49NISR Research GRANT
2007 Young Investigator Research Grant

Genomic engineering of Lactococcus lactis IL1403
Masayuki HASHIMOTO
International young researchers empowerment project, Shinshu University

Research aims
A set of single-gene deletion mutants of all nonessential
genes in Escherichia coli K-12 and Bacillus subtilis 168
have been constructed. In addition, we previously constructed a series of consecutive chromosomal deletion mutants of E. coli. From these results, around 300 essential
genes for growth in each organism were estimated1–3). In
the case of E. coli, which has around 4,400 genes on thechromosome, tens of thousands mutants would be needed
to disrupt all of the nonessential genes by using randommutagenesis methods. Therefore simple assay have to be
used for screening of the mutant library. On the other hand,a set of 4,100 single gene knockout mutants, which includes mutants defective in each of the nonessential genes,
can cover all of the nonessential genes of E. coli. Furthermore, 551 chromosomal deletion mutants cover the all of E.
coli genome. Since these mutant libraries can cover thenonessential genes by less number of mutants, it is possible
to screen them by complicated assay methods. Indeed, thesemutant libraries were applied to various screening forgenes, for instance, genes involved in biofilm formation,
swarming, or ATP generation.
Lactic acid bacteria (LAB) are a heterogeneous group of
microorganisms able to convert carbohydrates into lactic
acid. Useful LAB are industrially important that are used
all over the world in large variety of industrial food fermentations. In addition, LAB are also used to produce a material for poly lactic acid which is a kind of bioplastic, andare featured as probiotic bacteria. From such a point ofview, many complete genomic sequences and genome-scale
metabolic engineering of LAB have been reported. However, although random mutagenesis libraries of LAB wereFig. 1.

constructed by using transposon or insertion sequence, toour knowledge, there is no report about a set of single-gene
knockout mutants or consecutive chromosomal deletionmutants. Lactococcus lactis is a non-pathogen bacterium
and used by dairy industry as starters in cheese fermentation. L. lactis strain IL1403 was the first LAB whose complete genome sequence has been determined4). We constructed an efficient chromosomal deletion system for thisbacterium and are trying to construct a set of consecutive
chromosomal deletion mutants.
Methods and Results
The chromosomal deletion mutants were constructedwith a L. lactis homologous recombination system using
temperature sensitive replication plasmid (Fig. 1A). A vector plasmid pGhT000 was derived from pGh95). To construct pGhT000, a restriction enzyme XcmI site in an erythromycin resistance gene of pGh9 was point mutated to remove the site without amino acid substitution. Next, upp
gene from L. lactis IL1403 was cloned into the pGh9DXcmIto construct pGhT00. The upp gene encodes an uracil phosphoribosyltransferase enzyme catalyzing the conversion ofuracil to UMP. Whereas wild type L. lactis is sensitive togrowth inhibition by the toxic base analog 5-fluorouracil(5FU), a deletion mutant of upp is resistant6). Therefore theupp gene can be used as a negative selection marker. Then,a kanamycin resistant gene flanked by XcmI sites wascloned into the multicloning site of pGhT00 to construct
pGhT000. The plasmid was designed to produce single
thymidine overhangs at both ends of the linearized fragment when digested with XcmI, and thus enabled TAcloning of PCR products which have adenosine overhangs(A) The chromosomal deletion system. (B) Schematic drawing of a series of consecutive chromosomal deletion mutants.

50

at both ends. To make a deletion, a DNA fragment in whichchromosomal regions flanking the region to be deleted werejoined to the ends of a spectinomycin resistance gene wasconstructed by two round PCR, and it was cloned into
pGhT000. The resulting plasmid was then introduced into
L. lactis IL1403 upp. A spectinomycin resistant (SpR) transformant was cultured at 28°C for 150 min, followed by cultured at 35°C for 150 min. An aliquot of the culture wasplated and incubated at 35°C to obtain SpR recombinant.
Then, an aliquot of overnight culture of the SpR recombinant at 28°C were plated and incubated at 35°C to obtain
SpR and 5FU resistant clones, which were the expected
chromosomal deletion mutants. This system allowed for efficient construction of deletion mutants of L. lactis IL1403.
The obtained recombinants were confirmed for the presence of the expected deletion by PCR.
To construct a series of consecutive chromosomal deletion mutants efficiently, it is important that the region to bedeleted should not include essential genes. However, only
few essential genes of L. lactis are known. As describedabove, 271 genes of B. subtilis 168 was estimated as essential genes. Then, we compared the all genes of B. subtilis
168 and L. lactis IL1403, and identified around 300 genesof L. lactis as candidates for essential genes, which include
RNA coding genes. Considering the estimated number of
essential genes and chromosomal length (2.4 Mb) of L. lactis IL1403, the length of deletion to be made in each mutant
was set to around 10 kb (Fig. 1B). We are progressing toconstruct the series of consecutive deletion mutant of L.
lactis IL1403.

Conclusion
A plasmid vector pGhT000 for deletion of L. lactis
IL1403 chromosome was constructed. The vector plasmid
contains a temperature sensitive replication origin, upp
gene from L. lactis IL1403 as a negative selection marker,
and restriction enzyme XmaI sites for TA-cloning of PCR
product. By using the plasmid with the ts replication origin
and negative selection marker, we succeeded in efficiently
constructing chromosomal deletion mutants of L. lactis
IL1403. Essential genes of L. lactis were estimated fromthose of B. subtilis. Construction of a series of consecutive
chromosomal deletion mutants of L. lactis IL1403 is progressing.
References
1)

2)
3)
4)

5)

6)

Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. (2006) Construction ofEscherichia coli K-12 in-frame, single-gene knockout mutants. MolSyst Biol., 2, 2006.0008.
Kobayashi K et al. (2003) Essential Bacillus subtilis genes. Proc
Natl Acad Sci U S A., 100, 4678–4683.
Kato J, Hashimoto M. (2007) Construction of consecutive deletionsof the Escherichia coli chromosome. Mol Syst Biol., 3, 132.
Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, Ehrlich SD, Sorokin A. (2001) The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis
IL1403. Genome Res., 11, 731–753.
Maguin E, Prévost H, Ehrlich SD, Gruss A. (1996) Efficient insertional mutagenesis in lactococci and other gram-positive bacteria. JBacteriol., 178, 931–935.
Martinussen J, Hammer K. (1994) Cloning and characterization ofupp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis. J Bacteriol., 176, 6457–6463.