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The diversity of bacteria isolated from Antarctic freshwater reservoirs possessing the

ability to produce polyhydroxyalkanoates

Slawomir CIESIELSKI1*, Dorota GÓRNIAK2, Justyna MOŻEJKO2, Aleksander

ŚWIĄTECKI2, Jakub GRZESIAK3, Marek ZDANOWSKI3

1Department of Environmental Biotechnology, University of Warmia and Mazury in Olsztyn, ul. Sloneczna 45G, 10-718 Olsztyn, Poland

2Department of Microbiology, University of Warmia and Mazury in Olsztyn, ul. Oczapowskiego 1A, 10-719 Olsztyn, Poland

3Department of Antarctic Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Ustrzycka 10, 02 - 141 Warsaw, Poland

*Corresponding author: Slawomir Ciesielski,, Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, Sloneczna 45G, 10-719 Olsztyn, e-mail: slavcm@uwm.edu.pl, phone: (+48) (89) 5234144, fax: (+48)

(89) 5234131

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The diversity of bacteria isolated from Antarctic freshwater reservoirs possessing the ability to

produce polyhydroxyalkanoates

Abstract

The diversity of polyhydroxyalkanoates-producing bacteria in freshwater reservoirs in the Ecology

Glacier foreland, Antarctica, was examined by a cultivation-dependent method. Isolated strains were

analyzed phylogenetically by 16S rRNA gene sequencing, and classified as members of Alpha-, Beta-,

or Gammaproteobacteria classes. Polymerase chain reaction was used to detect PHA synthase genes.

Potential polyhydroxyalkanoates (PHAs) producers belonging mainly to Pseudomonas sp. and

Janthinobacterium sp. were isolated from all five sampling sites, suggesting that PHA synthesis is a

common bacterial feature at pioneer sites. All Pseudomonas strains had the genetic potential to

synthesize medium-chain-length PHAs whereas some isolated Janthinobacterium strains might

produce both short-chain-length PHAs and medium-chain-length PHAs. It is the first report revealing

that Janthinobacterium species could have the potential to produce medium-chain-length PHAs.

Keywords: biopolymers, glacier, horizontal gene transfer, Janthinobacterium sp., polyhydroxyalkanoates, Pseudomonas sp.

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Introduction

A wide variety of taxonomically different groups of microorganisms are known to produce

intracellular energy and carbon storage compounds, generally described as polyhydroxyalkanoates

(PHAs) [1]. It has been shown that bacterial cells with a higher content of PHAs can survive longer

than those with a lower PHAs content, because they can utilize their reserve material longer and more

efficiently [6]. Therefore, it was stated that the accumulation of PHAs might increase the survival

capabilities of these bacteria in extreme environments or when nutrient availability is poor [7].

PHAs are synthesized and stored in the form of granules by many bacteria and archaea [29].

The microorganisms usually do this when carbon is available, but other essential nutrients are not.

Polyhydroxyalkanoates are commonly divided into two groups based on the number of constituent

carbon atoms in their monomer units – short-chain-length (scl-) PHAs and medium-chain-length (mcl-

PHAs. The former consists of monomers with 3–5 carbon atoms and the latter of monomers with 6–14

carbon atoms. The most commonly produced is polyhydroxybutyrate P(3HB), an scl-PHA. The

physical properties of individual PHAs depend on the composition of the monomer units. P(3HB) is

highly crystalline, whereas mcl-PHAs are elastic with low melting temperature [30]. These variations in the PHA polymer family create the potential for a vast array of applications [13].

PHAs are accumulated in intracellular granules. The surface of PHA granules are coated with phospholipids and proteins which play a major role in PHA synthesis, degradation and even in the process of PHA synthesis regulation. The type of PHA produced and accumulated in the granules depends on the metabolic pathway of the particular microorganism. However, independently of the

type of synthesized PHAs, the enzyme called PHA synthase always plays the main role [18]. In scl-

PHA producing bacteria this enzyme is coded by the phaC gene that has only been found in a single

form in bacterial cells. In bacteria that synthesize mcl-PHAs, this gene is present in two similar forms

that are called phaC1 and phaC2. The nucleotide composition of phaC1 and phaC2 genes is very

similar but their substrate specificity may differ [9].

Extremophiles inhabiting polar areas are a source a novel enzymes, which has a great

economic potential in many industrial processes, including agricultural, chemical and pharmaceutical

applications [31]. Especially, shallow Antarctic reservoirs influenced by the forces of variable trophic

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conditions are underexploited sources of microorganisms with biotechnological potential. As a result

of selection pressures in these reservoirs, there is a potential for the discovery of novel biochemical

pathways leading to polyhydroxyalkanoates synthesis. Psychrotrophic bacteria, which are adapted to

variable temperatures and to other fluctuating conditions such as low nutrient availability and low

water quality, may offer important advantages for industrial PHAs production [8]. It is widely

accepted that microorganisms from unusual environments provide not only valuable resources for

exploiting novel biotechnological processes but also serve as models for investigating how these

biomolecules are stabilized when subjected to changing conditions [11]. The main purpose of this

study was to determine whether bacteria inhabiting freshwater reservoirs in glacier forefield areas

have the potential to synthesize polyhydroxyalkanoates.

Materials and methods

Collection sites

Samples of water and mats were collected in summer of 2011 year from five different aged, shallow

freshwater reservoirs and one stream. They were located in the foreland of Ecology Glacier near to the Polish Antarctic Station “Arctowski” (King George Island, West Antarctica) (Fig. 1).

Selected reservoirs were different in age, location and environmental parameters (Table 1). Located near to the front of Ecology Glacier pond 7 was two years old. This reservoir was located on the ground moraine and was directly supplied with water from the glacier, with no signs of vegetation. Stream 6 is receiving water flowing directly from the glacier. Pond 8 was located away from the front
of the glacier, with abundant bloom of microalgae. Reservoirs 10 and 11, which have been noticed

there since 1977, were situated on the top of lateral moraine. Both were situated close to each other

and connected with small stream (Fig. 1). Samples were taken with a sampler on a bracket into sterile

bottles and delivered to the laboratory within one hour. Microbial mat samples were collected as cores

(~1 x 5cm) and one gram of sample was aseptically weighed and homogenized in 9 ml sterile cold

(4oC) physiological saline (0.86% NaCl) using a vortex. Cultures were obtained by plating 100 µl of a

sample onto Antarctic Bacteriological Medium (ABM; pH 7.4) containing: peptone (0.5%, w/v), yeast

extract (0.2%, w/v), agar (2%, w/v) in three replicates. Plates were incubated at  10 °C for 14 days.

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The well separated colonies on medium were macroscopically described, photographed, than isolated

under binocular and transferred to slants of ABM in the tubes. After transportation to Poland the

microscopic purity evaluation of Gram stained strains was done.

Molecular biology procedures

In order to obtain DNA, cells were scraped from the surface of slants and washed in sterile water.

DNA extraction was performed using commercial kit Genomic Mini (A&A Biotechnology) according

to the manufacturers` instructions. The purified DNA was suspended in 50 µl of deionized, DNase free

water and stored in -20 °C. The partial 16S rRNA gene was amplified using the primers 341: 5’-

CCTACGGGAGGCAGCAG -3’ [14] and 16SR: 5’- TACCTTGTTACGACTTCACCCCA-3’ described previously by Rossau et al. [19]. In order to detect microorganism producing

polyhydroxyalkanoates, PCR was performed with two primer pairs. First one, recognizing both scl-

and mcl-PHA synthase genes was elaborated by Romo et al. [20] (G-D: 5'-GTGCCGCC(GC)(CT)

(AG)(GC)ATCAACAAGT-3'; G1-R: 5' GTTCCAG(AT)ACAG(GC)A(GT)(AG) TCG AA-3'). The

second primers pair ( I-179L: 5’-ACAGATCAACAAGTTCTACATCTT CGAC-3’; I-179R: 5’-

GGTGTTGTCGTTGTTCCAGTAGAGGATGTC - 3’) was specific for both genes responsible for mcl-

PHA synthesis [23]. PCR was performed in Eppendorf® Mastercycler Gradient (Eppendorf, Germany). The mixtures used for PCR amplification contained 50 ng of extracted DNA, 0.5 µM of each primer, 100 µM of deoxynucleoside triphosphate (Promega, Winsconsin, USA), 1 U of Taq DNA

polymerase (POLGEN, Poland), 5 µl reaction buffer (500 mM KCl, pH 8.5; Triton X-100). PHA

synthase gene fragment amplification was carried out using program given by the authors. The

temperature program for 16S rRNA gene amplification was as follows: 94 °C for 5 min; 30 cycles of

denaturation at 94 °C for 30s, annealing at 54 °C for 30s, extension at 72 °C for 1 min and single final

elongation at 72 °C for 5 min. The PCR amplicons of 16S rDNA and PHA synthase genes were

resolved on 1.5% agarose gels stained with ethidium bromide, and size of PCR products was estimated

using molecular weight marker (100bp, Promega, Winsconsin, USA). The sequencing of 16S rDNA

and scl-PHA synthase (phaC) PCR products was performed using the same PCR primers as for

amplification. Because PCR primers pair I-179L and I-179R amplifies both genes coding for mcl-PHA

synthase, we use primers orf2 (5’- CATGACAGCGGCCTGTTCACCTGG-3’ [4]) and I-179R [23]

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using the same conditions as described in the work of Ciesielski et al. [4]. Using this approach it was

possible to amplify and directly sequence a fragment of phaC gene about 720 bp long.  DNA

sequencing was performed using a Perkin Elmer ABI 373 Automated DNA Sequencer (PE Applied

Biosystems, Foster City, CA, USA) at the Institute of Biochemistry and Biophysics in Warsaw,

Poland. All reactions were run following the manufacturer’s protocols. The nucleotide sequences were

submitted to the GenBank database under accession numbers from KF301568 to KF301599.

Phylogenetic analysis

The sequences of genes coding for 16S rRNA and PHA synthases were compared with those from the

GenBank database using the NCBI blast program. Sequences were aligned using the ClustalW

program [28]. The evolutionary distances were inferred using the Neighbor-Joining method [21] with

the MEGA5 program [25]. To determine the degree of statistical support for branches in the

phylogeny, 1000 bootstrap replicates of data were analyzed. The gene sequences that were >94.0%

identical to sequences of cultured species in the NCBI database were assigned genus names [24].

Statistics

A canonical correspondence analysis (CCA) was conducted using a statistical package for Windows v. Canoco 4.5 [27]. The presence of bacteria possessing specific form of PHA synthase genes was analyzed in relation to the environmental background ‘all other data’.

Results

A total of 50 bacterial isolates from five ponds and one stream were characterized by

sequencing of 1000 bp long fragment of 16S rRNA gene. Twenty, unique DNA sequences were

phylogenetically analyzed altogether with their closest relatives derived from GenBank (Fig. 2).

Isolated  strains  belonged  mainly to the phylum of Proteobacteria and  represent  classes

Betaproteobacteria (9 isolates), Gammaproteobacteria (9 isolates), Alphaproteobacteria (2 isolates)

only two isolates belonged to Bacteroidetes phylum (Fig. 2). Nineteen strains were isolated from

ponds and only one (Pseudomonas sp. P45) from stream in the foreland of Ecology Glacier. The

highest number of strains were derived from pond 10.

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The most frequently recovered group of isolates were members of the Pseudomonas genus (7

isolates). Among them two strains (Pseudomonas sp. P5 and Pseudomonas sp. P45) possessed the

same DNA sequence of studied 16S rRNA gene fragment. The second numerous group of isolates

belonged to Janthinobacterium genus (5 isolates).

In  the  aim  of  detection  of  a  microorganism  possessing  the  potential  ability of

polyhydroxyalkanoates synthesis, PCR was performed with two primers pairs. The first one (G-D and

G-1R), is able to recognize both scl- and mcl-PHA synthase genes [20]. The second primers pair (I-

179L and I-179R) was specific only for mcl-PHA synthase genes [23]. The amplification using

primers G-D and G-1R gave a positive signal in 12 of the isolates corresponding to six pseudomonads

strains, four Janthinobacterium strains, and two undefined Betaproteobacteria (P21 and P25). The

obtained PCR amplicons possessed the expected length of 551 bp. The amplification with PCR

primers designed by Solaiman and co-workers [23] showed that six Pseudomonas strains (P1, P4, P10,

P27, P32, and P45) and two Janthinobacterium strains (P10 and P24) possessed genes responsible for

mcl-PHA synthesis. PCR products obtained by employing primer pairs G-D : G-1R and orf2 : I-179R

were directionally sequenced.

The results of DNA sequencing of PCR products obtained by using G-D and G-1R primers proved that studied DNA of two Janthinabacterium strains (P38 and P39), and two undefined Betaproteobacteria strains (P21 and P25) is responsible for scl-PHA synthesis. Nucleotide composition of PCR products amplified with orf2 : I-179R primers pair have provided evidence that all are typical for phaC1 gene coding for synthase responsible for mcl-PHA synthesis. For deeper analysis, all
obtained DNA sequences of phaC and phaC1 genes, after division into two group, were

phylogenetically examined altogether with their closest relatives found in GenBank. Evolutionary tree

presenting genetic distance between phaC gene DNA sequences is shown in the Figure 3. In the phaC

gene group (scl-PHA) very high similarity (genetic distance 0.236) was revealed between

Janthinobacterium sp. P39 and Antarctic Proteobacterium P21, inhabiting the same pond 7. Very close

to them DNA sequence of Janthinobacterium sp. P38 was placed, and all three sequences obtained in

this study were accompanied by DNA sequence of Zooglea ramigera. The fourth sequence, belonging

to Antarctic Proteobacterium P25 was grouped with Rhodoferax ferrireducens (genetic distance

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0.237). (Fig. 3). The second group, collecting DNA sequences of phaC1 gene (mcl-PHA), was

predominated by members of Pseudomonas species. The highest similarity (genetic distance 0.003),

was observed between Pseudomonas sp. P1 and Pseudomonas sp. P4 both inhabiting the mat from

lake 10. The next pair, revealing high identity, was composed of Janthinobacterium sp. P10 and

Pseudomonas sp. P19 (genetic distance 0.009) and was collected from different, although neighboring

ponds. Surprisingly, these two sequences were grouped together with Janthinobacterium sp. P24

sequence that came also from mat of pond 10. The rest of studied phaC1 gene sequences were

dispersed among sequences derived from GenBank. The most diverged phaC1 gene sequence

belonged to Pseudomonas sp. P45 that came from the glacier outlet stream (Fig. 4). Bacterial strains

possessing genes phaC and phaC1 are characterized in Table 2.

The statistical analysis of principal components (CCA) showed correlation between presence

of bacteria possessing particular PHA synthase gene and environmental conditions. Bacteria having

genetic potential to synthesize scl-PHA inhabited reservoirs with rather low trophy characterized by

low levels of total phosphorus and high amounts of pheophytins (Fig. 5).

Discussion

Most of isolates cultured directly from the samples taken from Antarctic ponds belonged to

Beta- and Gammaproteobacteria, among them Janthinobacterium spp. and Pseudomonas spp. predominated. Only two taxons belonging to Alphaproteobacteria were isolated (Fig. 2). Generally, the majority of 16S rDNA sequences from cultured bacteria were closely related to other sequences

found in cold environments [15, 32]. In particular, the presence of Janthinobacterium spp. in Antarctic

waters has previously been discussed [26]. Shivaji et al. [22] showed that strains of this genus isolated

from Antarctica could be divided into two groups; the first is “Jantinobacterium lividum group”,

whereas the second was called “atypical J. lividum group”. Our analysis also indicates that isolated

Janthinobacterium strains were divided into two groups on the base of gene coding for 16S rRNA (Fig.

2). Like Janthinobacterium genus members, bacteria belonging to Pseudomonas spp. are often

isolated from cold environments and are known for their metabolic versatility and high genome

plasticity [17].

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In the ponds that were studied, more than half of the isolated strains possessed genes

responsible for polyhydroxyalkanoates synthesis. Among them were mainly microbes belonging to the

Pseudomonas genus (six strains) and Janthinobacterium genus (five strains). Our results are in

concordance with results of Goh and Tan [7]. They isolated from Antarctic soil strains of bacteria

having the ability to produce PHAs, among them twelve belonging to Pseudomonas spp. and three to

Janthinobacterium genus.

The high number of isolates possessing genes responsible for PHAs synthesis suggests that

Antarctic bacteria evolved, or obtained in another way, this metabolic ability to endure the extreme

conditions typical for polar regions. Accordingly to our results, isolated strains possessed genes

responsible for scl- or mcl-PHA synthesis. It is worth pointing out that bacteria having phaC1 genes

were isolated both from mats and water, and those having phaC gene were only planktonic. It has

previously been reported that survival in harsh conditions can be increased by belonging to a complex

communities like microbial mats. Recent studies have evaluated the potential of microbial mats as

high-level polyhydroxyalkanoate production systems under natural conditions, and as a source of

bacterial polyhydroxyalkanoate producers [2, 12]. Accordingly to these studies, microorganisms inhabiting microbial mats from marine coastal zones are able to synthesize and accumulate high quantities of PHAs. Our results also show that microbial mats created in Antarctic ponds could be a potential source of PHA producing bacteria.

Our comparative analysis of DNA sequences of 16S rDNA and phaC/phaC1 genes suggested that some strains possess PHA synthesis genes that might have not been obtained by gradual

evolution. The best example are isolates belonging to Janthinobacterium spp. Among six members of

this genus, possessing genes responsible for PHAs synthesis, three of them had phaC genes, whereas

two had phaC1 genes. The phaC gene of Janthinobacterium spp. P21, P38 and P39 was similar to a

variant of this gene previously detected only in Zooglea ramigera, whereas the phaC1 genes of

Janthinobacterium sp. P10 and P24 were most similar to genes of Pseudomonas extremaustralis and

Pseudomonas fluorescences, respectively. The genes responsible for scl-PHA synthesis differ so much

from those responsible for mcl-PHA synthesis that their presence in these closely related bacteria is

most likely not the result of gradual evolution, but rather of horizontal gene transfer. It is interesting

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that isolates that had obtained these particular types of genes were divided into two subgroups on the

basis of 16S rDNA sequences (Fig. 2). It is possible that this evolutionary separation is related to some

physiological differences that make a particular PHA operon useful for these bacteria. Previously, only

scl-PHA producing Janthinobacterium spp. have been reported [7] and ours is the first report of

potential mcl-PHA synthesis by members of this genus.

We found genes coding for mcl-PHA synthase mainly in Pseudomonas species. 16S rDNA

gene analysis showed that the taxonomic position of each of the six Pseudomonas isolates having the

phaC1 gene was different (Table 2). Whereas, variation between phaC1 genes was lower, closest

DNA sequences of phaC1 gene of examined strains obtained by BLAST searching belonged only to

three genera. The DNA sequences of P1, P4, P19, and P27 were closest to the sequence of

Pseudomonas fluorescens, a well known mcl-PHA producer [5]. The structure of the phaC1 gene of

Pseudomonas sp. P32 was most similar to the gene of Pseudomonas sp. KBOS 04 isolated from

activated sludge by Ciesielski et al. [3]. In the same study, the same PHA operon was found in both

cells of Pseudomonas sp. KBOS04 and of Comamonas testosteroni, This observation may suggest that

these PHA operons have been horizontally transferred between cells. Pseudomonas sp. P45, inhabiting a glacial melt water stream, possessed a phaC1 gene most similar to that of Pseudomonas stutzeri, although the similarity was only 84%. It should be underlined that the nucleotide composition of the

Pseudomonas sp. P45 16S rRNA gene was most similar to P. fragii and identical to the DNA sequence of the Pseudomonas sp. P5 isolated from pond 8. In contrast to Pseudomonas sp. P45, in cells of strain P5 genes responsible for PHA synthesis were not detected. In general, comparative

analysis of 16S rRNA and phaC/phaC1 genes revealed that isolated bacteria gain the genetic ability to

synthesize PHA rather as a result of horizontal gene transfer than by gradual evolution. Furthermore, it

seems that some PHA operons are favored during this unusual ecological event.

Bacteria possessing PHA synthase coding genes were detected in each of the studied location

suggesting that PHAs accumulation could increase the survival capabilities of microorganisms in this

extreme environment. Bacteria having genes responsible for both scl- and mcl-PHAs synthesis were

found, but those able of scl-PHA synthesis were discovered only in ponds 7 and 10. These two

reservoirs characterized with high amounts of pheophytin and total chlorophyll. The relation between

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the presence of scl-PHA producing bacteria and environmental conditions is supported by CCA (Fig.

5). The highest number of different forms of phaC/phaC1 genes was founded in pond 10, which was

characterized by low pH (6.5), highest level of pheophytin (3.800 ug l-1), and total chlorophyll (4.700

ug l-1) and with the highest temperature measured (6.1 ºC). Only one strain having phaC1 gene was

isolated from pond 11 (Pseudomonas sp. P19) and what is interesting similar form of gene was found

in cells of Janthinobacterium sp. P10 inhabiting pond 10 (Fig. 1). Ponds 10 and 11 are connected with

the stream and it is likely, that this form of phaC1 was channeled from pond 11 to pond 10. A very

distant form of phaC1 gene was found in the mat taken from the stream 6, it might release from the

fact that environmental conditions in the streams are different from those in ponds. The spatial

distribution of microorganisms possessing particular form of genes in the area of glacier forefield

results mainly from glacier melting. The flowing water carries not only nutrients and minerals, but

also transfers microorganisms having accelerated ability to colonize new territories. This group can

include bacteria having ability to utilize PHAs produced by themselves or other members of microbial

community [10]. It seems that bacteria having genetic ability to store and degrade PHAs can work as a

pioneers during a succession process. Thus, glacier forefields provide a unique opportunity as a natural laboratory to study the succession of microorganisms.

To sum up, our study shows that many microorganisms inhabiting freshwater lakes charged with glacier meltwater possess the genes responsible for PHA synthesis. Most of the strains possessing PHA synthase genes belonged to Pseudomonas and Janthinobacterium species. All isolated
Pseudomonas species  had  the  phaC1 gene  responsible  for  mcl-PHA  synthesis,  whereas

Janthinobacterium isolates had phaC and phaC1 genes used for both scl- and mcl-PHA synthesis. To

our knowledge, this is the first evidence that Janthinobacterium spp. have the potential to synthesize

mcl-PHAs. Another interesting observation is the lack of correspondence between the evolutionary

history of the 16S rDNA genes and those coding for PHA synthases. Phylogenetic analyses of the

phaC and phaC1 genes, especially in Janthinobacterium sp., suggest that these genes could have been

acquired by horizontal gene transfer. The obtained results allow us to speculate that the possession of

these genes enabling PHA synthesis may improve the fitness and survival of bacteria in harsh

conditions.

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Acknowledgements

The study was financed under project no. N N304106940 of the Polish National Center for Science.

References

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31.	Van den Burg B (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6:213–218 

32.	Zdanowski MK, śmuda-Baranowska M, Świątecki A, Borsuk P, Górniak D, Grzesiak J (2013) Microbial community development in the post glacial soils of Ecology Glacier (King George Island, Antarctica) - culturable approach. Polar Biol 36:511-527 

Figures

Fig. 1. Map showing sampling location in the foreland of Ecology  Glacier, King Georg Island,

Antarctica. Sampling sites are characterized in Table 1. (Part of Admiralty Bay map modified from

Pudełko [16].

Fig. 2. Phylogenetic tree based on 16S rRNA sequences, generated by the neighbor-joining method

showing the genetic relationships among bacteria analyzed in this work. Thermotoga subterranea

(Acc. Nr NR_025969)was selected as the outgroup. The numbers on the branches refer to boostrap

values for 1,000 times. Accession numbers of DNA sequences are given in parentheses.

Fig. 3. Phylogenetic tree based on phaC gene coding for scl-PHA synthase, generated by the neighbor-

joining method showing the genetic relationships among bacteria analyzed in this work. The numbers

on the branches refer to boostrap values for 1,000 times. Accession numbers of DNA sequences are

given in parentheses.

Fig. 4. Phylogenetic tree based on phaC1 gene coding for mcl-PHA synthase, generated by the

neighbor-joining method showing the genetic relationships among bacteria analyzed in this work. The

numbers on the branches refer to boostrap values for 1,000 times. Accession numbers of DNA

sequences are given in parentheses.

Fig. 5. Canonical correspondence analysis showing relation between presence of bacteria possessing

specific form of PHA synthase genes and environmental data of sampling sites.

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58O 28’ W	58O 27’ W
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King George Island

200 M

							
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																																							Flavobacterium algicola	BACTEROIDETES		
																					Antarctic Alphaproteobacterium P35 (KF301583)			
																																100		(AB455265)			
																																					
																																								Flavobacterium sp. P14			
																																								(KF301573)			
100					Sphingomonadaceae bacterium N (DQ497241)			
																																								
100					Iodobacter fluviatilis (AB681839)			
																	Iodobacter sp. P11 (KF301572)			

																		88							Antarctic Proteobacterium P25 (KF301578)			
99																99							Antarctic Proteobacterium P36 (KF301584)			
																										
											100												
																			Rhodoferax sp. Asd M2A1 (FM955857)			
																												
																												Comamonas testosteroni (AY247415)	β-PROTEOBACTERIA		
																															
																															Burkholderia caryophylli (NR_040806)			
	71																													Janthinobacterium sp. P10 (KF301571)			
																											96						
																																								
																						81					Janthinobacterium sp. P24 (KF301577)			
	99																Janthinobacterium lividum (AM748811)			
																																					
																																		Janthinobacterium sp. P30 (KF301581)			

																						100									Burkholderia sp. CYEB-14 (FJ422391)			
																																						
																																				Antarctic Proteobacterium sp. P21 (KF301576)			
																						86														
																											54						Janthinobacterium sp. P39 (KF301586)			

																											64						Janthinobacterium sp. SMN 33.6 (JX624164)			

																																							
98																													61		Janthinobacterium sp. P38 (KF301585)			
																																		
	100																						Shewanella sp. P28 (KF301580)			
																																									
																															Escherichia coli (JX975437)			
																														
												100											Yersinia sp. P16 (KF301574)			
																										
		87			Pseudomonas antarctica (HE586386)			
								
		73					Pseudomonas sp. P27 (KF301579)			

		63						Pseudomonas marginalis (AF364098)			

		87								Pseudomonas veronii (AB056120)			

		66																																			
														Pseudomonas sp. P1 (KF301568)			
		39						Pseudomonas frederiksbergensis (JX401500)			

		22						Pseudomonas fluorescens (AJ278814)			

													Pseudomonas sp. P4 (KF301569)					
87															PROTEOBACTERIA		
																	
												99						Pseudomonas sp. P5 (KF301570)			
																					
	17						Pseudomonas sp. P19 (KF301575)			
											
	42																																					
		33							Pseudomonas migulae (AY047218)			
																																						
	43							Pseudomonas sp. P32 (KF301582)			

	50												Pseudomonas fragi (JX867753)	γ-		
		78																																					
									77			Pseudomonas sp. P45 (KF301587)			

																			
												Pseudomonas chlororaphis (JX082198)			
															
														Pseudomonas putida (X93997)					
																			
100									Pseudomonas stutzeri (AJ310484)			
																	
																				Pseudomonas oleovorans (NR_043423)			
94							78			Pseudomonas aeruginosa (AB126582)			
																			
48					99					Pseudomonas sp. OLB-1 (AJ387904)			

															
40											Pseudomonas resinovorans (AB021373)			
														
										Pseudomonas nitroreducens (D84022)			
													
58						Pseudomonas pseudoalcaligenes (Z76675)			

68									Pseudomonas mendocina (Z76674)			

Thermotoga subterranea (NR_025969)

																						
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3																										Antarctic Proteobacterium		
4																												
	40											P21 (KF301592)		
5																												
	51														Janthinobacterium sp. P39		
6																	
																											(KF301598)		
7	99																		
														Zoogloea ramigera (U66242)		
8																										

9	52																Janthinobacterium sp. P38 (KF301597)		
10																			

11																							Cupriavidus taiwanensis (GQ922054)		
12	42																			

13																										
							100			Cupriavidus necator (FJ897463)		
												
14	82																

15																									
																									Burkholderia sp. USM (JN022533)		
16	55																									

17																												
																		Alcaligenes latus (AF004933)		
18																													
19															Comamonas acidovorans (AB009273)		
20																	

21											Variovorax paradoxus (CP001635)		
22													
																													
23																					Polaromonas sp. (CP000529)		
24																							

25																				Rhodoferax ferrireducens (CP000267)		
																						
26	99														
																													
27	94																Antarctic Proteobacterium		
28																													
																													
																											P25 (KF301594)		
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																								99			Pseudomonas stutzeri (AY278219)		
																													
																								100									Pseudomonas pseudoalcaligenes (AF336848)		

															62																		Pseudomonas mendocina (AF311979)		

																															Pseudomonas nitroreducens (AF336849)		
78											86								

90																													Pseudomonas SP. P45		
																												P. resinovorans (AF129396)		
																																						
20																													Pseudomonas oleovorans (M58445)		

																		100							Pseudomonas putida (HQ662163)		

35																Pseudomonas SP. P32									

		100									Pseudomonas mediterranea (AY910768)		
													
																								Pseudomonas corrugata (AY910767)		

																		100			Pseudomonas SP. P1		
	39																						Pseudomonas SP.	P4		
																													
																								Pseudomonas fluorescens (FJ472656)		
	44																				
																					
																				Janthinobacterium SP. P24									
	39																						
																																	
																						Janthinobacterium SP. P10		
	90													
															100				Pseudomonas SP. P19									
																Pseudomonas extremaustralis (FN435843)		

																	Pseudomonas SP. KBOS04 (AY790328)		
90												

															Pseudomonas SP. P27									
91															
													Pseudomonas chlororaphis subsp. aureofaciens (AB049413)		

0.02																															

	

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Table 1. Characteristics of sampling sites

							Sampling site							
															
	Variables			6.		7.		8.		10.		11.			
										
	GPS position		62º48'26,4 S	62º48'25,2 S	62º10'02,3 S	62º09'23,2 S	62º09'37,0 S		
			058o28'53,9 W	058o28'50,1 W	058o2740,9 W	058o28'20,2 W	058o28'23,7 W		
	Elevation.	m a.s.l.		8		7		9	227	225			
	Area	m2			266.91	122.30	117.60	123.80			
	NH4-N	mg l-1	0.024	(±0.004)	0.035	(±0.011)	0.426	(±0.071)	0.161	(±0.018)	0.033	(±0.002)			
	PO4-P	mg l-1	0.148	(±0.003)	0.130 (±0.00)	0.120	(±0.042)	0.079 (±0.02)	0.154	(±0.044)			
	P-tot	mg l-1	0.03	(±0.00)	0.035	(±0.007)	0.050	(±0.028)	0.020 (±0.00)	0.070 (±0.00)			
	N-tot	mg l-1	0.650	(±0.071)	1.950	(±1.768)	4.100 (±0.00)	1.400 (±0.00)	1.700 (±0.00)			
	pH	mg l-1	7.6	(±0.5)	7.7	(±0.6)	8.4	(±0.7)	6.5	(±1.2)	7.1	(±0.8)			
	temp	oC	2.8	(±1.8)	2.9 (1.6)	5.0	(±0.2)	6.1	(±2.1)	5.8	(±0.6)			
	O2	mg l-1	11.85 (±0.35)	10.40 (±0.42)	10.86 (±1.22)	11.08 (±0.60)	10.92 (±0.23)			
	Conductivity	uS/cm	79.4 (±26.86)	84.7 (±41.37)	166.1	(±76.23)	135.0	(±32.85)	160.3	(±19.38)			
	Chlorophyll a	ug l-1	0.400	0.417	1.666	0.900	0.900			
	Pheophytin	ug l-1	2.800	2.791	2.125	3.800	1.800			
	Total chlorophyll	ug l-1	3.200	3.208	3.791	4.700	2.700			
	DOC	mg l-1	0.567	0.653	1.135	0.787	0.826			

								
1									
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3									
4									
5		Table 2. Molecular identification of isolated strains, their nearest neighbors based on 16S rRNA and phaC/phaC1 genes and their isolation location		
6									
7		Isolate	Putative	Closest relatives based on 16S rDNA	Closest cultured based on phaC/phaC1 gene	Isolation source		
8			PHA	sequence (Accession nr/identity %)*	sequence (Accession nr/identity %)*				
9		Pseudomonas sp. P1	Mcl	Pseudomonas extremaustralis SY11 (KC790323/99)	Pseudomonas fluorescens Pf0-1 (CP000094/91)	Mat (pond 10)		
10		Pseudomonas sp. P4	Mcl	Pseudomonas frederiksbergensis (JF343187/100)	Pseudomonas fluorescens Pf0-1 (CP000094/90)	Mat (pond 10)		
11		Pseudomonas sp. P19	Mcl	Pseudomonas putida C14 (JN228297/99)	Pseudomonas fluorescens BM07 (FJ472656/91)	Water (pond 11)		
12		Pseudomonas sp. P27	Mcl	Pseudomonas antarctica (HE586386/99)	Pseudomonas fluorescens SBW25 (AM181176/95)	Water (pond 8)		
13								
		Pseudomonas sp. P32	Mcl	Pseudomonas putida (HE586397/99)	Pseudomonas sp. KBOS04 (AY790328/92)	Water (pond 8)		
14								
		Pseudomonas sp. P45	Mcl	Pseudomonas fragi (AB685634/100)	Pseudomonas stutzeri 1317 (AY278219/84)	Stream 6		
15								
		Janthinobacterium sp. P10	Mcl	Janthinobacterium lividum (HQ824865/100)	Pseudomonas extremaustralis (FN435843/90)	Water (pond 10)		
16								
		Janthinobacterium sp. P24	Mcl	Janthinobacterium lividum (HQ824864/100)	Pseudomonas fluorescens BM07 (FJ472656/92)	Mat (pond 10)		
17								
								
18									
19		Antarctic Betaproteobacterium P21	Scl	Burkholderia sp. Era35(JQ977167/99)	Zoogloea ramigera (U66242/83)	Water (pond 7)		
20		Antarctic Betaproteobacterium P25	Scl	Rhodoferax ferrireducens (HG003356/99)	Comamonas testosteroni CNB-2 (CP001220/81)	Water (pond 10)		
21		Janthinobacterium sp. P38	Scl	Janthinobacterium sp. SMN 33.6 (JX624164/99)	Zoogloea ramigera (U66242/83)	Water (pond 10)		
22		Janthinobacterium sp. P39	Scl	Janthinobacterium sp. TMT4-26-6 (JX949991/99)	Zoogloea ramigera (U66242/83)	Water (pond 7)		
23								
									
24		Pseudomonas sp. P5	-	Pseudomonas fragii (AB685683/99)	-	Water (pond 8)		
25								
		Iodobacter sp P11	-	Iodobacter fluviatilis (KC213858/100)	-	Water (pond 7)		
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27		Flavobacterium sp. P14	-	Flavobacterium algicola (AB455265/99)	-	Water (pond 10)		
28		Yersinia sp. P16	-	Yersinia intermedia (NR_027545/99)	-	Water (pond 7)		
29		Shewanella sp. P28	-	Shewanella putrefaciens (KC607503/99)	-	Water (pond 8)		
30		Janthinobacterium sp. P30	-	Janthinobacterium lividum (HQ824941/99)	-	Water (pond 7)		
31		Antarctic Alphaproteobacterium P35	-	Sphingomonadaceae bacterium N (DQ497241/99)	-	Water (pond 7)		
32		Antarctic Betaproteobacterium P36	-	Rhodoferax ferrireducens (NR_074760/99)	-	Water (pond 8)		
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		* - only cultured strains were considered during BLAST searching					
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