Antonie van Leeuwenhoek

, Volume 76, Issue 1–4, pp 27–76

Low-redundancy sequencing of the entire Lactococcus lactis IL1403 genome

  • Alexander Bolotin
  • Stéphane Mauger
  • Karine Malarme
  • S. Dusko Ehrlich
  • Alexei Sorokin
Article

Abstract

Lactococcus lactis is an AT-rich gram positive bacterium phylogenetically close to the genus Streptococcus. Various strains of L. lactis are used in dairy industry as starters for cheese making. L. lactis is also one of the well characterized laboratory microorganisms, widely used for studies on physiology of lactic acid bacteria. We describe here a low redundancy sequence of the genome of the strain L. lactis IL1403. The strategy which we followed to determine the sequence consists of two main steps. First, a limited number of plasmids and λ-phages that carry random segments of the genome were sequenced. Second, sequences of the inserts were used for production of novel sequencing templates by applying Multiplex Long Accurate PCR protocols. Using of these PCR products allowed to determine the sequence of the entire 2.35 Mb genome with a very low redundancy, close to 2. The error rate of the sequence is estimated to be below 1%. The correctness of the sequence ass embly was confirmed by PCR amplification of the entire L. lactis IL1403 genome, using a set of 266 oligonucleotides. Anotation of the sequence was undertaken by using automatic gene prediction computer tools. This allowed to identify 1495 protein-encoding genes, to locate them on the genome map and to classify their functions on the basis of homology to known proteins. The function of about 700 genes expected to encode proteins that lack homologs in data bases cannot be reliably predicted in this way. The approach which we used eliminates high redundancy sequencing and mapping efforts, needed to obtain detailed and comprehensive genetic and physical maps of a bacterium.

Availability of detailed genetic and physical maps of the L. lactis IL1403 genome provides many entries to study metabolism and physiology of bacteria from this group. The presence of 42 copies of five different IS elements in the IL1403 genome confirms the importance of these elements for genetic exchange in Lact ococci. These include two previously unknown elements, present at seven and fifteen copies and designated IS1077 and IS983, respectively. Five potential or rudimentary prophages were identified in the genome by detecting clusters of phage-related genes. The metabolic and regulatory potential of L. lactis was evaluated by inspecting gene sets classified into different functional categories. L. lactis has the genetic potential to synthesise 20 standard amino acids, purine and pyrimidine nucleotides and at least four cofactors. Some of these metabolites, which are usually present in chemically defined media, can probably be omitted. About twenty compounds can be used by L. lactis as a sole carbon source. Some 83 regulators were revealed, indicating a regulatory potential close to that of Haemophilus influenzae, a bacterium with a similar genome size. Unexpectedly, L. lactis has a complete set of late competence genes, which may have concerted transcriptional regulation and unleadered po lycistronic mRNAs. These findings open new possibilities for developing genetic tools, useful for studies of gene regulation in AT-rich gram positive bacteria and for engineering of new strains for the diary industry.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Altschul SF, Gish W, Miller W, Myers EW, & Lipman DJ (1990) Basic local alignment search tool. J. Mol. Biol. 215(3): 403-410Google Scholar
  2. Andersen PS, Martinussen J & Hammer K (1996) Sequence analysis and identification of the pyrKDbF operon from Lactococcus lactis including a novel gene, pyrK, involved in pyrimidine biosynthesis. J Bacteriol 178(16): 5005-5012Google Scholar
  3. Anderson AW & Elliker PR (1953) The nutritional requirements of lactic streptococci isolated from starter cultures. I. Growth in a synthetic medium. J Dairy Science 36: 161-167Google Scholar
  4. Andersson SGE, Zomorodipour A, Andersson JO, Sicheritz-Pontén T, Alsmark UC, Podowski RM, Näslund AK, Eriksson A-S, Winkler HH & Kurland CG (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396: 133-140Google Scholar
  5. Bardowski J, Ehrlich SD & Chopin A (1992) Tryptophan biosynthesis genes in Lactococcus lactis subsp. lactis. J. Bacteriol. 174(20): 6563-7650Google Scholar
  6. Bardowski J, Ehrlich SD & Chopin A (1994) BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis. J. Bacteriol. 176(18): 5681-5685Google Scholar
  7. Beresford T & Condon S (1991) Cloning and partial characterization of genes for ribosomal ribonucleic acid in Lactococcus lactis subsp. lactis. FEMS Microbiol. Lett. 62(2-3): 319-323Google Scholar
  8. Biaudet V, el Karoui M & Grass A (1998) Codon usage can explain GT-rich islands surrounding Chi sites on the Escherichia coli genome. Mol. Microbiol. 29(2): 666-669Google Scholar
  9. Biswas I, Maguin E, Ehrlich SD & Grass A (1995) A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis. Proc. Natl. Acad. Sci. USA 92(6): 2244-2248Google Scholar
  10. Bolhuis A, Broekhuizen CP, Sorokin A, van Roosmalen ML, Venema G, Bron S, Quax WJ & van Dijl JM (1998) SecDF of Bacillus subtilis, a molecular Siamese twin required for the efficient secretion of proteins. J. Biol. Chem. 273(33): 21217-21224Google Scholar
  11. Bolotin A, Sorokin A & Ehrlich SD (1996) Mapping of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome using Long Accurate PCR and three yeast artificial chromosomes. Microbiology 142: 3017-3020Google Scholar
  12. Borodovsky M & McIninch J (1993) GENMARK: a parallel gene recognition for both DNA strands. Comput. Chem. 17: 123-133Google Scholar
  13. Burland V, Plunkett G, Daniels DL & Blattner F R (1993) DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication. Genomics 16(3): 551-561Google Scholar
  14. Campbell EA, Choi SY & Masure HR (1998) A competence regulon in Streptococcus pneumoniae revealed by genomic analysis. Mol. Microbiol. 27(5): 929-939Google Scholar
  15. Chedin F, Noirot P, Biaudet V & Ehrlich SD (1998) A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis. Mol. Microbiol. 29(6): 1369-1377Google Scholar
  16. Cheng S, Fockler C, Barnes WM & Higuchi R (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. U.S.A. 91(12): 5695-5699Google Scholar
  17. Chiarattini C & Milet M (1993) Gene organization, primary structure and RNA processing analysis of a ribosomal RNA operon in Lactococcus lactis. J. Mol. Biol. 230(1): 57-76Google Scholar
  18. Chopin A, Chopin MC, Moillo-Batt A & Langella P (1984) Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid 11(3): 260-263Google Scholar
  19. Chopin MC, Chopin A, Rouault A & Galleron N (1989) Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome. Appl. Environ. Microbiol. 55(7): 1769-1774Google Scholar
  20. Cocaign-Bousquet M, Garrigues C, Novak L, Lindley ND & Loubiere P (1995) Rational development of a simple synthetic medium for the sustained growth of Lactococcus lactis. J. Appl. Bacteriol. 79: 108-116Google Scholar
  21. Cocaign-Bousquet M, Garrigues C, Loubiere P & Lindley ND (1996) Physiology of pyruvate metabolism in Lactococcus lactis. Antonie Van Leeuwenhoek 70(2-4): 253-267Google Scholar
  22. Cole ST, Brosch R, Parkhill J, Gamier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S & Barry C.E. et al (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393(6685): 537-544Google Scholar
  23. Delorme C, Ehrlich SD & Renault P (1992) Histidine biosynthesis genes in Lactococcus lactis subsp. lactis. J. Bacteriol. 174(20): 6571-6579Google Scholar
  24. Delorme C, Godon J-J, Ehrlich SD & Renault P (1993) Gene inactivation in Lactococcus lactis: Histidine biosynthesis. J. Bacteriol. 175(14): 4391-4399Google Scholar
  25. De Vos WM & Vaughan EE (1994) Genetics of lactose utilisation in lactic acid bacteria. FEMS Microbiol. Rev. 15: 217-237Google Scholar
  26. De Vos WM (1996) Metabolic engineering of sugar catabolism in lactic acid bacteria. Antonie Van Leeuwenhoek 70(2-4): 223-242Google Scholar
  27. Dickely F, Nilsson D, Hansen EB & Johansen E (1995) Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector. Mol. Microbiol. 15(5): 839-847Google Scholar
  28. Dubnau D (1997) Binding and transport of transforming DNA by Bacillus subtilis: the role of type-IV pilin-like proteins — a review. Gene 192(1): 191-198Google Scholar
  29. Duong F & Wickner W (1997) The SecDFyajC domain of pre-protein translocase controls preprotein movement by regulating SecA membrane cycling. EMBO J. 16(16): 4871-4879Google Scholar
  30. El Karoui M, Ehrlich D & Gruss A (1998) Identification of the lactococcal exonuclease/recombinase and its modulation by the putative Chi sequence. Proc. Natl. Acad. Sci. USA 95(2): 626-631Google Scholar
  31. Engelke G, Gutowski-Eckel Z, Hammelmann M & Entian KD (1992) Biosynthesis of the [antibiotic nisin: genomic organization and membrane localization of the NisB protein. Appl. Environ. Microbiol. 58(11): 3730-3743Google Scholar
  32. Errington J (1986) A general method for fusion of the Escherichia coli lacZ gene to chromosomal genes in Bacillus subtilis. J. Gen. Microbiol. 132: 2953-2966Google Scholar
  33. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bull CJ, Tomb JF, Dougherty BA, Merrick JM et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269(5223): 496-512Google Scholar
  34. Gasson MJ (1983) Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154(1): 1-9Google Scholar
  35. Godon JJ, Chopin MC & Ehrlich SD (1992) Branched-chain amino acid biosynthesis genes in Lactococcus lactis subsp. lactis. J. Bacteriol. 174(20): 6580-6589Google Scholar
  36. Godon JJ, Delorme C, Bardowski J, Chopin MC, Ehrlich SD & Renault P (1993) Gene inactivation in Lactococcus lactis: Branched-chain amino acid biosynthesis. J. Bacteriol. 175(14): 4383-4390Google Scholar
  37. Griffin HG & Gasson MJ (1995) Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactis. Mol. Gen. Genet. 246(1): 119-127Google Scholar
  38. Grossiord B, Vaughan EE, Luesink E & de Vos WM (1998) Genetics of galactose utilisation via the Leloir pathway in lactic acid bacteria. Le Lait 78: 77-84Google Scholar
  39. Hahn J, Luttinger A & Dubnau D (1996) Regulatory inputs for the synthesis of ComK, the competence transcription factor of Bacillus subtilis. Mol. Microbiol. 21(4): 763-775Google Scholar
  40. Havarstein LS, Coomaraswamy G & Morrison DA (1995) An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 92(24): 11140-11144Google Scholar
  41. Havarstein LS, Gaustad P, Nes IF & Morrison DA (1996) Identification of the streptococcal competence-pheromone receptor. Mol. Microbiol. 21(4): 863-869Google Scholar
  42. Havarstein LS, Hakenbeck R & Ganstad P (1997) Natural competence in the genus Streptococcus: Evidence that Streptococci can change phenotype by interspecies recombinational exchanges. J. Bacteriol. 179: 6589-6594Google Scholar
  43. Hemila H, Pakkanen R, Heikinheimo R, Palva ET & Palva I (1992) Expression of the Erwinia carotovora polygalacturonase-encoding gene in Bacillus subtilis: role of signal peptide fusions on production of a heterologous protein. Gene 116(1): 27-33Google Scholar
  44. Huang DC, Novel M & Novel G (1991) A transposon-like element on the lactose plasmid of Lactococcus lactis subsp. lactis Z270. FEMS Microbiol. Lett. 61(1): 101-106Google Scholar
  45. Hutkins RW, Morris HA & McKay LL (1985) Galactokinase activity in Streptococcus thermophilus. Appl. Env. Microbiol. 50: 777-780Google Scholar
  46. Jensen PR & Hammer K (1993) Minimal requirements for exponential growth of Lactococcus lactis. Appl. Env. Microbiol. 59: 4363-4366Google Scholar
  47. Kilstrup M & Martinussen J (1998) A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis. J. Bacteriol. 180(15): 3907-3916Google Scholar
  48. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G et al. (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390: 249-256Google Scholar
  49. Lander ES & Waterman MS (1988) Genomic mapping by fingerprinting random clones: a mathematical analysis. Genomics 2(3): 231-239Google Scholar
  50. Lapidus A, Galleron N, Sorokin A & Ehrlich SD (1997) Sequencing and functional annotation of the Bacillus subtilis genes in the 200 kb rrnB-dnaB region. Microbiology 143: 3431-3441Google Scholar
  51. Lapujade P, Cocaign-Bousquet M & Loubiere P (1998) Glutamate biosynthesis in Lactococcus lactis subsp lactis NCDO 2118. Appl. Environ. Microbiol 64: 2485-2489Google Scholar
  52. Le Bourgeois P, Lautier M, Mata M & Ritzenthaler P (1992) Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL 1403. J. Bacteriol. 174(21): 6752-6762Google Scholar
  53. Le Bourgeois P, Lautier M, van den Berghe L, Gasson MJ & Ritzenthaler P (1995) Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion. J. Bacteriol. 177(10): 2840-2850Google Scholar
  54. Le Loir Y, Grass A, Ehrlich SD & Langella P (1998) A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. J. Bacteriol. 180(7): 1895-1903Google Scholar
  55. Lobry JR (1996a) Origin of replication of Mycoplasma genitalium. Nature 272: 745-746Google Scholar
  56. Lobry JR (1996b) Asymmetric substitution patterns in the two DNA strands of bacteria. Mol. Biol. Evol. 13: 660-665Google Scholar
  57. Lowe TM & Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25(5): 955-964Google Scholar
  58. Luesink EJ, van Herpen REMA, Grossiord BP, Kuipers OP & de Vos WM (1998) Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Mol. Microbiol. 30(4): 789-798Google Scholar
  59. Madsen SM, Albrechtsen B, Hansen EB & Israelsen H (1996) Cloning and transcriptional analysis of two threonine biosynthetic genes from Lactococcus lactis MG1614. J. Bacteriol. 178(13): 3689-3694Google Scholar
  60. Maeda S & Gasson MJ (1986) Cloning, expression and location of the Streptococcus lactis gene for phospho-beta-D-galactosidase. J. Gen. Microbiol. 132(2): 331-340Google Scholar
  61. Martinussen J, Andersen PS & Hammer K (1994) Nucleotide metabolism in Lactococcus lactis: salvage pathways of exogenous pyrimidines. J. Bacteriol. 176(5): 1514-1516Google Scholar
  62. Moll I, Resch A & Blasi U (1998) Discrimination of 5′-terminal start codons by translation initiation factor 3 is mediated by ribosomal protein S1. FEES Lett. 436(2): 213-217Google Scholar
  63. Nilsson D & Johansen E (1994) A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis. Biochim Biophys Acta 1219(1): 141-144Google Scholar
  64. Nilsson D & Kilstrup M (1998) Cloning and expression of the Lactococcus lactis purDEK genes, required for growth in milk. Appl. Environ. Microbiol. 64: 4321-4327Google Scholar
  65. Novak L, Cocaign-Bousquet M, Lindley ND & Loubiere P (1997) Metabolism and energetics of Lactococcus lactis during growth in complex or synthetic media. Appl. Environ. Microbiol. 63: 2665-2670Google Scholar
  66. Novak L, Cocaign-Bousquet M, Lindley ND & Loubiere P (1998) Cométabolisme sucre-acides aminés chez Lactococcus lactis (in French). Le Lait 78: 17-22Google Scholar
  67. O'Connell-Motherway M, Fitzgerald GF & van Sinderen D (1997) Cloning and sequence analysis of putative histidine protein kinases isolated from Lactococcus lactis MG1363. Appl. Environ. Microbiol. 63(6): 2454-2459Google Scholar
  68. Pearson WR, Wood T, Zhang Z & Miller W (1997) Comparison of DNA sequences with protein sequences. Genomics 46(1): 24-36Google Scholar
  69. Pogliano JA & Beckwith J (1994) SecD and SecF facilitate protein export in Escherichia coli. EMBO J. 13(3): 554-561Google Scholar
  70. Polzin KM, Romero D, Shimizu-Kadota M, Klaenhammer TR & McKay LL (1993) Copy number and location of insertion sequences ISS1 and IS981 in lactococci and several other lactic acid bacteria. J. Dairy Sci. 76(5): 1243-1252Google Scholar
  71. Poolman B (1993) Energy transduction in lactic acid bacteria. FEMS Microbiol. Rev. 12(1-3): 125-147Google Scholar
  72. Pouwels PH, Leer RJ, Shaw M, Heijne den Bak-Glashouwer MJ, Tielen FD, Smit E, Martinez B, Jore J & Conway PL (1998) Lactic acid bacteria as antigen delivery vehicles for oral immunization purposes. Int. J. Food. Microbiol. 41(2): 155-167Google Scholar
  73. Raya R, Bardowski J, Andersen PS, Ehrlich SD & Chopin A (1998) Multiple transcriptional control of the Lactococcus lactis trp operon. J. Bacteriol. 180(12): 3174-3180Google Scholar
  74. Resch A, Tedin K, Grundling A, Mundlein A & Blasi U (1996) Downstream box-anti-downstream box interactions are dispensable for translation initiation of leaderless mRNAs. EMBO J. 15(17): 4740-4748Google Scholar
  75. Riley M (1993) Functions of the gene products of Escherichia coli. Microbiol. Rev. 57(4): 862-952Google Scholar
  76. Schouler C, Gautier M, Ehrlich SD & Chopin MC (1998) Combinational variation of restriction modification specificities in Lactococcus lactis. Mol. Microbiol. 28(1): 169-178Google Scholar
  77. Simon D, Rouault A & Chopin MC (1985) Protoplast transformation of group N streptococci with cryptic plasmids. FEMS Microbiol. Lett. 26: 239-241Google Scholar
  78. Sorokin A, Lapidus A, Capuano V, Galleron N, Pujic P & Ehrlich SD (1996) A new approach using multiplex long accurate PCR and yeast artificial chromosomes for bacterial chromosome mapping and sequencing. Genome Res. 6(5): 448-453Google Scholar
  79. Stingele F, Neeser JR & Mollet B (1996) Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6. J. Bacteriol. 178(6): 1680-1690Google Scholar
  80. Tedin K, Rosch A & Blösi U (1997) Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5′ leader sequence. Mol. Microbiol. 25(1): 189-199Google Scholar
  81. Thomas TD & Craw VF (1984) Selection of galactose-fermenting Streptococcus thermophilus in lactose-limited chemostat cultures. Appl. Env. Microbiol. 48: 186-191Google Scholar
  82. Tracy RB, Chedin F & Kowalczykowski SC (1997) The recombination hot spot chi is embedded within islands of preferred DNA pairing sequences in the E. coli genome. Cell 90(2): 205-206Google Scholar
  83. Tulloch DL, Finch LR, Hillier AJ & Davidson BE (1991) Physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 and localization of six putative rRNA operons. J. Bacteriol. 173(9): 2768-7275Google Scholar
  84. Van de Guchte M, Ehrlich DS & Chopin A (1998) tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon. Mol. Microbiol. 29(1): 61-74Google Scholar
  85. Van Etten WJ & Janssen GR (1998) An AUG initiation codon, not codon-anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli. Mol. Microbiol. 27(5): 987-1001Google Scholar
  86. Van Kranenburg R, Maragg JD, van Swam II, Willem NJ & de Vos WM (1997) Molecular characterization of the plasmid-encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis. Mol. Microbiol. 24(2): 387-397Google Scholar
  87. Van Sinderen D, Karsens H, Kok J, Terpstra P, Ruiters MH, Venema G & Nauta A (1996) Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage rlt. Mol. Microbiol. 19(6): 1343-1355Google Scholar
  88. Van Sinderen D, Luttinger A, Kong L, Dubnau D, Venema G & Hamoen L (1995) comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis. Mol. Microbiol. 15(3): 455-462Google Scholar
  89. Vaughan EE & de Vos WM (1995) Identification and characterization of the insertion element IS 1070 from Leuconostoc lactis NZ6009. Gene 155(1): 95-100Google Scholar
  90. Vaughan EE, Pridmore RD & Mollet B (1998) Transcriptional regulation and evolution of lactose genes in the galactose-lactose operon of Lactococcus lactis NCDO 2054. J. Bacteriol. 180(18): 4893-4902Google Scholar
  91. Vellanoweth RL & Rabinowitz JC (1992) The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Mol. Microbiol. 6(9): 1105-1114Google Scholar
  92. Venter JC, Smith HO & Hood L (1996) A new strategy for genome sequencing. Nature 381: 364-366Google Scholar
  93. Wells JM, Wilson PW, Norton PM & Le Page RW (1993) A model system for the investigation of heterologous protein secretion pathways in Lactococcus lactis. Appl. Environ. Microbiol. 59(11): 3954-3959Google Scholar
  94. Wouters JA, Sanders JW, Kok J, de Vos WM, Kuipers OP & Abee T (1998) Clustered organization and transcriptional analysis of a family of five csp genes of Lactococcus lactis MG1363. Microbiology 144(10): 2885-2893Google Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Alexander Bolotin
    • 1
  • Stéphane Mauger
    • 1
  • Karine Malarme
    • 1
  • S. Dusko Ehrlich
    • 1
  • Alexei Sorokin
    • 1
  1. 1.Génétique Microbienne, INRAJouy en Josas cedexFrance

Personalised recommendations