Diversity, Ecological Distribution and Biotechnological Potential of Actinobacteria Inhabiting Seamounts and Non-Seamounts in the Tyrrhenian Sea

Diversity, ecological distribution and biotechnological potential of Actinobacteria inhabiting seamounts and non-seamounts in the Tyrrhenian Sea

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Authors Ettoumi, Besma; Chouchane, Habib; Guesmi, Amel; Mahjoubi, Mouna; Brusetti, Lorenzo; Neifar, Mohamed; Borin, Sara; Daffonchio, Daniele; Cherif, Ameur

Citation Diversity, ecological distribution and biotechnological potential of Actinobacteria inhabiting seamounts and non-seamounts in the Tyrrhenian Sea 2016 Microbiological Research

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DOI 10.1016/j.micres.2016.03.006

Publisher Elsevier BV

Journal Microbiological Research

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Title: Diversity, ecological distribution and biotechnological potential of Actinobacteria inhabiting seamounts and non-seamounts in the Tyrrhenian Sea

Author: Besma Ettoumi Habib Chouchane Amel Guesmi Mouna Mahjoubi Lorenzo Brusetti Mohamed Neifar Sara Borin Daniele Daffonchio Ameur Cherif

PII: S0944-5013(16)30081-7 DOI: http://dx.doi.org/doi:10.1016/j.micres.2016.03.006 Reference: MICRES 25870

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Received date: 9-12-2015 Revised date: 16-3-2016 Accepted date: 31-3-2016

Please cite this article as: Ettoumi Besma, Chouchane Habib, Guesmi Amel, Mahjoubi Mouna, Brusetti Lorenzo, Neifar Mohamed, Borin Sara, Daffonchio Daniele, Cherif Ameur.Diversity, ecological distribution and biotechnological potential of Actinobacteria inhabiting seamounts and non-seamounts in the Tyrrhenian Sea.Microbiological Research http://dx.doi.org/10.1016/j.micres.2016.03.006

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Besma Ettoumi1,2, Habib Chouchane3, Amel Guesmi1, Mouna Mahjoubi3, Lorenzo Brusetti4,

Mohamed Neifar3, Sara Borin2, Daniele Daffonchio2,5 and Ameur Cherif1,3*

1Laboratory of Microorganisms and Active Biomolecules, Faculty of Sciences of Tunis,

University of Tunis El Manar, 2092 Tunis, Tunisia, 2 Department of Food Environmental and

Nutritional Sciences (DeFENS), University of Milan, 20133 Milan, Italy, 3Univ. Manouba,

ISBST, BVBGR-LR11ES31, Biotechpole of Sidi Thabet, 2020, Ariana, Tunisia, 4 Faculty of

Science and Technology, Free University of Bozen/Bolzano, Bolzano, Italy, 5BESE,

Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia.

*Corresponding author. Mailing address: Prof. Ameur CHERIF: LR Biotechnology and Bio-

Geo Resources Valorization (LR11ES31) Higher Institute for Biotechnology - University of

Manouba Biotechpole of Sidi Thabet, 2020, Sidi Thabet, Ariana, Tunisia. Tel.: 00216-

70527882 / 71537040; Fax: 00216-70527882 / 71537044 Mobile: 00216-25 460 651/ 98 651

460; E-mail: [email protected]

Abstract

In the present study, the ecological distribution of marine Actinobacteria isolated from seamount and non-seamount stations in the Tyrrhenian Sea was investigated. A collection of

110 isolates was analyzed by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and

16S rRNA gene sequencing of representatives for each ARISA haplotype (n = 49).

Phylogenetic analysis of 16S rRNA sequences showed a wide diversity of marine isolates and clustered the strains into 11 different genera, Janibacter, Rhodococcus, Arthrobacter,

Kocuria, Dietzia, Curtobacterium, Micrococcus, Citricoccus, Brevibacterium,

Brachybacterium and Nocardioides. Interestingly, Janibacter limosus was the most encountered species particularly in seamounts stations, suggesting that it represents an endemic species of this particular ecosystem. The application of BOX-PCR fingerprinting on

J. limosus sub-collection (n = 22), allowed their separation into seven distinct BOX-genotypes suggesting a high intraspecific microdiversity among the collection. Furthermore, by screening the biotechnological potential of selected actinobacterial strains, J. limosus was shown to exhibit the most important biosurfactant activity. Our overall data indicates that

Janibacter is a major and active component of seamounts in the Tyrrhenian Sea adapted to low nutrient ecological niche.

Keywords: Deep sea sediments, Actinobacteria, ARISA fingerprinting, BOX-PCR, biosurfactant.

Introduction

Marine Actinobacteria have been recovered from various marine habitats such as marine sediments (Maldonado et al., 2009), seawater (Subramani and Aalbersberg, 2013), freshwater ecosystems (Allgaier and Grossart, 2006), Ocean sediments (Solano et al., 2009;

Jensen et al., 2005), marine sponges (Sun et al., 2010), hypersaline and saline sediments (Wu et al., 2009). In these environments, Actinobacteria contribute to the breakdown and recycling of organic materials and they play a significant role in mineralization of organic matter, immobilization of mineral nutrients and improvement of physical parameters and marine environmental protection (Manivasagan et al., 2014; Manivasagan et al., 2013). In addition to their ecological role, marine Actinobacteria have currently received considerable attention due to their capacity to produce novel secondary metabolites (antibiotics, exopolysaccharides, extremozymes, biosurfactants, etc.) with a wide range of biological and pharmaceutical properties such as antitumor, immunosuppressive, anti-inflammatory and antiviral activities

(Hong et al., 2009; Yuan et al., 2014; Olano et al., 2009; Dharmaraj, 2010). Besides their wide applications in bioremediation and hydrocarbon degradation, marine biosurfactants were recently investigated in medical and therapeutic applications (Kiran et al., 2010; Rodrigues et al., 2006; De Araujo et al., 2011; Gudina et al., 2013). The antiadhesive potential of marine biosurfactants and their involvement in the inhibition and disruption of pathogenic biofilm formation could also present a promising alternative to combat the development of antibiotic resistance caused by bacterial pathogens (Kiran et al., 2014; Padmavathi and Pandian, 2014).

Studying the diversity of marine environments leads to the selection of novel marine

Actinobacteria with new adaptive strategies and the synthesis of extremophilic secondary metabolites. According to Subramani and Aalbersberg (2013), in the period from 2007 to

2013, a total of 38 new actinobacterial species belonging to 15 different families have been isolated from marine sediments. The emergence of molecular techniques has increased the 3

recovery of many actinobacterial taxa different from their terrestrial counterparts (Subramani and Aalbersberg, 2013). Culture-independent studies such as metagenomic and metaproteomic as well as emerging “omics” technologies enable the investigation of marine environments and the access to metabolic pathways of microbial populations without cultivation (Rath et al., 2011). Although their wide application in the field of microbial ecology, molecular approach alone could not allow the whole microbial diversity of marine

Actinobacteria. Consequently, cultivation-dependent approaches remains, in combination with cultivation-independent techniques, the most practical method to access bacterial diversity in marine environment and to strengthen results. Moreover, cultivation-dependent studies have demonstrated that Actinobacteria were also indigenous to marine habitat after the description of the first obligate marine genus Salinispora by Maldonado et al. (2005) and other 14 novel actinobacterial genera discovered later by Goodfellow and Fiedler (2010). In fact, the use of appropriate isolation procedures could promote the growth of marine

Actinobacteria and provide an important source of novel metabolites (Subramani and

Aalbersberg, 2012).

The Mediterranean Sea is a complex semi-enclosed deep environment including two interacting basins characterized by strong energetic gradients and low nutrient concentrations, in which the eastern basin is more oligotrophic than the western (Barsanti et al., 2011;

Pusceddu et al., 2009). The biological production decreases from north to south and west to east and is inversely related to the increase in temperature and salinity (Coll et al., 2010). The topographic complexity of the Mediterranean Sea is responsible for the formation of eddies and circular currents affecting the primary productivity and the flux of organic matter settling to the seafloor (Danovaro et al., 1999; Danovaro et al., 2009). In the Mediterranean Sea, the deep Tyrrhenian Sea is spotted by at least 14 large and intermediate seamounts separated by great depths (Bo et al., 2011). These underwater mountains have been well investigated from 4

geographic point of view, but little is known about the biodiversity of their benthic prokaryotes (Danovaro et al., 2010). This biodiversity remains largely unexplored, and much work is needed to discover the contribution of seamounts to the biodiversity. In this study, a culture-dependent approach was applied to investigate the diversity of marine Actinobacteria by Automated Ribosomal Intergenic Spacer Analysis (ARISA), 16S rRNA gene sequencing and BOX-PCR typing. Moreover, we compared the bacterial distribution between seamounts and non-seamounts stations of the Tyrrhenian sediments. Finally, the biotechnological potential of marine isolates was evaluated by screening of efficient biosurfactant producing

Actinobacteria.

Material and methods

Sampling sites and bacterial isolation

Sediment sampling was carried out during the oceanographic campaign CIESM-SUB1 (R/V,

Universitatis, Naples from 21 to 30 July 2005) in the southern part of the Mediterranean Sea, called the southern Tyrrhenian area up to the Sardinia-Sicily channel (Fig. 1). A total of 13 sediment samples were collected at different depths (from 3430 to 3581m) from the seamounts (Station 6: Palinuro, station 2: Marsili) and non-seamounts stations (Station 4: non- seamount 1, station 8: non-seamount 2) (Fig. 1) (Danovaro et al., 2009; Danovaro et al., 2010;

Ettoumi et al., 2010) using multiple and box corers. Different levels of sampling were aseptically performed from the surface layer 0 cm, 10 cm, 20 cm and the 30 cm horizons.

Strains were isolated from marine sediments by the dilution of 1 g of each sample in sterile seawater, plating on marine agar (Supplementary data Table S1) and incubated at 25°C for more than 7 days. Colonies with different morphological characteristics were purified by repeating streaking and cryopreserved at -80°C in marine broth supplemented with 25% glycerol.

DNA extraction and PCR amplification 5

The DNA was extracted from marine collection following the method of Murray et al. (1998) adapted to pure isolates. As previously published ARISA was adapted to analyze intergenic spacer of pure strains (Ettoumi et al., 2010; Cardinale et al., 2004). Following primers were used to amplify bacterial intergenic transcribed spacers: ITSF

(5’GTCGTAACAAGGTAGCCGTA-3’) and ITSReub (5’-GCCAAGGCATCCACC-3’).

ITSReub was labelled at its 5’ end with HEX fluorchrome (6-carboxy-1,4 dichloro-

20,40,50,70-tetra-chlorofluorescein). ITS-PCR were migrated on 2% agarose gel and were prepared for ARISA analysis according to Cardinale et al. (2004) prior to their automated separation by capillary electrophoresis on an ABI Prism 310 Genetic analyser. ARISA electrophoregrams were analysed using the GeneScan 3.1 software program (Applied

Biosystems). BOX-PCR was performed using the BOXA1R primer as already described

(Ettoumi et al., 2013). Total DNA was extracted directly from marine sediments using the

Fast DNA Spin Kit for Soil (Bio 101) according to the manufacturer procedure and

Denaturing Gradient Gel Electrophoresis (DGGE) was performed as described by Ettoumi et al. (2010).

16S rRNA sequencing and phylogenetic analysis

The 16S rRNA gene from pure cultures was amplified using the following universal primers:

S-D-Bact-0008-a-S-20/ S-D-Bact-1495-a-A-20 according to the procedure described previously by Cherif et al. (2003). PCR products were subjected to electrophoresis in 1.5 % agarose gel in 0.5× tris-borate - EDTA buffer, stained for 10 min in a 0.5 mg L-1 solution of ethidium bromide, visualized by exposure to UV light and photographed with a digital capture system (Gel Doc, Bio-Rad).

The sequencing of 16S rRNA gene of representatives strains (n = 49) was performed at Primm

Biotech (Milano, Italy). Obtained sequences were initially compared to those available in

GenBank database using BLAST (http://www.ncbi.nlm.nih.gov) (Altschul et al., 1990). 6

Generated sequences and their homologous were aligned using ClustalW version 1.8

(Thompson et al., 1994). The method of Jukes and Cantor, was used to calculate evolutionary distances. Phylogenetic tree was constructed by the neighbour joining method and tree topology was evaluated by bootstrap analysis of 1000 data sets using MEGA 4.1 (Tamura et al., 2007).

Screening of biosurfactant producing marine Actinobacteria

The screening of biosurfactant production was carried out by the drop collapse test as described by Tugrul and Cansunar (2005). To induce biosurfactant production, bacterial isolates (n = 42) were cultivated in marine broth supplemented with glucose (2%). Seven microliters of diesel oil was added to each well of a 96-well microtiter plate, which was left to equilibrate for 24 h at room temperature. Then, 20 µl of the cultural supernatant was added to the oil surface of the microtiter. SDS (sodium dodecyl sulfate) and distilled water used as positive and negative controls, respectively. The shape of the drop on the oil surface was visually examined after 1 min and drops containing biosurfactants collapse, whereas non- surfactant-containing drops remain stable. Positive result for biosurfactant production was considered when the drop diameter was at least 1 mm larger than that produced by deionized water (negative control). The tests were done in triplicate to confirm the result.

Results and discussion

To assess the diversity of culturable marine bacteria, 323 bacterial colonies were isolated from

Tyrrhenian sediments. From this collection, 110 actinobacterial isolates were selected based on growth characteristics and the properties of the colonies (color, pigment formation and

Gram staining).

ARISA analysis of marine actinobacterial bacteria

ARISA was performed in triplicate and allowed us to define 43 different haplotypes

(Supplementary data Fig. S1) with one to five ARISA peaks. The isolate CS 4010116 (H36) 7

possessed the smallest intergenic space region with 152 bp, whereas, the isolates CS 632 (H2) and CS 621015 (H2) possessed the longest ITS region with 1000 bp. Most strains (n = 66) encompassed one intergenic spacer size ranging from 432 to 1000 bp. The remaining isolates

(n = 44) may harbour multiple copies of ribosomal operons showing at least two different intergenic spacer sizes (Table 1). This result indicated a high diversity especially with the presence of strain-specific haplotypes at seamount stations 6 and 2 (n = 15) and non-seamount station 4 (n = 17).

Bacterial identification and phylogenetic analysis of marine Actinobacteria

Isolates selected as representatives of ARISA types (n = 49) were subjected to 16S rRNA gene sequencing. Analysis and comparison of sequences with their closest relatives showed all isolates were assigned to high G+C Gram-positive bacteria sharing a phylogenetic affiliation with the order of Actinomycetales.

Partial 16S rRNA genes sequences of representatives isolates of ARISA types (n = 49) and their closest relatives in GenBank, were used to generate a neighbour joining phylogenetic tree (Fig. 2). The analysis of the phylogenetic tree showed that actinobacterial strains were clustered in 8 families (Intrasporangiaceae, Microbacteriaceae, Dermabacteraceae,

Brevibacteriaceae, Micrococcaceae, Nocardidoidaceae, Nocardiaceae and Dietziaceae) and

11 genera. Janibacter (64.5%) was the dominant genus, followed by Rhodococcus (8.2%),

Arthrobacter (5.5%), Kocuria (6.5%) and Dietzia (6.5%). While the remaining six genera

(Curtobacterium, Micrococcus, Citricoccus, Brevibacterium, Brachybacterium and

Nocardioides) were poorly represented, they were also recovered in diverse marine habitat.

Most isolates (n = 71, 64.5%) belonged to Intrasporangiaceae and displayed from 98% to

99% similarity with its closest relative type strain J. limosus (NR026362; Martin et al., 1997).

The majority of Intrasporangiaceae isolates were mainly collected from seamounts stations 6 and 2 (n = 53). This result suggests the extensive diversity observed at seamounts stations 6 8

and 2, particularly with the detection of 17 distinct ARISA haplotypes for the genus

Janibacter (Table 1).

The bacterial isolate CS 4120, showing a specific ARISA haplotype (H19), was found to be the only member of Microbacteriaceae (Fig. 2) and has shown 99% of 16S rRNA sequence similarity with the reference strain Curtobacterium flaccumfaciens (NR025467; Behrendt et al., 2002).

There were 14 sequences that clustered with four different genera of Micrococcaceae family.

Among them, the isolate CS 4116, (ARISA haplotype H18), was assigned to Micrococcus luteus (EU071594) with 100% of 16S rRNA identity. Recovered from the deepest layer of the non-seamount station 4, the strain CS 436 (H33) displayed 98% of 16S rRNA similarity with its closest relative Citricoccus parietis (Schafer et al., 2010).

Six marine isolates were related to the genus Arthrobacter, including one (CS 4038, H9), two

(CS 401089 and CS 4010116) and three isolates (CS 6315, 6015 and CS 6317) affiliated, respectively, to Arthrobacter crystallopoietes (NR026189), Arthrobacter subterraneus and

Arthrobacter agilis (KC788152). As it was shown previously, Arthrobacter subterraneus was collected from deep subsurface water of the South Coast of Korea, suggesting that these isolates could be well adapted to marine environment (Chang et al., 2007).

The isolates CS 434 (H38) and CS 6318 (H12), recovered respectively from the deepest layer of the seamount station 6 and the non-seamount station 4, showed 99% and 100% of 16S rRNA sequence similarity with the K. rosea (LN650464, JN084134). The remaining isolates

(n = 4, Fig. 2), collected from different layers of the non-seamount station 4, were also highly affiliated to K. rosea (99-100% of similarity) and showed interestingly four strain-specific

ARISA haplotypes (Table 1).

The two representative isolates of Brevibacteriaceae (CS 4111, H32) and Dermabacteraceae

(CS 8215, H5) were closely related to Brevibacterium casei (99%) and Brachybacterium arcticum (99%), respectively.

Within the Nocardiaceae family, one isolate (CS 602, H14) originated from superficial layers of the seamount station 6 exhibited high similarity with Rhodococcus fascians and four isolates (CS 621011, CS62107, CS62104 and CS 4029) showed 99% of 16S rRNA sequence similarity with the published reference strain Rhodococcus erythropolis (NR074622; Sekine et al., 2006). This species was of particular interest because it has potential in the exploration of new biosurfactants and could be used in bioremediation of marine oil pollution (Mahjoubi et al., 2013; Jain et al., 2013).

All recovered from the surface horizon of non-seamount stations 4 and 6, isolates CS 4010120

(H10), CS401076 (H10), CS6032 (H11), CS 401082 (H31) and CS601069 (H42) shared a high identity with Dietziacece, particularly with the published species Dietzia cercidiphylli

(NR044483; Li et al., 2008).

Finally, the two isolates CS 6061 (H6) and CS 6068 (H7) of Nocardidoidaceae, collected from superficial layer of seamount station 6 were clustered to Nocardioides jensenii. Both isolates could represent new marine species as their sequence identity with the closest reference strain Nocardioides jensenii (NR044981) was only 94%.

It has been shown that two ARISA haplotypes (H26, H28, Table 1) encompassed strains affiliated to two different species: J. limosus and R. erythropolis. This result indicated that

ARISA could not discriminate closely related species of Actinobacteria, since this technique was based on length variability and two different species could share the same length in their intergenic of the rrn operon.

Our study underlined that isolated strains displayed considerable diversity, which was attributed in 11 genera (Janibacter, Rhodococcus, Arthrobacter, Kocuria, Dietzia, 10

Curtobacterium, Micrococcus, Citricoccus, Brevibacterium, Brachybacterium and

Nocardioides) and 8 families (Intrasporangiaceae, Microbacteriaceae, Dermabacteraceae,

Brevibacteriaceae, Micrococcaceae, Nocardidoidaceae, Nocardiaceae and Dietziaceae). The majority of identified species in our work were also recovered in marine environment as recorded by previous studies (Goodfellow and Fiedler, 2010; Zhang et al., 2014; Duncan et al., 2015; Yuan et al., 2014). In comparison to our marine collection, the relative higher diversity observed in these studies and the lack of detection of some phylogenetic groups

(such as Streptomyces and Salinispora) could be explained as followed: (i) the application of highly selective isolation strategy to recover Actinobacteria from marine environment

(Duncan et al., 2015) and (ii) the presence of endemic species of Actinobacteria with restricted geographic distribution (Goodfellow and Fiedler, 2010).

As it was revealed by Jensen et al. (2015), that the recovery of marine Actinobacteria in marine sediments did not provide any explanation about what extend these bacteria are ecologically or evolutionary distinct from terrestrial relatives, particularly, because the genus of Streptomyces, known to be a common soil Actinobacteria, can be also metabolically active in the sea. Consequently, it remains unclear the mechanisms of actinobacterial marine adaptation suggesting that there is no common genetic basis for marine survival in marine environment among the available genome sequences of Actinobacteria (Penn and Jensen,

2012). In the same study of Penn and Jensen (2012), it was underlined the fact that the acquisition of genes involved in marine adaptation of Salinispora are fundamentally different from those reported from Gram-negative bacteria (Oh et al., 1991).

The application of molecular approach (DGGE) on the same marine samples as it was previously described by Ettoumi et al. (2010), showed the detection of only one uncultured

Actinobacterium clone and none of cultured strains were identified by culture-independent 11

approach. This finding could be explained by that (i) the majority of microorganisms are not easily cultured using standard microbiological techniques, (ii) the occurrence of these taxa in marine sediments as spores, which are particularly resilient to cell lysis and consequently, may be underrepresented in environmental DNA (Sun et al., 2010; Yang et al., 2013). We also suppose that the number of excised DGGE bands is not large enough to recover all Gram- positives clones inhabiting marine sediments and that DNA cannot be extracted from spores even when a specific lysis method is applied (Ettoumi et al., 2013).

Finally, our result supports also the view that, molecular and cultivation-based approaches lead to different features of actinobacterial diversity (Sun et al., 2010; Ettoumi et al., 2013).

Relationship between microdiversity of marine Actinobacteria as revealed by ARISA and their distribution in sediments horizons

Despite the fact that seamounts constitute biodiversity hot-spots, insights into their microbial diversity and particularly the culturable fraction are still limited. On the light of our data, there is a differential bacterial distribution between seamounts and non-seamounts stations. This was clearly observed when 76.8% of J. limosus strains retrieved in seamounts stations 2 and 6 and 90.5% were exclusively detected in deeper horizons of seamounts stations. Interestingly, except for the isolate (CS 213), all strains isolated from deep layers of seamounts station 2 were identified as J. limosus.

The difference in the bacterial affinities between seamounts and non-seamounts sites reflect the difference in biogeochemical conditions of these two habitats. As it is shown by the studies of Ettoumi et al. (2010), Pusceddu et al. (2009) and Danovaro et al. (2009), seamounts sediments were characterized by a high heterogeneity in terms of dimensions, species richness, physiochemical and trophic properties. Our data supports the fact that Janibacter strains dominate seamounts sites in response to oligotrophic conditions due to low organic matter. Consequently, these bacteria constitute an equilibrium community, which are more 12

adapted to low energy and nutritional demands (k-strategists, slow-growing). In contrast, fast- growing bacteria, called r-strategists, showed high affinity to high organic matter concentrations and could constitute an important component of non-seamounts stations.

According to previous studies, the genus Janibacter showed broad metabolic versatility toward a variety of aromatic compounds such as fluorene, carbazole (Oba et al., 2014;

Yamazoe et al., 2004), anthracene, phenanthrene, dibenzofuran (Jin et al., 2006), and polychlorinated biphenyls (Sierra et al., 2003).

There was also a relative higher phylogenetic diversity in non-seamounts stations (4 and 8) with respect to seamounts stations (2 and 6), and ten different genera were identified for these former sites (Table 1). Whereas, in seamounts stations, it has been shown that isolates were clustered to five genera of Janibacter, Rhodococcus, Dietzia, Kocuria and Nocardioides

(Table1). This relative greater diversity between seamounts and non-seamounts sediments could be related to the diverse aspects of biogeochemical cycles and organic matter fluxes which affect bacterial assemblages and microbial activity in such environment. As slow- growing bacteria, strains colonizing seamounts stations may harbour low rDNA copy number as result of their ecological strategies in the oligotrophic marine environment (Klappenbach et al., 2000). This is consistent with the observation that in our results 92.5% of Janibacter strains, detected in seamounts sites, showed only one or two ARISA peaks and consequently only one or few operons copies (Table 1). The highest number of J. limosus isolates was present in seamounts stations (75%) and less present in non-seamounts sediments (25%), leads to suggest that J. limosus represent the endemic species of seamounts sediments.

Originated from non-seamount station 4, Janibacter isolates CS 4210, CS 4I0 10 and CS 429 exhibited respectively two, three and four ARISA peaks, which could suggest that these isolates harboured multiple ribosomal operons. The apparent presence of J. limosus in non- seamounts sites could be the result of continuous interactions of seamounts with marine 13

currents and also the influence of eddies formation and bioturbation process (Danavaro et al.,

2009).

The application of ARISA has been shown to enable discrimination to the species level and demonstrate a high microdiversity among marine culturable Actinobacteria. Intergenic spacer regions length detected from these bacterial isolates ranged from 152 to 1000 bp (Table 1).

Isolates identified to J. limosus (n = 69) were the major component of culturable marine species and were distributed in seventeen distinct ARISA

profiles: 5 profiles with one peak (H1, H2, H21, H25 and H26), 5 profiles with 2 peaks (H15,

H16, H17, H23 and H28), 4 profiles with three peaks (H22, H30, H34 and H35) and 1 profile with 5 peaks (H43) (Table 1). Since the majority of actinobacterial strains harboured only one or two ARISA peaks (84.5%), it is assumed that they contained low copy number of ribosomal operons. As it has been reported previously by Brown et al. (2005) and

Klappenabach et al. (2000), where marine isolates may have low ribosomal copy number as a result of their ecological strategies, being slow-growing bacteria in oligotrophic marine sediments. According to Brown et al. (2005) the representatives of marine bacteria where multiple operons have been detected are identical, even within the highly intragenomic heterogeneity of rrn operons. In this work, it has been fully justified that ribosomal operon could constitute a suitable marker to support that genotypic features could reflect adaptive differences in the bacterial life strategies.

In marine environment, the ability of bacteria to cope with low amount of organic matter and stress salinity is due to their physiological and ecological plasticity. Microorganisms living in such habitat showed specific adaptation including new mechanisms and metabolic strategies such as transport and osmoregulatory compounds (Stan-Lotter and Fendrihan, 2013).

BOX-PCR analysis of major ARISA haplotype

Sequencing of the 16S rRNA gene could not differentiate among strains of J. limosus belonging to the same ARISA haplotype (H25). The intraspecific diversity of this sub- collection was assessed by BOX-PCR fingerprinting. This typing technique targeted repetitive

DNA elements leading to the amplification of different sizes that generated strain-specific fingerprint patterns. Consequently, BOX-PCR typing allowed the discrimination at the intraspecific level among different strains of the same species.

Genetic variability of the sub-collection of J. limosus (n = 22) belonging to the ARISA haplotype H25 was analyzed by BOX-PCR using the primer BOX-1AR (Cherif et al., 2003;

Ettoumi et al., 2013).

This fingerprinting typing method yielded 7 distinct BOX-PCR patterns (A to G,

Supplementary data Fig. S2 and Table 2) with PCR products ranging from 550 to 2000 bp.

Three BOX-genotypes B, E and A, were the most abundant patterns found accounting 50,

18.8 and 13.63% of the isolates, respectively. The first BOX-PCR profile with the largest number of strains (genotype B) comprised 11 isolates (50%) from seamount stations 2 and 6

(Table 2). The second dominant BOX-genotype E, comprised 4 strains isolated only from seamount station 6, whereas isolates belonging to genotype A (n = 3, 13.63%) were mainly originated from seamount station 2 (Table 2).

The remaining strains (n = 4) displayed strain-specific BOX patterns (genotypes C, D, F and

G, Table 2). A correlation was observed between the BOX groups and the geographic origin.

Five BOX-patterns included only strains originated from seamount station 6 (C, D, E and F) or from seamount station 2 (G), whereas BOX-PCR patterns A and B were detected in the two seamount stations 2 and 6. Thus BOX-PCR analysis indicated that the majority of strains had a widespread distribution (genotypes A and B), whereas others showed a station-dependent distribution (genotypes C, D, E, F and G).

Our results showed a genetic variability among the haplotype (H25) of J. limosus strains which may contribute to their adaptation to oligotrophic conditions in deep sea sediments.

Screening of biosurfactant production by marine Actinobacteria

Drop collapse test showed that 60.52% (n = 23) of strains were positive for biosurfactant production among the selected actinobacterial strains (n = 38). The highest biosurfactant activity (5 or 4 mm of diameter) was detected for 57.9% of the isolates, whereas the lowest activity (3.5 mm of diameter) was shown for 2.63% of the isolates. Our results revealed that strains identified as J. limosus exhibited an important biosurfactant activity: 5 strains of J. limosus showed 4 mm of diameter of drop collapse test and 2 strains showed 5 mm of diameter (Table 3). The biodegradation ability of Janibacter has been elucidated in previous studies. For example Janibacter sp. SB2 showed an efficient degradation ability of benzene, toluene, ethylbenzene and xylene in contaminated sea-tidal flat (Jin et al., 2013). Jin et al.

(2006) found that Janibacter terrae, isolated from sediment from East Lake in China, could completely degrade the dibenzofuran, which was used as sole source of carbon and energy.

Moreover, the study of Nguyen et al. (2013) reported the characterization of enzymes and regulatory genes involved in the degradation of dichloro-chlorophenyl-ethylene by Janibacter sp. TYM3221. In addition to biodegradation ability, J. limosus was also shown to produce new antibiotics (Asolkar et al., 2004), which highlight the valuable biotechnological potential of this actinobacterial species.

The remaining actinobacterial species displayed positive response for drop collapse test with 4 mm of diameter such as Dietzia dagingensis (n = 4), K. rosea (n = 3), R. erythropolis (n = 2),

R. fascians (n = 1) and Micrococcus luteus (n = 1). Our finding was in agreement with those of Mahjoubi et al. (2013) showing also that Rhodococcus and Kocuria species were biosurfactants producers. Moreover, the diversity of species producing biosurfactants and the diameter of drop collapse test reflected the structure diversity of microbial biosurfactants. 16

According to the study of Satpute et al. (2010), the biosurfactants produced by Rhodococcus showed two different structure and chemical composition such as glycolipid and free fatty acids.

Besides its biosurfactant activity (n = 4), the isolate CS 4116 identified as Micrococcus luteus displayed antibacterial activity against Bacillus cereus (data not shown). Similarly, it has been reported by Das et al., (2008), that a lipopeptide biosurfactant produced by marine B. circulans showed also antimicrobial properties against pathogenic microorganisms leading to their potential use in drug industry as new and potent therapeutic agents. Moreover, B. subtilis was well known by its ability to synthesize several lipopeptide biosurfactants with high antimicrobial properties such as surfactin, lichenysin, iturin, arthrofactin and pumilacidin

(Mukherjee et al., 2009).

It is interesting to note that antimicrobial activity of biosurfactants leads also to the inhibition of biofilm formation of bacterial and fungal species (Kiran et al., 2010; Dusane et al., 2013).

Their anti-adhesive and disruptive properties make them significant molecules playing an important role in the prevention and treatment of human chronic infections since the formation of microbial biofilm by pathogenic bacteria is associated with a decrease of antimicrobial susceptibility (Kiran et al., 2010). Although the antiadhesive activity of several biosurfactants produced by marine bacteria was investigated (Janek et al., 2012; Kavita et al.,

2014; Papa et al., 2013; Dusane et al., 2011), to the best of our knowledge, there was only one report on anti-biofilm potential of biosurfactants from Actinobacteria. As it was previously reported by Kiran et al. (2010), marine Brevibacterium casei produced a glycolipid biosurfactant compound involved in the disruption and the control of pathogenic biofilms.

Conclusion

The results of the present study highlight the broad diversity of marine Actinobacteria investigated in the Tyrrhenian Sea using mainly cultivation-dependent approach. The 17

application of ARISA showed a differential distribution of actinobacterial isolates between seamount and non-seamount stations. Due to their ecological behaviour and their adaptation to low nutritional exigency (k-strategists), Janibacter was considered as a major and active component of seamount stations in Tyrrhenian Sea. Whereas, non-seamounts stations were characterized by higher bacterial diversity, with members adapted to high organic matter (r- strategists). Marine Actinobacteria exhibited also a biotechnological potential as biosurfactant producers and J. limosus showed the most important activity. Further studies are necessary to explore the antimicrobial and biofilm disruption potentials of marine biosurfactants.

Acknowledgments

This work was performed in the frame of the CIESM-SUB programme (CIESM Sur-Un-

Bateau Cruise I). Pr. Laura Giuliano, who made possible this multidisciplinary research, Pr.

Giorgio Budillon, the Chief Scientist of the expedition, the captains Angelo Barca and crews of the R/V Universitatis, are gratefully acknowledged. We thank Dr. Mirko Magagnini for his help in samples collection. This work was partly supported by the Ministry of High Education and Scientific Research of the Tunisian Republic, Grant LR MBA206 and LR11ES31, and the

European Union in the ambit of project ULIXES (FP7-KBBE-2010-4, CP-FP-SICA under grant agreement no. 266473).

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Figure 1 Location of the four sampling sites relative to the oceanographic campaign

“CIESM-SUB1”.

Stations coordinates limits and depths: Station 6 (Palinuro), 39°52.820’ N, 012°46.070’ E,

3581 m; Station 4, 39°32.050’ N, 013°22.280’ E, 3453 m; Station 2 (Marsili), 39°07.470’ N,

6 014°06.430’ E, 3425 m and Station 8, 38°55.900’ N, 013°16.490’ E, 3460 m.

Figure 2 Neighbour-joining phylogenetic tree of 49 partial 16S rRNA sequences

(coordinates in E. coli 52 to 787 n) of marine Actinobacteria isolates with 17 closest NCBI relatives.

Phylogenetic dendrogram was evaluated by performing bootstrap analysis of 1000 data sets using MEGA 4.1. ARISA type (according to fig. S1 and Table 1) and the number of isolates per type are indicated. 16S rRNA sequence accession numbers of the reference strains are indicated in parentheses.

Table. 1 Phylogenetic affiliation, ARISA haplotypes and prevalence of marine Actinobacterial isolates

Genus Isolate Accession Closes relative Sequence Accession 16S rRNA ARISA ARISA bands

number length (bp) number sequence haplotype/ (bp)

similarity (%) number of

isolates

Janibacter CS 4I0 6 KT265333 Janibacter limosus strain DSM 11140 635 NR026362 99 H1 (14) 432

CS 621015 KT265286 Janibacter limosus strain DSM 11140 635 NR026362 99 H2 (13) 1000

CS 632 KT265287 Janibacter limosus strain DSM 11140 635 NR026362 99 H2 (13) 1000

CS 61109 KT265288 Janibacter limosus strain DSM 11140 635 NR026362 99 H15 (1) 229-432

CS 61107 KT265289 Janibacter limosus strain DSM 11140 634 NR026362 99 H16 (1) 230-434

CS 6152 KT265290 Janibacter limosus strain DSM 11140 634 NR026362 99 H17 (1) 434-442

CS 4210 KT265293 Janibacter sp. N59 635 HQ188604 98 H20 (1) 410-423

CS 61104 KT265294 Janibacter limosus strain DSM 11140 634 NR026362 99 H21 (1) 845

CS 621012 KT265291 Janibacter limosus strain DSM 11140 634 NR026362 99 H22 (1) 254-349-435

CS 621017 KT265292 Janibacter limosus strain DSM11140 635 NR026362 99 H23 (4) 254-435

CS 631034 KT265298 Janibacter limosus strain DSM 11140 635 NR026362 99 H26 (8) 433

CS 227 KT265295 Janibacter limosus strain DSM 11140 633 NR026362 99 H25 (22) 435

CS 621033 KT265296 Janibacter limosus strain DSM 11140 635 NR026362 99 H25 (22) 435

CS 6313 KT265297 Janibacter limosus strain DSM 11140 635 NR026362 99 H25 (22) 435

CS 631 KT265299 Janibacter limosus strain DSM 11140 635 NR026362 99 H28 (4) 254-432

CS 4I0 10 KT265334 Janibacter limosus strain DSM 11140 635 NR026362 99 H30 (2) 254-349-432

CS 228 KT265300 Janibacter limosus strain DSM 11140 634 NR026362 99 H34 (1) 232-241-435

CS 2211 KT265301 Janibacter limosus strain DSM 11140 635 NR026362 99 H35 (1) 377-424-435

CS 429 KT265302 Janibacter sp. N59 635 HQ188604 99 H37 (1) 254-264-349-

654

CS 6156 KT265303 Janibacter limosus strain DSM 11140 634 NR026362 99 H43 (1) 254-286-349- 432-442 Kocuria CS 4212 KT265304 Kocuria rosea 635 DQ060382 99 H3 (1) 671

CS 6318 KT265305 Kocuria rosea 635 JN084134 100 H12 (2) 232-696

CS 434 KT265306 Kocuria rosea 634 LN650464 99 H38 (1) 254-264-349- 671-677 CS 4211 KT265307 Kocuria rosea 635 JN084134 100 H39 (1) 230-254-349-

671

CS 4118 KT265308 Kocuria rosea 635 JN084134 100 H40 (1) 274-276-382-

424

CS 4119 KT265309 Kocuria rosea 634 DQ060382 99 H41 (1) 245-275-424

Arthrobacter CS 401089 KT265310 Arthrobacter subterraneus strain CH7 632 NR043546 99 H4 (1) 232-660

CS 6317 KT265311 Arthrobacter agilis 644 KC788152 99 H8 (1) 435-674

CS 4038 KT265312 Arthrobacter crystallopoietes strain DSM 632 NR026189 99 H9 (1) 544 20117 CS 6315 KT265314 Arthrobacter agilis 645 KC788152 99 H13 (1) 229-674

CS 6015 KT265313 Arthrobacter agilis 644 KC788152 99 H29 (1) 254-432-672

Arthrobacter CS 4010116 KT265315 Arthrobacter subterraneus strain CH7 633 NR043546 99 H36 (1) 152-254-349-

660

Rhodococcus CS 602 KT265316 Rhodococcus fascians 629 LN624624 100 H14 (1) 229-432-446

CS 4029 KT265317 Rhodococcus erythropolis 629 NR074622 99 H24 (1) 427-435

CS 62104 KT265318 Rhodococcus erythropolis 629 NR074622 99 H26 (8) 433

CS 621011 KT265319 Rhodococcus erythropolis 629 NR074622 99 H27 (1) 329-432

CS 62107 KT265320 Rhodococcus erythropolis 629 NR074622 99 H28 (4) 254-432

Dietzia CS 4010120 KT265321 Dietzia cercidiphylli strain YIM 65002 630 NR044483 100 H10 (4) 232-439

CS 401076 KT265322 Dietzia cercidiphylli strain YIM 65002 629 NR044483 100 H10 (4) 232-439

CS 6032 KT265323 Dietzia cercidiphylli strain YIM 65002 629 NR044483 99 H11 (1) 439

CS 401082 KT265324 Dietzia cercidiphylli strain YIM 65002 630 NR044483 99 H31 (1) 254-349-439

CS 401069 KT265325 Dietzia cercidiphylli strain YIM 65002 629 NR044483 100 H42 (1) 349-387-423-

516

Nocardioides CS 6061 KT265326 Nocardioides jensenii 630 NR044981 94 H6 (1) 252-613

CS 6068 KT265327 Nocardioides jensenii 628 NR044981 94 H7 (2) 613

Curtobacterium CS 4120 KT265328 Curtobacterium flaccumfaciens strain LMG 635 NR025467 99 H19 (1) 500-686 3645 Brevibacterium CS 4111 KT265329 Brevibacterium casei strain 60 636 EU086802 99 H32 (1) 254-349-472

Citricoccus CS 436 KT265330 Citricoccus parietis 635 NR104498 99 H33 (1) 257-677

Micrococcus CS 4116 KT265331 Micrococcus luteus strain EHFS-S11Hc 633 EU071594 100 H18 (1) 515-519

Brachybacterium CS 8215 KT265332 Brachybacterium arcticum strain Lac 5.2 635 AF434185 99 H5 (3) 456

Table 2. BOX-PCR genotypes of marine Janibacter limosus isolates.

BOX-genotype Length (bp) Percentage of isolates (%)

J. limosus A 650 13.63 B 2000, 650 50 C 2000, 800, 550 4.54 D 2000, 650, 550 4.54 E 2000, 850, 700, 550 18.8 F 850, 700, 650, 550 4.54 G 1100, 1000 4.54

Table 3. Biosurfactant production by Actinobacterial isolates Strain Affiliation Mean Value of diameter of drop collapse test (mm) ± Standard deviation CS 61109 Janibacter limosus 5 ± 0 CS 6313 Janibacter limosus 5 ± 0 CS 631 Janibacter limosus 4 ± 0.4 CS 61107 Janibacter limosus 4 ± 0 CS 621012 Janibacter limosus 4 ± 0.2 CS 227 Janibacter limosus 4 ± 0 CS 228 Janibacter limosus 4 ± 0 CS 4I0 6 Janibacter limosus 3 ± 0.2 CS 621015 Janibacter limosus 3 ± 0.2 CS 621017 Janibacter limosus 3 ± 0 CS 621033 Janibacter limosus 3 ± 0.2 CS 6152 Janibacter limosus 3 ± 0 CS 61104 Janibacter limosus 3 ± 0 CS 6156 Janibacter limosus 2 ± 0 CS 631034 Janibacter limosus 2 ± 0.2 CS 6317 Arthrobacter agilis 4 ± 0.2 CS 6315 Arthrobacter agilis 3 ± 0 CS 4010116 Arthrobacter subterraneus 3 ± 0.2 CS 6015 Arthrobacter agilis 2 ± 0 CS 4111 Brevibacterium casei 5 ± 0 CS 8215 Brachybacterium arcticum 3 ± 0.2 CS 436 Citricoccus sp. 3 ± 0 CS 4010120 Dietzia dagingensis 4 ± 0 CS 6032 Dietzia dagingensis 4 ± 0 CS 401082 Dietzia dagingensis 4 ± 0 CS 401069 Dietzia dagingensis 4 ± 0 CS 4212 Kocuria rosea 4 ± 0.2 CS 4211 Kocuria rosea 4 ± 0 CS 6318 Kocuria rosea 4 ± 0 CS 434 Kocuria rosea 3 ± 0 CS 6068 Nocardioides jensenii 5 ± 0.2 CS 62104 Rhodococcus erythropolis 4 ± 0.2 37

CS 621011 Rhodococcus erythropolis 4 ± 0.2 CS 4029 Rhodococcus erythropolis 3 ± 0 CS 62107 Rhodococcus erythropolis 3 ± 0 CS 602 Rhodococcus fascians 4 ± 0 CS 4116 Micrococcus luteus 4 ± 0.2

Figure 1

Italy

Tyrrhenian Sea

Station 6 (Palinuro) Station 4

Station 2 Station 8 (Marsili)

Mediterranean Sea

Sicily

Tunisia

Fig. 1. Ettoumi et al., 2015 Figure 2 Sequence ARISA type Number of isolates Assignation

CS 61109 15 1 CS 631034 26 8 CS 4I06 1 14 CS 6313 56 25 22 CS 2211 35 1 CS 631 28 4 CS 621015 2 13 67 CS 632 2 13 CS 61104 21 1 CS 227 25 22 Janibacter CS 621012 22 1 62 CS 61107 16 1 65 CS 6152 17 1 CS 228 34 1 CS 6156 43 1 96 CS 4I010 30 2 CS 621017 23 4 78 100 CS 621033 25 22 89

Janibacter limosus (NR026362) Janibacter sp. (HQ188604) 83 100 CS 4210 20 1 CS 429 37 1 69 100 CS 4120 19 1 Curtobacterium flaccumfaciens (NR025467) Curtobacterium 100 CS 436 33 1 55 70 Citricoccus parietis (NR104498) Citricoccus CS 4116 18 1 Micrococcus luteus (EU071594) Micrococcus 100 CS 6317 8 1 72 CS 6015 29 1 100 CS 6315 13 1 94 100 Arthrobacter agilis (KC788152) Arthrobacter Arthrobacter subterraneus (NR043546) CS 401089 4 1 100 CS 4010116 36 1 49 100 CS 4038 9 1 100 Arthrobacter crystallopoietes (NR026189) Kocuria rosea (LN650464) 81 34 CS 434 38 1 41 1 100 CS 4119 CS 6318 12 2 Kocuria 43 CS 4211 39 1

47 Kocuria rosea (DQ060382) Kocuria rosea (JN084134) 47 CS 4212 3 1 CS 4118 40 1 13 32 1 100 CS 4111 Brevibacterium 58 Brevibacterium casei (EU086802) 5 3 100 CS 8215 Brachybacterium Brachybacterium arcticum (AF434185) 66 CS 401082 31 1 21 CS 6032 11 1 23 CS 401069 42 1 73 Dietzia Dietzia cercidiphylli (NR044483) 100 CS 401076 10 4 10 4 67 CS 4010120 Rhodococcus erythropolis (NR074622) 98 CS 621011 27 1 26 8 99 CS 62104 28 100 CS 62107 4 Rhodococcus 24 CS 4029 1

100 Rhodococcus fascians (LN624624) CS 602 14 1 Nocardioides jensenii (NR044981) 100 7 2 100 CS 6068 Nocardioides 6 1 0,01 CS 6061

Fig. 2. Ettoumi et al., 2015