Darja Žgur-Bertok

The bacterial LexA transcriptional repressor

Abstract.Bacteria respond to DNA damage by mounting a coordinated cellular response, governed by the RecA and LexA proteins. In Escherichia coli, RecA stimulates cleavage of the LexA repressor, inducing more than 40 genes that comprise the SOS global regulatory network. The SOS response is widespread among bacteria and exhibits considerable variation in its composition and regulation. In some well-characterised pathogens, induction of the SOS response modulates the evolution and dissemination of drug resistance, as well as synthesis, secretion and dissemination of the virulence. In this review, we discuss the structure of LexA protein, particularly with respect to distinct conformations that enable repression of SOS genes via specific DNA binding or repressor cleavage during the response to DNA damage. These may provide new starting points in the battle against the emergence of bacterial pathogens and the spread of drug resistance among them.

Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance.

The nucleotide sequence of the Bacillus licheniformis bacitracin‐resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP‐binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro‐phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram‐negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide‐binding sites, BcrA protein and mammalian multidrug transporter or P‐glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide‐binding protein to determine the Importance of various regions in transport.

High Prevalence of Multidrug Resistance and Random Distribution of Mobile Genetic Elements Among Uropathogenic Escherichia coli (UPEC) of the Four Major Phylogenetic Groups

One hundred and ten UTI Escherichia coli strains, from Ljubljana, Slovenia, were analyzed for antibiotic resistances, mobile DNA elements, serotype, and phylogenetic origin. A high prevalence of drug resistance and multidrug resistance was found. Twenty-six percent of the isolates harbored a class 1 integron, while a majority of the strains (56%) harbored rep sequences characteristic of F-like plasmids. int as well as rep sequences were found to be distributed in a random manner among strains of the four major phylogenetic groups indicating that all groups have a similar tendency to acquire and maintain mobile genetic elements frequently associated with resistance determinants.

Virulence Potential of Escherichia coli Isolates from Skin and Soft Tissue Infections

ABSTRACT Escherichia coli strains frequently are isolated from skin and soft tissue infections (SSTI); however, their virulence potential has not yet been extensively studied. In the present study, we characterized 102 E. coli SSTI strains isolated mostly from surgical and traumatic wounds, foot ulcers, and decubitus. The strains were obtained from the Institute of Microbiology and Immunology, University of Ljubljana, Slovenia. Phylogenetic backgrounds, virulence factors (VFs), and antibiotic resistance profiles were determined. Correlations between VFs and phylogenetic groups were established and analyzed with regard to patient factors. Further, the associations of the three most prevalent antibiotic resistance patterns with virulence potential were analyzed. Our results showed that the majority of the studied strains (65%) belonged to the B2 phylogenetic group. The most prevalent VF was ompT (80%), while toxin genes cnf1 and hlyA were found with prevalences of 32 and 30%, respectively. None of the investigated bacterial characteristics were significantly associated with patient gender, age, type of infection, or immunodeficiency. The most prevalent antibiotic resistance pattern was resistance to ampicillin (46%), followed by resistance to tetracycline (25%) and fluoroquinolones (21%). Strains resistant to ciprofloxacin exhibited a significantly reduced prevalence of cnf1 (P < 0.05) and usp (P < 0.01). Our study revealed that E. coli isolates from SSTIs exhibit a remarkable virulence potential that is comparable to that of E. coli isolates from urinary tract infections and bacteremia.

DNA Damage Repair and Bacterial Pathogens

All species require DNA repair pathways to maintain the integrity of their genomes. Bacterial damage repair mechanisms have broader roles encompassing responses to stress, long-term colonization, as well as virulence. The SOS response regulates DNA repair and damage tolerance genes in many bacterial species. This article highlights the bacterial SOS response and its significance in bacterial adaptation and pathogenesis, as well as DNA damage responses provoked by bacterial pathogens in the mammalian host.

Genes regulated by the Escherichia coli SOS repressor LexA exhibit heterogenous expression

BackgroundPhenotypic heterogeneity may ensure that a small fraction of a population survives environmental perturbations or may result in lysis in a subpopulation, to increase the survival of siblings. Genes involved in DNA repair and population dynamics play key roles in rapid responses to environmental conditions. In Escherichia coli the transcriptional repressor LexA controls a coordinated cellular response to DNA damage designated the SOS response. Expression of LexA regulated genes, e.g. colicin encoding genes, recA, lexA and umuDC, was examined utilizing transcription fusions with the promoterless gfp at the single cell level.ResultsThe investigated LexA regulated genes exhibited heterogeneity, as only in a small fraction of the population more intense fluorescence was observed. Unlike recA and lexA, the pore forming and nuclease colicin activity genes as well as umuDC, exhibited no basal level activity. However, in a lexA defective strain high level expression of the gene fusions was observed in the large majority of the cells. All of the investigated genes were expressed in a recA defective strain, albeit at lower levels, revealing expression in the absence of a spontaneous SOS response. In addition, the simultaneous expression of cka, encoding the pore forming colicin K, and lexA, investigated at the single cell level revealed high level expression of only cka in rare individual cells.ConclusionLexA regulated genes exhibit phenotypic heterogeneity as high level expression is observed in only a small subpopulation of cells. Heterogenous expression is established primarily by stochastic factors and the binding affinity of LexA to SOS boxes.

Comparative analysis of yellow microbial communities growing on the walls of geographically distinct caves indicates a common core of microorganisms involved in their formation

Morphologically similar microbial communities that often form on the walls of geographically distinct limestone caves have not yet been comparatively studied. Here, we analysed phylotype distribution in yellow microbial community samples obtained from the walls of distinct caves located in Spain, Czech Republic and Slovenia. To infer the level of similarity in microbial community membership, we analysed inserts of 474 16S rRNA gene clones and compared those using statistical tools. The results show that the microbial communities under investigation are composed solely of Bacteria. The obtained phylotypes formed three distinct groups of operational taxonomic units (OTUs). About 60% of obtained sequences formed three core OTUs common to all three sampling sites. These were affiliated with actinobacterial Pseudonocardinae (30-50% of sequences in individual sampling site libraries), but also with gammaproteobacterial Chromatiales (6-25%) and Xanthomonadales (0.5-2.0%). Another 7% of sequences were common to two sampling sites and formed eight OTUs, while the remaining 35% were site specific and corresponded mostly to OTUs containing single sequences. The same pattern was observed when these data were compared with sequence data available from similar studies. This comparison showed that distinct limestone caves support microbial communities composed mostly of phylotypes common to all sampling sites.

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations.

Double locking of an Escherichia coli promoter by two repressors prevents premature colicin expression and cell lysis

The synthesis of Eschericha coli colicins is lethal to the producing cell and is repressed during normal growth by the LexA transcription factor, which is the master repressor of the SOS system for repair of DNA damage. Following DNA damage, LexA is inactivated and SOS repair genes are induced immediately, but colicin production is delayed and induced only in terminally damaged cells. The cause of this delay is unknown. Here we identify the global transcription repressor, IscR, as being directly responsible for the delay in colicin K expression during the SOS response, and identify the DNA target for IscR at the colicin K operon promoter. Our results suggest that, IscR stabilizes LexA at the cka promoter after DNA damage thus, preventing its cleavage and inactivation, and this cooperation ensures that suicidal colicin K production is switched on only as a last resort. A similar mechanism operates at the regulatory region of other colicins and, hence, we suggest that many promoters that control the expression of ‘lethal’ genes are double locked.

Escherichia coli Bacteriocins: Antimicrobial Efficacy and Prevalence among Isolates from Patients with Bacteraemia

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin.