Melghirimyces algeriensis gen . nov . , sp . nov . , a member of the family Thermoactinomycetaceae , isolated from a salt lake

Correspondence Marie-Laure Fardeau marie-laure.fardeau@univ-amu.fr Laboratoire de Microbiologie MIO-IRD, UMR 235, Aix-Marseille Université, case 925, 163 Avenue de Luminy, 13288 Marseille cedex 9, France Laboratoire de Biologie Cellulaire et Moléculaire (équipe de Microbiologie), Université des Sciences et de la Technologie, Houari Boumédiènne, Bab Ezzouar, Algiers, Algeria Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany

The genus Thermoactinomyces was described by Tsilinsky (1899) and accommodates Gram-positive, aerobic species with filamentous growth similar to that of actinomycetes (Hatayama et al., 2005).Thus, members of this genus were initially considered to be actinomycetes.However, due to similarities in the development, heat resistance and other properties of the endospores to those of Bacillus strains (Cross et al., 1968) and due to phylogenetic differences from actinomycetes, thermoactinomycetes have been affiliated to the 'Bacillales' (Stackebrandt & Woese, 1981).
Subsequently, the genus Thermoactinomyces has been divided into six genera on the basis of phylogenetic and chemotaxomomic studies: Thermoactinomyces sensu stricto (Yoon et al., 2005), Laceyella, Thermoflavimicrobium, Seinonella, Planifilum (Hatayama et al., 2005) and Mechercharimyces (Matsuo et al., 2006).These genera have been grouped into the same family, Thermoactinomycetaceae, which was described by Matsuo et al. (2006).Other genera in this family have recently been described, including Shimazuella (Park et al., 2007) and Desmospora (Yassin et al., 2009), which has made emendation of the description of the family Thermoactinomycetaceae necessary (Yassin et al., 2009).The description of this family was further emended by von Jan et al. (2011).The authors included the new genus Kroppenstedtia, members of which contain the isomer LL-diaminopimelic acid (DAP) in the cell wall; previous members of the family Thermoactinomycetaceae had only been found to contain meso-DAP.
Here, we characterize a novel member of the family Thermoactinomycetaceae isolated from soil of an Algerian salt lake during our investigations on extremophilic actinomycetes with the aim to explore their potential as producers of original bioactive metabolites.
Samples were collected from soil taken from an Algerian salt lake named Chott Melghir located in the south-east of Algeria.
A portion (1 g) of the collected soil sample was transferred into 100 ml ISP 2 liquid medium (Shirling & Gottlieb, 1966) supplemented with 10 % NaCl in 500 ml flasks and incubated in a rotary shaker incubator at 55 u C with shaking at 160 r.p.m. for 5 days.From this culture, an aliquot of 0.1 ml was taken daily and inoculated onto the surface of ISP 2 agar containing 10 % NaCl; plates were incubated at 55 u C and monitored after 48, 72 and 96 h.Strain NariEX T was isolated by this procedure and maintained on ISP 2 agar with 10 % NaCl.
The biomass used for chemotaxonomic and phylogenetic studies was obtained from 72-h-old liquid cultures of strain NariEX T in ISP 1 and 2 media supplemented with 10 % NaCl and incubated at 55 u C. Morphological characteristics and purity of strain NariEX T were checked under an Optiphot (Nikon) phase-contrast microscope.For transmission electron microscopy studies, cell preparations were negatively stained with sodium phosphotungstate, as described previously (Fardeau et al., 1997).For microscopic examination, cells were grown on nutrient broth and agar supplemented with 10 % NaCl for 3 days at 55 uC.The study of cultural characteristics was conducted as described by Shirling & Gottlieb (1966).The colours of aerial and substrate mycelia were determined by observation with the naked eye of cultures after 72 h incubation at 55 u C on the following different semi-solid media: tryptone-yeast extract agar (ISP 1); malt extractyeast extract agar (ISP 2); inorganic salt-starch agar (ISP 4); and glycerol-asparagine agar (ISP 5) (Shirling & Gottlieb, 1966).All media were supplemented with 10 % NaCl.Cultures on each medium were examined with respect to the development of growth, colour of aerial and substrate mycelia, and production of diffusible pigments.
The strain was tested for the utilization of 17 carbohydrates, 19 amino acids and 10 organic acids (Table 1) as sole carbon and energy sources by using the amended basal medium of Tsukamura (1966).The medium contained 10 % NaCl and the following (g l 21 ): (NH 4 ) 2 SO 4 , 2.64; KH 2 PO 4 , 0.5; MgSO 4 .7H 2 O, 0.5; agar, 20.Each of the organic acid sodium salts, carbohydrates and amino acids was added to the medium to a final concentration of 0.02 M. For the detection of acid production from carbohydrates, the same medium was used and supplemented with 0.2 g yeast extract l 21 and 20 ml 0.04 % bromocresol blue solution l 21 as pH indicator (Tsukamura, 1966).Acid production was demonstrated by a change in the pH indicator from purple to yellow, indicating acidification of the medium.Basal medium without carbon source was used as a control.ISP 9 liquid medium was used to confirm carbohydrate and organic compound utilization tests.Utilization of amino acids was tested in ISP medium 9 without a nitrogen source.HPLC was used to detect degradation of substrates.
The degradation of casein and tyrosine was determined according to the method of Staneck & Roberts (1974).Hydrolysis of starch was determined on nutrient agar medium supplemented with 1 % starch.After incubation at 55 u C for 5 to 7 days, cultures have been flooded with iodine solution.The disappearance of blue colour around the colonies is scored as positive result (Cowan, 1974).The ability of the strain to degrade aesculin was examined according to the method of Gordon et al. (1974) modified by using aesculin agar instead of aesculin broth.The medium was composed of (g per litre distilled water): aesculin, 1; ferric citrate, 0.5; peptone, 10; agar, 20.Hydrolysis of xylan (0.4 %, w/v) was detected on the basal medium of Tsukamura (1966).Degradation of xanthine, hypoxanthine, adenine and gelatin was determined according to the method of Gordon (1966) and Gordon & Mihm (1957).
To determine the range of temperatures tolerated, strain NariEX T was grown on ISP 2 medium supplemented with 10 % NaCl and incubated at 35, 37, 40, 50, 55 and 60 u C. The NaCl requirement for growth was determined on the ISP medium 2 by varying the salt concentration in the culture medium.NaCl concentrations tested were 0,2,5,7,10,12,15,16,17,18,20 and 21 % and cultures were incubated at 55 u C.
Catalase activity was tested by immersing one loopful of 3-day-old culture of strain NariEX T on ISP 2 medium supplemented with 10 % NaCl into an aqueous solution of H 2 O 2 and observing the occurrence of bubbling.Urease activity was determined according to the method of La ´nyı (1987).Indole production, Voges-Proskauer reaction and nitrate reduction/denitrification were investigated by using the methods of Gordon & Mihm (1957), Gue ´rin-Fauble ´e et al. (1992) and Joffin & Leyral (2006).previously (Ben Dhia Thabet et al., 2004).The partial sequences generated were assembled using BioEdit version 5.0.9 (Hall, 1999) and the consensus sequence of 1344 nt was manually corrected for errors.The most closely related sequences in GenBank (version 178), and the Ribosomal Database Project (release 10) were identified using BLAST (Altschul et al., 1997) and the Sequence Match program (Cole et al., 2009).These sequences were retrieved and aligned and the alignment was adjusted manually according to the 16S rRNA secondary structure using BioEdit.Evolutionary distances were calculated by using the Jukes and Cantor correction (Jukes & Cantor, 1969).Dendrograms were constructed with the program TREECON using the neighbour-joining method (Saitou & Nei, 1987).Tree topology was evaluated by the bootstrap method (1000 replications) of resampling (Felsenstein, 1985).Its topology was also supported by using the maximum-parsimony and maximum-likelihood algorithms.Phylogenetic trees were inferred by maximum-parsimony (Fitch, 1971), maximum-likelihood (Felsenstein, 1981) and neighbour-joining (Saitou & Nei, 1987) algorithms, using the program TREECON.Tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings.
Genomic DNA for analysis of DNA G+C content and for DNA-DNA hybridization was isolated after disruption of bacterial cells by using a French press (Thermo Spectronic) and purified by chromatography on hydroxyapatite using the procedure of Cashion et al. (1977).The G+C content was determined by HPLC as described by Mesbah et al. (1989).DNA-DNA hybridizations were carried out as described by De Ley et al. (1970) considering the modifications described by Huß et al. (1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltierthermostatted 666 multicell changer and a temperature controller with in situ temperature probe (Varian).
For analysis of cellular fatty acids, cells were cultivated on ISP 1 medium supplemented with 10 % NaCl at 55 u C for 72 h under aerobic conditions.Fatty acid methyl esters were obtained following the method of Stead et al. (1992) and were analysed by GC using the Microbial Identification System (MIDI, Sherlock Version 6.1; database, TSBA40; GC model 6890N, Agilent Technologies).Polar lipids were determined according to the method described by Minnikin et al. (1979) and separated by twodimensional TLC.To identify spots, ninhydrin reagent, Zinzadze reagent and molybdophosphoric acid were used (Embley & Wait, 1994).Isoprenoid quinones were extracted as described by Collins et al. (1977) and analysed by HPLC (Groth et al., 1996) On ISP 2 medium, strain NariEX T formed colonies of 3-5 cm in diameter that were yellow-coloured with wrinkles formed between the centre and the edge of the colony, with a small projection in the centre, dull, flat and dry.After prolonged incubation, the diameter of the colony could reach that of the Petri dishes containing the medium.
Colonies were inlaid in the agar and difficult to detach.
On ISP 1 medium, strain NariEX T formed pale yellow aerial and substrate mycelium after 48 to 72 h at 55 u C. Colonies were pale yellow, shiny, flat, smooth with regular margins, easily detachable from the agar and could be removed as a whole.No diffusible pigments were detected on this growth medium.The strain grew well on ISP 1 and ISP 2 media, but weak growth was observed on ISP 4, 5, 6 and 7.No melanoid pigments were produced on ISP 6 or 7 media.Microscopic examination revealed long, straight to flexuous, moderately branched septate hyphae that developed single endospores (Fig. S1, available in IJSEM Online).Electron microscopy showed a dense outer cell wall (Fig. S2).
On Tsukamura medium, strain NariEX T utilized glycerol, maltose, cellobiose, rhamnose, sucrose, myo-inositol, fructose ribose, mannose, glucose, raffinose, alanine, threonine, proline, glycine, asparagine, glutamic acid, aspartic acid, acetate, oxalate, succinate and malate as sole carbon sources for growth.No acid production was detected from the utilization of carbohydrate.Results of the tests carried out on ISP 9 liquid medium were difficult to interpret because of the weak growth and weak degradation of substrates in liquid medium.
Casein and gelatin were decomposed by the strain.Xanthine, hypoxanthine, tyrosine, adenine and starch were not hydrolysed.Urease and Voges-Proskauer reactions were negative.Indole was not produced.Nitrate was reduced to nitrite.Results of physiological and biochemical tests are summarized in Tables 1 and 2.
The DNA G+C content of strain NariEX T was 47.3 mol%.Hybridization was done with Kroppenstedtia eburnea DSM 45196 T and the two strains shared only 9.6 % DNA-DNA relatedness.Sequence alignment and subsequent comparisons with sequences of representative members of the domain 'Bacteria' consistently placed strain NariEX T within the phylum 'Firmicutes', class Bacilli, order Bacillales, family Thermoactinomycetaceae with Desmospora activa and K. eburnea as its closest phylogenetic neighbours (95.38 and 94.28 % similarities, respectively, with the type strains of these two species) (Fig. 1).
Based on 16S rRNA gene sequences, strain NariEX T was assigned to the family Thermoactinomycetaceae with K. eburnea and D. activa as the closest relatives.DNA-DNA hybridization between the type strain of K. eburnea and strain NariEX T revealed a relatedness value of only 9.6 %, thus indicating the great difference between the two strains.
The novel isolate could be distinguished from members of the Thermoactinomycetaceae by several characteristics in addition to phylogenetic data (Table 3).The most important distinguishing feature of strain NariEX T was the presence of the LL-DAP isomer in the cell wall; all described genera of the family Thermoactinomycetaceae, except K. eburnea, contain meso-DAP.K. eburnea and D. activa, the closest relatives of strain NariEX T based on 16S rRNA gene sequences, differed from strain NariEX T in their menaquinone profiles: strain NariEX T , in addition to MK-7 as major menaquinone, also contains MK-6 and MK-8, whereas K. eburnea and D. activa contain exclusively MK-7.In addition, strain NariEX T is the only member of the family Thermoactinomycetaceae that contains menaquinone MK-6, thus distinguishing it from all taxa of the family Thermoactinomycetaceae with validly published names.Like K. eburnea, which contains only glucose and ribose, D. activa lacks any characteristic cell-wall sugars; the presence of xylose, mannose and galactose in the cell wall sugars of strain NariEX T differentiated it from its two closest neighbours.The fatty acid profiles of strain NariEX T , K. eburnea and members of the genus Laceyella all contain iso-C 15 : 0 and anteiso-C 15 : 0 as major fatty acids, which distinguishes the strain from all other genera of the family Thermoactinomycetaceae.Laceyella strains differ from NariEX T by menaquinone, colour of aerial mycelium and presence of meso-DAP in the peptidoglycan.Another unique feature of strain NariEX T is its halotolerance; the strain was able to grow at NaCl concentrations of up to 21 %.This characteristic has not previously been reported for members of the family Thermoactinomycetaceae.Exhibits the following properties in addition to those given in the genus description.On ISP 2 medium, colonies are yellow, flat, dull with regular margins and radial wrinkles formed on the surface.Glycerol, maltose, cellobiose, rhamnose, sucrose, myo-inositol, fructose, ribose, mannose, glucose, raffinose, alanine, threonine, proline, glycine, asparagine, DNA purification, and PCR amplification and sequencing of the 16S rRNA gene were performed as described International Journal of Systematic and Evolutionary Microbiology 62 Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Thu, 07 Feb 2019 00:54:13

Fig. 1 .
Fig. 1.Neighbour-joining phylogenetic dendrogram based on 1344 unambiguous nucleotides showing the relationship between strain NariEX T and organisms belonging to the family Thermoactinomycetaceae.The dendrogram was reconstructed based 16S rRNA gene sequences of the type strains.GenBank accession numbers are shown in parentheses.Numbers at branch points specify the reliability of the branching order determined for 1000 resamplings; only bootstrap values ¢50 % are shown.Bar, 5 substitutions per 100 nucleotide positions.

Table 1 .
Morphological and physiological characteristics of strain NariEX T

Table 2 .
Fatty acid composition of strain NariEX T A. N. Addou and others International Journal of Systematic and Evolutionary Microbiology 62 Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Thu, 07 Feb 2019 00:54:13 Strainsof the genera Laceyella and Thermoflavimicrobium are not capable of growing in salinities greater than 1 %.Phenotypic and molecular studies indicate that strain NariEX T can be clearly distinguished from members of other genera of the family Thermoactinomycetaceae.Examination of the growth temperature, DNA G+C content, menaquinone composition and cellular fatty acids revealed that strain NariEX T did not belong to the genera Thermoactinomyces sensu stricto, Laceyella, Thermoflavimicrobium, Seinonella, Planifilum or Mechercharimyces.On the basis of the results presented in this study, strain NariEX T represents a novel species in a new genus in the family Thermoactinomycetaceae, for which the name Melghirimyces algeriensis gen.nov., sp.nov. is proposed.