Selection of glucose-assimilating variants ofAcinetobacter calcoaceticus LMD 79.41 in chemostat culture

Glucose metabolism has been studied in two strains ofAcinetobacter calcoaceticus. Strain LMD 82.3, was able to grow on glucose and possessed glucose dehydrogenase (EC 1.1.99.17). Glucose oxidation by whole cells was stimulated by PQQ, the prosthetic group of glucose dehydrogenase. PQQ not only increased the rate of glucose oxidation and gluconic acid production but also shortened the lag phase for growth on glucose. Strain LMD 79.41 also possessed glucose dehydrogenase but was unable to grow on glucose. Batch cultures and carbon-limited chemostat cultures growing on acetate in the presence of glucose oxidized the sugar to gluconic acid, which was not further metabolized. However, after prolonged cultivation on mixtures of acetate and glucose, carbon-limited chemostat cultures suddenly acquired the capacity to utilize gluconate. This phenomenon was accompanied by the appearance of gluconate kinase and a repression of isocitrate lyase synthesis. In contrast to the starter culture, cells from chemostats which had been fully adapted to gluconate utilization, were able to utilize glucose as a sole carbon and energy source in liquid and solid media.     

Paenibacillus isolates possess diverse dextran-degrading enzymes

AIMS: To isolate and identify dextran-degrading organisms from sugar mill and compost samples, and to examine the diversity of the dextranolytic enzymes produced. METHODS AND RESULTS: Fifteen dextranolytic prokaryotes were purified at various temperatures from sugar-mill or compost samples, using indicator plates containing blue dextran. A 16S rRNA gene sequence analysis showed that 12 isolates purified at 40, 50 or 70 degrees C were closely aligned to Paenibacillus spp. The three isolates purified at 60 degrees C had identical 16S rDNA sequences, with highest affinity to Bacillus spp. Liquid culture of the 11 isolates purified at 40 or 50 degrees C produced dextranolytic activity in the spent media with maximal activity at 40 or 45 degrees C under the assay conditions used. Hydrolysis of blue dextran in activity gels showed that the 12 Paenibacillus isolates produced from one to five dextranolytic proteins, ranging from 70 to 120 kDa. Based on 16S rDNA sequence, growth habit in liquid culture and dextranolytic enzyme pattern, the 12 Paenibacillus-like isolates could be differentiated into six distinct groups, one of which was capable of growth at 70 degrees C. CONCLUSIONS: The Bacillales, especially the Paenibacillus, are a valuable environmental repository for dextranolytic enzymes of diverse size and potentially diverse activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Dextranolytic enzymes produced by Paenibacillus spp. are an exploitable resource for those interested in modifying the structure of dextrans.     

PCR amplification of four genes coding for aminoglycoside-modifying enzymes in bacteria of clinical isolates from Jordan University Hospital

Three species of Gram-negative G(–) bacteria were chosen for this study: Escherichia spp. (29 isolates), Pseudomonas spp. (16 isolates) andEnterobacter spp. (17 isolates), utilizing colony PCR to detect genes coding for aminoglycoside-modifying enzymes. The 62 isolates were purified and cultured on nutrient broth media supplemented with 50 g kanamycin/ml. Only 17 out of the 62 isolates were resistant to kanamycin and were subjected to colony PCR protocol using 20 l cell lysate and eight sets of primers with each isolate. Sizing of the amplified fragments was carried out in order to determine the specificity of PCR. The 17 isolates were shown to carry the rrs, aacC2, aacA-aphD and aphA3 genes.     

Plasmids in clinical isolates ofStreptococcus pneumoniae

Forty clinical isolates ofStreptococcus pneumoniae with various antibiotic resistance profiles were screened for the presence of plasmids. Plasmids were demonstrated in five isolates. Three procedures for plasmid isolation were evaluated. A 10.8-kb plasmid was demonstrated by all three methods, but a further four plasmids were detected with one method only. The sizes of these plasmids were 11.5 kb, 10.8 kb, and 3.0 kb (two strains). Curing experiments were performed, but no plasmid/antibiotic correlation was observed. Tetracycline, erythromycin, and clindamycin resistance was lost in one strain, although the plasmid was still present.     

Genotypes of Candida dubliniensis in clinical isolates

Amplification of specific sequences of the ITS1 and ITS2 regions and the intervening 5.8S rRNA gene has lead to the identification of four separate genotypes in Candida dubliniensis. Using primers specific for each genotype, we have studied the prevalence of these genotypes among 68 clinical isolates, mostly from Spanish patients infected by HIV. The majority of the isolates tested belonged to genotype 1 (97%), while only one isolate each from genotypes 2 (1.5%) and 3 (1.5%) were detected in the oral cavity of two patients with HIV infection.     

Pyrimidine metabolism in Acinetobacter calcoaceticus

The metabolism of thymine, thymidine, uracil, and uridine has been investigated in five different strains of Acinetobacter calcoaceticus. Attempts to isolate thymine and thymidine auxotrophic mutants were not successful. Consistent with this finding was the observation that uptake of radioactive thymine or thymidine could not be demonstrated. Search for enzymes capable of transforming thymine via thymidine to thymidine-5'-monophosphate in crude extracts was performed, and the following enzymes were absent judging from enzyme assays: thymidine phosphorylase (EC 2.4.2.4), trans-N-deoxyribosylase (EC 2.4.2.6), and thymidine kinase (EC 2.7.1.21). The enzymes responsible for the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-triphosphate were present in crude extracts. Radioactive uracil was readily incorporated into both ribonucleic acid and deoxyribonucleic acid, the ratio being 6:1, and radioactivity was found only in pyrimidine bases. No uptake of uridine could be demonstrated. Uridine-5'-monophosphate pyrophosphorylase (EC 2.4.2.9) activity was detected in crude extracts, suggesting that uracil is converted directly to uridine-5'-monophosphate which is then phosphorylated to uridine-5'-triphosphate or transformed to other ribo- and deoxypyrimidine nucleotides.     

Actinobacterial chitinase-like enzymes: profiles of rhizosphere versus non-rhizosphere isolates

The objective of this study was to determine if antifungal actinomycetes isolated from rhizosphere and non-rhizosphere soils exhibit different chitinase-like production and (or) induction patterns. Selected isolates from both habitats were compared. Chitinase-like levels and isoform characteristic patterns were evaluated over time in culture fluids of isolates grown on media containing different combinations of colloidal chitin and fungal cell wall (FCW) preparation. Supernatants were also subjected to native and non-native polyacrylamide gel electrophoresis (PAGE), using glycol chitin amended gels. For non-native PAGE, protein samples were denatured by two different approaches. Multiple active bands, ranging from 20 to 53 kDa and present in varying amounts, were detected in gels for most strains. Different substrate preferences were observed among strains, and different chitinase-like enzymes were produced, depending upon the substrate combinations used. The presence of FCW in the medium induced specific chitinase-like enzymes not observed otherwise. Enzymatic activities and profiles of the isolates, however, were strain and substrate specific rather than habitat specific. However, a sagebrush rhizosphere soil had a larger actinomycete community with higher chitinolytic activities than the nearby bulk soil. The use of PAGE to compare chitinase-like proteins induced in media with and without FCW was useful for identifying chitinase-like enzymes potentially involved in antifungal activity.     

Hydrolytic enzymes and surfactants of bacterial isolates from lubricant-contaminated wastewater

Fifteen bacterial monocultures were isolated from lubricant-contaminated wastewater of an electric power station in Sofia. Six isolates showed best growth in liquid media with 1.5% hexadecane, and on mineral salt agar plates supplemented with one of the following hydrocarbons: n-hexadecane, paraffin, kerosene and samples of wastewater. The ability of all isolates to produce extracellular hydrolytic enzymes and surface-active glycolipids was assessed on the basis of their growth on hydrocarbons. The study of this relatively closed micro-ecosystem revealed the existence of well-balanced microbial consortium where different members have their own role and support each other. On this basis, an alternative approach is proposed for bioaugmented clean up of wastewater contaminated with hydrocarbons and organic polymers using a mixed culture of indigenous bacteria that combines the best producers of glycolipids and hydrolytic enzymes.     

Production of hydrolytic enzymes by oral isolates of Eikenella corrodens

Abstract Thermus thermophilus cells harboring an expression plasmid for the aqualysin I gene secrete the mature enzyme into the medium. In an Escherichia coli expression system, a precursor of the enzyme with the C-terminal pro-sequence is accumulated in the cells, and upon treatment at 65°C the active enzyme is produced. One- to 10-amino acid residue deletions, as well as complete 105-residue deletion of the C-terminal pro-sequence from the C-terminus, did not affect the production of the enzyme in T. coli cells. T. thermophilus cells harboring plasmids for mutant precursors with one- and three-residue deletions secreted the enzyme extracellularly. However, transformants harboring plasmids for mutant precursors with deletions of five or more amino acid residues could not be obtained. These results suggest that the C-terminal pro-sequence plays an important role in the extracellular secretion of the enzyme in T. thermophilus cells.     

Biological variability in clinical isolates of Trichomonas vaginalis

Eighteen clinical isolates of Trichomonas vaginalis were obtained from women who attended health centers of the Government of Madrid. A total of 1,848 vaginal specimens recovered during the gynaecological examination were seeded in culture tubes containing liquid Diamond medium. Pathogenicity to mice was determined after intraperitoneal inoculation of mice by quantification of mortality and gross damage to abdominal organs. As could be expected, a broad variability was obtained, being some of the isolates more virulent than the reference strain. Regarding to metronidazole susceptibility, none resistant isolate was found but different degrees of susceptibility were determined.     

Heavy metal resistance in clinical isolates ofPseudomonas aeruginosa

One hundred clinical isolates of Pseudomonas aeruginosa were checked for their sensitivity towards silver nitrate. Majority of the isolates were resistant at 20 mg/L and the resistance decreased with increasing concentration of silver nitrate, only 5% of the organisms showed resistance above 70 mg/L. These silver-resistant isolates were further checked for their resistance towards mercury and cadmium at 20 mg/L of concentration and the level of resistance was found to be 33 and 40%, respectively. A correlation between silver ion resistance and concurrent mercury and cadmium ion resistance was observed, suggesting a possible linkage between resistance towards various metal ions.     

UV-inducible DNA repair in Acinetobacter calcoaceticus

Bacterial mutation frequency after UV irradiation and phage mutation frequency under conditions of W-reactivation were determined in A. calcoaceticus. With the exception of streptomycin resistance, there was no increase in the frequency of the assayed markers above the background level. The increased survival of phage during W-reactivation was not followed by an increase in the frequency of mutation from turbid to clear plaque formers among phage survivors. The findings suggested that the UV-inducible repair pathway in A. calcoaceticus was error free. Post-irradiation incubation of UV-treated culture before phage infection resulted in a further increase of W-reactivation. As chloramphenicol inhibited this response, it was concluded that de novo protein synthesis was involved in the UV-inducible repair pathway in A. calcoaceticus.     

Phospholipase activities in clinical and environmental isolates of Acanthamoeba

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A(2) (PLA(2)) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA(2). Acanthamoeba exhibited optimal phospholipase activities at 37℃ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA(2)-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.     

Production of extracellular enzymes by isolates of Metarhizium anisopliae

Extracellular enzyme production in solid culture media was analyzed in order to determine the variability among different Metarhizium anisopliae isolates. Using specific substrates, amylase, lipase, chitinase, and protease production was tested in 11 isolates from different regions of Brazil. Enzyme production was determined by the formation of a halo around the colony, and the diameters of both halo and colony were measured. The enzymatic index was expressed by the colony diameter/halo diameter ratio. In general, the isolates from the same region had similar enzymatic indexes, although similar indexes were also found for isolates from geographically distinct regions. The different isolates were tentatively grouped according to index similarity.