Cloning of the replication region on the bacteriocinogenic plasmid pRJ9 of Staphylococcus aureus

Abstract A 5.8-kb Cla I fragment of pRJ9, a bacteriocinogenic plasmid of Sphylococcus aureus , was cloned in the unique Cla I site of pRJ5. The recombinant plasmid obtained, pRJ23, failed to confer bacteriocin production and immunity to bacteriocin on host cells. The cloned fragment was shown to contain the complete replicon of pRJ9. Attempts to clone the 4.4-kb Cla I fragment of pRJ9 were unsuccessful, apparently due to the inactivation of the basic replicon of the cloning vector. Therefore, plasmid pRJ5 cut at its Cla I site appears to be a suitable vector for cloning replication regions of plasmids that cab replicate in S. aureus .     

Mobilization functions of the bacteriocinogenic plasmid pRJ6 of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT _pRJ6 was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT _pRJ6, is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT _pRJ6 of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra _pC223 site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.     

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13 has been determined. The plasmid consists of 9957 base pairs (molecular weight 6.64 X 10(6] with a GC content of 54.4%. At this moment 16 identified biological functions can be assigned to the primary structure of the CloDF13 DNA. The functions include those of eight protein encoding genes, two untranslated RNA species, and six DNA sites. We discuss these functions in relation to the structure of CloDF13 DNA. For convenience we have divided the CloDF13 genome into five defined regions: region I (origin of vegetative replication, priming and control of replication, type I incompatibility), region II (cloacin DF13, cloacin immunity, cloacin release, cloacin operon control), region III (double-stranded DNA-phage interaction, type II incompatibility, multimer resolution), region IV (inhibition of male specific RNA phages and transfer of Flac), and region V (mobility proteins, basis of mobility).     

Description of a bacteriocinogenic plasmid in Beneckea harveyi.

A total of 795 strains of marine Vibrio species and Beneckea harveyi, a luminescent marine bacterium, were isolated from various sources in the area of Galveston Island, Tex., and screened for the production of bacteriocin-like substances. More than 8% of the Vibrio isolates produced low-molecular-weight (dialyzable) substances, which were lethal to a test strain of V. parahaemolyticus. Approximately 5% of the B. harveyi isolates produced higher-molecular-weight (nondialyzable) substances which were lethal to a test strain of B. harveyi. One of the B. harveyi strains (strain SY) produced a nondialyzable substance which was lethal to two of 39 strains of B. harveyi. The substance showed no activity toward 17 test strains drawn from the Vibrionaceae and Enterobacteriaceae. Strain SY showed no sensitivity to its own lethal agent and was shown by agarose gel electrophoresis and electron microscopy to harbor a single plasmid of 38 x 10(6) daltons. Variants of strain SY lacking the plasmid were produced by growth in the presence of the antibiotic novobiocin. These variants lacked both the ability to produce the lethal substance and the ability to survive in its presence. The lethal agent produced by strain SY is the first bacteriocin reported in marine bacteria. The term "harveyicin" is proposed to name this lethal substance.     

Mechanism of plasmid ColEl and pMB-9 mobilization by plasmid F'lac+ in Escherichia coli K-12]

The genetic control and mechanism of mobilization of the non-conjugative plasmids ColE1 and pMB-9 by the conjugative plasmids was orived to be recA-independent process in contrast to the mobilization of the chromosomal marker pro. Acridine orange and ethidium bromide curing data together with the results of electrophoretic analysis of plasmid DNA suggest that the plasmids F' lac+ and pMB-9 as well as F' lac+ and ColE1 remain autonomous after their contransfer to recipient cells. These data argue in favour of non-recombinational nature of the plasmid mobilization process. The possibility of transmission of a non-conjugative plasmid without transmission of a conjugative one from the donor strain carrying both plasmids was established. The results obtained are discussed with respect to the hypothesis on the effect of diffusible products encoded by the conjugative plasmid and required of the mobilization of the non-conjugative plasmid.     

Origin and direction of replication of the bacteriocinogenic plasmid Clo DF13.

Cairn's type replicative intermediates of both the wildtype Clo DF13 plasmid and the copy mutant CLO DF13 cop3 were isolated by dye-buoyant density centrifugation. Replicative intermediates were linearized at the HpaI or Sa1I cleavage site, and examined with the electron-microscope. The data show that replication of both the Clo DF13 wild type plasmid and the Clo DF13 cop3 plasmid, initiates at about 2.8% on the physical map. Replication proceeds unindirectionally and counterclockwise on this map.     

Replication of staphylococcal plasmid pT48

Insertion of a synthetic DNA linker into the repL gene of staphylococcal plasmid pT48 inactivates the replication system. This defect can be complemented in trans by the presence of a pT48 repL gene, but not by the rep genes of the related Staphylococcus areus plasmids pSN2 and pOX1000. Comparison of the sequences of the three replication proteins indicates that specificity may be determined by a putative helix-turn-helix region.     

In vitro construction of deletion mutants of the bacteriocinogenic plasmid Clo DF13.

The isolation and characterization of deletion mutants of the bacteriocinogenic plasmid Clo DF13 is described. To construct these deletion mutants, DNA of Clo DF13::Tn901 and Clo DF13-rep3::Tn901 plasmids was digested with restriction endonucleases, ligated with T4 ligase and introduced by transformation into Escherichia coli. The presence of the ampicilline transposon Tn901 facilitated the selection of plasmids. The resulting Clo DF13::Tn901 deletion mutants were analyzed by digestion with restriction endonucleases and electron microscopy. From the properties of the various deletion mutants it was concluded that a Clo DF13 DNA region, extending from 5 to 11.5% on the physical map, is essential for the replication of Clo DF13. This region, comprising about 600 base pairs, contains in addition to an origin of replication, DNA sequences which are involved in the regulation of Clo DF13 DNA replication. Furthermore it was observed that in case of the Clo DF13 copy mutant, Clo DF13-rep3, deletion of the 43% to 63% part of the plasmid genome, resulted in the generation of multimeric plasmid structures, accompanied with an impaired segregation of the plasmids to daughter cells.     

Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004

The copper-resistance determinant ( pco ) of Escherichia coli plasmid pRJ1004 was cloned and sequenced. Tn 1000 transposon mutagenesis identified four complementation groups, mutations in any of which eliminated copper resistance. DNA sequence analysis showed that the four complementation groups contained six open reading frames, designated pcoABCDRS . The protein product sequences derived from the nucleotide sequence show close homology between this copper-resistance system and the cop system of a plasmid pPT23D of Pseudomonas syringae pv. tomato . The PcoR and PcoS protein sequences show homology to the family of two-component sensor/responder phosphokinase regulatory systems. A seventh reading frame ( pcoE ) was identified from DNA sequence data, and lies downstream of a copper-regulated promoter. Transport assays with ⁶⁴Cu(II) showed that the resistant cells containing the plasmid had reduced copper accumulation during the log phase of growth, while increased accumulation had previously been observed during stationary phase. Chromosomal mutants defective in cellular copper management were obtained and characterized. In two of these mutants pco resistance was rendered totally inactive, whilst in another two mutants pco complemented the defective genes. These data indicate that plasmid-borne copper resistance in E. coli is linked with chromosomal systems for copper management.     

Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194.

The complete nucleotide sequence of pADB201, a 1.7-kilobase cryptic plasmid from Mycoplasma mycoides subsp. mycoides, is reported. The sequence contains a single large open reading frame capable of coding for a polypeptide of up to 198 codons long. The sequence of the putative polypeptide shows significant similarity to that of the repF gene product of staphylococcal plasmid pE194.     

Incompatibility-deficient derivatives of a small staphylococcal plasmid.

Incompatibility-deficient derivatives of a small staphylococcal plasmid, pT181, and one of its temperature-sensitive mutants, pSA0301, were isolated. These derivatives could not replicate autonomously and owed their survival to integration into another plasmid. They did, however, retain the plasmid repC gene, encoding a diffusible product required for autonomous replication of pT181, as shown by their ability to complement Tsr mutants of this plasmid. The results obtained suggest that all of five Tsr mutations isolated are located in a single rep cistron on pT181. As an intermediate step in the isolation of the incompatibility-deficient derivatives, a series of apparently defective plasmids, elements that can be maintained only in the presence of a “helper” plasmid, were obtained from pT181 and pSA0301 as a consequence of their cotransduction with other plasmids. These latter elements seem to have originated from the parental plasmids by the loss of a region necessary for their stable maintenance, different from the repC locus they still carry.     

A different path: Revealing the function of staphylococcal proteins in biofilm formation

Staphylococcus aureus and Staphylococcus epidermidis cause dangerous and difficult to treat medical device-related infections through their ability to form biofilms. Extracellular poly-N-acetylglucosamine (PNAG) facilitates biofilm formation and is a vaccination target, yet details of its biosynthesis by the icaADBC gene products is limited. IcaC is the proposed transporter for PNAG export, however a comparison of the Ica proteins to homologous exo-polysaccharide synthases suggests that the common IcaAD protein components both synthesise and transport the PNAG. The limited distribution of icaC to the Staphylococcaceae and its membership of a family of membrane-bound acyltransferases, leads us to suggest that IcaC is responsible for the known O-succinylation of PNAG that occurs in staphylococci, identifying a potentially new therapeutic target specific for these bacteria.     

Molecular structure of the immunity gene and immunity protein of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence of the Clo DF13 DNA region comprising the immunity gene has been determined. We also elucidated the aminoacid sequence of the 40 N-terminal and 7 C-terminal aminoacids of the purified immunity protein. From analysis of the data obtained we were able to locate the immunity gene between 11.7 and 14.5% on the Clo DF13 map, and to determine the complete aminoacid sequence of the immunity protein. It was observed that the Clo DF13 immunity gene encodes an 85 aminoacid protein and is transcribed in the same direction as the cloacin gene. These experimental data support our model, presented elsewhere, which implicates that the cloacin and immunity genes of Clo DF13 are coordinately transcribed from the cloacin promoter. We also present DNA sequence data indicating that an extra ribosome binding site precedes the immunity gene on the polycistronic mRNA. This ribosome binding site might explain the fact that in cloacinogenic cells more immunity protein than cloacin is synthesized. The comparison of the complete aminoacid sequence of the Clo DF13 immunity protein, with the aminoacid sequence data of the purified, comparable Col E3 immunity protein revealed that both proteins have extensive homologies in primary and secondary structure, although they are exchangeable only to a low extent in vivo and in vitro. It was also observed that a lysine residue was modified in immunity protein isolated from excreted bacteriocin complexes.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. II. Specific recombination functions involved in plasmid maintenance

Summary Clo DF13 plasmids that are present at high copy-number in bacterial cells, such as Clo DF13 cop1 Ts, cop2 and cop3 are not stably inherited in the progeny, when certain plasmid DNA regions have been deleted. We have localized two Clo DF13 DNA regions involved in stable maintenance through accurate partitioning (par) namely parA, located between 71% and 72% and parB, located between 45% and 50% on the Clo DF13 genome. The instability of these cop plasmids which is accompanied by the formation of high amounts of multimeric DNA molecules, could be abolished by the insertion of transposon Tn901 into the plasmid genome. In particular that part of Tn901, that encodes for the site-specific recombination/ resolution system, appeared to be essential for stabilizing plasmid molecules. Wild-type parA^- and/or parB^- Clo DF13 plasmids, in contrast to cop mutants lacking these regions, are stably maintained during subsequent cell division, indicating that other (host specified) functions contribute to plasmid stability. Analysis of the role of host recombination systems in plasmid partitioning revealed that the recA function has no influence and recBC contributes only weakly to plasmid stability. With respect to the recE pathway, however, we found that in a recE proficient host all plasmids, even those lacking parA and/or parB, are stably maintained, indicating that the function of parA and parB can be replaced not only by the site-specific resolution functions of transposon Tn901, but also by the recE system. The possible role of plasmid specified and host specified functions in plasmid partitioning will be discussed.     

Mutants of the Clo DF13 plasmid inEscherichia coli with a decreased bacteriocinogenic activity

Summary Three Clo DF13 mutant plasmids (designated asclp03, clp05 andclp21) that show a decreased cloacin activity were isolated. The decreased cloacin activity was not due to a reduced number of Clo DF13 copies per cell. The cloacins produced by theclp03 and theclp21 mutant plasmids have a strongly decreased killing activityin vivo in comparison with the wild type cloacin and the cloacin of theclp05 mutant plasmid. Furthermore no lacunae could be observed fromclp03 orclp21 harbouring strains, while strains harbouring theclp05 plasmid showed a 50–100 times decreased frequency of lacunae. In addition theclp05 mutant showed a decreased rate of RNA synthesis inclp05 harbouringEscherichia coli minicells. No complementation between the three mutant plasmids was observed. We suggest that theclp03 andclp21 mutations are located in the gene coding for the cloacin. Since the cloacin produced by theclp05 mutant plasmid has retained all the known wild type cloacin activities, the reduced inhibition zone in the stab test is probably caused by a mutation affecting the expression of the cloacin gene. The nature of this mutation is discussed.     

Identification and molecular genetic analysis of replication functions of the bacteriocinogenic plasmid pIP404 from Clostridium perfringens

The replication functions of the bacteriocinogenic plasmid pIP404, from Clostridium perfringens, were localized to a 2.8-kb EcoRI-EcoRV fragment by cloning into a vector deficient for replication in Bacillus subtilis. This fragment contains two genes, cop and rep, which encode proteins and an 800-bp noncoding segment of complex structure consisting of multiple tandemly repeated sequences. The Cop protein is involved in copy number control, whereas the rep gene product is essential for plasmid replication. By deletion analysis the minimal origin of replication was defined as the rep gene plus most of the repeated sequences. A powerful promoter producing a 150-nucleotide RNA molecule, RNA1, that could act as an anti-sense RNA to the rep gene was detected in the "origin-like" region. In contrast to most other small plasmids of gram-positive bacteria, pIP404, and its derivatives, does not appear to replicate via a single stranded intermediate in either C. perfringens or B. subtilis.