Genetic and Immunochemical Characterization of Thiocyanate-Degrading Bacteria in Lake Water

Natural aquatic and soil samples were screened for the presenceof thiocyanate-degrading bacteria. Using thiocyanate supplementation,we established an enrichment culture containing such bacteriafrom lake water. The dominant bacteria had the scnC-LS5 geneencoding thiocyanate hydrolase, which was closely related tothe enzyme found previously in Thiobacillus thioparus THI115isolated from activated sludge.

INTRODUCTION

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Introduction

Carbonyl sulfide (COS) is a major sulfur compound in the tropospherecontributing to sulfate aerosol, which accumulates in the stratosphereand influences the climate (3). About one-half of the COS originatesfrom marine and soil environments (11). Biological processeshave been considered to be responsible for the unique sulfurflow (14). However, little is known about the diversity andcomposition of the bacterial communities that are involved inthe production and degradation of COS in the environment. Ithas been observed that production of COS in soil is stimulatedby addition of thiocyanate (2, 14, 15). Katayama et al. (6,7) isolated Thiobacillus thioparus THI112 and THI115 from activatedsludge and showed that these bacteria are obligate chemolithotrophsthat utilize thiocyanate as a sole energy source and produceammonia and COS as the reaction products (8, 12). A unique enzymeresponsible for degradation of thiocyanate was purified fromthis bacterium and designated thiocyanate hydrolase (7). ThescnA, scnB, and scnC genes encoding the three subunits ( ${alpha}$ , ß,and ${gamma}$ ) of this enzyme have been cloned and sequenced. The deducedamino acid sequences exhibit significant homology to the sequencesof nitrile hydratases from various bacteria (10). Furthermore,thiocyanate-degrading activity has been found in the facultativechemolithotroph Paracoccus thiocyanatus (9), in which a thiocyanatehydrolase-like enzyme has been detected (K. Hatayama and Y.Katayama, unpublished data), indicating that microbes carryingthis enzyme may be distributed widely in water and soil environments.

In the present study, we detected thiocyanate-degrading andCOS-producing bacteria in lake water by using an enrichmentculture supplemented with thiocyanate. To quantify such bacteriain a natural aquatic environment, we used a fluorescent immunostainingtechnique with thiocyanate hydrolase-specific antibodies. Furthermore,scnC-specific primers were used to characterize the microbesharboring the related genes. To determine phylogenetic relationshipsamong thiocyanate-degrading bacteria, we analyzed 16S ribosomalDNA (rDNA) fragments by a PCR-denaturing gradient gel electrophoresis(DGGE) method.

Thiocyanate degradation and COS emission in environmental bacteria.

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Thiocyanate degradation and cos...

Water and soil samples were collected from various locationsin Japan (Table 1). The microorganisms in the samples were grownat 30°C in TC medium (7) containing 0.1 g of potassium thiocyanateper liter (TC1 medium). Consumption of thiocyanate in the mediumwas measured spectrophotometrically (7). To measure the amountof COS, a gas sample was collected from the headspace of a flaskequipped with a rubber stopper and analyzed by gas chromatography(7). As shown in Table 1, degradation of thiocyanate and COSemission in the cultures were evident in almost all samplesexamined. These results indicate that thiocyanate-degradingmicrobes are distributed widely in various natural and man-madeenvironments. Compared to soil and activated sludge samples,aquatic samples required a longer lag time before maximum degradationactivity was observed.

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TABLE 1. Thiocyanate degradation and COS emission in various environmental samples

To enrich thiocyanate-degrading bacteria, 100 ml of surfacewater from Lake Sagami was supplemented with 0.1 g of potassiumthiocyanate per liter and incubated in a 500-ml flask at 30°C.When the thiocyanate was degraded, the culture was supplementedwith additional potassium thiocyanate. The supplementation procedurewas repeated three times. Then 10 ml of the resultant culturewas inoculated into 90 ml of TC medium containing 0.5 g of potassiumthiocyanate per liter (TC5 medium), and the procedure was repeatedthree times. In the resultant enrichment culture, designatedculture LS5, the 0.45 mmol of thiocyanate initially presentin the medium was degraded in 38 h (Fig. 1). The concentrationof COS increased in the early phase of thiocyanate degradationbut decreased later. As observed with T. thioparus THI115 (7),COS molecules exported by the bacterial cells were subsequentlyingested by the same bacteria and utilized as an energy source.

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FIG. 1. Thiocyanate degradation (•) and COS emission ( ${circ}$ ) in enrichment culture LS5. The microorganisms in culture LS5 (10 ml) were inoculated into 90 ml of TC5 medium containing 5 mM potassium thiocyanate in a 500-ml flask. The ordinates indicate the residual amount of thiocyanate in the medium and the total amount of COS in the flask. The error bars indicate standard deviations based on duplicate experiments.

The production of thiocyanate hydrolase was analyzed by Westernblotting. As shown in Fig. 2, analysis of a crude extract ofthe microorganisms from culture LS5 grown in TC5 medium producedthree bands that were indistinguishable from the ${alpha}$ -, ß-,and ${gamma}$ -subunit bands of the thiocyanate hydrolase of T. thioparusTHI115. These protein bands were not obtained with a controlsample prepared from culture LS5 grown in 0.01x NBY medium (pH7.0) containing 0.1 g of meat extract (Kyokuto, Tokyo, Japan)per liter, 0.05 g of NaCl per liter, 0.1 g of Bacto Peptone(Difco) per liter, and 0.05 g of Bacto Yeast Extract (Difco)per liter.

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FIG. 2. Western blot analysis of the thiocyanate hydrolase. Crude extracts were prepared from culture LS5 grown in TC5 medium (LS5/TC5) or in 0.01x NBY medium (LS5/0.01x NBY). Thiocyanate hydrolase purified from T. thioparus THI115 was used as a standard.

The size of the bacterial population producing thiocyanate hydrolasewas estimated by fluorescent immunostaining (24) by using antiseraraised against the ${alpha}$ and ${gamma}$ subunits of the enzyme and fluoresceinisothiocyanate-labeled secondary antibody. To stain the cytoplasmicenzyme, the cell wall was permeabilized by treatment for 30to 60 min at 37°C with a lysozyme solution containing 50mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, lysozyme (40µg/ml), DNase I (1 mg/ml), and gelatin (10 µg/ml).The total cell number was determined by 4",6"-diamidino-2-phenylindole(DAPI) staining. As shown in Table 2, 93% of the cells in cultureLS5 were stained with the antibodies, while only 0.02% of thecells in the fresh surface water of Lake Sagami were stained.Moreover, the fluorescence intensities of the latter cells werequite low, indicating that enzyme synthesis may be inducibleby addition of thiocyanate. Since the thiocyanate level in thelake surface water was below the detection limit (data not shown),enzyme synthesis may be induced to only a limited extent inthe bacteria present in such an environment. There may be somelocalized habitat in the lake where certain populations of bacteriatake up the free form of thiocyanate liberated from thioglucoside(23) or cyanide (22) and survive as a reservoir.

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TABLE 2. Occurrence of thiocyanate-degrading bacteria in lake water and an enrichment culture

Characterization of scnC-related genes.

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Characterization of scnc-related...

For PCR analysis of the bacteria in culture LS5, we designedthe following degenerate primers: 5"-GTNGCNMRNGCNTGGBTNGAYCC-3"and 5"-GGICKIWSIGGIADIACNADRTA-3" (where B is C, G, or T; Dis A, G, or T; K is G or T; M is A or C; N is A, C, G, or T;R is A or G; S is C or G; W is A or T; and Y is C or T). Thisprimer set could be used to amplify part of the scnC gene encodingVal⁶⁸ to Pro¹⁹⁴ of the ${gamma}$ subunit of thiocyanate hydrolase, whichwas highly homologous to the corresponding parts of nitrilehydratases of various bacteria (10). Using this primer set,we amplified a DNA fragment of the same size ( $~$ 380 bp) from thegenomic DNA of the bacteria in culture LS5. Direct sequencingof the PCR product revealed a highly homologous gene, whichwe designated scnC-LS5. The nucleotide and amino acid sequencesof scnC-LS5 were 85% (284 of 333 residues) and 96% (107 of 111residues) identical to those of scnC, respectively. The amplifiedscnC-LS5 gene was cloned into the pCR2.1 vector (Invitrogen).We also observed nonspecific PCR bands at $~$ 350 and $~$ 290 bp whosesequences were not related to the scnC gene.

For Southern blot analysis, genomic DNA digested with restrictionenzymes were blotted onto Immobilon-Ny+ (Millipore) and incubatedwith the [ ${alpha}$ -³²P]dCTP-labeled scnC-LS5 probe. After hybridization,the filter was washed with 0.2x SSC-0.1% sodium dodecyl sulfatefor 30 min at 42°C (1x SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate). The hybridization signals were detected with BAS-1500(Fujifilm, Tokyo, Japan). EcoRI and HindIII digests of the genomicDNA of T. thioparus THI115 contained 5.0- and 9.0-kb singlebands, respectively, while EcoRI and HindIII digests of cultureLS5 DNA contained 7.0- and 7.2-kb single bands, respectively(Fig. 3). The results indicated that scnC and scnC-LS5 weresingle-copy genes and were distinct from each other.

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FIG. 3. Genomic Southern blot analysis of scnC and scnC-related genes. Genomic DNA (1 µg) extracted from T. thioparus THI115 or culture LS5 was digested with EcoRI or HindIII and separated on a 1.0% agarose gel. The positions of DNA size markers are indicated on the right.

Phylogenetic analysis of thiocyanate-degrading bacteria.

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Phylogenetic analysis of...

Using the bacterial genomic DNA samples, we performed a touchdownPCR (4) to amplify 16S rDNA. The forward primer(5"-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3")contained a part of the Escherichia coli 16S rDNA sequence (nucleotidepositions 341 to 357) with a 5" extension of a 40-bp GC clampthat was necessary for subsequent DGGE (17). The reverse primer(5"-CCCCGTCAATTCCTTTGAGTTT-3") was complementary to nucleotidepositions 907 to 928. DGGE analysis of the PCR products (16,19) was performed with a DCode universal mutation detectionsystem (Bio-Rad) according to the protocols provided by themanufacturer. Samples were subjected to electrophoresis for4 h at 200 V through a 6% polyacrylamide gel with a denaturantgradient. As shown in Fig. 4, the PCR products obtained fromculture LS5 grown in TC5 medium and from culture LS5 grown in0.01x NBY medium showed different migration patterns after DGGE.The DNA fragments were purified from the gel and subjected todirect sequencing. The 16S rDNA sequence of the major DNA bandfrom culture LS5 grown in TC5 medium, band B, indicated thatthe dominant bacteria in culture LS5 grown in TC5 medium, whichshould have had the scnC-LS5 gene, were closely related to astrain of T. thioparus (13) for which a 16S rDNA sequence (accessionno. M79426) has been reported. Minor DNA bands A, C, and D werephylogenetically related to the 16S rDNA of Cytophaga johnsonae,Aquaspirillum delicatum (21), and Rhodanobacter lindaniclasticus(18), respectively (Fig. 5), although the participation of thesebacteria in thiocyanate degradation has not been analyzed. Theposition of major band E from culture LS5 grown in 0.01x NBYmedium was close to the position of 16S rDNA of Burkholderiapyrrocinia (1), and this band was not obtained with cultureLS5 grown in TC5 medium. At least some of the accompanying heterotrophicpopulation might have utilized excretion products of the autotrophs.

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FIG. 4. DGGE patterns of the PCR products amplified with 16S rDNA primers. The template DNA was extracted from enriched bacteria from culture LS5 grown in TC5 medium (LS5/TC5) or from the control (culture LS5 grown in 0.01x NBY medium [LS5/0.01x NBY]). The denaturant gradient was formed by assuming that 7 M urea and 40% formamide provided 100% denaturing conditions at 60°C. The patterns of DNA stained with ethidium bromide are shown on the right.

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FIG. 5. Phylogenetic identities of the bacteria represented by the 16S rDNA bands obtained by DGGE (bands A, B, C, D, and E). The phylogenetic tree was constructed by using the CLUSTAL X search (20) and TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) programs. The 16S rDNA sequences of bacterial strains were obtained from GenBank; the accession numbers are indicated after the names of the organisms. The numbers at the nodes are bootstrap values (based on 1,000 trials). Scale bar = 0.1 change per nucleotide.

An enrichment procedure may enrich only some members of desiredbacterial groups (5). Alternative enrichment procedures mightmore effectively amplify other thiocyanate-degrading bacteriathat have not been found yet. Currently, we are collecting morescnC-related genes, including those derived from members ofother genera, such as P. thiocyanatus. This approach shouldbe helpful for designing versatile and specific degenerate primersfor analysis of the diverse thiocyanate-degrading bacteria thatmay be distributed widely in natural and man-made environments.

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The nucleotide sequence of scnC-LS5 has been deposited in theDDBJ database under accession number AB0074989.

ACKNOWLEDGMENTS

We thank Hiroyuki Yamamoto of the School of Medicine, St. MariannaUniversity, for guidance in the phylogenetic analyses.

This work was supported by a grant-in-aid from the Ministryof Education, Culture, Sports, Science and Technology.

FOOTNOTES

* Corresponding author. Mailing address: Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan. Phone: 81-42-367-5732. Fax: 81-42-367-5732. E-mail: katayama{at}cc.tuat.ac.jp.

REFERENCES

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