Contrasted Reactivity to Oxygen Tensions in Frankia sp. Strain CcI3 throughout Nitrogen Fixation and Assimilation

Reconciling the irreconcilable is a primary struggle in aerobic nitrogen-fixing bacteria. Although nitrogenase is oxygen and reactive oxygen species-labile, oxygen tension is required to sustain respiration. In the nitrogen-fixing Frankia, various strategies have been developed through evolution to control the respiration and nitrogen-fixation balance. Here, we assessed the effect of different oxygen tensions on Frankia sp. strain CcI3 growth, vesicle production, and gene expression under different oxygen tensions. Both biomass and vesicle production were correlated with elevated oxygen levels under both nitrogen-replete and nitrogen-deficient conditions. The mRNA levels for the nitrogenase structural genes (nifHDK) were high under hypoxic and hyperoxic conditions compared to oxic conditions. The mRNA level for the hopanoid biosynthesis genes (sqhC and hpnC) was also elevated under hyperoxic conditions suggesting an increase in the vesicle envelope. Under nitrogen-deficient conditions, the hup2 mRNA levels increased with hyperoxic environment, while hup1 mRNA levels remained relatively constant. Taken together, these results indicate that Frankia protects nitrogenase by the use of multiple mechanisms including the vesicle-hopanoid barrier and increased respiratory protection

Genomic analysis of Sinorhizobium meliloti IRAMC:0087 an halotolerant rhizobium isolated from the Tunisian desert

Arid and Saharan regions of Tunisia have great interest due to their endemic spontaneous legume’s diversity. The strain IRAMC:0087 was isolated from root nodules of a Saharan shrub Genista saharae growing in Southern Tunisia. Phenotypic characterization of this strain has revealed tolerance to high salinity levels, drought and high temperatures. To investigate the basis of this, we sequenced its complete genome and compared it to the genome of the closely related strain Sinorhizobium meliloti NBRC 14782T, an alfalfa (Medicago sativa) microsymbiont. The genome comprises 7,265,739 bp, which is comparable in size to other S. meliloti strains and contains a GC content of 61.94%. In total, 7536 protein-encoding sequences, 51 tRNAs and 5 rRNAs were identified. The genome encodes gene clusters supporting rhizosphere processes, secondary bioactive metabolites, plant growth-promoting activities and symbiosis. Gene distribution into COG functional categories revealed that the percentage of genes was assigned to amino acid transport and metabolism (10.92%), general function prediction (10.5%), carbohydrate transport and metabolism (9.52%), and transcription (9.21%). Despite the difference in size, IRAMC:0087 and NBRC 14782T genomes present a similar relative occurrence of functional protein encoding genes and do not show any gross genomic alterations. Interestingly, although IRAMC:0087 contains more protein encoding genes than NBRC 14782T (7536 vs 6696 genes), the major difference lies in the number of not functionally classifiable genes. Nevertheless, the number of genes for transposable elements (9 vs 2), for managing membrane transport (205 vs 147 genes) and for conducting iron acquisition and metabolism (41 vs 31 genes) are higher in IRAMC:0087 than NBRC 14782T. IRAMC:0087 is an interesting strain as it exhibits an endophytic and symbiotic behavior with hosts adapted to extreme climatic conditions. Comparative genomic analyses with other rhizobial strains have the potential to reveal novel factors mediating symbiosis under those conditions

Bacterial Endophytes from Legumes Native to Arid Environments Are Promising Tools to Improve <i>Mesorhizobium</i>–Chickpea Symbiosis under Salinity

Symbiotic nitrogen fixation is a major contributor of N in agricultural ecosystems, but the establishment of legume–rhizobium symbiosis is highly affected by soil salinity. Our interest is focused on the use of non-rhizobial endophytes to assist the symbiosis between chickpea and its microsymbiont under salinity to avoid loss of production and fertility. Our aims were (1) to investigate the impact of salinity on both symbiotic partners; including on early events of the Mesorhizobium-chickpea symbiosis, and (2) to evaluate the potential of four non-rhizobial endophytes isolated from legumes native to arid regions (Phyllobacterium salinisoli, P. ifriqiyense, Xanthomonas translucens, and Cupriavidus respiraculi) to promote chickpea growth and nodulation under salinity. Our results show a significant reduction in chickpea seed germination rate and in the microsymbiont Mesorhizobium ciceri LMS-1 growth under different levels of salinity. The composition of phenolic compounds in chickpea root exudates significantly changed when the plants were subjected to salinity, which in turn affected the nod genes expression in LMS-1. Furthermore, the LMS-1 response to root exudate stimuli was suppressed by the presence of salinity (250 mM NaCl). On the contrary, a significant upregulation of exoY and otsA genes, which are involved in exopolysaccharide and trehalose biosynthesis, respectively, was registered in salt-stressed LMS-1 cells. In addition, chickpea co-inoculation with LMS-1 along with the consortium containing two non-rhizobial bacterial endophytes, P. salinisoli and X. translucens, resulted in significant improvement of the chickpea growth and the symbiotic performance of LMS-1 under salinity. These results indicate that this non-rhizobial endophytic consortium may be an appropriate ecological and safe tool to improve chickpea growth and its adaptation to salt-degraded soils

Phylogenetic placement, floral anatomy, and morphological characterization of the North African pastoral halophyte Atriplex mollis Desf. (Amaranthaceae)

Atriplex mollis Desf. (Amaranthaceae), a North African endemic halophytic species, is further described in this study. Phylogenetic analysis based on a combined dataset of ITS and ETS rDNA and atpB-rbcL and trnK cpDNA showed that A. mollis is closely related to the Malta- and Gozo-endemic Cremnophyton lanfrancoi Brullo & Pavone. Given this close phylogenetic relationship, A. mollis is also considered among the oldest species of Atriplex, together with C. lanfrancoi. Molecular data also suggest that A. mollis in North Africa, C. lanfrancoi on Malta Island, and Atriplex cana Ledeb. in Eurasian semideserts constitute a separate clade within the tribe Atripliceae. As an 18-month-old shrub, A. mollis can reach a mean height of 44.06 ± 8.09 cm with a leaf area around 1.24 ± 0.15 cm2, and can produce seeds in order of 113.08 ± 28.52 g plant−1. The anatomy of A. mollis shows the presence of male and female developed flowers. Hermaphroditic flowers that may lead to the appearance of male flowers with underdeveloped female organs were rarely found. Three main shapes of ovule (campylotropous, amphitropous, and orthotropous) were found in A. mollis.This study was supported by the ERANETMED2-72-303 PACTORES project financed by the Ministry of Higher Education and Scientific Research of Tunisia and the University of Malta (Argotti Botanic Gardens).Peer reviewe

Diversity and Enzymatic Profiling of Halotolerant Micromycetes from Sebkha El Melah, a Saharan Salt Flat in Southern Tunisia

Twenty-one moderately halotolerant fungi have been isolated from sample ashes collected from Sebkha El Melah, a Saharan salt flat located in southern Tunisia. Based on morphology and sequence inference from the internal transcribed spacer regions, 28S rRNA gene and other specific genes such as β-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase, the isolates were found to be distributed over 15 taxa belonging to 6 genera of Ascomycetes: Cladosporium (n=3), Alternaria (n=4), Aspergillus (n=3), Penicillium (n=5), Ulocladium (n=2), and Engyodontium (n=2). Their tolerance to different concentrations of salt in solid and liquid media was examined. Excepting Cladosporium cladosporioides JA18, all isolates were considered as alkali-halotolerant since they were able to grow in media containing 10% of salt with an initial pH 10. All isolates were resistant to oxidative stresses and low temperature whereas 5 strains belonging to Alternaria, Ulocladium, and Aspergillus genera were able to grow at 45°C. The screening of fungal strains for sets of enzyme production, namely, cellulase (CMCase), amylase, protease, lipase, and laccase, in presence of 10% NaCl, showed a variety of extracellular hydrolytic and oxidative profiles. Protease was the most abundant enzyme produced whereas laccase producers were members of the genus Cladosporium

Cultivating the uncultured: growing the recalcitrant cluster-2 Frankia strains

The repeated failures reported in cultivating some microbial lineages are a major challenge in microbial ecology and probably linked, in the case of Frankia microsymbionts to atypical patterns of auxotrophy. Comparative genomics of the so far uncultured cluster-2 Candidatus Frankia datiscae Dg1, with cultivated Frankiae has revealed genome reduction, but no obvious physiological impairments. A direct physiological assay on nodule tissues from Coriaria myrtifolia infected with a closely-related strain permitted the identification of a requirement for alkaline conditions. A high pH growth medium permitted the recovery of a slow-growing actinobacterium. The strain obtained, called BMG5.1, has short hyphae, produced diazovesicles in nitrogen-free media, and fulfilled Koch's postulates by inducing effective nodules on axenically grown Coriaria spp. and Datisca glomerata. Analysis of the draft genome confirmed its close proximity to the Candidatus Frankia datiscae Dg1 genome with the absence of 38 genes (trehalose synthase, fumarylacetoacetase, etc) in BMG5.1 and the presence of 77 other genes (CRISPR, lanthionine synthase, glutathione synthetase, catalase, Na+/H+ antiporter, etc) not found in Dg1. A multi-gene phylogeny placed the two cluster-2 strains together at the root of the Frankia radiation

An integrated overview of the midgut bacterial flora composition of Phlebotomus perniciosus, a vector of zoonotic visceral leishmaniasis in the Western Mediterranean Basin

International audienceBackground: The Leishmania developmental life cycle within its sand fly vector occurs exclusively in the lumen of the insect's digestive tract in the presence of symbiotic bacteria. The composition of the gut microbiota and the factors that influence its composition are currently poorly understood. A set of factors, including the host and its environment, may influence this composition. It has been demonstrated that the insect gut microbiota influences the development of several human pathogens, such as Plasmodium falciparum. For sand flies and Leishmania, understanding the interactions between the parasite and the microbial environment of the vector midgut can provide new tools to control Leishmania transmission. Methodology/Principal findings: The midguts of female Phlebotomus perniciosus from laboratory colonies or from the field were collected during the months of July, September and October 2011 and dissected. The midguts were analyzed by culture-dependent and culture-independent methods. A total of 441 and 115 cultivable isolates were assigned to 30 and 11 phylotypes from field-collected and colonized P. perniciosus, respectively. Analysis of monthly variations in microbiota composition shows a species diversity decline in October, which is to the end of the Leishmania infantum transmission period. In parallel, a compilation and a meta-analysis of all available data concerning the microbiota of two Psychodidae genera, namely Phlebotomus and Lutzomyia, was performed and compared to P. perniciosus, data obtained herein. This integrated analysis did not reveal any substantial divergences between Old and New world sand flies with regards to the midgut bacterial phyla and genera diversity. But clearly, most bacterial species (>76%) are sparsely distributed between Phlebotominae species. Conclusion/Significance: Our results pinpoint the need for a more exhaustive understanding of the bacterial richness and abundance at the species level in Phlebotominae sand flies in order to capture the role of midgut bacteria during Leishmania development and transmission. The occurrence of Bacillus subtilis in P. perniciosus and at least two other sand fly species studied so far suggests that this bacterial species is a potential candidate for paratransgenic or biolological approaches for the control of sand fly populations in order to prevent Leishmania transmission

Collection of <i>Picoa</i> species studied in the present work.

<p>Collection of <i>Picoa</i> species studied in the present work.</p

SEM and LM images of some of the samples studied.

<p><b>a-b</b>. Autoptic specimen of <i>Picoa juniperi</i> from Vittadini’s herbarium WU10-145 ex PAD; <b>c</b>. BMBH1; <b>d</b>. BMBT26; <b>e</b>. AH 19584; <b>f</b>. M-0157945; <b>g</b>. AH 39246; <b>h</b>. AH 39286; <b>i</b>. AH 38931; <b>j</b>. AH 39206; <b>k</b>. BMBH9; <b>l</b>. BMBH5; <b>m</b>. BMBO19. Bars: <b>a</b> = 10 μm; <b>b</b> = 5 μm; <b>c-d</b> = 5 μm; <b>e-f</b> = 5 μm; <b>g</b> = 10 μm; <b>h-j</b> = 5 μm; <b>k-l-m</b> = 5 μm.</p

ITS consensus Bayesian phylogram of genus <i>Picoa</i> and its sister taxon <i>Geopora</i> reconstructed in MrBayes 3.1.

<p>Bold nodes are significantly supported by both inference methods employed (>70% BP and >95% PP). Nodes annotated were significantly supported by only one of these methods. Values represent Bayesian posterior probabilities, and RAxML bootstrap proportions, respectively.</p