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Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish
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International Journal of Food Microbiology 121 (2008) 116 – 121 www.elsevier.com/locate/ijfoodmicro
Short communication
Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish Shiro Itoi, Takeshi Abe, Sayaka Washio, Erika Ikuno, Yuna Kanomata, Haruo Sugita ⁎ Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa, 252-8510, Japan Received 21 December 2006; received in revised form 16 August 2007; accepted 6 November 2007
Abstract We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides (AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment. © 2007 Elsevier B.V. All rights reserved. Keywords: Halotolerant strain; Lactic acid bacteria; Lactococcus lactis; Marine fish; Takifugu niphobles
1. Introduction Lactococcus lactis is used worldwide for the manufacture of fermented dairy products, such as in the cheese-making process. L. lactis strains used for dairy production have been genetically subdivided into L. lactis subsp. lactis and L. lactis subsp. cremoris. It is necessary to differentiate L. lactis strains as either subsp. lactis or cremoris, since they show different characteristics in cheese production. This differentiation has traditionally been carried out on the basis of phenotypic traits such as growth temperature, salt tolerance and the ability to hydrolyze arginine (Mundt, 1986). Comparison of DNA sequence data has also shown genetic differences between the two subspecies (Ward et al., 1998). In addition, the two genetic types of L. lactis have
⁎ Corresponding author. Tel./fax: +81 466 84 3679. E-mail address: [email protected] (H. Sugita). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.11.031
been further divided into two or three phenotypes (Kelly and Ward, 2002). In these L. lactis types, the majority of strains used as dairy starters can be grouped into a small number of genetic lineages, and are not representative of the diversity found in L. lactis as a whole (Kelly and Ward, 2002). Actually, although new strains for starters have been screened primarily from milk products, the variability is low among strains used in industrial dairy fermentations (Salama et al., 1993). In spite of this low genetic diversity, these bacteria are also expected to function as probiotics (Kimoto et al., 2002). Isolation of new strains has been attempted from various environments including milk products and plant surfaces (Salama et al., 1995; Ulrich and Müller, 1999). Our recent study using the clone library method based on 16S ribosomal RNA (rRNA) gene sequences suggested the presence of lactic acid bacteria in the intestinal tract of the pufferfish Takifugu niphobles caught in the Shimoda, Shizuoka, Japan (Shiina et al., 2006). Results of this study indicated that L. lactis related
S. Itoi et al. / International Journal of Food Microbiology 121 (2008) 116–121
clones represented approximately 15% of the total intestinal microflora (Shiina et al., 2006). Lactic acid bacteria from marine environments are expected to show additional capabilities compared with milk- and plant-derived strains. Thus, in this study, L. lactis isolates were isolated from marine fish intestines, and were examined for their carbohydrate fermentation profiles and salt tolerance. 2. Materials and methods 2.1. Fish and bacteriological sampling Coastal fish specimens were collected with standard fishing gear at Shimoda, Shizuoka, Japan in water that was 17.5– 18.0 °C in temperature in April 2004, and 23.0 °C in June 2004. These specimens were composed of Ditrema temmincki (n = 1), Girella punctata (n = 14), Pseudolabrus japonicus (n = 1), Sebastes pachycephalus (n = 2), T. niphobles (n = 9) and Thalassoma cupido (n = 2). Each specimen was dissected aseptically after capture. The contents squeezed from the intestinal tracts were serially diluted with sterile 50% artificial seawater (ReiSea, Tokyo, Japan), inoculated in MRS broth (BD, Franklin Lakes, NJ, USA), and plated onto MRS-agar medium containing 2% agar for selection of lactic acid bacteria. Both the MRS broth and MRS-agar were prepared with 50% artificial seawater. The inoculated broth and plates were incubated at 25 °C for 5 days under aerobic conditions, since the intestinal bacteria of coastal puffer fishes were not predominantly colonized by obligate anaerobic 85 bacteria (Sugita et al., 1987). Subsequently, a loopful of bacterial culture from the 86 MRS broth was streaked on MRS-agar plates and incubated at 25 °C for 5 days. L. lactis subsp. lactis was also isolated from starter culture O-114 (Christian Hansen, Hoersholm, Denmark), which was purchased from Zao Dairy Center (Zao, Miyagi, Japan), using MRS-agar plates, and was tentatively named L. lactis subsp. lactis O-114 in this study. All isolates were examined for phenotypic properties including Gram reaction, spore formation, cellular morphology, motility, colony pigmentation, production of oxidase and catalase, and fermentation of glucose according to Ishida and Sugita (2000) for screening of lactic acid bacteria. L. lactis isolates were incubated on MRS-agar plates with or without 50% artificial seawater at 40 °C to identify the L. lactis subspecies. To test for gas production, the isolates were inoculated into phenol red broth (Ishida and Sugita, 2000) containing 1% glucose, 1% peptone, 0.5% sodium chloride and 0.0018% phenol red with a Durham tube (pH 6.28), and incubated at 25 °C for 5 days. L. lactis isolates were also incubated on MRSagar plates at 25 °C for 5 days under anaerobic conditions. Final pH of the MRS broth after 5-day culture was measured with a F22 pH meter (Horiba, Kyoto, Japan). The fermentation profile of 49 sugars and poly-alcohols (control) was recorded using the API 50 CH system (bioMérieux, Montalieu–Vercieu, France) according to the manufacturer's instructions. Since all of the L. lactis isolates were capable of growing at both 0% seawater and 0% sodium chloride at 25 °C, all tests were carried out in API 50-CHL medium at 25 °C.
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2.2. DNA extraction, PCR amplification and sequencing DNA was extracted from pure cultures according to the method of Sugita et al. (2005). The partial DNA fragments of bacterial 16S rRNA genes were amplified by PCR according to our previous report (Shiina et al., 2006) using a forward primer 20F (5′-AGAGTTTGATCCTGGCTCAG-3′, equivalent to positions 8 to 27 in Escherichia coli 16S rRNA) and a reverse primer 1500R (5′-GGTTACCTTGTTACGACTT-3′, equivalent to positions 1491 to 1509 in E. coli 16S rRNA) (Weisburg et al., 1991). PCR with a L. lactis-specific primer was carried out using two reverse primers, 685R (5′-TCTACGCATTTCACCGCTAC-3′, equivalent to positions 685 to 704 in E. coli 16S rRNA) (Lane, 1991) and LacreR specific to L. lactis (5′-GGGATCATCTTTGAGTGAT-3′, equivalent to positions 217 to 235 in E. coli 16S rRNA) (Pu et al., 2002) as follows: the reaction mixture contained genomic DNA as the template, 1 μl of 10 × Taq DNA polymerase buffer, 1.3 μl of 4.6 μM primers (20F, 685R and LacreR), 0.8 μl of 2.5 mM dNTP and 0.3 units of Taq DNA polymerase, and the total volume was brought up to 10 μl with sterile water. Direct sequencing of PCR products amplified with primers 20F and 1500R forbacterial isolates was performed for both strands with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). CLUSTALX version 1.83 (Thompson et al., 1997) was used to align the nucleotide sequences of the 16S rRNA genes of the intestinal bacterial isolates with those of the corresponding genes from other bacteria in the DDBJ/EMBL/GenBank databases that were found in BLAST searches (Altschul et al., 1997). 2.3. PCR-RFLP analysis To identify the subspecies of L. lactis, PCR-RFLP analysis was carried out essentially according to Ward et al. (1998). Briefly, a
Fig. 1. Electrophoretic patterns of PCR products amplified with three primers including a species-specific primer. The upper arrow indicates the band common to the bacterial 16S rRNA gene, whereas the lower one indicates the band specific to the Lactococcus lactis 16S rRNA gene. Lane M, 100 bp laddermolecular weight marker from 100 to 1000 bp; lanes 1 and 4, PCR product amplified with primers 20F and 685R; lanes 2 and 5, PCR product amplified with primers 20F and LacreR; lanes 3 and 6, PCR products amplified with primers 20F, 685R and LacreR.
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tions 361 to 338 in E. coli) for amplifying the partial 16S rRNA gene fragment were synthesized according to the report of Young et al. (1991). The PCR conditions employed for RFLP analysis were the same as those described previously. The RFLP analysis of the 16S rRNA gene product was performed by digesting 10 μl of the amplified product with 10 units of CfoI (Roche Diagnostics GmbH, Mannheim, Germany) or MboII (Nippongene, Tokyo, Japan). The digested samples were subjected to electrophoresis on a 3% agarose gel and stained with ethidium bromide. 2.4. Phylogenetic analysis
Fig. 2. PCR-RFLP profile of the 16S rRNA gene from the starter culture-derived L. lactis subsp. lactis and the pufferfish intestine-derived L. lactis isolates. PCR products for the partial 16S rRNA gene were treated with MboII (upper panel) and CfoI (lower panel). The restriction fragments are marked by arrows. Lane M, molecular size marker; lane 1–3, RFLP products of the pufferfish intestinederived isolates; lane 4, RFLP product of the cheese starter-derived L. lactis; U, untreated PCR product.
partial DNA fragment of the 16S rRNA gene was amplified by PCR. Primers Y1 (5′-TGGCTCAGGACGAACGCTGGCGGC3′, equivalent to positions 20 to 43 in E. coli) and Y2 (5′CCTACTGCTGCCTCCCGTAGGAGT-3′, equivalent to posi-
A phylogenetic tree was constructed by the neighbor-joining method (Saitou and Nei, 1987) based on paired alignments of nucleotide sequences of the bacterial 16S rRNA genes. A set of sequences for the 16S rRNA gene from Bacillus subtilis NCDO 1769T (DNA database accession number X60646) was used as the outgroup. 2.5. Growth in 0–200% seawater and 0–7% sodium chloride To examine the salt tolerance of isolates from marine fish and L. lactis subsp. lactis from the cheese starter culture, approximately 109 cells/ml of each of the four MRS cultures were cultured at 25 °C for 2 days. These cultures were inoculated onto MRS-agar plates prepared with 0, 20, 40, 60, 80, 100, 150 and 200% artificial seawater (final concentrations) corresponding to
Fig. 3. Phylogenetic tree constructed by the neighbor-joining method based on the partial nucleotide sequences of the 16S rRNA gene from bacteria related to lactic acid bacteria. Numbers at the branches denote the bootstrap percentages for 1000 replicates. AB278679–AB278680 and AB285124 in parentheses indicate the accession numbers deposited in the DDBJ/EMBL/GenBank databases in this study and the accession numbers for reference sequences are shown in parenthesis. The sequence of 16S rRNA gene from Bacillus subtilis NCDO 1769T was used as the outgroup. Only bootstrap probabilities N50% are represented. The scale indicates the evolutionary distance of the nucleotide substitutions per site.
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0, 0.6, 1.2, 1.9, 2.5, 3.1, 4.7 and 6.2% salt, respectively. In addition, these cultures were inoculated onto MRS-agar plates containing 0, 1, 2, 3, 3.5, 4, 5, 6 and 7% sodium chloride (final
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concentrations). The inoculated plates were incubated at 25 °C for 5 days under aerobic conditions and viable counts were obtained from the numbers of colonies on each medium. 3. Results and discussion
Table 1 Phenotypic characteristics of Lactococcus lactis subsp. lactis isolated from the intestinal tract of the pufferfish Takifugu niphobles and the cheese starter culture Characteristic or sugar assayed
Marine fish-isolates a
O-114 b
Lactococcus lactis subsp. lactis RO6 c
Gram stain Cell morphology Catalase Anaerobic growth Fermentations of: Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol β-Methyl-xyloside Galactose D-Glucose D-Fructose D-Mannose L-Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol α-Methyl-D-mannoside α-Methyl-D-glucoside N-Acetyl-D-glucosamine Amygdalin Arbutin Esculin Salicin Cellobiose Maltose Lactose Melibiose Saccharose Trehalose Insulin Melezitose D-Raffinose Starch Glycogen Xylitol β-Gentiobiose D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2-Ceto-gluconate 5-Ceto-gluconate Final pH
+d Cocci − +
+ Cocci − +
+ Cocci − +
− − − + + + − − − + + + + − − − − + − − − + + + + + + + + − + + − − − + − − + − − − − − − − + − − 4.05–4.07
− − − − + + − − − + + + + − − − − − − − − + − + + + + + + − − + − − − + − − + − − − − − − − − − − 4.36
− − − + + + − − − + + + + − − − − + − − − + + + + + + + + − + + − − − W − − + − − − − − − − W − − Nd
3.1. Isolation of L. lactis from marine fish A total of 668 isolates were isolated from various marine fish species, of which 227 isolates seemed to be lactic acid bacteria based on the phenotypic properties. As a result of PCR amplification using three primers 20F, 685R and LacreR, PCR products showed different electrophoretic patterns with two bands for L. lactis related bacteria and one band for other bacterial species (Fig. 1). Subsequently, 227 isolates of presumptive lactic acid bacteria were subjected to PCR analyses using the species-specific primer, and four isolates were detected as L. lactis-like bacteria. All isolates were derived from the intestinal contents of the pufferfish T. niphobles. Sequencing of part of the 16S rRNA gene was performed for four isolates detected with the L. lactis-specific PCR. As a result of direct sequencing of the 1500 bp region of the 16S rRNA gene, about 1200 bp of the 1500 bp region for four isolates were determined except for the 5′- and 3′-terminal regions. Comparison of the sequences using the BLAST search identified three isolates as L. lactis, whereas the other isolate was identified as L. mesenteroides. However, the genotypes of L. lactis subspecies could not be determined by direct sequencing in this study, because the differences between the two subspecies of lactis and cremoris are located in the 5′-terminus of the 16S rRNA gene (Ward et al., 1998). Subsequently, PCR products of about 350 bp were amplified with the primer set Y1 and Y2, and digested with MboII or CfoI, which have been reported to identify the subspecies of L. lactis (Ward et al., 1998). MboII produced no digestion fragments for three isolates from coastal fish and L. lactis subsp. lactis O-114 isolated from the starter culture, whereas CfoI produced fragments of approximately 300 and 50 bp from all 4 bacterial isolates (Fig. 2), indicating that the L. lactis isolates from marine fish were genetically identified as subspecies lactis. In addition, the tree generated by the neighborjoining method revealed that the isolates derived from the cheese starter and marine fish formed a cluster with L. lactis subsp. lactis (Fig. 3). The sequences from this study have been deposited in the DDBJ/GenBank/EMBL databases under accession numbers AB278679–AB278680 for marine fish-derived isolates and AB285124 for the cheese starter O-114-derived
Notes to Table 1 a Marine fish-isolates include strains E214, E219 and E230, which were isolated from the pufferfish Takifugu niphobles in this study. All isolates showed the same profiles. b O-114 represents the Lactococcus lactis subsp. lactis strain isolated from the cheese starter culture O-114. c The data for Lactococcus lactis subsp. lactis RO6 strain was cited from the report of Ennahar et al. (2003). d +, positive reaction; w, weakly positive reaction; −, negative reaction; and Nd, no data.
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counterpart. The 16S rRNA gene sequence (1371 bp) of E214 (AB278680) was identical to that of E230. Acidity of the MRS broth before culture was pH 6.28, whereas those after 5-day culture of the marine fish-derived L. lactis were pH 4.05–4.07 as in the case of cheese starter culture O-114 (pH 4.36) (Table 1). With the API 50 CH system and API 50-CHL medium, the three marine fish intestine-derived isolates, E214, E219 and E230, fermented L-arabinose, ribose, D-xylose, galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-D-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, saccharose, trehalose, starch, β-gentiobiose and gluconate (Table 1). These profiles completely coincided with that of the L. lactis subsp. lactis strain RO6 from whole-crop paddy rice silage (Ennahar et al., 2003), but were different from the starter culturederived L. lactis subsp. lactis in the fermentation of L-arabinose, mannitol, amygdalin, saccharose and gluconate (Table 1). None of the L. lactis isolates, including E214, E219, E230 and O-114 produced gas, but all grew well on MRS-agar plates with or without seawater at 40 °C. These characteristics are the same as those of L. lactis subsp. lactis but different from L. lactis subsp. cremoris (Teuber, 1995). 3.2. Salt tolerance of L. lactis The salt tolerance of L. lactis isolates derived from the pufferfish intestine (E214, E219 and E230) were compared with that of the cheese starter culture O-114. The highest viable counts are represented as 100% among the different cultures. The relative viable counts of isolates O-114 were 60–100% on MRS-agar plates containing 0–100% seawater and 30% on MRS-agar plates containing 150% seawater, but 0% on MRSagar plates containing 200% seawater (Fig. 4a). On the other hand, the relative viable counts of isolates E214, E219 and E230 were all over 60% even in 200% seawater (Fig. 4a). Similarly, isolate O-114 grew well (over 60%) in 0–4% sodium chloride, while isolates E214, E219 and E230 grew well in 0–6% sodium chloride (Fig. 4b). Thus, the O-114 isolate showed tolerance to 100% seawater and 4% sodium chloride (Fig. 4) as previously reported for L. lactis subsp. lactis (Mundt, 1986). On the other hand, the marine fish-derived L. lactis subsp. lactis isolates, E214, E219 and E230, were more salt tolerant than isolate O-114 (Fig. 4). Since the environments of coastal shores, especially the intertidal zone, have continuously changing conditions, with periodic differences in osmotic pressure, dissolved oxygen, and temperature, the organisms indigenous to these areas are typically capable of adapting to environmental changes. The high salt tolerance of marine fish-derived L. lactis subsp. lactis in the present study also suggested that these L. lactis subsp. lactis isolates have additional functions compared with the cheese starter-derived isolate. These novel L. lactis isolates might be used for the development of functional foods and novel food additives for preserved food products. Furthermore, the present results suggest that additional undetected L. lactis strains may be present in the intestinal tracts of marine fish and in marine environments, since the food preferences of marine fish are variable and their excreta are continuously being introduced to the environment.
Fig. 4. Effects of the varying concentrations of seawater and sodium chloride on the survival rates for the starter culture-derived Lactococcus lactis subsp. lactis and the pufferfish intestine-derived L. lactis isolates. Effects of the various seawater concentrations from 0 to 200% (a) and that of the various sodium chloride concentrations from 0 to 7% in MRS-agar plates (b) on the survival rate of L. lactis from different sources. Open circles represent relative viable count of O-114 (control), while closed circles, triangles and squares represent those of E214, E219 and E230, respectively.
The reason for the presence of L. lactis in marine environments is interesting to consider. L. lactis strains have been isolated not only from milk products but also from various environments such as plant surfaces (Salama et al., 1995; Ulrich and Müller, 1999), soil (Klijn et al., 1995), and termite hindguts (Bauer et al., 2000), suggesting that these bacteria have high adaptability to varied environments. Although viable cell of L. lactis had not been previously isolated from marine environments, the clone library method suggested the presence of high densities of L. lactis in the intestines of coastal fish (Shiina et al., 2006). Since L. lactis has rarely been isolated from marine environments, these results suggest that the cells may enter into a viable but non-culturable (VBNC) state. Alternatively, the presence of L. lactis strains in the intestinal tract of marine fish may result from the adaptation of bacteria from plant surfaces and milk products to marine environments. At this time, however, the origin of L. lactis in marine environments remains unexplained.
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Acknowledgments This study was supported, in part, by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and the Open Research Center Project by the Ministry of Education, Culture, Science, Sports and Technology. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bauer, S., Tholen, A., Overmann, J., Brune, A., 2000. Characterization of abundance and diversity of lactic acid bacteria in the hindgut of wood- and soil-feeding termite by molecular and culture-dependent techniques. Arch. Microbiol. 173, 126–137. Ennahar, S., Cai, Y., Fujita, Y., 2003. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl. Environ. Microbiol. 69, 444–451. Ishida, Y., Sugita, H. (Eds.), 2000. Methods in Microbiology for Marine Environmental Assessment. Koseisha-Koseikaku, Tokyo, pp. 1–198 (in Japanese). Kelly, W., Ward, L., 2002. Genotypic vs. phenotypic biodiversity in Lactococcus lactis. Microbiology 148, 3332–3333. Kimoto, H., Ohmomo, S., Okamoto, T., 2002. Cholesterol removal from media by lactococci. J. Dairy Sci. 85, 3182–3188. Klijn, N., Weerkamp, A.H., de Vos, W.M., 1995. Detection and characterization of lactose-utilizing Lactococcus spp. in natural ecosystems. Appl. Environ. Microbiol. 61, 788–792. Lane, D., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.), Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, NY, pp. 115–147. Mundt, J.O., 1986. Lactic acid streptococci. In: Sneath, P.H.A., et al. (Ed.), Bergey's Manual of Systematic Bacteriology, vol. 2. Williams & Wilkins, Baltimore, MD, pp. 1065–1066. Pu, Z.Y., Dobos, M., Limsowtin, G.K., Powell, I.B., 2002. Integrated polymerase chain reaction-based procedures for the detection and
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identification of species and subspecies of the Gram-positive bacterial genus Lactococcus. J. Appl. Microbiol. 93, 353–361. Saitou, N., Nei, N., 1987. A neighbor-joining method: a new method for constructing phylogenetic tree. Mol. Biol. Evol. 44, 406–425. Salama, M.S., Sandine, W.E., Giovannoni, S.J., 1993. Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes. Appl. Environ. Microbiol. 59, 3941–3945. Salama, M.S., Musafija-Jeknic, T., Sandine, W.E., Giovannoni, S.J., 1995. An ecological study of lactic acid bacteria: isolation of new strains of Lactococcus including Lactococcus lactis subsp. cremoris. J. Dairy Sci. 78, 1004–1017. Shiina, A., Itoi, S., Washio, S., Sugita, H., 2006. Molecular identification of intestinal microflora in Takifugu niphobles. Comp. Biochem. Physiol. D 1, 128–132. Sugita, H., Noguchi, T., Hwang, D.F., Furuta, M., Motokane, T., Sonoda, T., Hashimoto, K., Deguchi, Y., 1987. Intestinal microflora of coastal puffer fishes. Nippon Suisan Gakkaishi 53, 2201–2207. Sugita, H., Yamamoto, S., Asakura, C., Morita, T., 2005. Occurrence of Listonella anguillarum in seed production environments of Japanese flounder Palalichthys olivaceus (Temminck et Schlegel). Aquac. Res. 36, 920–926. Teuber, M., 1995. The genus Lactococcus. In: Wood, B.J.B., Holzapfel, W.H. (Eds.), The Lactic Acid Bacteria. The Genera of Lactic Acid Bacteria. Blackie Academic, Glasgow, Scotland, pp. 173–234. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882. Ulrich, A., Müller, T., 1999. Heterogeneity of plant-associated streptococci as characterized by phenotypic features and restriction analysis of PCRamplified 16S rRNA. J. Appl. Microbiol. 84, 293–303. Ward, L.J.H., Brown, J.C.S., Davey, G.P., 1998. Two methods for the genetic differentiation of Lactococcus lactis ssp. lactis and cremoris based on differences in the 16S rRNA gene sequence. FEMS Microbiol. Lett. 166, 15–20. Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703. Young, J.P., Downer, H.L., Eardly, B.D., 1991. Phylogeny of the phototrophic rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J. Bacteriol. 173, 2271–2277.
International Journal of Food Microbiology 121 (2008) 116 – 121 www.elsevier.com/locate/ijfoodmicro
Short communication
Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish Shiro Itoi, Takeshi Abe, Sayaka Washio, Erika Ikuno, Yuna Kanomata, Haruo Sugita ⁎ Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa, 252-8510, Japan Received 21 December 2006; received in revised form 16 August 2007; accepted 6 November 2007
Abstract We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides (AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment. © 2007 Elsevier B.V. All rights reserved. Keywords: Halotolerant strain; Lactic acid bacteria; Lactococcus lactis; Marine fish; Takifugu niphobles
1. Introduction Lactococcus lactis is used worldwide for the manufacture of fermented dairy products, such as in the cheese-making process. L. lactis strains used for dairy production have been genetically subdivided into L. lactis subsp. lactis and L. lactis subsp. cremoris. It is necessary to differentiate L. lactis strains as either subsp. lactis or cremoris, since they show different characteristics in cheese production. This differentiation has traditionally been carried out on the basis of phenotypic traits such as growth temperature, salt tolerance and the ability to hydrolyze arginine (Mundt, 1986). Comparison of DNA sequence data has also shown genetic differences between the two subspecies (Ward et al., 1998). In addition, the two genetic types of L. lactis have
⁎ Corresponding author. Tel./fax: +81 466 84 3679. E-mail address: [email protected] (H. Sugita). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.11.031
been further divided into two or three phenotypes (Kelly and Ward, 2002). In these L. lactis types, the majority of strains used as dairy starters can be grouped into a small number of genetic lineages, and are not representative of the diversity found in L. lactis as a whole (Kelly and Ward, 2002). Actually, although new strains for starters have been screened primarily from milk products, the variability is low among strains used in industrial dairy fermentations (Salama et al., 1993). In spite of this low genetic diversity, these bacteria are also expected to function as probiotics (Kimoto et al., 2002). Isolation of new strains has been attempted from various environments including milk products and plant surfaces (Salama et al., 1995; Ulrich and Müller, 1999). Our recent study using the clone library method based on 16S ribosomal RNA (rRNA) gene sequences suggested the presence of lactic acid bacteria in the intestinal tract of the pufferfish Takifugu niphobles caught in the Shimoda, Shizuoka, Japan (Shiina et al., 2006). Results of this study indicated that L. lactis related
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clones represented approximately 15% of the total intestinal microflora (Shiina et al., 2006). Lactic acid bacteria from marine environments are expected to show additional capabilities compared with milk- and plant-derived strains. Thus, in this study, L. lactis isolates were isolated from marine fish intestines, and were examined for their carbohydrate fermentation profiles and salt tolerance. 2. Materials and methods 2.1. Fish and bacteriological sampling Coastal fish specimens were collected with standard fishing gear at Shimoda, Shizuoka, Japan in water that was 17.5– 18.0 °C in temperature in April 2004, and 23.0 °C in June 2004. These specimens were composed of Ditrema temmincki (n = 1), Girella punctata (n = 14), Pseudolabrus japonicus (n = 1), Sebastes pachycephalus (n = 2), T. niphobles (n = 9) and Thalassoma cupido (n = 2). Each specimen was dissected aseptically after capture. The contents squeezed from the intestinal tracts were serially diluted with sterile 50% artificial seawater (ReiSea, Tokyo, Japan), inoculated in MRS broth (BD, Franklin Lakes, NJ, USA), and plated onto MRS-agar medium containing 2% agar for selection of lactic acid bacteria. Both the MRS broth and MRS-agar were prepared with 50% artificial seawater. The inoculated broth and plates were incubated at 25 °C for 5 days under aerobic conditions, since the intestinal bacteria of coastal puffer fishes were not predominantly colonized by obligate anaerobic 85 bacteria (Sugita et al., 1987). Subsequently, a loopful of bacterial culture from the 86 MRS broth was streaked on MRS-agar plates and incubated at 25 °C for 5 days. L. lactis subsp. lactis was also isolated from starter culture O-114 (Christian Hansen, Hoersholm, Denmark), which was purchased from Zao Dairy Center (Zao, Miyagi, Japan), using MRS-agar plates, and was tentatively named L. lactis subsp. lactis O-114 in this study. All isolates were examined for phenotypic properties including Gram reaction, spore formation, cellular morphology, motility, colony pigmentation, production of oxidase and catalase, and fermentation of glucose according to Ishida and Sugita (2000) for screening of lactic acid bacteria. L. lactis isolates were incubated on MRS-agar plates with or without 50% artificial seawater at 40 °C to identify the L. lactis subspecies. To test for gas production, the isolates were inoculated into phenol red broth (Ishida and Sugita, 2000) containing 1% glucose, 1% peptone, 0.5% sodium chloride and 0.0018% phenol red with a Durham tube (pH 6.28), and incubated at 25 °C for 5 days. L. lactis isolates were also incubated on MRSagar plates at 25 °C for 5 days under anaerobic conditions. Final pH of the MRS broth after 5-day culture was measured with a F22 pH meter (Horiba, Kyoto, Japan). The fermentation profile of 49 sugars and poly-alcohols (control) was recorded using the API 50 CH system (bioMérieux, Montalieu–Vercieu, France) according to the manufacturer's instructions. Since all of the L. lactis isolates were capable of growing at both 0% seawater and 0% sodium chloride at 25 °C, all tests were carried out in API 50-CHL medium at 25 °C.
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2.2. DNA extraction, PCR amplification and sequencing DNA was extracted from pure cultures according to the method of Sugita et al. (2005). The partial DNA fragments of bacterial 16S rRNA genes were amplified by PCR according to our previous report (Shiina et al., 2006) using a forward primer 20F (5′-AGAGTTTGATCCTGGCTCAG-3′, equivalent to positions 8 to 27 in Escherichia coli 16S rRNA) and a reverse primer 1500R (5′-GGTTACCTTGTTACGACTT-3′, equivalent to positions 1491 to 1509 in E. coli 16S rRNA) (Weisburg et al., 1991). PCR with a L. lactis-specific primer was carried out using two reverse primers, 685R (5′-TCTACGCATTTCACCGCTAC-3′, equivalent to positions 685 to 704 in E. coli 16S rRNA) (Lane, 1991) and LacreR specific to L. lactis (5′-GGGATCATCTTTGAGTGAT-3′, equivalent to positions 217 to 235 in E. coli 16S rRNA) (Pu et al., 2002) as follows: the reaction mixture contained genomic DNA as the template, 1 μl of 10 × Taq DNA polymerase buffer, 1.3 μl of 4.6 μM primers (20F, 685R and LacreR), 0.8 μl of 2.5 mM dNTP and 0.3 units of Taq DNA polymerase, and the total volume was brought up to 10 μl with sterile water. Direct sequencing of PCR products amplified with primers 20F and 1500R forbacterial isolates was performed for both strands with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). CLUSTALX version 1.83 (Thompson et al., 1997) was used to align the nucleotide sequences of the 16S rRNA genes of the intestinal bacterial isolates with those of the corresponding genes from other bacteria in the DDBJ/EMBL/GenBank databases that were found in BLAST searches (Altschul et al., 1997). 2.3. PCR-RFLP analysis To identify the subspecies of L. lactis, PCR-RFLP analysis was carried out essentially according to Ward et al. (1998). Briefly, a
Fig. 1. Electrophoretic patterns of PCR products amplified with three primers including a species-specific primer. The upper arrow indicates the band common to the bacterial 16S rRNA gene, whereas the lower one indicates the band specific to the Lactococcus lactis 16S rRNA gene. Lane M, 100 bp laddermolecular weight marker from 100 to 1000 bp; lanes 1 and 4, PCR product amplified with primers 20F and 685R; lanes 2 and 5, PCR product amplified with primers 20F and LacreR; lanes 3 and 6, PCR products amplified with primers 20F, 685R and LacreR.
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tions 361 to 338 in E. coli) for amplifying the partial 16S rRNA gene fragment were synthesized according to the report of Young et al. (1991). The PCR conditions employed for RFLP analysis were the same as those described previously. The RFLP analysis of the 16S rRNA gene product was performed by digesting 10 μl of the amplified product with 10 units of CfoI (Roche Diagnostics GmbH, Mannheim, Germany) or MboII (Nippongene, Tokyo, Japan). The digested samples were subjected to electrophoresis on a 3% agarose gel and stained with ethidium bromide. 2.4. Phylogenetic analysis
Fig. 2. PCR-RFLP profile of the 16S rRNA gene from the starter culture-derived L. lactis subsp. lactis and the pufferfish intestine-derived L. lactis isolates. PCR products for the partial 16S rRNA gene were treated with MboII (upper panel) and CfoI (lower panel). The restriction fragments are marked by arrows. Lane M, molecular size marker; lane 1–3, RFLP products of the pufferfish intestinederived isolates; lane 4, RFLP product of the cheese starter-derived L. lactis; U, untreated PCR product.
partial DNA fragment of the 16S rRNA gene was amplified by PCR. Primers Y1 (5′-TGGCTCAGGACGAACGCTGGCGGC3′, equivalent to positions 20 to 43 in E. coli) and Y2 (5′CCTACTGCTGCCTCCCGTAGGAGT-3′, equivalent to posi-
A phylogenetic tree was constructed by the neighbor-joining method (Saitou and Nei, 1987) based on paired alignments of nucleotide sequences of the bacterial 16S rRNA genes. A set of sequences for the 16S rRNA gene from Bacillus subtilis NCDO 1769T (DNA database accession number X60646) was used as the outgroup. 2.5. Growth in 0–200% seawater and 0–7% sodium chloride To examine the salt tolerance of isolates from marine fish and L. lactis subsp. lactis from the cheese starter culture, approximately 109 cells/ml of each of the four MRS cultures were cultured at 25 °C for 2 days. These cultures were inoculated onto MRS-agar plates prepared with 0, 20, 40, 60, 80, 100, 150 and 200% artificial seawater (final concentrations) corresponding to
Fig. 3. Phylogenetic tree constructed by the neighbor-joining method based on the partial nucleotide sequences of the 16S rRNA gene from bacteria related to lactic acid bacteria. Numbers at the branches denote the bootstrap percentages for 1000 replicates. AB278679–AB278680 and AB285124 in parentheses indicate the accession numbers deposited in the DDBJ/EMBL/GenBank databases in this study and the accession numbers for reference sequences are shown in parenthesis. The sequence of 16S rRNA gene from Bacillus subtilis NCDO 1769T was used as the outgroup. Only bootstrap probabilities N50% are represented. The scale indicates the evolutionary distance of the nucleotide substitutions per site.
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0, 0.6, 1.2, 1.9, 2.5, 3.1, 4.7 and 6.2% salt, respectively. In addition, these cultures were inoculated onto MRS-agar plates containing 0, 1, 2, 3, 3.5, 4, 5, 6 and 7% sodium chloride (final
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concentrations). The inoculated plates were incubated at 25 °C for 5 days under aerobic conditions and viable counts were obtained from the numbers of colonies on each medium. 3. Results and discussion
Table 1 Phenotypic characteristics of Lactococcus lactis subsp. lactis isolated from the intestinal tract of the pufferfish Takifugu niphobles and the cheese starter culture Characteristic or sugar assayed
Marine fish-isolates a
O-114 b
Lactococcus lactis subsp. lactis RO6 c
Gram stain Cell morphology Catalase Anaerobic growth Fermentations of: Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol β-Methyl-xyloside Galactose D-Glucose D-Fructose D-Mannose L-Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol α-Methyl-D-mannoside α-Methyl-D-glucoside N-Acetyl-D-glucosamine Amygdalin Arbutin Esculin Salicin Cellobiose Maltose Lactose Melibiose Saccharose Trehalose Insulin Melezitose D-Raffinose Starch Glycogen Xylitol β-Gentiobiose D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2-Ceto-gluconate 5-Ceto-gluconate Final pH
+d Cocci − +
+ Cocci − +
+ Cocci − +
− − − + + + − − − + + + + − − − − + − − − + + + + + + + + − + + − − − + − − + − − − − − − − + − − 4.05–4.07
− − − − + + − − − + + + + − − − − − − − − + − + + + + + + − − + − − − + − − + − − − − − − − − − − 4.36
− − − + + + − − − + + + + − − − − + − − − + + + + + + + + − + + − − − W − − + − − − − − − − W − − Nd
3.1. Isolation of L. lactis from marine fish A total of 668 isolates were isolated from various marine fish species, of which 227 isolates seemed to be lactic acid bacteria based on the phenotypic properties. As a result of PCR amplification using three primers 20F, 685R and LacreR, PCR products showed different electrophoretic patterns with two bands for L. lactis related bacteria and one band for other bacterial species (Fig. 1). Subsequently, 227 isolates of presumptive lactic acid bacteria were subjected to PCR analyses using the species-specific primer, and four isolates were detected as L. lactis-like bacteria. All isolates were derived from the intestinal contents of the pufferfish T. niphobles. Sequencing of part of the 16S rRNA gene was performed for four isolates detected with the L. lactis-specific PCR. As a result of direct sequencing of the 1500 bp region of the 16S rRNA gene, about 1200 bp of the 1500 bp region for four isolates were determined except for the 5′- and 3′-terminal regions. Comparison of the sequences using the BLAST search identified three isolates as L. lactis, whereas the other isolate was identified as L. mesenteroides. However, the genotypes of L. lactis subspecies could not be determined by direct sequencing in this study, because the differences between the two subspecies of lactis and cremoris are located in the 5′-terminus of the 16S rRNA gene (Ward et al., 1998). Subsequently, PCR products of about 350 bp were amplified with the primer set Y1 and Y2, and digested with MboII or CfoI, which have been reported to identify the subspecies of L. lactis (Ward et al., 1998). MboII produced no digestion fragments for three isolates from coastal fish and L. lactis subsp. lactis O-114 isolated from the starter culture, whereas CfoI produced fragments of approximately 300 and 50 bp from all 4 bacterial isolates (Fig. 2), indicating that the L. lactis isolates from marine fish were genetically identified as subspecies lactis. In addition, the tree generated by the neighborjoining method revealed that the isolates derived from the cheese starter and marine fish formed a cluster with L. lactis subsp. lactis (Fig. 3). The sequences from this study have been deposited in the DDBJ/GenBank/EMBL databases under accession numbers AB278679–AB278680 for marine fish-derived isolates and AB285124 for the cheese starter O-114-derived
Notes to Table 1 a Marine fish-isolates include strains E214, E219 and E230, which were isolated from the pufferfish Takifugu niphobles in this study. All isolates showed the same profiles. b O-114 represents the Lactococcus lactis subsp. lactis strain isolated from the cheese starter culture O-114. c The data for Lactococcus lactis subsp. lactis RO6 strain was cited from the report of Ennahar et al. (2003). d +, positive reaction; w, weakly positive reaction; −, negative reaction; and Nd, no data.
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counterpart. The 16S rRNA gene sequence (1371 bp) of E214 (AB278680) was identical to that of E230. Acidity of the MRS broth before culture was pH 6.28, whereas those after 5-day culture of the marine fish-derived L. lactis were pH 4.05–4.07 as in the case of cheese starter culture O-114 (pH 4.36) (Table 1). With the API 50 CH system and API 50-CHL medium, the three marine fish intestine-derived isolates, E214, E219 and E230, fermented L-arabinose, ribose, D-xylose, galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-D-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, saccharose, trehalose, starch, β-gentiobiose and gluconate (Table 1). These profiles completely coincided with that of the L. lactis subsp. lactis strain RO6 from whole-crop paddy rice silage (Ennahar et al., 2003), but were different from the starter culturederived L. lactis subsp. lactis in the fermentation of L-arabinose, mannitol, amygdalin, saccharose and gluconate (Table 1). None of the L. lactis isolates, including E214, E219, E230 and O-114 produced gas, but all grew well on MRS-agar plates with or without seawater at 40 °C. These characteristics are the same as those of L. lactis subsp. lactis but different from L. lactis subsp. cremoris (Teuber, 1995). 3.2. Salt tolerance of L. lactis The salt tolerance of L. lactis isolates derived from the pufferfish intestine (E214, E219 and E230) were compared with that of the cheese starter culture O-114. The highest viable counts are represented as 100% among the different cultures. The relative viable counts of isolates O-114 were 60–100% on MRS-agar plates containing 0–100% seawater and 30% on MRS-agar plates containing 150% seawater, but 0% on MRSagar plates containing 200% seawater (Fig. 4a). On the other hand, the relative viable counts of isolates E214, E219 and E230 were all over 60% even in 200% seawater (Fig. 4a). Similarly, isolate O-114 grew well (over 60%) in 0–4% sodium chloride, while isolates E214, E219 and E230 grew well in 0–6% sodium chloride (Fig. 4b). Thus, the O-114 isolate showed tolerance to 100% seawater and 4% sodium chloride (Fig. 4) as previously reported for L. lactis subsp. lactis (Mundt, 1986). On the other hand, the marine fish-derived L. lactis subsp. lactis isolates, E214, E219 and E230, were more salt tolerant than isolate O-114 (Fig. 4). Since the environments of coastal shores, especially the intertidal zone, have continuously changing conditions, with periodic differences in osmotic pressure, dissolved oxygen, and temperature, the organisms indigenous to these areas are typically capable of adapting to environmental changes. The high salt tolerance of marine fish-derived L. lactis subsp. lactis in the present study also suggested that these L. lactis subsp. lactis isolates have additional functions compared with the cheese starter-derived isolate. These novel L. lactis isolates might be used for the development of functional foods and novel food additives for preserved food products. Furthermore, the present results suggest that additional undetected L. lactis strains may be present in the intestinal tracts of marine fish and in marine environments, since the food preferences of marine fish are variable and their excreta are continuously being introduced to the environment.
Fig. 4. Effects of the varying concentrations of seawater and sodium chloride on the survival rates for the starter culture-derived Lactococcus lactis subsp. lactis and the pufferfish intestine-derived L. lactis isolates. Effects of the various seawater concentrations from 0 to 200% (a) and that of the various sodium chloride concentrations from 0 to 7% in MRS-agar plates (b) on the survival rate of L. lactis from different sources. Open circles represent relative viable count of O-114 (control), while closed circles, triangles and squares represent those of E214, E219 and E230, respectively.
The reason for the presence of L. lactis in marine environments is interesting to consider. L. lactis strains have been isolated not only from milk products but also from various environments such as plant surfaces (Salama et al., 1995; Ulrich and Müller, 1999), soil (Klijn et al., 1995), and termite hindguts (Bauer et al., 2000), suggesting that these bacteria have high adaptability to varied environments. Although viable cell of L. lactis had not been previously isolated from marine environments, the clone library method suggested the presence of high densities of L. lactis in the intestines of coastal fish (Shiina et al., 2006). Since L. lactis has rarely been isolated from marine environments, these results suggest that the cells may enter into a viable but non-culturable (VBNC) state. Alternatively, the presence of L. lactis strains in the intestinal tract of marine fish may result from the adaptation of bacteria from plant surfaces and milk products to marine environments. At this time, however, the origin of L. lactis in marine environments remains unexplained.
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Acknowledgments This study was supported, in part, by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and the Open Research Center Project by the Ministry of Education, Culture, Science, Sports and Technology. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bauer, S., Tholen, A., Overmann, J., Brune, A., 2000. Characterization of abundance and diversity of lactic acid bacteria in the hindgut of wood- and soil-feeding termite by molecular and culture-dependent techniques. Arch. Microbiol. 173, 126–137. Ennahar, S., Cai, Y., Fujita, Y., 2003. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl. Environ. Microbiol. 69, 444–451. Ishida, Y., Sugita, H. (Eds.), 2000. Methods in Microbiology for Marine Environmental Assessment. Koseisha-Koseikaku, Tokyo, pp. 1–198 (in Japanese). Kelly, W., Ward, L., 2002. Genotypic vs. phenotypic biodiversity in Lactococcus lactis. Microbiology 148, 3332–3333. Kimoto, H., Ohmomo, S., Okamoto, T., 2002. Cholesterol removal from media by lactococci. J. Dairy Sci. 85, 3182–3188. Klijn, N., Weerkamp, A.H., de Vos, W.M., 1995. Detection and characterization of lactose-utilizing Lactococcus spp. in natural ecosystems. Appl. Environ. Microbiol. 61, 788–792. Lane, D., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.), Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, NY, pp. 115–147. Mundt, J.O., 1986. Lactic acid streptococci. In: Sneath, P.H.A., et al. (Ed.), Bergey's Manual of Systematic Bacteriology, vol. 2. Williams & Wilkins, Baltimore, MD, pp. 1065–1066. Pu, Z.Y., Dobos, M., Limsowtin, G.K., Powell, I.B., 2002. Integrated polymerase chain reaction-based procedures for the detection and
121
identification of species and subspecies of the Gram-positive bacterial genus Lactococcus. J. Appl. Microbiol. 93, 353–361. Saitou, N., Nei, N., 1987. A neighbor-joining method: a new method for constructing phylogenetic tree. Mol. Biol. Evol. 44, 406–425. Salama, M.S., Sandine, W.E., Giovannoni, S.J., 1993. Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes. Appl. Environ. Microbiol. 59, 3941–3945. Salama, M.S., Musafija-Jeknic, T., Sandine, W.E., Giovannoni, S.J., 1995. An ecological study of lactic acid bacteria: isolation of new strains of Lactococcus including Lactococcus lactis subsp. cremoris. J. Dairy Sci. 78, 1004–1017. Shiina, A., Itoi, S., Washio, S., Sugita, H., 2006. Molecular identification of intestinal microflora in Takifugu niphobles. Comp. Biochem. Physiol. D 1, 128–132. Sugita, H., Noguchi, T., Hwang, D.F., Furuta, M., Motokane, T., Sonoda, T., Hashimoto, K., Deguchi, Y., 1987. Intestinal microflora of coastal puffer fishes. Nippon Suisan Gakkaishi 53, 2201–2207. Sugita, H., Yamamoto, S., Asakura, C., Morita, T., 2005. Occurrence of Listonella anguillarum in seed production environments of Japanese flounder Palalichthys olivaceus (Temminck et Schlegel). Aquac. Res. 36, 920–926. Teuber, M., 1995. The genus Lactococcus. In: Wood, B.J.B., Holzapfel, W.H. (Eds.), The Lactic Acid Bacteria. The Genera of Lactic Acid Bacteria. Blackie Academic, Glasgow, Scotland, pp. 173–234. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882. Ulrich, A., Müller, T., 1999. Heterogeneity of plant-associated streptococci as characterized by phenotypic features and restriction analysis of PCRamplified 16S rRNA. J. Appl. Microbiol. 84, 293–303. Ward, L.J.H., Brown, J.C.S., Davey, G.P., 1998. Two methods for the genetic differentiation of Lactococcus lactis ssp. lactis and cremoris based on differences in the 16S rRNA gene sequence. FEMS Microbiol. Lett. 166, 15–20. Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703. Young, J.P., Downer, H.L., Eardly, B.D., 1991. Phylogeny of the phototrophic rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J. Bacteriol. 173, 2271–2277.