Wide Distribution of Closely Related, Antibiotic-Producing Arthrobacter Strains throughout the Arctic Ocean

Abstract

We isolated 16 antibiotic-producing bacterial strains throughout the central Arctic Ocean, including seven Arthrobacter spp. with almost identical 16S rRNA gene sequences. These strains were numerically rare, as revealed using 454 pyrosequencing libraries. Arthrobacter spp. produced arthrobacilins A to C under different culture conditions, but other, unidentified compounds likely contributed to their antibiotic activity.

TEXT

Bioactive secondary metabolites, such as antibiotics, are key components in microbial interactions (15) and often have biotechnological potential (10). Relatively little is known about antibiotic compounds from Arctic or Antarctic bacteria, even though polar environments have been suggested as a source of novel natural products (22, 39). The purpose of the present study was to determine the occurrence and distribution of antibiotic-producing culturable bacteria in the central Arctic Ocean.

In Arctic and Antarctic sea ice, the culturable microbiota is dominated by Alphaproteobacteria such as Octadecabacter spp. (6), but their potential for secondary metabolite biosynthesis appears to be low (28). Among the Gammaproteobacteria and Bacteroidetes, which are abundant in sea ice (5), Glaciecola and Salegentibacter produce bioactive natural products (1, 41). Culturable bacteria in Arctic seawater are mainly represented by the Roseobacter clade, Gammaproteobacteria, and Actinobacteria (26), while the epibiotic flora of polar zooplankton includes Vibrionaceae-like strains (2). Many of these bacterial groups harbor strains with antagonistic traits (4, 12, 18, 25). Antibiotic activities from culturable polar bacteria have been reported for Actinobacteria (24, 29), Gammaproteobacteria (29, 38), bacilli (38), and cyanobacteria (3). Structure-elucidated antibiotics include phenazines from Pseudomonas (17), aromatic nitrogen compounds from Salegentibacter (1), and an angucyclinone from Streptomyces (7).

Here, we describe antibiotic-producing Arctic bacterial strains, including seven closely related Arthrobacter spp., collected during the LOMROG-II expedition (http://tinyurl.com/lomrogII). Terrestrial Arthrobacter spp. are common in soil and known antibiotic producers (19), but antibiotic compounds from aquatic or polar strains have not been studied in detail despite the existence of several putative producers (14, 23, 24, 33, 36, 41).

Isolation of Arctic bacteria.

Bacteria were isolated from environmental samples (Fig. 1) at both 5 and 20°C by spread-plating on marine agar (Difco catalog no. 212185), actinomycete isolation agar (Sigma catalog no. 17177), chitin agar (40), low-nutrient agar (8), and low-nutrient M3 agar (34). The latter three media were prepared with ambient Arctic seawater. In addition, enrichment cultures in half-strength marine broth (12) were prepared. Culturable counts were significantly (P < 0.05) higher in sea ice (average of 3.8 × 10⁴ CFU ml⁻¹) than in zooplankton (3.3 × 10² ml⁻¹ homogenate) and seawater (2.4 × 10²). Meltwater, snow, sediment, and frost flowers yielded counts below 10² CFU ml⁻¹. A number of randomly selected strains that were isolated on low-nutrient media and/or at 5°C were tested for their ability to grow at high nutrient concentrations and temperature, respectively. Twenty-seven of 30 strains (90%) isolated on low-nutrient media were able to grow on rich medium (marine agar), and 38 of 48 strains (80%) isolated at 5°C were able to grow at 20°C. This indicated a high rate of recovery of generalist species.

Fig 1.

Sampling sites for isolation of antibiotic-producing Arctic bacteria. At some sites, several samples were collected, i.e., seawater from different depths, different sections of a sea ice core, or different zooplankton organisms. In total, 58 samples were collected, including seawater (from sites 1, 3, 9, 10, 12, 19, 21, 22, and 26), sea ice (sites 2, 3, 7, 8, 11, 13, 15, 16, 18, 20, 23, and 25), zooplankton (sites 4, 5, 6, 10, 17, and 24), meltwater (sites 13, 16, 18, and 20), snow (sites 13, 18, and 20), sediment (site 14), and frost flowers (site 27). The map was created by use of the WGS 194 projection, displayed in North Pole Lambert Azimuthal Equal Area (using ArcGIS software).

Identification of antibiotic-producing strains.

Of 511 randomly selected strains representing a variety of colony morphotypes, 50 inhibited growth of Vibrio anguillarum 90-11-287 when spotted on pathogen-seeded agar (12). Additional replica plating of 32,385 colonies resulted in the isolation of further 61 inhibitory strains. Sixteen isolates retained considerable antibacterial activity upon repeated testing (Table 1). Sequencing of 16S rRNA gene fragments (20) showed that the antibiotic-producing isolates belonged to the genera Arthrobacter (7 strains), Pseudoalteromonas (4 strains), Vibrio (3 strains), and Psychrobacter (2 strains), with almost identical 16S rRNA gene sequences within each group (Fig. 2). The seven Arthrobacter spp. originated from different sources, including surface water, sea ice, zooplankton, the deep sea, and meltwater, at distant geographical sites (Table 1; Fig. 1). The antibacterial compound(s) were extractable in organic solvent only from those strains.

Table 1.

Sites of isolations, sources, genus affiliations, inhibitory spectra, and GenBank accession numbers of antibiotic-producing Arctic bacteria

Class	Strain	Site	Source	Genus	Inhibition ofa:		GenBank accession no.
					V. anguillarum	S. aureus
Actinobacteria	PP12	16	Sea ice	Arthrobacter	+	+	JF706632
	MB182	16	Sea ice		+	+	JF706644
	SS14	17	Copepods		+	+	JF706634
	TT4	18	Meltwater		+	+	JF706635
	ZZ3	18	Sea ice		+	+	JF706637
	LM7	22	Surface water		+	+	JF706639
	WX11	26	Deep sea		+	+	JF706642
Gammaproteobacteria	RR12	17	Amphipods	Vibrio	+	−	JF706633
	EF14	21	Deep sea		+	+	JF706638
	RS9	23	Sea ice		+	+	JF706640
	XX5	18	Sea ice	Psychrobacter	+	−	JF706636
	ST4	23	Sea ice		+	−	JF706641
	MB33	5	Copepods	Pseudoalteromonas	+	−	JF706643
	MB205	19	Surface water		+	−	JF706645
	MB220	18	Sea ice		+	−	JF706646
	MB240	20	Sea ice		+	−	JF706647

^aInhibition (+) defined as causing a clearing zone of >10 mm in pathogen-seeded agar.

Fig 2.

Neighbor-joining tree (1,000 bootstrap replicates) of aligned 16S rRNA gene sequences of antibiotic-producing Arctic bacteria. The closest cultured relatives (with GenBank accession numbers shown in parentheses) were included for comparison. Escherichia coli was used as the outgroup.

Antibiotic production by Arthrobacter spp.

Ethanolic extracts from Arthrobacter sp. WX11 grown in sea salt medium (43) inhibited growth of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus (Gram positives) and of Salmonella enterica, serovar Enteritidis, Aeromonas salmonicida, Vibrio vulnificus, V. parahaemolyticus, V. harveyi, V. anguillarum, Yersinia enterolitica, and Y. ruckeri (Gram negatives) in a well diffusion agar assay (16). Active ethanolic extracts were also obtained from cultures in media mimicking the habitats Arthrobacter spp. were isolated from, including 5.3% sea salt medium (mimicking high-salinity sea ice brine channels), marine minimal medium (31) containing 0.2% colloidal chitin (mimicking chitinous zooplankton), as well as AB medium (9) and salt-free LB medium (mimicking low-salinity meltwater). The antibiotic activity of Arthrobacter spp. grown with natural chitin was comparable to the behavior of an antagonistic Vibrio strain (42), but further studies are needed to determine whether antibiosis may occur in situ. Liquid chromatography (LC)-diode array-mass spectrometry (MS) performed according to the method described in reference 43 revealed almost identical metabolite profiles for all culture conditions (Fig. 3). Dereplication by high-resolution MS (1 ppm mass accuracy as defined by an internal standard) and comparison with published compounds (21) led to the tentative identification of arthrobacilins A, B and C (30), cyclic glycolipids each being present as two conformers (retention time [Rt] = 17.75/17.91 min for C₅₄H₉₆O₂₁, 19.22/19.36 min for C₅₆H₁₀₀O₂₁, and 20.55/20.62 min for C₅₈H₁₀₄O₂₁) (Fig. 3). Also, two potentially novel analogues (Rt = 14.85/14.92 min for C₅₀H₉₀O₂₁ and 16.24/16.33 min for C₅₂H₉₄O₂₁) were observed. However, arthrobacilins were also detected in ethyl acetate extracts that lacked antibacterial activity. The dual lipophilic and hydrophilic properties of the arthrobacilins made it impossible to assess the compounds quantitatively. We could therefore neither determine whether they had antibiotic activity nor address their potential mode of action, although they have been reported as weakly cytotoxic against human cancer cells and have been claimed to be antibiotic (30). Interestingly, the closest relative of our Arthrobacter isolates is able to reduce bacterial fish disease and is a patented fish vaccine (35), although this may be a result of immunostimulation rather than antibiotic activity (13, 36).

Fig 3.

LC-MS total ion chromatogram (electrospray ionization positive [ESI⁺]) of ethanolic Arthrobacter sp. WX11 culture extract. Peaks corresponding to retention times (Rt) of 17.91 (m/z 1,080), 19.22 (m/z 1,108), and 20.55 (m/z 1,136) min were tentatively identified as representing arthrobacilins A to C (inset). Molecular weight (MW) values were determined from the masses of the [M+NH₄]⁺ and [M+Na]⁺ ions. The two peaks with Rt = 14.90 (m/z 1,024) and 16.28 (m/z 1,052) min represent unknown arthrobacilin analogues.

Abundance of culturable antibiotic producers.

454 pyrosequencing libraries of sea ice and seawater originating from the same expedition (5) were checked for operational taxonomic units (OTUs) matching 16S rRNA gene fragments of antibiotic-producing isolates by alignment using the greengenes database (11) and the Needleman-Wunsch algorithm (27) in mothur (37). The full-length alignment was filtered to remove extraneous columns and the columns before and after the shorter reads, allowing a calculation of distance without bias between reads of similar lengths. Following the creation of a distance matrix, the reads were clustered (97% similarity). Although quantitative conclusions are hindered by potential PCR bias, this allowed estimation of the abundance of antibiotic-producing culturable isolates in the Arctic microbiota. 16S rRNA gene fragments of Psychrobacter XX5 and ST4 had three matching OTUs in the pyrosequencing libraries, which corresponds to an approximate abundance of 0.02%. Matching 16S rRNA gene fragments of antibiotic-producing Arthrobacter, Pseudoalteromonas, or Vibrio spp. were not detected, illustrating that these strains constitute only a minor fraction.

Concluding remarks.

Although 90% of the Earth's oceanic waters are cold (32), there is only limited knowledge about antibiotic production by Arctic marine bacteria. Here, we show that antibiotic-producing strains are present throughout the central Arctic Ocean and occur in different niches. The isolation of seven closely related, antagonistic Arthrobacter strains from diverse, distant habitats indicated a broad niche specificity and wide distribution. While we speculate that antibiosis may contribute to their widespread occurrence, it does not result in a considerable population size. Furthermore, the detection of arthrobacilins in fractions with and without antibiotic activity indicated that antibiosis may be dependent on the interplay of different compounds.

Nucleotide sequence accession numbers.

Data for sequences newly determined in this work are available under GenBank accession numbers JF706632 to JF706647.