Endosymbiotic bacteria in mycorrhizal fungi: from their morphology to genomic sequences

Arbuscular mycorrhizal (AM) fungi have been successful in time and space thanks to a long co-evolution with their host plants. In addition to this well known interaction, they also associate with bacteria that reside in the fungal cytoplasm. The chapter mostly focusses on endosymbionts belonging to the genus Burkholderia and found in many species of Gigasporaceae. We have used morphological and genetic approaches to investigate these intracellular microrganisms. Some genes related to metabolism, cell colonization events and nitrogen fixation have been characterized and suggest a potential role in the nutritional exchanges between endobacteria, fungi and plants.     

Rhizosphere micro-organisms in relation to the apple replant problem

Summary One of the factors giving rise to soil sickness in apple orchards is the rhizosphere microflora. The composition of the microbial coenosis in the rhizosphere changes with increasing age of the apple trees. An increase in the counts of micromycetes and actinomycetes and a decrease in bacterial counts was found in agreement with the decreasing pH of the rhizosphere soil. The presence of fluorescent pseudomonads in the rhizosphere of old apple trees was rare, but the planting of apple seedling into sick soil induced their proliferation. The relative proportion of individual genera of micromycetes changed according to the tree age; fungi of the genus Mucor were more often found in the rhizosphere of younger trees than in that of older ones while fungi of the genus Penicillium had an opposite trend. Biological tests showed that Penicillium fungi form the majority of the phytotoxic microflora. The amount of phytotoxic micromycetes was higher in ‘sick’ soil as compared with control soil in which apple trees had not been grown for at least 15 years. Higher numbers of phytotoxic micromycetes were isolated also from the rhizosphere of apple seedlings grown in ‘sick’ soil as compared with those growing in control soil. An increase in the amount of phytotoxic micromycetes in apple tree rhizosphere could be induced by mere addition of 5% (w/w) ‘sick’ soil to the soil in which apple trees were grown for the first time. By adding sterilized ‘sick’ soil, the amount of phytotoxic micromycetes in the apple seedling rhizosphere was not affected. Increased numbers of phytotoxic micromycetes affected negatively the growth of apple trees and the morphology of apple tree roots. This demonstrated the possibility of transfer of a factor participating in the etiology of soil sickness in apple orchards.     

A SINGLE ORIGIN OF PLASTIDS REVISITED: CONVERGENT EVOLUTION IN ORGANELLAR GENOME CONTENT1

In recent years a consensus has emerged from molecular phylogenetic investigations favoring a common endosymbiotic ancestor for all chloroplasts. It is within this conceptual framework that most comparative analyses of eukaryotic biochemistry and genetics now are interpreted. One of the first and most influential sources of data leading to this consensus is the remarkable similarity in genome content among all major plastid lineages. Here we report statistical analyses of two sequence data sets, genes encoding ribosomal proteins and transfer RNAs, from representatives of the three primary plastid lineages and a mitochondrion. The latter almost certainly originated in an independent endosymbiotic association and serves as a control for similarity due to convergent evolution. When genes related to organelle‐specific function are factored out, plastid genomes appear to be no more similar to each other than they are to the mitochondrion. Total similarities in gene content, measured as deviations from the expectation from a process of random gene loss, are correlated with the extent of reduction in the two genomes compared. They do not appear to reflect putative evolutionary relationships among plastids. These analyses indicate that similarities in plastid genome content are better explained by convergent evolution due to constraint on gene loss than by a shared evolutionary history. A review of other data cited as support for a single plastid origin suggests that the alternative hypothesis of multiple origins is at least equally consistent in most cases.     

Kefir micro-organisms: their role in grain assembly and health properties of fermented milk

Kefir is a homemade viscous and slightly effervescent beverage obtained by milk fermentation with kefir grains, which are built up by a complex community of lactic acid and acetic acid bacteria and yeasts confined in a matrix of proteins and polysaccharides. The present review summarizes the role of kefir micro-organisms in grain assembly and in the beneficial properties attributed to kefir. The use of both culture-dependent and independent methods has made possible to determine the micro-organisms that constitute this ecosystem. Kefir consumption has been associated with a wide range of functional and probiotic properties that could be attributed to the micro-organisms present in kefir and/or to the metabolites synthetized by them during milk fermentation. In this context, the role of micro-organisms in kefir health promoting properties is discussed with particular attention to the contribution of yeast as well as bioactive metabolites such as lactic and acetic acid, exopolysaccharides and bioactive peptides. Even though many advances on the knowledge of this ancient fermented milk have been made, further studies are necessary to elucidate the complex nature of the kefir ecosystem.     

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Decrease of enteric micro-organisms from rural sewage sludge during their composting in straw mixture

AIMS: To study the decrease of enteric micro-organisms including viable nematode eggs, enteroviruses, faecal indicators (Escherichia coli and enterococci) and pathogenic bacteria (Listeria monocytogenes, Salmonella sp. and Clostridium perfringens) of a rural sewage sludge when it is composted for 7 months in mixture with straw. METHODS AND RESULTS: Numbers of the test organisms and the physico-chemical parameters were measured on a monthly basis on the mixture, on the compost after being turned, and on the pile in three positions representing the part by which air is incoming, the bottom of the pile and the part through which air is outgoing. The lowest temperature in the pile was observed at the bottom, where it did not exceed 50 degrees C against 66 degrees C in the two other areas. There were no significant differences between the three areas in terms of micro-organism survival. Infectious enteroviruses were inactivated rapidly and were not found after the first turning whereas some genomes were detected until after the third turning. Escherichia coli and enterococci presented a similar survival rate and their number decreased by 4 log(10) whereas Salmonella decayed at a greater rate than L. monocytogenes. The numbers of C. perfringens decreased gradually to reach a final concentration in the mature compost of about 10(2) CFU g(-1) dry matter (d.m.), which was similar to that of the faecal indicators. CONCLUSIONS: The hygienic effect of sludge composting in mixture with straw results in a significant reduction of enteric micro-organisms, the concentration of the faecal indicators in the final product being < 64 most probable number g(-1) d.m. The concentrations of Salmonella, enteroviruses and viable nematode eggs in the final product were not detectable which is in accordance with the French legislation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results which pointed out the different behaviour of the test micro-organisms reflect the difficulty to propose a relevant indicator of hygienization. Otherwise, they show that composting is an efficient means for hygienization of sludge of rural wastewater treatment, where the straw is available close to their place of production.     

Experimental reconstruction of double‐stranded break repair‐mediated plastid DNA insertion into the tobacco nucleus

The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes. DNA from the endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a precursor of the nucleated eukaryotic host. An experimental confirmation regarding the molecular mechanisms responsible for organelle DNA incorporation into nuclei has not been performed until the present analysis. Here we introduced double‐stranded DNA breaks into the nuclear genome of tobacco through inducible expression of I‐SceI, and showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through double‐stranded DNA break repair. Microhomology‐mediated linking of disparate segments of chloroplast DNA occurs frequently during healing of induced nuclear double‐stranded breaks (DSB) but the resulting nuclear integrants are often immediately unstable. Non‐Mendelian inheritance of a selectable marker (neo), used to identify plastid DNA transfer, was observed in the progeny of about 50% of lines emerging from the screen. The instability of these de novo nu clear insertions of p last id DNA (nupts) was shown to be associated with deletion not only of the nupt itself but also of flanking nuclear DNA within one generation of transfer. This deletion of pre‐existing nuclear DNA suggests that the genetic impact of organellar DNA transfer to the nucleus is potentially far greater than previously thought.