Cellular response to horizontally transferred DNA in Escherichia coli is tuned by DNA repair systems

We studied how DNA divergence between recombining DNAs and the mismatch repair system modulate the SOS response in Escherichia coli. The observed positive log-linear correlation between SOS induction and DNA divergence, and the negative correlation between SOS induction and frequency of recombination, suggest that the level of SOS induction precisely reflects the difficulty of RecA protein to initiate a productive strand exchange process. Our results suggest that the mismatch repair system could contribute to this SOS induction more by affecting the RecA-catalyzed homology search than by acting on mismatched recombination intermediates. The propensity of the recombination machinery to promote recombination between the blocks of sequences with the highest identity results in the increasing ratios of merodiploids (partial diploids) over genuine recombinants (homologous replacements) with increasing DNA divergence. We discuss the role of molecular mechanisms involved in the control of the recombination between diverged DNA sequences in the maintenance of genomic stability and genome evolution.     

The rise and fall of mutator bacteria

Bacteria with elevated mutation rates are frequently found among natural isolates. This is probably because of their ability to generate genetic variability, the substrate for natural selection. However, such high mutation rates can lead to the loss of vital functions. The evolution of bacterial populations may happen through alternating periods of high and low mutation rates. The cost and benefits of high mutation rates in the course of bacterial adaptive evolution are reviewed.     

Posttranslational Modification of 6-Phosphofructo-1-Kinase in Aspergillus niger

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the V_max of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.     

Design,synthesis and biological evaluation of 4,5-dibromo-N-(thiazol-2-yl)-1H-pyrrole-2-carboxamide derivatives as novel DNA gyrase inhibitors

Development of novel DNA gyrase B inhibitors is an important field of antibacterial drug discovery whose aim is to introduce a more effective representative of this mechanistic class into the clinic. In the present study, two new series of Escherichia coli DNA gyrase inhibitors bearing the 4,5-dibromopyrrolamide moiety have been designed and synthesized. 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazole-2,6-diamine derivatives inhibited E. coli DNA gyrase in the submicromolar to low micromolar range (IC₅₀ values between 0.891 and 10.4 μM). Their “ring-opened” analogues, based on the 2-(2-aminothiazol-4-yl)acetic acid scaffold, displayed weaker DNA gyrase inhibition with IC₅₀ values between 15.9 and 169 μM. Molecular docking experiments were conducted to study the binding modes of inhibitors.     

High Frequency of Mutator Strains among Human Uropathogenic Escherichia coli Isolates

By using a panel of 603 commensal and pathogenic Escherichia coli and Shigella isolates, we showed that mutation rates of strains vary considerably among different ecotypes. Uropathogenic strains had the highest frequency of mutators, while strains from patients with bacteremia had the lowest mutation rates. No correlation between the mutation rates and antibiotic resistance was observed among the studied strains.     

Specificity of reversible ADP-ribosylation and regulation of cellular processes

Proper and timely regulation of cellular processes is fundamental to the overall health and viability of organisms across all kingdoms of life. Thus, organisms have evolved multiple highly dynamic and complex biochemical signaling cascades in order to adapt and survive diverse challenges. One such method of conferring rapid adaptation is the addition or removal of reversible modifications of different chemical groups onto macromolecules which in turn induce the appropriate downstream outcome. ADP-ribosylation, the addition of ADP-ribose (ADPr) groups, represents one of these highly conserved signaling chemicals. Herein we outline the writers, erasers and readers of ADP-ribosylation and dip into the multitude of cellular processes they have been implicated in. We also review what we currently know on how specificity of activity is ensured for this important modification.     

MCM9 Mutations Are Associated with Ovarian Failure,Short Stature,and Chromosomal Instability

Premature ovarian failure (POF) is genetically heterogeneous and manifests as hypergonadotropic hypogonadism either as part of a syndrome or in isolation. We studied two unrelated consanguineous families with daughters exhibiting primary amenorrhea, short stature, and a 46,XX karyotype. A combination of SNP arrays, comparative genomic hybridization arrays, and whole-exome sequencing analyses identified homozygous pathogenic variants in MCM9, a gene implicated in homologous recombination and repair of double-stranded DNA breaks. In one family, the MCM9 c.1732+2T>C variant alters a splice donor site, resulting in abnormal alternative splicing and truncated forms of MCM9 that are unable to be recruited to sites of DNA damage. In the second family, MCM9 c.394C>T (p.Arg132^∗) results in a predicted loss of functional MCM9. Repair of chromosome breaks was impaired in lymphocytes from affected, but not unaffected, females in both families, consistent with MCM9 function in homologous recombination. Autosomal-recessive variants in MCM9 cause a genomic-instability syndrome associated with hypergonadotropic hypogonadism and short stature. Preferential sensitivity of germline meiosis to MCM9 functional deficiency and compromised DNA repair in the somatic component most likely account for the ovarian failure and short stature.     

Reliable Detection of Dead Microbial Cells by Using Fluorescent Hydrazides

We have developed a new method for accurate quantification of dead microbial cells. This technique employs the simultaneous use of fluorescent hydrazides and nucleic acid dyes. Fluorescent hydrazides allow detection of cells that cannot be detected with currently used high-affinity nucleic acid dyes. This is particularly important for nongrowing bacterial populations and for multicellular communities containing physiologically heterogeneous cell populations, such as colonies and biofilms.Many different approaches are used to assess the viability of bacterial cells. Currently, the most-used method is determination of the number of CFU, which reflects the ability of cells to reproduce. However, this method is not reliable because a fraction of live bacterial cells cannot divide under standard growth conditions (2) and because some bacteria are killed due to oxidative stress that occurs upon plating (3). Another approach detects dead cells through staining with fluorescent dyes that have high affinities for nucleic acids (RNA and DNA). Examples of this class of dye include Sytox Green (SG) (7). Cells incorporating these stains are then identified using flow cytometry or fluorescence microscopy. Discrimination between live and dead cells is based on the selective entry of dyes such as SG into dead cells, whose membrane integrity is compromised. However, the accessibility of DNA to SG staining also depends on the bacterial cell cycle stage. Cells that die during stationary phase are poorly stained by SG, probably because the DNA topology is altered (7). In addition, it is not possible to detect dead cells whose nucleic acids are degraded.In order to detect dead cells that escape detection by the methods described above, we used an Alexa Fluor hydrazide (AFH) dye. AFH dyes are low-molecular-weight, bright, photostable fluorescent molecules that are generally used as cell tracers by microinjection into eukaryotic cells. Because hydrazine components interact with carbonyl groups (aldehydes and ketones), AFH can be used for the detection of carbonylated proteins (1, 10). The quantity of carbonylated proteins, which are irreversibly damaged, increases after various lethal stresses such as oxidative stress, heat shock, and acidic stress (4). In stationary phase, bacteria also accumulate carbonylated proteins (5). As AFH cannot pass freely across the functional membranes of living cells, we hypothesized that it can be used for the detection of dead cells by tagging carbonylated proteins even when cells are devoid of nucleic acids. For the development of this method, we used Escherichia coli as a model organism.We first compared the results of staining E. coli cells killed by heat treatment with SG and Alexa Fluor 633 hydrazide (AF633H; both purchased from Invitrogen, Carlsbad, CA). Cells from 1- and 15-day-old liquid cultures were killed by incubation at 95°C for 10 min, which reduced the CFU counts in the cultures from approximately 2 × 10⁸/ml to undetectable levels (below 10 CFU/ml). Aliquots of cells taken before and after heat treatment were stained with the two dyes and analyzed with a FACSAria cell sorter and flow cytometer (Becton Dickinson Biosciences, San Jose, CA) (Fig. ​(Fig.1).1). Among cells from the 1-day-old cultures, less than 1% collected before treatment and 99.5% collected after treatment were stained with the two dyes. The 15-day cultures contained large proportions of dead cells even before treatment, as indicated by a decrease in the CFU count from approximately 2 × 10⁹/ml on day 1 to 1 × 10⁸/ml on day 15 (a reduction of 95%). In 15-day cultures, 26 and 89% of cells before heat treatment were stained with SG and AF633H dyes, respectively. After heat treatment, CFU levels were again undetectable, and 99.9% of cells from 15-day cultures were stained with AF633H, while only 36% were stained with SG. This difference in staining of dead cells by SG and AF633H indicates that the ability of dead cells to be stained by SG decreases dramatically with cell age but that the staining of dead cells by AF633H increases with cell age. This may be due at least partly to the fact that AF633H can stain cells devoid of nucleic acids while SG cannot. Reconstruction experiments in which heat-treated and untreated cells from 1- and 15-day-old cultures were mixed in fixed ratios and stained by the two dyes gave expected values (Table ​(Table1),1), showing that AF633H can be used for precise quantification of dead cells. In addition, we observed that AF633H staining remained stable after cell fixation with paraformaldehyde (see Fig. S1 in the supplemental material).Open in a separate windowFIG. 1.Levels of fluorescence of heat-killed microbial cells stained by AF633H and SG are indicated by fluorescence intensity histograms for untreated microbial cells (white histograms) and heat-treated cells (brown histograms). Cells were stained with AF633H (left panels) and SG (right panels). E. coli (MG1665) cells were from 1- and 15-day-old cultures. D. radiodurans, B. subtilis, and S. cerevisiae cells were from 1-day-old liquid cultures. The presence of two peaks of fluorescence for cells stained with SG probably indicates the existence of cells with different numbers of copies of the chromosome.

TABLE 1.

Detection of dead E. coli cells in liquid cultures by AF633H and SG staining^a

Citrate Inhibition-Resistant Form of 6-Phosphofructo-1-Kinase from Aspergillus niger

Two forms of Aspergillus niger 6-phosphofructo-1-kinase (PFK1) have been described recently, the 85-kDa native enzyme and 49-kDa shorter fragment that is formed from the former by posttranslational modification. So far, kinetic characteristics have never been determined on the enzyme purified to near homogeneity. For the first time, kinetic parameters were determined for individual enzymes with respect to citrate inhibition. The native 85-kDa enzyme was found to be moderately inhibited by citrate, with the K_i value determined to be 1.5 mM, in the system with 5 mM Mg²⁺ ions, while increasing magnesium concentrations relieved the negative effect of citrate. An identical inhibition coefficient was determined also in the presence of ammonium ions, although ammonium acted as a strong activator of enzyme activity. On the other hand, the shorter fragment of PFK1 proved to be completely resistant to inhibition by citrate. Allosteric citrate binding sites were most probably lost after the truncation of the C-terminal part of the native protein, in which region some binding sites for inhibitor are known to be located. At near physiological conditions, characterized by low fructose-6-phosphate concentrations, a much higher efficiency of the shorter fragment was observed during an in vitro experiment. Since the enzyme became more susceptible to the positive control by specific ligands, while the negative control was lost after posttranslational modification, the shorter PFK1 fragment seems to be the enzyme most responsible for generating undisturbed metabolic flow through glycolysis in A. niger cells.