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E Grohmann

Publications and source records attributed to E Grohmann.

10 recordsLinked to original sources
PubMed1999

Expression of the mobM gene of the streptococcal plasmid pMV158 in Lactococcus lactis subsp. lactis.

The streptococcal plasmid pMV158 is not auto-transferable, but it can be mobilised between bacteria by the use of functions supplied by plasmids of the pIP501/pAM beta 1 family. Plasmid pMV158 encodes a protein, MobM, which is involved in its mobilisation. This process initiates when MobM specifically cleaves supercoiled pMV158 plasmid DNA at the origin of transfer, oriT. Plasmid pMV158 has been transferred to Lactococcus lactis by conjugation aided by plasmid pAM beta 1. In the lactococcal host, MobM-mediated specific pMV158-relaxed molecules were detected. The intracellular amount of MobM has been quantified by immunoblot analyses and shown to be about 3500 molecules per cell. In the same host, we have mapped the initiation point of transcription of mobM. Transcription of this gene is directed from a promoter with an extended--10 region which overlaps with the pMV158-oriT.

Bacterial Proteins↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1997

Determination of specific DNA strand discontinuities with nucleotide resolution in exponentionally growing bacteria harboring rolling circle-replicating plasmids.

Plasmid replication by the rolling circle mechanism and conjugative transfer of plasmids require the generation of a specific strand discontinuity in the DNA. In both processes cleavage at the so-called nic site is catalyzed by plasmid-encoded proteins. The strand discontinuities at the conjugative origins of transfer of plasmid pE194 and pMV158 were determined in Bacillus subtilis and Streptococcus pneumoniae, respectively, with a recently developed runoff DNA synthesis assay. The positions of intracellular cleavage within the respective transfer origins were shown to coincide with the site predicted for pE194 and with the nic site determined in vitro for pMV158. For pMV158, the influence of a mutation in the S. pneumoniae polA gene on the efficiency of replication was investigated. In addition, the nic site within the double-stranded origin of the-rolling circle-replicating plasmid pMV158 in S. pneumoniae as well as that of pFX2 in Escherichia coli was mapped with nucleotide resolution.

Bacillus subtilis↗
PubMed1997

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution.

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.

DNA, Bacterial↗
PubMed1995

Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1.

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.

Bacterial Proteins↗
PubMed1994

Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4.

The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.

Bacterial Proteins↗
PubMed1993

Stability of r-microbes: stabilization of plasmid vectors by the partitioning function of broad-host-range plasmid RP4.

The genes for biosynthesis of the biodegradable polymer poly-beta-hydroxybutyric acid (PHB) cloned from Alcaligenes eutrophus H16 were used for synthesis of PHB with recombinant Escherichia coli strains. It was recognized that the PHB-biosynthesis genes cause segregational instability to the plasmids used as vectors. Recombinant PHB-plasmids are rapidly lost from host cells and plasmid-free cells occur at high rates, even under conditions of selection for the plasmids. Cloning the partitioning region of plasmid RP4 onto such plasmids resulted in a high degree of stabilization. These par-stabilized recombinant PHB-plasmids could be maintained quite efficiently in batch cultivation experiments in the absence of any selection pressure.

Alcaligenes↗