Genes encoding synthesis of phenazine1-carboxylic acid in Pseudomonas batumici Текст научной статьи по специальности «Биологические науки»

UDC 579.841.1+577.18 https://doi.org/10.15407/biotech9.05.045

GENES ENCODING SYNTHESIS OF PHENAZINE-1-CARBOXYLIC ACID IN Pseudomonas batumici

V. V. Klochko1 1Zabolotny Institute of Microbiology and Virology

S. D. Zagorodnya1 of the National Academy of Sciences of Ukraine, Kyiv

O. N. Reva2 2Centre for Bioinformatics and Computational Biology,

University of Pretoria, South Africa

E-mail: [email protected]

Received 28.06.2016

The aim of this research was to elucidate the role of fenesin-1-carboxylic acid of Pseudomonas batumici and diversity of the genes encoding its synthesis in bacteria of the genus Pseudomonas. Phenazine-1-carboxylic acid in the concentration of 10 pg/ml stimulated the biofilm formation by batumin-producing strain. The presence of the corresponding gene in the genome of P. batumici was not successfully confirmed by PCR amplification with a set of primers designed for Pseudomonas. The complete genome sequencing of P. batumici has revealed a homologous gene that could encode synthesis of this compound. Comparative study of sequenced Pseudomonas genomes showed presence of at least two genetically diverse groups of phenazine coding orthologous genes. These genes could have distributed among rhizobacteria by the horizontal gene transfer.

Key words: Pseudomonas batumici, phenazine-1-carboxylic acid, biofilm formation.

The ability to synthesize the heterocyclic phenazine acids is a distinguishing feature of the metabolism of several species of the genus Pseudomonas. Data obtained in the recent years show that the importance of the phenazine for the producers is not limited by its antimicrobial activity. It acts also as an important regulator of gene expression and biofilm formation, participates in the redox reaction and induction of the systemic resistance of plants against pathogens and excites other important effects [1, 2]. Such versatility of the activities of these pigments in bacteria is consistent with an evolutionary hypothesis that the biosynthesis of energetically expensive metabolites in microbial cells is justified only if they are multifunctional [3].

Pseudomonas batumici, the producer of a medically important polyketide antibiotic batumin, synthesizes also the phenazine-1-carboxylic acid coloring the colonies and endowing the bacteria with a broad antibiotic activity,. Particularly, this compound inhibited the growth of phytopathogenic

fungi, agrobacteria, corynebacteria and phytopathogenic Pseudomonas at concentrations of 50-400 pg/ml indicating its contribution to the competitiveness of P. batumici in the rhizosphere. The ability to synthesize phenazine-1-carboxylic acid (PICA) is common for several Pseudomonas species. Synthesis of this bright yellow pigment was reported for P. aeruginosa, P. putida, P. fluorescens and P. chlororaphis. Genetic control of the biosynthesis of PICA in aforementioned species has been studied quite well. Nucleotide sequences of a fragment of phzABCDEFG, phzI and phzR operons have been determined. A schematic diagram of the biosynthetic pathway of synthesis of this antibiotic was suggested [4].

Synthesis of PICA was detected in the strains of P. batumici, which produced the polyketide antistaphylococcal antibiotic batumin [5]. Chloroform extracts from the culture liquid comprised the batumin and, depending on the composition of the medium, varying amounts of the phenazine pigment (from trace concentrations up to 250 mg/l).

Genetic encoding of phenazine biosynthesis by P. batumici had not been studied previously. The role of this pigment in the biology of P. batumici also remained unknown.

Aimes of the present work were to study the role of phenazine-1-carboxylic acid in P. batumici and to estimate the diversity of the genes encoding PICA synthesis in bacteria of the genus Pseudomonas.

Materials and Methods

The study object was the batumin-producing type strain P. batumici UCM B-321 from the Ukrainian collection of microorganisms (Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine).

Antagonistic activity of P. batumici against phytopathogenic fungi and bacteria was studied on the potato agar solid medium (per 1 liter of distilled water: 500 g potato broth; 5 g NaCl; 15 g agar); and on Gauze medium (per 1 liter of distilled water: 30 ml Hottinger bouillon; 5 g peptone; 10 g glucose; 5 g NaCl; 30 g agar-agar) against human opportunistic pathogens, which are listed below.

Phytopathogenic bacteria: Pseudomonas syringae pv. syringae, P. fluorescens, Pectobacterium carotovorum, Xanthomonas campestris, Clavibacter michiganensis, Agrobacterium tumefaciens, Erwinia aroidea;

Phytopathogenic fungi: Mucor plumbeus, Fusarium avenaceum, Drechslera graminea, Rhizopus arrhisus, Botrytis cynerea.

Opportunistic bacterial and fungal pathogens: Staphylococcus aureus UCM B-918, Escherichia coli UCM B-926, Pseudomonas aeruginosa UCM B-900, Bacillus subtilis UCM B-901, Candida albicans UCM Y-2681.

The strains of phytopathogenic species mentioned above were obtained from the collection of the Department of phytopathogenic bacteria and the Department of physiology and taxonomy of micromycetes of Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine, and the reference strains of the pathogenic microorganisms were obtained from the Ukrainian collection of microorganisms.

Phenazine-1-carboxylic acid was synthesized by P. batumici UCM B-321 in Erlenmeyer flasks with 100 ml of the King B medium on a rotary shaker, at 25 °C for 72 hours. The presence of P1CA in the culture liquid was assessed using the liquid chromatography-mass spectrometry (LC/MS), Agilent 1200 liquid chromatograph (Agilent

Technologies): XDB-C18 column (Zorbax 150 mmx4.6 pmx5 pm) in ACN:H2O mobile phase (55:45) with 0.5 mmol ammonium acetate at 30 °C with the flow rate 1 ml/min, injection volume of 5 ml under isocratic mode.

P1CA was extracted from the supernatant of the centrifuged cell-less culture liquid by acidifying it with 0.1 N HCl to pH 2-3. The yellow-green precipitate was separated by centrifugation for 10 min at 12.000 g and dissolved in chloroform, which was distilled in a rotary vacuum evaporator at 40 °C. Then the precipitated crystals were dissolved in benzene and extracted with 0.1 N K2HPO4 solution (pH 9.0). The resulting extract was again acidified and extracted with benzene. The resulted distilled crystals were re-crystallized from methanol.

Antimicrobial activity of P1CA was studied by the method of serial dilutions on meat peptone agar for bacteria and on beer wort for micromycetes.

Search for the phenazine biosynthesis operon was performed in the complete genome sequence of the strain Pseudomonas batumici UCM B-321 (RefSeqNZ_JXDG00000000.1). Genome annotation was processed by RASTServer (http://rast.nmpdr.org/) [6]. Homologous operons in the genomes of other microorganisms were searched for using the program AntiSMASH (http://antismash. secondarymetabolites.org/). Sequence alignment and inference of phylogenetic relationship between the operons of biosynthesis of phenazine-like compounds were carried out by Mauve 2.3.1 [7].

Transmission electron microscopy was performed using JEM-1400 microscope (Jeol, Japan) with an acceleration voltage of 80 kV. Suspension of P. batumici UCM B-321 cells was applied to a copper mesh (400 mesh). For contrasting, the samples were stained by 2% uranyl acetate solution. The cell sizes were estimated using JEM-1400 software.

Biofilm formation was studied according to O'Toole [8] by growing P. batumici UCM B-321 at 25 °C for 48 hours in 96-well plates on LB medium (per 1 liter of distilled water: 15 g/l peptone, 10 g/l yeast extract, 5 g/l NaCl). Initially, aliquots of 1 and 10 pg/ml P1CA were added to 0.25 ml per well of the cell suspensions of the culture medium (5107 cell/ ml). Biofilms were stained by 0.1% solution of gentian violet 2-F (bioMerieux, France) added in the amount of 0.1 ml per well. Then the microplate photometer Multiskan FC (ThermoFisherScientific, USA) was used to record the opacity at 540 nm.

Significance of the average values was controlled by the P < 0.05.

Results and Discussion

Yellow solid crystals were extracted from the culture liquid of P. batumici UCM B-321. , Compound's molecular weight determined by LC/MS-analysis (Fig. 1) was 224 (output time 6.9 min). Peaks of the absorption spectrum fell to 250 and 364 nm. The melting temperature was 238 °C. These data allowed to identify the extract as a phenazine-1-carboxylic acid.

A known precursor of phenazines synthesized by Pseudomonas is phenazine-1,6-dicarboxylate, which is formed by condensation of two molecules of chorismate. Transformation of 3-oxyanthranilate to phenazine-1,6-dicarboxylate is controlled by a gene with known nucleotide sequence, which is responsible also for the synthesis of isochorismatase [2]. Diagnostic primers developed previously for determination of the phenazine operon in P. fluorescens and P. chlororaphis were used in this study. PCR amplification with these primers results in synthesis of 620-kb fragments, but the amplification failed with the genomic DNA from P. batumici B-321 indicating specificity of the corresponding phenazine genes in this species [9].

The purpose of the further work was to identify an operon involved in the synthesis of the phenazine-like compound by P. batumici to carry out a comparative analysis with the known phenazine biosynthesis operons of Pseudomonas.

Annotation of the complete genome sequence of P. batumici revealed a secondary metabolite biosynthesis operon that showed a significant sequence similarity to the known phenazine operons of other Pseudomonas. Homology of these operons was confirmed also by ordering of the respective genes on the chromosomes (Fig. 2).

Other homologous phenazine synthesis operons were discovered in NCBI database by MegaBLAST. In total, ten phenazine synthesis operons including that of P. batumici В-321 with an average length of 8000 n.p. were aligned against each other By the program Mauve, which also inferred a phylogenetic tree shown in Fig. 3. Mauve algorithm accounts for both: mutations and genetic rearrangements in long DNA sequences.

It was discovered that the phenazine synthesis operons of Pseudomonas bacteria fell into two distinct groups. The sequence of P. batumici phenazine operon is more similar to the sequences of the homologous operons in P. aeruginosa, although the genome of the type strain P. aeruginosa PAO1 comprised two

Fig. 1. LC/MS-analysis and absorption spectra of phenazine-1-carboxylic acid isolated

from P. batumici В-321

Query sequence

4400Ü

AXQF01000004_cl: Pseudomonas aeruginosa BWHPSA023 adgel_-supercontl.l.C4, wh... (18% of genes show similarity)

-Hllllll 3HX>Oft3Cti>»iX=K*ttt(=>fr--

AXP001Q00020_c2: Pseudomonas aeruginosa BL12 adgfc-supercontl.3.C20, whole ... (18% of genes show similarity) AKJU0100005 5_cl: Pseudomonas sp, GM17 PMI20 contig 58.58, whole genome shot... (16% of genes show similarity)

^awwQwxHDtxocm—

AHOT01000028_c2: Pseudomonas chlororaphis 06 Contig0023, whole genome shotg... (16% of genes show similarity) AHHJ0100001 l_c2: Pseudomonas chlororaphis subsp. aureofaciens 30-84 ContigO... (16% of genes show similarity)

wiow M--[>i><KKKi«ODOira<=w<3

CP009290_cl: Pseudomonas chlororaphis subsp. aurantiaca strain JD37, comple... (16% of genes show similarity)

CP008696_cl2: Pseudomonas chlororaphis strain PA23, complete genome, (16% of genes show similarity)

CHOWWCKKJ 4M <ia KM t^ticwoaimraaaMCK;

ja-

L48616_cl: Pseudomonas fluorescens autoinducer synthase (phjl) gene, positi... (16% of genes show similarity)

■«•04CKKIIX]

AYÜDO 100000l_c8: Pseudomonas chlororaphis subsp. aurantiaca PB-St2 scaffol... (16% of genes show similarity)

Fig. 2. Phenazine biosynthesis operons similar to the operon identified in the P. batumici B-321 genome

4

1. CP006985.1 [3715166-3721387] Pseudomonas aeruginosa LESIike4 2 CP006985.1 [772602-778726] Pseudomonas aeruginosa LESIike4

3. CP004054.2 [792581-798705] Pseudomonas aeruginosa PA1

4. CP004054.2 [3542811-3548469] Pseudomonas aeruginosa PA1 - 5. Pseudomonas batumici UCMB-321

6. AE004091.2 [4713916-4720028] Pseudomonas aeruginosa PA01 AE004091.2 [2071277-2076923] Pseudomonas aeruginosa PA01 — 8. Pseudomonas fluorescers strain S2P5 phz biosynttietic gene locus AY960782 1 9. Pseudomonas chlororaphis subsp. aurantiaca strain StFRB508 phz gene cluster AB794886 1

-T

10. Pseudomonas chlororaphis subsp. chlororaphis strain G05 phz biosynthesis gene cluster KU517144.1

200

Fig. 3. Phylogenetic tree based on the nucleotide sequences of phenazine biosynthesis operons produced by Mauve 2.3.1 The branch labels inform about the organism, genome registration numbers and operon coordinates in the genome

copies of alternative phenazine operon similar to those in P. fluorescens and P. chlororaphis (Fig. 3). This mosaic distribution of the operons suggests a horizontal exchange of these genes between microorganisms.

The failure with the diagnostic amplification using the standard primers targeting the phenazine biosynthesis operons [9] could be explained by a variability of the target sequences in the two groups of these operons in Pseudomonas as shown in the alignment in Fig. 4.

It was found that the recommended primers were suitable to amplify only from the phenazine operons of P. fluorescens, P. chloraphis and the type strain P. aeruginosa PAO1. DNA segments in the corresponding genes of P. batumici and other P. aeruginosa strains varied in these regions by insertions and deletions of the targeted nucleotides that made amplification impossible. Analysis of the aligned sequences revealed areas alternative loci of the phzE gene (Fig. 5), which would better serve as targets for universal primers.

11

t1

[1

11

■ 11

i '

! 1

1 1

1 '

1 I 1

1 1

1 1 1

I '

Dir 3300 3310 3320 3330 3340 3350 3360 3370

1 cccaggcacaggctcagcacctggtggctcaggcacacggcgaggaacgg ccggcgctcgctgagca

2 cccaggcacaggctcagcacconsgt ggctcaggc acacg gc6ag gmcög ccggcgctcgctgfigcs

3 £ccagscscftggctcîigcaccïggtggctcaggcacacg3csaggaacsg cc gg c gc tc gct ga gca

4 ■.'i'Ci.GGCACSGGCTCAGCACCl'GGTGGCTCAGGCACSCGGCGAGGAACGG CCGGCGCTCGCTGAGCA

5 cccaggcagcggctgagcacctggtgactcaggcacacggcgagaaacgg ccgttgctcgtccagca

• • I. • • <I.>•>I< >• • I

3380 3390 3

GCGAACGGATGGC GCGAACGGATGGC GCGAACGGATGGC GCGAACGGATGGC GCGAACGGATGGC

6 ICCCGAGCGCAACCTT CCCCCTGGAGACCGTGGC ATGAGCGGCATTCCCGAAATCSCCGCCTATCCCTTGCCGACCGCCCAGCAACTGCCGGC

7 CCCGAGCGCAACCTT CCCCCTGGAGACCGTGGC ATGAGCGGCATTCCCGAAATCACCGCCTATCCCTTGCCGACCGCCCAGCAACTGCCGGC

8 CCCATCTCASCCCTTGCCTCCTTTTGSCTGGSGTTTSTCGACATGACCGGCATTCCaTCGSTCGTCCCCTaCGTCTTGCCTACTTCGCGCGSCCTGCCCST SCCCACCTCAACCCTTGCCTCCTTTTGACTGGAGT'TTGTCGACATGACCGGCATTCCATCGAl'CGTCCCTTACGTC'ITGCCTACÜTCGCGCGACCTGCCCGI! 10!"

Rev

i

2 3 A

5

6 7

e

9

gctcctccctcttttgtttccctt^ y'ccctt^cgctctgcct acttctcgcgacctgcccgc

3970 cagcgtgcgcc cagcgtgcgcc cagcgtgcgcc cagcgtgcgcc cagcgtgcgct

3980 3990 4000 4010 4020 4030 4040 4050 4060 ~

CGCCCTGGCCGTCACCGCCGATCAGCGCCGCCACCCCGCTGTAGTAGCC GCGACCCTGCGGCTCGTAGCGGCGGATCACCCGG CGCCCTGGCCGTCACCGCCGATCAGCGCCGCCACCCCGCTGTAGTAGCC GCGACCCTGCGGCTCGTAGCGGCGGATCACCCGG CG CCC TG Gc C GT CA CCG CC GA t CA GcGcc GC CAC CC CG c TG TAGTA GC C G CG a CC ct GCG Gc t CG ta GCG Gc GG a TC AC CCG G CG CC C TG GC C GT CA CCG CC GA T CA GCGCC GC CAC CC CGC TG TAGTA GC C GCGACCCTGCGGCT CG TAGC GGC GGA TCAC CCGG CGCCTTTTGCG-TCGCTGCCGATCAAGGCCGCGATGCCGCTGTASTAGGC ACGGCCCTTGGGCTCGTGACGGCGGATCACCCGG

cgccgccagccgctgcgcgatggtggtcaccaccgacgagg cg c cg cc a gc cg ctg cg cg a tg gtggt ca cc a cc ga c ga gg cgccgccagccgttgcgccatggtcätcaccaccgacgagg cgccgccagccgttgcgccatggtcatcaccsccgacgagg

10 ic g c cg cc a gc cg t tg cg cc a tg gt c at c a c ~'wwhimjirlrl

tg c tg ga a tg a a c tgctggaatgaac tggtgctatgagc tggtgctatgagc

GCCCTGCCCACATCGCTG gccctgcccacatcgctg c A agcc gc c cg tc t c A t c A ag cc gc c cg tc t c A t

IlMttJtMiMiJttM .■■.. ^"GCCGC^cgcctcat

ttgc ttgc gg gg gg

Fig. 4. Alignment of fragments of phzE targeted by the standard primers:

Dir — target area of the forward, and Rev — reverse primers; hereinafter: numbers of sequences: , 2 — P. aeruginosa LESlike 4; 3, 4 — P. aeruginosa PA1; 5 — P. batumici B-321; 6, 7 — P. aeruginosa PAO1; 8 — P. fluorescens S2P5; 9 — P. chlororaphis StFRB508; 10 — P. chlororaphis G05. Fragments complementary to primers are in black

Dir

Rev

1 ] 1

11 ■

5210 5220 5230 5240 5250 5260 5270 5200 5290 5300 5sJ

TGGCGTCGGCCACCAGGAACGGCTGGATGTCGTTGGAGTAGGCGTCGACCGTGGAGATCAGCACCCCGAC G T GG G CG TA T AC GC C G C ACA G C AC CAA CT t GG CG t C GGC c a CC a GG aa c GG CT GG a TG t CG tt GG a G ta G GCG TC GA c CG t G GSGAT CSG ca CC c CG AC GT GGG CG ts TSC GC c GCaca G cac caa CT T G G CG T C GGC CA CC a GG a A c GG CT GG a T A T CG TT GG a G T a G GCG T C GA c CG T G GAG AT c AG CA CC c CG AC GT GGG CG TA TAC GC c GCACa G cAC CAa CT TGGCGTCGGCCACCAGGAACGGCTGGATGTCGTTGGAGTAGGCGTCGACCGTGGAGATCAGCACCCCGAC GTGGGCGTATACGCCGCACAGCACCAACT t gg cg t cGGc CA cea GGaa t gg ctgga ta t CGtt cga G tAG GCa tc cac cg t G gagat cag ca cc c CGac at ggg cg ta cac cc c Gcaga GGat Caa CT CGACG GCCGCCTGCGC ATCGGCGTCGGC TCGACCATCGTG CGTCACTCCGATCCCCTGGGCG AGGCGGCGGAAAGCCG

cgACG GCCGCCTGCGC ATCGGCGTCGGC TCGACCATCGTG

cagcg gcgaggtgcgg atcagcgtgggc tcgaccatcgtg

CAGCG GCGAGGTGCGA ATCAGCGTGGGC TCGACCATCGTG

CTGCG GGCAGGTTCGG ATCAGTGTGGGT TCGACCATCGTG

cgtcactccgatcccctgggcg aggcggcggaaagccg

c gc ca t tc cgac ccgat gac cg aggccgccgaaagccg

cg c ca t tc cgacccgatgac cg aggctgccgaaagccg

cggcactccgaaccgctgaccg aagccgccgaaagccg

y dr cg t c ggc cr s ss s swe y g v ct gg a tr t cr t y vgh gt vg~v; "bjehjîhifcjrift?: -c rb ca y ¥ c cga h c c svt gr s cg tayav sc b gcvsa rv ry cra ct

I ' * " 1 ' ...................................................... " ' I ■ ' • .......*.......................„j

6120 6130 6140 6150 6160 6170 6180 6190 6200 6210 6TZ

ggtgcccggccgcgcgcaccgcctccaccagcggcggcaggcgc tcgccgaccttctg c gca cc ca tccgcgcgatcagcgtcag gcgccc

ggtgcccggccgcgcgcaccgcctccflccagcggcggcaggcgc tcgccgaccttctg cgcscccatccgcgcgatcagcgtcag gcgccc

ggtgcccggccgcgcgcaccgcctccaccagcggcggcaggcgc tcgccgaccttctg cgcgcccatccgcgcgatcagcgtcag gcgccc

ggtgcccggccgcgcgcaccgcctccaccagcggcggcaggcgc tcgccgaccttctg cgcgcccatccgcgcgatcagcgtcag gcgccc

tgtgccccgtggcgcggaccgcctccaccagcggcggcaggcgc tcggcgaccttctc ggcgcccatccgggcaatcagcgtcag gcggcc

cac cc cgaa t cg g tgctgacccgcgagggtccgcgcatcatcgccgacctgctgcgtcacgcgctggtc gagcggcggcc

cac cccgaatcgg tgctgacccgcgagggtccgcgcat cat cg ccgac ctgctgcgtcacgcgctggtc gagcggcggcc

Cat Gc cga gt cg c t gc t ga cc cag GAa gg tc cg c Gcat cat cg ccgac ctgct gcggcac gc c ct ga t ccaca ca cc tg t cgagaacaa cgc tt

catgccgagtcgc tgctgacccaggaaggtccgcgcatcatcgccgacctgctgcggcacgccctgatccacacacctgtcgagaacascgctt

catgccgagtcgc ttttgacccaggaaggcccgcgcatc-:-tc:gcr:g-?-cc~gc,tgcggcacgccctcatccacacgcctgtcgacagcagcgttt

b r y sc cs v r y sg s gc gsa c c g y b y y sa ccm r s gr v gg ym sg c gc at c.=, Mit KK MB EMaaCMig y c as gc v c y sr t cc rs k crm y y rkcgws a rc rr cgb y y

Fig. 5. Conserved loci of the phzE gene suitable for development of universal primers for a diagnostic amplification of fragments of phenazine synthesis operons in Pseudomonas

Sequences were numbered in the same order as in Fig. 4. An unnumbered line corresponds to the consensus

sequence

However, the applicability of such primers yet should be confirmed experimentally.

To conclude, the operon of the phenazine biosynthesis of P. batumici generally was similar to those of the fluorescent P. aeruginosa but the type strain of this species.

The aim of our study was to find out the role of PICA in ecology of the batumin producer. Considering that P. batumici was isolated from the rhizosphere, we made an attempt to evaluate the contribution of the phenazine pigment to the competitiveness of the strain B-321 in soil and rhizosphere. Also we were interested to study the effect of P1CA on opportunistic pathogens to estimate to which extent this activity of P. batumici was contributed by batumin and by other secondary metabolites (Table 1).

The highest growth inhibition by P. batumici UCM B-321 was exerted against Staphylococcus and Pseudomonas aeruginosa B-900, which obviously was caused by batumin. Inhibition of C. albicans and B. subtilis, which were not sensitive to batumin [8-10], may be explained by the activity of P1CA. Inhibition of the phytopathogenic bacteria also may be associated with the synthesis of the phenazine pigment as they are sensitive to this compound even at 50 pg/ml (Table 2).

Antimicrobial activity of P1CA is relatively weak compared to many other antibiotics

produced by Pseudomonas. However, phenazine was considered as an antifungal agent synthesized by the strains of the genus Pseudomonas used for plant protection against fungal diseases. Contrary, batumin does not suppress the growth of fungi at all. Activity of P. batumici against Agrobacterium, Corynebacterium, and phytopathogenic pseudomonads shown in Table 2 also may be attributed to phenazine.

P1CA may be an important factor of regulation of the processes of biofilm formation by Pseudomonas used for plant protection, as it is shown in Fig. 6 on an example with P. batumici B-321.

In this experiment, the biofilm formation by P. batumici was stimulated by supplementing of P1CA into the medium. Biofilm formation was observed as early as in 24 hours and continued up to 48 hours of cultivation. According to statistical analysis, there was no significant difference between the effects of P1CA The stimulation effect by P1CA on the biofilm formation was equally strong when the compound was applied in concentrations of 1 and 10 pg/ml. In both cases, the rate of biofilm formation uplifted with a statistical reliability ^ < 0.01) when compared to the biofilm formation rate on the medium without P1CA.

Also it was observed that the cells of P. batumici B-321 were 20% longer when grown

Table 1. Inhibition zones around colonies of P. batumici UCM B-321

Test strain Growth inhibition zone, mm

Phytopathogenic bacteria

Pseudomonas syringae pv. syringae 19

Pseudomonas fluorescens 22

Pectobacterium carotovorum 14

Xantomonas campestris 0

Clavibacter michiganensis 0

Agrobacterium tumefaciens 0

Opportunistic microorganisms

Staphylococcus aureus B-918 Total inhibition

Escherichia coli B-926 17

Pseudomonas aeruginosa B-900 0

Bacillus subtilis B-901 7

Candida albicansY-2681 10

Table 2. Activity of phenazine-1-carboxylic acid against several phytopathogenic fungi and bacteria

Minimum inhibitory concentration, ^g/ml

Fungi Bacteria

Muco rplumbeus 2GG Agrobacterium tumefaciens 2GG

Fusarium avenaceum 2GG Pseudomonas syringae 1GG

Drechslera graminea 5G Corynebacterium michiganense 2GG

Rhizopus arrhisus 1GG Erwinia aroidea 4GG

Fig. 6. Influence of phenazine-1-carboxylic acid on biofilm formation by P. batumici UCM B-321 strain

Statistical significance is given in the text

on the medium with 10 pg/ml PICA compared to the control growth: 2.81 ± 0.14 x 0.72 ± 0.07 pm and 2.34 ± 0.22 x 0.65 ± 0.02 pm, respectively. The observed elongation of the cells may be associated with a down-regulation of the rate of cell division by PICA [11].

The phenazine biosynthesis was detected in several unrelated taxa of microorganisms, including representatives of Proteobacteria, Actinobacteria and even phylogenetically distant Euryarcheota. Biological properties

of these various compounds are poorly studied despite a multitude of publications on practical importance of the phenazine synthesizing fluorescent bacteria of the genus Pseudomonas including opportunistic human pathogen P. aeruginosa and protecting plants soil saprophyte P. chlororaphis subsp. aureofaciens [2, 12].

Published earlier comparison of sequences of 16S rRNA [6] showed phylogenetic relatedness of P. batumici to P. chlororaphis,

which is another profoundly studied PICA producer. However, in contrast to the latter species, P. batumici is unable to synthesize the green fluorescent pigment pyoverdine. Search through the complete genome sequence confirmed absence of the corresponding genes in P. batumici. Hence, P. batumici is the only non-fluorescent species of Pseudomonas capable of producing PICA. The results signified that the phenazine operons of P. batumici was homologous to the phenazine synthesis operons of other representatives of the genus Pseudomonas. It resembled to some extent the ancestral variant of this gene linking the two groups of phenazine encoding operons (Fig. 3).

The carried out research demonstrated that the synthesis of PICA contributed to the competitiveness of P. batumici in soil and rhizosphere by supplementing of the antimicrobial activity of batumin. Moreover, PICA stimulated the biofilm formation by P. batumici. According to the literature data, biofilm is the state of the bacterial life cycle, when the microorganisms exert 95-99% of their activities in the natural habitats [13]. Biofilm formation by the fluorescent bacteria of the genus Pseudomonas and the role of phenazine in these processes were subjects of numerous studies. Regulated by the

quorum-sensing (QS) system, the synthesis of phenazine alters the expression levels of certain genes involved in cell adhesion during the development of biofilms. Phenzine also is involved in the ion reduction from Fe3+ to Fe2+. The rhizosphere-dwelling strain P. chlororaphis PCL1391 was proved to use phenazine-1-carboxamide to transform Fe3+ into easier mobilized Fe2+ ions allowing in this way the grow at microaerophilic conditions [14]. In P. chlororaphis strain P1CA acts conjointly with 2-oxiphenazine-1-carboxylic acid in regulation of the adhesion of cell in biofilms [15]. Similar effects of P1CA on the stimulation of biofilm formation and iron uptake was reported for the opportunistic pathogen P. aeruginosa [16]. It may be supposed that the phenazine plays a similar regulatory role in the life cycle of the non-fluorescent rhizobacterium P. batumici.

The data inform the literature and that obtained in the presented research suggests a manifold function of the phenazine substances in Pseudomonas That is still not fully understood and requires further studies.

The authors are grateful to O. A. Kipria-nova, Dr. Sc., for advice and and guidance during the preparation of materials for the article.

REFERENCES

1. Leland S., Pierson L., Pierson E. Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes. Appl. Microbiol. Biotechnol. 2010, V. 86, P. 1659-1670. doi: 10.1007/s00253-010-2509-3.

2. Bernd H., Rehm A. Pseudomonas: Model Organism, Pathogen, Cell Factory. Verlag GmbH.: Wiley-VCH, 2008, 403 p. doi: 10.1002/9783527622009.

3. Wink M. Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry. 2003, V. 64, P. 3-19.

4. Mavrodi D., Ksenzenko V., Bonsall R., Cook R., Boronin A. M., Thomashow L. A seven-gene locus for synthesis is of phenazine-1-carboxylic acid by Pseudomonas fluorescens 2-79. J. Bacteriol. 1998, N 180, P. 2541-2548.

5. Kiprianova E. A., Klochko V. V., Zelena L. B., Churkina L. N., Avdeeva L. V. Pseudomonas batumici sp.nov., the antibiotic-producing bacteria isolated from soil of the Caucasus Black Sea coast. Microbiol, zh. 2011, 73 (5), 3-8.

6. Klochko V. V., Zelena L. B., Kim J. Y., Avdeeva L. V., Reva O. N. Prospects of a new antistaphylococcal drug batumin revealed by molecular docking and analysis of the complete genome sequence of the batumin-producer Pseudomonas batumici UCM B-321. Int. J. Antimicrob. Agents. 2016, N 47, P. 56-61. doi: 10.1016/j.ijantimicag.2015.10.006.

7. Darling A., Mau B., Blattner F., Perna N. Mauve: multiple alignment of conserved Genomic sequence with rearrangements. Genome Res. 2004, N 14, P. 1394-1403. doi: 10.1101/gr.2289704.

8. O'Toole G., Kaplan H., Kolter R. Biofilm formation as microbial development. Ann. Rev. Microbiol. 2000, N 54, P. 49-79. doi: 10.1146/ annurev.micro.54.1.49.

9. Klochko V. V., Zelena L. B., Chugunova K. O., Avdeeva L. V. Phenazine complex analysis of strains Pseudomonas chlororaphis subsp. aureofaciens UCM B-111 and UCM B-306. Achievements and problems of genetics, selection, biotechnology. 2012, V. 4, P. 358-362 (In Ukranian).

10. Klochko V. V., Kiprianova E. A., Churkina L. N., Avdeeva L. V. Antimicrobial spectrum of antibiotic batumin. Microbiol. zh. 2008, 70 (5), 41-46. (In Russian).

11. Steinberger R., Allen A., Hansa H., Holden P. Elongation correlates with nutrient deprivation in Pseudomonas aeruginosa — unsaturates biofilms. Microb. Ecol. 2002, 43 (4), 416-423.

12. Chincholkar S., Thomashow L. Microbial Phenazines: Biosynthesis, Agriculture and Health. Springer-Verlag Berlin: Heidelberg. 2013, 248 p.

13. Nikolaev Yu. A., Plakunov V. K. Biofilm— "City of Microbes" or an Analogue of Multicellular Organisms? Microbiologiya. 2007, 76 (2), 149-163. (In Russian).

ГЕНИ, ЩО КОДУЮТЬ СИНТЕЗ ФЕНАЗИН-1-КАРБОНОВО1 КИСЛОТИ

У Pseudomonas batumici

В. В. Клочко1, С. Д. Загородня1, О. М. Рева2

Институт мшробмлогп i вiрусологil iM. Д. К. Заболотного НАН Укра1ни, Ки1в 2Centre for Bioinformatics and Computational Biology, University of Pretoria, South Africa

E-mail: [email protected]

Метою роботи було з'ясування рол1 феназин-1-карбоново1 кислоти Pseudomonas batumici та рiзноманiття гешв, що кодують 11 синтез у бактерш роду Pseudomonas. Феназин-1-карбонова кислота в концентраций 10 мкг/мл стимулювала формування бiоплiвки штамом-продуцентом. Проведення ПЦР з використанням специфiчних праймерiв, розроблених для Pseudomonas, не подтвердило наявнiсть у P. batumici гена синтезу феназину. Водночас сиквенування повного геному P. batumici виявило гомолопчний ген, який, iмовiрно, кодуе синтез цього антибштика. Порiвняльний аналiз геномiв бактерiй роду Pseudomonas показав ^нування, як мiнiмум, двох генетично розрiзнених груп ортологiчних генiв, як кодують синтез феназинiв. Припускають, що розповсюдження генiв синтезу феназинiв у ризобактерш пов'язано з горизонтальним перенесенням гешв.

Ключовi слова: гени Pseudomonas batumici, феназин-1- карбонова кислота,

бiоплiвкоутворення.

14. Hernandez M., Kappler A., Newman D. Phenazines and other redox-active antibiotics promote microbial mineral reduction. Applied and Environmental Microbiology. 2004, N 70, P. 921-928. doi: 10.1128/AEM.70.2.921-928.2004.

15. Maddula V., Pierson E., Pierson L. Altering the ratio of phenazines in Pseudomonas chlororaphis (aureofaciens) strain 30-84: effects on biofilm formation and pathogen inhibition. J. Bacteriol. 2008, N 190, P. 2759-2766. doi: 10.1128/JB.01587-07.

16. Wang Y., Wilks J., Danhorn T., Ramos I., Croal L., Newman D. Phenazine-1-Carboxylic Acid promotes bacterial biofilm development via ferrous iron acquisition. J. Bacteriol. 2011, 193 (14), 3606-3617. doi:10.1128/JB.00396-11.

ГЕНЫ, КОДИРУЮЩИЕ СИНТЕЗ ФЕНАЗИН-1-КАРБОНОВОЙ КИСЛОТЫ

У Pseudomonas batumici

В. В. Клочко1, С. Д. Загородня1, О. Н. Рева2

1Институт микробиологии и вирусологии им. Д. К. Заболотного НАН Украины, Киев 2Centre for Bioinformatics and Computational Biology, University of Pretoria, South Africa

E-mail: [email protected]

Целью работы было выяснение роли феназин-1-карбоновой кислоты Pseudomonas batumici и разнообразия генов, кодирующих ее синтез у бактерий рода Pseudomonas. Феназин-1-карбоновая кислота в концентрации 10 мкг/мл стимулировала формирование биопленки штаммом-продуцентом. Проведение ПЦР с использованием специфичных праймеров, разработанных для Pseudomonas, не подтвердило наличие у P. batumici гена синтеза феназина. В то же время секвениро-вание полного генома P. batumici выявило гомологичный ген, который, вероятно, кодирует синтез этого соединения. Сравнительный анализ геномов бактерий рода Pseudomonas показал существование, как минимум, двух генетически различимых групп ортологич-ных генов, кодирующих синтез феназинов. Высказано предположение, что распространение генов синтеза феназинов у ризобак-терий связано с горизонтальным переносом генов.

Ключевые слова: гены Pseudomonas batumici, феназин-1-карбоновая кислота, биопленко-образование.