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ArdB, ArdA, and Ocr proteins inhibit the endonuclease activity of the type I restriction-modification enzymes (RMI). In this study, we evaluated the ability of ArdB, ArdA, and Ocr to inhibit different subtypes of Escherichia coli RMI systems (IA, IB, and IC) as well as two Bacillus licheniformis RMI systems. Furthermore we explored, the antirestriction activity of ArdA, ArdB, and Ocr against a type III restriction-modification system (RMIII) EcoPI and BREX. We found that DNA- mimic proteins, ArdA and Ocr exhibit different inhibition activity, depending on which RM system tested. This effect might be linked to the DNA mimicry nature of these proteins. In theory, DNA-mimicmight competitively inhibit any DNA-binding proteins; however, the efficiency of inhibition depend on the ability to imitate the recognition site in DNA or its preferred conformation. In contrast, ArdB protein with an undescribedmechanismof action, demonstrated greater versatility against various RMI systems and provided similar antirestriction efficiency regardless of the recognition site. However, ArdB protein could not affect restriction systems that are radically different from the RMI such as BREX or RMIII. Thus, we assume that the structure of DNA-mimic proteins allows for selective inhibition of any DNA-binding proteins depending on the recognition site. In contrast, ArdB-like proteins inhibit RMI systems independently of the DNA recognition site. [ABSTRACT FROM AUTHOR]

ArdB, ArdA, and Ocr proteins inhibit the endonuclease activity of the type I restriction-modification enzymes (RMI). In this study, we evaluated the ability of ArdB, ArdA, and Ocr to inhibit different subtypes of Escherichia coli RMI systems (IA, IB, and IC) as well as two Bacillus licheniformis RMI systems. Furthermore we explored, the antirestriction activity of ArdA, ArdB, and Ocr against a type III restriction-modification system (RMIII) EcoPI and BREX. We found that DNA-mimic proteins, ArdA and Ocr exhibit different inhibition activity, depending on which RM system tested. This effect might be linked to the DNA mimicry nature of these proteins. In theory, DNA-mimic might competitively inhibit any DNA-binding proteins; however, the efficiency of inhibition depend on the ability to imitate the recognition site in DNA or its preferred conformation. In contrast, ArdB protein with an undescribed mechanism of action, demonstrated greater versatility against various RMI systems and provided similar antirestriction efficiency regardless of the recognition site. However, ArdB protein could not affect restriction systems that are radically different from the RMI such as BREX or RMIII. Thus, we assume that the structure of DNA-mimic proteins allows for selective inhibition of any DNA-binding proteins depending on the recognition site. In contrast, ArdB-like proteins inhibit RMI systems independently of the DNA recognition site.

Gene transfer in crenarchaea has been observed within natural and experimental populations of Sulfolobus. However, the molecular factors that govern how gene transfer and recombination manifest themselves in these populations is still unknown. In this study, we examine a plasmid-mediated mechanism of gene transfer in S. islandicus that results in localized high frequency recombination within the chromosome. Through chromosomal marker exchange assays with defined donors and recipients, we find that while bidirectional exchange occurs among all cells, those possessing the integrated conjugative plasmid, pM164, mobilize a nearby locus at a significantly higher frequency when compared to a more distal marker. We establish that traG is essential for this phenotype and that high frequency recombination can be replicated in transconjugants after plasmid transfer. Mapping recombinants through genomic analysis, we establish the distribution of recombinant tracts with decreasing frequency at increasing distance frompM164.We suggest the bias in transfer is a result of an Hfr (high frequency recombination)-like conjugation mechanism in this strain. In addition, we find recombinants containing distal non-selected recombination events, potentially mediated by a different host- encoded marker exchange (ME) mechanism. [ABSTRACT FROM AUTHOR]

Gene transfer in crenarchaea has been observed within natural and experimental populations of Sulfolobus. However, the molecular factors that govern how gene transfer and recombination manifest themselves in these populations is still unknown. In this study, we examine a plasmid-mediated mechanism of gene transfer in S. islandicus that results in localized high frequency recombination within the chromosome. Through chromosomal marker exchange assays with defined donors and recipients, we find that while bidirectional exchange occurs among all cells, those possessing the integrated conjugative plasmid, pM164, mobilize a nearby locus at a significantly higher frequency when compared to a more distal marker. We establish that traG is essential for this phenotype and that high frequency recombination can be replicated in transconjugants after plasmid transfer. Mapping recombinants through genomic analysis, we establish the distribution of recombinant tracts with decreasing frequency at increasing distance from pM164. We suggest the bias in transfer is a result of an Hfr (high frequency recombination)-like conjugation mechanism in this strain. In addition, we find recombinants containing distal non-selected recombination events, potentially mediated by a different host-encoded marker exchange (ME) mechanism.

The main mechanism of virulence in Klebsiella pneumoniae is the acquisition of K. pneumoniae virulence plasmids (KpVPs), which include two dominant types, namely, KpVP-1 (carrying iuc1, iro1, rmpA, and rmpA2) and KpVP-2 (carrying iuc2, iro2, and rmpA). Both are non-conjugative and associated with different hypervirulent clones. In contrast to KpVP-1 reported in K1, K2, and other serotypes of K. pneumoniae, KpVP-2 was only reported in K2 strains and rarely characterized. In this study, we identified a conjugative KpVP-2-type virulence plasmid from a clinical hypervirulent K. pneumoniae strain. This plasmid was generated by the integration of conjugative transfer genes into the KpVP-2-type plasmid Kp52.145 II and could be readily conjugated to Escherichia coli strain EC600 and K. pneumoniae strains of various types which are clinically existing, mediating hypervirulence. Furthermore, this kind of conjugative KpVP-2-type virulence plasmid has been disseminated in clinical settings in Hong Kong and other regions of the world. The generation of conjugative virulence plasmid may promote its transmission and explain the evolution of this type of virulence plasmid. [ABSTRACT FROM AUTHOR]

lsa(E) is a pleuromutilin, lincosamide, and streptogramin A (PLSA phenotype) resistance gene that was first described in S. aureus and was thought to have been transferred from Enterococcus sp. This study aimed to elucidate the prevalence of the lsa(E) gene among E. faecium isolates at a tertiary teaching hospital and to evaluate the transferability of the lsa(E) gene from E. faecium to S. aureus in vitro. A total of 96 E. faecium strains isolated from one hospital in Beijing in 2013 were analysed for quinupristin-dalfopristin (QDA) resistance genes, and multilocus sequence typing (MLST) was performed. The transferability of QDA resistance between ten E. faecium strains and four S. aureus strains was determined by filter mating. Genome sequencing of the transconjugant was performed. A total of 46 E. faecium isolates (46/96, 47.92%) tested positive for lsa(E), while two isolates (2/96, 2.08%) tested positive for lsa(A). Thirty-six lsa(E)-positive strains (36/46, 78.3%) belonged to ST78. Among 40 mating tests, lsa(E) was successfully transferred through one conjugation at a frequency of 1.125 × 10–7 transconjugants per donor. The QDA resistance of the transconjugant N7435-R3645 was expressed at a higher level (MIC = 16 mg/L) than that of the parent S. aureus strain (MIC = 0.38 mg/L). Next-generation sequencing (NGS) analysis of the transconjugant N7435-R3645 showed that the complete sequence of the lsa(E)-carrying plasmid pN7435-R3645 had a size of 92,396 bp and a G + C content of 33% (accession no. MT022086). The genetic map of pN7435-R3645 had high nucleotide similarity and shared the main open reading frame (ORF) features with two plasmids: E. faecium pMG1 (AB206333.1) and E. faecium LS170308 (CP025078.1). The rep gene of pN7435-R3645 showed 100% identity with that of pMG1, although it did not belong to the rep1-19 family but instead a unique rep family. Multiple antibiotic resistance genes, including lsa(E), aadE and lnu(B), erm(B), ant6-Ia, and lnu(B), were present on the plasmid. In conclusion, an lsa(E)-carrying plasmid that can be transferred by conjugation from E. faecium to S. aureus in vitro was identified. This multidrug resistance (MDR) pMG1-like plasmid may act as a vector in the dissemination of antimicrobial resistance among species.

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.

lsa (E) is a pleuromutilin, lincosamide, and streptogramin A (PLSA phenotype) resistance gene that was first described in S. aureus and was thought to have been transferred from Enterococcus sp. This study aimed to elucidate the prevalence of the lsa (E) gene among E. faecium isolates at a tertiary teaching hospital and to evaluate the transferability of the lsa (E) gene from E. faecium to S. aureus in vitro. A total of 96 E. faecium strains isolated from one hospital in Beijing in 2013 were analysed for quinupristin-dalfopristin (QDA) resistance genes, and multilocus sequence typing (MLST) was performed. The transferability of QDA resistance between ten E. faecium strains and four S. aureus strains was determined by filter mating. Genome sequencing of the transconjugant was performed. A total of 46 E. faecium isolates (46/96, 47.92%) tested positive for lsa (E), while two isolates (2/96, 2.08%) tested positive for lsa (A). Thirty-six lsa (E)-positive strains (36/46, 78.3%) belonged to ST78. Among 40 mating tests, lsa (E) was successfully transferred through one conjugation at a frequency of 1.125 × 10–7 transconjugants per donor. The QDA resistance of the transconjugant N7435-R3645 was expressed at a higher level (MIC = 16 mg/L) than that of the parent S. aureus strain (MIC = 0.38 mg/L). Next-generation sequencing (NGS) analysis of the transconjugant N7435-R3645 showed that the complete sequence of the lsa (E)-carrying plasmid pN7435-R3645 had a size of 92,396 bp and a G + C content of 33% (accession no. MT022086). The genetic map of pN7435-R3645 had high nucleotide similarity and shared the main open reading frame (ORF) features with two plasmids: E. faecium pMG1 (AB206333.1) and E. faecium LS170308 (CP025078.1). The rep gene of pN7435-R3645 showed 100% identity with that of pMG1, although it did not belong to the rep 1-19 family but instead a unique rep family. Multiple antibiotic resistance genes, including lsa (E), aadE and lnu (B), erm (B), ant6 -Ia, and lnu (B), were present on the plasmid. In conclusion, an lsa (E)-carrying plasmid that can be transferred by conjugation from E. faecium to S. aureus in vitro was identified. This multidrug resistance (MDR) pMG1-like plasmid may act as a vector in the dissemination of antimicrobial resistance among species. [ABSTRACT FROM AUTHOR]

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the tra R gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery. [ABSTRACT FROM AUTHOR]

Escherichia coli is one major cause of bacterial infections and can horizontally acquire antimicrobial resistance and virulence genes through conjugation. Because conjugative plasmids can rapidly spread among bacteria of different species, the plasmids carrying both antimicrobial resistance and virulence genes may pose a significant threat to public health. Therefore, the identification and characterization of these plasmids may facilitate a better understanding of E. coli pathogenesis and the development of new strategies against E. coli infections. Because iron uptake ability is a potential virulence trait of bacteria, we screened for E. coli conjugative plasmids able to confer both iron uptake ability and ampicillin resistance. The plasmid pEC41, which was derived from the bacteremia clinical isolate EC41, was identified. EC41, which carried the fimH 27 allele, belonged to sequence type (ST) 405 and phylogroup D. According to the sequencing analyses, pEC41 was 86 kb in size, and its backbone structure was almost identical to that of another highly conjugative plasmid, pCTX-M3, in which the extended-spectrum β-lactamase gene bla CTX–M–3 was originally identified. pEC41 carried bla CTX–M–3 and bla TEM–1. The ferric citrate uptake (fec) system was identified in pEC41 and was responsible for conferring iron uptake ability. The fec system contributes to the pathogenesis of EC41 in systemic infections but not in urinary tract infections (UTIs). However, this system promoted competitive fitness of a cystitis-associated clinical isolate to colonize urinary tracts. Additionally, the distribution of the fec system was related to E. coli isolates associated with human bacteremia and UTIs. In summary, the present study identified a novel conjugative plasmid, pEC41, which conferred both antimicrobial resistance and an extra iron uptake ability to E. coli. The iron uptake ability was encoded in the fec system and contributed to E. coli pathogenesis. This study is the first to show that the fec system is a virulence factor in E. coli. [ABSTRACT FROM AUTHOR]

This study aims to investigate and compare the complete nucleotide sequences of the multidrug resistance plasmids pVb0267 and pVb0499, which were recovered from foodborne Vibrio parahaemolyticus isolates, and analyze the genetic environment of blaCTX–M–55 to provide insight into the dissemination mechanisms of this resistance element. Analysis of the sequences of plasmids pVb0267 (166,467 bp) and pVb0499 (192,739 bp) revealed that the backbones of these two plasmids exhibited a high degree of similarity with pR148, a recognized type 1a IncC plasmid recovered from Aeromonas hydrophila (99% identity). The resistance genes, found in both plasmids, included qacH, aadB, arr2, blaOXA–10, aadA1, sul1, tet(A), and blaCTX–M–55, which were mostly arranged in a specific region designated ARI-A. Plasmid pVb0499 was found to possess a larger size of ARI-A than pVb0267, which lacked a mer determination region, a qnr A segment, an aacC3 gene and several mobility-encoding genes. Comparative analysis of resistance island (RI) of these plasmids and others revealed the potential evolution route of these RI sequences. In conclusion, plasmids harboring the blaCTX–M–55 gene has been recovered in Vibrio parahaemolyticus strains of food origin. It is alarming to find that IncC plasmids harboring resistance islands are disseminating in aquatic bacterial strains. The continuous evolution of resistance genes in conjugative plasmid in aquatic bacteria could be due to bacterial adaption to aquaculture environment, where antibiotics were increasingly used.

This study aims to investigate and compare the complete nucleotide sequences of the multidrug resistance plasmids pVb0267 and pVb0499, which were recovered from foodborne Vibrio parahaemolyticus isolates, and analyze the genetic environment of blaCTX–M–55 to provide insight into the dissemination mechanisms of this resistance element. Analysis of the sequences of plasmids pVb0267 (166,467 bp) and pVb0499 (192,739 bp) revealed that the backbones of these two plasmids exhibited a high degree of similarity with pR148, a recognized type 1a IncC plasmid recovered from Aeromonas hydrophila (99% identity). The resistance genes, found in both plasmids, included qacH, aadB, arr2, bla OXA–10 , aadA1, sul1, tet (A), and bla CTX–M–55, which were mostly arranged in a specific region designated ARI-A. Plasmid pVb0499 was found to possess a larger size of ARI-A than pVb0267, which lacked a mer determination region, a qnr A segment, an aacC3 gene and several mobility-encoding genes. Comparative analysis of resistance island (RI) of these plasmids and others revealed the potential evolution route of these RI sequences. In conclusion, plasmids harboring the bla CTX–M–55 gene has been recovered in Vibrio parahaemolyticus strains of food origin. It is alarming to find that IncC plasmids harboring resistance islands are disseminating in aquatic bacterial strains. The continuous evolution of resistance genes in conjugative plasmid in aquatic bacteria could be due to bacterial adaption to aquaculture environment, where antibiotics were increasingly used. [ABSTRACT FROM AUTHOR]

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.

This study investigates the evolution of an Escherichia coli sequence type 219 clone in a patient with recurrent urinary tract infection, comparing isolate EC974 obtained prior to antibiotic treatment and isolate EC1515 recovered after exposure to several β-lactam antibiotics (ceftriaxone, cefixime, and imipenem). EC974 had a smooth colony morphology, while EC1515 had a rough colony morphology on sheep blood agar. RAPD-PCR analysis suggested that both isolates belonged to the same clone. Antimicrobial susceptibility tests showed that EC1515 was more resistant to piperacillin/tazobactam, cefepime, cefpirome, and ertapenem than EC974. Comparative genomic analysis was used to investigate the genetic changes of EC974 and EC1515 within the host, and showed three plasmids with replicons IncI1, P0111, and IncFII in both isolates. P0111-type plasmids pEC974-2 and pEC1515-2, contained the antibiotic resistance genes aadA2, tetA, and drfA12. IncFII-type plasmids pEC974-3 and pEC1515-3 contained the antibiotic resistance genes blaTEM−1, aadA1, aadA22, sul3, and inuF. Interestingly, blaCMY−111 and blaCMY−4 were found in very similar IncI1 plasmids that also contained aadA22 and aac(3)-IId, from isolates EC974 (pEC974-1) and EC1515 (pEC1515-1), respectively. The results showed in vivo amino acid substitutions converting blaCMY−111 to blaCMY−4 (R221W and A238V substitutions). Conjugation experiments showed a high frequency of IncI1 and IncFII plasmid co-transference. Transconjugants and DH5α cells harboring blaCMY-4 or blaCMY-111 showed higher levels of resistance to ampicillin, amoxicillin, cefazolin, cefuroxime, cefotaxime, cefixime, and ceftazidime, but not piperacillin/tazobactam, cefpime, or ertapenem. All known genes (outer membrane proteins and extended-spectrum AmpC β-lactamases) involved in ETP resistance in E. coli were identical between EC974 and EC1515. This is the first study to identify the evolution of an IncI1 plasmid within the host, and to characterize blaCMY−111 in E. coli.

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae , pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence. [ABSTRACT FROM AUTHOR]

This study investigates the evolution of an Escherichia coli sequence type 219 clone in a patient with recurrent urinary tract infection, comparing isolate EC974 obtained prior to antibiotic treatment and isolate EC1515 recovered after exposure to several β-lactam antibiotics (ceftriaxone, cefixime, and imipenem). EC974 had a smooth colony morphology, while EC1515 had a rough colony morphology on sheep blood agar. RAPD-PCR analysis suggested that both isolates belonged to the same clone. Antimicrobial susceptibility tests showed that EC1515 was more resistant to piperacillin/tazobactam, cefepime, cefpirome, and ertapenem than EC974. Comparative genomic analysis was used to investigate the genetic changes of EC974 and EC1515 within the host, and showed three plasmids with replicons IncI1, P0111, and IncFII in both isolates. P0111-type plasmids pEC974-2 and pEC1515-2, contained the antibiotic resistance genes aadA2, tetA, and drfA12. IncFII-type plasmids pEC974-3 and pEC1515-3 contained the antibiotic resistance genes blaTEM−1, aadA1, aadA22, sul3, and inuF. Interestingly, blaCMY−111 and blaCMY−4 were found in very similar IncI1 plasmids that also contained aadA22 and aac(3)-IId, from isolates EC974 (pEC974-1) and EC1515 (pEC1515-1), respectively. The results showed in vivo amino acid substitutions converting blaCMY−111 to blaCMY−4 (R221W and A238V substitutions). Conjugation experiments showed a high frequency of IncI1 and IncFII plasmid co-transference. Transconjugants and DH5α cells harboring blaCMY-4 or blaCMY-111 showed higher levels of resistance to ampicillin, amoxicillin, cefazolin, cefuroxime, cefotaxime, cefixime, and ceftazidime, but not piperacillin/tazobactam, cefpime, or ertapenem. All known genes (outer membrane proteins and extended-spectrum AmpC β-lactamases) involved in ETP resistance in E. coli were identical between EC974 and EC1515. This is the first study to identify the evolution of an IncI1 plasmid within the host, and to characterize blaCMY−111 in E. coli. [ABSTRACT FROM AUTHOR]

pIP501 is a Gram-positive broad-host-range model plasmid intensively used for studying plasmid replication and conjugative transfer. It is a multiple antibiotic resistance plasmid frequently found in clinical Enterococcus faecalis and Enterococcus faecium isolates. Replication of pIP501 proceeds unidirectionally by a theta mechanism. The minimal replicon of pIP501 is composed of the repR gene encoding the essential rate-limiting replication initiator protein RepR and the origin of replication, oriR, located downstream of repR. RepR is similar to RepE of related streptococcal plasmid pAMβ1, which has been shown to possess RNase activity cleaving free RNA molecules in close proximity of the initiation site of DNA synthesis. Replication of pIP501 is controlled by the concerted action of a small protein, CopR, and an antisense RNA, RNAIII. CopR has a dual role: It acts as transcriptional repressor at the repR promoter and prevents convergent transcription of RNAIII and repR mRNA (RNAII), thereby indirectly increasing RNAIII synthesis. CopR binds asymmetrically as a dimer at two consecutive binding sites upstream of and overlapping with the repR promoter. RNAIII induces transcriptional attenuation within the leader region of the repR mRNA (RNAII). Deletion of either control component causes a 10- to 20-fold increase of plasmid copy number, while simultaneous deletions have no additional effect. Conjugative transfer of pIP501 depends on a type IV secretion system (T4SS) encoded in a single operon. Its transfer host-range is considerably broad, as it has been transferred to virtually all Gram-positive bacteria including filamentous streptomycetes and even the Gram-negative Escherichia coli. Expression of the 15 genes encoding the T4SS is tightly controlled by binding of the relaxase TraA, the transfer initiator protein, to the operon promoter, which overlaps with the origin of transfer (oriT). The T4SS operon encodes the DNA-binding proteins TraJ (VirD4-like coupling protein) and the VirB4-like ATPase, TraE. Both proteins are actively involved in conjugative DNA transport. Moreover, the operon encodes TraN, a small cytoplasmic protein, whose specific binding to a sequence upstream of the oriT nic-site was demonstrated. TraN seems to be an effective repressor of pIP501 transfer, as conjugative transfer rates were significantly increased in an E. faecalis pIP501ΔtraN mutant.