Enhanced Production of Polymyxin E in Paenibacillus polymyxa by Replacement of Glucose by Starch.

Polymyxin E or colistin, produced by Paenibacillus polymyxa, is an important antibiotic against Gram-negative pathogens. The objective of this study is to evaluate the effect of starch in fermentation medium on colistin biosynthesis in P. polymyxa. The results indicated that replacement of glucose by starch stimulated colistin production and biosynthesis rate. Overall, the stimulation extent was starch concentration-dependent. As expected, addition of starch induced the expression of amyE encoding amylase and increased amylase activity in fermentation solution. Additionally, replacement of glucose by starch resulted in residue reducing sugar and pH of fermentation mixture low relative to glucose as the sole sugar source. At the molecular level, it was found that replacement of glucose by starch has enhanced the relative expression level of ccpA encoding catabolite control protein A. Therefore, the repression of starch utilization by glucose could be probably relieved. In addition, use of starch stimulated the expression of regulatory gene spo0A but repressed the expression of another regulatory gene abrB. As a result, the expression of genes directly involved in colistin biosynthesis and secretion increased, indicating that at the transcriptional level spo0A and abrB played opposite roles in regulating colistin biosynthesis in P. polymyxa. Taken together, our data demonstrated that starch instead of glucose can promote colistin production probably by affecting the expression of colistin biosynthesis-related genes, as well as reducing the repression of glucose to a secondary metabolic product.

Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes.

Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab), L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes.

Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster.

Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.

Draft genome sequence of Paenibacillus polymyxa OSY-DF, which coproduces a lantibiotic, paenibacillin, and polymyxin E1.

Paenibacillus polymyxa OSY-DF is a Gram-positive rod-shaped bacterium isolated from a fermented vegetable food. This bacterial strain displays potent antimicrobial activities against Gram-positive and Gram-negative pathogenic bacteria, attributed to the production of the lantibiotic paenibacillin and the colistin peptide polymyxin E1. Here we report the draft genome sequence of Paenibacillus polymyxa OSY-DF.

Actividad in vitro de terapia combinada con colistina frente a Pseudomonas aeruginosa aisladas en Unidad de Cuidados Intensivos / In vitro activities of colistin combinations against Pseudomonas aeruginosa isolated from the intensive care unit

Introducción. Ante el incremento de aislados de Pseudomonasaeruginosa multirresistentes en la Unidad de CuidadosIntensivos (UCI) del Hospital Clínico San Carlos deMadrid y para determinar la posibilidad de que se tratase deun brote epidémico causado por la diseminación de unaúnica cepa, se recogieron 12 muestras consecutivas de distintospacientes que fueron identificadas y a las que posteriormentese determinó su sensibilidad a los antibióticosutilizados habitualmente para el tratamiento de las infeccionesproducidas por este microorganismo.Métodos. Mediante la amplificación de secuencia basadaen la reacción en cadena de la polimerasa (repetitive sequence-based polymerase chain reaction, rep-PCR) se determinó larelación clonal entre los aislados utilizando los primers BOX yERIC y se estudió la actividad in vitro frente a estas cepas decolistina, rifampicina, doxiciclina y azitromicina para determinaren qué casos la combinación de colistina con alguno de losotros tres antibióticos presentaba actividad sinérgica.Resultados. Los estudios de sensibilidad apuntan a la presenciade varias cepas de P. aeruginosa como responsables delas infecciones respiratorias producidas por este microorganismoen la UCI, hecho que fue corroborado mediante los estudiosclonales realizados. En los estudios de sinergia las asociacionesde colistina con doxiciclina y con azitromicina presentaron actividadsinérgica para alguno de los aislados.Discusión. Los resultados de los estudios clonales revelanla presencia de cinco clones diferentes entre los aisladosseleccionados, por lo que podemos concluir que no se tratade un brote de P. aeruginosa en la UCI. La actividad sinérgicade la asociación de colistina con azitromicina, doxiciclinay rifampicina ha sido menor de la esperada y se observa unelevado porcentaje de resultados indiferentes (AU)

Introduction. As the number of multidrug-resistantstrains of Pseudomonas aeruginosa has risen in the intensivecare unit (ICU) of the San Carlos Clinic Hospital,12 consecutive isolates from different patients were collectedto determine the possibility of an epidemic outbreakcaused by the spread of a single strain. We determinedthe antimicrobial susceptibility to the most commonagents used in the treatment of infections caused by thisbacteria. The results of susceptibility studies suggest thatdifferent strains of P. aeruginosa are responsible for therespiratory tract infections in ICU.Methods. The clonal relationship between the isolatesusing was determined using BOX and ERIC primersby means of repetitive sequence-based polymerase chainreaction (rep-PCR). The in vitro activity of these strainsagainst colistin, rifampicin, doxicycline and azythromycinwas studied to determine in which cases the combinationof colistin with any of the other three antibioticswas synergistic.Results. Sensitivity studies point out the presence ofseveral strains of P. aeruginosa as the causal agents ofrespiratory infections produced by this microorganism inthe ICU. Combinations of colistin with doxycicline andcolistin with azithromycin were synergistic for some isolatesin the synergy studies.Discussion. Clonal studies reveal the presence of fivedifferent clones among our isolates. Therefore we canconclude that there was no outbreak of P. aeruginosa inthe ICU. Synergistic activity of combinations of colistinplus azithromycin, colistin plus doxicycline and colistinplus rifampicin was less than expected and a high percentageof indifferent results was observed (AU)

Polymyxin E production by P. amylolyticus.

AIMS: To investigate antibiotic production by bacteria isolated from the hindgut of Tipula abdominalis, the aquatic crane fly. METHODS AND RESULTS: A group of five isolates with 99.1% 16S rRNA sequence similarity to Paenibacillus amylolyticus were identified as antibacterial producers using the cross-streak method against both Gram-positive and Gram-negative bacteria. For one isolate, P. amylolyticus C27, biochemical tests were performed to confirm 16S rRNA identification and the antibacterials were purified using chromatographic methods. Postsource decay (PSD) mass spectroscopy (MS) was used to identify the antimicrobials, which were found to be polymyxins E(1) and E(2). Investigation of the remaining four isolates using PSD MS revealed they all produce polymyxins E(1) and E(2) as well. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Although variants of the polymyxin antibiotics are known to be produced by several species within the Paenibacillus genus, this first investigation of antibacterial production by bacteria isolated from the hindgut of T. abdominalis describes a novel source for polymyxin E production as well as the first report of antibiotic production by P. amylolyticus.

Biosynthesis of polymyxin E by a cell-free enzyme system. IV. Acylation of enzyme-bound L-2,4-diaminobutyric acid.

An enzyme fraction, which catalyzes the ATP-PPi exchange reaction dependent on the three constituent amino acids of polymyxin E, was partially purified from crude extracts of Aerobacillus polyaerogenes. The approximate molecular weight was estimated to be 640,000 by Sepharose 4B gel filtration. Incubation of the enzyme with octanoyl coenzyme A and diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yielded octanoyldiaminobutyric acid thioesterified to the enzyme protein. On mild alkali treatment, octanoyldiaminobutyric acid, identified by paper chromatography, was released from the enzyme protein. From its acid hydrolyzate, diaminobutyric acid and octanoic acid were recovered in a molar ratio of 1 to 0.7. An ammonium sulfate fraction was required as the source of an acyltransferase for acylation of the enzyme-bound diaminobutyric acid. When [14C]-threonine was incubated with L-2,4-diaminobutyric acid in the presence of octanoyl coenzyme A, octanoyldiaminobutyrylthreonine bound to the enzyme protein was formed. These results suggest that acyldiaminobutyric acid bound to the enzyme protein is a possible initiation complex in the biosynthesis of polymyxin E.

Bacteriophages of Bacillus polymyxa.

Virulent bacteriophages of colistin--producing Bacillus polymyxa strains were studied. The phages were found to differ in lytic spectrum and were active only against strains of B. polymyxa. They did not attack other strains of the genus Bacillus. The virulent bacteriophages belong to two morphological groups differing in size. The size of the DNA of the bacteriophages of both groups is similar and ranges from 74.9 X 10(6) to 87.8 X 10(6) daltons. The cells of different B. polymyxa strains were also found to carry various defective phages which could be shown after mitomycin C induction of cell cultures. The antibacterial activity of mitomycin C induced cell lysates was not detected. Strains of B. polymyxa most probably devoid of defective bacteriophages (delysogenized) were isolated.

Biosynthesis of polymyxin E by a cell-free enzyme system. II. Synthesis of enzyme-bound octanoyldiaminobutyric acid.

A partially purified enzyme of Aerobacillus polyaerogenes, which produces polymyxin E, activates L-2,4-diaminobutyric acid and binds it as a thioester. The incubation of the enzyme preparation with octanoyl coenzyme A and L-2,4-diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yields octanoyldiaminobutyric acid thioesterified to the enzyme.

Partial purification and properties of L-2,4-diaminobutyric acid activating enzyme from a polymyxin E producing organism.

An L-2,4-diaminobutyric acid activating enzyme was found in crude extracts of Aerobacillus polyaerogenes, which produces polymyxin E1 and E2. The enzyme was partially purified by sonication of the cells, followed by ultracentrifugation, ammonium sulfate fractionation, and DEAE-cellulose column chromatography. In addition to L-2,4-diaminobutyric acid, the enzyme activated L-leucine and L-threonine, which are constituent amino acids of polymyxin E. All three amino acids were bound to the enzyme as thioesters. These results suggest that polymyxin is synthesized by a multienzyme thiotemplate mechanism, in the same way as gramicidin S, tyrocidines, bacitracins, and gramicidin A.