Genetics Of Lactobacilli: Plasmids And Gene Expression

Antonie van Leeuwenhoek 64: 85-107,1993. © 1993KluwerAcademic Publishers. Printedin the Netherlands.

Genetics of lactobacUli: plasmids and gene expression Peter H. Pouwels & Rob J. Leer Department Molecular Genetics and Gene-Technology, T N O Medical Biological Laboratory, Postbox 5815, 2280 H V Rijswijk, The Netherlands Received1July1993;accepted31 August1993

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 Grampositive 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 A U G 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, anaerobic 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 quality and to guarantee reproducibility of production processes, cultures of bacteria with selected and predictable properties are used as starters to inoculate 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 (Metchnikoff 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 decades 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-carcinogenic activity (Fernandes et al. 1987; Gurr 1987; Perdigon et al. 1988; Gerritse et al. 1991a, b). Some of these - assumed - properties of certain LactobaciUus 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 progressively 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 reproducible 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 fundamental 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 exhaustive 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 details on the chromosomal organization of the Lactobacillus 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).

87 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; Ishiwa & 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 & Savage 1985) have been reported. From these studies it has become clear that many, but not all species, harbour 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). Differences 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-Barba 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 chickens 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-

grees of sequence homology, as revealed by DNADNA 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 established, 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 phenotypical 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; ShimizuKadota 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 relative 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 sublethal 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 Grampositive bacteria, it seems fair to assume that the minimal replication region for all other Lactobacillus plasmids is comparable in size.

Replication protein

The replication proteins of RCR plasmids have nicking-closing activity at a specific site of the plusDNA 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 & Ehrlich 1989; Bron 1990; Ilyina & Koonin 1992), exemplified 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 Lactobacillus spp. either belong to the second or third category, as judged by this criterium (Table 1). Although Rep proteins and dso sequences from pLAB1000 and pUB110 display extensive similarity, no transcomplementation 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 (Iordanescu & 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-

89 T a b l e 1.

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

Plasmid

Strain

Size (kb)

dso

sso

Mob

Repressor

References

pLJ1 pLAB1000 p353-2 pAl pLPI p8014-2 pC30il pLB4

L. h e l v e t i c u s

3.3 3.3 2.4 2.8 2.1 1.9 2.1 3.5

? pC194 pC194 pE194 pC194 pC194 pC194 pE194

palL

-

-

?

+

-

paiL

-

-

?

-

+

pal L

-

-

palL

-

-

palL

-

-

palL

+

+

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

L. hilgardii L. p e n t o s u s L. p l a n t a r u m L. plantarum L. p l a n t a r u m L. p l a n t a r u m L. p l a n t a r u m

resent a new class of RCR replication proteins. Alternatively, pLJ1 might replicate by a different mechanism. A comparison of the structure of replication proteins of a variety of plasmids from Staphylococcus, Bacillus, Streptococcus and Lactobacillus indicates that the Rep proteins from Lactobacillus 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 singlestranded 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 Solar et al. 1987; Gruss et al. 1987; Boe et al. 1989). Except 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 comparisons, 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 singlestranded 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 horizontally transferred from and to other Lactobacillus 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 Staphylococcus 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 plasmidspecific targets, controlling plasmid replication in

an indirect way (Novick 1987; Thomas 1988). For many plasmids antisense RNA (also called countertranscript 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 CTRNAs 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, precluding 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).

91

Promoter region of repA gene of plasmid p353-2 ~900n|

~ 190hi

PrepA

~

-

m l l - . -

ATG

Pml I

Pml I

P CT

Pml

!

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 involve 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 (Projan & 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 genetransfer 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 Lactobacillus 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 kanamycin/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). Moreover, 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 estimated at 30-50 copies per cell. Copy-number mutants, showing a 3-5 fold elevated copy number, can arise spontaneously during vector construction,

92

/ & 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 safety 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-

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 organism. 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 & Anderson 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 stabilize ss-DNA intermediates and/or initiate replication at the minus origin. Recently, a series of broadhost-range vectors was constructed for Lactococcus based on the replication functions of the theta-type plasmid pAM[31 (Simon & Chopin 1988; O'Sullivan Table 2. Plasmid vectors with Lactobacillus replicon. Plasmid

Replicon

Origin

E. coli DNA

Size (kb)

Marker

Cloning sites

References

pLE16 pBG10 pLP3537

pLB10 pLJ1 p353-2

L. bulgaricus L. helveticus L. pentosus

pBR328 pBR329 pUC19

7.6 6.0 6.3

cml lacZ ery

Chagneaud et al. 1992 Hashiba et al. 1992

pLP317 pLP317cop pLPE323 pLPE350 pLPE23M pLEP24Mcop pLP3537xyl pLP825

p353-1 p353-1 p353-2 p353-2 p353-2 p353-2 p353-2 p8014-2

L. pentosus L. pentosus L. pentosus L. pentosus L. pentosus L. pentosus L. pentosus L. plantarum

no no no ** no no pUC pBR322

2.9 2.9 3.6 3.9 3.7 3.7 6.3 5.8

ery ery ery ery ery ery xyl, ery cml

HindlII BamHI, PstI BamHI, HindIII, KpnI, PstI, SphI (SphI) (SphI) EcoRI, XbaI HindIII, KpnI, PstI, Smal, SphI MCS* MCS BamHI, KpnI, SmaI SalI, Sph!

pLP82H pLPC37 pULP8/9 pSC10 pPSC20 pPSC22 pLUL634

p8014-2 p8014-2 pLP1

L. L. L. L. L. L. L.

pUC ** pUC no no no no

5.8 3.7 6.6 3.0 5.5 4.3 5.1

ery cml ery ery cml, ery cml, ery ery

pLUL631

plantarum plantarum plantarum plantarum plantarum plantarum reuteri

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

BamHI, SacI, Sphl, XbaI EcoRV, SphI HindIII

ClaI, Hpal

Posno et al. 1991a Posno et al. 1991a Posno et al. 1991a Posno et al. 1991a Leer et al. 1992 This paper, Fig. 3 This paper Posno et al. 1991b Leer et al. 1987; Posno et al. 1991a Posno et al. 1991a Leer et al. 1992 Bouia et al. 1989 Cocconcelli et aL 1991 Cocconcelli et al. 1991 Cocconcelli et al. 1991 Ahrn6 et al. 1992

93

Incompatibility

Fig. 2. Effect of plasmid copy number on the activity of the bacteriocin acidocin B. The acidocin B activity is visualized using Clostridium 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.

Lactobacillus strains are cured from the endogenous plasmid when transformed by a vector with a replicon from that plasmid (Bringel et al. 1989; Posno 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 exploited to cure strains from plasmids that are otherwise difficult to eliminate. Also functional relationships 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). 0

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, potentially, 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 (Hashiba 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).

J

3000

l

I

Pst l BspM I Stu 1 Bgl Cla 1 - Sph ]

]1

Xho I - Spo l - Bal I - Eae [ - Sal I - Pvu - Bbv I - Fsp I - Hind - Kpn l - Sma t Barn H I -

2000

I EcoR

I

"'i',,,,,,,,, ",,

II Ill

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 consisting 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.). Apparently, 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 Staphylococcus 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, harbours 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

95 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 decrease of the amount of ss-DNA intermediates (Shimizu-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.

1993). To obtain transformants with an insertion into 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. Alternatively, 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 Chromosomal integration

Transcription

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). Clostridium thermocellum endoglucanase and B. stearothermophylus 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.

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 Lactobacillus 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 originating from seven different species (Table 3). All promoters show typical - 35 and - 10 sequences which,

96 i:~ t,q

•5

.v.

0

s~

s ~4--~a,--I

S V

~

o

&a

o

='~,~

r;

gA

~s~

~ ~ ~v

~

< ~

q--4

0

l"q

r~

97 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 dinucleotide, 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).

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 (Vanderslice 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 relative to that of the promoter-proximal gene, or to allow for a difference in RNA turnover rates by dissection 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 antitermination, 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 comprise 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 multicopy plasmid, indicating that the element can function as an operator that titrates the repressor (Lokman 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 (Lokman 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 promoters in different lactobacilli. Promoter

Species

L. casei

L. pentosus Inducer

xylA xylR L-ldh

amyA

L. pentosus L. pentosus L. casei L. amylovorus

-

q-

-

-I-

n.d.* n.d. xxx -

n.d. n.d. xxx xx

x n.d. n.d.

xx 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 cultivated 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 translationinitiation sites of approximately seventy LactobaciUus genes are known. Despite the fact that the genes originate from more than ten species it is evident 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-Dalgarno 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 L. bulgaricus L. casei L. helveticus L. pentosus L. plantarum

6 6 17 7 9 11

GGAGG AGGA AGGAGG AGGAG GGAGG GGAGG

5-11 4- 9 3-11 5- 8 6- 9 6--12

The nucleotide indicated is the one occurring most frequently. Bold capital: > 75%; capital; 50%-75%. T h e 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'-phosphoribosylanthranilic acid isomerase) gene (Natori et al. 1990) and the purL (phosphoribosylformylglycinamidine 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 equimolar 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. U A A is most frequently used and is found in genes of all sPecies examined. The stopcodon UAG however is much more rare and is, except for an unidentified open reading frame in the insertion element IS1 (Shimizu-Kadota et al. 1985), not present in 17 genes belonging to the L. casei group. Whether all these genes are truly L. casei genes remains to be established (Collins et al. 1989). The codon A G A for arginine 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 (%)

Genes*

5'-end of gene**

L. acidophilus L. bulgaricus L. casei L. helveticus L. pentosus L. plantarum

Total gene

1st

2nd

3rd

Average

1st

2nd

3rd

Average

83 69 56 63 64 58

79 66 73 ' 89 64 65

80 67 67 88 66 85

81 67 65 80 65 69

56 46 43 54 49 56

60 61 63 69 64 69

70 39 53 71 60 73

62 49 53 65 58 66

* : n u m b e r of genes; ** : first five codons after the startcodon.

5 6 18 7 8 7

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 Lactobacillus species. L. bulgaricus

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.

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

M-E

N-E

65 86 77 85 76 67 1 73 12 1315

37 51 52 48 47 27 18 62 22 1464

25 25 30 33 29 22 28 40 42 442

L. plantarum C/U + C (%)

C/U + C (%)

E*

24 24 22 7 28 13 26 22 21 1621

Asp (GAG/U) Asn (AAC/U) Tyr (UAC/U) Phe (UUC/U) His (GAG/U) Ile (AUC/U) G/G + A (%) Glu (GAG/A) Lys (AAG/A) Gln (GAG/A) Total number codons

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

101 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 Staphylococcus (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

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 metabolic capacity of lactobacilli, it is of paramount importance to understand the mechanisms under-

Table 9. Heterologous gene expression in Lactobacillus. Protein

Origin

Species

Promoter

References

acidocin B levanase c~-Amylase Xylose isomerase 13-Galactosidase ~-Glucuronidase FMDV-[3-gal VP7-[~-gal FMDV-c~-amylase Chloramphenicol acetyltransferase

L. acidophilus B. subtilis L. amylovorus L. pentosus E. coli E. coli FMDV-E. coli Rotavirus-E. coli FMDV-L. amylovorus pC194

L. plantarum L. plantarum/L, casei L. plantarum/L, casei L. casei L. plantarum/L, casei L. plantarum L. casei L. plantarum L. casei L. casei L. pentosus

acdB tac amyA xylA xylR, cbh cbh xylR cbh arnyA amyA, L-ldh xylA, xylR

Leer, unpublished Wanker, pets. comm. Jore, pets. comm. Posno et al. 1991b Posno, pers. comm. Posno, pers. comm. Posno, per~. comm. Posno, pers. comm. Jore, pers. comm. This paper 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 conventional 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 inadvertent horizontal transfer of cloned genes takes place. This issue is an essential element in discussions about the acceptability of genetically engineered microorganisms in human and/or animal food. Despite these uncertainties, the dawning perspectives 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 biotechnologically 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.

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