Bacterial diversity obtained by culturable approaches in the gut of Glossina pallidipes population from a non sleeping sickness focus in Tanzania: preliminary results.

BACKGROUND: Glossina pallidipes is a haematophagous insect that serves as a cyclic transmitter of trypanosomes causing African Trypanosomiasis (AT). To fully assess the role of G. pallidipes in the epidemiology of AT, especially the human form of the disease (HAT), it is essential to know the microbial diversity inhabiting the gut of natural fly populations. This study aimed to examine the diversity of G. pallidipes fly gut bacteria by culture-dependent approaches. RESULTS: 113 bacterial isolates were obtained from aerobic and anaerobic microorganisms originating from the gut of G. pallidipes. 16S rDNA of each isolate was PCR amplified and sequenced. The overall majority of identified bacteria belonged in descending order to the Firmicutes (86.6%), Actinobacteria (7.6%), Proteobacteria (5.5%)and Bacteroidetes (0.3%). Diversity of Firmicutes was found higher when enrichments and isolation were performed under anaerobic conditions than aerobic ones. Experiments conducted in the absence of oxygen (anaerobiosis) led to the isolation of bacteria pertaining to four phyla (83% Firmicutes, 15% Actinobacteria, 1% Proteobacteria and 0.5% Bacteroidetes, whereas those conducted in the presence of oxygen (aerobiosis) led to the isolation of bacteria affiliated to two phyla only (90% Firmicutes and 10% Proteobacteria). Phylogenetic analyses placed these isolates into 11 genera namely Bacillus, Acinetobacter, Mesorhizobium, Paracoccus, Microbacterium, Micrococcus, Arthrobacter, Corynobacterium, Curtobacterium, Vagococcus and Dietzia spp.which are known to be either facultative anaerobes, aerobes, or even microaerobes. CONCLUSION: This study shows that G. pallidipes fly gut is an environmental reservoir for a vast number of bacterial species, which are likely to be important for ecological microbial well being of the fly and possibly on differing vectorial competence and refractoriness against AT epidemiology.

Unusual evolutionary mechanisms to escape effector-triggered immunity in the fungal phytopathogen Leptosphaeria maculans.

Leptosphaeria maculans is the fungus responsible for the stem canker disease of oilseed rape (Brassica napus). AvrLm3 and AvrLm4-7, two avirulence effector genes of L. maculans, are involved in an unusual relationship: AvrLm4-7 suppresses the Rlm3-mediated resistance. Here, we assessed AvrLm3 polymorphism in a collection of 235 L. maculans isolates. No field isolates exhibited deletion or inactivating mutations in AvrLm3, as observed for other L. maculans avirulence genes. Eleven isoforms of the AvrLm3 protein were found. In isolates virulent towards both Rlm3 and Rlm7 (a3a7), the loss of the Rlm3-mediated resistance response was due to two distinct mechanisms. First, when AvrLm4-7 was inactivated (deletion or inactivating mutations), amino acid substitutions in AvrLm3 generated virulent isoforms of the protein. Second, when only point mutations were observed in AvrLm4-7, a3a7 isolates still contained an avirulent allele of AvrLm3. Directed mutagenesis confirmed that some point mutations in AvrLm4-7 were sufficient for the fungus to escape Rlm7-mediated resistance while maintaining the suppression of the AvrLm3 phenotype. Signatures of positive selection were also identified in AvrLm3. The complex evolutionary mechanisms enabling L. maculans to escape Rlm3-mediated resistance while preserving AvrLm3 integrity, along with observed reduced aggressiveness of isolates silenced for AvrLm3, serves to emphasize the importance of this effector in pathogenicity towards B. napus. While the common response to resistance gene pressure is local selection of isolates depleted in the cognate avirulence gene, this example contributes to complexify the gene-for-gene concept of plant-pathogen evolution with a 'camouflaged' model allowing retention of nondispensable avirulence effectors.

Characterization of Halanaerocella petrolearia gen. nov., sp. nov., a new anaerobic moderately halophilic fermentative bacterium isolated from a deep subsurface hypersaline oil reservoir : New taxa: Firmicutes (Class Clostridia, Order Halanaerobiales, Halobacteroidaceae, Halobacteroides).

An anaerobic, halophilic, and fermentative bacterium, strain S200(T), was isolated from a core sample of a deep hypersaline oil reservoir. Cells were rod-shaped, non-motile, and stained Gram-positive. It grew at NaCl concentrations ranging from 6 to 26% (w/v), with optimal growth at 15% (w/v) NaCl, and at temperatures between 25 and 47°C with an optimum at 40-45°C. The optimum pH was 7.3 (range 6.2-8.8; no growth at pH 5.8 and pH 9). The doubling time in optimized growth conditions was 3.5 h. Strain S200(T) used exclusively carbohydrates as carbon and energy sources. The end products of glucose degradation were lactate, formate, ethanol, acetate, H(2), and CO(2). The predominant cellular fatty acids were non-branched fatty acids C(16:1), C(16:0), and C(14:0). The G + C mole% of the DNA was 32.7%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain S200(T) formed a distinct lineage within the family Halobacteroidaceae, order Halanaerobiales, and was most closely related to Halanaerobaculum tunisiense DSM 19997(T) and Halobacteroides halobius DSM 5150(T), with sequence similarity of 92.3 and 91.9%, respectively. On the basis of its physiological and genotypic properties, strain S200(T) is proposed to be assigned to a novel species of a novel genus, for which the name Halanaerocella petrolearia is proposed. The type strain of Halanaerocella petrolearia is strain S200(T) (=DSM 22693(T) = JCM 16358(T)).

Involvement of a single periplasmic hydrogenase for both hydrogen uptake and production in some Desulfovibrio species.

Various sulphate-reducing bacteria differing in the number of genes encoding hydrogenase were shown to ferment lactate in coculture with Methanospirillum hungatei, in the absence of sulphate. The efficiency of interspecies H2 transfer carried out by these species of sulphate-reducing bacteria does not appear to correlate with the distribution of genes coding for hydrogenase. Desulfovibrio vulgaris Groningen, which possesses only the gene for [NiFe] hydrogenase, oxidizes hydrogen in the presence of sulphate and produces some hydrogen during fermentation of pyruvate without electron acceptor. The hydrogenase of D. vulgaris was purified and characterized. It exhibits a molecular mass of 87 kDa and is composed of two different subunits (60 and 28 kDa). D. vulgaris hydrogenase contains 10.6 iron atoms, 0.9 nickel atom and 12 acid-labile sulphur atoms/molecule, and the absorption spectrum of the enzyme is characteristic of an iron-sulphur protein. Maximal H2 uptake and H2 evolution activities were 332 and 230 units/mg protein, respectively. D. vulgaris cells contain exclusively the [NiFe] hydrogenase, whatever the growth conditions, as shown by biochemical and immunological studies. Immunocytolocalization in ultrathin frozen sections of cells grown on lactate and sulphate, on H2 and sulphate and on pyruvate showed that the [NiFe] hydrogenase was located in the periplasmic space. Labelling was enhanced in cells grown on H2 and sulphate and on pyruvate. The results enable us to conclude that D. vulgaris Groningen contains a single hydrogenase of the [NiFe] type, located in the periplasmic space like that described for D. gigas. This enzyme appears to be involved in both H2 uptake and H2 production, depending on the growth conditions.

Effect of thiosulphate as electron acceptor on glucose and xylose oxidation by Thermoanaerobacter finnii and a Thermoanaerobacter sp. isolated from oil field water.

During glucose and xylose fermentation, Thermoanaerobacter finnii was observed to produce lactate, acetate, H2 and CO2, with ethanol being the major end product. Thermoanaerobacter strain SEBR 5268, an isolate from an oil field, also produced a similar range of end products from glucose and xylose fermentation, with the exception that both ethanol and lactate were the major products of sugar metabolism. Both these strains were able to reduce thiosulphate to sulphide in the presence of these two substrates, with acetate being the dominant metabolite in that case. In addition, a faster growth rate and increased cell yield were obtained in the presence of thiosulphate, than in its absence. The higher concentrations of acetate produced in the presence of thiosulphate rather than without any electron acceptor indicated that more ATP was generated from substrate-level phosphorylation. These results have implications for our understanding of the breakdown of carbohydrates present in organic matter found in the natural ecological niches of Thermoanaerobacter species (sulphide-, elemental sulphur- or sulphate-rich thermal hot springs and oil fields).

Methanoplanus petrolearius sp. nov., a novel methanogenic bacterium from an oil-producing well.

A disc-shaped methanogenic bacterium designated strain SEBR 4847T (T = type strain) was isolated from a sample collected from an African offshore oil field. Strain SEBR 4847T was non-motile, had a G + C content of 50 mol% and produced methane from H2 + CO2, formate, and CO2 + propanol. Strain SEBR 4847T grew optimally at 37 degrees C; no growth was observed at 25 degrees C or 45 degrees C. It grew in the presence of up to 50 g/l NaCl; 10-30 g/l was required for optimal growth. The optimum pH for growth was 7.0. Doubling time was about 10 h under optimal conditions. Based on 16S rRNA sequence analysis, the isolate was identified as a new species of the genus Methanoplanus and designated Methanoplanus petrolearius sp. nov. The type strain is SEBR 4847T (= OCM 486).

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field.

An obligately anaerobic spirochete designated strain SEBR 4228T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration) growth range 1.0-10%) at 37 degrees C (growth temperature range 20-40 degrees C) and pH of 7.0-7.2 (pH growth range pH 5.5-8.0). Strain SEBR 4228T grew on carbohydrates (glucose, fructose, ribose, D-xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H2S. Glucose was oxidised to lactate, acetate, CO2 and H2S in the presence of thiosulfate but in its absence lactate, ethanol, CO2 and H2 were produced. Fumarate was fermented to acetate and succinate. The G + C content of strain SEBR 4228T was 50%. Strain SEBR 4228T was spiral shaped measuring 5-30 by 0.3-0.5 micron and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228T possessed features typical of the members of the genus Spirochaeta. 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228T is a new species, which we have designated Spirochaeta smaragdinae. The type strain is SEBR 4228T (= DSM 11293).

Reevaluating the classification of Halobacteroides and Haloanaerobacter species based on sequence comparisons of the 16S ribosomal RNA gene.

The 16S rRNA gene (rDNA) sequence analysis of four halophilic anaerobes: Halobacteroides halobius, H. lacunaris. Haloanaerobacter (Hb.) chitinovorans and H. acetoethylicus confirmed that they were all members of the family Haloanaerobiaceae. H. lacunaris and H. halobius were found to be more closely related to each other and were distantly related to Sporohalobacter lortetti and the members of the genera Haloanaerobium and Halothermothrix. These data are in agreement with their assignment to the genus Halobacteroides. Further analysis indicated that Hb. chitinovorans was closely affiliated to members of the genus Halobacteroides, and therefore we propose to transfer it to the genus Halobacteroides as H. chitinovorans comb. nov. This transfer would invalidate the genus Haloanaerobacter, as Hb. chitinovorans is the only member of this genus. The 16S rDNA sequence analysis of H. acetoethylicum indicated that it was very closely related to members of the genus Haloanaerobium, viz. Haloanaerobium (Ha.) praevalens, Ha. salsugo, and Ha. alcaliphilum, and hence we propose to transfer it to the genus Haloanaerobium as Ha. acetoethylicus comb. nov.

Haloanaerobium congolense sp. nov., an anaerobic, moderately halophilic, thiosulfate- and sulfur-reducing bacterium from an African oil field.

A strictly anaerobic, moderately halophilic, Gram-negative, non-motile rod-shaped bacterium was isolated from an oil-well head sample of an offshore Congolese oil field. The strain, designated SEBR 4224T (T = type strain), grew optimally at 42 degrees C and pH 7.0 in a complex medium containing 10% NaCl with a generation time of 2.5 h. Strain SEBR 4224T grew on a range of carbohydrates including fructose, galactose, D-glucose, maltose, D-mannose, D-ribose, sucrose, and trehalose. Yeast extract and/or bio-Trypcase was required for growth on carbohydrates and could not be replaced with amino acids and/or vitamins. The end-products from glucose fermentation were acetate, H2, and CO2. Thiosulfate and elemental sulfur were used as electron acceptors. Thiosulfate improved carbohydrate utilization and biomass yields. The G + C content of the isolate was 34 mol%. Ribosomal 16S rRNA sequence analysis showed that strain SEBR 4224T is a new member of the genus Haloanaerobium. The lack of DNA homology with H. acetoethylicum, its closest relative, as determined by DNA-DNA hybridization supports the designation of strain SEBR 4224T as a new species, Haloanaerobium congolense sp. nov. The type strain is SEBR 4224T (= DSM 11287).

Mdx mouse skeletal muscle: could a mitochondrial factor be responsible for the absence of progressive necrosis?

We compared the myotoxic effect of chlorpromazine on mitochondria of gastrocnemius muscle in X-related muscular dystrophy (mdx) and control mice relative to changes in calmitine and calcium concentrations before and 3 and 6 days after a single injection of the drug. The results indicate that mdx mouse mitochondria are less sensitive to the myotoxic effect of chlorpromazine; calmitine and calcium binding were only slightly reduced compared to controls. Our observations indicate that the calmitine structure could differ in mdx and control mice with respect to calcium binding structures, and that the presence of calmitine in the mitochondria of mdx mouse skeletal muscle could explain why muscle degeneration does not occur in these animals. However, the muscles of patients with Duchenne muscular dystrophy (DMD) are lacking in calmitine and are subject to extensive progressive degeneration.

Calmitine: a calcium-binding mitochondrial protein specific for fast-twitch muscle fibers.

Mitochondrial fractions were isolated from fast-twitch (EDL), slow-twitch (soleus) and heart muscle of normal rat (WKY). Protein separation by electrophoresis and study of calcium-45 binding showed that a specific calcium protein (designated as calmitine) was present in the mitochondria of fast-twitch muscle but practically inexistent in slow-twitch and cardiac muscle. It seems to be related to calcium uptake by an energy-dependent mechanism.

Severe mitochondrial anomaly in dystrophic mouse skeletal muscle.

Mitochondrial fractions were isolated from skeletal muscle of control (C57 BL 6J dy/+) and dystrophic (C57 BL 6J dy/dy) mice, and enzymatic activities (cytochrome c oxidase, rotenone-insensitive NADH cytochrome c reductase) were determined. After electrophoretic separation, calcium-binding proteins were identified. An important anomaly was observed in the mitochondria of dystrophic muscle, i.e., a considerable reduction of a specific calcium-binding protein (61,000 Da mol. wt.).

Age-related calmitine distribution in mitochondria of normal and mdx mouse skeletal muscle.

In our study, mitochondria were isolated from skeletal muscle in 3-, 5-, 6- and 16-week-old mdx and control mice. A deficit was observed in a calcium-specific mitochondrial protein (named "calmitine") in 3-, 5- and 6-week-old mdx mice but only in 3-week-old control mice. In addition, there was a correlation between the amounts of calmitine and calcium uptake in mitochondria: the latter remained low in 3-, 5- and 6-week-old mdx mice and was similar to controls in 16-week-old mdx mice (as was calmitine). A relationship is suggested between the deficit in calmitine (and calcium uptake in mitochondria) and the important signs of fiber degeneration presented by mdx mice between 3 and 6 weeks of age (a return to normal was observed subsequently).

Muscular dystrophy: possible role of mitochondrial deficiency in muscle degeneration processes.

We isolated mitochondria from fast-twitch (extensor digitorum longus) and slow-twitch (soleus) skeletal muscle of the adult rat in normal conditions and 45 days after denervation as well as from skeletal muscle (gastrocnemius) of control and dystrophic (C57BL6J dy/dy and mdx) mice. We searched for the presence of a calcium-specific mitochondrial protein (calmitine) and measured calcium uptake in mitochondria. Our results indicate a possible correlation between the quantity of calmitine present and calcium entry into mitochondria. Both these parameters were elevated in rat fast-twitch and mouse mixed muscle and very low in slow-twitch muscle. They were also very low in dystrophic mouse muscle (C57BL6J dy/dy) with extensive muscle degeneration, but on the contrary elevated in muscle (mdx) with no important signs of degeneration. Finally, we found a normal calmitine concentration and very low calcium uptake in rat extensor digitorum longus after 45 days of denervation. On the basis of these results, it is hypothesized that calmitine synthesis could be subject to neural influence, thus specific for fast-twitch muscle, and that it could be linked to mitochondrial calcium uptake. A decrease in uptake could disturb certain enzymatic activities related to ATP synthesis and bring about muscle degeneration by inhibiting such synthesis. This would occur in the context of reduced calmitine synthesis in the case of genetic anomalies and of inactivation of calmitine after neural disturbance in the case of denervation.

Is there a maturation defect related to calcium in muscle mitochondria from dystrophic mice and Duchenne and Becker muscular dystrophy patients.

In our study, mitochondria were isolated from skeletal muscle in 2-, 3-, 4-, 6-, 8-, and 12-week-old normal (C57BL6j dy/+), and 4-, 8-, and 12-week-old dystrophic (C57BL6j dy/dy) mice and in normal subjects and patients with Duchenne or Becker muscular dystrophy. A deficit was observed in a calcium-specific mitochondrial protein in the very young control mouse, compared with the adult mouse. In the adult dystrophic mouse this deficit was found in clinically affected hindleg muscles as well as in apparently normal front leg muscles; it was also found in quadriceps muscles from patients with Duchenne and Becker muscular dystrophy. It is not observed in normal adult mice or in normal subjects. The body of our results suggests that in the forms of muscular dystrophy studied there would be a maturation defect in this calcium-binding mitochondrial protein ("calmitine"), a defect which might be generalized in the entire skeletal muscle system and conceivably could be the cause of muscle degeneration in certain myopathies such as Duchenne and Becker muscular dystrophy.

Effect of torbafylline on mitochondrial calmitine in mouse skeletal muscle regeneration after injection of a myotoxic drug.

The effect of torbafylline, a xanthine derivative (Hoechst, Werk Albert, Wiesbaden, Germany), was tested relative to changes in degeneration and subsequent regeneration processes in mouse gastrocnemius muscle induced by a single injection of chlorpromazine, a myotoxic drug. These processes were monitored by measuring changes in calmitine, a mitochondrial protein. We determined in our previous work that calmitine concentration decreases during degeneration and progressively increases during regeneration. In this study, we compared effects in torbafylline-treated mice with those in control mice treated with saline solution. The results show that regeneration is much faster with torbafylline treatment. Calmitine is decreased and returns quickly to normal in torbafylline-treated mice as compared to those treated with saline solution. Torbafylline might thus prove effective in stimulating muscle regeneration in myopathy.

Degeneration and subsequent regeneration of mouse skeletal muscle after a single injection of chlorpromazine: changes in mitochondrial calmitine and calcium content.

We studied experimental models capable of showing muscle degeneration and subsequent regeneration and observed the changes in calmitine, calcium uptake and calcium concentration in mitochondria during these processes. The results presented here are based on the study of mitochondria of mouse skeletal muscle after a single intramuscular injection of chlorpromazine. This drug induces myotoxic effects followed by muscle regeneration. Our results show that the muscle degeneration process, as shown by histological studies, was associated with some changes in mitochondria: a decrease in calmitine, a calcium overload and a decrease in calcium uptake; the subsequent regeneration process was associated with an increase in calmitine, a decrease in calcium concentration and an increase in calcium uptake, these 3 parameters returning to normal values. It seems that there is a correlation between a decrease in calmitine and muscle degeneration, and an increase in calmitine and muscle regeneration, as shown by our biochemical and histological observations.

Desulfovibrio aminophilus sp. nov., a novel amino acid degrading and sulfate reducing bacterium from an anaerobic dairy wastewater lagoon.

A mesophilic strain of sulfate-reducing bacterium, designated ALA-3T (T = type strain), was isolated from an anaerobic lagoon of a dairy wastewater treatment plant. The curved, Gram-negative, non-sporeforming cells (0.2 x 3.0-4.0 microns) existed singly or in chains, and were motile by single polar flagella. Optimum growth occurred at 35 degrees C and pH 7.5 on a medium containing lactate and sulfate. Thiosulfate or sulfite but not elemental sulfur, nitrate, or fumarate could also replace sulfate as an electron acceptor. Formate, alanine, aspartate, leucine, isoleucine, valine, and methionine, H2/CO2 and ethanol also served as electron donors with sulfate as an electron acceptor. Pyruvate, casamino acids, peptone, serine, glycine, cysteine and threonine were fermented. Sulfite and thiosulfate were disproportionated to sulfate and sulfide. The G + C content of the DNA was 66 mol % G + C. Phylogenetic analysis revealed that Desulfovibrio africanus was the nearest relative (similarity of 89%). Strain ALA-3T is physiologically and phylogenetically different from other Desulfovibrio species, and is designated Desulfovibrio aminophilus sp. nov. (DSM 12254).

Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle.

Two Ca2+ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca2+-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twitch muscle. Ca2+-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca2+-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased 45Ca-binding. These results indicate first that Ca2+-ATPase activity in EDL was under neural control, and that because of low Ca2+-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.

Serratia glossinae sp. nov., isolated from the midgut of the tsetse fly Glossina palpalis gambiensis.

We report the isolation of a novel bacterium, strain C1(T), from the midgut of the tsetse fly Glossina palpalis gambiensis, one of the vector insects responsible for transmission of the trypanosomes that cause sleeping sickness in sub-Saharan African countries. Strain C1(T) is a motile, facultatively anaerobic, rod-like bacterium (0.8-1.0 microm in diameter; 2-6 microm long) that grows as single cells or in chains. Optimum growth occurred at 25-35 degrees C, at pH 6.7-8.4 and in medium containing 5-20 g NaCl l(-1). The bacterium hydrolysed urea and used L-lysine, L-ornithine, citrate, pyruvate, D-glucose, D-mannitol, inositol, D-sorbitol, melibiose, amygdalin, L-arabinose, arbutin, aesculin, D-fructose, D-galactose, glycerol, maltose, D-mannose, raffinose, trehalose and d-xylose; it produced acetoin, reduced nitrate to nitrite and was positive for beta-galactosidase and catalase. The DNA G+C content was 53.6 mol%. It was related phylogenetically to members of the genus Serratia, family Enterobacteriaceae, the type strain of Serratia fonticola being its closest relative (99 % similarity between 16S rRNA gene sequences). However, DNA-DNA relatedness between strain C1(T) and S. fonticola DSM 4576(T) was only 37.15 %. Therefore, on the basis of morphological, nutritional, physiological and fatty acid analysis and genetic criteria, strain C1(T) is proposed to be assigned to a novel Serratia species, Serratia glossinae sp. nov. (type strain C1(T) =DSM 22080(T) =CCUG 57457(T)).