Lysozyme of the Mollusk Unio pictorum and the Sensitivity of Alkanotrophic Rhodococci to Its Effect

A preparation of lysozyme from a freshwater bivalve, Unio pictorum, has been isolated by sorption to chitin, and its physicochemical properties have been studied. An assessment of the sensitivity of 48 strains of rhodococci, belonging to the species Rhodococcus rubber, R. luteus, and R. erythropolis (Specialized Collection of Alkanotrophic Microorganisms of the Institute of Ecology and Genetics of Microorganisms, Ural Division of the Russian Academy of Sciences), which were isolated from diverse natural waters, to lysozyme of the mollusk Unio pictorum demonstrated that the three species differ in their sensitivity to its effects. The high resistance of rhodococci to lysozyme is indicative of their considerable permanence in hydrobiocenoses (and, therefore, ability to maintain self-purification of microbiocenoses from hydrocarbons).     

A possible evolutionary lag in the relationship between freshwater mussels and European bitterling

The coevolutionary dynamics between European bitterling Rhodeus amarus and freshwater unionid mussels, which the former parasitize by laying eggs on their gills, were tested. In a series of experiments fish preferences and mussel responses were compared in parasites and hosts of recent (Europe) and ancient (Asia) sympatry. Rhodeus amarus readily oviposited on the gills of all mussel species tested. Fish that laid their eggs on the gills of Asian Anodonta woodiana, however, suffered a dramatic reduction in reproductive success compared to fish that oviposited on the gills of European mussels: Unio pictorum , Unio tumidus , Anodonta anatina and Anodonta cygnea . This difference was the result of egg ejection behaviour by mussels rather than the unsuitability of the internal gill environment for European bitterling embryo development. The ejection response of mussels with a long sympatry with European bitterling was considerably more pronounced than that of mussels with a substantially shorter sympatry. The data support a coevolutionary arms race between bitterling and mussels and point to an evolutionary lag in the relationship between R. amarus and its European mussel hosts.     

Resource partitioning between unionid mussels in a Swedish lake outlet

Partitioning of food and space resources of two dominating unionid mussel species, Anodonta piscinalis and Unio pictorum , was investigated in a South Swedish lake outlet. Differences in diurnal activities were not considered to be a mechanism for niche segregation. The two species ingested particles of almost identical size distribution indicating a high overlap in food. Mussel distribution was correlated to environmental variables including substrate properties, water depth, current velocity, and distance from the lake, the latter being a relative measure of food quality and quantity, U. pictorum dominated near the lake and in the centre of the river while A. piscinalis had its peak density ca. 500 m below the lake and was relatively more abundant along the river margins. At maximum mussel densities, which were found near the lake, a separation in space was more obvious than downstream, implying possible interspecific interactions.     

Effect of mosquito killer insecticides on freshwater mussels

The effect of four insecticides (Fyfanon, K-Othrin, Unitox 7 and Unitox 20) was investigated on the freshwater mussels Anodonta cygnea L., Anodonta anatina L. and Unio pictorum L. The studies were performed in hard water (total salinity of 310 mg/l). The LC50 values for 24, 48, 72, 96 hr and 7 days were determined with a static test at 22 degrees C. All the three mussel species proved to be extremely tolerant against the insecticides tested. The effect of sublethal concentrations of these insecticides on the periodic activity of the mussels was also analyzed, comparing the changes of the active and resting periods. In a concentration of 0.1 microliter/l, all the four insecticides affected the periodic activity of U. pictorum, whereas in A. anatina three of them (Fyfanon, K-Othrin and Unitox 7) and in A. cygnea two of them (K-Othrin and Unitox 7) evoked alterations. This concentration is lower by three to five orders than those of the LC50 values of 96 hr.     

A proximate cue for oviposition site choice in the bitterling (Rhodeus sericeus)

1. We investigated two possible proximate cues used for oviposition site choice by females of the bitterling ( Rhodeus sericeus ), a freshwater fish that spawns on the gills of live unionid mussels. The two cues were the flow velocity and/or oxygen content of water emerging from the exhalant siphon of a mussel.
2. Field observations showed that female bitterling always inspected the exhalant siphons of mussels before they spawned in them. Siphon inspection was not always a prelude to spawning and it may serve as a means of assessing mussel quality. Female skimming behaviour, swimming over a mussel without spawning, may also be used to assess mussel quality, although the mechanism for this is unclear.
3. Measurements of the flow velocity of water emerging from the exhalant siphons of four mussel species ( Anodonta anatina, A. cygnea, Unio pictorum and U. tumidus ) showed a significant difference among species, with U. tumidus having the highest mean flow velocity and U. pictorum the lowest.
4. Measurements of the change in oxygen concentration of water entering a mussel inhalant siphon and leaving its exhalant siphon in field and laboratory studies showed a significant difference among the four mussel species, with A. cygnea exhibiting a significantly higher change in oxygen concentration than the other species.
5. The presence of bitterling embryos in the gills of a mussel significantly increased its oxygen consumption whereas larval glochidia had no significant effect. We discuss oxygen availability as a possible proximate cue for oviposition site choice in bitterling.     

Evaluation of ITS rDNA as a complement to mitochondrial gene sequences for phylogenetic studies in freshwater mussels: an example using Unionidae from north-western Europe

Mitochondrial inheritance in the Unionidae is complex since mitochondria can be inherited from both parents. An increased rate of recombination could lead to erroneous homology assessments, which could cause problems for phylogenetic reconstruction. For this reason we investigated the possibility of using a nuclear marker, the ribosomal internal transcribed spacer region, for phylogenetic studies in the Unionidae, as a complement and comparison to two of the most widely used mitochondrial genes today. The nuclear ribosomal internal transcribed spacer region ( ITS1 , 5.8S , ITS2 ) was sequenced from 72 specimens representing six of the seven species of Unionidae mussels occurring in NW Europe: Unio pictorum , U. tumidus , U. crassus , Anodonta anatina , A. cygnea and Pseudanodonta complanata. Sequences from Margaritifera margaritifera were used as an outgroup . The ITS sequences of all species were found to have low intragenomic and infraspecific variation. Compared to mitochondrial genes ( 16S and COI ) they show an intermediate genetic diversity. Phylogenetic analyses produce tree topologies that are congruent with those resulting from analyses of the mitochondrial sequences. Likewise, an incongruence length difference (ILD) test showed no significant incongruence between data sets, indicating that if recombination has occurred it has not produced any conflicting patterns. Best-resolved and supported trees are obtained when gaps are treated as a fifth character state. A combined analysis of the three gene regions shows that Unio crassus and U. pictorum are more closely related than either to U. tumidus . Pseudanodonta is nested within Anodonta as sister taxon to A. cygnea . Advice on how to best preserve mussel material for DNA studies is provided.     

Characterization of bacteria of the genera Mycobacterium, Rhodococcus and Nocardia isolated from environmental objects in the Republic of Daghestan

The bacteriological study of specimens taken from environmental objects, biomaterials from animals and sputa from tuberculosis patients revealed the ubiquity of Mycobacterium, Nocardia and rhodococci obtained from different kinds of environment in the Republic of Daghestan. In the identification 83.6% out of the isolated atypical Mycobacterium cultures were classified with Ranion groups II and IV. Among Nocardia, the most widespread organisms were N. asteroides (62.7%) and among rhodococci, R. erithropolis (61.9%).     

Mussels as bioindicators of trace metal pollution in the Danube Delta of Romania

Specimens of mussels species Anodonta anatina, Unio pictorum, U. tumidus and surfical sediment samples were collected in the summers of 1994 and 1995 from twelve lakes in the Danube Delta, Romania. Whole mussel tissues were analyzed for metals (Ag, As, Cd, Co, Cu, Cr, Ni, Pb, Se and Zn), and sediment samples were subjected to weak acid extraction and to a sequential extraction procedure, and analyzed for Cr, Cu, Ni and Zn. Total mean Cu and Ag concentrations were statistically greater in Unio species than in Anodonta species, but other metal levels did not significantly differ between mussel species. There was a ten fold variation for Cd and Pb and five fold variation for Zn, Ag and Co concentrations in mussels among lakes. Mean concentrations of As, Cu, Se and Zn in mussel tissues varied two fold among lakes. Zn, Co and Cd concentrations in mussels were significantly negatively correlated with distance gradients from the origin of the Delta and with distance from main channels of the Danube. Other metal concentrations in mussels did not correlate with either distance gradient. Trace metal levels in mussel tissues were correlated with those in weak acid and in sequential extractions of sediment. Zn concentrations in the carbonate fraction was the best predictor of Zn concentrations in mussels. Cu and Ni levels in the organic matter and sulfide fraction were the best estimators of Cu and Ni concentrations in mussels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence for the pathogenic role of Rhodococcus species in pulmonary diseases

Three hundred expectorated sputa from patients suffering from chest disorders were examined microscopically and cultured on various media. Among other micro-organisms isolated there were four strains of Rhodococcus aurantiacus (Gordona aurantiaca), one each of Rh. erythropolis, Rh. pellegrino, Rh. rubropertinctus and Rh. rhodnii. Suspensions in 5% hog gastric mucin were virulent for immuno-suppressed white mice and the pathology of their lungs was compatible with those produced in experimental nocardial and rhodococcus infections. The in-vitro antibiogram was similar to that of Nocardia asteroides. Two of the patients from whom Rh. aurantiacus and Rh. pellegrino were isolated responded bacteriologically and clinically to treatment with co-trimoxazole. The frequency with which these rhodococci were isolated and the clinical conditions of the patients strongly indicated a pathogenic role for some Rhodococcus species. It is suggested that rhodococci should be sought in chronic pulmonary infections, particularly in those who are immuno-compromised or debilitated, and their aetiologic role determined.     

Evidence for the pathogenic role of Rhodococcus species in pulmonary diseases

Three hundred expectorated sputa from patients suffering from chest disorders were examined microscopically and cultured on various media. Among other microorganisms isolated there were four strains of Rhodococcus aurantiacus ( Gordona aurantiaca ), one each of Rh. erythropolis, Rh. pellegrino, Rh. rubropertinctus and Rh. rhodnii. Suspensions in 5% hog gastric mucin were virulent for immuno-suppressed white mice and the pathology of their lungs was compatible with those produced in experimental nocardial and rhodococcus infections. The in-vitro antibiogram was similar to that of Nocardia asteroides. Two of the patients from whom Rh. aurantiacus and Rh. pellegrino were isolated responded bacteriologically and clinically to treatment with co-trimoxazole. The frequency with which these rhodococci were isolated and the clinical conditions of the patients strongly indicated a pathogenic role for some Rhodococcus species. It is suggested that rhodococci should be sought in chronic pulmonary infections, particularly in those who are immuno-compromised or debilitated, and their aetiologic role determined.     

Biochemical and immunological characterization of a glycosylated alpha-fucosidase from the invertebrate Unio: interaction of the enzyme with its in vivo binding partners

Mammalian alpha-fucosidase (EC 3.2.1.51) is a lysosomal enzyme that catalyzes the removal of fucose residues from glycosphingolipids and its absence in humans results in a rare metabolic disorder called fucosidosis. Among the invertebrates in the molluscs (Unio) two forms of the enzyme have been reported, a 68 kDa non-glycosylated form and a 56 kDa glycosylated form. The glycosylated form has been purified from the seminal fluid of Unio [Biochem. Biophys. Res. Commun. 234 (1997) 54]. In the present study, the 56 kDa glycosylated form has been purified to homogeneity from the whole body tissue of Unio using a series of chromatographic steps. The purified enzyme migrated as a single protein species in 10% SDS-PAGE. Antibodies to the purified enzyme were raised in a rabbit in order to study its biochemical and immunological properties. The purified enzyme is a glycoprotein that exhibits strong binding to Con A-Sepharose gel and can be deglycosylated by PNGase F enzyme suggesting it to be N-glycosylated. The enzyme has been shown to specifically interact with the mannose 6-phosphate receptor protein (MPR 300) purified from goat and Unio. This specific interaction is discussed in view of its possible in vivo binding partners.     

Ciliary activity of cells of gill and leg glimmeral epithelium of Unionidae invaded by trematodes of Aspidogaster conchicola and Bucephalus polymorphus

The paper provides data concerning the influence of the parasitic worms Aspidogaster conchicola and Bucephalus polymorphus on the rate and duration of ciliary beating of gill and leg in 7 species of Unionidae (Unio conus borysthenicus, U. tumidus falcatulus, U. rostratus rostratus, U. limosus graniger, U. pictorum ponderosus, Colletopterum piscinale falcatum, C. ponderosum rumanicum). The high level of infection of molluscs with B. polymorphus oppresses the glimmeral epithelium activity of gill and leg (by 4.2-32.3%). The weak and moderate levels of infection of molluscs with B. polymorphus do not decrease the glimmeral epithelium activity and sometimes even rise it (by 3.4-8.1%). The presence of few A. conchicola (1-3 individuals) in the organism of mollusc does not change the functioning of glimmeral epithelium.     

Fluorene degradation by bacteria of the genus Rhodococcus

Of the four investigated Rhodococcus strains (R. rhodochrous 172, R. opacus 4a and 557, and R. rhodnii 135), the first three strains were found to be able to completely transform fluorene when it was present in the medium as the sole source of carbon at a concentration of 12-25 mg/l. At a fluorene concentration of 50-100 mg/l in the medium, the rhodococci transformed 50% of the substrate in 14 days. The addition of casamino acids and sucrose (1-5 g/l) stimulated fluorene transformation, so that R. rhodochrous 172 could completely transform it in 2-5 days. Nine intermediates of fluorene transformation were isolated, purified, and structurally characterized. It was found that R. rhodnii 135 and R. opacus strains 4a and 557 hydroxylated fluorene with the formation of 2-hydroxyfluorene and 2,7-dihydroxyfluorene. R. rhodochrous 172 transformed fluorene via two independent pathways to a greater degree than did the other rhodococci studied.     

Selection of Microbial Population in Chlortetracycline-treated Ocean Perch (Sebastodes alutus)

Microorganisms isolated from chlortetracycline (CTC)-treated ocean perch were individually tested to determine their relative sensitivity to CTC by use of CTC-impregnated paper discs. The CTC sensitivity varied among isolates obtained from untreated fish. The proportion of CTC-resistant species increased with higher CTC concentration and the length of storage at 7 C. Among individual generic groups, "coryneforms" and yeasts were more resistant to CTC than other groups. Pseudomonas, Achromobacter, Flavobacterium, Bacillus, and Lactobacillus all contained species either resistant or sensitive to CTC. More CTC-resistant species in these genera accumulated with the increased CTC concentrations and with the length of storage in the presence of CTC.     

Characterization of LtsA from Rhodococcus erythropolis, an enzyme with glutamine amidotransferase activity

The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ltsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ltsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ltsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH(4)Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH(4)Cl, indicating that NH(3) can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.     

Host species preferences by bitterling, Rhodeus sericeus, spawning in freshwater mussels and consequences for offspring survival

Two hypotheses have been advanced to explain the evolution of host responses to parasites: the arms race-evolutionary lag and equilibrium hypotheses. We investigated predictions from these hypotheses based on interspecies host preferences and adaptations in an obligate spawning relationship between a freshwater fish, the European bitterling (Cyprinidae) and four species of freshwater mussels (Unionidae), which the fish use as hosts for their eggs. We found a significant trend in preference by the fish for mussels in the following order: Unio pictorum, U. tumidus, Anodonta anatina and A. cygnea. Male and female bitterling both showed this ranking and the clutch sizes deposited into each species also followed this trend. These host preferences proved to be adaptive in terms of egg ejection, which was lowest in the most preferred species (U. pictorum). Furthermore, these hierarchical host preferences were flexible, as females switched species when individuals of the preferred species ejected a greater number of eggs. The similarity in mussel defences between the U.K. population and a European population of ancient sympatry suggests that the absence of a defence in some mussel species may not be due to evolutionary lag. Mussel ejection behaviour may have reached an evolutionary equilibrium in each host species, or alternatively the fish may have evolved adaptive preferences that coincide with generalized mussel responses to foreign objects in their gills. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Physiology, biochemistry and taxonomy of deep-sea nitrile metabolising Rhodococcus strains

A collection of nitrile-hydrolysing rhodococci was isolated from sediments sampled from a range of deep coastal, and abyssal and hadal trench sites in the NW Pacific Ocean, as part of our programme on the diversity of marine actinomycetes. Nitrile-hydrolysing strains were obtained by batch enrichments on nitrile substrates with or without dispersion and differential centrifugation pre-treatment of sediments, and were recovered from all of the depths sampled (approximately 1100–6500 m). Two isolates obtained from the Ryukyu (5425 m) and Japan (6475 m) Trenches, and identified as strains of Rhodococcus erythropolis,were chosen for detailed study. Both of the deep-sea isolates grew at in situ temperature (4°C), salinities (0–4% NaCl) and pressures (40–60 MPa), results that suggest, but do not prove, that they may be indigenous marine bacteria. However, the absence of culturable Thermoactinomycespoints to little or no run off of terrestrial microbiota into these particular trench sediments. Nitrile-hydrolysis by these rhodococci was catalysed by a nitrile hydratase–amidase system. The hydratase accommodated aliphatic, aromatic and dinitrile substrates, and enabled growth to occur on a much wider range of nitriles than the only other reported marine nitrile-hydrolysing R. erythropolis which was isolated from coastal sediments. Also unlike the latter strain, the nitrile hydratases of the deep-sea rhodococci were constitutive. The possession of novel growth and enzyme activities on nitriles by these deep-sea R. erythropolisstrains recommends their further development as industrial biocatalysts.     

DNA base composition and homology values in the classification of some Rhodococcus species

Similar DNA homology values were recorded when a modified S1 nuclease technique and a standard nitrocellulose membrane filter method were applied to representative strains of Rhodococcus. The DNA homology data showed that R. globerulus, R. luteus and R. sputi form distinct genomic species. The congruence between the DNA homology and earlier numerical phenetic data was good, but there was evidence that some strains had been misclassified in the previous studies. In particular, the type strains of R. obuensis and R. sputi belong to a single genomic species. The former name is thus a later, subjective synonym of the latter. The guanine plus cytosine content of the DNA of the rhodococci fell within the range 61 to 72 mol%.     

Aquatic ecosystem as a bioreactor: water purification and some other functions.

A fundamental concept is proposed of aquatic ecosystem as a bioreactor that carries out the function of water purification in natural water bodies and streams. The ecosystem as a bioreactor has the following characteristic attributes: (1) it is a large-scale (large-volume) bioreactor; (2) it is a diversified (in terms of the number of taxa and the scope of functional activities) bioreactor; (3) it possesses a broad range of biocatalytic (chemical-transforming and degrading) capabilities. New experimental data on xenobiotics-induced inhibition of the functioning of the molluscs Unio tu- midus, U. pictorum, M. galloprovincialis and Limnaea stagnalis emphasize the potential ecological hazard from sublethal concentrations of pollutants (including those exemplified by synthetic surfactants and detergents).     

Lysis of modified walls from Lactobacillus fermentum.

The N and O substitution in wall peptidoglycan from Lactobacillus fermentum was studied in relation to growth phase, as well as the lytic activities and the effect of trypsin on them. The N-nonsubstituted sites were determined by dinitrophenylation techniques. The results indicate that an extensive substitution at the O groups takes place as cells go into the stationary growth phase, concomitant with a decrease in their lysozyme sensitivity. N-nonsubstituted residues, mainly glucosamine, occurred in both exponential-phase and stationary-phase walls but not in the corresponding peptidoglycans. Small amounts of N-nonsubstituted muramic acid were detected in walls and peptidoglycan from cells in the stationary growth phase only. N acetylation of isolated walls did not increase their lysozyme sensitivity but rather decreased it. Autolysis of walls was completely inhibited by the chemical modifications used. Trypsin stimulates the lysozyme sensitivity of native walls but has no effect on walls that had been O deacetylated and N acetylated. It is suggested that the effect of trypsin is due to its action as an esterase removing the O acetylation in lysozyme-resistant walls.