Phylogenetic diversity of T4-like bacteriophages in Lake Baikal, East Siberia

R E S E A R C H L E T T E R

Phylogeneticdiversityof T4-like bacteriophages inLakeBaikal,East SiberiaTatyana Vladimirovna Butina, Olga I. Belykh, Svetlana Yu. Maksimenko & Sergey I. Belikov

Limnological Institute, Siberian Branch of Russian Academy of Sciences, Irkutsk, Russia

Correspondence: Tatyana Vladimirovna

Butina, Limnological Institute, Siberian Branch

of Russian Academy of Sciences, PO Box 278,

Irkutsk 664033, Russia. Tel.: 17 395 2 51 18

74; fax: 17 395 2 42 54 05; e-mail:

[email protected]

Received 5 March 2010; revised 12 May 2010;

accepted 17 May 2010.

Final version published online 23 June 2010.

DOI:10.1111/j.1574-6968.2010.02025.x

Editor: Wolfgang Schumann

Keywords

T4-type phage; cyanophage; g23; diversity;

Lake Baikal.

Abstract

Among the tailed phages, the myoviruses, those with contractile tails, are wide-

spread and diverse. An important component of the Myoviridae family is the genus

‘T4-like viruses’. The present study was aimed at elucidating the molecular

diversity of T4-type bacteriophages in Lake Baikal by partial sequencing of g23

genes of T4-type bacteriophages. Our study revealed that the g23 gene sequences

investigated were highly diverse and different from those of T4-like bacteriophages

and from g23 clones obtained from different environments. Phylogenetic analysis

showed that all g23 fragments from Lake Baikal, except for the one sequence, were

more closely related to marine T4 cyanophages and to previously described

subgroups of uncultured T4 phages from marine and rice field environments.

Introduction

Tailed bacteriophages are the most abundant biological

entities in marine environments (Breitbart et al., 2002).

Among the tailed phages, the myoviruses, those with

contractile tails, are widespread and diverse. For example,

the environmental sequences belonging to the Myoviridae

family represent 11–23% of all sequences obtained from

metagenomic analysis of uncultured Pacific viral samples

(Breitbart et al., 2002). According to the virus taxonomy and

nomenclature approved by the International Committee on

Taxonomy of Viruses, the family of Myoviridae is composed

of seven genera (http://www.ncbi.nlm.nih.gov/ICTVdb/

Ictv/fs_index.htm). An important component of the Myo-

viridae family in particular from an ecological viewpoint is

the genus ‘T4-like viruses’. T4-like phages are a diverse

group of lytic bacterial viruses that share genetic homologies

and morphological similarities to the well-studied coliphage

T4 (Ackermann & Krisch, 1997). These phages have been

divided into subgroups (T-evens, PseudoT-evens, SchizoT-

evens and ExoT-evens) according to the sequences of their

virion genes (Monod et al., 1997; Hambly et al., 2001; Tetart

et al., 2001).

Recently, a set of degenerate PCR primers for the g23

gene, which encodes the major capsid protein in all of the

T4-type phages, has been designed (Filee et al., 2005).

Among T4 structural genes, g23 is thought to be a highly

reliable biomarker to study molecular diversity (Tetart et al.,

2001), because the phylogeny of T4-type bacteriophages

based on the partial g23 sequence is congruent with those

obtained from T4-type bacteriophage genomes (Desplats &

Krisch, 2003). These primers were used to amplify g23-

related sequences from diverse marine environments and

from paddy field agroecosystems (Filee et al., 2005; Jia et al.,

2007; Wang et al., 2009a, b). A majority of the sequences of

g23 PCR products from diverse marine environments be-

longed to five previously uncharacterized subgroups

(groups I–V) (Filee et al., 2005). The g23 gene sequences

from Japanese paddy fields were classified into six new

subgroups (Paddy groups I–VI) (Wang et al., 2009a). More-

over, Wang et al. (2009b) determined three additional paddy

T4 groups based on g23 gene analysis of the clone libraries

from Chinese paddy fields.

The first data on the presence and abundance of virus-like

particles in Lake Baikal were obtained in 2000. Staining with

SYBR Green revealed about 5.9 million virus-like particles per

FEMS Microbiol Lett 309 (2010) 122–129c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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mL (Belykh & Belikov, 2000). Later on, transmission electron

microscopy examinations showed a considerable morpholo-

gical diversity and seasonal dynamics of virioplankton in the

water of Lake Baikal. Viruses were represented by many

morphotypes of tailed phages, including phages of the family

Myoviridae (Drucker & Dutova, 2006, 2009). The abundance

of phages in the water of Lake Baikal suggested that they are an

essential component of this ecosystem.

The present study was aimed at elucidating the molecular

diversity of T4-type bacteriophages in Lake Baikal by target-

ing g23 genes of T4-type bacteriophages that could play

an important role in the food webs and in the evolution of

this ecosystem.

Materials and methods

Sampling and counting of bacteria

Water samples were collected from pelagic stations in

Northern (the Baikalskoe–Turali section, maximal depth

800 m, 55119.3090N, 109128.7300E) and Southern (the List-

vyanka–Tankhoy section, maximal depth 1450 m, 511

42.6530N 105101.6770E) basins of Lake Baikal. Water sam-

ples were taken at depths of 0–50 m on May 30 (Southern

Baikal) and June 2 (Northern Baikal), 2008.

For counting bacteria and picoplanktonic cyanobacteria,

samples were fixed with formalin and filtered through 0.22-

mm pore-size polycarbonate filters (Millipore). The filters

for bacteria counting were stained with 40,6-diamidino-

2-phenylindole (DAPI) solution. Picoplanktonic cyanobac-

teria were detected using the phycobilin autofluorescence as

described previously (Belykh & Sorokovikova, 2003).

The filters were examined under an Axiovert 200 micro-

scope (Zeiss, Germany). Bacteria and cyanobacteria cell sizes

and shapes were measured using microphotographs taken by a

Penguin 600CL camera (Pixera Corp.) and the VIDEOTEST-

RAZMER 5.0 software package (http://www.videotest.ru). The

biomass was estimated from the average cell volume and

abundance. For each station, a sample series, taken along the

vertical line (0, 5, 10, 15, 25 and 50 m), was counted as a

weighed arithmetic mean for 0–25 and 0–50-m layers.

Concentration of viral communitiesand DNA extraction

For T4-phage detection, the water samples (500 mL) from

depths between 5 and 10 m were used. The samples were

filtered sequentially. Most organisms and particles larger

than viruses were removed by filtration through polycarbo-

nate filters (Millipore) with pore diameters of 1.2, 0.45 and

0.22 mm. The filtered subsamples (100 mL) were then con-

centrated on 0.02-mm Anopore Inorganic Membranes

(Whatman). DNA was extracted from 0.02-mm filters using

a DNA-sorb kit (InterLabService, Russia) according to the

manufacturer’s protocol.

PCR amplification, cloning and sequencing

Degenerate g23 primers, MZIA1bis and MZIA6, were used

for PCR amplification (Filee et al., 2005). PCR was per-

formed using Amplisens kit (InterLabService). Two micro-

liters of DNA template was added to 8 mL of PCR mixture

containing 1.5 mM MgCl2, 0.20 mM concentration of each

deoxyribonucleoside triphosphate, 20 pmol each of the

primers and 1.0 U of Taq polymerase. PCRs were performed

as described by Filee et al. (2005).

Amplicons were initially visualized by 4% acrylamide gel

electrophoresis, followed by silver staining. Bands of the

appropriate molecular mass were excised from gels, rinsed

in plenty of water and frozen with 50 mL water. Water

extracts were used as the DNA template for PCR. All of the

reaction mixtures and conditions were the same as those in

the first amplification, except that the PCR reaction volume

was 50 mL. Purification of DNA fragments was performed by

0.8% agarose gel electrophoresis in 0.5�TAE buffer

(20 mM Tris-acetate, 5 mM EDTA, pH 8.0). PCR products

were extracted by freezing agarose plugs, which contained

the band, followed by centrifugation. The amplified DNA

fragments were cloned using the InsTAclone kit (Fermen-

tas). The positive clones were sequenced by the CEQ 8800

sequencer (Beckman Coulter).

Phylogenetic analysis

Sequences were aligned and formatted using CLUSTAL W

software BIOEDIT (v7.0.5) (Hall, 1999) and corrected manu-

ally with the help of the maximum-parsimony software

(MEGA 4) (Tamura et al., 2007). Translated sequences were

analyzed for the closest relatives by a BLAST search on the

NCBI web site. The alignment sequences were compared

with g23 fragments of known T4 phages obtained from the

T4-like genome database (http://phage.bioc.tulane.edu) and

with g23 clones of uncultured viruses of different origins.

Phylogenetic trees were reconstructed with the Bayesian

inference method using MRBAYES v3.1.2 (Huelsenbeck & Ron-

quist, 2001). An appropriate model of amino acid substitution

was selected previously by the PROTTEST v2.4 program (Abascal

et al., 2005) using the Bayesian information criterion. In

Bayesian inference, two parallel MCMC runs were carried out

for one million generations sampled every 100 generations for

a total of 5000 samples. The robustness of the trees was

estimated by posterior probabilities.

The nucleotide sequences reported in this paper

have been submitted to GenBank (FJ798929–FJ798951;

GU256228–GU256245).

FEMS Microbiol Lett 309 (2010) 122–129 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

123Phylogenetic diversity of T4-like phages in Lake Baikal

http://www.videotest.ru

http://phage.bioc.tulane.edu

Results

Abundance of bacteria and cyanobacteria

The abundances of picoplanktonic cyanobacteria and hetero-

trophic bacteria were different in the lake basins in early June,

2008. In Northern Baikal picoplanktonic cyanobacteria of the

genera Synechococcus, Cyanobium and Synechocystis developed

in huge numbers. They were dominated by an endemic

Baikalian autotrophic picoplankton species – Synechocystis

limnetica, which constituted 20% of the total picocyanobac-

terial number at depths of 0–25 m. As a whole, the numbers of

picocyanobacteria reached 268 000 cells mL�1 at a depth of

5 m; the abundance of heterotrophic bacteria was about

288 000 cells mL�1 in the upper 25-m layer (Fig. 1). Thus, the

share of picocyanobacteria in the total bacterial plankton

number was about 50%, in biomass – 68%.

At this time, the development of autotrophic picoplankton

in Southern Baikal was low, and the numbers of picocyano-

bacteria were 12 400 cells mL�1 in the 0–25-m layer (Fig. 1).

The main components of picocyanobacteria communities

were species of Synechococcus and Cyanobium genera, but, in

contrast to the Northern basin, the contribution of S. limnetica

to the total abundance did not exceed 4%. The abundance of

bacteria in the Southern basin was high and averaged

1 780 000 mL�1 in the 0–25-m layer (Fig. 1). The share of the

picocyanobacteria in total bacterial plankton abundance was

only 1%, in biomass – 3%.

Sequences of g23 fragments

PCR products were obtained from both Northern and South-

ern Baikal water samples: each sample exhibited five bands

that approximately ranged from 350 to 500 bp. All five bands

of g23 amplicons from Northern Baikal water samples and

only three bands from Southern Baikal were successfully

reamplified. We constructed clone libraries of the purified

g23 gene PCR products obtained from two stations. The

recovery efficiency of g23 gene fragments from Southern

Baikal was lower and only 70% of the clones contained correct

g23 inserts within this clone library. In total, 23 clones from

Northern Baikal and 18 from Southern Baikal were sequenced

and translated (g23 amino acid sequence from 118 to 289 in

the coliphage T4 sequence, Parker et al., 1984). The predicted

amino acid sequences from Lake Baikal were variable in length

from 105 to 143 residues. Each clone was designated as N0508

(Northern Baikal clone library) or S0508 (Southern Baikal),

followed by band and clone numbers.

The most similar based on BLAST hits were the g23 clones

from marine, paddy fields and T4 cyanophages (from 70%

and higher). The highest identity was observed between

S0508/2-4 clone and CS26 marine clone (89%) (Fig. 2). Two

highly conserved amino acid motifs of g23 marine sequences

uncovered by Filee et al. (2005) were also found in all g23

sequences from Lake Baikal (the first 11 and last 40 amino

acid residues shown in Fig. 2). For the phylogenetic analysis,

different T4-type phages and g23 clones of marine and

terrestrial environments from referred marine and paddy

T4 subgroups (Filee et al., 2005; Wang et al., 2009a, b)

including the closest relative clones were used.

Phylogenetic analysis of g23 clones

The Bayesian tree obtained in our study is shown in Fig. 3.

Our results revealed that neither of the Lake Baikal se-

quences was grouped into T-evens, PseudoT-evens or Schi-

zoT-evens. The majority of g23 clones from Lake Baikal

formed nine deep-branching clusters (B1–B9) with reliable

support (79–100%). Two Lake Baikal clusters (B3 and B4)

belonged to the ExoT-evens group of marine cyanophages.

Clusters B1, B5 and four separate Lake Baikal clones were

0 100 200 300 400 500

0

10

20

30

40

50

0 500 1000 1500 2000 2500

Abundance (thousand cells mL–1)

Dep

th (

m)

Northern Baikal Southern Baikal

Cyanobacteria

Heterotrophicbacteria

Fig. 1. Abundances of picoplanktonic

cyanobacteria and heterotrophic bacteria in

Northern and Southern Baikal in early June, 2008.

Bacteria and picocyanobacteria were counted

using an epifluorescence microscope: bacteria,

under UV excitation; cyanobacteria, under green

excitation.


124 T.V. Butina et al.

10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| T4 (T-evens) NSPTGQVFALRAVYGKDP--------VAAGAKEAFHPMYGPDAMFSGQGAAKK--------FPALAASTQTTVGDIYTHFFQETGTVYLQASVQVNT1 (SchizoT) TGPTSQVFTLRSIYGKDP--------L--NGVEAFHPTRQADASFSGQAGTGTAI----ADLPVSGAATDGTPYKAVVASVGGDADTVRYFLALG44RR (PseudoT) TGPTGQVFALRSVYGKDP--------LAAGAKEAFHPMYAPDAWHSSLATKGATTTTDGTPFAKLTAGQAIAEGDIVGHFFYESGTAFLQNVSGAS-PM2 (ExoT) SGPTGLIFAMRSRYENQ------------AGEEALFNEPDTGFTGGYDASQGD--------------------------------YAVRTGAGVGN0508/4-16 SGPTGLIFAMRARYENQ------------GGAEALYYEPDAGFSGGSDASQGA--------------------------------YGVRNAAGSGN0508/4-17 TGPTGLIFAMRARYENQ------------GGAEAMYYEPDAGFSGGSDASVGA--------------------------------YGVRNAAGSGP-SSM2 (ExoT) NGPTGLIFAMRSRYKTQ------------SGTEALFNEADTAFSGQPDGLDDT--------------------------SGFTATGANNVGLGTTN0508/5-21 NGPTGLIFAMRSRYSNQ------------TGTEAFFNESNSAFSGQNAALGLT--------------------------DGFSG---AAVGMGSTN0508/5-23 NGPTGLIFAMRSRYSNQ------------TGTEASFNESNSAFSGQNEALGLT--------------------------DGFSG---ATVGMGSTBC15 (P VII) SGPTGLIFCMKSRYSTQ------------AGTEALFNEADTDFTGTNGTGAHS------------------------------------------N0508/1-1 TQPTGLIFAMKSKYTSQ------------AGTEALFNEANTAFSGKASP-AHA------------------------------------------N0508/1-5 TQPTGLIFAMKSRYANQ------------TGTEALFDEAVTDHAGAASP-AHA------------------------------------------AL3 (M IV) TGPTGLVFAMRSRYTSQ------------TGTEALFNEANASFSGSAQGNTASI--------------------------------FVRDTTAAAS0508/2-1 TGPVGLIFALRSRYESQ------------TGSEALFNEANTTFTPSAAGNTASR--------------------------------FVVANTSNRS0508/2-6 TGPTGLIFAMRSRYTSQ------------SGTEALFNEANTTFGSSAKGNTASQ--------------------------------FVVANTSNRCS26 (M IV) TGPTGLIFAMR-SSLLS-----------QDGAEALVDESLPGAAGRSNQNHA------------------------------------GTIGGGDS0508/2-4 TGPTGLIFAMR-SSLVS-----------SDGAEALVDESMPGAAGRSNLNAA------------------------------------GTIGGGD3739 (M III) TGPSGLIFALRPQYSTQ------------GGTEALYNEADTDFSGSAA-GNT--------------------------------ASILVANGSAGS0508/2-5 TGLTGLIFAMRFALHQSD-----------WGTEALFNEANTFVHRFQLLVNT--------------------------------ASIQAANASSGFW-CM-32 XMPTGLIFAMKSKYGSG-------ATGPLTSTEALFNEADTDFSGTGTHQADT------------------------------------------S0508/1-5 NMPTGLIFAMKSKYGSG-------ATGPLTSTEALFNEADTDFAGTGTHTANT------------------------------------------KuCf-Jun12-17 TGPVGQIHTLRVRYANT-------AAGVTAGTEALGPFDIAKAYSGNEVQADP------------------------------------------S0508/1-1 IGPVGQIHTLRVRYAQSLTDSSAAATSVTAGSEALSPFTIAQAYSIVPQGTDT------------------------------------------N0508/2-6 TGPTGLIFAMRSTYITQ------------AGTEAFYNEANTGFGGVAGQQTAL------------------------------------------S0508/1-4 TGPTGPIFAMRSIYITQ------------AGTEAFYNEANTGFGGVAGQ-TAL------------------------------------------N0508/3-11 TGPTGLIFAMRTKYSGQ------------NGAEAFFNEANTGFSGLGTSGNAA------------------------------------------N0508/3-13 TGPTGLIFAMRTKYSSQ------------SGTEAFFNEANTGFAGANGGGAQVAL----------------------------------------N0508/3-14 TGPTGLIFAMRPVYATATAR------ANAPGGEALFTEANTGHSGNASNGN--------------------------------------------S0508/1-10 TGPTGLIFAMRTRFDTQ---------AN-NATEAFYNEALTTFSGTTSDMANV------------------------------------------S0508/1-7 TGPTGLIFAMRSRYTGQ---------AN-TNDEAFFNEANTTHAGDYPNDTQV-----------------------------------------AN0508/2-8 TGPTGLIFAMRSRYKTQ------------GGTEALFDVANTAFPSTAQSQTGS------------------------------------------

10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| T4 (T-evens) TIDAGATDAAK-LDAEIKKQMEAGALVEIAEGMATSIAE-LQEGFNGSTDNPWNEMGFRIDKQVIEAKSRQLKAAYSIELAQDLRAVHGMDADAENT1 (SchizoT) AVTVAV-E-GEMTVDEYTTAISSGLAVEIDAGMATSQAE-LQEAFNGSSNNEWNEMSFRIDKQVVEAKSRQLKAQYSIELAQDLRAVHGLDADAE44RR (PseudoT) SVTVGTNETGEALDKLINAAIGEGKLAEIAEGMATSIAE-LRQGFNGSNDNPWNEMSFRIDKQTVEAKSRQLKAQYSIELAQDLRAVHGMDADSES-PM2 (ExoT) GDSEGNNPALLNDAAPGTYEVGSK--------MPREDLERMGEAN-----RLFREMSFSIEKTSVTAQSRALKAEYTLELAQDLKAIHGLDAEQEN0508/4-16 GDAEGNNPAVLNDSSPGTYERGTT-------SLSRENSEILGEAG-----SLFREMSFSIEKTSVTAKTRALKADYTLELAQDLKAIHGLDAEQEN0508/4-17 GDAEGNNPAVLNDSSPGTYERGTT-------SLSRENSEILGEAG-----SLFREMSFSIEKTSVTAKTRALRADYTLELAQDLKAIHGLDAEQEP-SSM2 (ExoT) -AQQGSNPGLLNSTAAQTNAT---D-YNVGQGMRTDSAEDLGDGTG----DQFNQMAFSIEKVTVTAKSRALKAEYSLELAQDLKAIHGLNAEAEN0508/5-21 -AQGGTNPSILDGSNQANNAGTGSDQYNVGQGMRTDDSEDLGTSGGG----AFNEMAFSIEKVTVTAKSRALKAEYSLELAQDLKAIHGLNAEAEN0508/5-23 -AQGGTNPSILDGSNQANNAGTGSDQYNVGQGMRTDDSEDLGTSGGG----AFNEMAFSIEKVTVTAKSRALKAEYSLELAQDLKAIHGLNAEAEBC15 (P VII) ----GSNPVNGT----YTTG----------AGIATETAELED---------AFPEMAFSIEKTSVTAKTRLLKAEYTIELAQDLKAVHGLDAESEN0508/1-1 ----GSNPATGT----YTTG----------EAMTTAAAEDL----------TFAQMAFSIEKTTVTAQTRALKAEYTVELAQDLKAVHGLDAEAEN0508/1-5 ----GANPYAGT----YTTG----------VGQGTAAAESGD---------RFNEMAFSIEKTSVVAKSRQLKAEYSIELAQDLKSVHGLDAEGEAL3 (M IV) TGQTGADPSSLGNNANYSV--ST--------GMTTARAEKLGDSATG---NAFREMAFSIEKTAVTAVSRALKAEYTMELAQDLKAVHGLDAETES0508/2-1 SQGDETDPTGRVASGAAGYTVST--------GMTTARAEALGDGTT----NGFASMAFSVEKVAVTAVSRALKAEYTMELAQDLKAIHGLDAETES0508/2-6 -VQTGSDPTERVKAGAAGYNLST--------GMTTARAEALGDGST----NGFQEMAFSIEKVAVTAVSRALKAEYTMELAQDLKAIHGLDAEQECS26 (M IV) VGATETNPAVLNDSP-VGTYTS-------ATGMTRAQGEALGDSGAN----AFGEMAFSIEKSTVTAVSRALKAEYTMEPAQDLKAIHGLDAETES0508/2-4 VGATETNPAVLNDSPSAGTYTS-------AAGMTTAQGEALGDSGTN----AFAQMAFSIEKSTVTAVSRALKAEYTMELAQDLKAIHGLDAETE3739 (M III) TGHTGTDPNARASGSGYTVGQ----------GMSTASSEGLGKDTG----NEFNQMAFSIEKVTVTAVSRALKAEYPMELAQDLRAIHGLDAETES0508/2-5 SGHTGTDPTARASGSGYTVET----------GMTTAAAEQLGFGAN----HQFQEMAFSIEKIAVTAVSRALKAEYTMELAQDLKAVHGLDAETEFW-CM-32 ---------FQSNGNLGTFGT----------GMTTAAGEGF----------SPLNMGFSIEKVTVEAKTRALKAEYSLELAQDLKTVHGLDAESES0508/1-5 ---------FSSADALNTFGT----------GLTTASGEGF----------SPLNMGFSIEKVTVTAQTRALKAEYSLELAQDLKAMHNLDAESEKuCf-Jun12-17 ------------------------------AAASTARLEGV----------PGNKLSIQILKETVEAKTRKLSARWTFEAAQDANAIHGIDIEAXS0508/1-1 -----ATAY---------------------TGGNTAVMEGTG----------GKQISIQILKQAVEAKTRKLQARWTFESAQDAQAMHGIDVEAEN0508/2-6 AVGQAADANGTFVANAAAIA-----------GLGTTAAEDL----------TFKEMAFSIEKVTVTAKTRALKAEYSIELAQDLKAVHGLDAETES0508/1-4 AVGQAADANGTFVANAAA-A-----------GLGTTAAEDL----------TFKEMAFSIEKVTVTAKTRALKAEYSIELAQDLKAVHDLDAETEN0508/3-11 -FAEGSSPTEVFTSNAAPVG-----------AMTTARAEALGTASEAA--NACQEMAFSIEKVTVTAKTRALKAEYSMELAQDLKAVHGLDAETEN0508/3-13 -AAGGTLPTAMFTSNAAPIG-----------GMTTGSAEALGDGGVG---NTFQEMAFSIEKVTVTAKTRALKAEYSLELAQDLKAVHGLDAETEN0508/3-14 -ASTLSVNPANTNVFGLDNTG---------PGFSTSFGEAA----------NLAQMGFQIDRVAVTANTRGLQASYTLGLAQDLKAIHGLDAETES0508/1-10 -ANTLTNAGANIAAFTTANTG---------TGDSTANFET----------KNMANMAFTIERVSVTAKTRGLQASYTMELAQDLKAIHGLDAETES0508/1-7 LGVAGTANTTNTFVQNATGGS----------GLTTSQAESLGSS------VAMKEMAFAIEKVTVAALTRALKAEYTMELAQDLKAAHGLDAETEN0508/2-8 ------SPADLSAGTEYTRGT----------GFTTAQAEALGDGSG----QGFQEMAFSIEKIAVTARSRALKAEYTMEFAQDLKAVHGLDAEQE

Fig. 2. Amino acid alignments of a representative subset of g23 gene sequences from Lake Baikal with g23 sequences of some cultured T4-type phages

and with g23 clones from marine and paddy field environments whose identities with Baikalian sequences were the highest (4 70%). The groups of

sequences from Lake Baikal and previously described subgroups of T4 phage are separated by straight lines shown in the first column. The names of

reported subgroups are indicated in parentheses (M, marine group, P, paddy field group). The well-conserved amino acid residues within represented

subgroups are indicated by a color code. A black background indicates amino acid residues that are conserved for 4 90% of all known g23 sequences.

The well-conserved amino acid residues in the g23 sequences of uncultured T4 phages are marked by a grey background.



Paddy group IX

- Paddy group VIII

Paddy group III

Paddy group II

- Paddy group VIIIPaddy group V

Paddy group IV

Paddy group VI I

ExoT-evens

Marine group I

Paddy group I

Marine group IV

Marine group IV

Marine group III

Paddy group VI

Marine group II

Marine group V- Marine group I

SchizoT-evens

PseudoT-evens

T-evens

Fig. 3. Bayesian phylogenetic tree based on partial g23 amino acid sequences of T4-like bacteriophages. Bayesian posterior probabilities of branching

are given as percentages. Branches with Bayesian posterior probabilities o 70 are collapsed. The identified g23 gene fragments found in this study are

in bold. The braces delineate the clusters of Baikalian g23 clones. The g23 sequences from marine and paddy field environments are given with

accession numbers in parentheses. The reported subgroups of T4 phages are marked by triangle brackets.



grouped with marine or paddy soil T4 subgroups (Marine

groups III, IV; Paddy groups III, VI, VII). The rest of the

Baikalian clusters (B2, B6–B9) were separate and the acces-

sory of these clusters to any referred T4-type phage sub-

groups has not been determined.

The most unique sequence found in our study was a clone

S0508/1-1. It was clustered with two clones from Japanese

paddy fields (KuCf-Jun12-17 and Ch-Cf-Sep22-11) obtained

by Wang et al. (2009a). Apparently, this sequence had

originated from an ancestor other than Lake Baikal phage

sequences (Fig. 3).

Discussion

In this study, we analyzed the diversity of the T4-type

bacteriophages in Northern and Southern Baikal using a

PCR strategy based on the partial sequencing of the g23

gene. We also compared these data with the composition

and abundance of autotrophic picocyanobacteria and het-

erotrophic bacteria that are the most probable hosts for T4-

like phages. We found that the populations of both bacterial

and autotrophic plankton in Northern and Southern Baikal

basins were significantly different. Northern Baikal was

characterized by a high level of picocyanobacterial develop-

ment. In contrast to this basin, the predominant numbers of

heterotrophic bacteria were registered in Southern Baikal.

The differences in phytoplankton biomass were also re-

corded, and so the abundance of phytoplankton in Southern

Baikal was much higher (Sakirko et al., 2009). Our study

showed differences between the sequences of the T4 g23 gene

obtained from Northern and Southern Baikal. Five Lake

Baikal clusters (B1–B4, B7) were mainly composed of clones

from the Northern basin while B5, B6 and B8 generally

included clones from the Southern basin. Recently, Sandaa

& Larsen (2006) demonstrated pronounced seasonal dy-

namics of the viral populations in Norwegian coastal waters

and showed its correlation with the changes in the abun-

dance of possible hosts. Following from this, we supposed

that the biodiversity and quantity of bacterial plankton,

autotrophic plankton and phytoplankton in two basins of

Lake Baikal have determined a structure of viral commu-

nities in general and T4 bacteriophages in particular.

Our finding clearly showed that g23 genes from Lake

Baikal were different from g23 genes of T4-like bacterio-

phages and from g23 clones obtained from other environ-

ments (the highest identity was only 89%). Phylogenetic

analyses showed that g23 fragments from Lake Baikal, except

for the single sequence, were most closely related to the

ExoT-evens subgroup of marine T4 cyanophages and to

previously described subgroups of uncultured T4 phages

from marine and rice field environments. The ExoT evens

subgroup, all marine and paddy field subgroups, plus all

Baikalian clusters of g23 clones formed one large clade

reliably distant from the T-, PseudoT- and SchizoT-evens

subgroups of T4 bacteriophages (Fig. 3).

Two Lake Baikal clusters (B3, B4) composed of sequences

from the Northern basin were grouped with marine T4

cyanophages of the ExoT-evens subgroup. Cluster B4 was

more closely related to the g23 sequences of T4-type cyano-

phages S-PM2 and S-PWM3 isolated on Synechococcus sp.

Filee et al. (2005) found g23 sequences related to the ExoT-

even subgroup only in surface marine samples, in which

Synechococcus sp. are abundant. Short & Suttle (2005) ana-

lyzed the cyanophage diversity based on g20 gene sequences.

They concluded that half of the marine phage sequences

belonged to the group of T4-type cyanophages that infect

Synechococcus sp. In our case, water samples for T4-virus

examination were collected from the depth of 5–10 m, where

the abundance of picocyanobacteria is the highest (Belykh &

Sorokovikova, 2003; Belykh et al., 2007). Our sequences from

cluster B3 as well as from cluster B4 were also phylogenetically

close to cyanophages P-SSM2 and P-SSM4 isolated from

cyanobacterial Prochlorococcus strains. Cyanobacteria of this

genus are the dominant prokaryotic components of picophy-

toplankton in the ocean, but these cyanobacteria have never

been found in fresh waters. The sequences related to isolates

P-SSM2 and P-SSM4 were also obtained by Jia et al. (2007) in

a study of T4-phage diversity in Japanese rice fields, although

members of the genus Prochlorococcus have not been detected

in those rice fields. The sequences belonging to ExoT-evens

were found in the Northern Baikal sample, where picoplank-

tonic cyanobacteria were abundant. Therefore, it is most likely

that the sequences from clusters B3 and B4 belong to T4

cyanophages whose hosts belong to the genus Synechococcus.

A major portion of Baikalian sequences was closely related

(with 94–100% posterior probabilities) to uncultured T4

phages from marine and rice field environments (Fig. 3). The

cluster B1 composed by sequences from Northern Baikal was

close to the Paddy VII subgroup. Several g23 gene fragments

from the Southern basin clustered with Paddy groups III, VI

and Marine groups III and IV. The similarity of g23 sequences

from Lake Baikal and those from paddy soils and marine

environments suggests that T4 phages can survive and propa-

gate in diverse environments. Sano et al. (2004) showed that

viruses, in particular phages, are able to move between

different biomes (e.g. soil and seawater). Our finding con-

forms to their data and suggests that a broad host range of T4

phages probably provides them with a better possibility for

survival and moving between different ecosystems.

T4-like viruses, belonging to T-, PseudoT- and Schizo

T-evens subgroups, attack members of different genera of

Enterobacteriaceae family and genera Acinetobacter, Aeromo-

nas, Burkholderia, Pseudomonas and Vibrio of other families

(http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_index.htm).

The presence of potentially pathogenic bacteria of the listed

groups in Lake Baikal was shown previously using cultivating




methods (Drucker & Panasyuk, 2006) and by analysis of 16S

rRNA gene fragments (Bel’kova et al., 1996, 2003; Soutourina

et al., 2001). Enterobacteria and bacteria of the genus Pseudo-

monas were also detected in the samples used in our study (in

the Southern and Northern lake basins, respectively) (Parfe-

nova et al., 2009). However, we failed to detect structures

closely related to known T4 bacteriophages. T4-phage num-

bers, even if they were present in Lake Baikal water, were

probably extremely low due to the small concentrations of

their respective hosts. For example, enterobacteria were de-

tected at a concentration of 30 CFU mL�1 in a sample collected

in Southern Baikal (Parfenova et al., 2009).

As was noted above, one g23 clone from Lake Baikal

(S0508/1-1) was extremely different from other Baikalian

sequences and joined to a small group with two g23 sequences

from Japanese paddy soils. Two latter clones were obtained

from distant paddy fields in Northern and Southern Japan. In

spite of the geographical disconnected location, the Baikalian

clone and those from paddy fields had similar amino acid

changes in highly conserved motifs and similar sequences in

the hypervariable regions (Fig. 2). Phylogenetic analysis

showed their common origin with 100% posterior probability.

This group was quite distinct from other subgroups of T4

bacteriophages. Therefore, it is impossible to arrive at any

conclusion on the range of their hosts.

In conclusion, the present study demonstrated that g23

genes were highly diverse, suggesting a conceivable role of

T4 phages in the evolution of their hosts and in Lake Baikal

productivity. In general, the g23 gene sequences from Lake

Baikal, except for the single clone from Southern Baikal,

were closely related to marine T4 cyanophages and to

previously described subgroups of uncultured T4 phages

from marine and rice field environments. The composition

of T4 phages in Northern and Southern Baikal as well as the

populations of bacteria, phytoplankton and autotrophic

picoplankton differed. Further identification, isolation and

molecular characterization of T4-type bacteriophages from

various environments will allow us to obtain more accurate

information about the phylogenetic relations within the

genus ‘T4-like viruses’ and about the range of their hosts.

Acknowledgements

We are grateful to Dr Tatyana Sherbakova and Prof. Mikhail

Grachev (Limnology Institute, SB RAS), who helped in data

processing and execution of the paper. This work was

supported by the Russian Foundation for Basic Research,

project nos 10-04-01613 and 09-04-90420.

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Phylogenetic diversity of T4-like bacteriophages in Lake Baikal, East Siberia