- Volume 141, Issue 2, 1995

SUMMARY: Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of Pleurotus ostreatus. Immunoblot analysis revealed that antisera raised against laccase I were not reactive to laccase II. Laccase I, which exhibited faster mobility on nondenaturing polyacrylamide gel, was purified 42·9-fold with an overall yield of 10·8%. Gel filtration and SDS-PAGE revealed that laccase I is a single polypeptide with a molecular mass of approximately 64 kDa. Laccase I contained 12·5% carbohydrate by weight and 3·9 mol copper (mol protein)−1. The absorption spectrum of laccase I showed a type 1 signal at 605 nm and EPR spectra showed that the parameters of the type 1 and type 2 Cu signals were g ‖ = 2·197 and A ‖ = 0·009 cm−1, and g ‖ = 2·263 and A ‖ = 0·0176 cm−1, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose pK a values were 5·60–5·70 and 6·70–6·85, may play an important role in the active site of laccase I as the ligand of copper metal. The optimal pH and temperature for the activity of laccase I were 6·0–6·5 and 30–35 °C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the K m values for ferulic acid and syringic acid were 48 and 89 μM, respectively. EPR spectroscopic study of the action of laccase I on 3,5-dimethoxy-5-hydroxyacetophenone indicated that this enzyme catalyses single electron transfer with the formation of the phenoxy radical as an intermediate.

The mosquito pathogenic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) is a sterol auxotroph that can grow vegetatively in the absence of these compounds, but requires an exogenous source of sterols to enter its sexual and asexual reproductive cycles. Electrospray mass spectrometry (MS) and electrospray MS/MS were used to examine three major glycerophospholipid molecular species-glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE) and glycerophosphoinositol (GPI) - from fungal mycelium and nuclei grown in defined medium with and without isoprenoids which induce (cholesterol and ergosterol) or do not induce (squalene, cholestane) reproduction. Testosterone supplementation of defined media inhibited growth of L. giganteum, so the effect of this steroid on phospholipid metabolism could not be assessed. Mycelium grown in defined media supplemented with these isoprenoids produced significantly different quantities of total phospholipid relative to unsupplemented media and to each other, ranging from a mean of 292 μg phosphate per g wet weight for cholesterol-supplemented media to 56 μg phosphate per g wet weight for mycelium grown in the presence of squalene. A very large percentage of the GPC (69-80 mol%) and GPI (74-79 mol%) molecular species from mycelia and nuclei contained ether linkages. GPE molecular species had 13-20 mol% ethercontaining moieties. The elevated levels of ether lipids may be related to the sterol auxotrophic nature of the fungus. Isoprenoid supplementation of defined growth media resulted in many significant changes in molecular species for all three lipid classes. Significant differences (P < 005) in the percentage of total cell ether lipids in GPC and GPE were generated by isoprenoid supplements to culture media. Mycelium grown in the presence of the two sterols which induce asexual and sexual reproduction in L. giganteum, cholesterol and ergosterol, had a significantly greater percentage of ethercontaining GPE moieties. The glycerolipid species from nuclei isolated from cultures grown with cholesterol and ergosterol were similar to the composition of nuclei isolated from fungus cultured in defined medium without any supplement or supplemented with squalene. The nuclear membrane from mycelia grown in cholestane-supplemented media, however, had a very different glycerophospholipid composition relative to either whole cells or nuclei from cells grown on other media. It appears that one of the reasons that cyclic isoprenoids such as cholestane do not induce fungal reproduction is that they drastically alter the nuclear membrane glycerophospholipid composition.

SUMMARY: The formamidopyrimidine DNA glycosylase gene (fpg-L) of the Gram-positive microaerophilic bacterium Lactococcus lactis subsp. cremoris ML3 has been cloned, characterized and sequenced. Thefpg-L gene is composed of 819 bp encoding a protein of 31·3 kDa (Fpg-L). The deduced amino acid sequence of the Fpg-L protein shows 59% similarity and 38% identity with the Escherichia coli Fpg protein (Fpg-E). Polyclonal antibodies against Fpg-E react with the Fpg-L protein. The Fpg-L protein was purified to apparent homogeneity from the overproducing E. coli strain BH410 hosting plasmid pVE1064, which carries fpg-L under the control of the E. coli lacpromoter. In its active form, Fpg-L is a 30 kDa monomeric enzyme with a measured isoelectric point of 9·0. It contains one zinc per molecule and has a zinc finger motif localized at the carboxy-terminal end (Cys-X2-Cys-X16-Cys-X2-Cys-X3-COOH). The Fpg-L protein has two enzyme activities: DNA glycosylase, which excises 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine and 7,8-dihydro-8-oxoguanine, and DNA nicking at abasic sites. Furthermore, the expression of thefpg-L gene infpgandmutYmutants ofE. colisuppresses their spontaneous GC | TA mutator phenotype. The similarity of the activity of the two Fpg proteins and its conservation in evolutionarily distant bacteria may reflect the importance of its role in protecting bacterial DNA against oxidative free radicals.

The analysis of complex microbiota present in activated sludge is important for the understanding and possible control of severe separation problems in sewage treatment such as sludge bulking or sludge foaming. Previous studies have shown that nocardioform actinomycetes are responsible for these conditions, which not only affect the efficiency of sewage treatment but also represent a threat to public health due to spread of pathogens. However, isolation and identification of these filamentous, nocardioform actinomycetes is hampered by their fastidious nature. Most species are still uncultivable and their taxonomy is unresolved. To study the ecology of these micro-organisms at the molecular level, we have established a clone library of 16S rRNA gene fragments amplified from bulk sludge DNA. A rough indication of the predominant flora in the sludge was given by sequencing randomly chosen clones, which revealed a great diversity of bacteria from different taxa. Colony hybridization with oligonucleotide probe MNP1 detected 27 clones with 16S rDNA inserts from nocardioform actinomycetes and mycobacteria. The sequence data from these clones together with those from randomly chosen clones were used for comparative 16S rRNA analysis and construction of dendrograms. All sequences differed from those of previously sequenced species in the databases. Phenotypic characterization of isolates of nocardioform actinomycetes and mycobacteria cultivated in parallel from the same activated-sludge sample revealed a large discrepancy between the two approaches. Only one 16S rDNA sequence of a cultured isolate was represented in the clone library, indicating that culture conditions could select species which represent only a small fraction of the organisms in the activated sludge.

SUMMARY: The relationship between genes and enzymes in the methionine biosynthetic pathway has been studied in Pseudomonas aeruginosa. The first step is catalysed by an O-succinylhomoserine synthase, the product of the metA gene mapped at 20 min on the chromosome. The second step is achieved by direct sulfhydrylation, involving the enzyme encoded by a metZ gene that we have identified and sequenced, located at 40 min. Thus Pseudomonas appears to be the only organism so far described that uses O-succinylhomoserine as substrate for a direct sulfhydrylation. As in yeast, the two transsulfuration pathways between cysteine and homocysteine, with cystathionine as an intermediate, probably exist in parallel in this organism.

SUMMARY: The effect of glucose on a number of mitochondrial and cytoplasmic enzymes involved in carbon metabolism (L-lactate:ferricytochrome-c 2-oxidoreductase, malate dehydrogenase, β-galactosidase, invertase, maltase and NAD-glutamate dehydrogenase) has been analysed in two different strains of Kluyveromyces lactis (PM4-4B and JA6 strains). All the above mentioned enzymes were catabolite-repressible in a strain-dependent way. From this study differences in the regulation of some enzymes have been observed between K. lactis and Saccharomyces cerevisiae. To identify genes involved in glucose metabolism that may also control carbon catabolite repression, 2-deoxyglucose-resistant mutants (Dgr + ) of K. lactis that were unable to grow on glucose in the presence of antimycin A (Rag − ) were selected. In this way we identified four classes of mutants. Two of them define genes previously identified: RAG1 and RAG5 , encoding the low affinity glucose transport system and hexokinase, respectively. The two remaining classes of mutants define two new genes, DGR148 and DGR239 , that control the level of hexokinase, indicating that in K. lactis this enzyme is positively regulated by at least two genes. All the mutants devoid of hexokinase showed relief from carbon catabolite repression of several enzymes.

SUMMARY: The hydBGDA genes, which encode the four subunits β, γ, δ and α of the [Ni-Fe] hydrogenase from the archaeon Pyrococcus furiosus , have been isolated and sequenced using a PCR/IPCR-based strategy. From the sequence analysis it appears that the four structural genes are tightly linked and organized in a single transcription unit. The hydD and hydA gene products are related to the small and the large subunits of several archaeal and eubacterial [Ni-Fe] hydrogenases with an overall degree of sequence relatedness ranging from 35% to 50% (identity + similarity). In particular, the amino acid sequence motifs involved in the accommodation of nickel and iron-sulfur clusters are conserved. In addition, the database search revealed that the hydB and hydG gene products are homologous to the asrA - and asrB -encoded subunits of the sulfite reductase enzyme from Salmonella typhimurium . This is particularly interesting in view of the recent finding that the P. furiosus hydrogenase appears to be a bifunctional enzyme endowed with both proton- and sulfur- reducing activities.

Screening GenBank indicated that an esterase from Pseudomonas fluorescens had high sequence similarity with bacterial non-haem haloperoxides. However, this homology was limited to two distinct domains of the published esterase sequence. As errors in the published sequence were suspected, the esterase gene was sequenced again. The revised sequence displayed between 40 and 50% identical amino acids with the haloperoxidases, but distributed along the whole sequence. In addition to the structural homologies with haloperoxidases, the esterase also displayed functional homology. The recombinant esterase, purified from Escherichia coli cells, was capable of both ester hydrolysis and halogenation, as detected in situ by the formation of bromophenol blue or spectrophotometrically by the bromination of monochlorodimedon. The esterase is thus a bifunctional enzyme. The sequence analysis and the biochemical investigations show that the esterase belongs to the haloperoxidase family. It also possessed, however, a typical feature of serine-hydrolases, namely the consensus motif Gly-X-Ser-X-Gly around the active serine of the catalytic triad. By alignment of the esterase with different serine-hydrolase sequences, it was possible to identify the other two residues of the triad. The triad comprised the residues Ser95, Asp223 and His252. Interestingly, a structurally equivalent catalytic triad was also identified in the sequences of all bacterial non-haem haloperoxidases, in highly conserved domains. The presence of a catalytic triad in haloperoxidases is expected to be important in the mechanism of halogenation.

The location and organization of repetitive sequences, members of the RPS family, which are sequences specific to Candida albicans, were determined on each chromosome of C. albicans strain FC18. Using pulsed-field gel electrophoresis, we separated seven fractions from eight chromosomes. Each chromosome was cleaved by BamHI and XhoI to excise the RPSs, which were then detected by hybridization with an RPS probe. All chromosomes except chromosome 4 carried RPSs, and these RPSs were located within a limited region on each chromosome. From the digestion of each chromosome with Sfil and probing with the RPSs, we found that these recognition sites within the RPS region were conserved among all RPS-containing chromosomes. For further characterization of the RPSs, the locations and the boundary regions of the RPSs were examined on chromosome 6 of strain FC18 as a model chromosome. Using the restriction enzymes Sfil, Smal, Xhol, BamHl, Mlul and Nrul, we constructed a semi-macro physical map of the RPSs and their boundary regions on this chromosome. We also determined which part of the RPS was adjacent to each boundary by using sub-fragments of RPS as probes. The physical configuration around the RPSs and their boundary regions are presented. The results obtained should be useful for future analysis of the function of these regions.

SUMMARY: Three Xanthobacter Py2 mutants (M3, M8 and M10) lacking epoxyalkane-degrading activity were isolated and characterized. All mutants were able to grow on acetone, the degradation product of 1,2-epoxypropane conversions. Furthermore, they contained the unidentified ‘low molecular mass fraction’ (LMF) necessary for epoxyalkane-degrading activity. Three cosmids from a gene bank complemented the mutation in M10 and M8 but not in mutant M3. Epoxyalkane-degrading activity in crude extracts of 1,2-epoxypropane-grown complemented mutants was similar to the wild-type activity. Surprisingly, M10 transformed with complementing cosmid pEP9 showed a constitutively expressed epoxyalkane-degrading activity, which was not observed in the wild-type strain. The cosmid pEP9 was conjugated into Xanthobacter autotrophicus GJ10, which is not able to degrade 1,2-epoxypropane. In crude extracts of X . autotrophicus GJ10(pEP9), epoxyalkane-degrading activity was demonstrated, but only after the addition of the LMF from Xanthobacter Py2. Hybridization experiments demonstrated an overlap on complementing cosmids pEP1, pEP3 and pEP9. Subcloning revealed a 4.8 kb Eco RI- Hin dlll fragment to be necessary for complementing the mutant M10. In the sequence of this fragment four different ORFs were found.

The nucleotide sequence of the 3-chlorobenzoate 3,4-dioxygenase genes, designated cbaAB,from the transposon Tn5271 was determined. The function of the two sequenced open reading frames was evaluated by mutagenesis and expression in vivo to show that the cbaA and cbaB genes code for dioxygenase and reductase proteins, respectively. Comparison of the deduced amino acid sequences of the cbaAB genes with sequences for other oxygenases revealed a clearly defined lineage among the class IA oxygenases that shows several unique features. This lineage includes phthalate 4,5-dioxygenase (pht23), and based on the available NH3-terminal sequence of component A, also includes 4-sulphobenzoate 3,4-dioxygenase. Vanillate demethylase, encoded by the vanAB genes and formally a monooxygenase enzyme catalysing an oxidative demethylation, is also included in this lineage. The terminal chlorobenzoate dioxygenase (CbaA) component is characterized by a conserved Rieske-type [2Fe-2S]R ligand centre. The reductase component (CbaB) contains a plant-type ferredoxin [2Fe-2S]Fd',� FMN-isoalloxazine and NAD-ribose-binding domains and the orientation of these domains is conserved in all known class IA reductases. These results support the hypothesis that alternative fusions of the electron transfer modules of the reductases arose early in the divergence of oxygenase systems. The over-riding evolutionary constraint acting on the divergence of the class IA oxygenases would appear to be the requirement for a carboxyl group para to the site of oxygen insertion into the aromatic ring.

SUMMARY: ViableCoxiella burnetii organismswere isolated from the culture medium of persistently infected Baby Hamster Kidney (BHK-21) fibroblasts. When these organisms were incubated in host-cell-free medium at low pH, some of the de novo-synthesized protein made by the bacteria was translocated to the exterior of the cell. The exported protein was detectable after 2-7 h incubation at 37°C. No evidence was found to suggest that protein accumulation in the medium was due to leakiness caused by cell damage. Both DCCD (dicyclohexylcarbodiimide) and CCCP (carbonyl cyanide m-chlorophenylhydrazone) inhibited the process to some extent. Exported protein was represented largely by three polypeptides with molecular masses of 34, 24 and 12 kDa. De novo-synthesized proteins corresponding to these molecular masses were not detected in cytoplasmic fractions, but a membrane fraction might possess a similar form. It was concluded that a physiological process of protein translocation occurred in C. burnetii during acid activation in a defined medium. Organisms that were extracted directly from the cytoplasm of infected fibroblasts by a mechanical disruption procedure were also active in de novo protein synthesis; however they exported much less of the protein.

Minimal requirements of amino acids and vitamins were determined in chemically defined medium for five strains of Clostridium difficile. Cysteine, isoleucine, leucine, proline, tryptophan and valine were essential amino acids for growth of C. difficile. Arginine, glycine, histidine, methionine and threonine enhanced growth. Biotin, pantothenate and pyridoxine were essential vitamins. A defined medium containing the minimal requirements of amino acids and vitamins produced a rapid and heavy growth which was comparable to that in modified brain heart infusion, a complex medium. Adenine was able to substitute for glycine and threonine, suggesting that the two amino acids may be utilized as precursors of purine nucleotides. The defined medium developed here will assist physiological and biochemical studies on C. difficile.

Vibrio cholerae cells were incubated at 4 °C in nutrient-limited artificial seawater (ASW) microcosms. Plate counts declined from 8 × 105 to less than 2 c.f.u. ml−1 in about 23 d. When samples of microcosms were shifted to 30 °C, plate counts increased to 2·2×105 c.f.u. ml−1 in 72 h. An experiment was performed to determine whether culturable cells obtained after temperature upshifts were the result of ‘resuscitation’, or outgrowth, of nonculturable cells or of cell division and growth of the few culturable cells that remained in samples. Prior to temperature upshift, samples from the microcosms were diluted 10− and 100−fold in filter-sterilized (0·1 μm) ASW from the microcosms. Undiluted, 1/10, and 1/100 diluted samples recovered culturability to about 2·2×105 c.f.u. ml−1 within 72 h of temperature upshift. If resuscitation of nonculturable cells had occurred, the resultant number of culturable cells in diluted samples would have been 1/10 and 1/100 that of undiluted samples, respectively. In microcosms where plate counts had declined to less than 1 c.f.u. ml−1, 1/100 diluted samples did not regain culturability, i.e. no culturable cells remained from which growth could occur. Our conclusions are that in the experiments reported here, recovery of culturable cells on temperature upshifts resulted from growth and that there were no growthinhibiting factors in the spent growth medium, supported by the finding that about 102 recovered V. cholerae cells ml−1 inoculated into filter-sterilized microcosm ASW grew to about 6·2 ×105 c.f.u. ml−1 in 24 h, confirming that V. cholerae is capable of significant growth in ASW.