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Novel Exopolysaccharides Produced by Lactococcus Lactis Subsp

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Novel Exopolysaccharides Produced by Lactococcus Lactis Subsp Biosci. Biotechnol. Biochem., 77 (10), 2013–2018, 2013 Novel Exopolysaccharides Produced by Lactococcus lactis subsp. lactis, and the Diversity of epsE Genes in the Exopolysaccharide Biosynthesis Gene Clusters y Chise SUZUKI, Miho KOBAYASHI, and Hiromi KIMOTO-NIRA NARO Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan Received April 19, 2013; Accepted June 26, 2013; Online Publication, October 7, 2013 [doi:10.1271/bbb.130322] To characterize novel variations of exopolysacchar- ovalbumin-sensitized mice.12) Strain C59 also showed ides (EPSs) produced by dairy strains of Lactococcus significant activity as a plasminogen activator.13) This lactis subsp. lactis and subsp. cremoris, the EPSs of five activity was extracted from bacterial cells by 0.1 M dairy strains of L. lactis were purified. Sugar composi- sodium carbonate buffer (pH 10.0), suggesting that tion analysis showed two novel EPSs produced by cell-surface materials of strain C59 are involved in it. strains of L. lactis subsp. lactis. One strain produced EPS and CPS synthesis is usually controlled by eps EPS lacking galactose, and the other produced EPS operons located on a chromosome or plasmid, and by containing fucose. Among the eps gene clusters of these genes outside the operon.6,14–16) Clusters can differ strains, the highly conserved epsD and its neighboring greatly among strains belonging to the same species, but epsE were sequenced. Sequence and PCR analysis the first six genes, epsRXABCD, in lactococcal eps revealed that epsE genes were strain-specific. By South- operons are highly conserved among strains.6,15,17,18) A ern blot analysis using epsD, the eps gene cluster in each tyrosine (Tyr) phosphorylation regulatory system is strain was found to locate to the chromosome or a very involved in the modulation of polysaccharide synthesis. large plasmid. This is the first report on the identifica- Lactococcal EpsA, EpsB, and EpsC are the predicted tion of two novel EPSs in L. lactis subsp. lactis. The homologs of CpsC (Chain-length determinant protein), strains can be detected among other strains by using CpsD (the Tyr kinase) and CpsB (phospho-Tyr-protein epsE genes specific to them. phosphatase) of streptococcal capsule biosynthesis sys- tems, suggesting that the lactococcal homologs function Key words: exopolysaccharide; sugar composition; in the regulation of CPS/EPS biosynthesis.17,19) EpsD of fucose; Lactococcus lactis; dairy strains lactococci is an undecaprenylphosphate glucosephos- photransferase, and a homolog of CpsE/WchA of Certain strains of Lactococcus lactis produce extrac- Streptococcus pneumoniae. It is essential for CPS/EPS ellular polysaccharides (EPSs) that are associated with biosynthesis,20) and deletion mutants of epsD do not the cell surface in the form of capsular polysaccharides produce EPSs.18,19) Except for such conserved regions, (CPSs), or are secreted into the extracellular environ- the genes following epsD vary among strains. In ment.1) EPS of L. lactis subsp. cremoris, but not of S. pneumonia, 90 individual serotypes are recognized subsp. lactis, have been well characterized. These EPS at present, and the sequences of the capsular biosyn- are heteropolysaccharides composed of repeating units thetic genes of all 90 serotypes have been analyzed.21) In of sugars, among which galactose, glucose, and rham- L. lactis subsp. cremoris, several polysaccharide struc- nose are the most common monosaccharides. The tures of repeated units have been described: pentasac- structures and compositions of several EPSs from charide with galactose,3) pentasaccharide with glucose, L. lactis subsp. cremoris, as well as the sequences and galactose, and rhamnose (ratio 2:2:1),2,22) pentasacchar- organization of the eps genes, have also been re- ide with glucose and galactose (ratio 3:2),4,16) and solved.2–5) The eps gene cluster of L. lactis subsp. lactis heptasaccharide with glucose, galactose, and rhamnose strain KF147, isolated from plants, has been character- (ratio 2:3:2),5) and others,18) but those of L. lactis subsp. ized by complete genome sequence analysis,6) but lactis have not yet been understood. Here, to explore the neither EPS structure nor sugar composition has been variety of Lactococcus EPSs, the sugar compositions of investigated. CPSs/EPSs are thought to protect bacteria EPSs from five dairy strains of L. lactis subsp. lactis and against both environmental and host factors that may be of subsp. cremoris and the DNA sequences of epsD and detrimental to survival. As food materials, EPSs are its flanking epsE genes from these strains were analyzed. useful for increasing viscosity and improving the texture 7,8) of fermented dairy products. CPSs/EPSs have been Materials and Methods found to have properties beneficial to human health, both immunostimulatory9,10) and immunomodulatory.11) Strains, media, and culture preparation. The strains of L. lactis Our group found, for example, that oral administration used in this study are listed in Table 1. L. lactis strains were grown at of live CPS-producing L. lactis subsp. lactis C59 30 C for 18 h in M17 broth (Difco, Detroit, MI, USA) containing 0.5% significantly reduced total IgE antibody levels in (wt/vol) glucose (GM17) or in chemically defined medium23) y To whom correspondence should be addressed. Fax: +81-29-838-8606; E-mail: csuzuki@affrc.go.jp Abbreviations: EPS, exopolysaccharide; CPS, capsular polysaccharide; CDMG, chemically defined medium containing glucose; GM17, M17 broth containing glucose; DSMG, digested skim milk medium containing glucose; ABEE, p-aminobenzoic acid ethyl ester; TCA, trichloroacetic acid; PFGE, pulsed-field gel electrophoresis 2014 C. SUZUKI et al. Table 1. L. lactis Strains Used in This Study a 50-mL reaction volume containing 1 Â LA PCR buffer II, 1.5 mM MgCl2, 200 mM dNTPs, 2.5 units of LA Taq DNA polymerase (Takara Strain Species Source Bio, Otsu), 0.5 mM 50- and 30-primers, and 30 ng of template DNA 0 C59a L. lactis subsp. lactis bv. Laboratory collection48) where not otherwise specified. To detect epsB genes, primers 5 B83 diacetylactis and 30B1074 were used. Amplifications were performed in a DNA C60a L. lactis subsp. cremoris Laboratory collection48) thermal cycler (2400, Perkin-Elmer, Waltham, MA) under the DRC2a L. lactis subsp. lactis bv. NIRDa following cycling conditions: initial 94 C for 5 min; 35 cycles at diacetylactis 94 C for 30 s, 62 C for 30 s, and 72 C for 4 min; and final 72 C for a a Hc-1 L. lactis subsp. cremoris NIRD 8 min. For vectorette PCR, DNA fragments digested with an a 48) O22 L. lactis subsp. cremoris Laboratory collection appropriate restriction enzyme (Dra I, Alu I) were ligated to a 49) H61 L. lactis subsp. cremoris Laboratory collection synthetic oligonucleotide duplex (annealed anchor bubble-1 and -2). H-17 L. lactis subsp. cremoris Laboratory collection49) Three mL of the ligation mixture was used as template, along with one 341 L. lactis subsp. cremoris Laboratory collection48) of the epsD primers and a universal vectorette primer. For inverse Q14 L. lactis subsp. lactis Laboratory collection48) Q20 L. lactis subsp. lactis Laboratory collection48) PCR, DNA fragments digested by means of an appropriate restriction G50 L. lactis subsp. lactis Laboratory collection48) enzyme were self-ligated and used as PCR templates. The PCR J50 L. lactis subsp. cremoris Laboratory collection48) products were separated by 1.0% (wt/vol) agarose gel electrophoresis, O7 L. lactis subsp. lactis bv. Laboratory collection48) purified, and sequenced with an ABI Prism 3700 Genetic Analyzer diacetylactis (Applied Biosystems, Foster City, CA). Sequence data were assembled Ho-6 L. lactis subsp. cremoris Laboratory collection49) and analyzed with an AutoAssembler 2.1 (Applied Biosystems). H41-21 L. lactis subsp. cremoris Laboratory collection49) Databases were searched by BLASTN and BLASTP algorithms.28) H41-61 L. lactis subsp. cremoris Laboratory collection49) 49) F-16 L. lactis subsp. cremoris Laboratory collection Phylogenetic tree analysis. Multiple sequence alignments were b 924 L. lactis subsp. cremoris NIRD done using the program CLUSTAL W.29,30) The evolutionary history ATCC19257 L. lactis subsp. cremoris ATCCc was inferred by the neighbor-joining method31) with 1,000 bootstrap aStrains used in the preparation of EPS and the sequence of epsE are trials. Evolutionary distances were computed by the p-distance underlined. method.32) Evolutionary analyses were conducted in MEGA5.33) bNIRD, National Institute for Research in Dairying (UK) Pulsed-field gel electrophoresis (PFGE) of the lactococcal genome. containing 2% glucose (CDMG). For comparison, digested skim milk The genomic DNAs of the lactococcal strains were purified and 34) medium containing 2% glucose (DSMG)24) was also used in EPS digested with Sma I in agarose plugs. PFGE was done by a CHEF preparation. DRIII system (Bio-Rad). Agarose gels (1%) were prepared from Pulsed-field Certified agarose (Bio-Rad) and then run in 1 Â TAE at Preparation of EPS. L. lactis cells were grown in CDMG. EPS in 14 C at an initial switch time of 3.0 s, and a final switch time of 21.8 s the CDMG culture filtrates was precipitated by the addition of 2 (run time 18 h, angle 120 , voltage 6.0 V/cm). volumes of ethanol, and was dissolved in water. After deproteinization by the addition of trichloroacetic acid (TCA, final concentration, 12%), Southern blot analysis. The DNA fragments, separated by PFGE EPS was re-precipitated with 2 volumes of ethanol and dissolved in were transferred onto nylon membrane (Hybond N, Amersham water, followed by dialysis and lyophilization. A Shimadzu LC10AD Biosciences, Buckinghamshire, England) by means of a vacuum blot HPLC system (Shimadzu, Kyoto) was used for all HPLC analyses. apparatus (Vacugene, Amersham Biosciences). An epsD fragment High molecular weight EPSs were fractionated with a TSKgel (460 bp) was amplified with primers 50D1318 and 30D104, purified G5000PW column (600 Â 7:5 mm i.d., Tosoh, Tokyo) equilibrated with a PCR purification kit (QIAGEN), and labeled with a Digoxigenin with 0.1 M ammonium hydrogen carbonate (pH 8.0) at 0.8 mL minÀ1.
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