genetics of lactobacilli: plasmids and gene expression

Antonie van Leeuwenhoek 64: 85-107, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Genetics of lactobacUli: plasmids and gene expression

Peter H. Pouwels & Rob J. Leer Department Molecular Genetics and Gene-Technology, TNO Medical Biological Laboratory, Postbox 5815, 2280 H V Rijswijk, The Netherlands

Received 1 July 1993; accepted 31 August 1993

Key words: Lactobacillus, plasmid vector, DNA replication, structural stability, segregational stability, chromosomal integration, transcription, promoter, antisense RNA, translation, codon usage, protein secretion, heterologous gene expression

Abstract

This paper reviews the present knowledge of the structure and properties of small (< 5 kb) plasmids present in Lactobacillus spp. The data show that plasmids from Lactobacillus spp., like many plasmids from other Gram- positive bacteria, display a modular organization and replicate by a mechanism of rolling circle replication. Structurally, plasmids from lactobacilli are closely related to plasmids from other Gram-positive bacteria. They contain elements (plus- and minus origin of replication, element(s) for control of plasmid replication, mobilization function) showing extensive similarity to analogous elements in plasmids from these other organisms. It is believed that lactobacilli have acquired such elements by intra- and/or intergenic transfer mechanisms. The first part of the review is concluded with a description of plasmid vectors with a Lactobacillus replicon and integrative vectors, including data concerning their structural and segregational stability.

In the second part of this review we describe the progress that has been made during the last few years in identifying and characterizing elements that control expression of genetic information in lactobacilli. Based on the sequence of eleven identified and twenty presumed promoters, some preliminary conclusions can be drawn regarding the structure of Lactobacillus promoters. A typical Lactobacillus promoter shows significant similarity to promoters from E. coli and B. subtilis. An analysis of published sequences of seventy genes indicates that the region encompassing the translation start codon AUG also shows extensive similarity to that of E. coli and B. subtilis. Codon usage of Lactobacillus genes is not random and shows interspecies as well as intraspecies heterogeneity. Interspecies differences may, in part, be explained by differences in G + C content of different lactobacilli. Differences in gene expression levels can, to a large extent, account for intraspecies differences of codon usage bias. Finally, we review the knowledge that has become available concerning protein secretion and heterologous gene expression in lactobacilli. This part is concluded with a compilation of data on the expression in Lactobacillus of heterologous genes under the control of their own promoter or under control of a Lactobacillus promoter.

Introduction

The genus Lactobacillus belongs to the family of Lactobacteriaceae also known as lactic acid bacteria, a group of microorganisms that have been used

for centuries in the preparation and processing of foods and beverages (Rose 1982; Kandler 1984; Chassy 1985, Chassy 1987). These rod-shaped, an- aerobic to micro-aerophilic, non-pathogenic bacteria, which produce lactic acid as their major end-

86

product, are nowadays used in numerous fermentation processes, either alone or together with other organisms such as streptococci, pediococci, lactococci, leuconostocs and yeasts (Kilara & Treki 1984). Particularly in dairy industry these bacteria are of vital importance. Fermented dairy products represent about 20% of the total economic value of fermented foods worldwide (Sharpe 1979). In addition, lactobacilli are used in the production and preservation of sausages and meat, in fermentation of olives and vegetables, in baking, and in the preparation of silage. To maintain products of high qual- ity and to guarantee reproducibility of production processes, cultures of bacteria with selected and predictable properties are used as starters to inoc- ulate food or feed.

Some fermented milk products for many years are believed to have certain health promoting properties for humans. This assumption was originally largely based upon Metchnikoff's theory (Metchni- koff 1908) that harmful effects of undesired bacteria can be overcome by establishing a new balance between intestinal bacteria, through ingestion of lactobacilli or fermented products made by these organisms. Research carried out during the last few dec- ades has resulted in additional claims of health and/ or nutritional benefits for humans and animals associated with the consumption of fermented milk products. Such claims include: control of intestinal infections, improved nutritional value of some foods, control of serum cholesterol levels, improvement of lactose metabolism, induction of a-specific and specific immune responses, and anti-carcino- genic activity (Fernandes et al. 1987; Gurr 1987; Perdigon et al. 1988; Gerritse et al. 1991a, b). Some of these - assumed - properties of certain Lactoba- ciUus species have also focused interest on their use as probiotics to improve the growth potential of livestock.

Due to its great economic importance for the agro-feed sector and its alleged importance for human and animal health, research on selection and characterization of Lactobacillus strains, on metabolism, physiology and genetics of these organisms, and investigations aimed at the improvement of the characteristics of such strains have increased pro- gressively over the last decade. Based on knowl-

edge and methods applied to the development of genetic manipulation systems for other microbes, in particular Escherichia coli, Bacillus subtilis and Lactococcus, rapid progress has been made in developing the methodology for genetic manipulation of lactobacilli by recombinant DNA techniques. Initially, research was focused on the characterization of Lactobacillus DNA by cloning and expression of DNA fragments in E. coli, and on the isolation and characterization of phages and plasmid DNA molecules. Aided by the development of a re- producible system to transform Lactobacillus strains (Chassy & Flickinger 1987; Aukrust & Nes 1988; Luchansky et al., 1988), methods became available to (re)introduce DNA into these organisms. This has prompted studies of more funda- mental processes like DNA replication and control of gene expression, as well as studies aimed at the improvement of specific properties of lactobacilli. As a result of the efforts carried out in a number of laboratories, Lactobacillus research has come of age. Methods for the introduction and stable maintenance of DNA into Lactobacillus are routine now and can be applied to almost any Lactobacillus species. Both broad host-range and narrow host-range multi-copy plasmid vectors based on a variety of replicons have become available for the introduction and expression of homologous and heterologous genes. Finally, methods have been developed to insert genes at specific sites of the chromosome allowing genes to be mutated at will.

No attempts will be made in this review to be ex- haustive with respect to the genetics of lactobacilli. In this review the state of the art will be presented of our knowledge concerning plasmid structure and plasmid replication. Secondly, a description will be given of our present knowledge of gene structure and gene expression of lactobacilli. For further de- tails on the chromosomal organization of the Lacto- bacillus genome, on the genetics of phages and phage resistance, and gene-transfer systems the reader is referred to recent review articles (Chassy & Murphy 1993; Le Bourgeois et al. 1993; Mercenier et al. 1993; Pouwels et al. 1992).

Plasmids

Occurrence

Plasmids have been found in many Lactobacillus spp. since they were first discovered in 1976 by Chassy and co-workers (Chassy et al. 1976; Hofer 1977; Chassy et al., 1978; Smiley & Fryder 1978; Ishi- wa & Iwata 1980; Klaenhammer & Sutherland 1980; Vescovo et al. 1981; Vescovo et al. 1982; Morelli et al. 1983a, b; Klaenhammer 1984; Nes 1984; West & Warner 1985; Jewell & Collins-Thompson 1989; Rinckel & Savage 1990). Several studies concerning plasmid contents of lactobacilli isolated from plant material (Daeschel et al. 1987), meat (Ahrn6 et al. 1989; Ahn & Stiles 1990), silage (Hill & Hill 1986), sour dough (Spicher & L6nner 1985; L6nner et al. 1990) and the gastro-intestinal tract (Klaenhammer & Sutherland 1980; Vescovo et al. 1982; Lin & Sav- age 1985) have been reported. From these studies it has become clear that many, but not all species, har- bour one or more plasmids. Only a fraction of L. plantarum strains isolated from different sources was reported to contain plasmids (Klaenhammer 1984; Nes 1984; West & Warner 1985; Hill & Hill 1986; Von Hysby & Nes 1986; Mayo et al. 1989). Dif- ferences in plasmid content of Lactobacillus strains reported by different investigators, may in part be explained by differences in plasmid extraction methods. However, also differences in source of strains can account for such differences. In contrast with most of the aforementioned studies, Ruiz-Bar- ba et al. (Ruiz-Barba et al. 1991) detected a variety of plasmids in each of the 35 L. plantarum strains from olives and many of the plasmids were found to be of considerably higher molecular weight than those previously observed. In Lactobacillus strains isolated from the gastro-intestinal tract from chick- ens and mice, plasmids were detected in a minor fraction of strains analyzed (Lin & Savage 1985; Posno, pers. comm.). Plasmids are also rarely found in L. bulgaricus strains (Chassy, pers. comm.). L. bulgaricus strain 10 (Culture Collection at North Carolina State University) and L. bulgaricus strain M-878 (Meiji Institute of Health Science, Japan) appear to form the exception (Chagneaud et al. 1992; Sasaki, pers. comm.). Plasmids with variable de-

87

grees of sequence homology, as revealed by DNA- DNA hybridization and/or nucleotide sequence analysis, are found in several species (Bringel et al. 1989; Josson et al., 1989; Vogel et al. 1991; Leer et al. 1992), suggesting that horizontal transfer of plasmids and/or recombination between plasmids are rather frequent events.

Although their ubiquitous presence is well estab- lished, little is known about the function of plasmids. Unlike in lactococci, where the occurrence on plasmids of genes involved in essential functions like proteolysis (Kok 1990) or sugar metabolism (Gasson 1990), and other traits like phage resistance and conjugal transfer (Sanders et al. 1986; McKay & Baldwin 1984; de Vos et al. 1984) and bacteriocin production (Klaenhammer 1993) is quite common, few such plasmid-borne traits have been found in lactobaciUi. During the last few years extensive research has been carried out to link plasmid content with specific bacterial characteristics. Except for cases where plasmid content could be correlated with such phe- notypical properties as drug resistance (Ishiwa & Iwata 1980; Vescovo et al. 1982; Morelli et al. 1983a, b; Axelsson et al. 1988), N-acetyl-glucosamine and slow acid formation (Smiley & Fryder 1978), glucidic metabolism (Liu et al. 1988), carbohydrate metabolism (Chassy et al. 1978; Chassy & Rokaw 1981; Shimizu- Kadota 1987; Kanatani et al. 1992), aminoacid metabolism (Shay et al. 1988), bacteriocin production and immunity (Muriana & Klaenhammer 1987; Schillinger & Lficke 1989; Van der Vossen et al., in preparation) or slime production (Ahrn6 et al. 1989), most plasmids in Lactobacillus, in particular small plasmids, are cryptic.

Segregational stability

Most indigenous Lactobacillus plasmids are segregationally stable. A number of strains have already been propagated under experimental conditions for many years without noticeable changes in plasmid content. Some exceptions have, however, been found. For example, maltose utilization which is a plasmid-borne trait in some lactobacilli present in meat, was found to be unstably inherited (Liu et al. 1988). Three consecutive transfers in the presence

88

of acriflavine resulted in an almost complete loss of the plasmid. Similar phenomena where observed for plasmid-linked galactose utilization markers from a strain of L. acidophilus (Kanatani et al. 1992) and plasmid-borne lactose markers (Chassy et al. 1978). In these cases the markers were found to be present on large plasmids (40-80 MDa). The rela- tive instability of large plasmids contrasts that of the small cryptic plasmids. A number of plasmids found in strains of e.g.L, plantarum or L. pentosus cannot be eliminated by cultivation of the bacteria at sub- lethal temperatures in the presence of acriflavine or novobiocin (Bringel et al. 1989; Leer, unpublished observations), conditions that are effective in curing of most other bacterial strains of endogenous plasmids. The presence of these small plasmids might confer a selective advantage over strains lacking them under conditions used outside the laboratory, although they appear not to carry essential genes.

Plasrnid structure

The nucleotide sequence of eight small cryptic plasmids from Lactobacillus spp. has been determined. Plasmids pLB4 (Bates & Gilbert 1989), pC30il (Skaugen 1989), pLPI (Bouia et al. 1989), p8014-2 (Leer et al. 1992) and pAl (Vujcic & Topisirovic 1993) are from L. plantarum, p353-2 (Leer et al. 1992) from L. pentosus, pLJ1 (Takiguchi et al. 1989) from L. helveticus and pLAB1000 (Josson et al. 1990) from L. hilgardii. The organization of the plasmids, which vary in size from 1.9-3.5 kb, is very similar. All plasmids, except pLJ1, code for a protein showing similarities to the replication proteins (Rep) of plasmids from Gram-positive bacteria. The involvement of this protein in replication has been demonstrated by in trans complementation assays (Bringel et al. 1989; Josson et al. 1990). Based on the functionality of these proteins as replication proteins and their structural similarity to replication proteins from other plasmids, it was assumed that small plasmids from Lactobacillus spp. replicate by a mechanism of Rolling Circle Replication (RCR). Direct proof for this hypothesis has been obtained for pLAB1000 and p353-2, by showing the

accumulation of single-stranded DNA replication intermediates (Josson et al. 1990; Leer et al. 1992). The minimal replication region has been determined for pLAB1000 in Bacillus, Enterococcus and Lactobacillus strains as being 1.5 kb (Josson et al. 1990). Based on the similarity of the structure of pLAB1000 and other plasmids from Lactobacillus spp. with that of other RCR plasmids from Gram- positive bacteria, it seems fair to assume that the minimal replication region for all other Lactobacil- lus plasmids is comparable in size.

Replication protein

The replication proteins of RCR plasmids have nicking-closing activity at a specific site of the plus- DNA strand, called plus-origin of replication, dso (double-stranded DNA origin, previously called ori (+)) as was first demonstrated for pT181 (Koepsel et al. 1987). RCR plasmids have been grouped into four families based on structural similarities of the replication proteins and cognate dso (Gruss & Ehr- lich 1989; Bron 1990; Ilyina & Koonin 1992), exem- plified by pT181 (Khan & Novick 1983), pUBll0 and pC194 (McKenzie et al. 1986, 1987; Horinouchi & Weisblum 1982b), pLS1 and pE194 (Lacks et al. 1986; Horinouchi & Weisblum 1982a) and pSN2 (Khan & Novick 1982). All plasmids from Lactoba- cillus spp. either belong to the second or third cate- gory, as judged by this criterium (Table 1). Although Rep proteins and dso sequences from pLAB1000 and pUB110 display extensive similarity, no trans- complementation is observed (Josson et al. 1990), indicating that Rep proteins show strict specificity for their target nicking site, even towards members of the same family. A similar phenomenon was observed for plasmids from the pT181 family (Iorda- nescu & Projan 1988).

The Rep proteins from pAl and pLB4 are similar to pE194 and other members of the pLS1 family of plasmids. Plasmid pLJ1 is a 3.3 kb plasmid containing one open reading frame (ORF) which could code for a protein of 41 kDa, presumed to be the replication protein. No sequence similarity with other known (replication) proteins has been found, suggesting that the Rep protein of pLJ1 might rep-

Table 1. Characteristic features of L a c t o b a c i l l u s plasmids.

89

Plasmid Strain Size (kb) d s o s so Mob Repressor References

pLJ1 L. h e l v e t i c u s 3.3 ? p a l L - -

pLAB1000 L. h i lgardi i 3.3 pC194 ? + - p353-2 L. p e n t o s u s 2.4 pC194 p a i L - -

pAl L. p l a n t a r u m 2.8 pE194 ? - + pLPI L. p l a n t a r u m 2.1 pC194 p a l L - -

p8014-2 L. p l a n t a r u m 1.9 pC194 p a l L - -

pC30il L. p l a n t a r u m 2.1 pC194 p a l L - -

pLB4 L. p l a n t a r u m 3.5 pE194 p a l L + +

Takiguchi et al. 1989 Josson et al. 1990 Leer et al. 1992 Vujcic & Topisirovic 1993 Bouia et al. 1989 Leer et al. 1992 Skaugen 1989 Bates & Gilbert 1989

resent a new class of RCR replication proteins. Al- ternatively, pLJ1 might replicate by a different mechanism. A comparison of the structure of replication proteins of a variety of plasmids from Sta- phylococcus, Bacillus, Streptococcus and Lactoba- cillus indicates that the Rep proteins from Lactoba- cillus plasmids pLAB1000, pC30il, pLP1, p8014-2 and p353-2 form a cluster that is closely related to the Rep protein of pUB110 but more distantly related to that of pC194 (Alonso pers. comm.).

Minus-origin of replication

The formation of complementary DNA on single- stranded DNA intermediates is initiated at a specific site of the plus-DNA strand, the minus-origin of replication, sso (single-stranded DNA origin), displaying complex secondary DNA structure (Del Solar et al. 1987; Gruss et al. 1987; Boe et al. 1989; Devine et al. 1989). Minus-origins of replication are usually not essential for plasmid replication, but in their absence the amount of single-stranded DNA intermediates increases, which may result in a decreased copy number and segregational instability (Del Solar et al. 1987; Gruss et al. 1987; Deng et al. 1988). Their functioning is host dependent (Del So- lar et al. 1987; Gruss et al. 1987; Boe et al. 1989). Ex- cept for the minus-origin of pUBll0, which is functional in more than one host, most minus-origins present on staphylococcal plasmids like pT181, pC194 and pE194 are not functional in B. subtilis (Gruss et al. 1987). Based on sequence compari- sons, at least three families of minus-origins of rep-

lication have been found, designated palA, palT and palU (Bron 1990).

The presence of a minus-origin of replication in plasmid pLAB1000 from L. hilgardii was predicted from an analysis of the secondary structure (Josson et al. 1990). For plasmid p353-2 the functionality of an inverted repeat sequence in conversion of single- stranded to double-stranded DNA was determined by mutation deletion analysis. The inverted repeat in p353-2 shows strong similarity to similar inverted repeats in pLB4, p8014-2, pLP1, pC30il and pLJ1, but not to any of the minus-origin sequences found in plasmids from other bacterial genera, and thus represents a new class of minus-origins of replication, named palL (Leer et al. 1992). The high level of similarity (74-98 %) among minus-origins of replication of Lactobacillus plasmids mentioned above is even more striking, because these plasmids show otherwise no sequence similarity. The finding that the same type of minus-origin of replication is found in plasmids with different types of replication protein and cognate dso, suggests that also plasmids from Lactobacillus spp. have a modular organization, comprising sequence elements that are hori- zontally transferred from and to other Lactobacil- lus strains and possibly even other genera. Such a phenomenon has already been observed for DNA cassettes from other plasmids (Projan & Novick 1988; Gruss & Ehrlich 1989).

The observation that minus-origins of replication, which are virtually identical (> 98% similarity), are found in two plasmids (p8014-2 and pLJ1) from different Lactobacillus species, suggests that the palL type of minus-origin is functional in different hosts, unlike most other minus-origin of repli-

90

cation. Conversely, the palU type of minus origin from pUBll0, which is functional in both Bacillus and in Staphylococcus, was found not to be functional in L. casei (Shimizu-Kadota et al. 1991). These authors also reported that the palA type of minus origin from pC194 and phage M13 decreased the accumulation of ss-DNA intermediates in L. casei, indicating that, besides palL, also other types of minus origin may be functional in Lactobacillus.

Mobilization functions

Plasmids pLB4 and pLAB1000 carry a gene coding for a 42-46 kDa protein which is presumably involved in plasmid mobilization. The protein encoded by pLAB1000 was shown not to influence DNA replication in Lactobacillus, Bacillus and Staphylo- coccus strains (Josson et al. 1990). The mob gene product is thought, by analogy with similar proteins from pE194, pT181 and pUB110, to fuse plasmids at a specific co-integration site called, RSa, allowing mobilization of non-conjugative plasmids. An R S A

site showing considerable similarity to that present in pE194 and pT181, has been found in plasmids pLB4 and pLAB1000 (Bates & Gilbert 1989; Josson et al. 1990), as well as in pAl (Vujcic & Topisirovic 1993). interestingly, pAl does not code for a Mob protein but contains an ORF which could code for a protein of 103 amino acids showing extensive similarity to the N-terminal region of Mob proteins from other plasmids. This truncated mob gene is probably not expressed as no expression signals were found 5' to the gene and no protein of the expected size could be demonstrated when the plasmid was expressed in vitro (Vujcic & Topisirovic 1993). The R S A site in pLAB1000 overlaps the promoter of the mob gene as was found for plasmid pT181 (Gennaro et al. 1987).

Control of D NA replication

Plasmid DNA replication is regulated through negative control mechanisms by plasmid-borne repressors. These trans-acting repressors bind to plasmid- specific targets, controlling plasmid replication in

an indirect way (Novick 1987; Thomas 1988). For many plasmids antisense RNA (also called counter- transcript RNA or CT-RNA) has been implicated in the mechanism of control of plasmid DNA replication. These CT-RNAs, which are complementary to the 5' end of Rep protein encoding RNA, interfere with the expression of the rep gene, by a mechanism of transcription attenuation (Novick et al. 1989) or by inhibition of translation initiation (Alonso & Taylor 1987; Maciag et al. 1988; Del Solar & Espinosa 1992).

In only one of the plasmids from Lactobacillus spp., pLB4 from L. plantarum, has a gene been found which might encode a repressor protein. The protein shows similarity to repressors encoded by members of the pLS1 family of plasmids. By analogy with pLS1, the pLB4 repressor might repress the synthesis of rep RNA and, in addition, control its own synthesis (Del Solar & Espinosa 1992). These authors suggested that control of DNA replication in pLB4 is also exerted at the level of translation initiation by a CT-RNA which sequesters the Shine-Dalgarno sequence of rep RNA (Shine & Dalgarno 1974).

Plasmid p353-2 of L. pentosus codes for two CT- RNAs of ~ 75 and ~ 250 nucleotides transcribed from the region encoding the 5' end of the rep gene and in the opposite direction (Pouwels et al. 1993). CT-RNA negatively controls plasmids DNA replication. Control of plasmid replication is exerted by a mechanism involving transcription attenuation of rep RNA at a site just in front of the rep gene. In the presence of CT-RNA (wild-type plasmid) more than 90% of transcription initiated at an upstream promoter is prematurely terminated at this attenuator, whereas in the absence of CT-RNA nearly all transcripts reach a size corresponding to that of the rep gene (Fig. 1). A computer-assisted analysis of RNA secondary structures indicates the presence near the 5' end of rep RNA of a sequence which can hybridize to part of the attenuator sequence, pre- cluding the formation of the attenuator stem-loop structure. Secondary structure predictions also suggest that CT-RNA induces a conformational change of the 5' untranslated region of rep RNA, resulting in the formation of the transcription terminator (Pouwels et al. 1993).

Promoter region of repA gene of plasmid p353-2

91

~ 1 9 0 h i

PrepA ATG

Pml I Pml I P CT Pml

~900n|

~ - m l l - . -

!

Fig. 1. Promoter region of repA gene of plasmid p353-2 from L. pentosus, repA RNA synthesis is controlled by an antisense RNA (CT-RNA) which induces the formation of a transcription attenuator, just in front of the ATG codon. As a result, more than 90% of all transcripts initiated at the promoter of repA are arrested at the attenuator. Deletion of the promoter of CT-RNA (Pm/I fragments) results in readthrough at the attenuator which is accompanied by a 5-10-fold increase of the plasmid copy number.

The dissimilarity of the replication proteins but similarity of the organization of the replication regulatory region and mode of regulation of DNA replication of p353-2 and members of the pT181 family of plasmids is of importance with respect to the evolutionary relationship between the two plasmids. In contrast to p353-2 and pT181, but similar to plasmid pLS1, control of replication of pC194 and pUB110 takes place post-transcriptionally and might in- volve sequestration of the translation-initiation signals (Alonso & Taylor 1987; Maciag et al. 1988). These data indicate that the mode of control of DNA replication is different for different members of the pUB110 family of plasmids. Apparently, replication proteins and replication control region are found in different combinations, presumably as a result of horizontal transfer of DNA elements (Pro- jan & Novick 1988; Gruss & Ehrlich 1989; Ilyina & Koonin 1992; Pouwels et al. 1993).

Plasmid vectors

General properties

Broad-host-range vectors like the lactococcal plasmid pGK12, which has been shown to replicate in a variety of bacterial strains (Kok et al. 1984), can also replicate in different Lactobacillus species (Bringel et al. 1989; Posno et al. 1991a). These vectors have been instrumental in the development of gene- transfer systems for Lactobacillus, at the time that

vectors based on Lactobacillus replicons were not yet available. To date a spectrum of plasmid vectors with Lactobacillus replicons has been constructed, allowing genetic manipulation of a wide variety of Lactobacillus species. Most plasmid vectors are derived from small cryptic plasmids of different Lac- tobacillus species, which replicate through a mechanism of RCR. This may affect the stability of recombinant plasmids, as will be shown in a subsequent section. Table 2 presents a list of plasmid vectors currently in use in various laboratories. Vectors contain either the erythromycin-resistance (ery) gene from pE194 or pAM~I, or the chloramphen- |col-resistance (cml) gene from pC194 or pBR328 as selection marker. Since lactobacilli are intrinsically resistant to relatively high concentrations of kana- mycin/neomycin, these markers cannot be used for vector construction. Also ampicillin resistance cannot be used as selection marker in Lactobacillus (Shimizu-Kadota et al. 1991).

Most vectors, as for example pLP825 and pLPE323, display a wide-host-range phenotype, since they can be propagated in a wide variety of Lactobacillus species (Posno et al. 1991a). More- over, their copy number in different Lactobacillus strains does not significantly differ, indicating that also control mechanisms for DNA replication in different host bacteria operate in a similar way. The average copy number of these plasmid vectors is es- timated at 30-50 copies per cell. Copy-number mutants, showing a 3-5 fold elevated copy number, can arise spontaneously during vector construction,

92 /

probably as a result of selection pressure (Fig. 2). The mutations, which give rise to the cop phenotype, have not been mapped. A cop mutant of pLPE323, which was purposely made by deletion of the replication repressor gene, shows a 5-10-fold increase of the copy number (Pouwels et al. 1993).

Some of the Lactobacillus vectors lacking E. coli sequences can nevertheless replicate in this host or- ganism. For example, plasmids of the pPSC series and pAl-derived vectors are promiscuous plasmids that can be propagated in different Lactobacillus species, in Bacillus and in E. coli (Cocconcelli et al. 1991; Vujcic & Topisirovic 1993). Similar findings have been reported for lactococcal plasmids like pSH71 and pWV01 (Kok et al. 1984; Gasson & An- derson 1985; de Vos 1987). Replication proteins of RCR plasmids generally are expressed in E. coli, suggesting that replication of these plasmids in E. coli depends on the presence of host factors that sta- bilize ss-DNA intermediates and/or initiate replication at the minus origin. Recently, a series of broad- host-range vectors was constructed for Lactococcus based on the replication functions of the theta-type plasmid pAM[31 (Simon & Chopin 1988; O'Sullivan

& Klaenhammer 1993). They might be useful for cloning in Lactobacillus as plasmids replicating by a theta-type mechanism show structural and segregational stability (Swinfield et al. 1991; Br0ckner 1992).

Recently, an improved version of vector pLPE323, named pLPE23M (Fig. 3) was obtained by introduction of a multi-linker region with 19 unique restriction enzyme sites. The usefulness of the vector has been demonstrated by cloning and overexpression of several proteins in Lactobacillus. Also a vector with narrow host-range has been described. Plasmid pLUL631 from L. reuteri carrying an erythromycin-resistance gene was found to replicate in L. reuteri and in a strain of L. fermentum among several lactobacilli and other Gram-positive bacteria tested (Ahrn6 et al. 1992). Similarly, a 3.6 kb plasmid replicon from L. crispatus was found to replicate only in the host strain from which it was derived (Posno pers. comm.). The latter type of vectors offers attractive properties with regard to safe- ty aspects associated with the use of live recombinant DNA organisms in e.g. food products. Vectors with a narrow host range are less likely to be hori-

Table 2. Plasmid vectors with Lactobacillus replicon.

Plasmid Replicon Origin E. coli Size Marker Cloning sites References DNA (kb)

pLE16 pLB10 L. bulgaricus pBR328 7.6 cml HindlII Chagneaud et al. 1992 pBG10 pLJ1 L. helveticus pBR329 6.0 lacZ BamHI, PstI Hashiba et al. 1992 pLP3537 p353-2 L. pentosus pUC19 6.3 ery BamHI, HindIII, KpnI, PstI,

SphI Posno et al. 1991a

pLP317 p353-1 L. pentosus no 2.9 ery (SphI) Posno et al. 1991a pLP317cop p353-1 L. pentosus no 2.9 ery (SphI) Posno et al. 1991a pLPE323 p353-2 L. pentosus no 3.6 ery EcoRI, XbaI Posno et al. 1991a pLPE350 p353-2 L. pentosus ** 3.9 ery HindIII, KpnI, PstI, Smal, SphI Leer et al. 1992 pLPE23M p353-2 L. pentosus no 3.7 ery MCS* This paper, Fig. 3 pLEP24Mcop p353-2 L. pentosus no 3.7 ery MCS This paper pLP3537xyl p353-2 L. pentosus pUC 6.3 xyl, ery BamHI, KpnI, SmaI Posno et al. 1991b pLP825 p8014-2 L. plantarum pBR322 5.8 cml SalI, Sph! Leer et al. 1987;

Posno et al. 1991a

pLP82H p8014-2 L. plantarum pUC 5.8 ery BamHI, SacI, Sphl, XbaI Posno et al. 1991a pLPC37 p8014-2 L. plantarum ** 3.7 cml EcoRV, SphI Leer et al. 1992 pULP8/9 pLP1 L. plantarum pUC 6.6 ery HindIII Bouia et al. 1989 pSC10 L. plantarum no 3.0 ery Cocconcelli et aL 1991 pPSC20 L. plantarum no 5.5 cml, ery Cocconcelli et al. 1991 pPSC22 L. plantarum no 4.3 cml, ery Cocconcelli et al. 1991 pLUL634 pLUL631 L. reuteri no 5.1 ery ClaI, Hpal Ahrn6 et al. 1992

* MCS, multi-cloning site. ** 0.3 kb pBR

Fig. 2. Effect of plasmid copy number on the activity of the bacteriocin acidocin B. The acidocin B activity is visualized using Clos- tridium sporogenes as indicator bacteria, immobilized in agar. To the wells were applied neutralized supernatants of (A) parent strain L. acidophilus M46 with acidocin B gene on a low-copy number plasmid, (B) L. plantarum with low-copy vector (pGKV21) containing acidocin B gene, (C) L. plantarum with high-copy mutant (pLPE24M) containing acidocin B gene. In L. acidophilus M46 and L. plantarum/pGKV12-acidocin B, the copy number is 5-15. The copy number in L. plantarum/ pLPE24M-acidocin B is 50-100.

zontally transferred to other bacterial species than vectors based on broad-host-range replicons, and are, consequently, intrinsically more safe.

Vectors have also been described which, poten- tially, are useful for the development of food-grade vectors. Plasmid pBG10, which carries the L. bulgaricus gene encoding J3-galactosidase under control of the promoter of the ery gene from pAM[31, might be useful for the selection of transformants in milk where lactose is the sole energy source (Hashi- ba et al. 1992). Plasmid pLP3537xyl, which contains genes from L. pentosus involved in the catabolism of xylose, is capable of conferring to lactobacilli the capacity to utilize xylose as sole energy source, a trait which is infrequently found in lactobacilli (Posno et al. 1991b).

93

Incompatibility

Lactobacillus strains are cured from the endogenous plasmid when transformed by a vector with a replicon from that plasmid (Bringel et al. 1989; Pos- no et al. 1991a; Leer et al. 1992). This finding, which can be easily explained by selective advantage of vector DNA over the resident plasmid, can be ex- ploited to cure strains from plasmids that are otherwise difficult to eliminate. Also functional relation- ships between replication functions in different plasmids can be assessed in this way. For example, the lactococcal plasmid pGK12 was found to cure L. pentosus MD353 from an 1.7 kb plasmid, indicating that the plasmids share replication functions, although they show little DNA homology (Posno et al. 1991a). L. plantarum ATCC8014 and L. pentosus MD353 were reciprocally cured of the endogenous plasmids p8014-2 and p353-2, when they were transformed by vectors based on these plasmids. The assumption that the two plasmids have similar replication functions was verified by sequence analysis, showing that the two plasmids encode replication proteins displaying 94 % similarity and have identical target sites for these proteins (Leer et al. 1992).

3000

l 0

J

I

2000 I "'i',,,,,,,,, EcoR I ",,

P s t l B s p M I S t u 1 B g l ]1 Cla 1

- S p h ] - X h o I

- S p o l - B a l I - E a e [ - S a l I

- P v u II - B b v I - Fsp I - H ind Ill - K p n l - S m a t

Barn H I

Fig. 3. Structure of plasmid pLPE23M. Plasmid pLPE23M is constructed by insertion of an erythromycin-resistance gene from pE194 and a multi-cloning oligonucleotide into the XbaI site of plasmid p353-2 from L. pentosus. In plasmid pLPE24M (see Fig. 2) the erythromycin-resistance gene was cloned in the opposite orientation.

94

Structural stability

For application in industrial fermentation processes, it is essential that the vector remains structurally intact (structural stability) and can be maintained in the host cell in the absence of selective pressure during the fermentation process (segregational stability). Most plasmid vectors remain structurally intact when the bacteria are cultivated in the presence or absence of the selective agent. No structural instability in L. casei or L. pentosus was observed after insertion into a Lactobacillus vector of DNA fragments derived from bacteriophage )~ varying in size from 2 to 9 kb, irrespective of whether the bacteria were cultivated in the presence or absence of the selective agent (Leer et al. 1992). Complete structural stability was also observed when a 4 kb DNA fragment coding for a hybrid protein consist- ing of a foot-and-mouth disease virus epitope and E. coli [3-galactosidase or a 7.2 kb DNA fragment from L. pentosus comprising genes involved in xylose catabolism were cloned in a Lactobacillus vector (Posno et al. 1991b). However, attempts to clone the Hepatitis delta surface protein under control of a Lactobacillus promoter in L. casei did result in transformants with plasmids of the expected size only, when the cloned gene lacked proper translation-initiation signals (Jore, pers. comm.). Appar- ently, the expression of a protein which is harmful to lactobacilli can be obviated by the occurrence of deletions. Recombinant DNA could also be stably maintained without structural changes when DNA was inserted into the chromosome (Scheirlinck et al. 1989; Bhowmik & Steel 1993; Leer et al. 1993). It appears that except for cases where expression of the cloned gene results in a deleterious protein, cloning of homologous and heterologous DNA into Lactobacillus offers no serious problems. Even relatively large fragments can be stably maintained without the occurrence of detectable deletions or rearrangements.

Segregational stability

Accurate copy number control is required for stable plasmid maintenance. Since RCR plasmids ap-

pear to lack a partitioning function, plasmids most probably are randomly distributed over daughter cells (Novick 1987). Large fluctuations in copy number as a result of a disturbance of copy number control systems may lead to daughter cells receiving no plasmid molecules. For the development of vectors that can be stably maintained, knowledge about plasmid or host factors that contribute to copy number control thus is of paramount importance.

Most vectors are rapidly lost (50-> 95 % loss after 100 generations) when lactobacilli are cultivated in the absence of the selective agent (Bringel et al. 1989; Posno et al. 1991a; Shimizu-Kadota et al. 1991). Vectors with a replicon from Lactococcus or Sta- phylococcus are even less stable in Lactobacillus, showing segregation rates of several percent per generation (Posno et al. 1991a; Shimizu-Kadota et al. 1991). Some vectors with Lactobacillus replicons can, however, be stably maintained for more than 100 generations in Lactobacillus in the absence of selective pressure (Posno et al. 1991a; Cocconcelli et al. 1991; Leer et al. 1992). Of particular interest is plasmid pLPE323 which was found to be segregationally fully stable in all except one Lactobacillus strain (Leer et al. 1992). Plasmid pLP3537, which is rapidly lost under non-selective conditions, har- bours the same replicon as pLPE323 but differs from it by the presence of E. coli vector sequences. The difference in stability between the two plasmids can be fully accounted for by E. coli sequences. After removal of ~ 95% of the E. coli sequences from pLP3537, the resulting vector had become segregationally completely stable. A similar result has been obtained for pLP825 which comprises pBR322 sequences and a replicon from L. plantarum. Segregational instability increases as the size of the inserted DNA fragment increases (Leer et al. 1992), like a phenomenon also observed in B. subtilis for plasmids derived from pUB110 (Bron & Luxen 1985; Bron et al. 1988a, b).

In studies with RCR plasmids from S. aureus (Gruss et al. 1987), Streptococcus pneumoniae (Del Solar et al. 1987), Streptomyces lividans (Deng et al. 1988) and B. subtilis (Bron 1990), a functional minus origin has been implicated in plasmid segregational stability, probably because of inefficient synthesis of the minus strand of the plasmid. A plasmid with a

deletion removing one-half of the stem-loop, which is involved in the conversion of ss-DNA to ds-DNA, is considerably less stable than the parent plasmid in two Lactobacillus species, indicating that also in Lactobacillus a functional minus-origin of replication is important for segregational stability (Leer et al. 1992). Likewise, Shimizu-Kadota and coworkers have observed that introduction of a palA-type or M13 minus origin into a pUB110-derived vector increases the segregational stability of the vector in L. casei (Shimizu-Kadota et al. 1991). Increased segregational stability was positively correlated with a de- crease of the amount of ss-DNA intermediates (Shi- mizu-Kadota et al. 1991; Leer et al. 1992), indicating that inefficient conversion of ss-DNA into ds-DNA in Lactobacillus disturbs copy number control and, consequently, the segregational stability.

Chromosomal integration

Segregational stabilization of cloned genes by integration into the chromosome offers an alternative to replicating plasmid vectors, especially in cases where recombinant vectors are highly unstable, as was observed in other bacteria (Raibaud et al. 1984; Prozorov et al. 1987; Chopin et al. 1989; Leenhouts et al. 1991). Chassy has demonstrated that a plasmid encoding the Lactobacillus Lac-PTS is integrated into the chromosome upon transformation of a lactose-negative L. casei strain (Chassy 1987). Clostri- dium thermocellum endoglucanase and B. stearoth- ermophylus a-amylase genes have been inserted at an unknown site of the L. plantarum chromosome by means of a homologous single-cross-over recombination event. The genes introduced could be stably maintained and expressed under non-selective conditions (Scheirlinck et al. 1989). Replacement of chromosomal genes by mutant alleles was demonstrated in L. helveticus and L. plantarum after transformation with vectors that do not replicate in these organisms and contain an internal part of the target gene or a gene in which an antibiotic-resistance marker was spliced into the target gene (Bhowmik & Steele 1993; Leer et al. 1993). The mutant phenotype is stably maintained for more than 100 generations under non-selective conditions (Leer et al.

95

1993). To obtain transformants with an insertion in- to the chromosome, a high efficiency of transformation with replicating vectors is required, as the efficiency of transformation is three to four orders of magnitude lower than with replicating vectors. Al- ternatively, vectors may be used carrying a thermosensitive replicon. The broad-host-range plasmids pGhost (Maguin et al. 1992) and pE194ts (GrycZan et al. 1982) carrying a thermo-sensitive replicon derived from pGK12 and pE194, respectively, have been used for insertion of DNA fragments into the chromosome of Bacillus and Lactococcus. Since these vectors are also able to replicate in lactobacilli, they might be used for gene-tagging in these organisms as well.

Gene expression

Transcription

To date more than sixty-five Lactobacillus genes have been cloned and sequenced. They originate from more than ten different species, which are evolutionary related but significantly distinct. This is, amongst others, reflected by considerable differences in overall G + C content among different lactobacilli (L. divergens 33-35%; L. fermentum 52%). This evolutionary difference should be kept in mind when comparing expression signals such as promoters, terminators and ribosome binding sites. Evidence has been obtained indicating that expression of Lactobacillus genes takes place when such genes are transferred to other Lactobacillus species or to E. coli (Lerch et al. 1989a, b; Natori et al. 1990; Toy & Bognar 1990; Posno et al. 1991b). These results have allowed a preliminary analysis of Lacto- bacillus transcription and translation signals that are sufficiently similar to be recognized in other hosts, even although control of gene expression might be different.

Promoter sequences To date, transcription start sequences have been identified for eleven Lactobacillus genes originat- ing from seven different species (Table 3). All promoters show typical - 35 and - 10 sequences which,

96

r;

0

r ~

S

o &a o

~ g A

= ' ~ , ~ ~ ~ ~ v ~

~ s ~ < ~

l"q

i:~ t,q

• 5 .v.

0

s ~

s

~ a 4--~ ,--I

V

q--4

when aligned, allow to define a consensus sequence. This consensus sequence resembles that of promoters in E. coli and Bacillus. Also, - 35 and - 10-like sequences with a spacer of ~ 18 nucleotides have been identified upstream of the coding region of nineteen other chromosomal and plasmid-borne Lactobacillus genes (Table 3). Conclusions concerning promoter structure of these genes should as yet be taken with some caution, since the - 35 and - 1 0 sequences were assigned on the assumption that they would conform to a consensus sequence. The length of the untranslated region of most transcripts is relatively small (< 100 nucleotides), comparable to that found in E. coli and Bacillus. The region 5' to the - 35 sequence is rich in A residues, as has previously been observed for Bacillus promoters that are recognized by (y-70 factors (Moran et al. 1982; Graves & Rabinowitz 1986). The spacer region between the two conserved hexanucleotides is rich in T and A residues. A conserved dinucleo- tide, TG was found 5' to the - 10 region as was observed for B. subtilis promoters (Moran et al. 1982). The distance between the - 10 sequence and actual startsite of transcription varies considerably and no specific sequence element can as yet be observed.

Several Lactobacillus promoters have been shown to drive expression of genes fused to such promoters. Expression of the cat gene (encoding chloramphenicol acetyl transferase) was demonstrated in L. casei for the L-ldh promoter of L. casei and the a-amy promoter from L. amylovorus, and for the L. pentosus xyIR and xylA promoters in L. pentosus. Considerable differences in expression levels were observed, suggesting differences in 'strength' of the promoters (Table 4). Expression of the cat gene driven by Pamy is regulated in a similar fashion as is the amy gene in L. amylovorus. The results indicate that these promoters are expressed in heterologous hosts, and are subjected to the same regulation mechanism in lactobacilli as in their chromosomal environment in the original host when the promoter is placed on a multi-copy plasmid. Constitutive expression of the cat gene with these promoters could be demonstrated in E. coli. The promoters Pamy and PxylR have also been used succesfully to express foreign genes in L. casei (see Section 5.5).

97

Terminator sequences Rho-independent-like terminators have been observed at the 3' end of most Lactobacillus genes. The results of Northern blot analyses of several transcripts are consistent with the notion that these sites function as true terminators of transcription (Copeland et al. 1989; Lokman, pers. comm.). The conclusion that these sequences do indeed function as transcription terminators is reinforced by the observation that insertion of the presumed terminator sequence of the xylA/B operon of L. pentosus downstream (3') of a marker gene results in a transcript that is terminated at the site of the terminator.

In the hdcA/B operon of Lactobacillus 30a (Van- derslice et al. 1986) and the xylA/B operon of L. pentosus (Lokman et al. 1991), palindromic sequences have been found in the intergenic region that might function as transcription attenuators and/or RNA processing sites. The function of such elements might be to reduce the efficiency with which the promoter-distal gene is transcribed rela- tive to that of the promoter-proximal gene, or to allow for a difference in RNA turnover rates by dis- section of the two transcripts (Higgins et al. 1982; Belasco & Higgins 1988; McCormick et al. 1991). Palindromic structures believed to function as sites for transcription attenuation and transcription anti- termination, have been found in the 5' untranslated region of the replication protein (repA) encoding gene of the L. pentosus plasmid p353-2 and upstream of the lacEGF genes from L. casei, respectively (Pouwels et al. 1993; Alpert & Siebers, pers. comm.).

Regulation of transcription

Knowledge concerning regulation of gene expression in lactobacilli is scarce and is mainly limited to lactose and xylose fermentation routes. In L. casei the lacEGF genes are preceded by a sequence which has been tentatively identified as the promoter. This promoter is followed (50 nt) by a sequence capable of forming a stable stem-loop structure in the RNA that resembles a rho-independent transcription terminator. Downstream (25 nt) of the terminator starts an 879nt open reading frame

98

which is terminated 120 nt before lacE. Northern blot experiments suggest that the ORF is transcribed together with the genes lacEGF as a 4.4 kb polycistronic mRNA, which is initiated at the promoter located in front of the ORE The data also suggest that the transcriptional unit does not com- prise genes coding for tagatose fermentation, as is the case in Lactococcus lactis and Staphylococcus aureus (Oskouian & Stewart 1990; Van Rooijen et al. 1991a; Van Rooijen et al. 1991b). The putative translation product of the ORF (34 kDa) shows significant similarity to the anti-terminator proteins, SacT, SacY, BglG and ArbB (Debarbouill6 et al. 1991). These data may indicate that transcription of the lac-PTS genes in L. casei is at least partly regulated by an anti-termination mechanism similar to that observed for the ~-glucoside operon in E. coli.

The enzymes involved in catabolism of D-xylose in L. pentosus and L. brevis are induced by xylose. Control of xyl gene expression in L. pentosus has been shown to take place at the level of initiation of transcription, which is induced by xylose and negatively controlled by a repressor protein. The N-terminal region of the repressor protein displays a helix-turn-helix motif characteristic for DNA-binding proteins. In the intergenic region between xyIR and xylA a palindromic structure was found immediately after the transcription-initiation site. The element renders expression of the xyl genes constitutive, when introduced into L. pensosus on a multi- copy plasmid, indicating that the element can function as an operator that titrates the repressor (Lok- man et al., in preparation).

The xyl genes in L. pentosus and L. brevis appear to be subject to catabolite repression as no enzyme synthesis is observed in a medium containing glucose. During growth on a medium containing xylose, high levels of xylP/Q mRNA and xylA/B mRNA are found in L. pentosus, however these levels are greatly reduced when the bacteria are cultivated in a glucose-containing medium or when both xylose and glucose are present in the medium (Lok- man et al., in preparation). In the intergenic region between xylR and xylA, a second palindromic structure is present which overlaps the - 35 element of the promoter PxylA/B. This element shows similarity to the catabolite responsive element found near

Table 4. Expression of cat gene under control of different promo-

ters in different lactobacilli.

Promoter Species L. casei L. pentosus

Inducer

- q - - -I-

xylA L. pentosus n.d.* n.d. - x x

xylR L. pentosus n.d. n.d. x x

L-ldh L. casei xxx xxx n.d. n.d.

amyA L. amylovorus - x x n.d. n.d.

* n.d., not determined; the inducer of the promoters of xylA and

xylR was xylose, that of promoter amyA, cellobiose. Bacteria

with a plasmid carrying the promoter of the L-ldh gene were cul-

tivated in glucose (-) or cellobiose (+) containing medium.

x-xxx, weak to strong expression.

the 5' end of a number of genes in Bacillus (Weicker & Chambliss 1990). The hypothesis that the element is also involved in catabolite repression in L. pentosus is supported by the finding that glucose repression is partially alleviated when the element is introduced on a multi-copy vector. Presumably, a protein which interacts with the element is titrated, resulting in levels of expression of xylA/B in the presence of glucose that are comparable to those found in the presence of xylose (Lokman et al., in preparation).

Translation

The nucleotide sequences around the translation- initiation sites of approximately seventy Lactoba- ciUus genes are known. Despite the fact that the genes originate from more than ten species it is evi- dent that the sequences show some common features. The preferred startcodon is AUG. While four genes start with GUG and three with UUG, all others start with AUG. A highly conserved sequence (AGGAGG), resembling the Shine-Dal- garno motif is found in most of the genes at a distance of 6-10 nucleotides 5' to the startcodon (Table 5). No specific motifs were found in the spacer region, nor was a significant deviation from the A/T content or the Pu/Py ratio observed in the spacer region. The A/T content of the region immediately downstream from the translation startcodon, how-

99

Table 5. Translation initiation region of Lactobacillus genes.

Species No. of genes RBS Spacer (nt)

L. acidophilus 6 G G A G G 5-11

L. bulgaricus 6 A G G A 4 - 9

L. casei 17 A G G A G G 3-11

L. helveticus 7 A G G A G 5 - 8

L. pentosus 9 G G A G G 6 - 9

L. plantarum 11 G G A G G 6--12

The nucleotide indicated is the one occurring most frequently.

Bold capital: > 75%; capital; 50%-75%. The spacer is the

region between the RBS and the translation initiation codon.

ever, deviates from that of the overall composition of the genes. From Table 6 it is clear that the average A/T content of the first five codons after the startcodon is significantly higher than that of the total genes. This holds both for species with high A/T content (e.g.L. helveticus) as well as for species with low A/T content (L. bulgaricus). When the data are analysed for individual positions in the codons, similar conclusions can be inferred. At each position of the codon the A/T content is significantly higher in codons near the 5' end of the genes than in other codons. These data may be interpreted to mean that a high G/C content of the gene 3' of the startcodon might favour intra- or intermolecular RNA-RNA formation, which would interfere with translation initiation.

Translational coupling of contiguous genes can increase the efficiency of translation of the downstream gene. Overlapping start/stop codons, or partly overlapping reading frames, were observed for thefgs (folylpoly-gamma-glutamate synthetase)

gene (Toy & Bognar 1990), the trpF (N-5'-phospho- ribosylanthranilic acid isomerase) gene (Natori et al. 1990) and the purL (phosphoribosylformylglyci- namidine synthetase II) gene (Gu et al. 1992) from L. casei. Interestingly, the lacL/lacM (Chassy & Flickinger 1993), trpB/A (Natori et al. 1990) and purQ/purL (Gu et al. 1992) genes of L. casei are partially overlapped, presumably ensuring the equi- molar synthesis of the two subunits of the enzymes (Oppenheim & Yanofsky 1980). In three genes translation termination is effected by two consecutive stopcodons.

Codon usage Codon usage of Lactobacillus genes shows a clear bias for specific codons. All possible codons are used by lactobacilli, but it appears not by all species. This also applies to stopcodons. UAA is most frequently used and is found in genes of all sPecies ex- amined. The stopcodon UAG however is much more rare and is, except for an unidentified open reading frame in the insertion element IS1 (Shimi- zu-Kadota et al. 1985), not present in 17 genes be- longing to the L. casei group. Whether all these genes are truly L. casei genes remains to be estab- lished (Collins et al. 1989). The codon AGA for ar- ginine is frequently used in L. helveticus and L. plantarum but is rarely or not at all used in most other species. The same applies to the isoleucine codon AUA which is frequently used in L. acidophilus but rarely used in all other species. Table 7 shows that codon usage in two-codon sets, and in isoleucine codons, that use GNN as anticodon, is clearly

Table 6. A/T content of 1st, 2nd and 3rd nucleotide of codons in 5' region of Lactobacillus genes.

Species A/T content (%)

5 ' -end of gene** Total gene

1st 2nd 3rd Average 1st 2nd 3rd Average

Genes*

L. acidophilus 83 79 80 81 56 60 70 62

L. bulgaricus 69 66 67 67 46 61 39 49 L. casei 56 73 ' 67 65 43 63 53 53

L. helveticus 63 89 88 80 54 69 71 65 L. pentosus 64 64 66 65 49 64 60 58

L. plantarum 58 65 85 69 56 69 73 66

5

6 18

7 8

7

* : numbe r of genes; ** : first five codons after the startcodon.

100

different for L. bulgaricus and L. plantarum. C is preferentially used over U, and G over A in L. bulgaricus, whereas the reverse it true for L. plantarum. These results may - in part - be explained by differences in G + C content of the species (L. bulgaricus 50%, L. plantarum 45%, Osawa et al. 1990; Pouwels & Leunissen, in preparation). It seems likely, however, that also the constraint imposed by tRNA affects codon usage. Efficiently expressed genes show a marked preference for C over T, and G over A residues (except for Glu and Gln) in the third position of the codon in two-codon sets, and in isoleucine codons (Table 8). These results indicate that in highly expressed genes in Lactobacillus more NNC codons are chosen than NNU as a result of selection pressure by GNN anticodons. In weakly expressed genes codon choice apparently is much less affected by tRNA.

Protein secretion

Excretion of proteins into the culture fluid is of considerable importance from the biotechnological point of view, as the purification of secreted proteins will in general be easier than that of intra-cellular proteins. Although lactobacilli do not appear to be prodigious protein secretors, several species are known to produce extra-cellular enzymes like a.amylase, insulinase and proteinase (Nakamura & Crowel11979; Nakamura 1981; Szil~igi, pers. comm.; Holck & Naes 1992) and bacteriocins (Joerger & Klaenhammer 1990; Muriana & Klaenhammer

Table 7. Codon usage in two-eodon sets, and Ile of different Lac-

tobacillus species.

L. bulgaricus L. plantarum

C/U + C (%) Asp (GAG/U) 77 Ash (AAC/U) 71 Tyr (UAC/U) 75 Phe (UUC/U) 58 His (CAC/U) 80 Ile (AUG/U) 69

G/G + A (%) Glu (GAG/A) 11 Lys (AAG/A) 75 Gln (GAG/A) 58

Total number codons 2919

24 24 22

7 28 13 26 22 21

1621

1991). The genes coding for an a-amylase from L. amylovorus, a proteinase from L. paracasei and bacteriocins, lactacin F and acidocin B from L. acidophilus have recently been cloned and sequenced (Jore et al., pers. comm.; Muriana & Klaenhammer 1991; Holck & Naes 1992; Van der Vossen et al., pers. comm.). The a-amylase from L. amylovorus and proteinase from L. paracasei contain signal sequences that are typical for secreted proteins. The signal sequence of a-amylase from L. amylovorus is 49 amino acids long, somewhat larger than the B. subtilis enzyme (Jore et al., in preparation). The signal sequences of the two bacteriocins conform only loosely to those associated with secreted proteins, but show resemblance to signal sequences of bacteriocins from other species (Leer et al., in preparation; Klaenhammer 1993). The genes of two surface-layer proteins from L. brevis and L. acidophilus have recently been cloned and their sequence determined (Vidgr6n et al. 1992; Boot et al. 1993). The proteins contain signal sequences that are typical for exported proteins and are 30 and 24 amino acids long, respectively (Pugsley 1989).

Heterologous gene expression

A number of studies have appeared describing the expression of heterologous genes in Lactobacillus spp. The successful transfer of conjugal plasmids from heterologous hosts into Lactobacillus, and the propagation of various plasmid vectors in a variety

Table 8. Codon usage in two-codon sets, and Ile of highly and weakly expressed Lactobacillus genes.

E* M-E N-E

C/U + C (%) Asp (GAG/U) 65 37 25 Asn (AAC/U) 86 51 25 Tyr (UAC/U) 77 52 30 Phe (UUC/U) 85 48 33 His (GAG/U) 76 47 29 Ile (AUC/U) 67 27 22

G/G + A (%) Glu (GAG/A) 1 18 28 Lys (AAG/A) 73 62 40 Gln (GAG/A) 12 22 42

Total number codons 1315 1464 442

* E, efficiently; M-E, moderately-efficiently, N-E, not efficiently.

of Lactobacillus spp., were the first demonstrations that heterologous genes involved in plasmid replication and antibiotic-resistance are expressed in lactobacilli. Besides these genes, which are expressed under the control of their own regulatory elements, also a number of genes from other bacterial sources encoding intra- or extracellular enzymes have been expressed in lactobacilli from their own promoter. Amongst the genes expressed in lactobacilli are a-amylases and glucanases from Clostridium and Bacillus (Jones & Warner 1990; Leer, unpublished observations; Chassy, pets. comm.; Bates et al. 1989; Scheirlinck et al. 1989; Baik & Pack 1990; Thompson & Collins 1991), the lux gene from Vibrio fischeri (Ahmad et al. 1988), and the lipase and lysostaphin genes from Staphylo- coccus (Vogel et al. 1990; Gaier et al. 1992).

Expression of heterologous proteins under the transcriptional control of Lactobacillus promoters was recently demonstrated for a number of enzymes and fusion proteins (Table 9). For example, the E. coli lacZ gene as well as fusion genes coding for a foot-and-mouth disease epitope, or an epitope of rotavirus capsid protein VP7 fused to lacZ, were expressed under the control of the promoter of the xylose repressor gene, xylR. The expression levels are relatively low (up to 1.0%), but might be considerably increased by making use of stronger promoters.

Expression and secretion of extra-cellular proteins into the culture fluid was reported for c~-amylase from L. amylovorus in L. casei under control of

Table 9. Heterologous gene expression in Lactobacillus.

101

its own promoter (Jore et al., in preparation). The regulation of expression was the same in the two organisms. Also genes from more distantly related organisms like S. aureus and E. coli are expressed in Lactobacillus (Table 9).

Conclusions

At the time of the first international congress on Lactic Acid Bacteria in 1984, our knowledge of genetics of lactobacilli was non-existent while the development of gene-transfer and gene-expression systems for lactoccoci was well under way and potential applications of that research were already within reach. During the last few years, however, considerable progress has been made in developing the tools for genetic manipulation of lactobacilli. Knowledge of the structure and mode of replication of plasmids from Lactobacillus spp., and the use of this knowledge for gene-transfer experiments in Lactobacillus has come to a stage where applications in strain improvement programmes can be foreseen. Also our insight into the question of how to stably introduce and express foreign genes in lactobacilli has reached a level similar to that previously demonstrated for other microorganisms.

A major limitation for a number of applications is, however, the lack of detailed knowledge on control of gene expression. In order to exploit the me- tabolic capacity of lactobacilli, it is of paramount importance to understand the mechanisms under-

Protein Origin Species Promoter References

acidocin B L. acidophilus L. plantarum acdB Leer, unpublished levanase B. subtilis L. plantarum/L, casei tac Wanker, pets. comm. c~-Amylase L. amylovorus L. plantarum/L, casei amyA Jore, pets. comm. Xylose isomerase L. pentosus L. casei xylA Posno et al. 1991b 13-Galactosidase E. coli L. plantarum/L, casei xylR, cbh Posno, pers. comm. ~-Glucuronidase E. coli L. plantarum cbh Posno, pers. comm. FMDV-[3-gal FMDV-E. coli L. casei xylR Posno, per~. comm. VP7-[~-gal Rotavirus-E. coli L. plantarum cbh Posno, pers. comm. FMDV-c~-amylase FMDV-L. amylovorus L. casei arnyA Jore, pers. comm. Chloramphenicol pC194 L. casei amyA, L-ldh This paper acetyltransferase L. pentosus xylA, xylR Lokman, pets. comm.

102

lying control of gene expression under conditions prevailing in situ. For example, the use of lactobacilli in the gastro-intestinal tract will require knowledge about control of expression of LactobaciUus

genes in vivo. A second major handicap is our lack of information regarding transfer of genes to and from Lactobacillus by mechanisms other than transformation. Improving knowledge of conven- tional methods of gene-transfer such as conjugation will facilitate strain improvement. This technology can be used to make engineered Lactobacillus

strains available for application in food and feed industry. Such knowledge will also provide information concerning the question whether or not inad- vertent horizontal transfer of cloned genes takes place. This issue is an essential element in discus- sions about the acceptability of genetically engineered microorganisms in human and/or animal food. Despite these uncertainties, the dawning per- spectives for using lactobacilli with improved properties also in areas other than the food/feed sector such as in oral vaccination programmes or in controlling bile acid metabolism warrant further investigations into this scientifically and biotechnologi- cally highly rewarding field.

Acknowledgement

We thank Dr Bruce Chassy for critical reading of the manuscript and Jack Leunissen for help with computer analysis of nucleotide sequences.

References

Ahmad KA & Stewart GSAB (1988) Cloning of the lux genes into Lactobacillus casei and Streptococcus lactis: phosphate- dependent light production. Biochem. Soc. Trans. 1068

Ahn C & Stiles ME (1990) Antibacterial activity of lactic acid bacteria isolated from vacuum-packaged meats. J. Appl. Bac- teriol. 69:302-310

Ahrn6 S, Molin G & Axelsson L (1992) Transformation of Lacto- bacillus reuteri with electroporation studies on the erythromycin resistance plasmid pLUL631. Curr. Microbiol. 24:199-205

Ahrn6 S, Molin G & Stahl S (1989) Plasmids in Lactobacillus isolated from meat and meat products. Syst. Appl. Microbiol. 11: 320-325

Alonso JC & Tailor RH (1987) Initiation of plasmid pC194 repli-

cation and its control in Bacillus subtilis. Mol. Gen. Genet. 210: 476-484

Aukrust T & Nes IF (1988) Transformation of Lactobacillus plantarum with the plasmid pTV1 by electroporation. FEMS Microbiol. Lett. 52:127-132

Axelsson LT, Ahrn6 SEI, Andersson MC & Stahl SR (1988) Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Laetobacillus reuteri. Plasmid 20: 171-174

Baik BH & Pack MY (1990) Expression of a Bacillus subtilis endoglucanase gene in Lactobacillus acidophilus. Biotechnol. Lett. 12:919-924

Bates EEM & Gilbert HJ (1989) Characterization of a cryptic plasmid from Lactobacillus plantarum. Gene 85:253-258

Bates EEM, Gilbert H J, Hazlewood GP, Huckle J, Laurie JI & Mann SP (1989) Expression of a Clostridium thermocellurn endoglucanase gene in Lactobacillus plantarum. Appl. Environ. Microbiol. 55:2095-2097

Belasco JG & Higgins CF (1988) Mechanisms of mRNA decay in bacteria: a perspective. Gene 72:15-23

Boe L, Gros MF, Riele H te, Ehrlich SD & Gruss A (1989) Repli- cation origins of single-stranded DNA plasmid pUB110. J. Bacteriol. 171:3366-3372

Boot H, Kolen KPAM, Noort JM van & Pouwels PH (1993) S- layer protein of Lactobacillus acidophilus ATCC 4356: Puri- fication, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J. Bacteriol. 175:6089~096

Bor YC, Moraes C, Lee SP, Crosby WL, Sinskey AJ & Batt CA (1992) Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase. Gene 114:127-132

Bouia A, Bringel E Frey L, Kammerer B, Belarbi A, Goyon- varch A & Hubert JC (1989) Structural organization of pLP-1, a cryptic plasmid from Lactobacillus plantarum CCM 1904. Plasmid 22:185-192

Bringel F, Frey L & Hubert JC (1989) Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobaeillus plantarum CCM 1904 and its use in shuttle vector construction. Plasmid 22:193-202

Bron S (1990) Plasmids. In: Harwood CR & Cutting SM (Eds) Molecular Biology Methods for Bacillus (pp 75-174) John Wi- ley & Sons, Chichester UK

Bron S & Luxen E (1985) Segregational instability of pUBll0- derived recombinant plasmids in Bacillus subtilis. Plasmid 14: 235-244

Bron S, Haima P, Belkum M van & Luxen E (1988a) Segregation- al plasmid instability in Bacillus subtilis. In: Ganesan AT & Hoch JA (Eds) Genetics and Biotechnology of Bacilli, Vol. 2 (pp 305-309) Academic Press, Inc., San Diego

Bron S, Luxen E & Swart P (1988b) Instability of recombinant pUBll0 plasmids in Bacillus subtilis, plasmid-encoded stability function and effects of DNA inserts. Plasmid 19:231-241

Bhowmik T & Steele JL (1993) Development of an electroporation procedure for gene disruption in Lactobacillus helveticus CNRZ 32. J. Gen. Microbiol. (In press)

Brackner R (1992) A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene 122:187-192

Chagnaud P, Chion CKNCK, Duran R, Naouri E Arnaud A & Gatzy P (1992) Construction of a new shuttle vector for Lacto-

bacillus. Can. J. Microbiol. 38:69-74 Chassy BM (1985) Prospects for improving economically signif-

icant Lactobacillus strains by 'genetic technology'. Trends in Biotechnology 3:273-275

- - (1987) Prospects for the genetic manipulation of lactobacilli. FEMS Microbiol. Rev. 46:297-312

Chassy BM, Gibson E & Giuffrida A (1976) Evidence for extra- chromosomal elements in Lactobacillus. J. Bacteriol. 127: 1576-1578

- - (1978) Evidence for plasmid-associated lactose metabolism in Lactobacillus casei. Current Microbiol. 1:141-144

Chassy BM & Flickinger JL (1987) Transformation of Lactoba- cillus casei by electroporation. FEMS Microbiol. Lett. 44:173- 177

Chassy BM, Flickinger JL & Thompson J (1993) The [3-galactosidase of Lactobacillus casei is a heterodimer encoded by overlapping genes. J. Bacteriol. Submitted

Chassy BM & Rokaw E (1981) Conjugal transfer of lactose plas- raids in Lactobacillus casei. In: Levy S, Clowes R & Koenig E (Eds) Molecular Biology, Pathogenesis and Ecology of Bacte- rial Plasmids (pp 590) Plenum Press, New York

Chassy BM & Murphy C (1993) The Lactococci and the Lactoba- cilli. In: Gram Positive Bacteria. American Society for Micro- biology (In press)

Chopin MC, Chopin A, Rouault A & Galleron N (1989) Inser- tion and amplification of foreign genes in the Lactococcus lac-

tis subs. lactis chromosome. Appl. Environm. Microbiol, 55: 1769-1774

Christiaens H, Leer RJ, Pouwels PH & Verstraete W (1992) Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum using a direct plate assay. Appl. Environ. Microbiol. 58:3792-3798

Cocconcelli PS, Gasson MJ, Morelli L & Bottazzi V (1991) Sin- gle-stranded DNA plasmid, vector construction and cloning of Bacillus stearotherrnophilus c~-amylase in Lactobacillus. Res. Microbiol. 142:643-652

Collins MD, Phillips BA & Zanoni P (1989) Deoxyribonucleic acid homology studies of Lactobacillus casei, Lactobacillus

paracasei sp. nov., subsp, paracasei and subsp, tolerans, and Lactobacillus rhamnosus sp. nov., comb. nov. Int. J. Syst. Bac- teriol, 39:105-107

Copeland WC, Domena JD & Robertus JD (1989) The molecular cloning, sequence and expression of the hdc B gene from Lactobacillus 30A. Gene 85:259-266

Daeschel MA, Andersson RE & Fleming HP (1987) Microbial ecology of fermenting plant materials. FEMS Microbiol. Rev. 46:357-367

Damiani G, Romagnoli S, Ferretti L, Morelli L, Bottazzi V & Sgaramella V (1987) Sequence and functional analysis of a di- vergent promoter from a cryptic plasmid of Lactobacillus acidophilus 168S. Plasmid 17:69-72

Debarbouille M, Martin-Verstraete I, Arnaud M, Klier A & Rapport G (1991) Positive and negative regulation controlling

103

expression of the sac genes in Bacillus subtilis. Res. Microbiol. 142:757-764

Del Solar GH, Puyet A & Esponosa M (1987) Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1. Nucleic Acids Res. 15:5561-5580

Del Solar GH & Espinosa M (1992) The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products-the antisense RNA II and repressor protein, RepA. Mol. Microbiol. 6:83-94

Deng Z, Keiser T & Hopwood D (1988) 'Strong incompatibility' between derivatives of the Streptomyces multicopy plasmid plJ101. Mol. Gen. Genet. 214:286-294

Devine KM, Hogan ST, Higgins DG & McConnell DJ (1989) Replication and segregational stability of the Bacillus plasmid pBAA1. J. Bacteriol. 171:1166-1172

Fernandes CE Shahani KM & Amer MA (1987) Therapeutic role of dietary lactobacilli and lactobacillic fermented dairy products. FEMS Microbiol. Rev. 46:343-356

Gaier W, Vogel RF & Hammes WP (1992) Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobacil- lus casei. Lett. Appl. Microbiol. 14:72-76

Gasson MJ & Anderson PH (1985) High copy number plasmid vectors for use in lactic streptococci. FEMS Microbiol. Lett. 30:193-196

Gasson MJ (1990) In vivo genetic systems in lactic acid bacteria. FEMS Microbiol. Rev. 87(1-2): 43-60

Gennaro ML, Kornblum J & Novick RP (1987) A site-specific recombination function in Staphylococcus aureus plasmids. J. Bacteriol. 169:2601-2610

Gerritse K, Posno M, Schellekens MM, Boersma WJA & Claas- sen E (1991a) Oral administration of TNP-Lactobacillus conjugates in mice: A model for evaluation of mucosal and systemic immune responses and memory formation elicited by transformed lactobacilli. Res. Microbiol. 141:955-962

Gerritse K, Posno M, Fasbender MJ, Schellekens MM, Boersma WJA & Claassen E (1991b) Mucosal immune responses and systemic immunological memory after oral administration of TNP-Lactobacillus conjugates in mice. Lymphatic tissues and in vivo immune responses. Adv. Exp. Med. & Biol. 84:497-504

Gilliland SE (1990) Health and nutritional benefits from lactic acid bacteria. FEMS Microbiol. Rev. 87:175-188

Graves MC & Rabinowitz JC (1986) In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. J. Biol. Chem. 261:11409-11415

Gruss AD, Ross HF & Novick RP (1987) Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids. Proc. Natl. Acad. Sci. USA 84: 2165-2169

Gruss A & Ehrlich D (1989) The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol. Rev. 53:231-241

Gryczan TJ, Hahn J, Contente S & Dubnau D (1982) Replication and incompatibility properties of plasmid pE194 in Bacillus subtilis. J. Bacteriol. t52:722-735

Gu ZM, Martindale DW & Lee BH (1992) Isolation and com-

104

plete sequence of the pur L gene encoding FGAM synthase II in Lactobacillus casei. Gene 119:123-126

Gurr MI (1987) Nutritional aspects of fermented milk products. FEMS Microbiol. Rev. 46:337-342

Hashiba H, Takiguchi R, Jyoho K & Aoyama K (1992) Establish- ment of a host-vector system in Lactobacillus helveticus with ~-galactosidase activity as a selection marker. Biosci. Biotech- nol. Biochem. 56:190-194

Heaton MP & Neuhaus FC (1992) Biosynthesis of D-alanyl lipo- teichoic acid: cloning, nucleotide sequence and expression of the Lactobacillus casei gene for the D-alanine activating enzyme. J. Bacteriol. 174:4707-4717

Higgins CF, Ames GFL, Barmes WM, Clement JM & Hofnung M (1982) A novel intercistronic regulatory element of prokaryotic operons. Nature 298:760-762

Hill HA & Hill JE (1986) The value of plasmid profiling in mon- itoring Lactobacillus plantarum in silage fermentations. Curr. Microbiol. 13:91-94

Hofer F (1977) Involvement of plasmids in lactose metabolism in Lactobacillus casei suggested by genetic experiments. FEMS Microbiol. Lett. 1:167-170

Holck A & Naes H (1992) Cloning, sequencing, and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp, paracasei NCDO 151. J. Gen. Microbiol. 138:1353-1364

Horinouchi S & Weisblum B (1982a) Nueleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J. Bacteriol. 150:804-814

- - (1982b) Nucleotide sequence and functional map of pC194, a plasmid that specifies chloramphenicol resistance. J. Bacte- riol. 150:815-825

HiSttinger H, Ohgi T, Zwahlen M-C, Dhamija S & Soell D (1987) Allele-specific complementation of an Escherichia coli leuB mutation by a Lactobacillus bulgaricus tRNA gene. Gene 60: 75-83

Hybsy KO yon & Nes IF (1986) Changes in the plasmid profile of Lactobacillus plantarum obtained from commercial meat starter cultures. J. Appl. Bacteriol. 60:413-417

Ilyina TV & Koonin EV (1992) Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archae- bacteria. Nucl. Acids. Res. 20:3279-3285

Iordanescu S & Projan SJ (1988) Replication termination for staphylococcal plasmids: plasmids pT181 and pC221 crossreact in the termination process. J. Bacteriol. 170:3427-3434

Ishiwa H & Iwata S (1980) Drug resistance plasmids in Lactoba- cillus fermentum. J. Gen. Appl. Microbiol. 26:1-74

Iwata M (1988) Characterization of a pAMI31 deletion derivative isolated from Lactobacillus casei after conjugation. Biochimie 30:553-558

Jewell B & Collins-Thompson DL (1989) Characterization of chloramphenicol resistance in Lactobacillus plantarurn catC2. Curr. Microbiol. 19:343-346

Joerger MC & Klaenhammer TR (1990) Cloning, expression and nucleotide sequence of the Lactobacillus helveticus 481 gene

encoding the bacteriocin helveticin. J. Bacteriol. 172: 6339- 6347

Jones S & Warner PJ (1990) Cloning and expression of alpha- amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum. Lett. Appl. Microbiol. 11: 214-219

Josson K, Scheirlinck T, Michiels F, Platteeuw C, Stanssens P, Joos H, Dhaese P, Zabeau M & Mahillon J (1989) Character- ization of a Gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii. Plasmid 21:9-20

Josson K, Soetaert E Michiels E Joos H & Mahillon J (1990) Lac- tobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other Gram-positive organisms. J. Bacteriol. 172:3089-3099

Kanatani K, Tahara T, Yoshida K, Miura H, Sakamoto M & Osh- imura M (1992) Plasmid-linked galactose utilization by Lacto- bacillus acidophilus TK8912. Biosci. Biotech. Biochem. 56: 826-827

Kandler O (1984) Current taxonomy of lactobacilli. Dev. Indust. Microbiol. 25:109-123

Khan S & Novick R (1982) Structural analysis of plasmid pSN2 in Staphylococcus aureus: no involvement in enterotoxin B production. J. Bacteriol. 149:642-649

- - (1983) Complete nucleotide sequence of pT181, a tetracycline- resistance plasmid from Staphylococcus aureus. Plasmid 10: 251-259

Kilara A & Treki N (1984) Uses of lactobaeilli in foods. Unique benefits. Dev. Indust. Microbiol. 25:125-.138

Kim SF, Baek SJ & Pack MY (1991) Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene. Appl. Environ. Microbiol. 57:2413-2417

Klaenhammer TR (1984) A general method for plasmid isolation in lactobacilli. Curr. Microbiol. 10:23-28

- - (1993) Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12:39-85

Klaenhammer TR & Sutherland SM (1980) Detection of plasmid deoxyribonucleic acid in an isolate of Lactobacillus acidophilus. Appl. Environ. Microbiol. 35:592-600

Knauf H J, Vogel RF & Hammes WP (1992) Cloning, sequence, and phenotypic expression of katA which encodes the catalase of Lactobacillus sake LTH677. Appl. Environ. Microbiol. 58: 832-839

Koepsel R & Khan S (1987) Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein. Nucleic Acids Res. 15: 4085-4097

Kok J (1990) Genetics of the proteolytic system of the lactic acid bacteria. FEMS Microbiol. Rev. 87:15-42

Kok J, Vossen JMBM van der& Venema G (1984) Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl. Envi- ron. Microbiol. 48:726-731

Lacks SA, Lopez P, Greenberg B & Espinosa M (1986) Identifi- cation and analysis of genes for tetracycline resistance and replication functions in the boad-host-range plasmid pLS1. J. Mol. Biol. 192:753-765

Le Bourgeois P, Lautier M & Ritzenthaler P (1993) Chromo-

some mapping in lactic acid bacteria. FEMS Micro. Rev. 12: 109-124

Leenhouts K J, Gietema J, Kok J & Venema G (1991) Chromoso- mal stabilization of the proteinase genes in Lactococcus lactis. Appl. Environ. Microbiol. 56:2568-2575

Leer R, Christiaens H, Verstraete W, Peters L, Posno M & Pou- wels PH (1993) Gene-disruption in Lactobacillus plantarum strain 80 by site-specific recombination: isolation of a mutant strain deficient in conjugated bile salt hydrolase activity. Mol. Gen. Genet. 239:26%272

Leer RJ, Luijk N van, Posno M & Pouwels PH (1992) Structural and functional analysis of two cryptic plasmids from Lactoba- cillus pentosus MD353 and Lactobacillus plantarurn ATCC 8014. Mol. Gen. Genet. 234:265-274

Leer RJ, Posno M, Rijn JMM van, Lokman BC & Pouwels PH (1987) Transformation of Lactobacillus plantarum by plasmid DNA. FEMS Microbiol. Rev. 46:P20

Leong-Morgenthaler R Zwahlen MC & H6ttinger H (1991) Lac- tose metabolism in Lactobacillus bulgaricus: Analysis of the primary structure and expression of the genes involved. J. Bacteriol. 173:1951-1957

Lerch H-R Frank R & Collins J (1989a) Cloning, sequencing and expression of the L-2-hydroxyisocaproate dehydrogenase-encoding gene of LactobaciIlus confusus in Escherichia coli. Gene 83:263-270

Lerch HE Blocker H, Kallwas H, Hoppe J, Tsai H & Collins J (1989b) Cloning, sequencing and expression in Eschericia coli of the D-2-hydroxyisocaproate dehydrogenase gene of Lacto- bacillus casei Gene 78:47~7

Lin JHC & Savage DC (1985) Cryptic plasmids in Lactobacillus strains isolated from the murine gastrointestinal tract. Appl. Environ. Microbiol. 49:1004-1006

Liu ML, Kondo JK, Barnes MB & Bartholomeu DT (1988) Plas- mid-linked maltose utilization in Lactobacillus ssp.. Biochimie 70:351-355

Lokman BC, Santen P van, Verdoes JC, Kruse J, Leer R J, Posno M & Pouwels PH (1991) Organization and characterization of three genes involved in D-xylose catabolism in Lactobacillus pentosus. Mol. Gen. Genet. 230:161-169

L6nner C, Preve-Akeson K & A h r n 6 0 (1990) Plasmid contents of lactic acid bacteria isolated from different types of sour doughs. Curr. Microbiol, 20:201-207

Luchansky JB, Muriana PM & Klaenhammer TR (1988) Appli- cation of electroporation for transfer of ptasmid DNA to Lac- tobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacteri- urn. Mol. Microbiol. 2:637-646

Maciag IE, Virel J-F & Alonso JC (1988) Replication and incompatibility properties of plasmid pUB110 in Bacillus subtilis. Mol. Gen. Genet. 212:232-240

Maguin E, Duwat R Hege T, Ehrlich D &Gruss A (1992) New thermosensitive plasmid for gram-positive bacteria. J. Bacte- riol. 174:5633-5638

Mayo B, Hardisson C & Brana AF (1989) Selected characteristic of several strains of Lactobacillus plantarum. Microbiologia 5: 105-122

105

McCormick JR, Zengel JM & Lendahl L (1991) Intermediates in the degradation of mRNA from the lactose operon of Escher- ichia coll. Nucleic Acids Res. 19:2767-2776

McKay LL & Baldwin KA (1984) Conjugative 40-megadalton plasmid in Streptococcus lactis subsp, diacetylactis DRC3 is associated with resistance to nisin and bacteriophage. Appl. En- viron. Microbiol. 47:68-74

McKenzie T, Hoshino T, Tanaka T & Sueoka N (1986) The nucleotide sequence of pUBll0: some salient features in relation to replication and its regulation. Plasmid 15:93-103

McKenzie T, Hoshino T, Tanaka T & Sueoka N (1987) A revision of the nucleotide sequence and functional map of pUBll0. Plasmid 17:83-85

Mercenier A, Pouwels PH & Chassy BM (1993) Genetic engi- neering of lactobacilli, leuconostocs, and Streptococcus ther- mophilus. In: Vos W de & Gasson M (Eds) Applied Genetics of Lactic Acid Bacteria. Blackie & Son, Ltd, Glasgow (In press)

Metchnikoff E (1908) The prolongation of life. G.R Putnam's & Sons, New York

Moran CP Jr, Lang N, LeGrice SFJ, Lee G, Stephens M, Sonesh- ein AL, Pero J & Losick R (1982) Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol. Gen. Genet. 186:339-346

Morelli L, Vescovo M & Bottazzi V (1983a) Plasmids and antibiotic resistance in Lactobacillus helveticus and Lactobacillus bulgaricus isolated from natural whey culture. Microbiologia 6:145-154

- - (1983b) Identification of chloramphenicol resistance plasmids in Lactobacillus reuteri and Lactobacillus acidophilus. Int. J. Microbiol. 1:1-5

Muriana P & Klaenhammer TR (1987) Conjugal transfer of plasmid-encoded determinants for bacteriocin production and immunity in Lactobacillus acidophilus 88. Appl. Environ. Micro- biol. 53:553-560

- - (1991) Cloning, phenotypic expression, and DNA sequence of the gene for Lactocin F, an antimicrobial peptide produced by Lactobacillus spp.. J. Bacteriol. 173:1779-1788

Nakamura LK (1981) Lactobacillus amylovorus, a new starch- hydrolyzing species fi'om cattle waste-corn fermentations. Int. J. Syst. Bacteriol. 31:56-63

Nakamura LK & Crowell CD (1979) Lactobacillus amylophilus, a new starch-hydrolyzing species from swine waste-corn fermentation. Dev. Indust. Microbiol. 20:531-540

Natori Y, Kano Y & Imamoto F (1990) Nucleotide sequences and genomic constitution of five tryptophan genes of Lactoba- cillus casei. J. Biochem. (Tokyo) 107:248-255

Nes IF (1984) Plasmid profiles of ten strains of Lactobacillus plantarum. FEMS Microbiol. Lett. 21:35%361

Novick RP (1987) Plasmid incompatibility. Microbiol. Rev. 51: 381-395

Novick RR lordanescu S, Projan SJ, Kornblum J & Edelman I (1989) pT181 plasmid replication is regulated by a countertran- script-driven transcriptional attenuator. Cell 59:395404

Oppenheim DS & Yanofsky C (1980) Translational coupling

106

during expression of the tryptophan operon of Escherichia coli. Genetics 95:785-795

Osawa S, Muto A, Ohama T, Andachi Y, Tanaka R & Yamao F (1990) Prokaryotic genetic code. Experientia 46:1097-1106

O'Sullivan DJ & Klaenhammer TR (1993) High and low copy number Lactococcus shuttle cloning vectors with feature for clone selection. Submitted

Oskouian B & Stewart GC (1990) Expression and catabolic repression of the lactose operon of Staphyloccus aureus. J. Bac- teriol. 172:3804-3812

Perdigon G, Macias MEN de, Alvarez S, Oliver G, Ruiz Holgado AP de (1988) Systemic augmentation of the immune response in mice by feeding fermented milks with Lactobacillus casei

and Lactobacillus acidophilus. Immunology 63:17-23 Pinter K, Davisson VJ & Santi DV (1988) Cloning, sequencing,

and expression of the Lactobacillus casei thymidylate synthase gene. DNA 7:235-241

Porter EV & Chassy BM (1988) Nucleotide sequence of the [3-D- phospho-galactoside galactohydrolase gene of Lactobacillus casei: comparison to analogouspbg genes of other Gram-positive organisms. Gene 62:263-276

Posno M, Leer RJ, Luijk K van, Giezen MJF van, Heuvelmans PTHM & Pouwels PH (1991a) Incompatibility of Lactobacil-

lus vectors with replicons derived from small cryptic Lactoba- cillus plasmids and segregational instability of the introduced vectors. Appl. Environ. Microbiol. 57:1822-1828

Posno M, Heuvelmans PTHM, Giezen MJF van, Lokman BC, Leer RJ & Pouwels PH (1991b) Complementation of the in- ability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus. Appl. Environ. Microbiol. 57:2764-2766

Pouwels PH, Leer RJ & Posno M (1992) Genetic modification of Lactobacillus: A new approach toward strain improvement. In: Actes du Colloque Lactic 91:133-148

Pouwels PH, van Luijk N, Leer RJ & Posno M (1993) Control of replication of the Lactobacilluspentosus plasmid p 353-2: Evi- dence for a mechanism involving transcription attenuation of the gene coding for the replication protein. Molec. Gen. Ge- net. in press

Projan S & Novick R (1988) Comparative analysis of five related staphylococcal plasmids. Plasmid 19:203-221

Prozorov AA, Poluektova EU, Sachenko GV, Nezametdivona VZ & Khasanov FK (1987) Various means of integration of the expressible human dihydrofolate reductase gene into the Bacillus subtilis genome. Gene 57:221-227

Pugsley AP (1989) Protein targeting. Academic Press, Inc. Raibaud O, Mock M, Schwartz M (1984) A technique for inte-

grating any DNA fragment into the chromosome of Escher- ichia coli. Gene 29:231-241

Rinckel LA & Savage DC (1990) Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33. Plasmid 23:119-125

Rooijen RJ van & Vos WM de (1991a) Molecular cloning, transcriptional analysis, and nucleotide sequence of lacR, a gene encoding the repressor of the lactose phosphotransferase system of Lactobacillus lactis. J. Biol. Chem. 265:18499-18503

Rooijen RJ van, Schalkwijk S van & Vos WM de (1991b) Mole- cular cloning, characterization, and nucleotide sequence of the tagatose-6-phosphate pathway gene cluster of the lactose operon of Lactobacillus lactis. J. Biol. Chem. 266:7176-7181

Rose AH (1982) History and scientific basis of microbial activity in fermented foods. In: Rose AH (Ed) Fermented Foods (pp 1-13) Academic Press, New York

Ruiz-Barba JL, Piard JC & Simenez-Diaz R (1991) Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations. J. Appl. Bacteriol. 71: 417-421

Sanders ME, Leonhard P J, Sing WD & Klaenhammer TR (1986) Conjugal strategy for construction of fast acid-producing, bacteriophage-resistant lactic streptococci for use in dairy fermentations. Appl. Environ. Microbiol. 52:1001-1007

Scheirlinck T, Mahillon J, Joos H, Dhaese P & Michiels F (1989) Integration and expression of a-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome. Appl. En- viron. Microbiol. 55:2130-2137

Schillinger U & LOcke FK (1989) Antimicrobial activity of Lac-

tobacillus sake isolated from meat. Appl. Environ. Microbiol. 55:1901-1906

Sharpe ME (1979) Lactic acid bacteria in the dairy industry. J. Soc. Dairy Technol. 32:9-18

Shay B J, Egan A, Wright M & Rogers P (1988) Cysteine metabolism in an isolate of Lactobacillus sake: plasmid composition and cysteine transport. FEMS Microbiol. Lett. 56:183-188

Shimizu-Kadota M (1987) Properties of lactose plasmid pLY101 in Lactobacillus casei. Appl. Environ. Microbiol. 53: 2987- 2991

Shimizu-Kadota M, Kiwaki M, Hirokawa H & Tsuchida N (1985) ISLI: a new transposable element in Lactobacillus casei. Mol. Gen. Genet. 200:193-198

Shimizu-Kadota M, Shibahara-Sone H & Ishiwa H (1991) Shut- tle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin. Appl. Environ. Microbiol. 57:3292-3300

Shine J & Dalgarno L (1974) The 3'-terminal sequence of Es-

cherichia coli 16S RNA: complementarity to non-sense triplets and ribosome binding sites. Proc. Natl. Acad. Sci USA 71: 5463-5467

Simon D & Chopin A (1988) Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70:559-566

Skaugen M (1989) The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum. Plasmid 22: 175-179

Smiley MB & Fryder V (1978) Plasmids, lactic acid production, and N-acetyl-D-glucosamine fermentation in Lactobacillus

helveticus subsp, jugurtL Appl. Environ. Microbiol. 35: 777- 781

Spicher G & L6nner C (1985) Die Mikroflora des Sauerteiges XXI. die in Sauerteigen schwedischer B~ckereien vorkom- menden Lactobacillen. Zu Lebensm. unters. Forsch. 181:9-13

Swinfield MJ, Janniere L, Ehrlich SD & Minton NP (1991) Char- acterization of a region of the Enterococcus faecalis plasmid pAMI31 which enhances the segregational stability of pAMJ31-

derived cloning vectors in Bacillus subtilis. Plasmid 26: 209- 221

Taguchi H & Ohta T (1991) D-lactate dehydrogenase is a mem- ber of the D-isomer-specific 2-hydroxyacid dehydrogenase family: Cloning, sequencing and expression in Escherichia coli of the L-lactate dehydrogenase gene of Lactobacillus plantarum. J. Biol. Chem. 266:12588-12594

Takiguchi R, Hashiba H, Aoyama K & Ishii S (1989) Complete nucleotide sequence and characterization of a cryptic plasmid from Lactobacillus helveticus subsp, jugurti. Appl. Environ. Microbiol. 55:1653-1655

Thomas CM (1988) Recent studies on the control of plasmid replication. Biochim. Biophys. Acta 949:253-263

Thompson K & Collins M (1991) Molecular cloning in Lactoba- cillus helveticus by plasmid pSA3 : pVA797 co-integrate formation and conjugal transfer. Appl. Microbiol. Biotech. 35: 334-338

Toy J & Bognar AL (1990) Cloning and expression of the gene encoding Lactobacillus casei Folylpoly-gamma-glutamate synthetase in Escherichia coli and determination of its primary structure. J. Biol. Chem. 265:2492-2499

Vanderslice P, Copeland W & Robertus J (1986) Cloning and nucleotide sequence of wild type and a mutant histidine decar- boxylase from Lactobacillus 30a. J. Biol. Chem. 261: 15186- 15191

Vescovo M, Morelli L & Bottazzi V (1981) Evidence of plasmid DNA in Lactobacillus. Microbiologia 4:413-419

107

- - (1982) Drug resistance plasmids in Lactobacillus acidophilus

and Lactobacillus reuteri. Appl. Environ. Microbiol. 43:50-56 Vidgr6n G, Palva I, Pakkanen R, Lounatmaa K & Palva A (1992)

S-layer of Lactobacillus brevis: PCR cloning and determination of the nucleotide sequence. J. Bacteriol. 174:7419-7427

Vogel RE Gaier W & Hammes WP (1990) Expression of the lipase gene from Staphylococcus hyicus in Lactobacillus curvatus Lc2-c. FEMS Microbiol. Lett. 69:289-292

Vogel RF, Lohmann M, Weller AN, Hugas M & Hammes WP (1991) Structural similarity and distribution of small cryptic plasmids of Lactobacillus curvatus and L. sake. FEMS Micro- biol. Lett. 84:183-190

Vos WM de (1987) Gene cloning and expression in lactic streptococci. FEMS Microbiol. Rev. 46:281-295

Vos WM de, Underwood HM & Davies FL (1984) Plasmid encoded bacteriophage resistance in Streptococcus cremoris SKll. FEMS Microbiol. Lett. 23:175-178

Vujcic M & Topisirovic L (1993) Molecular analysis of the rolling-circle replicating plasmid pAl of Lactobacillus plantarum Al12. Appl. Environ. Microbiol. 59:274-280

Weicker MJ & Chambliss GH (1990) Site-directed mutagenesis of the catabolite repression operator sequence in Bacillus sub-

tilis. Proc. Natl. Acad. Sci. USA 87:6238-6242 West CA & Warner PJ (1985) Plasmid profiles and transfer of

plasmid-encoded antibiotic resistance in Lactobacillus planta-

rum. Appl. Environ. Microbiol. 50:1319-1321

genetics of lactobacilli: plasmids and gene expression

Documents