Effect of amino acids on the growth of a B. polymyza 1538 culture and the biosynthesis of polymyxin B]

The effect of a number of the most important amino acids on the growth of B. polymyxa 1538 and polymyxin B biosynthesis was studied. It was found that all of the amino acids tested except D-L-threonin and L-alpha-gamma-DABA inhibited the initial growth of the organism. D-L-threonine and L-alpha-gamma-DABA had some stimulating effect on the culture growth without affecting the final accumulation of the biomass. The antibiotic synthesis proceeded more vigorously when the medium contained D-L-threonine, L-alpha-gamma-DABA, L-asparagine, L-proline, L-glutamine, D-L-asparaginic acid and L-glutamic acid in the concentrations tested. Neither of the tested amino acids used alone or in combination provided the levels of polymyxin B biosynthesis observed on the media containing rich sources of organic nitrogen.     

The activity of polymyxins against dense populations of Escherichia coli.

The activities of polymyxin B sulphate, colistin (polymyxin E) sulphate and their sulphomethyl derivatives were compared by continuous turbidimetric monitoring of dense cultures of an Escherichia coli strain exposed to these agents. Judged by the concentration of antibiotic which caused a rapid fall in opacity of the culture, polymyxin B sulphate and colistin sulphate had similar activities, but the sulphomethyl compounds differed considerably: sulphomyxin sodium induced lysis of the culture at a concentration four times that of the parent compound, whereas colistin sulphomethate sodium induced a delayed fall in opacity consistent with recruitment of activity as the inactive sulphomethyl derivative was broken down to the parent compound. Durign overnight incubation, regrowth of cultures which had initially succumbed to polymyxin action occurred, apparently due to the selection of phenotypically resistant variants from within the population. In this way cultures could easily be adapted to growth in concentrations of antibiotic well above the conventionally-determined minimum inhibitory concentration. The comparative ease of adaptation was in the order: colistin sulphomethate greater than sulphomyxin greater than colistin sulphate greater than polymyxin B sulphate.     

Mechanism of Polymyxin B Resistance in Proteus mirabilis

The lipids from three types of organisms-a Proteus mirabilis wild type highly resistant to polymyxin B, a polymyxin B-sensitive mutant derived from the wild type, and the wild type grown in the presence of sulfadiazine resulting in phenotypic conversion to polymyxin B sensitivity-were examined to determine the nature of polymyxin B resistance. The phospholipid compositions were nearly identical; each organism contained similar small amounts of N-methyl phosphatidylethanolamine in addition to comparable quantities of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. the fatty acid compositions were similar in the exponential phase of growth; in the stationary phase, sulfadiazine markedly inhibited the synthesis of cyclopropane fatty acids. Liposomes prepared from the dried lipids of the three types of organisms were extensively and similarly disrupted by the polymyxin. These findings suggest that polymyxin B resistance in P. mirabilis is determined by the cell envelope which prevents access of the antibiotic to the susceptible lipid target sites.     

Growth requirements and the effect of organic components of the synthetic medium on the biosynthesis of the antibiotic nisin in Streptococcus lactis strain.

A synthetic medium SM-3 has been elaborated for growth of Streptococcus lactis strain 51, which contains the minimal number of organic components required for the growth of this strain and nisin production. This medium contains 9 amino acids, 4 vitamins from B group, glucose and mineral salts. Addition of biotin to the medium stimulated the growth of the strain, while the addition of purines and/or pyrimidines had no effect. Hitherto biotin has been considered to be necessary for the growth of S. lactis and purines and pyrimidines were believed to stimulate the growth of these bacteria. In strain 51 the minimal requirements for growth were also the minimal requirements for nisin biosynthesis. Strain 51 produced 3-4 times less nisin in medium SM-3 than in a complex medium. The addition of one of four amino acids (serine, proline, cysteine or cystine) to SM-3 medium increased the amount of antibiotic produced. The addition of all four amino acids simultaneously, caused formation of nisin amounts similar to those produced in complex medium.     

Selection of Lysine Plus Threonine-Resistant Mutant of Maize

Resistance to certain amino acids or amino acid analogs can lead to overproduce specific 'free amino acids. By selection-Mutagenic treatment-Selection, lysine plus threonine-resistant mutant (RLT) was obtained from tissue culture of maize, W77-R3019V The resistance of RLT was 20 times higher than that of wild type. The levels of all free aspartate family amino acids in RLT were higher than those in wild type. Especially, threonine was 20 times higher. The resistance was inheritable and segregation in progenies, RLT1 and F1, was approximate to 3:1 and 1:1 resistant/sensitive ratio, respectively. The resistance was inherited as a single dominant or semidominant nuclear gene. In RLT2 embryo cultures, the resistance and free threonine levels in resistant callus were 20 and 23 times higher than those in sensitive one, respectively. In the homozygous seeds of RLT2, the levels of free threonine, arginine, lysine, methionine and isoleucine were 11, 8, 5, 5 and 3 times higher than those of wild type.     

Comparison of the inhibitory effects of diverse amino acids and amino acid analogs on 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity in isolated epidermal cells

At a concentration of 1.25 mM, 14 amino acids were capable of inhibiting the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in isolated epidermal cells. The greatest percentages of inhibition of TPA-induced epidermal ornithine decarboxylase activity were as follows: cysteine, 98%; tryptophan, 74%; methionine, 64%; phenylalanine, 51%; glycine, 44%; asparagine, 43%; glutamic acid, 42%; leucine, 40%; and arginine, 39%. These amino acid treatments did not alter the time- and concentration-response curves for induction of ornithine decarboxylase activity by TPA. Moreover, there was no difference between the rates at which [3H]arginine, [3H]leucine, [3H]phenylalanine, [3H]methionine, [3H]tryptophan and [14C]cysteine were taken up by freshly isolated epidermal cells or incorporated into epidermal proteins. Arginine, phenylalanine and methionine inhibited the induction of ornithine decarboxylase activity by the tumor promoter to degrees comparable to those elicited by their analogs canavanine and homoarginine, beta-2-thienyl-DL-alanine, and ethionine, respectively. These amino acids and amino acid analogs did not alter the overall rate of protein synthesis. In contrast, both the amino acids and their analogs increased the rates of proteolysis in isolated epidermal cells, an effect which correlated well with the abilities of these different compounds to inhibit TPA-induced ornithine decarboxylase activity. Moreover, both methionine and phenylalanine decreased the half-life and increased the rate of heat denaturation of the TPA-induced enzyme, a result identical to that obtained after treatment with the analogs ethionine and beta-2-thienyl-DL-alanine, respectively. Taken together, these results suggest that millimolar concentrations of exogenous amino acids might induce the synthesis of abnormal proteins and nonfunctional enzymes. Therefore, it is speculated that the uptake of unbalanced amounts of amino acids into the epidermal target cells might alter the stability and the ultrastructure of the TPA-stimulated enzyme just as the amino acid analogs do.     

Effect of polymyxin B on outer membranes of Serratia marcescens: morphological alterations of the outer membranes and their lipopolysaccharide components.

The in vitro or the in vivo treatment of outer membranes and their lipopolysaccharide (LPS) components from Serratia marcescens with the antibiotic polymyxin B appeared to alter their normal morphology in a sequential manner. The normal spherodial morphology was destablized into a flattened structure after the in vitro treatment of either the resistant strain 08 or the sensitive strain Bizio. The more severe in vivo treatment of the outer membranes from the resistant strain converted the flattened forms further into spheres with undefined periphery and diminished sizes. On the other hand, the same treatment of the outer membranes from the sensitive strain resulted in numerous incomplete spheres and short rods, which were similar to the various morphological forms of the LPS components after polymyxin B treatment. The difference in the morphological changes of the outer membranes and their LPS components of the resistant and sensitive strains after polymyxin B treatment may be explained by the variation of the susceptiblity of the membrane components to the degradative effects of the antibiotic.     

影响多粘菌素B对绿脓杆菌杀菌效力的主要环境因素的研究

本文通过改变温度,水活度,气体条件和营养含量等影响绿脓杆菌生长的主要环境因素,测定多粘菌素B对绿脓杆菌的最小杀菌浓度(MBC)。结果表明环境因素导致或显著影响绿脓杆菌对抗生素的生态耐受性。实验表明多粘菌素B对绿脓杆菌的杀菌效力,除药物对细菌特有的药理学作用外,还取决于细菌的生长环境。结合冷休克率试验表明,环境影响细菌群体处于分裂状态的菌数。若分裂状态菌数下降表明生长速度减慢。提示了多粘菌素B对绿脓杆菌的效力指数,定量分析可以作为其综合效力作用的表现。以同步培养法确定在单个细胞周期中的抗生素敏感阶段。同时以冷休克率试验资料证明细菌处于分裂状态和幼龄期是其敏感阶段。初步阐述了生长速度缓慢与药物的生态耐受性密切相关。     

Correlation of Bacterial Lipid Composition with Antibiotic Resistance

The correlation of bacterial lipid composition with antibiotic resistance was investigated with particular emphasis on those organisms in which resistance may be related to membrane or envelope structure or function, as in resistance to tetracyclines and polymyxin. Chloroform-methanol-extractable lipids, phosphatidyl ethanolamine fractions, and both fatty acids of these lipid fractions and total fatty acids were studied by using thin-layer chromatography, gas chromatography, and infrared spectroscopy. Consistent quantitative differences were found between the fatty acid compositions of sensitive and resistant strains. Most notable was the fact that, in gram-negative organisms, resistant strains showed decreases in cyclopropane acids as compared with sensitive strains. These changes were found to be inherent in the strains and not due to growth stage or culture age. No significant qualitative differences were noted. In contrast, no such variation in fatty acid content was observed in penicillin-sensitive and resistant strains of gram-positive cocci. As significant alterations of fatty acid composition were noted in gram-negative strains resistant to antibiotics, we suggest that resistance is correlated to membrane or envelope lipid composition.     

Induction of polymyxin resistance in Pseudomonas fluorescens by phosphate limitation

Shift of Pseudomonas fluorescens NCMB 129 from a phosphate rich into a phosphate limited medium results in a reduction of the membrane phospholipids phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Concomitantly a positively charged ornithine amide lipid is synthesized. The gradual increase of this lipid is paralleled by an increasing resistance to polymyxin B. The binding capacities of intact cells, and isolated inner and outer membranes for the antibiotic are reduced in the resistant organisms. It is discussed that the observed effect could be circumstantial evidence that the positively charged polymyxin B needs negatively charged receptors in biological membranes in order to exert its antibiotic activity.List of Abbreviations PE phosphatidylethanolamine - PG phosphatidylglycerol - CL cardiolipin - PX polymyxin B     

Effects of polymyxin B sulfate and polymyxin B nonapeptide on growth and permeability of the yeast Saccharomyces cerevisiae

Summary Polymyxin B, a toxic, membrane-affecting antibiotic, can be rendered harmless to yeast cells by enzymatic removal of its fatty acyl moiety. The remaining cyclic peptide portion, polymyxin B nonapeptide, has no significant effect on growth and viability but it drastically reduces mating efficiency. In addition, the cyclic peptide enhances sensitivity of cells to several drugs, presumably by increasing membrane permeability. Mutants resistant to polymyxin B are simultaneously less responsive to the combination of the nonapeptide and the drugs. This indicates that the peptide portion of polymyxin B is the moiety responsible for the permeability changes. The resistance is inherited as a simple recessive trait. The mutation has been mapped to chromosome XV of Saccharomyces cerevisiae.     

Isolation,structural characterization,and properties of mattacin (polymyxin M), a cyclic peptide antibiotic produced by Paenibacillus kobensis M

Mattacin is a nonribosomally synthesized, decapeptide antibiotic produced by Paenibacillus kobensis M. The producing strain was isolated from a soil/manure sample and identified using 16 S rRNA sequence homology along with chemical and morphological characterization. An efficient production and isolation procedure was developed to afford pure mattacin. Structure elucidation using a combination of chemical degradation, multidimensional NMR studies (COSY, HMBC, HMQC, ROESY), and mass spectrometric (MALDI MS/MS) analyses showed that mattacin is identical to polymyxin M, an uncommon antibiotic reported previously in certain Bacillus species by Russian investigators. Mattacin (polymyxin M) is cyclic and possesses an amide linkage between the C-terminal threonine and the side chain amino group of the diaminobutyric acid residue at position 4. It contains an (S)-6-methyloctanoic acid moiety attached as an amide at the N-terminal amino group, one D-leucine, six L-alpha,gamma-diaminobutyric acid, and three L-threonine residues. Transfer NOE experiments on the conformational preferences of mattacin when bound to lipid A and microcalorimetry studies on binding to lipopolysaccharide showed that its behavior was very similar to that observed in previous studies of polymyxin B (a commercial antibiotic), suggesting an identical mechanism of action. It was capable of inhibiting the growth of a wide variety of Gram-positive and Gram-negative bacteria, including several human and plant pathogens with activity comparable with purified polymyxin B. The biosynthesis of mattacin was also examined briefly using transpositional mutagenesis by which 10 production mutants were obtained, revealing a set of genes involved in production.     

Isolation and characteristics of polymyxin-resistant mutants of Shigella flexneri

Some characteristics of S. flexneri 2a mutants resistant to various concentrations of polymyxin M were studied. The data indicate that mutations resulting in low (50 microgram/ml) and high (300 microgram/ml) levels of the antibiotic resistance were determined by different genes. Polymyxin resistance led to changed permeability of the outer membrane with respect to detergents and some antibiotics, such as aminoglycosides, penicillins, chloramphenicol and amphotericin B but did not change sensitivity of the strains to some bacteriophages, except phage PI. Mutants resistant to 50 microgram/ml of polymyxin M preserved their ability to induce keratoconjunctivitis in guinea pigs. Part of the strains resistant to 300 microgram/ml of the antibiotic lost this property. No correlation between the polymyxin M resistance level, loss of the pathogenic properties and toxicity of the bacterial cells was found. It was confirmed that though inactivation of endotoxin by polymyxins is associated with their capacity for interaction with lipid A, this component does not participate in development of resistance to these antibiotics.     

Isolation and identification of an antibiotic from culture 8-86]

An antibiotic complex was isolated from culture 8-86 referred to Bacillus. The complex consisted of components 8-86A and 8-86B active against gram-negative organisms. By its physico-chemical properties such as IR and UV spectra, amino acid composition, specific rotation and fatty acid composition component 8-86B was shown to be close to polymyxin F.     

Susceptibility of Recent Isolates of Pseudomonas aeruginosa to Gentamicin, Polymyxin, and Five Penicillins, with Observations on the Pyocin and Immunotypes of the Strains

The susceptibilities of recently isolated strains of Pseudomonas aeruginosa to gentamicin, polymyxin B, carbenicillin, ampicillin, penicillin G, and two newer penicillins were tested with the inocula-replicating technique by using undiluted and 10(-3) dilutions of the cultures. With either inoculum, polymyxin B was the most active agent, and a comparison with previous data from this laboratory showed that the susceptibility of P. aeruginosa to this antibiotic had not changed over the past 20 years. Gentamicin was nearly as active as polymyxin, all but 2 of the 141 strains tested with the diluted inoculum being inhibited by 6.25 mug/ml or less. AB-2288, an agent resembling carbenicillin, was four times more active than carbenicillin or BLP-1654; the last two were equally active against the 10(-3) inoculum. A more marked inoculum effect was noted with the penicillin analogues tested, the increase in minimum inhibiting concentration with the undiluted culture being eight-fold for carbenicillin and at least 16-fold for AB-2288 and BLP-1654. Pyocin typing and serotyping failed to demonstrate any clearly predominating types.     

Analysis of human IL-6 mutants expressed in Escherichia coli. Biologic activities are not affected by deletion of amino acids 1-28

We have constructed and analyzed amino terminally deleted analogs of IL-6. Progressively shortened variants of mature IL-6 were constructed at the cDNA level and expressed in Escherichia coli. Mutant proteins were recovered from refractile bodies by solubilizing in 6 M guanidine-HCl. The mutant protein concentration in these preparations was estimated by Western blotting by using an IL-6-specific mAb and the biologic activity was measured in the B9 (hybridoma growth factor) assay. The first 28 amino acids of mature IL-6 could be removed without significantly affecting biologic activity. A further removal of amino acids 29 and 30 resulted in an approximately 50-fold decrease, whereas removal of amino acids 31 to 34 virtually abolished the activity. The mutants showed the same reaction pattern in three other IL-6 assays: induction of murine thymocyte proliferation, induction of fibrinogen synthesis by a human hepatoma cell line (HepG2), and the induction of IgM synthesis by an EBV-transformed B cell line. This suggests that a single functional domain might be responsible for all four activities of IL-6.     

Chemical Communication of Antibiotic Resistance by a Highly Resistant Subpopulation of Bacterial Cells

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.     

Growth and Initiation of Protein Synthesis in Escherichia coli in the Presence of Trimethoprim

Escherichia coli grew exponentially at a reduced rate in the presence of 50 or 100 mug of trimethoprim/ml if the low-molecular-weight products of folate metabolism or their precursors (thymidine, purines, methionine, glycine, and pantothenate) were supplied in the medium. Folate metabolism was inhibited 99.9% by these concentrations of trimethoprim, but a low level of formylation of methionyl transfer ribonucleic acid (met-tRNA(F)) could be detected. However, in a medium containing all major amino acids, nucleosides, and vitamins, formylation of met-tRNA(F) was undetectable in the presence of trimethoprim. No other amino-masked amino acids were detected, and methionine remained a major amino-terminal amino acid of mature proteins. met-tRNA(F) was rapidly labeled with exogenous methionine and was associated with 30s ribosomal subunits and 70s ribosomes. It was concluded that initiation of protein synthesis can occur with unformylated met-tRNA(F) in E. coli. Changes in macromolecular composition were associated with the lack of formylation, in particular a fourfold increase in both met-tRNA(F) and ribosomal subunits. These changes would tend to compensate for the low specific rate of initiation with unformylated met-tRNA(F).     

Interactions among amino acid transport systems in snail Helix aspersa intestine

Interactions among the transport of diverse amino acids in everted intestine of snail Helix aspersa have been studied. The uptake of 0.5 mM methionine is clearly inhibited by high concentrations (40 mM) of leucine, and not by proline or lysine, whereas the last two amino acids inhibit cycloleucine uptake. Methionine strongly inhibits proline and lysine uptake, which is significantly inhibited by their analogs hydroxiproline and arginine, respectively. Results suggest that in Helix intestine the transport systems for basic amino acids and iminoacids are shared with high affinity by methionine whereas the neutral amino acids transport systems do not seem to be shared, or are so very weakly, by the basic ones or by the imino acids.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.