Transfer of plasmids by conjugation in Streptococcus pneumonias

Transfer of resistance plasmids occurred by conjugation in Streptococcus pneumoniae (pneumococcus) similarly to the process in other streptococcal groups. The 20-megadalton plasmid pIP501 mediated its own DNase-resistant transfer by filter mating and mobilized the 3.6-megadalton non-self-transmissible pMV158. Pneumococcal strains acted as donors or as recipients for intraspecies transfers and for interspecific transfers with Streptococcus faecalis. Transconjugants contained the plasmids expected from their phenotypes and acted as donors for further transfers. Deficiency in an endonuclease essential for entry of transforming DNA did not affect the frequency of transfer. Transfer-deficient mutants of pIP501 have been found.     

Construction of the mobilizable plasmid pMV158GFP,a derivative of pMV158 that carries the gene encoding the green fluorescent protein

Plasmid pMV158 has been employed to construct cloning non-mobilizable vectors for various Gram-positive organisms. Here we report the construction of a mobilizable pMV158-based plasmid that harbors the gene encoding the green fluorescent protein under the control of a promoter inducible by maltose. The plasmid was mobilized between strains of Streptococcus pneumoniae as well as from S. pneumoniae to Lactococcus lactis or Enterococcus faecalis at the same frequency as its parental. Transconjugant that received the GFP-tagged plasmid could be detected by their fluorescence, which was especially high in E. faecalis cells.     

Use of the Lactococcal nisA Promoter To Regulate Gene Expression in Gram-Positive Bacteria: Comparison of Induction Level and Promoter Strength

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.     

Small cryptic plasmids of Streptococcus pneumoniae belong to the pC194/pUB110 family of rolling circle plasmids

The DNA sequences of two related plasmids pPR1 and pPR3 described previously in Streptococcus pneumoniae isolates from Germany and Spain were now determined. Both plasmids belong to a family of rolling circle (RC) plasmids found in a variety of bacteria. Their GC content with 32% is lower than that of the S. pneumoniae chromosomal DNA. The plasmid pPR3 has a molecular size of 3160 bp with four putative open reading frames, whereas pPR1 contained a deletion of 313 bp that included the 5′-part of ORF2 and upstream regions and differed by three bp from pPR3. The predicted protein of ORF1 showed high similarity to replication proteins of RC plasmids with 74% identical amino acids to RepA of Streptococcus thermophilus plasmids. Sequences similar to the plus origin of replication of ssDNA plasmids were present in both plasmids. They also contained a 152-bp region with over 83% identity to the minus origin of replication of the Streptococcus agalacticae plasmid pMV158.     

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 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.     

Large-scale filter mating assay for intra- and inter-specific conjugal transfer of the promiscuous plasmid pMV158 in Gram-positive bacteria

Many conjugative or mobilizable plasmids from Gram-positive bacteria can be transferred mainly on filter-based surfaces. Such is the case of the streptococcal promiscuous plasmid pMV158 which can be mobilized by functions provided by auxiliary plasmids of the Inc18 family. We have developed a new large scale method consisting of a 96-well microtiter plate coupled with a filter so that we could perform several mating assays between strains of Streptococcus pneumoniae and between S. pneumoniae and Enterococcus faecalis cells. Transfer was monitored by employment of a derivative plasmid from pMV158 tagged with the gene encoding the Green Fluorescent Protein. Several experimental conditions, like different ratios donor to recipient cells, could be tested, thus allowing us to perform multiple mating assays in a single experiment.     

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformation

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10⁵ transformants/g DNA) was achieved. Correspondence to: J. Nevera     

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.     

The bacterial flora of the midgut of two Danish populations of healthy fifth instar larvae of the turnip moth, Scotia segetum

Examination of the midgut bacteria of two Danish populations of healthy fifth instar turnip moth larvae, Scotia (=Agrotis) segetum, living on potatoes and celery gave the following results. The total number of living microorganisms in the midgut varied between 1.0 × 10⁴ and 4.0 × 10⁵. Larvae from celery in N. W. Zeeland always contained Streptococcus faecalis and six members of Enterobacteriaceae, viz., Citrobacter freundii, Klebsiella pneumoniae, Hafnia alvei, Proteus mirabilis, P. vulgaris, and Erwinia amylovora. In larvae from potatoes in E. Jutland, the species consistently present were Streptococcus faecalis and four species of Enterobacteriaceae, viz., Escherichia coli, Erwinia amylovora, E. carotovora var. atroseptica, and one other, probably a member of the E. carotovora group. Streptococcus faecalis is supposed to occur as a mutualist in the alimentary tract, suppressing Gram-positive bacteria.     

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     

Narrow host range of some streptococcal R plasmids

Nine R plasmids originally harbored by Streptococcus faecalis (pIP614, pIP655, pIP685, pIP686, pIP 1075, pIP1017),S. faecium (pIP716, pIP991), and group B Streptococcus (pMV120) wild-type hosts were transferred by conjugation into various recipients in order to study the extent of their intraspecies, interspecies, and intergeneric host range. Recipients were streptococci of groups A, B, C, D (S. faecalis, S. faecium, S. durans, S. bovis), and G, S. sanguis, two S. pneumoniae strains (encapsulated and nonencapsulated), and two strains of different genera, Staphylococcus aureus and Listeria inocua. The plasmids carried different antibiotic resistance markers: tetracycline, high levels of gentamicin and kanamycin or of streptomycin and kanamycin, and chloramphenicol. These R plasmids displayed narrow host ranges. They transferred into S. faecalis recipients and plasmid DNA could be detected in these transconjugants. Occasionally, the R plasmids also transferred into one or more other recipients, but no detectable plasmid DNA could be demonstrated in the new hosts.