Karima Hezbri

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.

Stone-dwelling actinobacteria Blastococcus saxobsidens , Modestobacter marinus and Geodermatophilus obscurus proteogenomes

The Geodermatophilaceae are unique model systems to study the ability to thrive on or within stones and their proteogenomes (referring to the whole protein arsenal encoded by the genome) could provide important insight into their adaptation mechanisms. Here we report the detailed comparative genome analysis of Blastococcus saxobsidens (Bs), Modestobacter marinus (Mm) and Geodermatophilus obscurus (Go) isolated respectively from the interior and the surface of calcarenite stones and from desert sandy soils. The genome-scale analysis of Bs, Mm and Go illustrates how adaptation to these niches can be achieved through various strategies including ‘molecular tinkering/opportunism’ as shown by the high proportion of lost, duplicated or horizontally transferred genes and ORFans. Using high-throughput discovery proteomics, the three proteomes under unstressed conditions were analyzed, highlighting the most abundant biomarkers and the main protein factors. Proteomic data corroborated previously demonstrated stone-related ecological distribution. For instance, these data showed starvation-inducible, biofilm-related and DNA-protection proteins as signatures of the microbes associated with the interior, surface and outside of stones, respectively.

Geodermatophilus poikilotrophi sp. nov.: A Multitolerant Actinomycete Isolated from Dolomitic Marble

A novel Gram-reaction-positive, aerobic actinobacterium, tolerant to mitomycin C, heavy metals, metalloids, hydrogen peroxide, desiccation, and ionizing- and UV-radiation, designated G18T, was isolated from dolomitic marble collected from outcrops in Samara (Namibia). The growth range was 15–35°C, at pH 5.5–9.5 and in presence of 1% NaCl, forming greenish-black coloured colonies on GYM Streptomyces agar. Chemotaxonomic and molecular characteristics of the isolate matched those described for other representatives of the genus Geodermatophilus. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diaminoacid. The main phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and small amount of diphosphatidylglycerol. MK-9(H4) was the dominant menaquinone and galactose was detected as diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids iso-C16:0 and iso-C15:0 and the unsaturated C17:1 ω8c and C16:1 ω7c. The 16S rRNA gene showed 97.4–99.1% sequence identity with the other representatives of genus Geodermatophilus. Based on phenotypic results and 16S rRNA gene sequence analysis, strain G18T is proposed to represent a novel species, Geodermatophilus poikilotrophi. Type strain is G18T (= DSM 44209T = CCUG 63018T). The INSDC accession number is HF970583. The novel R software package lethal was used to compute the lethal doses with confidence intervals resulting from tolerance experiments.

Blastococcus capsensis sp. nov., isolated from an archaeological Roman pool and emended description of the genus Blastococcus, B. aggregatus, B. saxobsidens, B. jejuensis and B. endophyticus.

A novel Gram-reaction-positive actinobacterium, designated BMG 804T, was isolated from an archaeological Roman pool located in Gafsa, Tunisia. The strain grew as dry bright orange colonies at 30 °C and pH 6.0-8.0. It contained meso-diaminopimelic acid in the cell wall. The whole-cell sugars consisted of glucose, rhamnose and ribose. Polar lipids present were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, an unidentified glycolipid and two unidentified phospholipids. MK-9(H4) was the predominant menaquinone. The fatty acid profile contained major amounts (>5 %) of C17 : 1ω8c, C16 : 1ω7c, iso-C15 : 0, iso-C16 : 0 and iso-C16 : 1H. The 16S rRNA gene sequence of BMG 804T showed 99.4 % as highest sequence similarity with Blastococcussaxobsidens. DNA-DNA hybridization between strain BMG 804T and B.saxobsidens DSM 44509T was 48.6±6.6 %. The G+C content of the DNA was 73.7 mol%. On the basis of the phenotypic and genotypic characteristics, including DNA-DNA hybridization results, BMG 804T (=DSM 46835T=CECT 8876T) is proposed as the type strain of a novel species Blastococcuscapsensis sp. nov. Emended descriptions of the genus Blastococcus and the species Blastococcus aggregatus, B. saxobsidens, Blastococcus jejuensis and Blastococcus endophyticus are also proposed.

Geodermatophilus pulveris sp. nov., a gamma-radiation-resistant actinobacterium isolated from the Sahara desert

A black-pigmented, aerobic actinobacterium, tolerant to ionizing radiation, designated BMG 825T, was isolated from desert limestone dust in Tunisia. The strain grew within the temperature range 10-40 °C, at pH 5.5-11.0 and in the presence of 2 % NaCl. The DNA G+C content was 75.7 mol%, and its cell-wall peptidoglycan contained meso-diaminopimelic acid. Sugars of whole-cell hydrolysates were galactose, glucose, and trace amounts of ribose and mannose. The predominant menaquinone was MK-9(H4), and the major fatty acids were iso-C16 : 0 and C16 : 1ω7c. The polar lipid profile comprised phosphatidylcholine, phosphatidylinositol, diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine and an unspecified glycolipid. 16S rRNA gene sequence analysis revealed that the strain fell into the genus Geodermatophilus, showing the highest similarity with Geodermatophilus poikilotrophus DSM 44209T (99.1 %). DNA-DNA hybridization results, phylogenetic distinctiveness and phenotypic properties supported the classification of this strain as a representative of a novel species of the genus Geodermatophilus, for which the name Geodermatophilus pulveris sp. nov. is proposed. The type strain is BMG 825T (=CECT 9003T=DSM 46839‏T).

Geodermatophilus sabuli sp. nov., a γ-radiation-resistant actinobacterium isolated from desert limestone.

A novel γ-radiation-resistant and Gram-staining-positive actinobacterium designated BMG 8133T was isolated from a limestone collected in the Sahara desert of Tunisia. The strain produced dry, pale-pink colonies with an optimum growth at 35–40 °C and pH 6.5–8.0. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. The main polar lipids were phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine and one unspecified glycolipid. MK-9(H4) was the dominant menaquinone. Galactose and glucose were detected as diagnostic sugars. The major cellular fatty acids were branched-chain saturated acids iso-C16 : 0 and iso-C15 : 0. The DNA G+C content of the novel strain was 74.5 %. The 16S rRNA gene sequence showed highest sequence identity with Geodermatophilus ruber (98.3 %). Based on phenotypic results and 16S rRNA gene sequence analysis, strain BMG 8133T is proposed to represent a novel species, Geodermatophilus sabuli sp. nov. The type strain is BMG 8133T ( = DSM 46844T = CECT 8820T).

Permanent Improved High-Quality Draft Genome Sequence of Nocardia casuarinae Strain BMG51109, an Endophyte of Actinorhizal Root Nodules of Casuarina glauca

ABSTRACT Here, we report the first genome sequence of a Nocardia plant endophyte, N. casuarinae strain BMG51109, isolated from Casuarina glauca root nodules. The improved high-quality draft genome sequence contains 8,787,999 bp with a 68.90% GC content and 7,307 predicted protein-coding genes.

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.

Permanent Draft Genome Sequences of Three Frankia sp. Strains That Are Atypical, Noninfective, Ineffective Isolates

ABSTRACT Here, we present draft genome sequences for three atypical Frankia strains (lineage 4) that were isolated from root nodules but are unable to reinfect actinorhizal plants. The genome sizes of Frankia sp. strains EUN1h, BMG5.36, and NRRL B16386 were 9.91, 11.20, and 9.43 Mbp, respectively.

Permanent Draft Genome Sequence of Nocardia sp. BMG111209, an Actinobacterium Isolated from Nodules of Casuarina glauca

ABSTRACT Nocardia sp. strain BMG111209 is a non-Frankia actinobacterium isolated from root nodules of Casuarina glauca in Tunisia. Here, we report the 9.1-Mbp draft genome sequence of Nocardia sp. strain BMG111209 with a G + C content of 69.19% and 8,122 candidate protein-encoding genes.