Plasmid Replication Research Articles

Persistence of plasmids targeted by CRISPR interference in bacterial populations

CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy and microfluidics analyses we demonstrate that plasmids can persist for multiple generations in some Escherichia coli cell lineages at conditions of continuous targeting by the type I-E CRISPR-Cas system. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to continuous maintenance of mobile genetic elements in some cells, which leads to diversification of phenotypes in the entire community and allows rapid changes in the population structure to meet the demands of a changing environment.

Synchrotron Radiation Circular Dichroism, a New Tool to Probe Interactions between Nucleic Acids Involved in the Control of ColE1-Type Plasmid Replication

Hfq is a bacterial master regulator which promotes the pairing of nucleic acids. Due to the high molecular weight of the complexes formed between nucleic acids and the amyloid form of the protein, it is difficult to analyze solely by a gel shift assay the complexes formed, as they all migrate at the same position in the gel. In addition, precise kinetics measurements are not possible using a gel shift assay. Here, we used a synchrotron-based biophysical approach, synchrotron radiation circular dichroism (SRCD), to probe the interaction of the Escherichia coli Hfq C-terminal amyloid region with nucleic acids involved in the control of ColE1-like plasmid replication. We observed that this C-terminal region of Hfq has an unexpected and significant effect on the annealing of nucleic acids involved in this process and, more importantly, on their alignment. Functional consequences of this newly discovered property of the Hfq amyloid region are discussed in terms of the biological significance of Hfq in the ColE1-type plasmid replication process and antibiotic resistance.

Maximizing Photosynthesis-Driven Baeyer–Villiger Oxidation Efficiency in Recombinant Synechocystis sp. PCC6803

Photosynthesis-driven whole-cell biocatalysis has great potential to contribute to a sustainable bio-economy since phototrophic cells use light as the only energy source. It has yet to be shown that phototrophic microorganisms, such as cyanobacteria, can combine the supply of high heterologous enzyme levels with allocation of sufficient reduction equivalents to enable efficient light-driven redox biocatalysis. Here, we demonstrated that the heterologous expression of an NADPH-dependent Baeyer–Villiger monooxygenase (BVMO) gene from Acidovorax sp. CHX100 turns Synechocystis sp. PCC6803 into an efficient oxyfunctionalization biocatalyst, deriving electrons and O2 from photosynthetic water oxidation. Several expression systems were systematically tested, and a PnrsB-(Ni2+)–controlled expression based on a replicative plasmid yielded the highest intracellular enzyme concentration and activities of up to 60.9 ± 1.0 U gCDW−1. Detailed analysis of reaction parameters, side reactions, and biocatalyst durability revealed—on the one hand—a high in vivo BVMO activity in the range of 6 ± 2 U mgBVMO−1 and—on the other hand—an impairment of biocatalyst performance by product toxicity and by-product inhibition. Scale-up of the reaction to 2-L fed-batch photo-bioreactors resulted in the stabilization of the bioconversion over several hours with a maximal specific activity of 30.0 ± 0.3 U g CDW−1, a maximal volumetric productivity of 0.21 ± 0.1 gL−1 h−1, and the formation of 1.3 ± 0.1 gL−1 of ε-caprolactone. Process simulations based on determined kinetic data revealed that photosynthesis-driven cyclohexanone oxidation on a 2-L scale under high-light conditions was kinetically controlled and not subject to a limitation by photosynthesis.

Harnessing plasmid replication mechanism to enable dynamic control of gene copy in bacteria

Dynamic regulation has been proved efficient in controlling gene expression at transcriptional, translational, and post-translational level. However, the dynamic regulation at gene replication level has been rarely explored so far. In this study, we established dynamic regulation at gene copy level through engineering controllable plasmid replication to dynamically control the gene expression. Prototypic genetic circuits with different control logic were applied to enable diversified dynamic behaviors of gene copy. To explore the applicability of this strategy, the dynamic gene copy control was employed in regulating the biosynthesis of p-coumaric acid, which resulted in an up to 78% increase in p-coumaric acid titer to 1.69 g/L in shake flasks. These results indicated the great potential of applying dynamic gene copy control for engineering biosynthesis of valuable compounds in metabolic engineering.

Carbapenemase Production and Epidemiological Characteristics of Carbapenem-Resistant Klebsiella pneumoniae in Western Chongqing, China.

BackgroundThis study aimed to determine the molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in a hospital in western Chongqing, southwestern China.MethodsA total of 127 unique CRKP isolates were collected from the Yongchuan Hospital of Chongqing Medical University, identified using a VITEK-2 compact system, and subjected to microbroth dilution to determine the minimal inhibitory concentration. Enterobacteriaceae intergenic repeat consensus polymerase chain reaction and multilocus sequence typing were used to analyze the homology among the isolates. Genetic information, including resistance and virulence genes, was assessed using polymerase chain reaction. The genomic features of the CRKP carrying gene bla KPC-2 were detected using whole-genome sequencing.ResultsST11 was the dominant sequence type in the homology comparison. The resistance rate to ceftazidime-avibactam in children was much higher than that in adults as was the detection rate of the resistance gene bla NDM (p < 0.0001). Virulence genes such as mrkD (97.6%), uge (96.9%), kpn (96.9%), and fim-H (84.3%) had high detection rates. IncF (57.5%) was the major replicon plasmid detected, and sequencing showed that the CRKP063 genome contained two plasmids. The plasmid carrying bla KPC-2, which mediates carbapenem resistance, was located on the 359,625 base pair plasmid IncFII, together with virulence factors, plasmid replication protein (rep B), stabilizing protein (par A), and type IV secretion system (T4SS) proteins that mediate plasmid conjugation transfer.ConclusionOur study aids in understanding the prevalence of CRKP in this hospital and the significant differences between children and adults, thus providing new ideas for clinical empirical use of antibiotics.

A Structural Perspective of Reps from CRESS-DNA Viruses and Their Bacterial Plasmid Homologues.

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

Undetectable Production of the VIM-1 Carbapenemase in an Atlantibacter hermannii Clinical Isolate.

The differential expression of VIM-1 in Atlantibacter hermannii WEB-2 and Enterobacter hormaechei ssp. hoffmannii WEB-1 clinical isolates from a rectal swab of a hospitalized patient in France was investigated. A. hermannii WEB-2 was resistant to all β-lactams except carbapenems. It produced ESBL SHV-12, but the Carba NP test failed to detect any carbapenemase activity despite the production of VIM-1. Conversely, E. hormaechei WEB-1, previously recovered from the same patient, was positive for the detection of carbapenemase activity. The blaVIM–1 gene was located on a plasmid and embedded within class 1 integron. Both plasmids were of the same IncA incompatibility group and conferred the same resistance pattern when electroporated in Escherichia coli TOP10 or Enterobacter cloacae CIP7933. Quantitative RT-PCR experiments indicated a weaker replication of pWEB-2 in A. hermannii as compared to E. hormaechei. An isogenic mutant of A. hermannii WEB-2 selected after sequential passages with increased concentrations of imipenem possessed higher MICs for carbapenems and cephalosporins including cefiderocol, higher levels of the blaVIM–1 gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding blaVIM–1 gene occurred in the A. hermannii mutant with detectable carbapenemase activity. The lack of detection of the VIM-1 carbapenemase activity in A. hermannii WEB-2 isolate was likely due to a weak replication of the IncA plasmid harboring the blaVIM–1 gene. Imipenem as selective pressure led to a duplication of this gene on the plasmid and to the restoration of a significant carbapenem-hydrolyzing phenotype.

Paclitaxel and its derivative facilitate the transmission of plasmid-mediated antibiotic resistance genes through conjugative transfer

The rapid dissemination of antibiotic resistance by horizontal gene transfer (HGT) renders the global resistance crisis more tense and urgent as few effective antimicrobials are available to combat multidrug-resistant (MDR) pathogens at present. Conjugation is one of the most dominant and representative pathways of HGT. Antibiotic residue in environment is recognized as an important accelerator for conjugal transfer, whereas the roles of non-antibiotic pharmaceuticals in this process are not fully understood. Here we found that environmentally relevant concentrations of paclitaxel as well as its derivative docetaxel, two commonly used anticancer drugs, remarkably facilitated the conjugative transfer of resistance plasmids carrying multiple antibiotic resistance genes (ARGs). The underlying mechanisms accounting for the enhanced conjugation were investigated by detecting the activity of RpoS regulon, membrane permeability, SOS response and gene expression of conjugative transfer systems. Our results showed that paclitaxel induced a series of cellular responses, including up-regulation of rpoS expression, activated SOS response, increased cell membrane permeability, enhanced plasmid replication and mating pilus formation. Collectively, our data provide new insight on the roles of paclitaxel and its derivative in promoting the conjugal transfer of ARGs, highlighting the importance of good antimicrobial stewardship.

A genetic toolkit for co-expression of multiple proteins of diverse physiological implication

Construction of plasmids is crucial for expression of functional proteins of diverse physiological impact in E. coli. Here, we first designed and constructed a novel pair of bacterial expression vectors, i.e., pAS01 and pAS02, to be co-transformed with pQE30 for the co-expression of three target genes. The three plasmids contain ColE1, p15A and pSC101 origin of replication for high, medium and low copy plasmids, respectively, and same promoter (T5) and RBS. We then cloned genes encoding three reporter proteins (GFPuv, TurboRFP, and EYFP) in each of these plasmids and co-expressed in E. coli in six different combinations. Each of these reporter proteins exhibited diverse impact on growth, plasmid copy number and stability, and expression of other reporter proteins. Our results indicate that GFP and RFP were the most and the least favorable proteins for the cells, respectively, in terms of these parameters, especially on impacting expression of other co-expressed proteins.

RFPlasmid: predicting plasmid sequences from short-read assembly data using machine learning.

Antimicrobial-resistance (AMR) genes in bacteria are often carried on plasmids and these plasmids can transfer AMR genes between bacteria. For molecular epidemiology purposes and risk assessment, it is important to know whether the genes are located on highly transferable plasmids or in the more stable chromosomes. However, draft whole-genome sequences are fragmented, making it difficult to discriminate plasmid and chromosomal contigs. Current methods that predict plasmid sequences from draft genome sequences rely on single features, like k-mer composition, circularity of the DNA molecule, copy number or sequence identity to plasmid replication genes, all of which have their drawbacks, especially when faced with large single-copy plasmids, which often carry resistance genes. With our newly developed prediction tool RFPlasmid, we use a combination of multiple features, including k-mer composition and databases with plasmid and chromosomal marker proteins, to predict whether the likely source of a contig is plasmid or chromosomal. The tool RFPlasmid supports models for 17 different bacterial taxa, including Campylobacter , Escherichia coli and Salmonella , and has a taxon agnostic model for metagenomic assemblies or unsupported organisms. RFPlasmid is available both as a standalone tool and via a web interface.

Introduction: the secret lives of microbial mobile genetic elements.

Introduction: the secret lives of microbial mobile genetic elements.

Expression and activity of heterologous hydroxyisocaproate dehydrogenases in Synechocystis sp. PCC 6803 ΔhoxYH

Exploiting light to drive redox reactions is currently a hot topic since light is considered as an environmentally friendly source of energy. Consequently, cyanobacteria, which can use light e.g., for generating NADPH, are in the focus of research. Previously, it has been shown that various heterologous redox enzymes could be expressed in these microorganisms. Here we demonstrated the successful inducer-free expression of α-keto-acid dehydrogenases (L-HicDH and D-HicDH) from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008 in Synechocystis sp. PCC 6803 ΔhoxYH mutant using replicative plasmids. While the L-HicDH showed poor activity limited by the amount of expressed enzyme, the D-HicDH was applied both in vivo and in vitro, transforming the selected α-keto acids to the corresponding optically pure (R)-α-hydroxy acids (ee >99%) in up to 53% and 90% conversion, respectively.

Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates.

Simple SummaryDuring replication, DNA molecules undergo topological changes that affect supercoiling, catenation and knotting. To better understand this process and the role of topoisomerases, the enzymes that control DNA topology in in vivo, two-dimensional agarose gel electrophoresis were used to investigate the efficiency of three type II DNA topoisomerases, the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α, on partially replicated bacterial plasmids containing replication forks stalled at specific sites. The results obtained revealed that despite the fact these DNA topoisomerases may have evolved to accomplish specific tasks, they share abilities. To our knowledge, this is the first time two-dimensional agarose gel electrophoresis have been used to examine the ability of these topoisomerases to relax supercoiling in the un-replicated region and unlink pre-catenanes in the replicated one of partially replicated molecules in vitro. The methodology described here can be used to study the role of different topoisomerases in partially replicated molecules.DNA topoisomerases are the enzymes that regulate DNA topology in all living cells. Since the discovery and purification of ω (omega), when the first were topoisomerase identified, the function of many topoisomerases has been examined. However, their ability to relax supercoiling and unlink the pre-catenanes of partially replicated molecules has received little attention. Here, we used two-dimensional agarose gel electrophoresis to test the function of three type II DNA topoisomerases in vitro: the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α. We examined the proficiency of these topoisomerases on a partially replicated bacterial plasmid: pBR-TerE@AatII, with an unidirectional replicating fork, stalled when approximately half of the plasmid had been replicated in vivo. DNA was isolated from two strains of Escherichia coli: DH5αF’ and parE10. These experiments allowed us to assess, for the first time, the efficiency of the topoisomerases examined to resolve supercoiling and pre-catenanes in partially replicated molecules and fully replicated catenanes formed in vivo. The results obtained revealed the preferential functions and also some redundancy in the abilities of these DNA topoisomerases in vitro.

Assessment of the plasmidome of an extremophilic microbial community from the Diamante Lake, Argentina

Diamante Lake located at 4589 m.a.s.l. in the Andean Puna constitutes an extreme environment. It is exposed to multiple extreme conditions such as an unusually high concentration of arsenic (over 300 mg L−1) and low oxygen pressure. Microorganisms thriving in the lake display specific genotypes that facilitate survival, which include at least a multitude of plasmid-encoded resistance traits. Hence, the genetic information provided by the plasmids essentially contributes to understand adaptation to different stressors. Though plasmids from cultivable organisms have already been analyzed to the sequence level, the impact of the entire plasmid-borne genetic information on such microbial ecosystem is not known. This study aims at assessing the plasmidome from Diamante Lake, which facilitates the identification of potential hosts and prediction of gene functions as well as the ecological impact of mobile genetic elements. The deep-sequencing analysis revealed a large fraction of previously unknown DNA sequences of which the majority encoded putative proteins of unknown function. Remarkably, functions related to the oxidative stress response, DNA repair, as well as arsenic- and antibiotic resistances were annotated. Additionally, all necessary capacities related to plasmid replication, mobilization and maintenance were detected. Sequences characteristic for megaplasmids and other already known plasmid-associated genes were identified as well. The study highlights the potential of the deep-sequencing approach specifically targeting plasmid populations as it allows to evaluate the ecological impact of plasmids from (cultivable and non-cultivable) microorganisms, thereby contributing to the understanding of the distribution of resistance factors within an extremophilic microbial community.

Making sense of the natural antisense transcript puzzle.

In plants, thousands of genes are associated with antisense transcription, which often produces noncoding RNAs. Although widespread, sense-antisense pairs have been implicated in a limited variety of functions in plants and are often thought to form extensive dsRNA stretches triggering gene silencing. In this opinion, we show that evidence does not support gene silencing as a major role for antisense transcription. In fact, it is more likely that antisense transcripts play diverse functions in gene regulation. We propose a general framework for the initial functional dissection of antisense transcripts, suggesting testable hypotheses relying on an experiment-based decision tree. By moving beyond the gene silencing paradigm, we argue that a broad and diverse role for natural antisense transcription will emerge.

Host age increased conjugal plasmid transfer in gut microbiota of the soil invertebrate Caenorhabditis elegans

Plasmid conjugation contributes greatly to the spread of antibiotic resistance genes (ARGs) in soils. However, the spread potential in the gut of soil fauna remains poorly studied, and little was known about the impact of host age on ARGs dissemination in the gut microbiota of soil animals. Here, the typical nematode-Caenorhabditis elegans was employed as the model soil animal, aiming to investigate transfer of broad-host-range IncP-1ɛ from Escherichia coli MG1655 to gut microbiota within 6 days under varied temperature gradients (15, 20 and 25 °C) using qPCR combined with plate screening. Results showed that conjugation rates increased with incubation time and rising temperature in the gut of C. elegans, sharing a similar trend with abundances of plasmid conjugation relevant genes such as trbBp (mating pair formation) and trfAp (plasmid replication). Incubation time and temperature significantly shaped the gut microbial community of C. elegans. Core microbiota in the gut of C. elegans, including Enterobacteriaceae, Lactobacillaceae and Leuconostocaceae, constituted a large part of transconjugal pool for plasmid IncP-1ɛ. Our results highlight an important sink of gut microbiota for ARGs dissemination and upregulation of ARGs transfer in the gut microbiota with host age, further potentially stimulating evolution of ARGs in terrestrial environments.

USING A NONVIRAL AND NONINTEGRATING DNA VECTOR TO GENERATE SAFE CAR-T CELLS

CAR-T cell therapy has proven its importance in cancer treatment and currently there are five approved products on the market. However, these products are based on viral vectors, which presents a risk of insertional mutagenesis, it can also be immunogenic and its manufacture is extremely expensive. The development of a nonviral vector, suitable for an efficient and stable transfection of NK and T cells is the goal of this study. In this study we use a special episomal vector (extrachromosomal vectors) with i) scaffold/matrix attachment region (S/MAR) motifs that can actively mediate the episomal maintenance, ii) a plasmid replication enhancer, the replication-Initiation Region (IR) from the β-globin locus (pEP-IR) and iii) a CAR anti CD19 (pEP-IR-CAR). We electroporated activated and non-activated T cells and NK cells with different plasmid concentrations. We evaluated transfection efficiency and viability. Our findings showed that unstimulated T cells are transfected more efficiently than activated T cells. Transfection of unstimulated T cells with 20 ug of supercoiled pEP-IR-GFP plasmid reached 22 to 63% of GFP and viability around 54% depending on the culture condition used after electroporation (FBS% and IL2). We then accessed the efficacy of pEP-IR-CAR and CAR expression, confirmed in 3,2% of the cells with 89% of viability. Cells expressing CAR on their surface were marked with anti-CAR antibody Biotin-SP-conjugated and selected by anti-Biotin microbeads, achieving an enrichment of 54,9% CAR+ cells. Our results showed that we established an optimized protocol resulting in an acceptable transfection efficiency and the transfected cells can be selected. So, the next step is to perform in vitro cytotoxicity and in vivo assays with the episomal CAR+ cells. Financially supported by FAPESP 2020/09206-4, FAPESP 2019/25309-0, CTC Center for Cell-based Therapy (FAPESP 2013/08135-2), and National Institute of Science and Technology in Stem Cell and Cell Therapy (CNPq 573754-2008-0 and FAPESP 2008/578773).

Ubiquitous Conjugative Mega-Plasmids of Acinetobacter Species and Their Role in Horizontal Transfer of Multi-Drug Resistance.

Conjugative mega-plasmids play a special role in adaptation since they carry a huge number of accessory genes, often allowing the host to develop in new niches. In addition, due to conjugation they are able to effectively spread themselves and participate in the transfer of small mobilizable plasmids. In this work, we present a detailed characterization of a recently discovered family of multiple-drug resistance mega-plasmids of Acinetobacter species, termed group III-4a. We describe the structure of the plasmid backbone region, identify the rep gene and the origin of plasmid replication, and show that plasmids from this group are able not only to move between different Acinetobacter species but also to efficiently mobilize small plasmids containing different mob genes. Furthermore, we show that the population of natural Acinetobacter strains contains a significant number of mega-plasmids and reveal a clear correlation between the living conditions of Acinetobacter strains and the structure of their mega-plasmids. In particular, comparison of the plasmids from environmental and clinical strains shows that the genes for resistance to heavy metals were eliminated in the latter, with the simultaneous accumulation of antibiotic resistance genes by incorporation of transposons and integrons carrying these genes. The results demonstrate that this group of mega-plasmids plays a key role in the dissemination of multi-drug resistance among Acinetobacter species.

Bacterial YedK represses plasmid DNA replication and transformation through its DNA single-strand binding activity

The SOS response-associated peptidase (SRAP) is an ancient protein superfamily in all domains of life. The mammalian SRAP was recently reported to covalently bind to the abasic sites (AP) in single stranded (ss) DNA to shield the chromosome integrity. YedK, the Escherichia coli SRAP, is not functionally characterized. Here we report the fortuitous pull-down of YedK from bacterial cell lysates by short (<20 bp) double stranded (ds) DNAs, further enrichment of YedK was observed when single stranded (ss) DNA was added. YedK can bind multiple DNA substrates, particularly with a high affinity to DNA duplex with single strand segment. As a SRAP protein, the involvement of YedK in SOS response was extensively examined, however yedK mutant of Escherichia coli showed no difference from the wild type strain upon the treatments with UV and various DNA damaging reagents, indicating its non-essentiality or redundancy in E. coli. Surprisingly, yedK mutants derived from Escherichia coli and Samonella enterica both showed an increased plasmid DNA transformation efficiency compared to the wild types. In accordance with this, induction of YedK effectively decreased the copy number of plasmid DNA. Site-directed mutagenesis of YedK demonstrated that residues involved in single strand DNA binding and cysteine residue at position 2 from N-terminus can discharge the repression of the plasmid transformation efficiency.

Differential Chromosome- and Plasmid-Borne Resistance of Escherichia coli hfq Mutants to High Concentrations of Various Antibiotics.

The Hfq protein is a bacterial RNA chaperone, involved in many molecular interactions, including control of actions of various small RNA regulatory molecules. We found that the presence of Hfq was required for survival of plasmid-containing Escherichia coli cells against high concentrations of chloramphenicol (plasmid p27cmr), tetracycline (pSC101, pBR322) and ampicillin (pBR322), as hfq+ strains were more resistant to these antibiotics than the hfq-null mutant. In striking contrast, production of Hfq resulted in low resistance to high concentrations of kanamycin when the antibiotic-resistance marker was chromosome-borne, with deletion of hfq resulting in increasing bacterial survival. These results were observed both in solid and liquid medium, suggesting that antibiotic resistance is an intrinsic feature of these strains rather than a consequence of adaptation. Despite its major role as RNA chaperone, which also affects mRNA stability, Hfq was not found to significantly affect kan and tet mRNAs turnover. Nevertheless, kan mRNA steady-state levels were higher in the hfq-null mutant compared to the hfq+ strain, suggesting that Hfq can act as a repressor of kan expression.This observation does correlate with the enhanced resistance to high levels of kanamycin observed in the hfq-null mutant. Furthermore, dependency on Hfq for resistance to high doses of tetracycline was found to depend on plasmid copy number, which was only observed when the resistance marker was expressed from a low copy plasmid (pSC101) but not from a medium copy plasmid (pBR322). This suggests that Hfq may influence survival against high doses of antibiotics through mechanisms that remain to be determined. Studies with pBR322Δrom may also suggest an interplay between Hfq and Rom in the regulation of ColE1-like plasmid replication. Results of experiments with a mutant devoid of the part of the hfq gene coding for the C-terminal region of Hfq suggested that this region, as well as the N-terminal region, may be involved in the regulation of expression of antibiotic resistance in E. coli independently.