- Volume 136, Issue 8, 1990

Two enzymes that hydrolysed lactose were purified essentially to homogeneity from cell extracts of the oleaginous yeast Trichosporon cutaneum. One enzyme of M r 120000 had properties typical of a β-galactosidase (EC 3.2.1.23). It hydrolysed lactose, lactulose and nitrophenyl-β-d-galactosides. The enzyme required K+ or Rb+ for activity, and other monovalent cations tested were not effective. Enzyme activity was abolished by EDTA and stimulated by Mg2+, Mn2+ and Ca2+. The β-galactosidase was induced by lactose, galactose, lactulose and lactobionic acid. The other enzyme, a β-glycosidase (EC 3.2.1.21) of M r 52000 showed no ionic requirements and it hydrolysed lactose, nitrophenyl-β-d-galactosides, 4-nitrophenyl-β-d-glucoside, cellobiose, laminaribiose, laminaritriose and sophorose, but not gentiobiose, 4-nitrophenyl-β-d-mannoside or sucrose. This enzyme was induced by lactose, galactose and lactulose, and also by cellobiose.

The soil yeast Trichosporon cutaneum was grown in continuous culture on a minimal medium containing phenol or glucose, or both. The utilization rate of the carbon substrate and the specific activities of catechol 1,2-dioxygenase (EC 1.13.11.1), pyruvate kinase (EC 2.7.1.40), alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1) were determined. Both substrates were utilized simultaneously, the rate of glucose utilization being much higher than that of phenol. Basal activity of catechol 1,2-dioxygenase [3–5 mU (mg protein)−1] was found in glucose-grown cells. High glucose concentrations (20 g l−1) did not prevent the induction of phenol metabolism as reflected in the derepression of catechol 1,2-dioxygenase and the consumption of phenol. However, glucose partially repressed the overall phenol metabolism when T. cutaneum was grown on phenol together with glucose: phenol utilization and catechol 1,2-dioxygenase activity were reduced by 60% and 75% respectively. Complete derepression of catechol 1,2-dioxygenase did not occur until the glucose was depleted. Catechol 1,2-dioxygenase was repressed, but not inactivated, by glucose, as judged from the wash-out profile during transition from growth on phenol to growth on glucose. Pyruvate kinase activity was about twice as high in glucose-grown cells as in phenol-grown cells, and the level of the enzyme during growth on glucose was unaffected by the presence of phenol. Aspartate aminotransferase activity was higher in cells grown with phenol, or phenol together with glucose, than in cells grown on glucose alone. Alanine aminotransferase activity increased only when both substrates were present.

2-Oxoglutarate decarboxylase from the lactic acid bacterium Leuconostoc oenos was purified by precipitation with PEG and ion-exchange chromatography. The strictly thiamin-pyrophosphate-dependent enzyme decarboxylated 2-oxoglutarate to succinic semialdehyde. Oxalacetate was metabolized to a lesser extent. The measured K m values for 2-oxoglutarate and thiamin pyrophosphate were 1 mm and 0·03 mm respectively. The enzyme had a molecular mass in the range 65–70 kDa, did not consist of subunits and showed significant similarities to the corresponding mitochondrial enzyme of Euglena gracilis.

Enzyme preparations from Xanthomonas campestris incubated in the presence of UDP-[14C]GlcA and Mg2+ produced a lipophilic galacturonide with unusual properties. It was easily degraded by both mild acid treatment (0·01 m-HCl, 100 °C, 10 min) and mild alkali treatment (0·06 m-NaOH, room temperature, 5 min) releasing free [14C]galacturonic acid. The galacturonide appeared to be a single compound with one negative charge, as judged by TLC, paper electrophoresis and chromatography, LH-20 gel filtration and DEAE-cellulose column chromatography. Competition experiments indicated that the true glycosyl donor was UDP-GalA, in agreement with the detection of UDP-GlcA-4-epimerase activity in the crude enzyme preparation. The transglycosidase activity was located mainly in the membrane fraction. UDP inhibited the reaction and even produced some loss of label, suggesting an easily reversible reaction. UMP had almost no effect.

The kinetics of carboxylic acid methylation by chloromethane (CH3Cl) in mycelia of the fungus Phellinus pomaceus were examined. Substantial incorporation of C2H3-into ester was observed within 5 min of addition of C2H3Cl to washed mycelia in the presence of the non-physiological acceptor butyric acid, rendering it unlikely that CH3Cl was converted to a diffusible intermediate before acting as methyl donor. The rate of methyl butyrate biosynthesis attained a maximum of 0·14 mol g−1 h−1 at 1·5 mm-butyric acid, with higher concentrations causing increasing inhibition. Exogenous CH3Cl did not affect methyl butyrate production implying that the rate of CH3Cl biosynthesis did not limit methylation. However, C2H3,-incorporation from exogenous C2H3Cl into methyl butyrate rose sharply from 20 to 60% between 15 and 4 mm-butyric acid, suggesting inhibition of CH3Cl biosynthesis by the acid, an interpretation supported by the rapid decline in gaseous CH3Cl release by mycelia between 1·5 and 2 mm-butyric acid. With the natural acceptor benzoic acid as substrate a significant increase in the rate of ester biosynthesis was obtained in the presence of exogenous CH3Cl. Ester biosynthesis was maximal (0·18 μmol g−1 h−1) at 0·5 mm-benzoic acid but fell extremely rapidly with increasing concentration. As with butyric acid supraoptimal concentrations halted CH3Cl release and increased C2H3-incorporation from exogenous C2H3Cl. Studies on C2H3-incorporation from exogenous C2H3Cl into ester revealed a linear relationship between the logarithm of the percentage C2H3-incorporation and the logarithm of C2H3Cl concentration with both butyric and benzoic acids as substrate, suggesting that exogenous C2H3Cl competed with endogenously synthesized CH3Cl for adsorption at a solid interface, possibly a membrane within the cell, prior to reaction of the compound at the active site. When mycelia were grown in the presence of different halide ions, greater methylating activity was found in Br−- and I−-grown mycelia. The system had a higher affinity for CH3Br and CH3I as methyl donors than CH3Cl. Fluoromethane was not a substrate for the methylating system nor did it act as a competitive inhibitor.

Conditions for obtaining stable protoplasts from Sclerotium glucanicum and their reversion to hyphal growth were determined. 1,3-β-Glucan synthase activity was detected in particulate enzyme fractions from mycelium and protoplasts of Scl. glucanicum. UDP-[U-14C]glucose was linearly incorporated into a β-glucan for about 20 min at 25 °C. Optimum pH and temperature values, as well as thermal stabilities of the 1,3-β-glucan synthase activity, were determined. High concentrations of EDTA were inhibitory. Enzyme activity was stimulated by ATP and GTP. The apparent K m value for UDP-glucose was 0·54 mm. The reaction product was characterized as 1,3-β-glucan by 13C NMR spectroscopy and hydrolysis products of an exo-1,3-β-glucanase.

Differences were detectable among strains of the opportunist fungal pathogen Aspergillus fumigatus when water-soluble (WS) preparations were analysed by combined SDS-PAGE and Western blotting procedures. A wide range of molecules of apparent molecular masses from approximately 20 to > 100 kDa showed specific binding to antibodies raised in rabbits to A. fumigatus wall and cytoplasmic components. The ability to bind antibody was markedly reduced by treatment of these antigens with sodium periodate or with specific proteases or glucanases. Pretreatment of blotted antigens with either concanavalin A (ConA) or wheat germ agglutinin (WGA) did not, however, inhibit subsequent antibody binding. The antigens of subfractions prepared from a single strain of A. fumigatus WS material were also susceptible to periodate oxidation and enzymic hydrolysis. Slight cross-reactivity was apparent when crude preparations of cellular or culture filtrate antigens, used in this laboratory to detect antibodies to Candida albicans, Coccidioides immitis and Cryptococcus neoformans, were probed with hyperimmune rabbit antisera to A. fumigatus. Efforts were made to characterize the WS preparations of A. fumigatus, used as diagnostic antigens in many laboratories. The electrophoretically separated antigenic moieties were shown to be predominantly glycoproteins. Binding of cytoplasmic antigens to antibodies raised to wall material showed the presence of many common components in both wall and cytosol. Antiserum to wall components revealed most differentiation among A. fumigatus strains.

Fumarate hydratase (EC 4.2.1.2) from the extremely thermophilic archaeobacterium Solfolobus solfataricus has been purified to homogeneity by a rapid purification procedure using affinity chromatography and high-performance size-exclusion chromatography, and the enzyme’s physical and biochemical properties have been determined. The native enzyme has a molecular mass of 170 kDa and is composed of identical subunits with a molecular mass of 45 kDa, thus indicating a tetrameric structure similar to fumarases isolated from other organisms. The enzyme was active at temperatures ranging from 40 °C to 90 °C, with a maximum activity at 85 °C. The pH optimum for generation of fumarate was found to be pH 8·0. The enzyme showed high stability to denaturation by heat and organic solvents.

A Lactobacillus sake strain L45 isolated from naturally fermented dry sausage, produced a bacteriocin designated lactocin S. The bacteriocin was moderately heat-stable and its activity was sensitive to proteases. Bacteriocin activity was found in the growth medium during the late exponential phase of growth and was directed against selected strains within the closely related genera Lactobacillus, Leuconostoc and Pediococcus. During propagation in liquid medium, non-bacteriocin-producing bacteria (Bac−) appeared with high frequency. Some isolates had also lost immunity to the bacteriocin (Imm−). Analyses of L45 Bac+Imm+ isolates revealed two plasmids of about 50 kb (pCIM1) and 34 kb (pCIM2). All of the L45 Bac+Imm+ variants had lost pCIM1. Three Bac−Imm+ isolates were found which still contained pCIM1. However, DNA restriction enzyme analyses disclosed differences between pCIM1 in the Bac+Imm+ and Bac−Imm+ isolates. These experiments strongly suggest that the pCIM1 plasmids are involved in production of the bacteriocin and in immunity to the bacteriocin.

Streptomyces coelicolor A3(2) bldB mutants are blocked in the formation of aerial hyphae. A phage library of wildtype S. coelicolor DNA was used to isolate recombinant phages which restore wild-type morphological development to several bldB mutants. Of several mutations, one, bld-28, previously mapped at bldB was not complemented by the cloned region, indicating that the bldB locus is composed of at least two distinct genes. Partial localization of bldB-complementing activity showed that a 1·5 kb fragment is sufficient for complementation of the bld-15 mutation whereas bld-17 requires the same region as well as additional sequences. Under stringent conditions, genomic DNA hybridizing to the cloned sequences was absent from other Streptomyces species, including the closely related Streptomyces lividans 66. DNA sequences causing marked plasmid structural instability in S. coelicolor, but not in S. lividans, are also located in this region.

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis α-amylase signal peptide and that coding for the mature part of the extracellular thermostable α-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of α-amylase was enhanced more than 1·7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable α-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable α-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.

The major outer-membrane protein (MOMP) of Chlamydia trachomatis is a promising candidate antigen for- andchlamydial vaccine development. We have sequenced the MOMP genes for a serovar A and a serovar B isolate and have compared these new sequences with those already reported. Intra-serovar changes in the inferred amino acid sequences of the surface-exposed variable segments known to be responsible for binding of neutralizing antibody were observed. Nevertheless, epitope mapping with solid-phase peptides showed that these intra-serovar changes did not affect the binding of serovar- and subspecies-specific, potentially protective antibodies. Variable segment 1 of C. trachomatis serovar A contained two adjacent antibody-binding sites, one of which was C-subspecies specific while the other was serovar A specific. Therefore the subspecies binding site for C-complex organisms is in variable segment 1, whilst that for B-complex organisms is in variable segment 4. This work shows that MOMP sequences are relatively stable within the serovar categorization for isolates taken decades apart from different continents. Within a given serovar, however, limited interchange of functionally related amino acids may occur without impairing the binding of serovar-specific antibody.

The cloning, expression and nucleotide sequence of a 3·74 kb DNA segment on pLS215 containing a β-glucosidase gene(bglA) from Butyrivibrio fibrisolvens H17c was investigated. The B. fibrisolvens bglA open reading frame (ORF) of 2490 bp encoded a β-glucosidase of 830 amino acid residues with a calculated M r of 91800. In Escherichiu coli C600(pLS215) cells the β-glucosidase was localized in the cytoplasm and these cells produced an additional protein with an apparent M r of approximately 94000. The bglA gene was expressed from its own regulatory region in E. coli and a single mRNA initiation point was identified upstream of the bglA ORF and adjacent to a promoter consensus sequence. The primary structure of the β-glucosidase showed > 40 % similarity with a domain of 237 amino acids present in the β-glucosidases of Kluyveromyces fragilis and Clostridium thermocellum. The B. fibrisolvens β-glucosidase hydrolysed cellobiose to a limited extent, cellotriose to cellobiose and glucose, and cellotetraose and cellopentaose to predominantly glucose.

Fourteen mutants of the fungus Phycomyces blakesleeanus, showing high levels of resistance to copper, were isolated. In all the mutants, copper resistance behaved as a very variable and unstable trait. In the mutant strain MU102, the mutation was demonstrated to be cytoplasmically inherited. In addition, this mutant strain differed from the wild-type in growth, respiration rate, and shape and viability of spores.

The two xylE genes for catechol 2,3-oxygenase, encoded by TOL plasmid pWW53, carry a common SalI restriction site within the reading frame. Each gene was cut at the SalI site and the 5′ end of each gene spliced to the 3′ end of the other to form hybrid genes, from both of which catalytically active catechol 2,3-oxygenase activities were expressed. The kinetic parameters were determined for the gene products of both the hybrid and the wild-type xylE genes with catechol, 3-methylcatechol and 4-methylcatechol as substrates. Comparison of the results suggested firstly, that the C-terminal regions of the enzymes determined both the binding and the catalytic specificity, and, secondly, that the N-terminal region of one of the enzymic gene products contained a secondary binding site which caused inhibition by excess substrate for methylcatechol substrates but not for catechol. One of the wild-type enzymes appeared to have an intrinsically higher activity for all three substrates than the other. This higher activity depended on the presence of both its C- and N-terminal regions, and in both hybrid enzymes, which contained only one of these regions, activity was significantly reduced.

The genetic basis of bacteriocin (Bac) production by six strains of Staphylococcus aureus was examined. Gene transfer experiments (in which the plasmids were tagged with the erythromycin resistance transposon Tn551) and plasmid-elimination experiments by growth at 43 °C associated bacteriocin production with a particular plasmid in each strain. The Bac plasmids could be separated into two distinct groups: the first comprised plasmids larger than 40 kb, which did not specify immunity to bacteriocins; the second comprised small plasmids (8·0–10·4 kb) which also specified immunity to bacteriocins. The sequence relations among the small plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) were investigated by comparing restriction enzyme digest patterns and by hybridization. Plasmids pRJ10 and pRJ11 were indistinguishable and very closely related to plasmid pRJ9. Plasmid pRJ6, although different from the others, shared regions of sequence homology with them. No homology was found between plasmids pRJ6 or pRJ9 and the large Bac plasmids.

Binding of purified K99 fimbriae to cryostat sections of pig small intestine was detected. Binding sites were located in the mucus layer, but not in the submucosal connective tissue. High-M r mucin glycopeptides from pig small intestine were found to bind to K99-fimbriated enterotoxigenic Escherichia coli, in contrast to non-fimbriated cells. Sialic acid specificity of K99 fimbriae was demonstrated by the significant reduction in binding upon desialylation of mucin glycopeptides. The binding was saturable and the dissociation constant was estimated to be 6×10−7 M. Fimbriated bacteria were calculated to possess 2·3×103 binding sites per cell.

The efficacy and selectivity of chaotropic and phase-partitioning procedures for the extraction of membrane proteins from Brucella ovis were compared with a standard Sarkosyl method. Major group 1, 2 and 3 outermembrane proteins (OMPs) of B. ovis stained by Coomassie blue in SDS-PAGE gels had, respectively, apparent molecular masses of 81/82 kDa, 39–41 kDa and 30–32 kDa. The presence of these bands in the Sarkosyl extract of total membrane vesicles (TMVs) indicate that the procedure failed to selectively solubilize only inner-membrane proteins (IMPs). SDS-PAGE analyses also revealed the presence of OMPs and other additional bands following extraction of B. ovis TMVs by butanol phase-partitioning or with extraction solutions based on the chaotropic reagents potassium thiocyanate (KSCN), sodium salicylate (SSC) and lithium acetate (LAE). OMPs are therefore not selectively extracted by any one of these procedures. Based on the number and staining intensity of extracted membrane-associated polypeptides, the efficacy of different extraction procedures could be graded in decreasing order as follows: KSCN, SSC, butanol and LAE. Both butanol and SSC were particularly effective in extracting group 3 OMPs. Sera from chronic excretor rams were used to identify zones of seroreactivity in immunoblots. Essentially, two reactivity patterns were seen: strong antibody binding against polypeptides in zones A (46-85 kDa), C (28–32 kDa) and D (18–22 kDa) in one, and additional reactivity against zones B (34–44 kDa) and E (13–18 kDa) polypeptides in the other. Irrespective of the method of membrane extraction, immunoblots using pooled ram sera collected from flocks with ovine brucellosis, and grouped on the basis of increasing complement fixation titre, displayed more intense reactivity against an increasing number of polypeptide bands. Membrane antigens extracted with KSCN and Sarkosyl possessed strong immunoblot reactivity in all five zones while OMPs generally showed the weakest reactivity. In contrast, immunoblot activity of polypeptides in SSC, butanol and LAE extracts were clustered in zones A, C, D and E. It is concluded that chaotropic and organic phase-partitioning procedures do not selectively extract OMPs from B. ovis but represent simple and efficacious methods for generating antigen preparations enriched in membrane polypeptides.

To determine if the host-modulated adherence characteristics of the intracellular bacterial pathogen Chlamydia trachomatis were due to the acquisition of altered surface-exposed proteins, highly purified chlamydiae grown in two different host cells were analysed. Two serovars, L1 and E, were grown for multiple passages in both HeLa and McCoy host cells. Numerous protein differences in the chlamydial elementary bodies (EB) of each serovar grown in the two different hosts were detected by two-dimensional (2-D) gel electrophoresis and fluorography of radioactively labelled proteins. At least four to six serial passages in the alternative host were necessary before the changes were apparent. Iodination of suspensions of purified chlamydiae and 2-D electrophoresis revealed several surface proteins that were determined by the host cells in which the bacteria had replicated. These iodinated chlamydial proteins were removed by treatment of the iodinated EB with trypsin, indicating their location at the bacterial surface. Two of the major constituents of the outer-membrane complex, the cysteine- and methionine-rich 60 kDa and 40 kDa proteins, remained unchanged in both molecular mass and charge during the host adaptation. Several chlamydial proteins capable of binding iodinated host membrane preparations also exhibited host-dependent alterations. Immunoblotting experiments with a rabbit and a human polyclonal sera indicated that distinct host-specified chlamydial proteins were reactive with the two sera.

The gefS gene, coding for a glucosyltransferase which synthesizes water-soluble glucan and previously cloned from Streptococcus downei strain MFe28 (mutans serotype h) into a bacteriophage vector, was subcloned into a plasmid vector. The gtfS gene products expressed in Escherichia coli were compared to the primer-independent, oligoisomaltosaccharide synthase in Streptococcus sobrinus strain AHT (mutans serotype g) and shown to resemble it closely in molecular mass, isoelectric point, immunological properties, optimum pH and K m values. The glucans produced from sucrose by the gtfS gene products are α-1,6-linked linear oligo-isomaltosaccharides without any branching sites. A similar gtfS gene was also detected on chromosomal DNA from S. sobrinus strain AHT.