AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1991, 0099-2240/91/092764-03$02.00/0 Copyright C) 1991, American Society for Microbiology

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Complementation of the Inability of Lactobacillus Strains To Utilize D-Xylose with D-Xylose Catabolism-Encoding Genes of Lactobacillus pen tosus M. POSNO,* P. T. H. M. HEUVELMANS, M. J. F. VAN GIEZEN, B. C. LOKMAN, R. J. LEER, AND P. H. POUWELS TNO Medical Biological Laboratory, P.O. Box 45, 2280 AA Rijswijk, The Netherlands Received 31 December 1990/Accepted 4 July 1991

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.

Lactobacillus strains are particularly known for their widespread application in industrial fermentation processes (11). Consequently, strain improvement of Lactobacillus spp. has always received considerable attention. During the past few years, major breakthroughs have been established in the genetic engineering of Lactobacillus strains. Vectors based on cryptic, endogenous Lactobacillus plasmids and antibiotic resistance genes have been constructed (1, 6, 13). Furthermore, methods for the introduction of (plasmid) DNA into almost every Lactobacillus species have been developed (1, 3, 6, 10, 13). In view of these recent achievements and the rapid increase in knowledge of gene expression in Lactobacillus spp. (2), construction by recombinant DNA techniques of Lactobacillus strains with improved or novel properties actually has come within reach. As the first step in the process of making a genetically modified Lactobacillus strain accepted by regulatory authorities, substitution of antibiotic resistance markers by food-grade markers seems obligatory. Up to now, food-grade selection systems for Lactobacillus spp. have not been described. Since only a few Lactobacillus species can utilize D-xylose as an energy source (8), we decided to exploit the potential of D-xylose fermentation as a food-grade selection marker for Lactobacillus spp. Lactobacillus strains were routinely cultivated at 37°C in MRS broth (4) with 2% (wt/vol) glucose or 2% (wt/vol) D-xylose. For plating, MRS was solidified with 1.5% agar (Difco). Erythromycin was used at 5 ,ug/ml. Escherichia coli JM109 was used as the host strain for cloning in E. coli. Standard recombinant DNA techniques were carried out according to the methods of Sambrook et al. (14). Electroporation of Lactobacillus ATCC 393 and L. plantaruim NCDO 1193 and isolation of Lactobacillus plasmid DNA were carried out as described elsewhere (12). We have isolated two plasmids, pXH5OA and pXH37A (9), which together contain at least three genes involved in D-xylose catabolism in L. pentosus MD353. The genes are organized in a cluster on the chromosome (xyl cluster) in the order 5'-xylR (encoding the presumed regulatory protein)xylA (encoding D-xylose isomerase)-xylB (encoding D-xylulose kinase)-3' (Fig. 1). To enable a functional analysis of the L. pentosus MD353 xyl cluster in Lactobacillus spp., the

genes were inserted in the E. coli-Lactobacillus shuttle vector pLP3537 according to the following strategy. First,

the 2.4-kb PstI-HindIII fragment of pXH5OA (encompassing xylR and the 5' part of xylA) was cloned in E. coli between the PstI and Hindlll sites of pLP3537 (Fig. 1). Subsequently, the hybrid plasmid (pLP3537-1) was linearized with Hindlll and ligated with the 3.7-kb Hindlll fragment of pXH37A (containing the remaining part of xylA and xylB). We at first transformed E. coli with the ligation mixture. The resulting recombinant plasmid, however, appeared to be highly unsta-

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FIG. 1. Cloning of three genes involved in D-xylose catabolism in a Lactobacillus vector. The genetic organization of the L. pentosus MD353 chromosomal DNA region containing xylR, xylA, and xylB (represented by thick arrows) is shown in the lower part of the figure. The number of amino acid residues (aa) in each protein is

given in parentheses. The E. coli-Lactobacillus shuttle vector *

pLP3537, shown in the upper part of the figure, consists of pUC19 (thin line), an erythromycin resistance gene (ery), and a 2.3-kb plasmid (p353-2) of L. pentosus MD353.

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electroporation, the cell suspension was spread on MRS agar with D-xylose and bromocresol purple as an indicator for acid production. After 3 to 4 days of anaerobic incubation at 37°C, a few erythromycin-resistant transformants surrounded by a yellow halo were obtained. As shown in Fig. 2, in all of these transformants a plasmid (designated pLP3537xyl) of the expected size and restriction enzyme pattern was present. The copy number of pLP3537-xyl is similar to that of pLP3537 (Fig. 2). When L. casei ATCC 393 and another non-D-xylose-fermenting strain, L. plantarum NCDO 1193, were retransformed with pLP3537-xyl isolated from L. casei ATCC 393, many (>104 CFU/p.g of DNA) erythromycinresistant transformants were obtained that were all able to ferment D-xylose. Erythromycin-resistant transformants with pLP3537 or pLP3537-1, used as controls, did not produce enough acid from D-xylose for halo formation. Apparently, the D-xylose catabolism-encoding genes (xyl genes) present on pLP3537-xyl contain all of the information necessary to convert D-xylose into D-xylulose-5-phosphate, an intermediate that can be further metabolized both by D-xylose-fermenting and non-D-xylose-fermenting Lactobacillus strains (5, 7). Plasmid DNA analysis of D-xylosefermenting L. casei ATCC 393 and L. plantarum NCDO 1193 cells revealed that in all transformants pLP3537-xyl was present and structurally stable. As can be deduced from the analysis of the growth rates of the different Lactobacillus strains in MRS medium and in MRS medium with glucose or D-xylose (Fig. 3), the efficiency with which Lactobacillus strains transformed with pLP3537-xyl can utilize D-xylose is comparable to that of the L. pentosus MD353 wild-type strain. In addition, the growth rate in the presence of D-xylose of L. pentosus MD353 or Lactobacillus strains transformed with pLP3537-xyl is always lower than that in the presence of glucose (Fig. 3).

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FIG. 2. Agarose gel electrophoresis of plasmid DNA isolated from L. casei ATCC 393 transformed with pLP3537 (6.1 kb) (lane 1), pLP3537-1 (8.5 kb) (lane 2), and pLP3537-xyl (12.2 kb) (lane 3) and E. coli transformed with pXH37A (6.3 kb) (lane 4). Lane 5, L. casei ATCC 393. HindIll digests of pLP3537, pLP3537-1, and pLP3537xyl isolated from L. casei ATCC 393 and of pXH37A isolated from E. coli are present in lanes 6, 7, 8, and 9, respectively. The size reference for linear DNA fragments is lambda DNA digested with BstEII (lane 10). A similar result was obtained for L. plantarum NCDO 1193 transformed with pLP3537 or pLP3537-xyl (not shown).

ble in this host. Possibly, reconstruction of a functional L. pentosus MD353 xylA gene is detrimental to E. coli, as the (parent) plasmids pLP3537-1 and pXH37A are stable. Therefore, the ligation mixture was used to transform L. casei ATCC 393, a non-D-xylose-fermenting strain that can be efficiently transformed with plasmid DNA (3). Following

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FIG. 3. Growth curves of L. pentosus MD353 (A), L. casei ATCC 393 (B), L. casei ATCC 393 transformed with pLP3537 (C), and L. casei ATCC 393 transformed with pLP3537-xyl (D) in MRS medium (A), MRS medium with D-xylose (0), or MRS medium with glucose (*). L. casei transformants were cultivated in the presence of erythromycin. Because of components present in the MRS medium, we consistently observed some background growth of non-D-xylose-fermenting strains in medium with D-xylose. Similar results were obtained for L. plantarum NCDO 1193 transformed with pLP3537 or pLP3537-xyl (not shown).

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In theory, there are several possible ways to improve the ability of L. casei ATCC 393 and L. plantarum NCDO 1193 transformed with pLP3537-xyl to ferment D-xylose. For example, improvement of the system may be achieved by the optimization of the expression of the L. pentosus MD353 xyl genes in the heterologous hosts and/or by the introduction of specific D-xylose transport function(s) in pLP3537xyl. D-Xylose transport function(s) is not present on pLP3537-xyl (9). Sequence analysis of the region upstream from L. pentosus MD353 xylR has indicated the presence of a putative D-xylose transport gene. Currently, experiments are being carried out aimed at the characterization and functional analysis of this gene and at the unraveling of the molecular mechanisms underlying the regulation of the expression of xyl genes in L. pentosus MD353. Together, these experiments will contribute to the development of a food-grade selection system for Lactobacillus spp. which is based on D-xylose fermentation. We are greatly indebted to R. A. Baan for critically reading the manuscript and for constructive discussions. REFERENCES 1. Bringel, F., L. Frey, and J.-C. Hubert. 1989. Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction. Plasmid 22:193-202. 2. Chassy, B. M. 1987. Prospects for the genetic manipulation of lactobacilli. FEMS Microbiol. Rev. 46:297-312. 3. Chassy, B. M., and J. L. Flickinger. 1987. Transformation of Lactobacillus casei by electroporation. FEMS Microbiol. Lett. 44:173-177.

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4. DeMan, J. C., M. Rogosa, and M. E. Sharpe. 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130-135. 5. Jeffries, T. W. 1983. Utilization of xylose by bacteria, yeasts, and fungi. Adv. Biochem. Eng. Biotechnol. 27:1-32. 6. Josson, K., T. Scheirlinck, F. Michiels, C. Platteeuw, P. Stanssens, H. Joos, P. Dhaese, M. Zabeau, and J. Mahillon. 1989. Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii. Plasmid 21:9-20. 7. Kandler, 0. 1983. Carbohydrate metabolism in lactic acid bacteria. Antonie van Leeuwenhoek 49:209-224. 8. Kandler, O., and N. Weiss. 1986. Regular, nonsporing grampositive rods: Lactobacillus, p. 1209-1234. In P. H. A. Sneath, N. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore. 9. Lokman, B. C., et al. Mol. Gen. Genet., in press. 10. Luchansky, J. B., P. M. Muriana, and T. R. Klaenhammer. 1988. Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus, and Propionibacterium. Mol. Microbiol. 2:637-646. 11. McKay, L. L., and K. A. Baldwin. 1990. Applications for biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbiol. Rev. 87:3-14. 12. Posno, M., R. J. Leer, N. van LuiJk, M. J. F. van Giezen, P. T. H. M. Heuvelmans, B. C. Lokman, and P. H. Pouwels. 1991. Incompatibility of Lactobacillus vectors with replicons derived from small cryptic Lactobacillus plasmids and segregational instability of the introduced vectors. Appl. Environ. Microbiol. 57:1822-1828. 13. Posno, M., R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels. 1988. Transformation of Lactobacillus plantarum by plasmid DNA, p. 397-401. In A. T. Ganesan and J. A. Hoch, (ed.), Genetics and biotechnology of bacilli, vol. 2. Academic Press, Inc., New York. 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose ...
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