Construction of a Tightly Regulated Plasmid Vector for Streptococcus pneumoniae: Controlled Expression of the Green Fluorescent Protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P_M promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P_M promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P_M-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.     

Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae: controlled expression of the green fluorescent protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P(M) promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P(M) promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P(M)-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.     

Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis

Promoter-probe and terminator-probe plasmid vectors make possible to rapidly examine whether particular sequences function as promoter or terminator signals in various genetic backgrounds and under diverse environmental stimuli. At present, such plasmid-based genetic tools are very scarce in the Gram-positive pathogenic bacteria Streptococcus pneumoniae and Enterococcus faecalis. Hence, we developed novel promoter-probe and terminator-probe vectors based on the Streptococcus agalactiae pMV158 plasmid, which replicates autonomously in numerous Gram-positive bacteria. As reporter gene, a gfp allele encoding a variant of the green fluorescent protein was used. These genetic tools were shown to be suitable to assess the activity of promoters and terminators (both homologous and heterologous) in S. pneumoniae and E. faecalis. In addition, the promoter-probe vector was shown to be a valuable tool for the analysis of regulated promoters in vivo, such as the promoter of the pneumococcal fuculose kinase gene. These new plasmid vectors will be very useful for the experimental verification of predicted promoter and terminator sequences, as well as for the construction of new inducible-expression vectors. Given the promiscuity exhibited by the pMV158 replicon, these vectors could be used in a variety of Gram-positive bacteria.     

Lagging-Strand Replication from the ssoA Origin of Plasmid pMV158 in Streptococcus pneumoniae: In Vivo and In Vitro Influences of Mutations in Two Conserved ssoA Regions

The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RS_B] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication in Streptococcus pneumoniae. Cells containing plasmids with mutations in the RS_B accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RS_B region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

Fitness of the pMV158 replicon in Streptococcus pneumoniae

Promiscuous, rolling-circle replication plasmid pMV158 determines tetracycline resistance to its host and can be mobilized by conjugation. Plasmid pLS1 is a deletion derivative of pMV158 that has lost its conjugative mobilization ability. Both plasmids replicate efficiently and are stably inherited in Streptococcus pneumoniae. We have analyzed the effect of pMV158 and pLS1 carriage on the bacterial growth rate. Whereas the parental plasmid does not significantly modify the cell doubling time, pLS1 slows down the growth of the bacterial host by 8-9%. The bases of the differential burden caused by pMV158 and pLS1 carriage are not yet understood. The negligible cost of the pMV158 parental natural plasmid on the host might explain the prevalence of small, multicopy, rolling-circle replication plasmids, even though they lack any selectable trait.     

Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01

Mycobacterium neoaurum NwIB-01 exhibits powerful ability to cleave the side chain of soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). The difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. To enhance ADD quantity in products, 3-ketosteroid Δ¹-dehydrogenase genes (kstD _M and kstD _A) were obtained from M. neoaurum NwIB-01 and Arthrobacter simplex respectively. Using replicating vector pMV261, kstD _M and kstD _A were overexpressed in M. neoaurum NwIB-01. For foreign gene stable expression, the integration vector pMV306 was used for kstD _M/kstD _A overexpression and the relevant sequences of promoter and kanamycin antibiotic resistance gene sequences were amplified by PCR to verify plasmid integrity. The resultant plasmid and mutant strain were verified and the kstD augmentation mutants were good ADD-producing strains. The ADD producing capacity of NwIB-04 and NwIB-05 was 0.1401 and 0.1740 g/l (cultured in shake bottles with 0.4 g/l phytosterols), and the molar ratio of ADD in products was 98.34 and 98.60 %, respectively. This study on the manipulation of the main kstD_M gene in Mycobacterium sp. provides a feasible way to achieve excellent phytosterol-transformation strains with high product purity.     

Direction of transcription affects the replication mode of lambda in an in vitro system

Summary pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably mantained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30° C. The optimal concentration of template DNA was 40 g/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its serivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.     

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro.     

Lagging-Strand DNA Replication Origins Are Required for Conjugal Transfer of the Promiscuous Plasmid pMV158

The promiscuous streptococcal plasmid pMV158 is mobilizable by auxiliary plasmids and replicates by the rolling-circle mechanism in a variety of bacterial hosts. The plasmid has two lagging-strand origins, ssoA and ssoU, involved in the conversion of single-stranded DNA intermediates into double-stranded plasmid DNA during vegetative replication. Transfer of the plasmid also would involve conversion of single-stranded DNA molecules into double-stranded plasmid forms in the recipient cells by conjugative replication. To test whether lagging-strand origins played a role in horizontal transfer, pMV158 derivatives defective in one or in both sso''s were constructed and tested for their ability to colonize new hosts by means of intra- and interspecies mobilization. Whereas either sso supported transfer between strains of Streptococcus pneumoniae, only plasmids that had an intact ssoU could be efficiently mobilized from S. pneumoniae to Enterococcus faecalis. Thus, it appears that ssoU is a critical factor for pMV158 promiscuity and that the presence of a functional sso plays an essential role in plasmid transfer.Conjugation of bacterial plasmids is, together with transposition, the most important source of horizontal gene transfer among bacteria of the same or of different species (42). Conjugation implies the unidirectional transfer of one plasmid DNA strand from a donor to a recipient cell. This is initiated by the activity of a plasmid-encoded protein generically termed relaxase, in a process that resembles replication by the rolling-circle mechanism (13, 29). In the case of numerous, small plasmids (<10 kb) isolated primarily from gram-positive bacteria, two pioneer findings led to the discovery of the rolling-circle mechanism of replication (RCR) plasmids (reviewed in references 20 and 21). First, the strand-specific single-stranded DNA (ssDNA) molecules which act as replication intermediates were identified (41) and, second, the relaxing activity on the supercoiled DNA via the recognition of a specific sequence (the double-strand origin) of the Rep initiator proteins were described (22). Most RCR plasmids are not self-transmissible; instead, they encode not only the Rep topoisomerase-like initiator but also a Mob protein with relaxase activity involved in mobilization mediated by auxiliary plasmids. Such is the case of the promiscuous plasmid pMV158, which can be mobilized between various bacterial species by the pMV158-encoded MobM protein and by helper conjugative plasmids belonging to the Inc18 plasmid family, such as pAMβ1 (15), or even by IncP plasmids, such as RP4 (11). The relaxing activity of MobM on supercoiled DNA of pMV158 and the site of cleavage were first demonstrated in vitro (5, 17), and later the same activity of the MobA protein of the Staphylococcus aureus RCR plasmid pC221 was demonstrated (3, 39).Initiation of transfer, like initiation of RCR, involves cleavage of the phosphodiester bond of a specific dinucleotide on one of the plasmid strands. Cleavage is mediated either by the plasmid-encoded Mob protein at the origin of transfer (oriT) during conjugation or by the plasmid-encoded Rep protein at the dso during replication. In both processes, this initial stage is followed by displacement of the cleaved strand in a unidirectional manner (8, 21, 29, 36). Thus, RCR and conjugal transfer are equivalent processes in the sense that they generate strand-specific ssDNA plasmid intermediates that correspond only to the cleaved strand (9, 16, 41). The ssDNA intermediates are generated in the plasmid host by the activity of the Rep initiator protein (replication) or generated and transferred to the recipient cell (T-DNA) and closed by the Mob relaxase (conjugation), where they are converted into double-stranded plasmid DNA (dsDNA) molecules by lagging-strand synthesis. Replication of the lagging strand is initiated at the single-strand origins (sso) by the host RNA polymerase (RNAP), upon recognition of a specific site on ssDNA and synthesis of a short RNA primer (pRNA). The pRNA is used by DNA polymerase I for limited extension synthesis, followed by replication of the lagging strand by DNA polymerase III (27). Features of the sso include the potential to generate stem-loop structures on ssDNA (9, 16, 41) that can conform a ssDNA promoter, which is inactive in the dsDNA configuration. This kind of promoter was described in the coliphage N4 (18), as recognized by the virion RNAP (4, 14). A different kind of ssDNA promoter, F_rpo, was reported for the Escherichia coli plasmid F and was demonstrated to be used for gene expression and appeared to play a role during plasmid conjugation (34). The presence of ssDNA promoters has also been demonstrated in plasmids pMV158 (27) and ColI-P9 (1, 35). The organization of this kind of promoters showed that they are placed on the DNA strand that is partially complementary to the template strand.The first sso was described in the staphylococcal RCR plasmid pT181, in which a deletion located out of the replicon led to instability, reduction in copy number, and accumulation of ssDNA intermediates (16). Plasmid pMV158 exhibits two sso''s, ssoA and ssoU (23). Two conserved regions were found in the ssoA of pLS1 plasmid (a nonmobilizable pMV158 derivative lacking ssoU): a short region termed recombination site B, RS_B, supposedly involved in plasmid cointegration (16, 38), and a 6-nucleotide (nt) consensus sequence (5′-TAGCGT-3′, termed CS-6). Determination of the roles of these two conserved sites showed that, whereas RS_B was the primary site of RNAP binding (located at the stem of the hairpin), CS-6 was the termination site for the synthesis of a 20-nt pRNA in the loop of the hairpin (27). The predicted intrastrand pairings in the pMV158-ssoA showed the presence of an ssDNA promoter in the vicinity of the RS_B, which would have a consensus −35 region (5′-TTGACA-3′) but a weak −10 region (5′-TAcgcT-3′). With this situation, RNA synthesis should start and proceed in the direction toward the binding site of RNAP, being thus opposite to RNA synthesis from classic promoters (27) (see Fig. ​Fig.1A).1A). Sites homologous to RS_B and CS-6 were later observed in pMV158-ssoU (24).Open in a separate windowFIG. 1.Features of pMV158 and its two lagging-strand origins. (A) Schematic map of the plasmid transfer module indicating relevant restriction sites and the relative positions (shadowed) of the two lagging-strand origins of replication (ssoA and ssoU). Plasmid-encoded MobM protein (arrow below the map) and the position of the oriT are depicted. Direction of DNA transfer is indicated. The EcoRI fragment deleted to construct the pLS1 derivative (28) and the positions of primers used are also shown. A representation of the secondary structures of ssoA (left) and of ssoU (right) indicates the positions of the CS-6 and the RS_B regions (boxed). The locations of the G3 and G7 mutations in the ssoA and of the restriction sites used to generate deletions in the ssoU are shown. The start point and direction for the RNA primer (pRNA) synthesis, downstream to CS-6 sequence, is indicated by a wavy arrow. (B) Relevant sequence features of the two pMV158 sso. The RS_B and CS-6 sequences are shown in boxes. The restriction sites of BsaI and DraI used to generate ssoU-ΔBD mutant are also indicated, as well as the nucleotides changed (boldface) to construct the ssoA-G3G7 mutant (sequence indicated beneath).In the present study we have addressed the question of whether and, eventually, which of the two pMV158-ssos plays a role in conjugal transfer. With this objective, we constructed pMV158-derivatives defective in one or both sso''s and tested their role on intra- and interspecies mobilization. Whereas either sso supported transfer between strains of Streptococcus pneumoniae with the same efficiency as the parental pMV158, only the ssoU could do so when conjugal transfer was assayed between S. pneumoniae and Enterococcus faecalis. Our findings show that the functionality of ssoU is a critical factor in the colonization of a broad range of gram-positive bacteria for the pMV158 promiscuous plasmid and demonstrate that efficient transfer and replication in enterococci depend upon a functional ssoU. We suggest that sso''s lacking functionality for vegetative replication in a specific host should not be efficient in conjugative transfer and vice versa, since both events are mechanistically identical. As far as we know, this is the first report that shows the effect of sso functionality on horizontal gene transfer by plasmid conjugation, as well as the efficiency of the ssoA and ssoU in E. faecalis.     

Interspecific plasmid transfer between Streptococcus pneumoniae and Bacillus subtilis

Summary The streptococcal plasmids pMV158 and pLS1, grown in Streptococcus pneumoniae, were transferred to Bacillus subtilis by DNA-mediated transformation. The plasmids were unchanged in the new host; no deletions were observed in 80 instances of transfer. Their copy number was similar to that in S. pneumoniae. Two B. subtilis plasmids, pUB110 and pBD6, could not be transferred to S. pneumoniae. Hybrid plasmids were produced by recombining the EcoRI fragment of pBD6 that confers Km^r with EcoRI-cut pLS1, which confers Tc^r. The simple hybrid, pMP2, was transferable to both species and expressed Tc^r and Km^r in both. A derivative, pMP5, which contained an insertion in the pBD6 component, expressed a higher level of kanomycin resistance and was more easily selected in S. pneumoniae. Another derivative, pMP3, which contained an additional EcoRI fragment, presumably of pneumococcal chromosomal DNA, could not be transferred to B. subtilis. Previous findings that monomeric plasmid forms could transform S. pneumoniae but not B. subtilis were confirmed using single plasmid preparations. Although plasmids extracted from either species were readily transferred to S. pneumoniae, successive passage in B. subtilis increased the ability of plasmid extracts to transfer the plasmid to a B. subtilis recipient. This adaptation was tentatively ascribed to an enrichment of multimeric forms in extracts of B. subtilis as compared to S. pneumoniae. A review of host ranges exhibited by plasmids of Gram-positive bacteria suggested differences in their ability to use particular host replication functions. The pMP5 plasmid, with readily selectable Km^r and Tc^r markers in both hosts, and with the potential for inactivation of Km^r by insertion in the Bg/II site, could be a useful shuttle vector for cloning in S. pneumoniae and B. subtilis.     

Efficient expression of a promoter-controlled gene: Tandem promoters of lambda PR and PL functional in enteric bacteria

We describe an expression vector that functions in enteric bacteria. The vector contains the coliphage λ promoters P_R and P_L and entire P_R and P_L operators in tandem upstream from the multiple cloning sites containing the kanamycin-resistant gene. The vector also specifies a ribosome binding site and a thermolabile repressor, cI₈₅₇, and the P_RM promoter. These promoters as well as lacUV5 and trp promoters were inserted into the EcoRI site of pKO-1 plasmid so that they drove the expression of a reporter gene, galactokinase (galK). The P_RP_L promoter showed the highest efficiency of galK expression in the Escherichia coli strain K12ΔH1Δtrp; it was strong in Klebsiella aerogenes, and weak in Serratia marcescens and Citrobacter freundii.     

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RS_R.     

Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis.

The Bacillus plasmid pLS11 partitions faithfully during cell division. Using a partition-deficient plasmid vector, we randomly cloned DNA fragments of plasmid pLS11 and identified the locus that regulates plasmid partition (par) by cis complementation in Bacillus subtilis. The cloned par gene conferred upon the vector plasmid a high degree of segregational stability. The par locus was mapped to a 167-base-pair segment on pLS11, and its nucleotide sequence was determined. The cloned par fragment regulated the partition of several different Bacillus replicons, and it only functioned in cis; it did not contain the replication function nor elevate the plasmid copy number in B. subtilis. The expression of par was orientation specific with respect to the replication origin on the same plasmid. We propose that the pLS11-derived par functions as a single-stranded site that interacts with other components involved in plasmid partition during cell division.