Impact of metal pollution on fungal diversity and community structures

The impact of metal pollution on plant communities has been studied extensively in the past, but little is known about the effects of metal pollution on fungal communities that occur in metal‐polluted soils. Metal‐tolerant ecotypes of the ectomycorrhizal fungus Suillus luteus are frequently found in pioneer pine forests in the Campine region in Belgium on metal‐polluted soils. We hypothesized that metal pollution would play an important role in shaping below‐ground fungal communities that occur in these soils and that Suillus luteus would be a dominant player. To test these hypotheses, the fungal communities in a young pine plantation in soil polluted with zinc, and cadmium were studied using 454 amplicon pyrosequencing. Results show that zinc, cadmium and soil organic matter content were strongly correlated with the fungal community composition, but no effects on fungal diversity were observed. As hypothesized, S. luteus was found to be a dominant member of the studied fungal communities. However, other dominant fungal species, such as Sistotrema sp., Wilcoxina mikolae and Cadophora finlandica were found as well. Their presence in metal‐polluted sites is discussed.     

Culture‐dependent and culture‐independent characterization of potentially functional biphenyl‐degrading bacterial community in response to extracellular organic matter from Micrococcus luteus

Biphenyl (BP)‐degrading bacteria were identified to degrade various polychlorinated BP (PCB) congers in long‐term PCB‐contaminated sites. Exploring BP‐degrading capability of potentially useful bacteria was performed for enhancing PCB bioremediation. In the present study, the bacterial composition of the PCB‐contaminated sediment sample was first investigated. Then extracellular organic matter (EOM) from Micrococcus luteus was used to enhance BP biodegradation. The effect of the EOM on the composition of bacterial community was investigated by combining with culture‐dependent and culture‐independent methods. The obtained results indicate that Proteobacteria and Actinobacteria were predominant community in the PCB‐contaminated sediment. EOM from M. luteus could stimulate the activity of some potentially difficult‐to‐culture BP degraders, which contribute to significant enhancement of BP biodegradation. The potentially difficult‐to‐culture bacteria in response to EOM addition were mainly Rhodococcus and Pseudomonas belonging to Gammaproteobacteria and Actinobacteria respectively. This study provides new insights into exploration of functional difficult‐to‐culture bacteria with EOM addition and points out broader BP/PCB degrading, which could be employed for enhancing PCB‐bioremediation processes.     

Deploying strain specific hypersensitive resistance to diminish temporal virus spread

Spread of necrotic and non‐necrotic strains of Bean yellow mosaic virus (BYMV) was compared when aphid vectors moved both types from external or internal virus sources to plots of Lupinus spp. (lupin). Regardless of whether virus sources were internal or external, removed or left in place, and spread was within plots with homologous sources or across buffers to plots containing the opposite type of virus source, non‐necrotic BYMV always spread faster than necrotic BYMV in plots of L. angustifolius (narrow‐leafed lupin). When necrotic BYMV spread from external sources into plots sown with two L. angustifolius genotypes differing in their necrosis responses to different BYMV strain groups and one genotype of L. luteus (yellow lupin) giving only non‐necrotic responses, differing symptom reactions in the two L. angustifolius genotypes revealed presence of two distinct necrotic BYMV strain groups and overall virus spread was greater in this species than in L. luteus. Spread of non‐necrotic BYMV in L. angustifolius was always polycyclic in nature. However, when it came initially from external sources, spread of necrotic BYMV was largely monocyclic. This work demonstrates how temporal virus spread can be diminished when hypersensitive (necrotic) resistance is deployed and the limitations associated with employing hypersensitivity that is strain specific.     

Species-specific EcoRI repetitive elements of at least 16 kb in length are present in Lupinus luteus

Summary A genomic DNA library of Lupinus luteus cv. Ventus was constructed in the phage vector EMBL3 using Mb oI-digested DNA. Screening with a 1070 bp labelled repetitive unit from L. luteus yielded several DNA clones. The repetitive family is composed of elements whose length is at least 16 kb. The average copy number of the cloned fragments is 5.0 × 10⁴ per haploid genome and constitutes approximately 3% of the total L. luteus genome. The homologous repeats were found in all ten cultivars of L. luteus tested but were not detected in two cultivars each of the closely related species Lupinus albus and Lupinus angustifolius. The EcoRI family fragments could thus be considered as species-specific DNA elements. These fragments may be useful as molecular markers in the genetic manipulation of L. luteus.     

The biogeographic legacy of an imperilled taxon provides a foundation for assessing lineage diversification,demography and conservation genetics

Aim To test alternative biogeographic hypotheses related to the diversification of a montane mammal (Zapus hudsonius luteus) endemic to the American Southwest. Location South‐western United States. Methods We used statistical phylogeographic analyses of mitochondrial DNA (1512 bp; two genes) from 93 individuals from six geographic regions to test diversification hypotheses. Species distribution models of climate and fossil records were integrated to assess contemporary and historical distributions and barriers to gene flow. We calculated dates of divergence and examined historical demography using coalescent simulations. Results We documented monophyly of Z. h. luteus represented by 19 segregated haplotypes. Predicted current distribution generally coincided with known localities, while predicted paleodistributions suggested that this lineage was widespread throughout lower elevations of the American Southwest and on the Edwards Plateau (as documented by the fossil record). Population size did not change substantially during a westward shift in range that occurred in the last 100 k generations. Results supported fragmentation of a common ancestor during the Holocene as the most plausible explanation for genetic structure. Main conclusions Monophyletic Z. h. luteus reflects fragmentation of a common ancestor with recent (Holocene) upslope colonization of disjunct montane areas. We refute the hypotheses of in situ divergence or origins from a Colorado Piedmont ancestor. Instead, westward colonization from the Edwards Plateau during the Wisconsin followed by Holocene fragmentation, which serves as a generalized biogeographic hypothesis for species associated with mesic graminoid habitats in the American Southwest. Further exploration of these signatures using independent nuclear DNA is warranted. Key conservation implications are (1) Z. h. luteus is a monophyletic lineage on an independent evolutionary trajectory; (2) Z. h. luteus shared a recent common ancestor with Z. h. pallidus (not Z. h. preblei); (3) mtDNA does not reflect recent population declines; and (4) coalescent simulations and species distribution models reflect Holocene fragmentation.     

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

Growth promoting and inhibiting effects of extracellular substances of soil microalgae and cyanobacteria on Escherichia coll and Micrococcus luteus

Different taxa of chlorophycean, trebouxiophycean and xanthophycean soil microalgae and of cyanobacteria have been tested for the release of substances that inhibit the growth of either Echerichia coli (Migula) Castellani et Chalmersor Micrococcus luteus (Schroeter) Cohn. Experiments suggest two types of antibacterial effects: one type is constitutive; that is, the antibacterial activity is always present in the algal culture medium, as is the case with the Chroococcus turgidus (medium that inhibits the growth of Escherichia coli). The other type is induced; that is, the antibacterial activity occurs only when algae are in contact with bacteria. This is the case when growth of Micrococcus luteus is inhibited in co‐culture with Chroococcus turgidus (Kützing) Nägeli or with Xanthonema debile (Vischer) Silva and when growth of Escherichia coll is inhibited in co‐culture with Tetracystis sp. As well as inhibition, promotion of bacterial growth was observed. This was probably an unspecific effect resulting from soluble organic and inorganic substances, such as carbohydrates, that are generally present in algal cultures.     

ATP- and polyphosphate-dependent bacterial NAD+ kinases

Measurable levels of activity of NAD⁺ kinases of actinomycetesMicrococcus luteus andCoryne-bacterium ammoniagenes were observed after substituting inorganic tripolyphosphate for ATP, whereas the enzyme from the eubacteriumEscherichia coli was not active with this substrate. Gradient PAGE found two molecular isoforms of NAD⁺ kinase inC. ammoniagenes andE. coli; four forms were found inM. luteus. All isoforms of this enzyme found inC. ammoniagenes andM. luteus displayed NADP-synthesizing activity in the presence of either ATP or tripolyphosphate. Because of its capability of utilizing inorganic tripolyphosphate,M. luteus is the most promising NADP producer organism.     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus,Peridinium balticum,and Crypthecodinium cohnii 1, 2

Chromatin from a uninucleate dinoflagellate, Crypthecodinium cohnii, a binucleate dinoflagellate, Peridinium balticum, and a chromophyte, Olisthodiscus luteus, was examined by nuclease digestion and the results were compared to those from vertebrates. Gel analysis of the products of staphylococcal (micrococcal) nuclease digestion revealed a DNA repeat unit of 220(±5) base pairs for O. luteus and 215(±5) for P. balticum. Limit digestion gave a core particle of 140 base pairs, revealing that these longer repeat sizes are due to longer linker regions. No repeating subunit structure was found upon electrophoresis of digests of C. cohnii nuclei. Examination of the DNA fragments produced by DNAse I digestion of nuclei isolated from P. balticum and O. luteus showed the same ladder of ten base multiples as seen in chromatin from other eukaryotes. Examination of the kinetics of digestion by DNAse II of Peridinium chromatin revealed less susceptibility when compared to DNAse I digestions while 70% of Olisthodiscus chromatin and 35% of C. cohnii chromatin was sensitive to DNAse II. These data, taken together with previous results from Euglena, indicate that while algal chromatin is similar to that of higher eukaryotes in regard to DNAse I and II action, it differs in that the linker DNA is longer. In addition, the Hl-like histone from O. luteus and P. balticum is located in the linker DNA as in higher eukaryotes.     

Iron-dependent growth of and siderophore production by two heterotrophic bacteria isolated from brackish water of the southern Baltic Sea

Iron is indispensable to the growth and metabolism of all marine organisms, including bacteria. In this work, we investigated and compared the influence of iron(III) concentration on the growth of and siderophore production by two heterotrophic bacteria – Micrococcus luteus and Bacillus silvestris.Our results showed that the iron concentration strongly influences the growth of both species. The growth curves were different for each iron concentration and each strain. M. luteus grew more rapidly than B. silvestris, but produced a roughly four times smaller quantity of siderophores. Both M. luteus and B. silvestris secreted hydroxamate-type siderophores and α-keto/α-hydroxy acids, but did not produce catecholates.This paper is probably the first to report on siderophore production by B. silvestris and M. luteus isolated from seawater. Moreover, the influence of different iron concentrations on the growth of and siderophore production in these bacteria has been documented. This provides further evidence indicating iron bioavailability as the actual reason for siderophore release by biota.     

Antimicrobial and Cytotoxic Activity of Extracts of Ferula heuffelii Griseb. ex Heuff. and Its Metabolites

The antimicrobial and cytotoxic activities of isolates (CHCl₃ and MeOH extracts and selected metabolites) obtained from the underground parts of the Balkan endemic plant Ferula heuffelii Griseb . ex Heuff . were assessed. The CHCl₃ and MeOH extracts exhibited moderate antimicrobial activity, being more pronounced against Gram‐positive than Gram‐negative bacteria, especially against Staphylococcus aureus (MIC=12.5 μg/ml for both extracts) and Micrococcus luteus (MIC=50 and 12.5 μg/ml, resp.). Among the tested metabolites, (6E)‐1‐(2,4‐dihydroxyphenyl)‐3,7,11‐trimethyl‐3‐vinyldodeca‐6,10‐dien‐1‐one ( 2 ) and (2S*,3R*)‐2‐[(3E)‐4,8‐dimethylnona‐3,7‐dien‐1‐yl]‐2,3‐dihydro‐7‐hydroxy‐2,3‐dimethylfuro[3,2‐c]coumarin ( 4 ) demonstrated the best antimicrobial activity. Compounds 2 and 4 both strongly inhibited the growth of M. luteus (MIC=11.2 and 5.2 μM , resp.) and Staphylococcus epidermidis (MIC=22.5 and 10.5 μM , resp.) and compound 2 additionally also the growth of Bacillus subtilis (MIC=11.2 μM ). The cytotoxic activity of the isolates was tested against three human cancer cell lines, viz., cervical adenocarcinoma (HeLa), chronic myelogenous leukemia (K562), and breast cancer (MCF‐7) cells. The CHCl₃ extract exhibited strong cytotoxic activity against all cell lines (IC₅₀<11.0 μg/ml). All compounds strongly inhibited the growth of the K562 and HeLa cell lines. Compound 4 exhibited also a strong activity against the MCF‐7 cell line, comparable to that of cisplatin (IC₅₀=22.32±1.32 vs. 18.67±0.75μM ).     

Biosorption of Strontium from Aqueous Solutions by Micrococcus luteus Sr02

Although strontium (Sr²⁺) is found in soils, sediments and surface waters, there is limited evidence about its biosorption. In this study, a surface water strain being highly tolerant to Sr²⁺ was isolated and identified as Micrococcus luteus Sr02 by using 16S rRNA sequencing. Biosorption behavior and mechanisms of Sr²⁺ by M. luteus Sr02 were investigated through batch experiments, zeta potential measurements, Fourier transform infrared spectroscopy and scanning electron microscopy. The results showed that M. luteus Sr02 have potential to sorb Sr²⁺ and can be used efficiently for the removal of Sr²⁺ from aqueous solutions.     

Nitroxyl compounds in bacteria: Search for functions

A method was developed for incorporation of two deuterium atoms into the reactive group of a new type of nitroxyl compounds from Micrococcus luteus (a lysodektose which can be transformed into a long-living free radical). This compound was found to persist in the cells treated with gentamycin and chloramphenicol, while treatment with gramicidin C, ampicillin, benzyl viologen, furadonin, and mercury chloride resulted in a drastic decrease of its intracellular content. Cultivation of M. luteus in a medium with EDTA resulted in accumulation of much higher amounts of lysodektose (up to 300%), and this phenomenon is interpreted as an indication of the possible siderophore function of the compound. Since the M. luteus genome was recently sequenced, this may help to understand the fate and role of lysodektose in bacterial metabolism.     

STEROLS OF DICTYOCHA FIBULA (CHRYSOPHYCEAE) AND OLISTHODISCUS LUTEUS (RAPHIDOPHYCEAE)1

Sterols from cultured Dictyocha fibula Ehrenberg and Olisthodiscus luteus Carter were extracted and identified for comparison with sterols from other Chromophycota species. Although orientation at C-24 was not absolutely determined, the sterols of Dictyocha, a silicoflagellale, appeared to consist of only 24-methyl-22-dehydrocholesterol and 24-methylenecholesterol, a sterol composition not known in any other alga. This is in keeping with the taxonomic isolation Dictyocha has among algae. On the other hand, Olisthodiscus luteus contained 24-ethylcholesterol, 24-ethylcholestanol, 24-methylcholesterol, and cholesterol. This composition is in accord with sterols of members of the Xanthophyceae and Raphidophyceae.     

Digestion by serine proteases enhances salt tolerance of glutaminase in the marine bacterium <Emphasis Type="Italic">Micrococcus luteus</Emphasis> K-3

Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase.     

Bioassay‐Guided Fractionation,Chemical Compositions and Antibacterial Activity of Extracts from Rhizomes of Globba schomburgkii Hook.f.

Bioassay‐guided fractionation was conducted on dichloromethane extract from the rhizomes of Globba schomburgkii Hook.f., which have previously been reported as the part with the highest antibacterial activity. 10 fractions and 20 sub‐fractions were obtained and evaluated for their potency against various strains of bacteria. The most active sub‐fractions were 8 times more effective against Staphylococcus aureus and Micrococcus luteus than the original crude extract. Moreover, two pure compounds, namely petasol and (E)‐15,16‐dinorlabda‐8(17),11‐dien‐13‐one, were successfully isolated and characterized for the first time from this plant species. Untargeted compound analysis of all fractions and sub‐fractions was performed by gas chromatography hyphenated with mass spectrometry, leading to positive identification of 167 compounds according to comparison with the mass spectrum and retention index database, 137 of which have never been reported for G. schomburgkii. The correlation between antibacterial activity and composition of each fraction suggests that the bioactive compounds could be 4,8‐β‐epoxycaryophyllene, methyl isocostate, (E)‐labda‐8(17),12‐diene‐15,16‐dial, α‐kessyl acetate, zederone, clovanediol, ledene oxide‐(I), alantolactone, or 8α,11‐elemadiol.