Association of virulence gene expression with colistin-resistance in Acinetobacter baumannii: analysis of genotype, antimicrobial susceptibility, and biofilm formation.

BACKGROUND: Acinetobacter baumannii causes difficult-to-treat nosocomial infections, which often lead to morbidity due to the development of antimicrobial drug resistance and expression of virulence genes. Data regarding the association of resistance to colistin, a last treatment option, and the virulence gene expression of A. baumannii is scarce. METHODS: We evaluated the MLVA genotype, antimicrobial resistance, and biofilm formation of 100 A. baumannii isolates from burn patients, and further compared the in vitro and in vivo expression of four virulence genes among five colistin-resistant A. baumannii (Cst-R-AB) isolates. Five Cst-R-AB isolates were tested; one from the present study, and four isolated previously. RESULTS: Our results showed that reduced expression of recA, along with increased in vivo expression of lpsB, dnaK, and blsA; are associated with colistin resistance among Cst-R-AB isolates. Differences in virulence gene expressions among Cst-R-AB isolates, may in part explain common discrepant in vitro vs. in vivo susceptibility data during treatment of infections caused by Cst-R-AB. CONCLUSIONS: Our findings highlight the intricate relationship between colistin-resistance and virulence among A. baumannii isolates, and underscore the importance of examining the interactions between virulence and antimicrobial resistance toward efforts to control the spread of multidrug-resistant A. baumannii (MDR-AB) isolates, and also to reduce disease severity in burn patients with MDR-AB infection.

Influences of epigallocatechin gallate and citric acid on Escherichia coli O157:H7 toxin gene expression and virulence-associated stress response.

Citric acid and EGCG at their minimum inhibitory concentrations were tested in this study. Logarithmic phase cells of Escherichia coli O157:H7 (ATCC 43895) were exposed to EGCG and citric acid respectively. The results of RT-real time PCR showed that both EGCG and citric acid increased stx2 and oxyR expression and decreased stx1, recA and Q expression. The result of Western blotting for RecA protein further indicated that both EGCG and citric acid decreased RecA production. Both EGCG and citric acid increased the level of intracellular reactive oxygen species and H2 O2 production and decreased superoxide dismutase activity. Therefore, EGCG and citric acid might induce stx2 production by increasing oxidative stress response and inhibit stx1 production by suppressing SOS response. In our study, the differential effects of the two antimicrobials were observed. EGCG reduced ompC and rpoS expression. However, citric acid caused an increase in ompC and rpoS expression. Membrane permeability is associated with toxin release. Citric acid increased the outer membrane permeability of E. coli O157:H7. However, the outer membrane of E. coli O157:H7 remained unaffected by EGCG. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxins are the major virulence factors of Escherichia coli O157:H7. The use of antimicrobials triggering Shiga toxin production is controversial. (-)-epigallocatechin-3-gallate (EGCG) citric acid are often used singly or in combination to prevent micro-organisms in some food products. This study evaluated toxin induction in E. coli O157:H7 in response to EGCG and citric acid and investigated the potential mechanism of action. The findings may contribute to the proper use of EGCG and citric acid as antimicrobials.

[The Engineering of a Yarrowia lipolytica Yeast Strain Capable of Homologous Recombination of the Mitochondrial Genome].

None of the studied eukaryotic species has a natural system for homologous recombination of the mitochondrial genome. We propose an integrated genetic construct pQ-SRUS, which allows introduction of the recA gene from Bacillus subtilis into the nuclear genome of an extremophilic yeast, Yarrowia lipolytica. The targeting of recombinant RecA to the yeast mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibits affinity to the outer mitochondrial membrane and thus provides cotranslational transport of RecA to the inner space of the mitochondria. The Y. lipolytica strain bearing the pQ-SRUS construct has the unique ability to integrate DNA constructs into the mitochondrial genome. This fact was confirmed using a tester construct, pQ-NIHN, intended for the introduction of the EYFP gene into the translation initiation region of the Y. lipolytica ND1 mitochondrial gene. The Y. lipolytica strain bearing pQ-SRUS makes it possible to engineer recombinant producers based on Y. lipolytica bearing transgenes in the mitochondrial genome. They are promising for the construction of a genetic system for in vivo replication and modification of the human mitochondrial genome. These strains may be used as a tool for the treatment of human mitochondrial diseases (including genetically inherited ones).

Kinetics of recA and recX induction in drug-susceptible and MDR clinical strains of Mycobacterium tuberculosis.

OBJECTIVES: To investigate and compare the expression of recA and recX, components of the SOS pathway, following rifampicin treatment in drug-susceptible and MDR clinical strains of Mycobacterium tuberculosis. METHODS: Strains (M. tuberculosis and Mycobacterium smegmatis) were subjected to rifampicin- and mitomycin-induced stress for 36 h followed by RNA extraction. recA and recX in the RNA extract were estimated using qRT-PCR. RESULTS: The MDR clinical strain induced faster (24 h) and higher (7-fold) levels of recA as compared with the drug-susceptible strain (36 h) in response to rifampicin. recX levels were found to rise with an increase in levels of recA; however, the levels were relatively higher than recA. CONCLUSIONS: Drug-susceptible and MDR strains have different kinetics of induction of DNA repair.

Opposing effects of aminocoumarins and fluoroquinolones on the SOS response and adaptability in Staphylococcus aureus.

OBJECTIVES: RecA is the key enzyme involved in DNA repair, recombination and induction of the SOS response and is central to the development of antibiotic resistance. Here we assessed the interaction of two different gyrase inhibitors, ciprofloxacin (a fluoroquinolone) and novobiocin (an aminocoumarin), on RecA activity and the SOS response in Staphylococcus aureus. METHODS: The influence of different gyrase inhibitors on the SOS response of S. aureus (including recA and lexA mutants) was analysed by northern blot analysis, real-time RT-PCR, western blot analysis and promoter activity assays. Recombination as well as mutation frequencies were determined for the different antibiotic combinations. RESULTS: We verified that ciprofloxacin leads to RecA activation and therefore induction of the SOS response. In contrast, novobiocin treatment resulted in an inhibition of recA transcription independent of LexA. When novobiocin and ciprofloxacin were added simultaneously, recA was reduced to the same level as with novobiocin alone. In combination, novobiocin also partially reduces the ciprofloxacin-mediated induction of the LexA target gene umuC (error-prone polymerase). Apart from reducing recA and umuC expression, novobiocin also inhibited the frequency of recombination, mutation and the formation of non-haemolytic variants. CONCLUSION: In summary, aminocoumarins inhibit recA expression in S. aureus and probably delay the process of developing antibiotic resistance and gene transfer. A clinical re-evaluation of these compounds as well as designing more applicable derivatives should be considered.

A novel endogenous induction of ColE7 expression in a csrA mutant of Escherichia coli.

Carbon storage regulator A (CsrA) is an important regulator that controls central metabolic pathways and a variety of physiological functions. We found that disruption of csrA in cells containing the ColE7 operon caused a 12-fold increase in colicin E7 production. Moreover, real-time RT-PCR demonstrated a decrease of around 50 % in the lexA mRNA of the csrA mutant. However, the cellular level of RecA protein and its mRNA were not significantly different from the wild type strain. Our results suggest that a novel induction mechanism might exist in E. coli that allows the expression of ColE7 operon in response to a metabolic shift. Proteomic analysis suggested that csrA deficient mutant may adapt PEP-glyoxylate cycle for energy production. Thus, the physiological changes in the csrA mutant may be similar to carbon source limitation for initiating the expression of ColE7 operon in response to stringent environmental conditions.

Stimulation of the Streptococcus pneumoniae RecA protein-promoted three-strand exchange reaction by the competence-specific SsbB protein.

The effect of the transformational competence-specific Streptococcus pneumoniae single-stranded DNA binding protein, SpSsbB, on the ATP-dependent three-strand exchange activity of the SpRecA protein was investigated. Although SpRecA exhibited only a trace level of strand exchange activity in the absence of SpSsbB, an extensive strand exchange reaction was observed when SpSsbB was added to the reaction solution after SpRecA. A more limited strand exchange reaction was observed, however, when SpSsbB was added to the reaction solution before SpRecA. This dependence on the order of addition, together with additional DNA-dependent ATP hydrolysis experiments, indicated that the mechanism of stimulation may involve the postsynaptic binding of SpSsbB to the displaced linear single-stranded DNA reaction product. When dATP was provided in place of ATP as the nucleotide cofactor (to suppress a potentially inhibitory effect of SpSsbB on the interaction of SpRecA with the circular ssDNA reaction substrate), the stimulatory effect of SpSsbB on the strand exchange reaction was apparent regardless of the order in which it was added to the reaction solution. These findings suggest that SpSsbB may be able to facilitate SpRecA-promoted DNA recombination reactions during natural transformation in S. pneumoniae.

Topoisomerase I function during Escherichia coli response to antibiotics and stress enhances cell killing from stabilization of its cleavage complex.

OBJECTIVES: To explore the role of topoisomerase I in gene activation and increased RecA levels during the bacterial SOS response, as well as the effect of antibiotic treatment and stress challenge on cell killing initiated by trapped topoisomerase I cleavage complex. METHODS: A mutant Escherichia coli strain with a ΔtopA mutation was used to investigate the role of topoisomerase I function in the SOS response to trimethoprim and mitomycin C. Induction of the recA and dinD1 promoters was measured using luciferase reporters of these promoters fused to luxCDABE. An increase in the RecA level following trimethoprim treatment was quantified directly by western blotting. The effect of stress challenge from trimethoprim and acidified nitrite treatments on cell killing by topoisomerase I cleavage complex accumulation was measured by the decrease in viability following induction of recombinant mutant topoisomerase I that forms a stabilized cleavage complex. RESULTS: Topoisomerase I function was found to be required for efficient transcriptional activation of the recA and dinD1 promoters during the E. coli SOS response to trimethoprim and mitomycin C. The role of topoisomerase I in the SOS response was confirmed with quantitative western blot analysis of RecA following trimethoprim treatment. The bactericidal effect from topoisomerase I cleavage complex accumulation was shown to be enhanced by stress challenge from trimethoprim and acidified nitrite. CONCLUSIONS: Bacterial topoisomerase I function is actively involved in the SOS response to antibiotics and stress challenge. Cell killing initiated by the topoisomerase I cleavage complex would be enhanced by antibiotics and the host response. These findings provide further support for bacterial topoisomerase I as a therapeutic target.

Overexpression of the LexA-regulated tisAB RNA in E. coli inhibits SOS functions; implications for regulation of the SOS response.

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage lambda and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene.

[Transgenic tomato plants expressing recA and NLS-recA-licBM3 genes as a model for studying meiotic recombination].

Homologous DNA recombination in eukaryotes is necessary to maintain genome stability and integrity and for correct chromosome segregation and formation of new haplotypes in meiosis. At the same time, genetic determination and nonrandomness of meiotic recombination restrict the introgression of genes and generation of unique genotypes. As one of the approaches to study and induce meiotic recombination in plants, it is recommended to use the recA gene of Escherichia coli. It is shown that the recA and NLS-recA-licBM3 genes have maternal inheritance and are expressed in the progeny of transgenic tomato plants. Plants expressing recA or NLS-recA-licBM3 and containing one T-DNA insertion do not differ in pollen fertility from original nontransgenic forms and can therefore be used for comparative studies of the effect of bacterial recombinases on meiotic recombination between linked genes.

[Expression of gene recA of Deinococcus radiodurans in Escherichia coli cells].

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of Deinococcus radiodurans recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations at genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 times and is essentially to the contribution of plasmid pUC 19-recA1.1 harboring the E. coli recA+ gene in the recovery of resistance of the deltarecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.

Precise temporal modulation in the response of the SOS DNA repair network in individual bacteria.

The SOS genetic network is responsible for the repair/bypass of DNA damage in bacterial cells. While the initial stages of the response have been well characterized, less is known about the dynamics of the response after induction and its shutoff. To address this, we followed the response of the SOS network in living individual Escherichia coli cells. The promoter activity (PA) of SOS genes was monitored using fluorescent protein-promoter fusions, with high temporal resolution, after ultraviolet irradiation activation. We find a temporal pattern of discrete activity peaks masked in studies of cell populations. The number of peaks increases, while their amplitude reaches saturation, as the damage level is increased. Peak timing is highly precise from cell to cell and is independent of the stage in the cell cycle at the time of damage. Evidence is presented for the involvement of the umuDC operon in maintaining the pattern of PA and its temporal precision, providing further evidence for the role UmuD cleavage plays in effecting a timed pause during the SOS response, as previously proposed. The modulations in PA we observe share many features in common with the oscillatory behavior recently observed in a mammalian DNA damage response. Our results, which reveal a hitherto unknown modulation of the SOS response, underscore the importance of carrying out dynamic measurements at the level of individual living cells in order to unravel how a natural genetic network operates at the systems level.

A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR.

Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes.

Sulfamethoxazole - Trimethoprim represses csgD but maintains virulence genes at 30°C in a clinical Escherichia coli O157:H7 isolate.

The high frequency of prophage insertions in the mlrA gene of clinical serotype O157:H7 isolates renders such strains deficient in csgD-dependent biofilm formation but prophage induction may restore certain mlrA properties. In this study we used transcriptomics to study the effect of high and low sulfamethoxazole-trimethoprim (SMX-TM) concentrations on prophage induction, biofilm regulation, and virulence gene expression in strain PA20 under environmental conditions following 5-hour and 12-hour exposures in broth or on agar. SMX-TM at a sub-lethal concentration induced strong RecA expression resulting in concentration- and time-dependent major transcriptional shifts with emphasis on up-regulation of genes within horizontally-transferred chromosomal regions (HTR). Neither high or low levels of SMX-TM stimulated csgD expression at either time point, but both levels resulted in slight repression. Full expression of Ler-dependent genes paralleled expression of group 1 pch homologues in the presence of high glrA. Finally, stx2 expression, which is strongly dependent on prophage induction, was enhanced at 12 hours but repressed at five hours, in spite of early SOS initiation by the high SMX-TM concentration. Our findings indicate that, similar to host conditions, exposure to environmental conditions increased the expression of virulence genes in a clinical isolate but genes involved in the protective biofilm response were repressed.

Analysis of the stress effects of endocrine disrupting chemicals (EDcs) on Escherichia coli.

In this study, three of the representative EDCs, 17beta-estradiol, bisphenol A, and styrene, were employed to find their mode of toxic actions in E. coli. To accomplish this, four different stress response genes, recA, katG, fabA, and grpE genes, were used as a representative for DNA, oxidative, membrane, or protein damage, respectively. The expression levels of these four genes were quantified using a real-time RT-PCR after challenge with three different EDCs individually. Bisphenol A and styrene caused high-level expression of recA and katG genes, respectively, whereas 17beta-estradiol made no significant changes in expression of any of those genes. These results lead to the classification of the mode of toxic actions of EDCs on E. coli.

Involvement of inorganic polyphosphate in expression of SOS genes.

Inorganic polyphosphate (poly(P)) is a linear polymer that has been found in every organism so far examined. To elucidate the functions of poly(P) in the regulation of gene expression, the level of cellular poly(P) in Escherichia coli was reduced to a barely detectable concentration by overproduction of exopolyphosphatase (exopoly(P)ase) with a plasmid encoding yeast exopoly(P)ase (Shiba et al., Proc. Natl. Acad. Sci. USA 94 (1997) 11210-11215). It was found that exopoly(P)ase-overproducing cells were more sensitive to UV or mitomycin C (MMC) than were control cells. Poly(P) accumulation was observed after treatment with MMC, whereas the poly(P) level was below the detectable level in cells that overproduced exopoly(P)ase. When exopoly(P)ase-overproducing cells were transformed again by a multiple copy number plasmid that carries the polyphosphate kinase gene (ppk), the cells accumulated a great amount of poly(P) and restored the UV and MMC sensitivities to the level of control cells. In exopoly(P)ase-overproducing cells, the expression of recA and umuDC were not induced by MMC. In addition, a strain containing multiple copies of ppk accumulated not only a large amount of poly(P) but also recA mRNA. Since recA expression was induced in a recA-deletion strain harboring a plasmid with the ppk gene, poly(P) could be necessary for regulating the expression of SOS genes without depending on the RecA-LexA regulatory network.

Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA.

We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional RecA protein to yield presynaptic filaments. Here, electron microscopy has been used to further explore the parameters of this assembly process. The optimal extent of presynaptic filament formation required at least one RecA protein monomer per three nucleotides, high concentrations of ATP (greater than 3 mM in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein assembly.

The curious case of protein splicing: mechanistic insights suggested by protein semisynthesis.

The gradual accumulation of examples of protein splicing, in which a nested intervening sequence is spliced out of the interior of a polyprotein precursor, suggests that this curious phenomenon might prove to have universal phylogenetic distribution and biological significance. The known examples are reviewed, with the aim of establishing underlying patterns, and a generalized mechanism of autocatalytic protein splicing is proposed. The testable consequences of such a proposal and the possible evolutionary origins of the phenomenon are discussed.

The Azotobacter vinelandii recA gene: sequence analysis and regulation of expression.

The nucleotide (nt) sequence of the Azotobacter vinelandii recA gene (Av-recA) was determined and compared with the recA sequences from Pseudomonas aeruginosa (Pa-recA), a soil bacterium, and Escherichia coli (Ec-recA), an enteric bacterium. The Av-recA gene and the deduced aa sequence were found to be more similar to their Pa-recA counterparts than to the Ec-recA gene and protein. Expression of Av-recA was found to be autoregulatory. Unlike Ec-recA and Pa-recA, however, expression of Av-recA was weakly enhanced upon DNA damage. In E. coli, expression of an Av-recA::lacZ fusion was poor, but its autoregulation was similar to that of Ec-recA. Av-recA expression, however, could not induce the repair system response in E. coli.

Sequencing, targeted mutagenesis and expression of a recA gene required for the extreme radioresistance of Deinococcus radiodurans.

Deinococcus radiodurans and other members of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. A DNA damage-sensitive and natural transformation-deficient strain generated by chemical mutagenesis (strain rec30) was found to be defective in a gene that has extended homology with recA of Escherichia coli. Upon transformation with a chromosomal DNA fragment that contained this deinococcal recA gene from wild-type (wt) D. radiodurans both DNA damage resistance and full transformation competence were restored in the rec30 mutant. Targeted insertional mutagenesis of the deinococcal recA gene was used to construct a mutant isogenic with the wt. The insertional mutant was phenotypically indistinguishable from strain rec30, indicating that the recA defect alone was responsible for observed phenotypic alterations. For example, in the case of ionizing radiation, the D37 of the wt was about 1.75 Mrad, while the D37 of rec30 and the insertional mutant were both 25 krad, a 70-fold decrease. Evidence is presented that expression of the deinococcal recA gene in E. coli is lethal, suggesting that the mode of interaction of the deinococcal RecA protein with nucleic acids or other cellular proteins differs at least in part from RecA of E. coli.