Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce

International Journal of Food Microbiology 148 (2011) 60–65

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce Masataka Satomi a,⁎, Manabu Furushita b, Hiroshi Oikawa c, Yutaka Yano a a b c

National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa-ku, Yokohama 236-8648, Japan Department of Food Science and Technology, National Fisheries University, 2-7-1 Nagata-Honmachi, Shimonoseki 759-6595, Japan National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan

a r t i c l e

i n f o

Article history: Received 14 December 2010 Received in revised form 14 April 2011 Accepted 29 April 2011 Available online 7 May 2011 Keywords: Fish sauce Halophilic lactic acid bacteria Histamine Histidine decarboxylase Plasmid Tetragenococcus halophilus

a b s t r a c t Nineteen isolates of histamine producing halophilic bacteria were isolated from four fish sauce mashes, each mash accumulating over 1000 ppm of histamine. The complete sequences of the plasmids encoding the pyruvoyl dependent histidine decarboxylase gene (hdcA), which is harbored in histamine producing bacteria, were determined. In conjunction, the sequence regions adjacent to hdcA were analyzed to provide information regarding its genetic origin. As reference strains, Tetragenococcus halophilus H and T. muriaticus JCM10006 T were also studied. Phenotypic and 16S rRNA gene sequence analyses identified all isolates as T. halophilus, a predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR, Southern blot, and complete plasmid sequencing) of the histamine producing isolates confirmed that all the isolates harbored approximately 21–37 kbp plasmids encoding a single copy of the hdc cluster consisting of four genes related to histamine production. Analysis of hdc clusters, including spacer regions, indicated N 99% sequence similarity among the isolates. All of the plasmids sequenced encoded traA, however genes related to plasmid conjugation, namely mob genes and oriT, were not identified. Two putative mobile genetic elements, ISLP1-like and IS200-like, respectively, were identified in the up- and downstream region of the hdc cluster of all plasmids. Most of the sequences, except hdc cluster and two adjacent IS elements, were diverse among plasmids, suggesting that each histamine producers harbored a different histamine-related plasmid. These results suggested that the hdc cluster was not spread by clonal dissemination depending on the specific plasmid and that the hdc cluster in tetragenococcal plasmid was likely encoded on transformable elements. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Histamine, one of the primary causative agents of food related intoxication, occasionally accumulates in fermented foods such as fermented fish products, wine, and cheese (Ladero et al., 2010; Spano et al., 2010; Silla Santos, 1996). Its production is the result of bacterial decarboxylation of L-histidine (van Poelje and Snell, 1990) which in the case of fermented foods, is performed by gram-positive microbes known as histamine producing bacteria (Calles-Enríquez et al., 2010; Hernandez-Herrero et al., 1999; Kimura et al., 2001; Kobayashi et al., 2000; Konagaya et al., 2002; Landete et al., 2005; Le Jeune et al., 1995; Lonvaud-Funel, 2001; Lonvaud-Funel and Joyeux, 1994; Sato et al., 1995; Satomi et al., 1997, 2008; Taylor, 1986; Udomsil et al., 2010; Yatsunami and Echigo, 1991; Yokoi et al., 2011). These bacteria are

⁎ Corresponding author at: Biochemistry and Food Technology Division, National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan. Tel.: + 81 45 788 7670; fax: + 81 45 788 5001. E-mail address: [email protected] (M. Satomi). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.04.025

typified by the presence of pyruvoyl dependent histidine decarboxylase (HDC) (Coton et al., 1998; De las Rivas et al., 2008; Huynh and Snell, 1985; Joosten and Northolt, 1989; Recsei et al., 1983; Vanderslice et al., 1986; van Poelje & Snell, 1990) encoded by the structural gene hdcA. This gene, along with hdcP, hdcB, and hdcRS, comprise the hdc cluster, a sequence region related to histamine production shown to be conserved in several lactic acid bacteria (Lucas et al., 2005; Martín et al., 2005; Satomi et al., 2008). Fish sauce is a common, traditional fermented condiment in Southeast and East Asia. Recently, Japan has dramatically increased fish sauce production as its fishing industry tries to reduce waste by making full use of fish materials (Funatsu et al., 2000). Halophilic lactic acid bacteria, primarily Tetragenococcus spp., were isolated as the dominant microbe during Japanese fish sauce fermentation where they play a crucial role in reducing the pH of fish sauce mash (Ito et al., 1985a, b; Sato et al., 1995; Taira et al., 2007; Fujii et al., 2008). However, it has been shown that certain Tetragenococcus strains also cause histamine accumulation in fish sauce production (Kobayashi et al., 2000; Sato et al., 1995; Satomi et al., 1997). Our previous report (Satomi et al., 2008) demonstrated that a) a histamine producing

M. Satomi et al. / International Journal of Food Microbiology 148 (2011) 60–65

strain harbored an approximately 30 kbp plasmid (pHDC) encoding a single copy of the hdc cluster including hdc A; b) the nucleotide sequence of hdc cluster shared N99% sequence similarity with that of Lactobacillus hilgardii 0006, L. sakei LTH2076, Oenococcus oeni 9204, three non-halophilic lactic acid bacteria phylogenetically distinct from Tetragenococcus spp.. These results suggested that the origin of hdc cluster in these lactic acid bacteria was the same, that gene transfer occurred over a relatively short time, and that the hdc cluster was able to be transferred between genera. However studies for gene prevalence of the histamine producing factor among halophilic lactic acid bacteria isolated from fish sauce have only recently begun. The aims of this study were to determine the complete sequences of the plasmids encoding hdc cluster in histamine producing bacteria and to analyze the sequence region adjacent to hdc cluster. This analysis should provide information regarding the origin of the hdc cluster, as its highly conserved sequence is present in several different genera of lactic acid bacteria suggesting horizontal genetic transfer.

2. Materials and methods 2.1. Samples and isolation of histamine producing bacteria Samples were obtained from four different lots of fish sauce mashes (each consisting of deep sea smelt or flying fish meat, Koji starter, and sodium chloride) manufactured in Japan (Table 1). All of the fish sauce mashes accumulated over 1000 ppm of histamine. Enrichment culture and limiting dilution methods using histidine broth (HB; 1% glucose, 1% peptone, 0.2% yeast extract, 0.1% L-histidine, 10% NaCl and 50% of sea water, pH 6.5) were employed to isolate histamine producing bacteria from the fish sauce mashes as described previously (Satomi et al., 2008). Samples were diluted 10 7-fold with HB and 100 μl of cell suspension were dispensed into 96-well plates and incubated at 30 °C for 6 days. Histamine production was evaluated using a commercial histamine measurement kit (Kikkoman, Noda, Japan). Histamine producing isolates were purified from histamine positive wells by streaking onto HA (1.5% of agar was supplemented with HB). Unless otherwise indicated, growth conditions for halophilic bacteria were at 30 °C, for 3 days in HB or for 6 days on HA. T. halophilus H (Satomi et al., 2008) and T. muriaticus JCM10006 T were studied as reference strains of histamine producer.

2.2. Bacterial identification Morphological characteristics of the new isolates were observed by microscope using a commercial gram staining kit (Merck, Darmstadt, Germany). Growth characteristic tests, temperature, pH, and NaCl tolerance, were performed as described previously (Satomi et al., 2008). 16S rRNA gene was amplified by using the PCR method with a universal primer set (Weisburg et al., 1991) and sequenced. The BLAST algorithm (Altschul et al., 1997) was used to examine the phylogenetic relationship of isolates to known sequences in the Genbank, EMBL, and DDBJ databases.

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2.3. Gene location and copy number of hdcA Isolates grown in HB were suspended in TE buffer (pH 8.0) and treated with lysozyme (final conc. 2 mg/ml; Wako, Osaka, Japan) for cell wall lysis. After lysozyme treatment, total DNA was extracted and purified by a standard method (Johnson, 1981). Plasmid DNA (up to 50 kbp in size) was extracted using the QIAprep spin miniprep kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. The presence of hdcA was determined using PCR with specific primer sets as described previously (Satomi et al., 2008). To determine hdcA location and copy number in bacterial DNA, southern blotting utilizing a specific detection probe amplified with PCR was performed as previously detailed (Satomi et al., 2008). Native and Not I digested DNA from the new isolates and T. muriaticus JCM10006 T were loaded into 0.3% agarose gel (Nippon gene, Toyama, Japan) for electrophoresis using TAE buffer at 4 °C for 24 h. After electrophoresis, DNA fragments in agarose gel were transferred onto a Hybond-N+ membrane (GE healthcare, Hino, Japan) and hybridized with the hdcA specific DNA probe according to manufacturer's protocol for ECL kit (GE healthcare). The DNA probe corresponded to a 563 bp internal region of the hdcA amplified by PCR from the total DNA of histamineproducing T. halophilus H using the HmF-HmR primer set. The probe was labeled with alkali phosphatase using the AlkPhos direct kit (GE healthcare), and detected by chemiluminescence with CDP-star (GE healthcare) according to manufacturer's protocol. Unless otherwise indicated, Taq polymerase for PCR and restriction enzymes were purchased from Takara bio, Shiga, Japan. 2.4. Gene diversity of plasmid encoding hdcA To study gene diversity among histamine producing bacteria, plasmid typing was performed by restriction fragment length polymorphism (RFLP) analysis with HindIII digestion of plasmid DNA. Plasmid groups were determined based on banding patterns of electrophoresis. A total of 21 isolates including 19 new isolates and two reference strains were classified (Table 2) and a representative isolates was selected from each group for the whole sequence analysis. 2.5. Plasmid sequence To determine the complete sequence of plasmids, shotgun cloning was performed and a DNA library covering the whole plasmid encoding hdcA was constructed. The EcoRI or HindIII-digested plasmid DNA fragments were ligated with pUC118 (Takara), and transformed into E. coli DH5α (Takara). Positive clones for hdcA were selected using colony hybridization with hdcA specific probe and insert sequences were determined with sequence primers, M13 (−21) (5′-GTAAA ACGAC GGCCA GT-3′) and M13RV (5′-CAGGA AACAG CTATG AC-3′). DNA sequences were determined using an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA) with the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit following the manufacturer's directions (ABI). Gaps in the plasmid molecule were closed by primer walking. To determine the sequence

Table 1 List of the fish sauce samples. Sample

Fish Fish Fish Fish Fish a

sauce sauce sauce sauce sauce

Lot

Aa Ba Ca Da E

A B C D E

Same fish sauce manufacture.

Product year

Material and fermentation methods Fish material

Mold starter (koji)

Salt conc. (w/v)

Fermentation period

Reference

2002 2002 2007 2007 1997

Deep sea smelt Flying fish Deep sea smelt Deep sea smelt Squid liver

Added Added Added Added –

20.5% 18.9% 17.6% 17.6% 20.7%

8 months 8 months 8 months 8 months 24 months

Taira et al., 2007 Taira et al., 2007 – – Fujii et al., 2008

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M. Satomi et al. / International Journal of Food Microbiology 148 (2011) 60–65

Table 2 Summary of the characteristic of plasmids encoding hdc cluster in harbored in histamine producing Tetragenococcus strains. Strainsa

Plasmid designation

Sourceb

H F A I RO HA NI HO HE TO TI RI NU RU WO WA KA YO TA RE JCM10006T

pHDC-H pHDC-F pHDC-A pHDC-I pHDC-RO pHDC-HA pHDC-NI pHDC-HO pHDC-HE pHDC-TO pHDC-TI pHDC-RI pHDC-NU pHDC-RU pHDC-WO pHDC-WA pHDC-KA pHDC-YO pHDC-TA pHDC-RE pHDC-Tm

Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish

a b c d e f g

sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce sauce

A A B C C C C C C C C D D D D D D D D D E

Identificationc

RFLPd typing

Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. halophilus Tc. muriaticus

I I II III III III III IV IV IV IV V V V V V V V V V VI

Full sequence

Length (bp)e

putative replication type (closest sequence)f

+ + + + – – – + – – – + – – – – – – – – +

29,924⁎ 29,924⁎ 32,501⁎ 23,071⁎ 23 k 23 k 23 k 36,638⁎

Theta Theta RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR RCR

37 k 37 k 37 k 21,231⁎ 21 k 21 k 21 k 21 k 21 k 21 k 21 k 21 k 21,516⁎

Ped. pentosaceus pMD136 Ped. pentosaceus pMD136 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4 St. haemolytics pLNU4

oriT + + – – – – – – – – – – – – – – – – – – –

Tng

Reference

Accession number

+ + + + + + + + + + + + + + + + + + + + +

Satomi et al., 2008 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

AB362339 – AB588176 AB588179 – – – AB588177 – – – AB588180 – – – – – – – – AB588178

All of strains produced histamin in histidine broth. Fish sauce A, B, C, and D were produced by same manufacturer. Isolates were identified based on 16S rRNA gene sequence and phenotypic characteristics. Tc; Tetragenococcus. RFLP; Restriction fragment length polymorphism. *; Data from full length sequence, no mark; length was estimated based on an agarose electrophoresis analysis. Ped.; Pediococcus, St.; Staphylococcus, RCR; Rolling circle replication. Transposons were coded upstream and downstream of hdc cluster.

gaps resulting from shotgun cloning, primers were designed to the contig ends; PCR amplicons generated with these primers were sequenced as described previously. Unless otherwise indicated, all of the genetic procedures were carried out according to the standard methods (Sambrook et al., 1989). Sequence data were edited and analyzed using the GENETYX computer software (Software Development Company, Tokyo, Japan).

21–37 kbp in size (data not shown), were subsequently digested using EcoRI and HindIII separately and southern blotting performed with the hdcA specific detection probe. A single clear band was shown from digested DNA at 2.5 kbp and 7.0 kbp (EcoRI and HindIII digests, respectively; data not shown) demonstrating that a single copy of hdcA was present on the plasmid in the histamine producing isolates. 3.3. Typing of plasmids encoding hdcA

3. Results 3.1. Isolation and identification of histamine producing bacteria A total of 19 histamine producing isolates were isolated from the four different lots of Japanese fish sauce used in this study (Table 2). All of the isolates were identified as Tetragenococcus halophilus based on phenotypic characteristics (gram-positive, halophilic cocci, and lactic acid production) and sequence similarity of 16S rDNA (N99% similarity with T. halophilus: accession number D88668). 3.2. Gene location and copy number of hdcA The presence of hdcA was demonstrated by using PCR with genespecific primer sets as described previously (Satomi et al., 2008); histamine producing isolates were judged to contain hdcA based on appropriate banding visualized using gel electrophoresis (data not shown). Amplified hdcA fragments from the new isolates were sequenced and compared with sequences in the database indicating a high sequence similarity (N99%) with the same region of hdcA of lactic acid bacteria. To determine the gene location and copy number of hdcA in bacterial DNA, southern blotting was performed for new isolates and reference strains utilizing an hdcA specific detection probe amplified using PCR. Undigested and Not I digested DNA (total and plasmid) were loaded into the same agarose gel in different lanes and analyzed. A single band of hdcA was present at a position separate from genomic DNA band as described previously (Satomi et al., 2008). The plasmids, estimated by mobility of Not I digested plasmid to be approximately

The result of RFLP analysis indicated that the 21 plasmids (19 new isolates and 2 reference strains) encoding hdcA extracted from tetragenococcal isolates were divided into six genetic groups (Table 2). With the exception of those isolated from fish sauce C, all bacterial plasmids isolated from a given fish sauce were categorized into the same genetic group; plasmids from fish sauce C were separated into two genetic groups. One isolate was selected from each genetic group as the representative plasmid (Table 2) and designated as follows: Group I, pHDC-F from isolate F; Group II, pHDC-A from isolate A; Group III, pHDC-I from isolate I; Group IV, pHDC-HO from isolate HO; Group V, pHDC-RI from isolate RI; and Group VI, pHDC-Tm from T. muriaticus JCM10006 T. 3.4. Plasmid sequence Complete sequences of the five representative plasmids nominated from each genetic group were performed as previously described (Satomi et al., 2008); a summary of sequence analysis is shown in Fig. 1. Detailed information of each plasmid is presented in Supplementary Tables 1–3. The plasmids could be distinguished from each other by size, ranging from 21,231 to 36,638 bp, as indicated from RFLP analysis. All of the plasmids encoded the four known genes related to histamine production, hdcP, hdcA, hdcB, and hdcRS; these hdc clusters, including the internal spacer regions, shared sequence similarity to the previously reported sequences of T. halophilus H (Satomi et al., 2008) and L. hilgardii 0006 (Lucas et al., 2005) with N99% homology. Additionally, the order of genes within hdc cluster was identical to the sequences of L. buchneri B301, L.

M. Satomi et al. / International Journal of Food Microbiology 148 (2011) 60–65

these genes in pHDC-I and pHDC-RI were different from the others. Almost all ORFs and functional sequences found in this study corresponded to that of lactic acid bacteria deposited in the database. The plasmid pHDC-F and pHDC-H, which was reported previously (Satomi et al., 2008), coded putative oriT and mob gene cluster related to plasmid transfer, but putative oriT region and its related genes were not determined from other plasmids.

hilgardii 0006, L. reuteri F275, L. sakei LTH2076, Oenococcus oeni 9204, and T. halophilus H. Two putative insertion sequences (IS) positioned in the adjacent regions to the hdc cluster were found as conserved sequences, although many transposase genes including truncated genes were determined around the hdc cluster from sequenced plasmids. They shared high sequence similarity with known IS's including ISLP1-like (Nicoloff and Bringel, 2003) and putative truncated IS200-like (Beuzón et al., 2004), respectively. Although IS1216V (Heatona et al., 1996) was also found upstream of ISLP1-like on most plasmids, it was not determined on pHDC-A. Two putative IS, ISLP1-like and IS1216V, were positioned at the upper region of hdc cluster accompanied with putative inverted repeat sequence (IR) at both side of genes (Fig. 1). Interestingly, the replication region of plasmids showed diversity, indicating that the origin of the plasmids was different. The sequence types of putative plasmid replication were divided into two groups; theta type replication (pHDC-H and pHDC-F) and rolling circle replication (pHDC-A, pHDC-HO, pHDC-Tm, pHDC-I and pHDC-RI). The sequence of rolling circle replication type was identical to the same region of Staphylococcus haemolytics plasmid pLNU4 (Lüthje et al., 2007) sharing 100% sequence similarity. The sequence of pHDC-H and pHDC-F was completely identical, indicating that both isolates were clones or plasmid transferred. The plasmids that coded the same putative replication system of pHDC-A shared the same plasmid maintenance genes, although other parts of the plasmids were different. The plasmid pHDC-I and pHDC-RI had the same plasmid maintenance systems (parA, pinR, and soj) as that of pHDC-A, pHDC-HO, and pHDC-Tm, but the order and disposition of

pHDC-H, -F

IS lp1

IS 1216V

Sugar metabolism

ori+rep

IS lp1

Amino acid metabolism

(Sample B) 32 501bp 32,501bp

IR

IR

IS 4

Tn 11

DNA modify

Plasmid maintenance

IR

IS 1216V

IS lp1

IS 200

HDC

Tn 11

Plasmid maintenance

IS 200

IR

IR

Amino acid metabolism

ND

IS 1216V

Plasmid maintenance IR

HDC

Tn 11

IS 200

Plasmid maintenance

DNA modify

IR

IR

ori+rep

(Sample D) 21,231bp

IS lp1

IS 1216V

IR

(Sample E) 21,516bp

oriT

ori+rep

(Sample (S l C) 36,638bp

pHDC-Tm HDC T

IS 200

HDC

IR

pHDC-RI pHDC RI

Plasmid maintenance

ori+rep

((Sample p C)) 23,071bp

pHDC-HO

IS 200

HDC

IR

IR

pHDC-I

The full sequences of tetragenococcal plasmids encoding hdcA were determined and analyzed, focusing on adjacent sequences to the hdc cluster. A summary of plasmid sequence data is shown in Fig. 1 and Supplementary Tables 1–3. Lucas et al. (2005) suggested the possibility of gene transfer among lactic acid bacteria based on the high sequence homology of hdcA. Our previous study (Satomi et al., 2008) showed that pHDC-H coded oriT and mob genes, which were related to plasmid transfer (Giacomini et al., 2000; Kantor et al., 1997) and hypothesized that the spread of the hdc cluster among isolates in the same manufactured fish sauces could be due to plasmid transfer. In this study, histamine producing bacteria were isolated from manufactured fish sauce in different product lots and we investigated the possibility of plasmid transfer using mob gene determined from pHDC-H. Sequence similarity of the hdc clusters was almost identical among plasmids isolated from the fish sauce preparations in this study as well as to plasmids obtained from T. muriaticus JCM10006 T previously isolated from Japanese squid liver sauce. Moreover, the

oriC+rep

(Sample A) 29,924bp

pHDC-A

4. Discussion

IR

IR

IR

63

IS lp1

HDC

IS 200

Tn 11

IS 4

ND

IR

IR

ori+rep

IS 1216V

IS lp1

HDC

IS 200

Tn 11

Plasmid maintenance

DNA modify

Fig. 1. Physical and genetic maps of plasmids encoding hdc in Tetragenococcus isolates. A box indicates putative function predicted from sequence similarity. Arrows indicate transcriptional direction of gene clusters. The box “sugar metabolism” represents the six genes related to sugar metabolism (Sugar phosphatase isomerase/epimerase, H+/gluconate symporter related permease, 2-dehydro-3-deoxygluconokinase, Orotidine 5′-phosphate decarboxylase, Sugar phosphatase isomerise, and KDPG and KHG aldolase); “HDC” represents the four genes of the hdc pathway (hdcP, hdcA, hdcB and hdcRS); “Plasmid maintenance” represents the two genes of plasmid maintenance (pinR and parA or soj); “oriT” represents the four genes related to plasmid mobilization (oriT, mobC, mobA, and mobB); “amino acid metabolism” represents the four genes of amino acid metabolism (L-alanine dehydrogenase, threonine dehydratase, and two amino acid permease); “DNA modify” represents the two genes of DNA modification (DNA restriction subunit type III and methyltransferase type III R–M system). The highest degree of similarity of each ORF is shown in Supplementary Tables 1, 2, and 3. IR, inverted repeat sequence. ND, unidentified orfs. Region coding ISLP1, HDC, and IS200 was conserved among all plasmids with 99% of sequence similarity.

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M. Satomi et al. / International Journal of Food Microbiology 148 (2011) 60–65

sequence of pHDC-Tm was significantly close to pHDC-A, in spite of the geographically disparate locations where the hosts were isolated. It was expected that all plasmids would code genes related to plasmid conjugation, such as mob genes and oriT. However, with the exception of pHDC-H and -F, these genes did not appear on plasmids sequenced in this study. Instead, all of the plasmids shared traA which encodes for a protein that makes nicks on DNA chains to initiate conjugation (Horii et al., 2002). It is known that plasmid conjugation operates via a type IV secretion system and is managed by a complicated relationship of several genes (Kurenbach et al., 2006). It is nearly impossible that traA could carry out plasmid conjugation alone, and the existence of traA alone on the plasmids suggested that these were originally conjugative plasmids. Supporting these results, analysis of the full sequences of representative hdc cluster encoded plasmids demonstrated a variety of plasmid lengths and gene constructions suggesting that the spread of histamine production during individual fish sauce manufacturing was not a clonal property. For instance, although the sequence length of pHDC-HO was 4 kbp longer than pHDC-A, almost all sequences in the two plasmids, except one transposase gene and an unidentified ORF, were identical to each other. Likewise, pHDC-Tm encoded the same putative replication region as pHDC-A, but it lacked amino acid metabolite genes, indicating that pHDC-Tm consisted of only the hdc cluster, plasmid maintenance genes, and three transposase genes. The hosts of pHDC-I and pHDC-HO were isolated from the same fish sauce production lot, but structure of plasmids were different each other. These results indicated that hdc cluster was not coded on the specific plasmid. Previous reports (Lucas et al., 2005, 2008; Satomi et al., 2008) indicated that hdc clusters in the lactic acid bacteria have phylogenetic relationships among them and may even be transferred between genera. Yet, as mentioned previously, the possibility of whole plasmid transfer by mob genes was low for the bacteria isolated in this study. Transposons or DNA recombination represent possible mechanisms for the spread of hdc cluster among lactic acid bacteria based on the high sequence homology of hdc cluster shared within the group and the several insertion sequences including truncated genes identified in plasmids encoding the hdc cluster. In particular, two putative IS positioned in the up- and downstream regions of hdc cluster were predicted as candidates of causative genes for transport; as with the hdc cluster, both putative transport genes were conserved among plasmids sequenced in this study. Insertion sequence element ISLP1like may function as a mobile genetic element as IR sequences were present on both sides of the genes and it shared high sequence similarities with that of the known transposase, ISLpl1, which has previously been shown to transfer among lactic acid bacteria and may play a role in LAB genome plasticity and adaptation to their environment (Nicoloff and Bringel, 2003). Insertion sequence IS200like was also found downstream of hdc cluster among all of plasmids, but it was a putatively truncated gene. If both IS were still active and maintained ability of gene transfer, the IR sequences would be necessary. On the plasmids related to pHDC-A, 24 bases of the IR sequence were maintained, which seems a meaningful length. However 12 bases of 5′ end of the IR on pHDC-H, -F, -I, -HO, -RI and -Tm were defective. Almost all IR sequences of ISLP1-like transposons deposited in the database consist of more than eight bases; there was no information about active IR sequence consisting of less than eight bases. It is possible that these short IR sequence on the plasmids may not be recognized as IR elements by transposons. Insertion sequence IS1216V also was found upstream of ISLP1-like on all plasmids with the exception of pHDC-A and a gene similar to the transposon 11 superfamily was determined downstream of IS200-like gene from all plasmids except pHDC-H. However neither mobile genetic element was considered related to transfer of the hdc cluster as they were not shared among all of plasmids. The present data does not prove that transposons were able to transfer the hdc cluster to other plasmid or genomes. Additionally, the hdc clusters found in the lactic acid

bacteria L. buchneri B301 (Martín et al., 2005), L. reuteri F275 (NC_009513), and Streptococcus thermophilus (Calles-Enríquez et al., 2010) were encoded on the chromosome. In the case of Streptococcus thermophilus (Calles-Enríquez et al., 2010) and Staphylococcus epidermidis TYH1 (Yokoi et al., 2011), evidence of horizontal transfer was reported. According to these data, gene transfer of hdc cluster coded in tetragenococcal plasmid may be caused by DNA recombination rather than transposon. Supporting this, it was observed that aside from the hdc cluster, the order and arrangement of genes in each plasmid was different, though the sequences of each corresponding ORF among the plasmids were the same. Furthermore, pHDC-I, -RI, and -Tm coded only the hdc cluster as a useful gene for survival strategy, though two other kinds of plasmids coded sugar metabolism or amino acid utilization genes. Histamine production of lactic acid bacteria is known as one of the stress responses against the decreasing pH by lactic acid production during fermentation (Kimura et al., 2001). Therefore, hdcA may be an important gene for bacterial survival in low pH environment. When bacteria obtain the hdc cluster from others, either strategically or accidentally, they may positively keep it under high selective pressures. It was known that antimicrobial reagent resistant genes distributed on bacterial DNA are transferred as clusters composed of several genes (Rice and Carias, 1998). However in most cases, the mechanisms of these gene transfers remain unclear. In order to resolve the origin of hdc cluster and its transfer mechanism, further study focusing DNA recombination among lactic acid bacteria is necessary. Supplementary files mentioned in the text, no alt-e-component provided at doi:10.1016/j.ijfoodmicro.2011.04.025.

Acknowledgments The authors thank Dr. Funatsu, T. Takano, and K. Shouzen for sample supply. The technical assistance of N. Hatano and N. Sakai is gratefully acknowledged. Dr. James Bruckner and Shariff Osman are acknowledged for their helpful discussions.

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