Species-Specific Distribution of a Modular Family 19 Chitinase Gene In

FEMS Microbiology Ecology 37 (2001) 135^141 www.fems-microbiology.org

Species-speci¢c distribution of a modular family 19 chitinase gene in Burkholderia gladioli

Hyesuk Kong a, Makoto Shimosaka b, Yasuo Ando c, Kouji Nishiyama a, Takeshi Fujii a, Kiyotaka Miyashita a;* Downloaded from https://academic.oup.com/femsec/article/37/2/135/473702 by guest on 04 October 2021

a Laboratory of Soil General Microbiology, National Institute for Agro-Environmental Sciences, Tsukuba 305-8604, Japan b Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan c Japan International Research Center for Agricultural Sciences, Tsukuba 305-8686, Japan

Received 5 February 2001; received in revised form 25 June 2001; accepted 2 July 2001

First published online 9 August 2001

Abstract

The chitinase gene chiB of Burkholderia gladioli strain CHB101 is the first family 19 chitinase gene found in bacteria other than Streptomyces. The presence of chitinase genes homologous to the chiA (family 18) and chiB (family 19) of B. gladioli strain CHB101 was investigated in closely related species Burkholderia andropogonis, Burkholderia caryophylli, Burkholderia glumae, Burkholderia plantarii, and Burkholderia cepacia as well as in other strains of B. gladioli. Twenty-seven of the 30 strains of B. gladioli examined produced chitinases and were all positive when probed with chiA and chiB of CHB101, although the restriction fragment length polymorphism patterns in these strains were not necessarily identical to that of CHB101. The remaining three strains of B. gladioli and all strains of the other Burkholderia species tested showed no chitinase activity and gave no signal with the chiA or chiB probes. Analysis of the 16S rDNA sequence indicated that the three chitinase-negative strains of B. gladioli were phylogenetically closer to B. glumae or B. cepacia than to B. gladioli, and should therefore be reassigned. Apparently, possession of a modular family 19 chitinase gene homologous to chiB of CHB101 is a species-specific characteristic of B. gladioli. In this article, we show the species-specific distribution of a family 19 chitinase gene in B. gladioli. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Family 19 chitinase gene; Southern hybridization; 16S rDNA sequence; Phylogenetic analysis; Burkholderia gladioli

1. Introduction ily 18 and family 19 chitinases di¡er in amino acid sequences, three-dimensional (3D) structures, and hydrolytic Chitin, one of the most abundant natural polymers, is mechanisms [5]. The catalytic domains of family 18 chiti- an important structural component of many organisms nases have an (K/L) eight-barrel fold as demonstrated by including the cuticles of insects, shells of crustaceans, 3D structural analysis of hevamine [6] and Serratia mar- and cell walls of many fungi [1]. Chitinases (EC cescens chitinase A [7]. On the other hand, the catalytic 3.2.1.14), the enzymes that degrade chitin (poly-L-1,4-N- domains of family 19 chitinases have a high K-helical con- acetylglucosamine), have been receiving attention because tent and structural similarity, including a conserved core, of their possible application for the biological control of with chitosanase and lysozyme [8,9]. Family 18 and family chitin-producing organisms [2]. Chitinase genes have been 19 chitinases are therefore thought to have di¡erent evolu- isolated from many di¡erent organisms including bacteria, tionary origins. Family 18 contains chitinases from many viruses, higher plants, and animals. Based on amino acid chitin-producing organisms. sequence similarities of their catalytic domains, chitinases Plant chitinases are divided into classes I^V, classes III are divided into two di¡erent families (families 18 and 19) and V belonging to family 18, while classes I, II, and IV of the glycosyl hydrolases classi¢cation system [3,4]. Fam- belong to family 19 [10]. Family 19 chitinases had only been reported from higher plants until chitinase C of Streptomyces griseus HUT6037 was discovered [11]. The chitinase C of S. griseus HUT6037 has a catalytic domain * Corresponding author. Tel.: +81 (298) 38 83 09; Fax: +81 (298) 38 81 99. homologous to those of plant class I, II, and IV chitinases, E-mail address: [email protected]¡rc.go.jp (K. Miyashita). and has an N-terminal chitin-binding domain. Although

FEMSEC 1270 9-10-01 136 H. Kong et al. / FEMS Microbiology Ecology 37 (2001) 135^141 genes homologous to chiC of S. griseus HUT6037 have et al. [19] and plasmid DNA preparations were made using been found in the other species of Streptomyces, distribu- Qiagen spin mini-prep kits (Qiagen) according to the in- tion of the family 19 chitinase gene was still limited to the structions of the manufacturer. Restriction enzyme diges- genus Streptomyces [12,13]. tions and DNA ligations were performed following the Strain CHB101, isolated as a chitinase and chitosanase recommendations of suppliers (Takara, Toyobo, and Nip- producer, was identi¢ed as Burkholderia gladioli. The ge- ponGene). Competent cells of E. coli DH5K for transfor- nus Burkholderia belongs to the L-subclass of the Proteo- mation by 42³C heat shock were prepared using the meth- bacteria [14]. B. gladioli is known as a plant pathogen and ods of Hanahan [20]. All DNAs for ligation (vectors and also as an opportunistic pathogen of humans. To our inserts) were puri¢ed from agarose gel using GeneClean II knowledge, no strain of B. gladioli has ever been reported (BIO 101, USA). to produce chitinase. ChiB of CHB101 is the ¢rst family For Southern hybridization, restriction enzyme-digested 19 chitinase identi¢ed in bacteria other than Streptomyces DNA fragments were separated on a 0.7% agarose gel, [15]. then transferred to a Hybond-N nylon membrane (Amer- Phylogenetic analysis of family 19 chitinases strongly sham Pharmacia Biotech). The membranes were probed Downloaded from https://academic.oup.com/femsec/article/37/2/135/473702 by guest on 04 October 2021 supported the possibility that Streptomyces family 19 chi- with two fragments of B. gladioli strain CHB101 chitinase tinases were acquired from plants by horizontal gene genes: a 2.1-kb EcoRI fragment, which contains chiA transfer [13]. The predicted amino acid sequence of the (family 18 chitinase), and a 1.2-kb NruI fragment contain- chitinase gene, chiB, cloned from CHB101 shows 60^ ing chiB (family 19 chitinase). Probe labeling and Southern 70% identity with family 19 chitinases of Streptomyces. blotting were carried out using the Gene Images Alkphos The high similarity between chiB of CHB101 and family Direct labeling and detection system (Amersham Pharma- 19 chitinases of Streptomyces might indicate a gene trans- cia Biotech) as instructed by the manufacturer. Hybridiza- fer between these two distantly related bacteria [15]. tion was performed as described by Sambrook et al. [16]. The unpredicted discovery of the family 19 chitinase gene in the strain of B. gladioli raised the question whether 2.3. Chitinase assay the family 19 chitinase gene is speci¢c to strain CHB101, or is widely distributed among the strains of B. gladioli Chitinase production was assayed both on agar plates and the closely related species. The elucidation of the dis- and liquid culture using inorganic salts medium with tribution of family 19 chitinase genes in and/or around 0.15% colloidal chitin. For chitinase production in liquid B. gladioli is expected to bring about useful information medium, B. gladioli cells were grown in inorganic salts about the evolution of family 19 chitinase genes in eubac- medium with 0.15% colloidal chitin at 30³C and harvested teria. after 72 h of culture. Chitinase activity in the supernatant was measured as described previously using 4-methylum- belliferyl-NP,NQ,NQP-triacetylchitotriose (Sigma, USA) as 2. Materials and methods the substrate [17].

2.1. Bacterial strains, plasmid, and media 2.4. PCR and 16S rDNA sequencing

Escherichia coli DH5K was used as the host organism The 16S rDNA sequences of B. gladioli strain CHB101, for recombinant plasmids, and plasmid pUC19 was used B. gladioli type strain MAFF 311021, and other strains of as a vector for DNA cloning and sequencing. E. coli B. gladioli and Burkholderia cepacia were determined. To DH5K carrying pUC19 or its derivatives were grown in determine the sequences of extended 16S rDNA gene re- Luria^Bertani (LB) medium (broth or semi-solidi¢ed with gions corresponding to the region of nucleotides 28^1491 1.5% agar) [16] containing 50 Wg of ampicillin per ml. An in the E. coli sequence, polymerase chain reactions (PCR) inorganic salts medium [17] containing 0.15% colloidal were carried out using the primers 27f (5P-AGAGTTT- chitin was used for chitinase production of Burkholderia GATCTGGCTCAG-3P) and 1492r (5P-TACGGYTAC- cells. CTTGTTACGACTT-3P) as previously described [21]. The strains of Burkholderia species used in this study are PCR was performed directly in a 50-Wl reaction mixture listed in Table 1. The chitinase production of all strains of containing 1 Wl total DNA, 5 Wl bu¡er (60 mM

B. gladioli and Burkholderia spp. (obtained from the Min- (NH4)2SO4, 100 mM KCl, 1.2 M Tris^HCl, pH 8.0, 1% istry of Agriculture, Forestry and Fisheries of Japan Triton X-100, 0.01% BSA), 2 mM of each dNTP, 25 mM 31 (MAFF)) [18] was compared with that of B. gladioli strain MgCl2, 0.5 WM of each primer and 2.5 U Wl TaqI DNA CHB101. polymerase. Ampli¢cations were carried out on a dry block thermocycler (Gene Amp PCR system 9700) using 2.2. Recombinant DNA techniques the following program: 30 cycles of denaturation (30 s at 94³C), annealing (15 s at 55³C) and extension (30 s at Genomic DNAs were prepared as described by Ausubel 74³C). PCR products were cloned into the pUC19, trans-

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Table 1 List of Burkholderia strains used in this study Species Strain designation MAFF accession No. Other strain designation GenBank accession No. Source B. gladioli 311021T ATCC10248 X67038 Gladiolus (USA) 302408, 302410 Vigna angularis (Japan) 302418 Vigna radiata (Japan) 302424, 302427, 302430, 302433 Cymbidium (Japan) 302515, 302518, 302521, 302524 Tulip (Japan) 302533 Soil (Japan) 302911, 302918, 302386, 302543 Oryza sativa (Japan) 301064, 301065 Freesia (Japan) 301580, 301582 Dendrobium (Japan) 301584, 301586 Vuylstekeara (Japan) 301588 Cymbidium (Japan) 301728 Vanda (Japan) Downloaded from https://academic.oup.com/femsec/article/37/2/135/473702 by guest on 04 October 2021 302385 Gladiolus (Japan) 302537 Onion (Japan) EB 8779-1a, EB 8992-1a, EB 81236-3a Grasses (Japan) B. andropogonis 302151T ATCC23061 X67037 Sorghum (USA) 301005 Sorghum (Japan) 301006 Sorghum (Japan) 301116, 301191 Carnation (Japan) 301154 Sorghum (Japan) 302546 Baby's breath (Japan) 302667 Zea mays (Japan) 302673 Sorghum (Japan) 311010 Ruscus (Japan) B. caryophylli 311024T ATCC25418 X67039 Carnation (USA) 301192 Carnation (Japan) 302881 Limonium sinuatum (Japan) B. glumae 307769T ATCC33617 U96931 O. sativa (Japan) 302412 O. sativa (Japan) 302417 V. radiata (Japan) 302462 V. radiata (Japan) B. plantarii 301723T LMG9035 U96933 O. sativa (Japan) 311030 O. sativa (Japan) 302381 Soil (Japan) B. cepacia 311023T ATCC25416 U96927 Onion, Allium cepa 302528, 302529, 302530 Soil (Japan) 302531, 302532 Soil (Japan) 302798, 302799 Hot spring (Japan) Type strains are indicated by a superscript T; MAFF, Ministry of Agriculture, Forestry and Fisheries Genebank, Japan; ATCC, American Type Cul- ture Collection, Rockville, MD, USA; LMG, BCCM/LMG Culture Collection Laboratorium voor Microbiologie Gent, Universiteit Gent, Belgium. aIsolated in this study. formed into E. coli DH5K, and sequenced. Sequencing 3. Results reactions were performed with a thermo sequenase cycle sequencing kit (Amersham Pharmacia Biotech), and DNA 3.1. Chitinase production by B. gladioli and related sequences were determined and analyzed with a Li-Cor Burkholderia spp. 4000 Automated DNA Sequencer (Aloka). Thirty strains of B. gladioli, and strains of closely re- 2.5. Phylogenetic analysis lated species (Burkholderia andropogonis, 10 strains; Burk- holderia caryophylli, three; Burkholderia glumae, four; The 16S rDNA sequences of B. gladioli strain CHB101, Burkholderia plantarii, three; B. cepacia, eight) were inves- B. gladioli type strain MAFF 311021 and other strains of tigated for extracellular chitinase production on 0.15% B. gladioli and B. cepacia were compared with those of colloidal chitin agar plates and compared with that of other L-subclass Proteobacteria obtained from the Gen- B. gladioli CHB101. B. gladioli CHB101 and 27 other Bank/EMBL/DDBJ databases. The sequences determined strains of B. gladioli including the type strain MAFF over 1400 bp were adopted for this phylogenetic analysis. 311021 were able to degrade chitin as indicated by clear Phylogenetic trees were constructed using the UPGMA haloes on chitin-containing agar plates, whereas the three method [22] of the Genetyx-Win program (version 4.0, remaining strains of B. gladioli (MAFF 301584, MAFF Software Development Co., Japan). 301586, and MAFF 301728) and all strains of closely re-

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Table 2 Chitinase production of B. gladioli and related Burkholderia spp. Strain Chitinase Strain Chitinase Strain Chitinase Strain Chitinase productiona productiona productiona productiona B. gladioli MAFF 301580 + MAFF 301116 3 B. plantarii MAFF 302408 + MAFF 301582 + MAFF 301154 3 MAFF 301723 3 MAFF 302410 + MAFF 301588 + MAFF 301191 3 MAFF 302381 3 MAFF 302418 + MAFF 302385 + MAFF 302151 3 MAFF 311030 3 MAFF 302424 + MAFF 302386 + MAFF 302546 3 B. cepacia MAFF 302427 + MAFF 302537 + MAFF 302667 3 MAFF 302528 3 MAFF 302430 + MAFF 302543 + MAFF 302673 3 MAFF 302799 3 MAFF 302433 + MAFF 311021 + MAFF 311010 3 MAFF 311023 3 MAFF 302515 + EB 8779-1 + B. caryophylli MAFF 302529 3 MAFF 302518 + EB 8982-1 + MAFF 301192 3 MAFF 302530 3 MAFF 302521 + EB 81236-3 + MAFF 302881 3 MAFF 302531 3 Downloaded from https://academic.oup.com/femsec/article/37/2/135/473702 by guest on 04 October 2021 MAFF 302524 + MAFF 301584 3 MAFF 311024 3 MAFF 302532 3 MAFF 302533 + MAFF 301586 3 B. glumae MAFF 302798 3 MAFF 302911 + MAFF 301728 3 MAFF 301169 3 MAFF 302918 + B. andropogonis MAFF 302412 3 MAFF 301064 + MAFF 301005 3 MAFF 302417 3 MAFF 301065 + MAFF 301006 3 MAFF 302462 3 aChitinase production was estimated by the ability to form clear haloes around the colony on inorganic salts agar medium containing 0.15% colloidal chitin. +, positive; 3, negative. lated species were not (Table 2). The 27 strains of B. Burkholderia species possess genes homologous to chiA gladioli also produced chitinase in inorganic salts liquid and chiB genes of B. gladioli strain CHB101, Southern medium containing 0.15% colloidal chitin but not in LB hybridization was carried out using fragments of chiA (data not shown). and chiB of CHB101 as probes. Genomic DNA was ex- tracted from various Burkholderia strains listed in Table 1 3.2. Detection of genes homologous to the family 18 and 19 and digested with EcoRI. Some of the results are shown in chitinases of B. gladioli strain CHB101 Fig. 1. Strong signals were visible with the strains of B. gladioli except the chitinase negative strains, MAFF To determine whether strains of B. gladioli and other 301584, MAFF 301586, and MAFF 301728. Signals for

Fig. 1. Southern hybridization analysis to detect genes homologous to the family 18, 19 chitinase genes of B. gladioli CHB101 in various strains of B. gladioli and B. cepacia. Chromosomal DNAs were prepared from various strains of B. gladioli and B. cepacia, digested with EcoRI, then fragments were separated on a 0.7% agarose gel and transferred to a Hybond-N nylon membrane. The membranes were probed with the following fragments of B. gladioli strain CHB101 chitinase genes: a 2.1-kb EcoRI fragment, which contains chiA (family 18 chitinase), and a 1.2-kb NruI fragment containing chiB (family 19 chitinase). Lanes: 1, MAFF 302408; 2, MAFF 302911; 3, MAFF 301064; 4, MAFF 301584; 5, MAFF 301586; 6, MAFF 301588; 7, MAFF 301728; 8, MAFF 311021; 9, MAFF 302529; 10, MAFF 302530; 11, MAFF 301582; 12, MAFF 301065; 13, CHB101; 14, EB 8982-1; 15, EB 8779-1; 16, EB 81236-3. The sizes of the DNA marker depicted in lane M are 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12 kb (kb DNA ladder, Stratagene).

FEMSEC 1270 9-10-01 H. Kong et al. / FEMS Microbiology Ecology 37 (2001) 135^141 139 chiA were identical among B. gladioli MAFF 302408, for chiB (family 19 chitinase) gene were identical (Fig. 1B) MAFF 302911, MAFF 301582, EB 8982-1, EB 8779-1 in all strains tested except for MAFF 311021. The remain- and EB 8123-3, and among B.gladioli MAFF 301064, ing B. gladioli strains were also positive for chiA and chiB MAFF 301065, MAFF 311021, and CHB101. These B. probes (data not shown). The closely related Burkholderia gladioli strains, positive for the chiA probe, were also pos- species B. andropogonis, B. caryophylli, B. glumae, B. plan- itive for the chiB probe, indicating that these strains pos- tarii, and B. cepacia were negative for both probes (data sess family 19 chitinase genes. The patterns of the signals not shown). Downloaded from https://academic.oup.com/femsec/article/37/2/135/473702 by guest on 04 October 2021

Fig. 2. Phylogenetic tree obtained with the 16S rDNA sequences of members of Burkholderia species. The phylogenetic tree was calculated by the UP- GMA method. Bootstrap values shown are 80%. The scale bar indicates substitutions per site (s/s). GenBank accession numbers are enclosed in paren- theses.

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3.3. rDNA sequence analysis of B. gladioli and The high sequence similarity between family 19 chiti- related strains nases of bacteria and those of plants along with the limited distribution of the genes in prokaryotic organisms The 16S rDNA sequence (about 1.4-kb region) was de- appear to support the idea that the acquisition of family termined for the strains of B. gladioli (CHB101, MAFF 19 chitinase by bacteria occurred relatively recently [13]. 301064, MAFF 301588, MAFF 302911, MAFF 302408, The high similarity between Streptomyces and B. gladioli MAFF 301728, MAFF 301584, MAFF 301586, EB 81236- family 19 chitinases also suggests that the second gene 3, EB 8779-1, and EB 8982-1) and of B. cepacia (MAFF transfer which occurred directly or indirectly between 302529, MAFF 302530). The 16S rDNA sequence was these distantly related bacteria, was a more recent event. also determined for the type strain MAFF 311021. The The fact that chiA (family 18) and chiB (family 19) of sequences were aligned and compared with the closely re- strain CHB101 are found together indicates that B. gla- lated sequences in the GenBank database. A phylogenetic dioli concurrently acquired both genes. While chiA and tree was inferred using the UPGMA method (Fig. 2). chiB have been cloned independently from strain Strains MAFF 301064, MAFF 301588, MAFF 302911, CHB101 [15], they may be located close together in the Downloaded from https://academic.oup.com/femsec/article/37/2/135/473702 by guest on 04 October 2021 MAFF 302408, EB 81236-3, EB 8779-1, CHB101, and genome. Chromosomal walking from/to chiA or chiB in EB 8982-1 form a cluster with the B. gladioli type strain these strains might reveal the evidence of the mode of their MAFF 311021. Burkholderia cocovenenans and B. gladioli transfer. represent the same taxon [23]. At this point, the question arises as to why the chitinase The three remaining strains assigned as B. gladioli, genes have been retained stably in B. gladioli. B. gladioli MAFF 301728, and MAFF 301584 and MAFF 301586, strains have been isolated from pathogenic lesions of appeared to be closer to B. glumae and B. cepacia, respec- plants. B. gladioli strains EB 8779-1, EB 8982-1, and EB tively. It is, therefore, indicated that these three `B. gla- 81236-3 have been isolated from guinea grass (Panicum dioli' strains, which are negative in chitinase production maximum) as endophytic bacteria by smashing the stems. and lack a family 19 chitinase gene, should be reas- Thus B. gladioli strains seem to have a close relationship signed. with plants as plant pathogens or endophytic bacteria. Production of chitinase might be advantageous for B. gladioli to survive in/on plants by competing with 4. Discussion plant-associated fungi. Family 19 chitinases are di¡erent from family 18 chitinases in many respects. For example Chitinases are produced by various bacteria including the antifungal activity found in family 19 chitinases is the genera Serratia, Vibrio, Bacillus, Streptomyces, etc. considered to be more e¡ective than that in family 18 Genes for chitinases from these bacteria have been ana- chitinases. Chitinase C of S. griseus HUT6037, the ¢rst lyzed. To our knowledge, however, Burkholderia have not family 19 chitinase found in bacteria, exhibited a remark- been reported as chitinase producers. The identi¢cation of able ability to inhibit hyphal extension of Trichoderma strain CHB101 of B. gladioli, the ¢rst example of a Gram- reesei [13]. The antifungal activity of other plant family negative bacterium producing family 19 chitinase [15], 19 chitinases (classes I, II, and IV) has been demonstrated prompted us to examine the distribution of the gene ho- [24^26]. It is, therefore, implied that antifungal activity mologous to the chiB of CHB101, a family 19 chitinase may be a general property of family 19 chitinases. B. gla- gene, in strains of B. gladioli and closely related species. dioli CHB101 has been isolated as an antagonist of plant In Streptomyces, possession of family 19 chitinase genes pathogenic fungi. The production of family 19 chitinase seems to be a common characteristic of the genus [11,12]. thus is expected to contribute to the antagonistic ability of In contrast, the distribution of family 19 chitinase genes in this strain. Burkholderia is shown to be restricted to the species B. gladioli has attracted attention as a clinical micro- B. gladioli. As shown in Fig. 2, B. gladioli strains can be organism. B. gladioli colonizes the respiratory tracts of divided into two subgroups according to their rDNA se- patients with cystic ¢brosis and chronic granulomatous quence similarity. One subgroup contains CHB101, the disease [27]. It is important for clinical microbiology labo- type strain MAFF 311021, and B. cocovenenans. The other ratories to distinguish B. gladioli from B. cepacia complex. subgroup consists of strains isolated from various plants However, because of the high degree of phenotypic simi- in Japan. These two subgroups do not represent geological larity between these two species, accurate identi¢cation is di¡erences since strain CHB101 was isolated in Japan. di¤cult. The presence of genes homologous to chiA and Because neither B. glumae nor B. plantarii has a family chiB of CHB101 is a highly speci¢c characteristic of 19 chitinase gene, it is apparent that chiB, a family 19 B. gladioli. By hybridization with gene probes or by de- chitinase gene, was transferred after the divergence of signing PCR primers for the genes, B. gladioli should be the B. glumae^B. plantarii branch and the B. gladioli able to be distinguished from other closely related species branch had occurred. with a high degree of speci¢city and sensitivity.

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