Kamila Plháčková

Innovative methods for soil DNA purification tested in soils with widely differing characteristics.

ABSTRACT Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO3 or purification of extracted DNA by CaCl2. The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl2 purification and in 93% of cases by the method based on CaCO3 pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

Development of a high-expression system for penicillin G acylase based on the recombinant Escherichia coli strain RE3 (pKA18)

Abstract A high-expression system for penicillin G acylase (PGA) was developed using the high PGA-producing strain Escherichia coli RE3 as a host and a recombinant plasmid pKA18 which was constructed by cloning the chromosomal pga gene coding for PGA in the strain RE3 on multicopy vector pK19. One particular objective for the elaboration of this expression system was the selection of a convenient host strain and modification of the growth conditions. From a total of 15 hosts, the strain RE3 cultured in a mineral medium exhibited the highest expression of a pga -encoding gene recombinant plasmid and high segregational plasmid stability. The high-expression system led to an increase in the specific activity of PGA up to 1,000 U g −1 cell dry weight and a total activity of about 4,500 U l −1 of the culture in a laboratory fermentor.

Mutasynthesis of Lincomycin Derivatives with Activity against Drug-Resistant Staphylococci

ABSTRACT The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl-l-proline. Feeding this mutant strain with precursor derivatives resulted in production of 4′-butyl-4′-depropyllincomycin and 4′-pentyl-4′-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4′-pentyl-4′-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.

Cloning of penicillin G acylase-encoding gene from Escherichia coli high-producing strain RE3

SummaryPga gene from the industrial strain of E. coli RE3 hyperproducing penicillin G acylase (PGA) was cloned using a simple and rapid NIPAB-based chromogenic method for the detection of PGA-positive recombinant clones. Heterogeneous genetical material was prepared by subcloning pga in vectors pACYC184, pBR322-2 and pK19. The highest constitutive expression of pga was observed in a strain bearing the recombinant plasmid pKA18 synthesizing 2.5 times more of enzyme than the parent strain.

Lincosamide Synthetase—A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

Ultra-high-performance liquid chromatography fingerprinting method for chemical screening of metabolites in cultivation broth.

A fingerprinting method for chemical screening of microbial metabolites, potential antibiotics, in spent cultivation broths is described. The method is based on high-throughput ultra-high-performance liquid chromatography (UHPLC) separation with UV detection (photodiode array detector). Thirteen antibiotic standards and four cultivation broths were used for the method development. The comparison of ten liquid-liquid and solid phase extraction protocols for sample clean-up and pre-concentration revealed that Oasis HLB C18 sorbent gives the best recoveries. The Acquity BEH C18 chromatographic column was chosen for the samples separation with respect to its universality, selectivity, efficiency and robustness. The method is presented by two 3D fingerprints for every sample that was obtained under different, acidic and alkaline, UHPLC conditions. The acidic mobile phase consisted of 0.5% phosphoric acid with methanol and the alkaline mobile phase of 1mM ammonium formate, pH 9 with acetonitrile. Each pair of 3D fingerprints includes the following physico-chemical information: polarity (retention time), presence and characterization of chromophores (UV spectra), compound concentration (detector response), and acid-base properties (influence of different pH of the aqueous parts of mobile phases on retention times). The sample extraction and method validation were assessed with relative standard deviation (RSD) of 0.5, 5.0 and 20.0% for retention times, peak areas and minor compound peak areas, respectively.

Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes

In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence.