Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The groE operon consists of two genes, groES and groEL; point mutations in either gene conferred the same phenotype, reducing Lac+ adaptive mutation 10- to 20-fold. groE mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In recG+ strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in recG mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.     

Differential epigenetic compatibility of qnr antibiotic resistance determinants with the chromosome of Escherichia coli

Environmental bacteria harbor a plethora of genes that, upon their horizontal transfer to new hosts, may confer resistance to antibiotics, although the number of such determinants actually acquired by pathogenic bacteria is very low. The founder effect, fitness costs and ecological connectivity all influence the chances of resistance transfer being successful. We examined the importance of these bottlenecks using the family of quinolone resistance determinants Qnr. The results indicate the epigenetic compatibility of a determinant with the host genome to be of great importance in the acquisition and spread of resistance. A plasmid carrying the widely distributed QnrA determinant was stable in Escherichia coli, whereas the SmQnr determinant was unstable despite both proteins having very similar tertiary structures. This indicates that the fitness costs associated with the acquisition of antibiotic resistance may not derive from a non-specific metabolic burden, but from the acquired gene causing specific changes in bacterial metabolic and regulatory networks. The observed stabilization of the plasmid encoding SmQnr by chromosomal mutations, including a mutant lacking the global regulator H-NS, reinforces this idea. Since quinolones are synthetic antibiotics, and since the origin of QnrA is the environmental bacterium Shewanella algae, the role of QnrA in this organism is unlikely to be that of conferring resistance. Its evolution toward this may have occurred through mutations or because of an environmental change (exaptation). The present results indicate that the chromosomally encoded Qnr determinants of S. algae can confer quinolone resistance upon their transfer to E. coli without the need of any further mutation. These results suggest that exaptation is important in the evolution of antibiotic resistance.     

Baicalein suppresses the SOS response system of Staphylococcus aureus induced by ciprofloxacin

Numerous antibiotics can induce an SOS repair system in bacteria that leads to antibiotic-resistant mutation of the bacterium. Therefore, searching for drugs that can prevent the SOS response and thus improve the long-term viability of some antibiotics is important. In this study, we aimed to detect the suppressive effects of baicalein on the SOS system and rifampin-resistant mutation in Staphylococcus aureus. We determined the reactive oxygen species (ROS) formation and intracellular ATP level in S. aureus with baicalein treatment to investigate the mechanisms involved in its effects on the SOS system. The results showed that baicalein was a potent inhibitor of the expression of the SOS genes RecA, LexA and SACOL1400. The rifampin-resistant mutation rate of S. aureus induced by ciprofloxacin was significantly reduced after treatment with baicalein. Treatment with baicalein led to a significant decrease in intracellular reactive oxygen species (ROS) formation and ATP level. Our findings indicate that baicalein may be an SOS-response inhibitor in S. aureus through inhibiting ROS formation and ATP production and may be used to prevent excessive mutation induced by antibiotics.     

细菌DNA损伤修复的诱导、调控及结局的研究进展

DNA损伤修复(SOS反应)是细菌适应环境、抵抗外界压力和修复自身损伤的重要机制.为了解SOS反应的过程,全面揭示细菌生存机制,本研究对DNA损伤修复的过程、调节及适应性变化进行文献综述.结果 表明,内源和外源的诸多压力都可以激活SOS反应,抗生素是激活该反应的主要因素.RecA在感知外界压力和系统启动过程中发挥重要作...     

Lex marks the spot: the virulent side of SOS and a closer look at the LexA regulon

The SOS response that responds to DNA damage induces many genes that are under LexA repression. A detailed examination of LexA regulons using genome-wide techniques has recently been undertaken in both Escherichia coli and Bacillus subtilis. These extensive and elegant studies have now charted the extent of the LexA regulons, uncovered many new genes, and exposed a limited overlap in the LexA regulon between the two bacteria. As more bacterial genomes are analysed, more curiosities in LexA regulons arise. Several notable examples include the discovery of a LexA-like protein, HdiR, in Lactococcus lactis, organisms with two lexA genes, and small DNA damage-inducible cassettes under LexA control. In the cyanobacterium Synechocystis, genetic and microarray studies demonstrated that a LexA paralogue exerts control over an entirely different set of carbon-controlled genes and is crucial to cells facing carbon starvation. An examination of SOS induction evoked by common therapeutic drugs has shed new light on unsuspected consequences of drug exposure. Certain antibiotics, most notably fluoroquinolones such as ciprofloxacin, can induce an SOS response and can modulate the spread of virulence factors and drug resistance. SOS induction by beta-lactams in E. coli triggers a novel form of antibiotic defence that involves cell wall stress and signal transduction by the DpiAB two-component system. In this review, we provide an overview of these new directions in SOS and LexA research with emphasis on a few themes: identification of genes under LexA control, the identification of new endogenous triggers, and antibiotic-induced SOS response and its consequences.     

The increased sensitivity of rifamycin-resistant mutants of Methylobacterium AM1 to a variety of antimicrobial agents

Rifamycin-resistant strains of Methylobacterium AM1 are more sensitive to β-lactam antibiotics, chloramphenicol, tetracycline, spectinomycin, puromycin, nalidixic acid, benzalkonium chloride and p -fluorophenylalanine than are rifamycin-sensitive strains. This suggests that mutation to rifamycin resistance is associated with a general increase in membrane permeability and may have a bearing on the use of rifamycin-resistant mutants in genetic and biochemical studies of this organism.     

Multidrug therapy and evolution of antibiotic resistance: when order matters

The evolution of drug resistance among pathogenic bacteria has led public health workers to rely increasingly on multidrug therapy to treat infections. Here, we compare the efficacy of combination therapy (i.e., using two antibiotics simultaneously) and sequential therapy (i.e., switching two antibiotics) in minimizing the evolution of multidrug resistance. Using in vitro experiments, we show that the sequential use of two antibiotics against Pseudomonas aeruginosa can slow down the evolution of multiple-drug resistance when the two antibiotics are used in a specific order. A simple population dynamics model reveals that using an antibiotic associated with high costs of resistance first minimizes the chance of multidrug resistance evolution during sequential therapy under limited mutation supply rate. As well as presenting a novel approach to multidrug therapy, this work shows that costs of resistance not only influences the persistence of antibiotic-resistant bacteria but also plays an important role in the emergence of resistance.     

Impact of mutation rate on the adaptation of gut bacteria

To study the role of mutator bacteria in the evolution of bacterial populations, we followed the impact of the mutation rate of Escherichia coli strains in the colonisation of the gut of axenic mice and the evolution of the mutation rate of bacterial populations living in the gut. We show that mutator bacteria have an advantage during the colonization. This adaptive advantage comes from their ability to generate adaptive mutations faster than wild type strains, mutations that allow their maintenance in the ecosystem. However, while mutator bacteria are becoming specialised to the environment they are living in, they accumulate mutations that may be deleterious or lethal in secondary environments. By following the evolution of the mutation rate of bacterial populations living in the gut of mice receiving antibiotics, we show that this therapy selects not only for antibiotic resistant mutants but also for mutator alleles that enhance mutation rates and are responsible for the appearance of the resistance. The costs of a high mutation rate, due to the accumulation of mutations, is seen in environments where changes are recurrent. In an ever-changing situation where every change is new, mutator bacteria might help the evolution of bacterial populations.     

Biological Cost of Pyocin Production during the SOS Response in Pseudomonas aeruginosa

LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs.     

DNA Gyrase Is the Target for the Quinolone Drug Ciprofloxacin in Arabidopsis thaliana

The Arabidopsis thaliana genome contains four genes that were originally annotated as potentially encoding DNA gyrase: ATGYRA, ATGYRB1, ATGYRB2, and ATGYRB3. Although we subsequently showed that ATGYRB3 does not encode a gyrase subunit, the other three genes potentially encode subunits of a plant gyrase. We also showed evidence for the existence of supercoiling activity in A. thaliana and that the plant is sensitive to quinolone and aminocoumarin antibiotics, compounds that target DNA gyrase in bacteria. However, it was not possible at that time to show whether the A. thaliana genes encoded an active gyrase enzyme, nor whether that enzyme is indeed the target for the quinolone and aminocoumarin antibiotics. Here we show that an A. thaliana mutant resistant to the quinolone drug ciprofloxacin has a point mutation in ATGYRA. Moreover we show that, as in bacteria, the quinolone-sensitive (wild-type) allele is dominant to the resistant gene. Further we have heterologously expressed ATGYRA and ATGYRB2 in a baculovirus expression system and shown supercoiling activity of the partially purified enzyme. Expression/purification of the quinolone-resistant A. thaliana gyrase yields active enzyme that is resistant to ciprofloxacin. Taken together these experiments now show unequivocally that A. thaliana encodes an organelle-targeted DNA gyrase that is the target of the quinolone drug ciprofloxacin; this has important consequences for plant physiology and the development of herbicides.     

mucA基因突变的黏液型铜绿假单胞菌PA17与PD0300生物被膜形态观察和耐药性比较

目的研究新的mucA基因缺失突变的黏液型铜绿假单胞菌PA17和经典mucA基因点突变的黏液型铜绿假单胞菌PD0300在生物被膜状态下对临床常用抗菌药物的耐药性变化,探讨mucA基因突变对铜绿假单胞菌生物被膜形态及细菌耐药性的影响。方法改良的平板法建立生物被膜,将含绿色荧光蛋白的pUCP／UV质粒转化两株铜绿假单胞菌,激光共聚焦显微镜下观察生物被膜形态;采用琼脂二倍稀释法测定常用β-内酰胺类,氨基甙类,喹诺酮类抗菌药物对铜绿假单胞菌菌株PA17、PD0300的最低抑菌浓度（MIC）;利用96孔板建立生物被膜测定抗菌药物对第五天成熟生物被膜内细菌的最低杀菌浓度（MBC）。结果新的mucA基因缺失突变的黏液型PA17成熟生物被膜呈薄膜状、经典mucA基因点突变的黏液型PD0300成熟生物被膜呈蘑菇状;在浮游状态下PA17、PD0300对头孢他啶（CAZ）、妥布霉素（TOB）、庆大霉素（GEN）、亚胺培南（IPM）敏感,而对左氧氟沙星（LVX）、环丙沙星（CIP）不敏感,两者具有一致的耐药性;生物被膜状态下两者对抗菌药物敏感性降低20—8000倍;黏液型PD0300成熟生物被膜对抗菌药物敏感性低于黏液型PA17。结论黏液型铜绿假单胞菌在相同条件下能形成不同形态的生物被膜;生物被膜状态下较浮游状态下对常用抗菌药物敏感性明显下降,同时生物被膜形态也影响抗菌药物敏感性。     

Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda

Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83 --> Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83 --> Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42 degrees C.     

Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein.

Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The LexA repressor, which controls the expression of the sfiA gene, was present in hupA hupB mutant bacteria in concentrations half of those of the parent bacteria, but this decrease was independent of the specific cleavage of the LexA repressor by activated RecA protein. One possibility to account for the filamentous morphology of hupA hupB mutant bacteria is that the lack of HU protein alters the expression of specific genes, such as lexA and fts cell division genes.     

LexA-independent expression of a mutant mucAB operon.

pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene products of which are required for the SOS-dependent processing of damaged DNA necessary for most mutagenesis. Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit, with LexA acting as a direct repressor. pGW16 is a pKM101 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increase in spontaneous mutation rate. In this report, we show that pGW16 differs from pKM101 in being able to enhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3 host. The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includes the mucAB coding region and approximately 0.6 kb of the 5'-flanking region. We determined the sequence of a 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in that region, a G.C-to-A.T transition that alters a sequence homologous to known LexA-binding sites. DNA gel shift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation, whereas a fragment containing the wild-type sequence is efficiently bound by LexA. This mutation also alters an overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closer to the consensus sequence. The observation that the synthesis of pGW16-encoded mucAB proteins in maxicells is increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmid suggests that this mutation also increases the activity of the mucAB promoter.