Antonie van Leeuwenhoek DOI 10.1007/s10482-015-0461-z

ORIGINAL PAPER

Geodermatophilus aquaeductus sp. nov., isolated from the ruins of Hadrian’s aqueduct Karima Hezbri . Faten Ghodhbane-Gtari . Maria del Carmen Montero-Calasanz . Haı¨tham Sghaier . Manfred Rohde . Cathrin Spro¨er . Peter Schumann . Hans-Peter Klenk . Maher Gtari

Received: 23 March 2015 / Accepted: 16 April 2015 Ó Springer International Publishing Switzerland 2015

Abstract An orange-black, Gram-positive, aerobic and gamma-ray resistant actinobacterium was isolated from the ruins of a Roman aqueduct located in Northern Tunisia. The optimal growth for the strain was found to be at 25–35 °C and at pH 6.0–9.5. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The peptidoglycan was found to contain meso-diaminopimelic acid as diagnostic diaminoacid. The main polar lipids were identified as phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, an unidentified glycolipid and an unidentified

Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0461-z) contains supplementary material, which is available to authorized users. K. Hezbri  F. Ghodhbane-Gtari  M. Gtari (&) Laboratoire Microorganismes et Biomole´cules Actives, Universite´ de Tunis El Manar (FST) & Universite´ de Carthage (INSAT), 2092 Tunis, Tunisia e-mail: [email protected] M. del Carmen Montero-Calasanz  C. Spro¨er  P. Schumann  H.-P. Klenk Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany

aminophospholipid; MK-9(H4) was found to be the dominant menaquinone and galactose was detected as the diagnostic sugar, with glucose, ribose and mannose also present. The major cellular fatty acids were identified as branched-chain saturated acids iso-C16:0, iso-C15:0 and iso-H-C16:0. The 16S rRNA gene showed 95.4–99.6 % sequence identity with the type strains of the genus Geodermatophilus. DNA–DNA relatedness values with closely related species were 39.9 ± 4.9, 33.9 ± 1.9, 27.0 ± 2.5 and 13.2 ± 1.35 % with Geodermatophilus amargosae, G. normandii, G. saharensis and G. tzadiensis respectively. Based on phenotypic results and 16S rRNA gene sequence analysis, strain BMG801T (=DSM 46834T = CECT 8822T) is proposed to represent the type strain of a novel species, Geodermatophilus aquaeductus sp. nov.

M. Rohde Central Facility for Microscopy, HZI – Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany H.-P. Klenk School of Biology, Newcastle University, Ridley Building, Newcastle upon Tyne NE1 7RU, UK

H. Sghaier Centre National des Sciences et Technologies Nucle´aires, Poˆle Technologique, BP 72, 2020 Sidi Thabet, Tunisia

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Antonie van Leeuwenhoek

Keywords Geodermatophilus  Gamma ray  Monument  Taxonomy

Materials and methods Strain isolation, growth and morphological characterization

Introduction The order Geodermatophiliales was recently described based on complete genome sequence data (Sen et al. 2014) and contains only a single family, Geodermatophilaceae (Normand 2006; Normand et al. 2014). This family was emended five years ago by Zhi et al. (2009) to include the three genera Geodermatophilus, Blastococcus and Modestobacter, members of which were initially isolated from desert soils (Luedemann 1968), sea water (Ahrens and Moll 1970) and Antarctic regolith (Mevs et al. 2000), respectively. Members of these three genera form rudimentary hyphae and have in common a modest growth requirement and the ability to grow as pioneers on poor substrates (Normand and Benson 2012). The type genus Geodermatophilus contains actinobacteria that form dark, light-red or coral-pink pigmented and non-aerial rudimentary mycelia that eventually develop complex sporangia (Normand and Benson 2012). The genus currently comprises 17 species mainly isolated from arid and hyperarid environments such as sand and stone, although some were also isolated from rhizospheric soils and lake sediments. Beside Geodermatophilus obscurus G-20T that was isolated by Luedemann (1968) and genome-sequenced by Ivanova et al. (2010), seventeen validly named species have been classified in the genus within the last 4 years (Bertazzo et al. 2014; Jin et al. 2013; Montero-Calasanz et al. 2012, 2013a, b, c, d, e, f; Montero-Calasanz et al. 2014a, b; Nie et al. 2012; Qu et al. 2013). Members of the genus Geodermatophilus have been reported to be markedly resistant to adverse environmental conditions such as ultraviolet (UV) light, ionizing radiation (IR), desiccation and heavy metals (Rainey et al. 2005; Gtari et al. 2012; Montero-Calasanz et al. 2013b; Montero-Calasanz et al. 2014b). Moreover, some strains were revealed to produce remarkably resistant enzymes such as esterases (Essoussi et al. 2010; Jaouani et al. 2012; Normand et al. 2014). Here we describe a new species of this genus, Geodermatophilus aquaeductus sp. nov., isolated from the surface of an altered calcarinite stone found in the ruins of a Roman aqueduct located in northern Tunisia.

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Strain BMG801T was isolated during a screening of microorganisms from the surface of an altered calcarinite stone found in the ruins of a Roman aqueduct, called the aqueduct of Hadrian, which supplied water to the ancient city of Carthage from Zaghouan city [Northern Tunisia longitude 10°90 000 E Latitude 36°240 000 N]. Portions of the sample were treated with saline solution (NaCl 0.9 % (w/v)), shaken overnight at 28 °C before the solution was streaked out on Luedemann plates and incubated at 28 °C for up to 20 days. A moist orange-black colony of the strain was isolated, maintained on Luedemann medium (DSMZ medium 877) and designated as BMG801T. Cultural characteristics were noted on GYM Streptomyces medium (DSMZ medium 65), R2A medium (DSMZ medium 830) and Luedemann medium for 5 days at 28 °C. Morphological characterisation of the bacterial cells was assessed on 7 days old cultures grown in broth using a field-emission scanning electron microscope (FE-SEM Merlin, Zeiss, Germany). Motility of cells was tested on modified ISP2 (Shirling and Gottlieb 1966) swarming agar (0.3 %, w/v) at pH 7.2 supplemented with (l-1) 4.0 g dextrin, 4.0 g yeast extract and 10.0 g malt extract. The Gram type of the strain was determined by the KOH test (Gregersen 1978). Oxidase and catalase activities were analysed as previously described (Montero-Calasanz et al. 2012). Culture of the strain was tested on modified ISP2 medium under variable pH values from 4.0 to 12.5 (in increments of 0.5 pH units) and on GYM Streptomyces medium for different temperatures ranging from 10 to 60 °C at 5 °C increments. The ability of the strain to degrade specific substrates was examined using agar plates with various basal media: casein degradation was tested on plates containing milk powder (5 % w/v), NaCl (0.5 %) and agarose (1 %); tyrosine degradation was determined as previously described by Gordon and Smith (1955) on plates containing peptone (0.5 %), beef extract (0.3 %), L-tyrosine (0.5 %) and agarose (1.5 %). Xanthine and hypoxanthine decompositions (0.4 %) were examined using the same basal medium, while starch degradation was tested on plates containing nutrient broth (0.8 %).

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Enzymatic activities were tested using API ZYM galleries according to the instructions of the manufacturer (bioMe´rieux). The oxidation of carbon compounds was tested at 28 °C using API 20 NE strips (bioMe´rieux) and GEN III Microplates in an Omnilog device (BIOLOG Inc., Hayward, CA, USA). Data was then compared with the reference strains Geodermatophilus africanus DSM 45422T, Geodermatophilus amargosae DSM 46136T, Geodermatophilus arenarius DSM 45418T, Geodermatophilus nigrescens DSM 45408T, Geodermatophilus normandii DSM 45417T, G. obscurus DSM 43160T, Geodermatophilus saharensis DSM 45423T, Geodermatophilus telluris DSM 45421T, Geodermatophilus tzadiensis DSM 45416T and Geodermatophilus dictyosporus DSM 43161T in parallel assessments. The GEN III Microplates were analysed for BMG801T as well as for the reference strains in duplicate and cells suspensions were prepared in a viscous inoculating fluid (IF C) provided by the manufacturer at 70 % Transmittance (T) for G. amargosae DSM 46136T, at 75–79 % T for G. africanus DSM 45422T, at 90 % T for G. arenarius DSM 45418T, at 80–83 % T for G. dictyosporus DSM 43161T and all other reference strains and at 82–85 %

for strain BMG801T. Data were exported and analysed using the opm package for R (Vaas et al. 2012, 2013) v.1.0.6. Reactions with a distinct behaviour between the two replicates were regarded as ambiguous. Chemotaxonomic analysis Whole-cell amino acids and sugars were extracted following a standard protocol (Lechevalier and Lechevalier 1970) and analysed by thin layer chromatography (TLC) according to Staneck and Roberts (1974). Separation by two-dimensional TLC and identification of polar lipids were performed according to procedures outlined by Minnikin et al. (1984). Additionally, choline-containing lipids were detected by spraying with Dragendorff’s reagent (Merck) (Tindall 1990). Extraction of menaquinones (MK) was carried out from freeze-dried cell material using methanol (Cashion et al. 1977) and further analysed using high-performance liquid chromatography (HPLC) (Kroppenstedt 1982). Cellular fatty acids were extracted in two independent repetitions from biomass grown on GYM agar plates held at 28 °C for 4 days and harvested always from the same sector (the last quadrant streak). Fatty acids analysis was

Fig. 1 Scanning electron micrograph of strain BMG801T grown for 7 days on GYM Streptomyces medium at 28 °C. Cells are pleiomorphic, singular or associated in cauliflower-like aggregates

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Light-orange, black

Moist

6.0–9.5

25–35

Colony colour on GYM

Colony surface on GYM

pH optimum

Temperature optimum (°C)

±

?

±

-

?

?

?

±

±

D-Cellobiose

Turanose

a-D-Lactose

D-Mannose

D-Galactose

L-Rhamnose

Sodium lactate

D-Arabitol

D-Glucose-6-

?

-

-

Methyl pruvate

Acid

Acid

Pectin

L-Lactic

D-Malic

MK-9(H4)

PC, DPG, PI, PE, APL

i-C16:0, i-C15:0, i-H-C16:1

Predominant menaquinone(s)a

Polar lipidsb

Major fatty acidsc

-

-

±

L-Histidine

-

L-Glutamic

acid

L-Arginine

Phosphate

±

D-Maltose

Utilization of:

1

Characteristics

i-C15:0, i-C16:0

PC, DPG, PI, PE

MK-9(H4), MK9(H0)

?

-

-

-

-

-

?

?

-

-

?

?

?

?

?

?

?

25–35

6.5–8.0

Dry

Black

2

i-C15:0, i-C16:0, C17:1x8c

DPG, PC, PE, PI, PG

MK-9(H4), MK9(H2), MK8(H4)

?

?

-

?

-

?

-

-

?

±

-

?

?

-

?

?

?

24–28

–

Dry

Black

3

i-C15:0, ai-C15:0 i-C16:0, ai-C17:0, C17:1x8c

DPG, PE, PC, PI, 2PL,PG

MK-9(H4)

?

?

?

-

?

?

-

-

-

?

-

?

-

-

?

?

?

20–32

7.0–7.5

Moist

Light-red, red

4

i-C15:0, i-C16:0

DPG, PE, PC, PI, PG

MK-9(H4)

±

-

±

?

-

?

-

?

±

?

?

±

?

±

?

?

?

37

7.0

Moist

Light-red, black

5

Table 1 Phenotypic characteristics of strain BMG801T and related Geodermatophilus species

i-C15:0, i-C16:0

PE, PC, DPG, PI, PG,

MK-9(H4), MK8(H4), MK-9 (H0)

-

?

?

-

-

?

-

-

-

?

?

?

?

?

?

?

?

28–40

6.0–8.5

Moist

Light-red, brown

6

i-C15:0, i-C16:0, C17:1x8c

PE, PC, PI, DPG, PG

MK-9(H4), MK8(H4), MK9(H0)

?

?

?

?

-

?

?

-

?

?

-

?

?

-

-

-

?

20–37

6.0–8.5

Moist

Light-red, black

7

i-C15:0, i-C16:0, i-H-C16:1

DPG, PC, PI, PE, PG

MK-9(H4), MK-8(H4)

?

±

?

?

?

?

?

-

?

?

?

?

?

?

?

?

?

20–35

6.0–8.0

Moist

Light-red, black

8

i-C15:0, i-C16:0

DPG, PC, PE, PI, PG

MK-9(H4), MK-9(H0)

±

?

-

?

-

-

?

-

-

-

?

?

?

-

?

?

?

25–37

6.0–8.5

Moist

Light-red, greenishblack

9

i-C15:0, i-C16:0

DPG, PC, PE, PI, APL, PG

MK-9(H4)

?

?

?

?

-

?

?

-

?

?

?

?

?

-

?

?

?

20–35

6.0–8.5

Dry

Black

10

Antonie van Leeuwenhoek

–

D-Glucose-6-

?

acid

acid

L-Lactic

D-Malic

MK-9(H4), MK9(H0),MK-9(H2)

DPG, PME, PE, PI, 3PL

Predominant menaquinone(s)a

Phospholipidsb

?

?

Methyl pyruvate

–

?

L-Histidine

Pectin

–

?

L-Glutamic

Acid

L-Arginine

Phosphate

–

D-Arabitol

?

?

±

D-Galactose

±

?

D-Mannose

L-Rhamnose

?

a-D-Lactose

Sodium Lactate

?

?

Turanose

DPG, PME, PE, PI, 5PL

MK-9(H4), MK9(H0)

?

?

?

?

–

?

–

±

–

?

?

?

?

?

?

?

?

30

D-Cellobiose

30

Temperature optimum (°C)

7.0

Moist

Light red

12d

D-Maltose

Moist

7.0

pH optimum

Light red

Colony colour on GYM

Colony surface on GYM

11d

Characteristics

Table 1 continued

DPG, PC, PE, PI, PG

MK-9(H4)

–

–

±

–

–

–

–

–

–

?

–

–

–

–

–

±

–

25–35

6.0–8.0

Dry

Black

13

DPG, PC, PE, PI, PG

MK-9(H4)

–

–

?

?

–

?

–

±

–

–

?

?

?

–

?

?

?

25–40

6.0–10.0

Moist

Light-red, greenish-black

14

DPG, PE, PI, PIM

MK-9 (H4), MK-9(H0)

–

–

?

?

–

?

–

–

?

?

–

–

–

±

?

?

?

28

7.0

Moist

Coral pink

15e

DPG, PC, PE, PI, PG

MK-9(H4)

–

±

±

–

±

±

–

–

–

±

–

±

±

±

?

–

–

25–35

6.0–12.0

Dry

Black

16

DPG, PE, PE-OH, PI, GPL, PL

MK-9(H4), MK-9(H0), MK10(H4), MK-9(H2)

–

–

?

?

–

?

–

±

±

?

?

–

?

?

?

?

?

20–30

6.5–8.5

Moist

Coral pink

17

PE, PC, PI, DPG

MK-9(H4)

?

±

?

?

–

–

?

–

–

–

?

?

?

±

–

?

?

25–30

7.0–9.5

Moist

Light-red, greenish-black

18

Antonie van Leeuwenhoek

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conducted using the Microbial Identification System (MIDI) Sherlock Version 6.1 (method TSBA40, ACTIN6 database) as described by Sasser (1990) and their annotation was performed according to IUPAC nomenclature as previously outlined by Montero-Calasanz et al. (2012). The peptidoglycan content was hydrolysed by HCl (6 N) at 100 °C for 16 h followed by TLC analysis as described by Schleifer and Kandler (1972). All chemotaxonomic analyses were conducted under standardized conditions for BMG801T strain and using a set of closely related reference strains.

Results taken from Jin et al. (2013)

Results taken from Qu et al. (2013) e

d

The components are listed in decreasing order of quantity

Only components making up C /10 % peak area ratio are shown c

b

Only components making up C /5 % peak area ratio are shown

DNA extraction, identification, PCR amplification, sequencing and phylogenetic analysis

a

All physiological data are from this study ? positive reaction; - negative reaction; ± ambiguous; MK menaquinones; DPG diphosphatidylglycerol; PE phosphatidylethanolamine; PG phosphatidylglycerol; PC phosphatidylcholine; PI phosphatidylinositol; APL unknown amino-phospholipid

C18:1x9c

Strains 1 G. aquaeductus sp. nov. BMG801T, 2 G. dictyosporus DSM 43161T, 3 G. obscurus DSM 43160T, 4 G. ruber DSM 45317T, 5 G. nigrescens DSM 45408T, 6 G. arenarius DSM 45418T, 7 G. siccatus DSM 45419T, 8 G. saharensis DSM 45423T, 9 G. tzadiensis DSM 45416T, 10 G. telluris DSM 45421T, 11 G. soli DSM 45843T, 12 G. terrae DSM 45844T, 13 G. africanus DSM 45422T, 14 G. normandii DSM 45417T, 15 G. taihuensis DSM 45962T, 16 G. amargosae DSM 46136T, 17 G. brasiliensis DSM 44526T, 18 G poikilotrophi DSM 44209T

C17:1x8c

i-C15:0,

i-C16:0, C17:1x8c

i-C16:0,

i-C15:0,

i-C15:0,

i-C16:0

i-C15:0, i-C15:0, i-C16:0 i-C15:0,

i-C16:0,

i-C15:0, Major fatty acidsc

i-C16:0, i-C17:0

i-C16:0

i-C16:0, C17:1x8c

17 13 Characteristics

Table 1 continued

11d

12d

14

15e

16

18

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Genomic DNA extraction, PCR amplification and sequencing of the 16S rRNA gene were performed as described previously (Montero-Calasanz et al. 2012). Pairwise similarities between the almost full length 16S rRNA gene sequence of isolate BMG801T and corresponding sequences of the most closely related type strains were calculated as recommended by Meier-Kolthoff et al. (2013). The evolutionary distance between the sequences was calculated using Kimura’s two parameter model (Kimura 1980). Phylogenetic trees based on the aligned sequences were inferred using the maximum-likelihood (Felsenstein 1981), maximum parsimony (Fitch 1971) and neighbour-joining (Saitou and Nei 1987) trees generated with MEGA6 (Tamura et al. 2013) and 1000 bootstrap replicates. For DNA–DNA hybridization tests, cells were disrupted by using a Constant Systems TS 0.75 KW (IUL Instruments, Germany). DNA in the crude lysate was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA– DNA hybridization was carried out as described by De Ley et al. (1970) and modified by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6 9 6 multicell changer and a temperature controller with in situ temperature probe (Varian). Gamma-ray experiments Prior to testing their tolerance to ionizing-radiation, non-sporulating cultures of strains BMG801T and G. obscurus G-20T (used as control; (Gtari et al. 2012)), were prepared by growing them in Luedemann

Antonie van Leeuwenhoek 57

BMG801 Geodermatophilus amargosae DSM 46163T (HF679056) Geodermatophilus normandii DSM45417T (HE654546) Geodermatophilus tzadiensis DSM 45416T (HE654545) Geodermatophilus dictyosporus DSM 43161T (HF970584)

78

Geodermatophilus saharensis DSM 45423T (HE654551) 92

Geodermatophilus telluris DSM 45421T (HE815469) 98

61

Geodermatophilus arenarius DSM 45418T (HE654547) Geodermatophilus nigrescens DSM 45408T (JN656711)

Geodermatophilus africanus DSM 45422T (HE654550)

57

Geodermatophilus ruber DSM 45317T (EU438905)

63

Geodermatophilus poikilotrophi DSM 44209T (HF970583)

84 63

Geodermatophilus obscurus DSM 43160T (X92356) Geodermatophilus siccatus DSM 45419T (HE654548) Geodermatophilus terrae DSM 45844T(JN033773)

100 64

100

Geodermatophilus soli DSM 45843T (JN033772) Geodermatophilus taihuensis DSM45862T (JX294478) Geodermatophilus brasiliensis DSM 44526T (DQ029102)

76

Modestobacter marinus DSM 45201T (EU181225)

96

Modestobacter roseus DSM 45764T (JQ819258) 87

Modestobacter versicolor DSM 16678T (AJ871304) Modestobacter multiseptatus DSM 44406T (Y18646)

Blastococcus endophyticus DSM45413T (GQ494034)

60

Blastococcus saxobsidens DSM 44509T (FN600641)

71 85

Blastococcus jejuensis DSM 19597T(DQ200983) Blastococcus aggregatus DSM 4725T (L40614)

Frankia alni ACN14a (CT573213)

0.005

Fig. 2 Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences, showing the phylogenetic position of strain BMG801T within the family Geodermatophilaceae. Only bootstrap values higher than 50 % are shown above the branches

medium supplemented with tryptose (Difco, Detroit, USA) (Ishiguro and Wolfe, 1970) for 5 days and at 28 °C. Ionizing-radiation experiments were carried out according to the protocol outlined by Gtari et al. (2012).

Results and discussion Strain BMG801T was observed to grow well on GYM Streptomyces and Luedemann media, but did not grow on R2A medium. Growth of BMG801T was observed at pH 5.0-10.5 (optimum: pH 6.0–9.5) and a temperature range from 10 to 45 °C (optimum 25–35 °C). Growth was observed in the presence of 1 % NaCl but not in the presence of 4 and 8 % NaCl. Colonies were observed to be moist black and orange at the border. The cells were observed to be Gram positive and pleiomorphic, appearing under microscopy as individual cells or associated in cauliflower-like

aggregates (Fig. 1). Results from phenotype microarray analysis are shown as a heatmap in the supplementary material (Fig. S1). A summary of selected differential phenotypic characteristics is presented in Table 1. Analysis of cell-wall components revealed the presence of meso-diaminopimelic acid (Cell wall type III), consistent with the descriptions of members of other species of the genus Geodermatophilus (Lechevalier and Lechevalier 1970; Montero-Calasanz et al. 2014). Strain BMG801T was found to contain mainly menaquinone MK-9(H4) (99.1 %), in agreement with values reported for the family Geodermatophilaceae (Normand 2006). The major fatty acids identified were the saturated branched-chain acids iso-C16:0 (47.1 ± 0.8 %), iso-C15:0 (19.1 ± 0.4 %) and iso-H-C16:0 (15.0 ± 0.02 %). The phospholipid pattern was found to consist of phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine (Supplementary Fig. S2), which is in accordance with profiles obtained

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Antonie van Leeuwenhoek Fig. 3 Estimation of survival following exposure to gamma-radiation for strain BMG801T and G. obscurus DSM 43160T as positive control. The mean c.f.u. ml-1 per strain is given together with the LD50 and LD10 values in the upper panel of the figure; y-axis is on a logarithmic scale. The lower panel depicts LD10 and LD50 values per strain and the differences between strains together with confidence intervals. Confidence intervals that do not contain zero (dashed vertical line) indicate significant differences to zero

for the type strains of other Geodermatophilus species investigated in this study (Table 1). An unidentified glycolipid and an unidentified aminophospholipid were additionally detected. Whole-cell sugar analysis revealed galactose as the diagnostic sugar (Lechevalier and Lechevalier 1970) but also glucose, ribose and mannose. Strain BMG801T, together with type strains of all members of the genus Geodermatophilus, were placed within the same phylogenetic group based on 16S rRNA gene sequence and Maximum Likelihood estimation (Fig. 2). Even though high 16S rRNA gene sequence similarities were observed between strain BMG801T and G. amargosae (99.6 %), G. normandii (99.6 %), G. saharensis (99.2 %) and G. tzadiensis (99.1 %) type strains, low DNA–DNA relatedness were measured as being respectively 39.9 ± 4.9, 33.9 ± 1.9, 27.0 ± 2.5 and 13.2 ± 1.35. Accordingly strain BMG801T clearly represents a member of a separate and new bacterial species (Wayne et al. 1988).

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Gamma-ray tolerance Gamma-radiation survival of strain BMG801T (Fig. 3) showed insignificantly different inactivation kinetics when compared with G. obscurus DSM 43160T, a highly resistant strain according to Gtari et al. (2012). Strain BMG801T and G. obscurus DSM 43160T exhibited LD10 s of 8 and 9 kGy, respectively, which are comparatively high values when compared with those displayed by the highly radioresistant strain D. radiodurans R1 (*7 kGy) (Battista et al. 1999), although other authors have reported a LD10 around 13 kGy using the same strain (Daly 2009). Apart from the phylogenetic analysis based on 16S rRNA gene sequences, together with DNA–DNA relatedness, several phenotypic features support the distinctiveness of strain BMG801T from representatives of all other Geodermatophilus species (Table 1). Based on the phenotypic and genotypic data presented, we propose that strain BMG801 T represents the type

Antonie van Leeuwenhoek

strain of a novel Geodermatophilus species with the name Geodermatophilus aquaeductus sp. nov.

directly or indirectly to this research exist that may be important to readers to be disclosed.

Description of Geodermatophilus aquaeductus sp. nov

References

Geodermatophilus aquaeductus (a.quae.duc’tus. L. gen. n. aquaeductus, of an aqueduct, pertaining to the Roman aqueducts that were used to carry water, referring to the location where the type strain of the species was sampled). Colonies are orange black light coloured, regular, with a moist surface. Cells are Gram- positive, catalase positive and oxidase negative. Cell growth range is from 10 to 45 °C and from pH 5.0 to 10.5. No diffusible pigments are produced on any of the tested media. Utilises several carbon and nitrogen sources (Table 1). Positive for aesculin degradation, but negative for the reduction of nitrate and denitrification, indole production and casein, tyrosine, starch, xanthine gelatin and hypoxanthine degradation. Test for alkaline phosphatase is positive but those for leucine arylamidase, esterase (C4), esterase lipase (C8), valine arylamidase, ß-galactosidase, a- and ß-glucosidase, acid phosphatase, Naphthol-AS-BI-phosphohydrolase, lipase (C14), cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, ß-glucuronidase, N-acetyl-ß-glucosamidase, a-mannosidase, urease and a-fucosidase are negative. The peptidoglycan in the cell wall contains meso-diaminopimelic acid as diamino acid, with galactose as the diagnostic sugar. The predominant menaquinone is MK-9(H4). The main polar lipids are phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol, and phosphatidylethanolamine. Cellular fatty acids consist mainly of the branched-chain saturated acids iso-C16:0, iso-C15:0 and iso-H-C16:0. The type strain is BMG801T (=DSM 46834T = CECT 8822 T). The INSDC accession number of the 16S rRNA gene sequence of the type strain is LN626272. Acknowledgments This research was supported by The Ministe`re de l’Enseignement Supe´rieur et de la Recherche Scientifique, Tunisia (LR03ES03). Chemotaxonomic work was done at the DSMZ (German Collection of Microorganisms and Cell Cultures) Braunschweig. Conflict of interest Authors disclose that there are no conflicts of interest. No research involving human participants and/ or animals was performed. No non-financial interests tied

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