RESUMEN
Bacteria have adapted to phage predation by evolving a vast assortment of defence systems1. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data2. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library3 with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.
Asunto(s)
Bacterias , Bacteriófago T4 , ADN Glicosilasas , Bacterias/clasificación , Bacterias/enzimología , Bacterias/genética , Bacterias/inmunología , Bacterias/virología , Bacteriófago T4/crecimiento & desarrollo , Bacteriófago T4/inmunología , Bacteriófago T4/metabolismo , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Escherichia coli/genética , Escherichia coli/virología , Biblioteca de Genes , Metagenómica/métodos , Microbiología del Suelo , Replicación ViralRESUMEN
Novel antibiotics are in constant demand to combat a global increase in antibiotic-resistant infections. Bacterial natural products have been a long-standing source of antibiotic compounds, and metagenomic mining of environmental DNA (eDNA) has increasingly provided new antibiotic leads. The metagenomic small-molecule discovery pipeline can be divided into three main steps: surveying eDNA, retrieving a sequence of interest, and accessing the encoded natural product. Improvements in sequencing technology, bioinformatic algorithms, and methods for converting biosynthetic gene clusters into small molecules are steadily increasing our ability to discover metagenomically encoded antibiotics. We predict that, over the next decade, ongoing technological improvements will dramatically increase the rate at which antibiotics are discovered from metagenomes.
Asunto(s)
Productos Biológicos , ADN Ambiental , Antibacterianos/farmacología , Bacterias/genética , Metagenómica , Familia de MultigenesRESUMEN
The capture of metagenomic DNA in large clone libraries provides the opportunity to study microbial diversity that is inaccessible using culture-dependent methods. In this study, we harnessed nuclease-deficient Cas9 to establish a CRISPR counter-selection interruption circuit (CCIC) that can be used to retrieve target clones from complex libraries. Combining modern sequencing methods with CCIC cloning allows for rapid physical access to the genetic diversity present in natural ecosystems.
Asunto(s)
Ecosistema , Metagenómica , Células ClonalesRESUMEN
Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs. Our solution simultaneously captures large numbers of BGCs by cloning the genomes of a strain collection in a large-insert library and uses the CONKAT-seq (co-occurrence network analysis of targeted sequences) sequencing pipeline to efficiently localize clones carrying intact BGCs which represent candidates for heterologous expression. Our discovery of several natural products, including an antibiotic that is active against multi-drug resistant Staphylococcus aureus, demonstrates the potential of leveraging economies of scale with this approach to systematically interrogate cryptic BGCs contained in strain collections.
Asunto(s)
Productos Biológicos , Staphylococcus aureus Resistente a Meticilina , Antibacterianos , Productos Biológicos/metabolismo , Vías Biosintéticas/genética , Genoma Bacteriano/genética , Staphylococcus aureus Resistente a Meticilina/genética , Familia de MultigenesRESUMEN
Mycobacterium abscessus is a rapidly emerging mycobacterial pathogen causing dangerous pulmonary infections. Because these bacteria are intrinsically multidrug resistant, treatment options are limited and have questionable efficacy. The current treatment regimen relies on a combination of antibiotics, including clarithromycin paired with amikacin and either imipenem or cefoxitin. Tigecycline may be added when triple therapy is ineffective. We initially screened a library containing the majority of clinically available antibiotics for anti-M. abscessus activity. The screen identified rifabutin, which was then investigated for its interactions with M. abscessus antibiotics used in drug regimens. Combination of rifabutin with either clarithromycin or tigecycline generated synergistic anti-M. abscessus activity, dropping the rifabutin MIC below concentrations found in the lung. Importantly, these combinations generated bactericidal activity. The triple combination of clarithromycin, tigecycline, and rifabutin was also synergistic, and clinically relevant concentrations had a sterilizing effect on M. abscessus cultures. We suggest that combinations including rifabutin should be further investigated for treatment of M. abscessus pulmonary infections.
Asunto(s)
Antibacterianos/farmacología , Claritromicina/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Mycobacterium abscessus/efectos de los fármacos , Rifabutina/farmacología , Tigeciclina/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Ensayos Analíticos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/crecimiento & desarrollo , Mycobacterium abscessus/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Diseases caused by various Mycobacterium sp., especially Mycobacterium tuberculosis, are a major burden on global health care. Due to high intrinsic antibiotic resistance, treatment options are severely limited. In mycobacteria, WhiB7 coordinates intrinsic resistance to a broad range of antibiotics. While WhiB7 has been established as an auto-regulatory transcriptional activator, the signals and genes needed to induce its expression are poorly understood. Using Mycobacterium smegmatis as a model, we coupled transposon mutagenesis and next generation sequencing with WhiB7-specific antibiotic selection to identify genes that contribute to WhiB7 regulation and function. We showed that whiB7 expression was regulated by two coordinated processes: early termination of the whiB7 transcript and increased whiB7 promoter activity. Early termination was irreversibly maintained by constitutive expression of a putative aspartate aminotransferase gene, MSMEG_4060. A pair of hypothetical genes, MSMEG_3637 and MSMEG_3638, were identified as important contributors to whiB7 promoter induction on antibiotic challenge. Expansion of our understanding of the WhiB7-resistance pathway may lead to identification of inhibitors that allow the use of previously ineffective antibiotics to treat mycobacterial diseases.
Asunto(s)
Mycobacterium smegmatis/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Reguladores/genética , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Regiones Terminadoras Genéticas/genética , Regiones Terminadoras Genéticas/fisiología , Factores de Transcripción/metabolismoRESUMEN
Combinations of antibiotics, each individually effective against Mycobacterium abscessus, are routinely coadministered based on the concept that this minimizes the spread of antibiotic resistance. However, our in vitro data contradict this assumption and instead document antagonistic interactions between two antibiotics (clarithromycin and amikacin) used to treat M. abscessus infections. Clinically relevant concentrations of clarithromycin induced increased resistance to both amikacin and itself. The induction of resistance was dependent on whiB7, a transcriptional activator of intrinsic antibiotic resistance that is induced by exposure to many different antibiotics. In M. abscessus, the deletion of whiB7 (MAB_3508c) resulted in increased sensitivity to a broad range of antibiotics. WhiB7 was required for transcriptional activation of genes that confer resistance to three commonly used anti-M. abscessus drugs: clarithromycin, amikacin, and tigecycline. The whiB7-dependent gene that conferred macrolide resistance was identified as erm(41) (MAB_2297), which encodes a ribosomal methyltransferase. The whiB7-dependent gene contributing to amikacin resistance was eis2 (MAB_4532c), which encodes a Gcn5-related N-acetyltransferase (GNAT). Transcription of whiB7 and the resistance genes in its regulon was inducible by subinhibitory concentrations of clarithromycin but not by amikacin. Thus, exposure to clarithromycin, or likely any whiB7-inducing antibiotic, may antagonize the activities of amikacin and other drugs. This has important implications for the management of M. abscessus infections, both in cystic fibrosis (CF) and non-CF patients.
Asunto(s)
Amicacina/farmacología , Antibacterianos/farmacología , Claritromicina/farmacología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Mycobacterium abscessus/genética , Amicacina/antagonistas & inhibidores , Proteínas Bacterianas/genética , Antagonismo de Drogas , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/aislamiento & purificaciónRESUMEN
Complex continuous wavelet coherence (WTC) can be used for non-stationary signals, such as electroencephalograms. Areas of the WTC with a coherence higher than the calculated optimal threshold were obtained, and the sum of their areas was used as a criterion to differentiate between groups of experienced insight-focused meditators, calm-focused meditators and a control group. This method demonstrated the highest accuracy for the real WTC parts in the frontal region, while for the imaginary parts, the highest accuracy was shown for the frontal occipital pairs of electrodes. In the frontal area, in the broadband frequency, both types of experienced meditators demonstrated an enlargement of the increased coherence areas for the real WTC parts. For the imaginary parts unaffected by the volume conduction and global artefacts, the most significant increase occurred for the frontal occipital pair of electrodes.
Asunto(s)
Corteza Cerebral/fisiología , Electroencefalografía/métodos , Meditación/psicología , Adulto , Femenino , Humanos , Imaginación/fisiología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the host's repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimer's absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Proteínas Hierro-Azufre/genética , Mycobacterium smegmatis/metabolismo , Rhodococcus/metabolismo , Streptomyces lividans/metabolismo , Factores de Transcripción/genética , Actinobacteria , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Proteínas Hierro-Azufre/metabolismo , Metiltransferasas/metabolismo , Mutagénesis , Mycobacterium smegmatis/genética , Rhodococcus/genética , Especificidad de la Especie , Streptomyces lividans/genética , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , Activación Transcripcional/genéticaRESUMEN
Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7's ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the -35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7-SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium smegmatis/genética , Factor sigma/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , ADN/química , ADN/metabolismo , Farmacorresistencia Bacteriana , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium smegmatis/efectos de los fármacos , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Factor sigma/química , Transactivadores/química , Transactivadores/genéticaRESUMEN
Current tuberculosis treatment regimens are notoriously limited, lengthy and becoming increasingly ineffective due to the emergence of drug-resistant mutant strains of Mycobacterium tuberculosis. The intrinsic resistance of M. tuberculosis to the majority of available drugs relies both on the impermeability of its cell envelope, and its ability to activate specific genes and physiological states. WhiB7 is a transcriptional regulatory protein underlying this adaptive process. Transcription of the whiB7 gene is upregulated in response to a variety of antibiotics having different structures and targets, as well as in response to metabolic signals. The whiB7 regulon activates various systems of intrinsic drug resistance involving antibiotic export, antibiotic inactivation (by chemical modifications of the drug or its target) and significant changes to thiol redox balance. Drugs have been identified that inactivate resistance determinants in the whiB7 regulon, thereby potentiating the activities of diverse antibiotics against M. tuberculosis.
Asunto(s)
Proteínas Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/efectos de los fármacos , Factores de Transcripción/fisiología , Antituberculosos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Oxidación-Reducción , Regulón/efectos de los fármacos , Regulón/fisiología , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Intrinsic drug resistance in Mycobacterium tuberculosis limits therapeutic options for treating tuberculosis. The mycobacterial transcriptional regulator whiB7 contributes to intrinsic resistance by activating its own expression and many drug resistance genes in response to antibiotics. To investigate whiB7 activation, we constructed a GFP reporter to monitor its expression, and we used it to investigate the whiB7 promoter and to screen our custom library of almost 600 bioactive compounds, including the majority of clinical antibiotics. Results showed whiB7 was transcribed from a promoter that was conserved across mycobacteria and other actinomycetes, including an AT-rich sequence that was likely targeted by WhiB7. Expression was induced by compounds having diverse structures and targets, independent of the ability of whiB7 to mediate resistance, and was dependent on media composition. Pretreatment with whiB7 activators resulted in clinically relevant increases in intrinsic drug resistance. Antibiotic-induced transcription was synergistically increased by the reductant dithiothreitol, an effect mirrored by a whiB7-dependent shift to a highly reduced cytoplasm reflected by the ratio of reduced/oxidized mycothiol. These data provided evidence that intrinsic resistance resulting from whiB7 activation is linked to fundamental changes in cell metabolism.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Homeostasis/efectos de los fármacos , Homeostasis/genética , Mycobacterium/genética , Mycobacterium/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Farmacorresistencia Bacteriana/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genética , Genes Reporteros/genética , Datos de Secuencia Molecular , Mycobacterium/efectos de los fármacos , Motivos de Nucleótidos/genética , Oxidación-Reducción/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Gene clusters found in bacterial species classified as Streptomyces encode the majority of known antibiotics as well as many pharmaceutically active compounds. A site-specific recombination system similar to those that mediate plasmid conjugation was engineered to catalyze tandem amplification of one of these gene clusters in a heterologous Streptomyces species. Three genetic elements were known to be required for DNA amplification in S. kanamyceticus: the oriT-like recombination sites RsA and RsB, and ZouA, a site-specific relaxase similar to TraA proteins that catalyze plasmid transfer. We inserted RsA and RsB sequences into the S. coelicolor genome flanking a cluster of 22 genes (act) responsible for biosynthesis of the polyketide antibiotic actinorhodin. Recombination between RsA and RsB generated zouA-dependent DNA amplification resulting in 4-12 tandem copies of the act gene cluster averaging nine repeats per genome. This resulted in a 20-fold increase in actinorhodin production compared with the parental strain. To determine whether the recombination event required taxon-specific genetic effectors or generalized bacterial recombination (recA), it was also analyzed in the heterologous host Escherichia coli. zouA was expressed under the control of an inducible promoter in wild-type and recA mutant strains. A plasmid was constructed with recombination sites RsA and RsB bordering a drug resistance marker. Induction of zouA expression generated hybrid RsB/RsA sites, evidence of site-specific recombination that occurred independently of recA. ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic, degradative, and photosynthetic pathways in a wide variety of organisms.
Asunto(s)
Antibacterianos/biosíntesis , Genes Bacterianos/genética , Familia de Multigenes , Streptomyces coelicolor/genética , Antraquinonas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Southern Blotting , ADN Bacteriano/genética , Electroforesis en Gel de Agar , Escherichia coli/genética , Escherichia coli/metabolismo , Amplificación de Genes , Datos de Secuencia Molecular , Plásmidos/genética , Recombinación Genética , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces coelicolor/metabolismoRESUMEN
The psychrophilic bacterium Flavobacterium psychrophilum is a rapidly emerging, virulent pathogen of a variety of commercially important finfish species, including salmonids. No vaccines against F. psychrophilum are currently available, partly due to its recalcitrant growth in vitro. Consequently, we explored the possibility of constructing recombinant vaccines in Escherichia coli as a prophylactic biotechnological strategy to counter F. psychrophilum infections. An immunoreactive clone from a F. psychrophilum expression library was found to express a approximately 16 kDa protein antigen. A proteomics approach was taken to identify the ORF encoding the approximately 16 kDa protein. Tryptic fragments of the approximately 16 kDa protein were analyzed by MALDI-TOF mass spectrometry and compared to theoretical (in silico) tryptic fragments of translated ORFs predicted within the cloned DNA. The target protein was identified as a 166 amino acid protein (named 7-166) with homology to rplJ which encodes bacterial ribosomal protein L10. Whenhighly expressed in E. coli as an N-terminal fusion protein, this chimera reacted with convalescent rainbow trout serum. When adjuvanted and administered intraperitoneally to immature rainbow trout a high level of protection (82% RPS) was afforded against virulent F. psychrophilum challenge; thus establishing F. psychrophilumrplJ homologue 7-166 as a promising vaccine candidate for RTFS.
Asunto(s)
Vacunas Bacterianas/inmunología , Enfermedades de los Peces/inmunología , Flavobacterium/inmunología , Oncorhynchus mykiss/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Vacunas Bacterianas/genética , Western Blotting , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/inmunología , Infecciones por Flavobacteriaceae/microbiología , Flavobacterium/genética , Flavobacterium/metabolismo , Biblioteca de Genes , Oncorhynchus mykiss/microbiología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteína Ribosómica L10 , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/inmunología , Proteínas Ribosómicas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Flavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome, an economically important disease of immature salmonid fish for which there is no vaccine. Convalescent serum from the host, rainbow trout (Oncorhynchus mykiss), reacted strongly with a approximately 20 kDa, Flavobacterium-specific protein antigen (subsequently named FspA) from F. psychrophilum. Protein-enriched, detergent-partitioned samples were separated by two-dimensional gel electrophoresis and the protein target was excised, proteolytically cleaved and the resulting peptides analysed by MS. Quadrupole-time-of-flight MS was used to generate a fragmented peptide spectrum. The resulting peptide sequences were then used to design degenerate PCR primers to amplify the gene (fspA) of interest: 612 bp encoding 203 aa, including a putative 19 aa N-terminal signal sequence which predicted a processed 19 303.6 Da protein. FspA proved to be unique and only homologous to two unspecified sequences reported from Flavobacterium johnsoniae, although weakly homologous to a Yersinia pseudotuberculosis adhesin. An amplified gene fragment (537 bp, encoding 179 aa) was further cloned into an expression vector, expressed as a approximately 30 kDa N-terminal fusion protein and found to retain its strong reactivity with host serum antibodies. These results suggest that the surface-localized FspA may be an important subunit vaccine candidate antigen against F. psychrophilum.
Asunto(s)
Antígenos Bacterianos/inmunología , Enfermedades de los Peces/inmunología , Flavobacterium/inmunología , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/sangre , Flavobacterium/química , Datos de Secuencia MolecularRESUMEN
The Escherichia coli plasmid pKL1 is one of the smallest bacterial plasmids. It encodes a single, autoregulating structural gene, repA, responsible for replication and copy number control. The oligomerization of RepA was previously proposed as the basis of a strategy for pKL1 copy number control. To elucidate the oligomerization properties of RepA in solution, RepA was expressed in E. coli; purified by ion exchange and hydrophobic chromatography; and examined in solution by spectrapolarimetry, light scattering, sedimentation velocity, and equilibrium ultracentrifugation. RepA behaved as a concentration-dependent equilibrium of dimers and hexamers. Conformational parameters of the RepA hexameric complex were determined. These results support the proposed autogenous regulatory model whereby RepA hexamers negatively regulate repA expression thereby affecting the copy number control of pKL1. RepA of pKL1 is the first plasmid replication initiation protein documented to be in dimeric-hexameric forms.