied with composition of CM and with strain. All strains cultivated in CM showed higher hydrophobicity and higher elastase, protease and lipase (with the exception of one strain) activity in comparison with bacteria incubated in MM. Even no production of elastase was detected in the strains after growth in MM. Motility of bacteria was affected by culture media the least. In vitro composition of growth media influenced some potential virulence factors of P. aeruginosa.     

Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors

We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis.     

Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa

We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2-oxa-1, 3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370-24374). This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent Km of 139 microM, and a Vmax of 5.3 micromol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.     

Molecular cloning, sequencing, purification, and characterization of Pseudomonas aeruginosa ribosome recycling factor

Ribosome recycling factor (RRF) is required for release of 70S ribosomes from mRNA on reaching the termination codon for the next cycle of protein synthesis. The RRF-encoding gene (frr) of Pseudomonas aeruginosa PAO1 was functionally cloned by using a temperature-sensitive frr mutant of Escherichia coli and sequenced. The P. aeruginosa frr was mapped at 30 to 32 min of the P. aeruginosa chromosome. The deduced amino acid sequence of RRF showed a 64% identity to that of E. coli RRF. In an assay including E. coli polysome and elongation factor G, purified recombinant RRF of P. aeruginosa released monosomes from polysomes. This is the first case in which an RRF homologue was found to be active in heterogeneous ribosome recycling machinery. The genes for ribosomal protein S2 (rpsB), elongation factor Ts (tsf), and UMP kinase (pyrH) are located upstream of frr. The arrangement of the genes, rpsB-tsf-pyrH-frr, resembles those reported for E. coli and Bacillus subtilis. Even in the cyanobacterium genome, the arrangement pyrH-frr is conserved. Although RRF homologues are found in eukaryotic cells, phylogenetic analysis suggests that they were originally present within the members of the phylogenetic tree of prokaryotic RRF. This finding suggests that the ribosome recycling step catalyzed by RRF is specific for prokaryotic cells and that eukaryotic RRF is required for protein synthesis in organelles, which are believed to be phylogenetically originated from prokaryotes.     

Acetylation of amikacin, a new semisynthetic antibiotic, by Pseudomonas aeruginosa carrying an R factor

A clinical isolate Pseudomonas aeruginosa GN315 resistant to amikacin (AK), a new semisynthetic antibiotic, inactivated AK by acetylation. The acetylating enzyme was purified approximately 146-fold from a crude extract of GN315 by affinity chromatography. Fractionated samples obtained by affinity chromatography showed almost the same inactivation curves toward 3',4'-dideoxykanamycin B (DKB) and AK. Partially purified AK-acetylating enzyme inactivated DKB and kanamycin A but could not inactivate gentamicin C(1). The optimal pH for their inactivation was 6.0 to 7.0, and the pH curves for the inactivation of both drugs were almost the same. These facts indicate that AK and DKB are inactivated by the same aminoglycoside-acetylating enzyme. Through elemental analysis, the inactivated AK was found to be a monoacetylated product of AK. A sample of inactivated AK was purified and compared with a synthetic 6'-N-acetyl AK by thin-layer chromatography, and the results indicated that AK was inactivated by acetylation of the 6'-NH(2) group. The ultraviolet, infrared, and nuclear magnetic resonance spectra of the inactivated AK showed that AK was inactivated by the enzyme through acetylation of the amino group of 6'-amino-6'-deoxy-d-glucose moiety of AK. This enzyme, mediated by R factor, is capable of conferring resistance to AK, DKB, kanamycin, gentamicin, and sulfanilamide.     

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.     

An extracellular protease of Streptococcus gordonii hydrolyzes type IV collagen and collagen analogues

Streptococcus gordonii is a frequent cause of infective bacterial endocarditis, but its mechanisms of virulence are not well defined. In this study, streptococcal proteases were recovered from spent chemically defined medium (CDM) and fractionated by ammonium sulfate precipitation and by ion-exchange and gel filtration column chromatography. Three proteases were distinguished by their different solubilities in ammonium sulfate and their specificities for synthetic peptides. One of the enzymes cleaved collagen analogs Gly-Pro 4-methoxy-beta-naphthylamide, 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), and p-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (pZ-peptide) and was released from the streptococci while complexed to peptidoglycan fragments. Treatment of this protease with mutanolysin reduced its 180- to 200-kDa mass to 98 kDa without loss of enzymatic activity. The purified protease cleaved bovine gelatin, human placental type IV collagen, and the Aalpha chain of fibrinogen but not albumin, fibronectin, laminin, or myosin. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. Maximum production of the 98-kDa protease occurred during growth of S. gordonii CH1 in CDM containing 0.075% total amino acids at pH 7.0 with minimal aeration. Higher initial concentrations of amino acids prevented the release of the protease without reducing cell-associated enzyme levels, and the addition of an amino acid mixture to an actively secreting culture stopped further enzyme release. The purified protease was stored frozen at -20 degreesC for several months or heated at 50 degreesC for 10 min without loss of activity. These data indicate that S. gordonii produces an extracellular gelatinase/type IV collagenase during growth in medium containing minimal concentrations of free amino acids. Thus, the extracellular enzyme is a potential virulence factor in the amino acid-stringent, thrombotic, valvular lesions of bacterial endocarditis.     

The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP+ cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP+ cells, all present within the CD3+ population, with CD4+ and CD8+ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34+ cells were also susceptible, varying from 6% to 10% GFP+ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP+ T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP+ cells did not correlate with the percentage of S/G2/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.     

Crystal structure of the unliganded alkaline protease from Pseudomonas aeruginosa IFO3080 and its conformational changes on ligand binding

Since volatile anesthetics, barbiturates, and local anesthetics have been reported to inhibit endothelium-dependent relaxation, we hypothesized that any drug with anesthetic action would suppress this relaxation. In the present study, using rat thoracic aortae, we attempted to determine whether nonbarbiturate intravenous anesthetics, including midazolam, propofol, and ketamine, suppress endothelium-dependent relaxation, and to clarify the mechanism(s) involved. Acetylcholine-induced, endothelium-dependent relaxation was significantly attenuated by propofol and ketamine, but was unaffected by midazolam. Sodium nitroprusside (SNP)-induced relaxation was attenuated by propofol, but not by midazolam or ketamine. The acetylcholine-stimulated 3',5'-cyclic guanosine monophosphate (cGMP) level was reduced by pretreatment with propofol and ketamine but not by midazolam, and that stimulated by SNP was reduced by propofol but not by ketamine or midazolam. We conclude that propofol and ketamine suppress endothelium-dependent relaxation, whereas midazolam has no influence. Moreover, the suppressive effect of ketamine on endothelium-dependent relaxation is mediated by suppression of nitrous oxide (NO) formation, whereas that of propofol may be mediated at least partly by suppression of NO function.     

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.     

Molecular characterization of OXA-20, a novel class D beta-lactamase, and its integron from Pseudomonas aeruginosa

The Pseudomonas aeruginosa Mus clinical isolate produces OXA-18, a pI 5.5 class D extended-spectrum beta-lactamase totally inhibited by clavulanic acid (L. N. Philippon, T. Naas, A.-T. Bouthors, V. Barakett, and P. Nordmann, Antimicrob. Agents Chemother. 41:2188-2195, 1997). A second beta-lactamase was cloned, and the recombinant Escherichia coli clone pPL10 expressed a pI 7.4 beta-lactamase which conferred high levels of amoxicillin and ticarcillin resistance and which was partially inhibited by clavulanic acid. The 2.5-kb insert from pPL10 was sequenced, and a 266-amino-acid protein (OXA-20) was deduced; this protein has low amino acid identity with most of the class D beta-lactamases except OXA-2, OXA-15, and OXA-3 (75% amino acid identity with each). OXA-20 is a restricted-spectrum oxacillinase and is unusually inhibited by clavulanic acid. OXA-20 is a peculiar beta-lactamase because its translation initiates with a TTG (leucine) codon, which is rarely used as a translational origin in bacteria. Exploration of the genetic environment of oxa20 revealed the presence of the following integron features: (i) a second antibiotic resistance gene, aacA4; (ii) an intI1 gene; and (iii) two 59-base elements, each associated with either oxa20 or aacA4. This integron is peculiar because it lacks the 3' conserved region, and therefore is not a sul1-associated integron like most of them, and because its 3' end is located within tnpR, a gene involved in the transposition of Tn5393, a gram-negative transposon. P. aeruginosa Mus produces two novel and unrelated oxacillinases, OXA-18 and OXA-20, both of which are inhibited by clavulanic acid.     

Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion

The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin-like Xcp proteins was investigated using bifunctional crosslinking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT-containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PIlC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N-terminus or PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be crosslinked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine-tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.     

Pseudomonas aeruginosa lipopolysaccharides and pathogenesis

Pseudomonas aeruginosa lipopolysaccharide (LPS) plays a key role in pathogenesis. In acute infections, a smooth LPS protects the organism from complement-mediated killing and, during chronic lung infections, an altered rough LPS helps the organism evade host defense mechanisms.     

OXA-16, a further extended-spectrum variant of OXA-10 beta-lactamase, from two Pseudomonas aeruginosa isolates

Pooled whey from the production of one variety of ovine cheese and two varieties of caprine cheeses was studied for gross composition and individual whey protein composition over one production season. Individual proteins were quantified by sodium dodecyl sulfate-PAGE and digital imaging technology. The mean proportion of alpha-lactalbumin (LA) from caprine wheys from the manufacture of Chevre and Cheddar-type cheeses was higher than values previously reported for bovine whey from Cheddar cheese; proportions of serum albumin, immunoglobulin (Ig)G, and beta-lactoglobulin (LG) were lower. Ovine whey from Manchego-type cheese showed a higher proportion of beta-LG, about the same proportion of alpha-LA, and lower proportions of serum albumin and IgG than did the bovine whey. Relative amounts of alpha-LA decreased throughout the season, but beta-LG rose in midlactation and then gradually decreased toward the end of lactation. Relative proportions of serum albumin remained fairly stable throughout the year, and IgG decreased.     

Preliminary crystallographic study of cyclohexadienyl dehydratase from Pseudomonas aeruginosa

Single crystals of cyclohexadienyl dehydratase from Pseudomonas aeruginosa have been obtained by vapour diffusion from ammonium sulphate solution (pH 6.0) at 4 degrees C. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2 with a = b = 105.5 A and c = 165.0 A. The asymmetric unit contains at least one dimeric protein molecule with M(r) = 72 kDa. The crystals diffract to 3 A resolution and are suitable for an X-ray analysis.