Biochemistry and genetics of chloramphenicol production

In Streptomyces venezuelae, chloramphenicol is derived by an unusual diversion of chorismate, the branchpoint intermediate of the pathway involved in the biosynthesis of aromatic amino acids. In the chloramphenicol-producing organism, the DAHP synthetase was neither feedback inhibited nor repressed. Chorismate mutase was not repressed or inhibited by the intermediates or end-products of the shikimate-chorismate pathway. However, anthranilate synthetase and prephenate dehydratase are feedback inhibited by tryptophan and phenylalanine, respectively. During growth, when primary metabolism is not perfectly coordinated, decreasing demand for aromatic amino acids results in shunting of chorismate towards chloramphenicol biosynthesis.The endogenous synthesis of chloramphenicol produced by Streptomyces venezuelae is inhibited by the increasing concentration of chloramphenicol in the medium. Arylamine synthetase, the first enzyme involved in chloramphenicol biosynthesis, is repressed by the secreted chloramphenicol, by dl-p-aminophenylalanine and l-threo-p-aminophenylserinol. The excess intracellular chorismate pool is diverted to other aromatic shunt metabolites if biosynthesis of chloramphenicol is inhibited. There appears to be a glutamine binding protein subunit which is shared by several enzymes involved in amination of the aromatic ring of chorismate.Chloramphenicol producing organism also inactivated intracellular chloramphenicol. However, the resistance of the streptomycetes is due to inducible impermeability of the organism to chloramphenicol during antibiotic production. Streptomyces venezuelae is sensitive to chloramphenicol when it is not engaged in antibiotic production. The resistance to and production of chloramphenicol are induced simultaneously.A linkage map for 17 marker loci using Streptomyces venezuelae has been constructed. Restriction enzyme map of a plasmid from the chloramphenicol-producing streptomycetes has also been developed. The role of the plasmid in chloramphenicol biosynthesis and the life-cycle of the Streptomyces venezuelae is not yet understood.     

Trial of Chloramphenicol for Meningitis in Northern Savanna of Africa

In a controlled trial chloramphenicol proved as effective and much cheaper than penicillin for the treatment of group A meningococcal meningitis in Zaria, Nigeria. A short course of five days cured most patients. Adults and older children were soon able to take chloramphenicol by mouth, which reduced the cost and simplified treatment.It is suggested that chloramphenicol is a suitable alternative to sulphonamides for the treatment of meningococcal meningitis in those parts of Africa where the organism is sulphonamide-resistant.     

Effects of addition of chloramphenicol on the growth and ultrastructure of Streptomyces venezuelae

The effects of adding chloramphenicol before inoculation and during exponential growth of Streptomyces venezuelae (3022a) in fermentors were studied. The responses of the organism during synthesis of chloramphenicol (in a glycerol-serine-lactate medium) were compared with those in media supporting less (glycerol-nutrient broth-yeast extract) or no synthesis (glucosemineral salts). In systems where little or no synthesis of the chloramphenicol occurred, addition of the antibiotic induced micromorphological and ultrastructural abnormalities similar to those reported for sensitive bacteria. There was also an increase in the frequency of mesosomes and electron-light areas. It was suggested that the former may be associated with activity of chloramphenicol hydrolase and the latter with storage and/or excretion of the breakdown product; N-acetyl p-nitro-phenylserinol. When chloramphenicol synthesis occurred, addition of the antibiotic had less effect on the micromorphology or ultrastructure of S. venezuelae as permeability barriers to external chloramphenicol had been established. Electron-light areas were frequent, possibly being associated with storage and excretion of precursors of chloramphenicol.     

Action of hydroxyurea on Anabaena variabilis

Summary Low concentrations of hydroxyurea stimulated the growth of the blue-green alga Anabaena variabilis that had been pretreated with sublethal concentrations of chloramphenicol or of certain nucleic acid base analogues. When supplemented to the culture medium, hydroxyurea also counteracted the growth inhibitory effect of chloramphenicol on this organism. In contrast, when A. variabilis cells grown in the presence of hydroxyurea were subsequently treated with chloramphenicol, they were found to have become highly susceptible to the growth inhibitory effects of chloramphenicol. The growth of hydroxyurea pretreated cells in basal medium was attended by a lag that was shorter than that of untreated controls; on the other hand, when hydroxyurea pretreated cells were inoculated into chloramphenicol-supplemented medium, they exhibited a longer lag than that shown by untreated cells in chloramphenicol.The results obtained are discussed in terms of the probable effects of hydroxyurea and chloramphenicol on certain enzyme systems.     

Naturally occurring antibiotic resistance inBacillus sphaericus andBacillus licheniformis

Bacillus sphaericus is an aerobic, spore-forming, gram-variable bacillus. Certain strains of this organism are extremely pathogenic for mosquito larvae. Strains from four different serotypes ofB. sphaericus were found to be naturally resistant to streptomycin and to chloramphenicol. Both entomocidal and nonentomocidal strains of this organism exhibited similar patterns of resistance to these antimicrobial agents. In contrast, other members of the genusBacillus, includingB. subtilis, B. thuringiensis, B. laterosporus, andB. amyloliquefaciens, proved to be quite sensitive to these antibiotics. Four strains ofB. licheniformis were found to be resistant to chloramphenicol but sensitive to streptomycin.     

Construction and characterization of plasmid vectors for cloning in the entomocidal organism Bacillus sphaericus 1593

A plasmid vector for cloning in Bacillus sphaericus 1593 was constructed in B. subtilis from two parent plasmids, pBC16 and pBD64. When characterized, the 3.9-MDa chimeric plasmid pNN101 was found to consist of the MspI fragment containing the chloramphenicol acetyltransferase (CAT) gene from pBD64 inserted into an MspI site in pBC16. pNN101 was shown to replicate, express, and be stably maintained in B. sphaericus 1593 without affecting the mosquito larvicidal activity of this organism. A derivative of this plasmid, pNN302, was constructed in which a unique HindIII site was introduced into the CAT gene without loss of chloramphenicol resistance.     

Relationship between Dimorphology and Respiration in Mucor genevensis Studied with Chloramphenicol

Growth of Mucor genevensis, a facultatively anaerobic dimorphic mold, in high concentrations of chloramphenicol (4 mg/ml) leads to increased numbers of yeast-like cells and small club-like mycelial forms. This change in morphology is accompanied by a threefold increase in the mass doubling time, the loss of cyanide-sensitive respiration, and the development of cyanide-insensitive respiration. Associated with these changes is the absence of cytochromes aa(3) and b and the inability of the organism to utilize ethanol; in addition, mitochondria appear more numerous and have less internal membrane. A further inhibitory action of the antibiotic, other than eliminating functional mitochondria, appears likely since microaerobic cultures which lack respiratory ability have twice the mass doubling time in the presence of the drug. Although a small inhibition of amino acid incorporation by cytoplasmic ribosomes is found with a high chloramphenicol concentration, it is insufficient to account for the effect on growth of the microaerobic culture. The nature of this additional effect of chloramphenicol remains to be determined, but it has been shown that increasing the glucose concentration can partially reverse this action of the antibiotic. The effect of the drug on the morphology of the organism is not as dramatic as that of phenethyl alcohol in producing yeast-like forms. However, in view of the action of chloramphenicol in eliminating functional mitochondria in M. genevensis the suggestion that phenethyl alcohol exerts its effect in promoting yeast-like morphology by uncoupling oxidative phosphorylation should be re-examined.     

The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture

A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain.     

Specificity and mechanism of tetracycline resistance in a multiple drug resistant strain of Escherichia coli

Izaki, Kazuo (University of Tokyo, Tokyo, Japan), Kan Kiuchi, and Kei Arima. Specificity and mechanism of tetracycline resistance in a multiple drug resistant strain of Escherichia coli. J. Bacteriol. 91:628-633. 1966.-A decrease in the uptake of tetracycline occurred concurrently with a rise in the level of resistance of a multiple drug resistant strain of Escherichia coli grown in the presence of tetracycline. Although the strain was also resistant to streptomycin and chloramphenicol, growth in the presence of these two antibiotics did not influence the uptake of tetracycline. The induction of resistance, or decreased uptake of tetracycline, was dependent on growth of the organism in the presence of the drug. Decreased uptake of tetracycline could not be induced in a sensitive strain of the same organism under conditions suitable for induction of the resistant strain. The decrease in accumulating power of the resistant organism cultured in the presence of tetracycline does not appear to be due to selection of a resistant strain from cultures containing both resistant and sensitive strains.     

Transformation of the <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis> chloroplast genome: prospects for understanding chloroplast function in extreme environments

Key message

We have successfully transformed an exthemophilic red alga with the chloramphenicol acetyltransferase gene, rendering this organism insensitive to its toxicity. Our work paves the way to further work with this new modelorganism.

Abstract

Here we report the first successful attempt to achieve a stable, under selectable pressure, chloroplast transformation in Cyanidioschizon merolae—an extremophilic red alga of increasing importance as a new model organism. The following protocol takes advantage of a double homologous recombination phenomenon in the chloroplast, allowing to introduce an exogenous, selectable gene. For that purpose, we decided to use chloramphenicol acetyltransferase (CAT), as chloroplasts are particularly vulnerable to chloramphenicol lethal effects (Zienkiewicz et al. in Protoplasma, 2015, doi: 10.1007/s00709-015-0936-9). We adjusted two methods of DNA delivery: the PEG-mediated delivery and the biolistic bombardment based delivery, either of these methods work sufficiently with noticeable preference to the former. Application of a codon-optimized sequence of the cat gene and a single colony selection yielded C. merolae strains, capable of resisting up to 400 µg/mL of chloramphenicol. Our method opens new possibilities in production of site-directed mutants, recombinant proteins and exogenous protein overexpression in C. merolae—a new model organism.

     

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.     

Plasmid-borne resistance to arsenate, arsenite, cadmium, and chloramphenicol in a Rhodococcus species

Summary A primarily genetic approach was employed to obtain plasmids in Rhodococcus erythropolis ATCC 12674 which carried genes conferring increased resistance to sodium arsenate and arsenite, cadmium chloride, and chloramphenicol. The plasmids were large, migrating more slowly than chromosomal DNA in agarose gels, and were made up of resistance determinants from the host organism together with part of the genome of nocardiophage Q4. Purified plasmid was used to transform a suitable recipient to increased resistance to sodium arsenate, sodium arsenite, and cadmium chloride.     

Multiple drug resistance inAerobacter aerogenes

Multiple drug resistance resulting from continued growth in various drugs in turn followed by serial subculture in the simultaneous presence of all the drugs has been studied. In this way strains ofA. aerogenes resistant, in considerable measure, to 3, 4 and 5 drugs have been obtained. Although the final state was always a compromise between the various new properties it was often a good and very effective compromise and for this reason kinetic properties of the cells such as growth rate had to be intensively followed. Tests for presence or absence of growth after a fixed interval of time would not have sufficed. At the 5-drug stage a good compromise was obtained when the agents used were streptomycin, sulphanilamide, chloramphenicol, ampicillin and 5-aminoacridine. When tretamine (triethylene melamine) replaced 5-aminoacridine as the fifth drug, the rate of growth in the various drugs both singly and in admixture was not as good.Continued subculture in sulphanilamide medium led to a condition of hypersensitivity to chloramphenicol. Ampicillin-resistant strains were more inhibited by 2-phenylethanol than the ampicillin-sensitive parent organism. Strains resistant to chloramphenicol were also resistant to tretamine and vice versa. Such cross-resistance did not occur with any of the other drugs at the concentrations used.     

Induction of biohydrogenation of oleic acid in Bacillus cereus by increase in temperature

A system catalyzing the biohydrogenation of oleate to stearate is present in a strain of Bacillus cereus which has been incubated at 37°. but not at 20°. The appearance of the system is blocked by the addition of chloramphenicol or rifampin. These findings indicate the biohydrogenation system present in this organism is induced by an elevation in temperature.     

EVIDENCE THAT MICROCYSTIN IS A THIO-TEMPLATE PRODUCT1

The hepatotoxic cyclic heptapeptide toxins of cyanobacteria, collectively termed microcystins, are potent inhibitors of protein phosphatases PP1 and PP2A. The structure of microcystins resembles small, cyclic peptide secondary metabolites from fungi and eubacteria. Many of these metabolites are manufactured via a nonribosomal thio-template mechanism. We submit evidence that microcystin is synthesized by a similar mechanism. The organism used in this study was Microcystis aeruginosa PCC7820. Using the traditional ATP-³²PP_i exchange assay for thio-template activity, we found activity in the presence of the substrate d -amino acids occurring in microcystin. Thio-template mechanisms are known to be unaffected by protein synthesis inhibitors such as chloramphenicol. We subjected cultures in exponential and stationary growth to chloramphenicol and monitored culture health versus toxicity. Although the health of the treated cultures declined, the toxicity of the remaining cells increased. We developed an in vitro assay to measure microcystin synthesis in cell lysates in the presence of chloramphenicol. By supplementing the lysates with ATP and the substrate amino acids present in microcystin, we detected a fourfold increase in total microcystins over the course of 20 min.     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO₂ of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CAP chloramphenicol     

Nanocomplexes on the basis of Taunit associated with biocides as effective anti-cyanobacterial agents

Cyanobacteria are photoautotrophic bacteria that are known also as blue-green algae. They accumulate on different surfaces and objects and contribute to their biodegradation. Moreover, cyanobacteria produce toxins, which lead to harmful environmental and human health impacts. Hence, cyanobacterial growth control problem is very vital. The goal of the study was to obtain new nanocomplexes on the basis of a modern nanomaterial Taunit associated with antibiotic chloramphenicol and herbicide diuron and to test their antimicrobial effect against a model organism such as the unicellular cyanobacterium Synechocystis sp. PCC 6803. A nanomaterial made of multiwalled carbon nanotubes (MWCNTs) called Taunit was used for the first time to obtain nanocomplexes coupled either with herbicide diuron (DCMU (3-(3,4-dichlorophenyl)- 1,1-dimethylurea) or with antibiotic chloramphenicol. A small amount of Taunit (~1 mg) was needed to adsorb micrograms of diuron or chloramphenicol. The new formed nanocomplexes differentiate in their antimicrobial activity, which could be explained by the difference in their chemical mechanism of action. Taunit ? diuron complex showed a higher biocide action against cyanobacterium than the Taunit ? chloramphenicol complex. The results allow to discuss the prospects of research on the use of Taunit ? diuron complex as a coating for various surfaces exposed to cyanobacteria fouling.     

Preferential utilization of aromatic compounds over glucose by Pseudomonas putida CSV86

Pseudomonas putida CSV86, a naphthalene-degrading organism, exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase. Glucose supplementation did not suppress the activity of degrading enzymes, which were induced upon addition of aromatic compounds. The induction was inhibited by chloramphenicol, suggesting that de novo protein synthesis was essential. Cells showed cometabolism of aromatic compounds and organic acids; however, organic acids suppressed glucose utilization.