ISSN 00262617, Microbiology, 2015, Vol. 84, No. 3, pp. 433–441. © Pleiades Publishing, Ltd., 2015. Original Russian Text © V.A. Demakov, D.M. Vasil’ev, Yu.G. Maksimova, Yu.A. Pavlova, G.V. Ovechkina, A.Yu. Maksimov, 2015, published in Mikrobiologiya, 2015, Vol. 84, No. 3, pp. 369–378.
EXPERIMENTAL ARTICLES
Activated Sludge Bacteria Transforming Cyanopyridines and Amides of Pyridinecarboxylic Acids V. A. Demakova, b, D. M. Vasil’eva, Yu. G. Maksimovab, 1, Yu. A. Pavlovaa, b, G. V. Ovechkinaa, and A. Yu. Maksimova, b a
Institute of Ecology and Genetics of Microorganisms, Ural Branch, Russian Academy of Sciences, Perm, Russia b Perm State National Research University, Perm, Russia Received March 28, 2014
Abstract—Species diversity of bacteria from the activated sludge of Perm biological waste treatment facilities capable of transformation of cyanopyridines and amides of pyridinecarboxylic acids was investigated. Enrich ment cultures in mineral media with 3cyanopyridine as the sole carbon and nitrogen source were used to obtain 32 clones of gramnegative heterotrophic bacteria exhibiting moderate growth on solid and liquid media with 3 and 4cyanopyridine. Sequencing of the 16S rRNA gene fragments revealed that the clones with homology of at least 99% belonged to the genera Acinetobacter, Alcaligenes, Delftia, Ochrobactrum, Pseudomonas, Stenotrophomonas, and Xanthobacter. PCR analysis showed that 13 out of 32 isolates contained the sequences (~1070 bp) homologous to the nitrilase genes reported previously in Alcaligenes faecalis JM3 (GenBank, D13419.1). Nine clones were capable of nitrile and amide transformation when grown on mini mal salt medium. Acinetobacter sp. 11h and Alcaligenes sp. osv transformed 3cyanopyridine to nicotinamide, while most of the clones possessed amidase activity (0.5 to 46.3 mmol/(g h) for acetamide and 0.1 to 5.6 mmol/(g h) for nicotinamide). Nicotinamide utilization by strain A. faecalis 2 was shown to result in excretion of a secondary metabolite, which was identified as dodecyl acrylate at 91% probability. Keywords: nitrileutilizing bacteria, cyanopyridines, amides of pyridinecarboxylic acids, activated sludge, nit rilase, amidase DOI: 10.1134/S0026261715030030
Biological purification of wastewater is based on aerobic and/or anaerobic degradation and mineraliza tion of organic matter by microorganisms [1, 2]. The activated sludge of biological waste treatment facilities carrying out the process of wastewater treatment in aerotanks under aerobic conditions is a community of microorganisms of different taxonomic groups coex isting with certain multicellular animalcules. Expo sure to the constant action of high concentrations of organic compounds, toxic substances, and heavy met als in wastewater is a selective factor favoring accumu lation of the microorganisms possessing a high biodeg radation potential. Nitrile compounds are widespread in nature; they are mainly present in plants as cyanoglycosides, as well as cyanolipids, cyanoalkaloids, ricin, phenyl acetoni trile, etc. More than 2000 species of plants accumulate cyanoglycosides in seeds, roots, and leaves and secrete nitrile metabolites as the main component of the root rhizosphere exudate. This process probably affects the evolution and propagation of microorganisms capable of assimilating nitriles [3]. Moreover, the chemical industry makes wide use of different organic nitriles for producing a number of polymers, chemicals, and 1
Corresponding author; email:
[email protected]
pharmaceuticals. Many nitriles are highly toxic, pro duce a mutagenic and carcinogenic effect, and inacti vate the respiratory system by binding to cytochrome c oxidase. Microbial degradation is an efficacious mode of removal of highly toxic nitriles from the environ ment [4]. The capacity for hydrolysis of nitriles may be used for biocatalytic synthesis of compounds which are important in the chemical and pharmaceutical industries [5, 6]. Nitrile transformation by microorganisms can occur in two different ways: (1) onestep hydrolysis of nitriles to the corresponding carboxylic acid and ammonium mediated by nitrilase (EC 3.5.5.1) and (2) twostep nitrile catabolism involving nitrile hydra tion to amides by nitrile hydratase (EC 4.2.1.84) and subsequent transformation into carboxylic acids and ammonium by amidase (EC 3.5.1.4) [7]. The microor ganisms utilizing nitriles were revealed among different taxonomic groups; in particular, the capacity for hydrolysis of aromatic nitriles has been observed in the representatives of the following genera of actino and proteobacteria: Rhodococcus, Nocardia, Agrobacterium, Microbacterium, Arthrobacter, Alcaligenes, Comamo nas, Klebsiella, Bacillus, Pseudomonas, Acinetobacter, etc. [5, 8], as well as in the fungi Fusarium, Aspergillus, Giberella, and Penicillum [9].
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Table 1. Primers for amplification of the nitrilase genes Name
5'–3' sequence
Specificity
NPF EBCF
GAAATTCCATATGACGGTGCATAAAAAACAG
Nitrilase Pseudomonas fluorescens EBC
NPF EBCR NPF 5F NPF 5R NK22F NK22R Acidv1 Acidv2 AlcJM31 AlcJM32 Comt1 Comt2
CGGGATCCCTTGTCGCCTTGCTCTTCT TTGGATCCATGCCCAAGTCCGTTG TATAAGCTTGGTAAAGCGCACCCCGGGAC ATGTCCAGCAATCCAGAGCTCAAGTACAC CTGGCCTCCGCCTTGGCCC ATGGTTTCGTATAACAGCAAGTTCCTCG CTACTTTGCTGGGACCGGTTCTTCA ATGCAGACAAGAAAAATCGTCCGGG TCAGGACGGTTCTTGCACCAGTAGC ATGAAAAATTATCCTACAGTCAAGGTAGC TCACGCTGTGGCTACGCGCCTC
(AY885240.1) Nitrilase Pseudomonas fluorescens Pf5 (YP_260015.1) Nitrilase Rhodococcus rhodochrous K22 (D12583.1) Nitrilase Acidovorax facilis 72w (AX384691) Nitrilase Alcaligenes faecalis JM3 (D13419) Nitrilase Comamonas testosteroni (L32589)
Investigation of nitrilehydrolyzing bacteria is mainly aimed at comprehensive characterization of the strains possessing the biotechnological potential, regulation of the biosynthesis of biodegradation enzymes, and selection of cultivation conditions [10– 13]. Insufficient attention is given to ecological aspects: the works on the propagation of nitrileutiliz ing microorganisms are scarce [14], and such an important kind of aquatic ecosystems formed at the junction of human economic activity and the environ ment as aerobic activated sludge has hardly been touched upon. The goal of the present work was to study the bio logical diversity and the nitrile and amidetransform ing capacity of gramnegative bacteria isolated from the activated sludge of biological waste treatment facilities on medium with 3cyanopyridine. MATERIALS AND METHODS The study subject was activated sludge sampled from the aerotanks of the biological waste treatment facilities of Perm municipal wastewater (OOO NovogorPrikam’e) in June 2011. The enrichment cultures of nitrilehydrolyzing bacteria were obtained on the nitrogenfree mineral medium of the following composition (g/L): KH2PO4, 1.0; K2HPO4 · 3H2O, 3.7; NaCl, 0.5; MgSO4 · 7H2O, 0.5; FeSO4 · 7H2O, 0.005; CoCl2 · 6H2O, 0.01; pH 7.2–7.4 with 3cyanopyridine (10–50 mM) as the only carbon and nitrogen source. Activated sludge was introduced at a ratio of 2 mL per 50 mL of the medium. The enrichment cultures were incubated at 30°C on a shaker at 100 rpm for 14 days, plated onto the synthetic agar medium of the same composition, and cultivated in the thermostat at 30°C. The transfers were performed at least three times.
The isolates were identified based on their morpho logical, biochemical, and chemotaxonomic charac teristics according to Bergey’s Manual of Determina tive Bacteriology (Gram reaction, cell and colony morphology, motility, tests for the presence of cata lase, oxidase, and growth on sugars). The species were definitively identified using PCR analysis with the 16S rRNA gene primers constructed on the basis of the sequences represented in the GenBank database and synthesized by Evrogen (Moscow). Chromosomal DNA preparations were obtained using the phenol method [15]. DNA amplification was carried out using thermo stable Taq polymerase (SibEnzim Ltd., Novosibirsk, Russia) using a T3 thermocycler (Biometra, Ger many). The oligonucleotide primers specific to the DNA of the genes of known nitrilases were designed according to the nitrilase gene 5' and 3'sequences of the gramnegative bacteria available in the GenBank database (Table 1). The PCR products were electrophoretically sepa rated in 1.2–1.5% agarose gel in Trisborate buffer at a field intensity of 5 V/cm. The 1kb and 100b molec ular markers were used to assess the molecular mass of the DNA fragments (OOO Sibenzim and Axigen®). The bands were visualized and the data were docu mented after staining the gel with ethidium bromide using the BioDocAnalyze geldocumentation system (Biometra, Germany). Prior to sequencing, the PCR products were puri fied by two methods: using the ExoSAMPix enzyme mixture (Fermentas Life Sciences) and by means of an EGel apparatus (Invitrogen, United States) accord ing to the manufacturer’s instruction. Sequencing was carried out on a Genetic Analyzer 3500xL (Applied Biosystems, United States) accord ing to the manufacturer’s instructions. MICROBIOLOGY
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The homology of 16S rRNA and nitriliase gene sequences with known genes of microorganisms was analyzed using the BLAST software package (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) and the Chromas lite 2.1 package. The nucleotide sequences were compared using the ClustalW 2.0.9 and YACWGUI 1.2 software packages. The nitrile and amidehydrolyzing activity of the isolated clones was determined during transformation of aliphatic and aromatic nitriles and amides by sus pensions of the cells grown in synthetic mineral medium with 0.1 M acetamide as a source of carbon and nitrogen. The biomass was centrifuged for 20 min at 10000 g, washed thrice with 0.01 M potassium phos phate buffer, pH 7.2 ± 0.2. The transformation reac tion of acrylonitrile and 3cyanopyridine was per formed at 30°C for 6 h; the reaction mixture was cen trifuged and quickly frozen at –18°C. The reaction products were analyzed by HPLC on an LC10 chro matograph (Shimadzu, Japan). Acrylic acid nitrile, acrylamide, and acrylic acid were determined on the Synergi 4u HydroRP 80A 250 × 4.6mm column. NaH2PO4 (25 mM) was used as the mobile phase; the flow rate was 0.75 mL/min at 25°C; detection was carried out at a wavelength of 200 nm. Detection of 3cyanopyridine, nicotinamide, and nicotinic acid was carried out on a Luna 5u C 18(2) 100A (250 ± 4.6 mm) column. KH2PO4 (10 mM) and 25% aceto nitrile were used as the mobile phase; the flow rate was 0.5 mL/min at 25°C; detection was carried out at a wavelength of 200 nm. Acetamide and acetic acid were determined by gas chromatography on a GC2014 chromatograph (Shimadzu, Japan) equipped with a flame ionization detector. The 2m chromatographic column with an inner diameter of 3 mm was filled with the solid sup port polysorb1, the fraction 0.25–0.5 mm (Rea khim, Russia). The carrier gas nitrogen was supplied at a volume flow of 35 cm3/min. The thermostat tem perature was 190 ± 3°C; the evaporator temperature was 220 ± 10°C. The solutions of pure nitriles, amides, and carboxylic acids were used as the stan dards for HPLC and GC. Changes in the composition of the culture liquid during growth were analyzed using chromatographic– mass spectral analysis on a 689/573T MSD apparatus (AgilentHewlett Packard) with an RTX5MS capil lary column and a 5m HP5MS precolumn. Sample extraction with ethyl acetate was preliminarily carried out. The initial column temperature was 90°C (3 min); it was heated to 250°C at a rate of 15°C/min. The evaporator temperature was 280°C; helium was used as a carrier gas. RESULTS AND DISCUSSION Enrichment cultures on synthetic mineral medium with 3cyanopyridine were used to obtain 32 clones of MICROBIOLOGY
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gramnegative heterotrophic bacteria from the acti vated sludge and sediment samples of the Perm biolog ical waste treatment facilities. Grampositive bacteria were not isolated from the enrichment cultures. The isolated clones retained the capacity for moderate growth in liquid and on solid mineral medium with 3cyanopyridine as the substrate for several sequential transfers. Based on the analysis of their 16S rRNA gene sequences, the isolates were assigned (with homology of at least 99%) to the genera Acinetobacter, Alcaligenes, Delftia, Ochrobactrum, Pseudomonas, Stenotrophomonas, and Xanthobacter (Table 2). The capacity of bacteria for utilization of nitrile compounds is known to require the presence of the nitrilase, nitrile hydratase, and amidase genes. One step hydrolysis of cyanopyridines to the corresponding pyridinecarboxylic acids, which is carried out by nit rilases, is of interest for biocatalytic preparation of the intermediate compounds for a number of important pharmaceuticals [16]. In order to reveal the nitrilase encoding genes, the clones isolated were screened with the primers specific to the nitrilase genes of the known types, which have been previously reported in gram negative bacteria, and the amplicons obtained were sequenced. PCR analysis showed that 13 out of 32 iso lates contained the sequences (~1070 bp) homologous to the nitrilase genes reported previously in Alcaligenes faecalis JM3 (GenBank, D13419.1) (Fig. 1). Of these, based on 16S rRNA homology, 12 isolates were assigned to A. faecalis and one isolate exhibited 100% homology to P. putida (Table 2). Sequencing the nit rilase genes of the isolates studied revealed that eight of them contain nitrilase of one type, while five, includ ing the P. putida strain, contained nitrilase of a differ ent type. The nitrilase sequences within each group were identical; the intergroup gene homology was 91.1%; the similarity to amino acid sequences was 96.6% (Tables 3, 4). Comparison with known homologous genes repre sented in the GenBank database established that the nucleotide sequence of the first type was 99.9% homologous to that from Alcaligenes sp. ECU0401 (FJ943638.1), with the coded protein being absolutely identical. The nitrilase gene of the second type had the closest similarity to the D13419.1 sequence from A. faecalis (94.3%), while the amino acid sequence was 98.3% identical (Tables 3, 4). Transformation of aliphatic and aromatic nitriles and amides by the isolates obtained was investigated. It was found that only two isolates, Acinetobacter sp. 11h and Alcaligenes sp. osv, exhibited nitrile hydratase activity and transformed 3cyanopyridine into nicoti namide (Table 5). At the same time, all the strains studied hydrolyzed aliphatic and aromatic amides to the corresponding carboxylic acids. According to the literature data, cyanopyridines may affect amidase activity [17]. Although 13 isolates were found to con tain the nitrilase genes and the cultures were main tained on 3cyanopyridine as the sole source of carbon
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Table 2. Phylogenetic diversity of the microbial community isolated on 3cyanopyride from the activated sludge of Perm biological treatment facilities and the presence of nitrilases in the isolated clones Isolate 11h osv 8h 13 12 2 3 11 4h 9 13h A6 9h A10 8 A8 2h 3h A7h A7 A6h visev 12h 1 5 1h 5h A5 A3 il 10h 4
Number Similarity of nucleotides between read 16S rRNA genes, %
Closest validly described species according to the GenBank Acinetobacter guillouiae CIP 63.46T (APOS01000028) Alcaligenes aquatilis LMG 22996T ( AJ937889) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Alcaligenes faecalis subsp. parafaecalis GT (AJ242986) Delftia lacustris DSM 21246T ( EU888308) Ochrobactrum anthropi ATCC 49188T ( CP000758) Pseudomonas monteilii CIP 104883T (AF064458) Pseudomonas monteilii CIP 104883T (AF064458) Pseudomonas monteilii CIP 104883T (AF064458) Pseudomonas monteilii CIP 104883T (AF064458) Pseudomonas monteilii CIP 104883T (AF064458) Pseudomonas monteilii CIP 104883T (AF064458) Pseudomonas putida NBRC 14164T (AP013070) Stenotrophomonas acidaminiphila AMX19T (AF273080) Xanthobacter flavus 301T (X94199)
99.17 100 100 99.88 99.77 99.77 99.77 99.77 99.76 99.76 99.76 99.76 99.76 99.76 99.76 99.75 99.75 99.75 99.72 99.70 99.57 100 100 100 100 100 100 100 100 100 99.89 99.88
Nitrilases, groups
846 568 853 852 860 856 856 853 882 847 847 825 844 824 823 811 810 796 713 673 797 854 858 835 858 844 843 636 606 820 870 847
2 1 1 2 2 1 2 1 1 1 1
1
2
Type strains of a genus are designated with the superscript index T.
and nitrogen, no transformation of this substrate by nitrilases was revealed. It may be suggested that moderate growth and persistence of the cultures on 3cyanopyridine occurred due to spontaneous hydrolysis of this substrate to the corresponding amide and carboxylic acid. The isolated clones grew on nicotinamide as the sole source of carbon and energy. It was shown that the bacterial cultures attained the maximal optical density by the third day of growth, which indicated complete
consumption of nicotinamide (Fig. 2). Utilization of this compound was confirmed by the chromatogra phy–mass spectrometry of the liquid culture of A. faecalis 2 sampled daily. An increase in the concen tration of the substance identified as dodecyl acrylate with 91% probability was revealed to occur simulta neously with the utilization of nicotinamide (Fig. 3). Emergence of the secondary metabolite, an acrylic acid derivative, implies a rupture of the pyridine ring in the process of utilization. The fact of the release of this MICROBIOLOGY
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5
4h
4
3h
3
2h
2
1h
1
1 kb
ACTIVATED SLUDGE BACTERIA TRANSFORMING CYANOPYRIDINES
1071 bp
13h
13
12h
12
11h
11
10h
10
9h
9
8h
1 kb
1000 bp
1071 bp
A5h
A5
A4h
A4
A3
A2h
A1h
A1
il
vis
osv
1 kb
1000 bp
1071 bp
A10
A9
A8
A7h
A7
A6h
A6
A5h
A5
1 kb
1000 bp
1071 bp
1000 bp
Fig. 1. Electrophoresis of the DNA PCR products corresponding in size to the nitrilase gene of A. faecalis JM3 (GenBank, D13419.1).
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Table 3. Comparison between the nitrilase gene nucleotide sequences Alcaligenes faecalis Pseudomonas putida (D13419.1) MTCC5110 Alcaligenes faecalis (D13419.1) Pseudomonas putida MTCC5110 Alcaligenes sp. (ECU0401) Alcaligenes faecalis 12 Alcaligenes faecalis 4h
Alcaligenes sp. (ECU0401)
Alcaligenes faecalis 12
Alcaligenes faecalis 4h
1
0.996
0.898
0.897
0.943
0.996
1
0.894
0.893
0.940
0.898
0.894
1
0.999
0.910
0.897 0.943
0.893 0.940
0.999 0.910
1 0.911
0.911 1
Table 4. Comparison between the nitrilase amino acid sequences Alcaligenes faecalis Pseudomonas Alcaligenes sp. putida MTCC5110 (ECU0401) (D13419.1) Alcaligenes faecalis (D13419.1) Pseudomonas putida MTCC5110 Alcaligenes sp. (ECU0401) Alcaligenes faecalis 12 Alcaligenes faecalis 4h
Alcaligenes faecalis 12
Alcaligenes faecalis 4h
1
1
0.963
0.963
0.983
1
1
0.963
0.963
0.983
0.963
0.963
1
1
0.966
0.963 0.983
0.963 0.983
1 0.966
1 0.966
0.966 1
Table 5. Activity of the enzymes of nitrile metabolism in the isolates obtained on 3cyanopyridine
Bacterial genus
Isolate
Nitrile hydratase activity, mmol/(g h) with 3cyanopyridine as a substrate
Amidase activity, mmol/(g h) nicotinamide as a substrate
acetamide as a substrate
Acinetobacter sp.
11h
4.4
5.6
0.6
Alcaligenes sp.
2
0
0.2
7.0
2h
0
0.1
1.0
4h
0
0.4
2.4
A6
0
0.2
3.1
A7h
0
0.7
0.5
A8
0
0.2
46.3
osv
0.4
0.2
1.6
A5
0
0.8
1.4
Pseudomonas sp.
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mM 14 12
2
1
1.0
10
0.8
8
0.6
6
0.4
4
0.2
2
0 0
1
3
2
5
4
6
7
Days Fig. 2. Growth of A. faecalis 2 on nicotinamide as the sole source of carbon and nitrogen: nicotinamide content in the culture medium (1) and the dynamics of the optical density of the culture (2).
5
As early as in 1955–1957, Hughes [18] and Behr man et al. [19] proposed the metabolic pathway of transformation of nicotinic acid into aliphatic com pounds associated with the cleavage of the pyridine ring. The first intermediate of both enzymatic and oxi dative dissimilation was 6hydroxynicotinic acid. Fur ther oxidation of 6hydroxynicotinic acid results in the formation of 2,5dihydroxypyridine; the degradation of this heterocyclic compound requires simultaneous absorption of two oxygen atoms to yield an aliphatic product with a double bond in the cisconfiguration. The microorganisms possessing the enzymes for nitrile hydrolysis enzymes (nitrile hydratase, amidase, and nitrilase) are probably capable of transforming aro matic nitriles and amides into the corresponding acids (c) TIC: 9.D
8.81
Abundance 10000 9000 8000 7000 6000 5000 4.43 5.68 4000 5.22 3000 4.86 6.12 2000 6.68 1000
1
6
8
9 10 11 12 13 14 15 16 Time (b) TIC: 7.D
7
8.90
Abundance 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 5
secondary metabolite into the culture medium upon growth on nicotinamide and the possible metabolic pathways resulting in its formation will further be stud ied in detail.
(а) TIC: 6.D
Abundance 380000 360000 340000 320000 300000 280000 260000 240000 220000 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000
439
5
6
7
11.13
2
12.86
8
Abundance
9
10 11 12 13 14 15 Time (d) TIC: 10.D 11.13
35000 30000 1
25000
2
20000 15000 10000
5.67 5.22 6.11
5000 4.83 6
7
8
9 10 11 Time
12 13
5
6
7
8
9 10 11 12 13 14 15 16 Time
Fig. 3. Nicotinamide decrease (1) and the appearance of dodecyl acrylate (2) in the culture medium: on the first (a); second (b); fourth (c); and seventh day of growth (d). MICROBIOLOGY
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yd r N
itr il
eh
Nicotinamide
CN
e as id Am
at as e
N
COOH
COOH
Nitrilase
N
N
3Cyanopyridine
+1/2O2
Nicotinic acid
HO
HO +1/2O2
N
6Hydroxynicotinic acid
HO
N
2,5Dihydroxypyridine +O2 2,5Dihydroxypyridine
oxidase
H HOOC
C C
COOH
H
H
H
Fumaric acid
C C
H
COOH COOH
+H2O –NH3
Maleic acid
H
C C
COOH +H2O –HCOOH
CONH2
O
N H
COOH CHO
NFormyl maleic acid
Fig. 4. Metabolic pathway of utilization of nicotinic acid.
(Fig. 4), which are then catabolized to simpler ali phatic compounds utilized by the cell to obtain energy. Thus, we studied the phylogenetic diversity of het erotrophic gramnegative bacteria from the activated sludge of Perm biological waste treatment facilities isolated on cyanopyridine and growing on amides of pyridinecarboxylic acids as the sole carbon and nitro gen source. Although the cultures could be maintained on 3cyanopyridine and moderate growth occurred on this substrate, no utilization of this compound was found, whereas the isolated clones grew actively on the amides of pyridinecarboxylic acids and utilized nicoti namide. Amidase activity was also revealed in the clones containing the nitrilase genes in their genomes, which indicates the possibility that these bacteria may possess both metabolic pathways. At the same time, further selection aimed at increasing the nitrile and amidehydrolyzing activity is necessary for the devel opment of the biotechnological potential of the iso lated clones. ACKNOWLEDGMENTS The work was supported by the “Molecular and Cellular Biology” program of the Presidium of the Russian Academy of Sciences and the State Assign ment for Research Work, project no. 6.2635.2014/K.
3.
4.
5.
6. 7. 8.
9.
10.
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