Effects of Mitomycin C on Macromolecular Synthesis in Escherichia coli

When cells of Escherichia coli B growing in a glucose-synthetic medium were treated with mitomycin C, the effects produced by the antibiotic varied, depending on the concentration. When the concentration was reduced to less than 0.1 mug/ml, the action of the antibiotic was bacteriostatic; cell elongation resulted, but no effect on the synthesis of cellular macromolecules was apparent. At higher levels (more than 5 mug/ml), mitomycin C was highly bactericidal and inhibited deoxyribonucleic acid synthesis almost completely. The exposure of growing cells to a bactericidal level of mitomycin C resulted also in a delayed inhibition of the synthesis of ribonucleic acid (RNA) and protein. The capacity of the treated cells to synthesize beta-galactosidase inducibly in a medium free from a carbon source remained constant for the first 30 min and then was destroyed progressively with time. Prolonged incubation with the bactericidal level of mitomycin C caused a degradation of cellular nucleic acids, particularly RNA. The degraded nucleic acid components were eventually released into the medium.     

Antimicrobial peptides (AMPs): peptide structure and mode of action

Antimicrobial peptides (AMPs) have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum. Their amino acid composition, amphipathicity, cationic charge, and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Although these models are helpful for defining mechanisms of AMP activity, their relevance to resolving how peptides damage and kill microorganisms still needs to be clarified. Moreover, many AMPs employ sophisticated and dynamic mechanisms of action to carry out their likely roles in antimicrobial host defense. Recently, it has been speculated that transmembrane pore formation is not the only mechanism of microbial killing by AMPs. In fact, several observations suggest that translocated AMPs can alter cytoplasmic membrane septum formation, reduce cell-wall, nucleic acid, and protein synthesis, and inhibit enzymatic activity. In this review, we present the structures of several AMPs as well as models of how AMPs induce pore formation. AMPs have received special attention as a possible alternative way to combat antibiotic-resistant bacterial strains. It may be possible to design synthetic AMPs with enhanced activity for microbial cells, especially those with antibiotic resistance, as well as synergistic effects with conventional antibiotic agents that lack cytotoxic or hemolytic activity.     

体细胞胚发生的生化基础

在胚性细胞分化和分裂过程中ATP酶活性和分布的动态变化表明,这些胚性细胞进行着旺盛的主动物质吸收和活跃的新陈代谢过程。在多种植物的体细胞胚发生中过氧化物酶的活性与同工酶的种类都高于对照,而且在大麦中发现过氧化物酶、酯酶和酸性磷酸酶同工酶的结合应用可以作为体细胞胚发生的标志酶。胚性愈伤组织中可溶性蛋白质含量与组分远高于或多于非胚性愈伤组织。大多数材料中都存在45kD-55kD的胚胎发生特异性蛋白质组分。而且在体细胞胚发生中蛋白质和核酸代谢动态呈规律性变化,首先是RNA合成速率增加,继而是蛋白质的迅速合成,并在胚性细胞分化和发育过程中一直保持相对较高水平,其中mRNA种类丰富,不同发育时期mRNA种类不同,因此转译形成多种蛋白质。DNA的代谢相对较稳定,但在胚性细胞系中DNA合成量仍高于非胚性细胞系。加入蛋白质或核酸合成抑制剂,不仅抑制了蛋白质和核酸的合成,同时也抑制了体细胞胚的发生与发育,而且抑制剂加和时间愈早,影响愈严重。由此表明,蛋白质与核酸的合成为体细胞胚的分化和发育奠定了分子基础。     

Mode of Action of Antibiotic U-24,544

Antibiotic U-24,544, a new antibacterial agent, was found to be an effective uncoupler of phosphorylation associated with the oxidation of glutamate and succinate in rat liver mitochondria. Respiration was inhibited during glutamate oxidation but not during succinate oxidation. In a medium deficient in inorganic phosphate, the agent showed slight stimulation of mitochondrial glutamate oxidation. Mitochondrial swelling induced by inorganic phosphate was suppressed. The antibiotic inhibited protein, nucleic acid, and cell wall synthesis in Mycobacterium avium cells nearly equally without a predominant inhibition of any one of these macromolecular biosynthetic processes. Nucleic acid and polypeptide synthesis remained unaffected, but respiration was inhibited in cell-free bacterial systems. It was thus concluded that the antibiotic interfered primarily with the cellular energy-generating processes.     

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.     

Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins

Daptomycin is a lipopeptide antibiotic used clinically for the treatment of certain types of Gram-positive infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Details of the mechanism of action of daptomycin continue to be elucidated, particularly the question of whether daptomycin acts on the cell membrane, the cell wall, or both. Here, we use fluorescence microscopy to directly visualize the interaction of daptomycin with the model Gram-positive bacterium Bacillus subtilis. We show that the first observable cellular effects are the formation of membrane distortions (patches of membrane) that precede cell death by more than 30 min. Membrane patches are able to recruit the essential cell division protein DivIVA. Recruitment of DivIVA correlates with membrane defects and changes in cell morphology, suggesting a localized alteration in the activity of enzymes involved in cell wall synthesis that could account for previously described effects of daptomycin on cell wall morphology and septation. Membrane defects colocalize with fluorescently labeled daptomycin, DivIVA, and fluorescent reporters of peptidoglycan biogenesis (Bocillin FL and BODIPY FL-vancomycin), suggesting that daptomycin plays a direct role in these events. Our results support a mechanism for daptomycin with a primary effect on cell membranes that in turn redirects the localization of proteins involved in cell division and cell wall synthesis, causing dramatic cell wall and membrane defects, which may ultimately lead to a breach in the cell membrane and cell death. These results help resolve the longstanding questions regarding the mechanism of action of this important class of antibiotics.     

Evidence for the lack of relationship between inhibition of nucleic acid synthesis and cytotoxicity of adriamycin

The ability of adriamycin-sensitive and -resistant Sarcoma 180 cells to incorporate thymidine and uridine into macromolecular material following exposure to this antibiotic was directly compared to the degree of cell survival by measuring in the same population precursor incorporation and cloning efficiency in soft agar after different intensities of drug exposure. The concentration and time dependence of inhibition of these processes by adriamycin wer compared. No correlation between the ability to incorporate radioactive precursors into DNA and RNA and the extent of cell survival was observed except ay very toxic drug concentrations. The results indicate that the extent of inhibition of precursor incorporation into DNA and RNA following drug exposure is not predictive of cell survival. This finding implies that the effect of adriamycin on nucleic acid synthesis is not directly coupled to cytotoxicity.     

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A-T and G-C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another. —Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G-C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A-T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A-T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G-C specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.—The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.     

Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell

Cell shape in most eubacteria is maintained by a tough external peptidoglycan cell wall. Recently, cell shape determining proteins of the MreB family were shown to form helical, actin-like cables in the cell. We used a fluorescent derivative of the antibiotic vancomycin as a probe for nascent peptidoglycan synthesis in unfixed cells of various Gram-positive bacteria. In the rod-shaped bacterium B. subtilis, synthesis of the cylindrical part of the cell wall occurs in a helical pattern governed by an MreB homolog, Mbl. However, a few rod-shaped bacteria have no MreB system. Here, a rod-like shape can be achieved by a completely different mechanism based on use of polar growth zones derived from the division machinery. These results provide insights into the diverse molecular strategies used by bacteria to control their cellular morphology, as well as suggesting ways in which these strategies may impact on growth rates and cell envelope structure.     

Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.     

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.     

Induction of autolysis in nongrowing Escherichia coli

Unless relaxation of the stringent response is achieved, all nongrowing bacteria rapidly develop resistance to autolysis induced by a variety of agents, including all classes of cell wall synthesis inhibitors. We now describe inhibitors of cell wall synthesis which were unusual in that they could continue to effectively induce autolysis in relA+ Escherichia coli even after prolonged amino acid starvation. The process of cell wall degradation seems to be catalyzed by similar hydrolytic enzymes in nongrowing and growing cells, yet the activity of these new agents capable of inducing autolysis in the nongrowing relA+ cells did not involve relaxation of RNA or peptidoglycan synthesis. We propose that the suppression of autolysis characteristic of nongrowing cells can be bypassed by a novel mechanism of autolytic triggering which is independent of the relA locus.     

Alginate gel microchip for real-time monitoring of intracellular processes in bacterial and yeast cells

A method of alginate-based hydrogel cell microchip manufacturing is proposed. The development of mild conditions for cell immobilization in microvolumes of non-toxic alginate gel allows extending the range of microorganisms used. Different approaches to cell analysis using microchip have been approved in pilot studies. By the example of Escherichia coli, Bordetella bronchiseptica and Saccharomyces cerevisiae it is shown that cell microchip can be successfully applied for monitoring of nucleic acid and protein synthesis in growing cells simultaneously using two fluorescent dyes. The influence of chloramphenicol on the nucleic acids and protein synthesis in five bacterial strains has been studied on the microchip. The microchip was also applied for the analysis of inducible fluorescent protein EGFP synthesis in E. coli cells, the correlation between the level of EGFP synthesis and concentration of the inductor in the medium has been established.     

Inhibition of Deoxyribonucleic Acid Synthesis and Bud Formation by Nalidixic Acid in Hyphomicrobium neptunium

The relationship between chromosome replication and morphogenesis in the budding bacterium Hyphomicrobium neptunium has been investigated. Nalidixic acid was found to completely inhibit deoxyribonucleic acid synthesis, but not ribonucleic acid synthesis. The antibiotic was bacteriostatic to the organism for the initial 5 h of exposure; thereafter it was bacteriocidal. Observation of inhibited cultures revealed cells that had produced abnormally long stalks, but no buds. These results indicate that bud formation is coupled to chromosome replication in H. neptunium. They do not exclude the possibilities that cross wall formation and bud separation may also be coupled to chromosome replication.     

Lysis of growing fissin-yeast cells induced by aculeacin A,a new antifungal antibiotic

Cells of Schizosaccharomyces pombe grown in the presence of aculeacin A, a peptide antibiotic, were lysed resulting the death of cells. Under high osmolarity, the cellular lysis induced by aculeacin A was considerably reduced. The use of synchronous-culture systems distinguished cell elongation from cell division revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis. Aculeacin A-resistant survivors exhibited morphological alterations which were swollen at one or both ends of the cell and appeared drumstick or dumbbel like; the wall of the bulge region was observed to be stained with a fluorescent brightener, as well as that of the cell plate region. These effects of aculeacin A are discussed as compared with effects of 2-deoxy-D-glucose.     

Ultrastructure of Staphylococcus in disruption of the integrity of the cell wall

The effects of penicillin, lithium chloride and homologous antiserum with complement on S. aureus after a single exposure to these agents and in subsequent subculturing were studied. The viability of the altered forms obtained in these experiments was evaluated by the number of colony-forming units per ml. The action of all above-mentioned agents resulted in the appearance of staphylococcal forms with the altered cell wall. The lesions in the submicroscopic organization of the cell wall, produced by the action of the above-mentioned agents, differed in the appearance of porosity and ruptures in the wall under the action of penicillin and antiserum, thinning and peeling-off of the wall under the action of lithium chloride. The damage of the cell wall is accompanied by the disorganized septal development and mitosis, and sometimes by the formation of elementary bodies in the cytoplasm.     

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming

Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting these rare events. Here, we describe a protocol to induce formation of bi-species mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies in vitro. This procedure can be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming.     

Molecular probes of DNA bulges: functional assay and spectroscopic analysis

Bulged structures in DNA and RNA have been linked to biomolecular processes involved in numerous diseases, thus probes with affinity for these nucleic acid targets would be of considerable utility to chemical biologists. Herein, we report guided chemical synthesis of small molecules capable of binding to DNA bulges by virtue of their unique (spirocyclic) geometry. The agents, modeled on a natural product congener, show pronounced selectivity for specific bulged motifs and are able to enhance slipped DNA synthesis, a hallmark functional assay of bulge binding. Significantly, bulge-agent complexes demonstrate characteristic fluorescent signatures depending on bulge and flanking sequence in the oligo. It is anticipated that these signature patterns can be harnessed as molecular probes of bulged hotspots in DNA and RNA.     

Inhibition by Chloramphenicol of Glucose Transfer in Teichoic Acid Biosynthesis

CHLORAMPHENICOL is widely accepted as a highly effective inhibitor of protein synthesis in bacteria, both in whole cells and at the subcellular level. Although some of the details of its mechanism of action are still unsettled, it has been shown to bind selectively to the 50S ribosomal subunit¹ and to inhibit peptide formation possibly by preventing binding between the ribosome and raRNA². It is well established that the bacteriostatic action of the antibiotic results from inhibition of protein synthesis and its use as a tool in the study of cellular biochemistry is frequently based on the view that its action is highly specific. In fact, there are reports of its inhibitory action on other cell processes³, but these are either relatively unimportant or the effects are only shown at concentrations of antibiotic much higher than those required for inhibition of protein synthesis. Anraku and Landman⁴ have reported that chloramphenicol inhibits a late stage in the reversion of protoplasts of Bacillus subtilis to the osmotically stable bacillary form and this is accompanied by inhibition of synthesis of a phosphorylated wall polymer believed to be a teichoic acid. It was suggested that inhibition of synthesis of wall polymers, including the teichoic acid, was an indirect effect arising from inhibition of synthesis of the appropriate enzyme proteins. We now report that chloramphenicol powerfully inhibits the biosynthesis of a wall teichoic acid in a cell-free system of fragmented cytoplasmic membrane from B. licheniformis ATCC 9945 (B. subtilis NCIB 8062); this occurs through direct action on the teichoic acid synthesizing system and is unrelated to protein synthesis. Although this may provide an alternative explanation of the effect observed by Anraku and Landman, a more detailed study of their system would be required.