Penicillium Arizonense as a Novel Producer Strain for Mycophenolic Acid and Expression Analysis of Biosynthesizing Gene Clusters

A novel potent mycophenolic acid (MPA) producer strain of the genus Penicillium was isolated from refrigerated Mozzarella cheese and identified as P. arizonenseHEWt1. The molecular mechanism of MPA production by this new isolate was our main target. To achieve this objective, we first isolated three MPA overproducer mutants by exposing the wild type to different doses of gamma-rays, and the fermentation conditions for the highest production of MPA by both the wild type and mutants were optimized. Then, orthologs of MPA gene clusters in P. brevicompactum were cloned and predicted from the genome of P. arizonense. Sequencing and bioinformatic analysis proved the presence of a cluster containing five putative genes in the P. arizonense HEWt1 genome ortholog to the MPA cluster, mpaA, mpaC, mpaF, mpaG, and mpaH. All predicted genes displayed 96-97% similarity with the related hypothetical protein of P. arizonense. The genes, mpaG, mpaC, and mpaF. represented 69%, 82%, 84%, respectively, similarity with their orthologous genes in P. brevicompactum, whereas mpaG and mpaA represented 75% and 79%, respectively, similarity to their orthologous genes in P. roqueforti. Gene expression analysis through quantitative rPCR indicated an increase in the transcription value of all annotated genes in the three mutants over the wild type. A highly significant increase in the gene expression of mpaC, mpaF, and mpaH was observed, with 8.4561±1.02, 5.6569±0.87, and 4.6268±0.18-fold increases, respectively, in P. arizonense-MT1 compared with wild-type. These results confirmed the potential participation of these genes in MPA biosynthesis and are the first report regarding the molecular mechanism of MPA production by P. arizonense.

Genome Sequence of Lysobacter sp. Strain BMK333-48F3, the Producer Strain of Potent Lipopeptide Antibiotics of the Tripropeptin Family

Lysobacter sp. strain BMK333-48F3 is known primarily for its production of the antibiotically active tripropeptins. Here, we report its draft genome sequence, which will give insight into the biosynthesis of tripropeptins and enable genome mining for further secondary metabolites.

N-acetyl-cysteinylated streptophenazines from Streptomyces

ABSTRACTHere, we describe two N-acetyl-cysteinylated streptophenazines (1 and 2) produced by soil-derived Streptomyces sp. ID63040 and identified through a metabolomic approach. These metabolites attracted our interest due to their low occurrence frequency in a large library of fermentation broth extracts and their consistent presence in biological replicates of the producer strain. The compounds were found to possess broad-spectrum antibacterial activity while exhibiting low cytotoxicity. The biosynthetic gene cluster from Streptomyces sp. ID63040 was found to be highly similar to the streptophenazine reference cluster in the MIBiG database, which originates from the marine Streptomyces sp. CNB-091. Compounds 1 and 2 were the main streptophenazine products from Streptomyces sp. ID63040 at all cultivation times, but were not detected in Streptomyces sp. CNB-091. The lack of obvious candidates for cysteinylation in the Streptomyces sp. ID63040 biosynthetic gene cluster suggests that the N-acetyl-cysteine moiety derives from cellular functions, most likely from mycothiol. Overall, our data represent an interesting example on how to leverage metabolomics for the discovery of new natural products and point out to the often-neglected contribution of house-keeping cellular functions to natural product diversification.Graphical abstract

Obtaining Multiprotein Antigens Of Brucella And Study Of Their Immunoreactivity

Rose-Bengal test, complement fixation test and the agglutination test are mainly used for the diagnosis of animal brucellosis. These tests are characterized by low sensitivity and specificity, which is one of the main reasons for the low effectiveness of measures aimed at eradicating brucellosis. The use of modern highly sensitive serological tests requires the availability of antigens specific to Brucella spp. The aim of the study was to obtain multiproteins of the pathogen by recombinant DNA technology and to study their antigenic properties. In the course of the study, three types of multiproteins were obtained, constructed from diagnostically important peptides that form B.abortus and B.melitensis outer membrane proteins. All target products were synthesized by the producer strain in a form that is authentic to natural proteins, and showed immunogenicity in mouse model. Antibodies produced against the multiproteins were specific for the single proteins of the pathogen's cell wall. The data obtained indicate the need to continue studies to determine the possibility of using multiproteins as an antigen in an enzyme-linked immunosorbent assay to detect anti-Brucella specific antibodies.

Bacteriocins: Recent advances in application as an antimicrobial alternative

: Due to the emergence and development of antibiotic resistance in the treatment of bacterial infections, efforts to discover new antimicrobial agents have increased. One of these antimicrobial agents is a compound produced by a large number of bacteria called bacteriocin. Bacteriocins are small ribosomal polypeptides that can exert their antibacterial effects against bacteria close to their producer strain or even non-closely strains. Adequate knowledge of the structure and functional mechanisms of bacteriocins and their spectrum of activity, as well as knowledge of the mechanisms of possible resistance to these compounds will lead to further development of their use as an alternative to antibiotics. Furthermore, most bacteria that live in the gastrointestinal tract (GIT) have the ability to produce bacteriocins, which spread throughout the GIT. Despite antimicrobial studies in vitro, our knowledge of bacteriocins in the GIT and the migration of these bacteriocins from the epithelial barrier is low. Hence, in this study, we reviewed general information about bacteriocins, such as classification, mechanism of action and resistance, emphasizing their presence, stability, and spectrum of activity in the GIT.

Targeting riboswitches with synthetic small RNAs for metabolic engineering

Our growing knowledge of the diversity of non-coding RNAs in natural systems and our deepening knowledge of RNA folding and function have fomented the rational design of RNA regulators. Based on that knowledge, we designed and implemented a small RNA (sRNA) tool to target bacterial riboswitches and activate gene expression. The synthetic sRNA is suitable for the regulation of gene expression both in cell-free and in cellular systems. It targets riboswitches to promote the antitermination folding regardless the cognate metabolite concentration. Therefore, it prevents transcription termination increasing gene expression up to 103-fold. We successfully used sRNA arrays for multiplex targeting of riboswitches. Finally, we used the synthetic sRNA to engineer an improved riboflavin producer strain. The easiness to design and construct, and the fact that the riboswitch-targeting sRNA works as a single genome copy, make it an attractive tool for engineering industrial metabolite-producing strains.

Ecological Aspect of Antibiotic Batumin Synthesis by Pseudomonas batumici

The species Pseudomonas batumici, isolated from the rhizosphere of eucalyptus in the humid subtropical zone, is a producer of the polyketide antibiotic batumin with highly selective activity against staphylococci. Batumin biosynthesis operon includes 28 genes or 74 151 bp. According to modern notions, the biosynthesis of energy-intensive metabolites, which probably includes batumin, is justified in the case of its multifunctionality for producers. The species P. batumici, as a representative of rhizosphere bacteria, must interact with plants and compete with the surrounding microbiota. Aim. To determine the role of batumin in the ecology of the rhizosphere producer strain P. batumici UCM B-321. Methods. The batumin producing strain P. batumici UCM B-321T was obtained from the Ukrainian Collection of Microorganisms. Antibiotic batumin was obtained by fermentation of P. batumici UCM B-321. Extraction was carried out from acidified P. batumici fermentation broth by chloroform (1:2). Chromatographic analysis of fermentation broth obtained after centrifugation was carried out by HPLC using liquid chromatograph Agilent 1200 with mass spectrometric detector Agilent G1956B. Batumin derivatives were obtained after the extraction of the fermentation broth of P. batumici using thin layer chromatography (TLC) on silica gel plates (Merck, USA) in the benzene-isopropanol system (5:1). Disc-diffusion method on phytopathogenic test-strains was used for bioautography. Biofilm formation by P. batumici strain was studied according to O’Toole by growing strain B-321 at 25 0C for 48 hours in 96-well plates on LB medium. Batumin effect upon bacterial mobility was studied using Volf and Berg method in Petri dishes with 0.5% semisolid bacterial agar. To research chemotaxis the soil strain Bacillus subtilis IMV B-7023 and the following concentrations of batumin were used: 20, 50, and 150 μg/mL. The studies were performed using Tso and Adler method. Results. Growth inhibition zones for phytopathogenic bacteria strains were the following (in mm): Pseudomonas syringae pv. syringae UCM B-1027T – 19±3, P. fluorescens IMV 8573 – 22±3, Pectobacterium carotovorum UCM B-1075T – 17±2. Activity against Xantomonas campestris pv. campestris UCM B-1049, Clavibacter michiganensis subsp. michiganensis IMV 102, Agrobacterium tumefaciens UCM B-1000 was not detected. Minimum inhibitory concentrations (MIC) in the range from 8 to 64 μg/mL for P. carotovorum UCM B-1075T, Erwinia aroidea IMV 1058, Proteus vulgaris UCM B-905 and P. fluorescens IMV 8573 are hardly comparable with the discovered against staphylococci. TLC analysis of its broth extract revealed five separate compounds with different values of retention factors: Rf1=0.42; Rf2=0.38; Rf3=0.31; Rf4=0.28; Rf5=0.25. The main component of extract was batumin, other four substances were present in minor quantities. All found substances had similar absorption maxima with the minimum differences between isomeric forms: descarbamoyl batumin-enol (Mr=505, λ=226 nm), descarbamoyl batumin-keto (Mr=505, λ=231 nm), batumin (Mr=548, λ=231 nm), batumin-enol (Mr=548, λ=228 nm) and 17-hydroxy-batumin (Mr=550, λ=229 nm). The largest inhibition zone (P. carotovorum UCM B-1075T) was on the third compound placement which represents of batumin, tiny inhibition zones were found around keto and enol form of descarbamoyl batumin. Observation of live bacterial cells in light microscope confirmed a serious disruption of motility in all these bacteria by batumin in the concentration far lower than the MIC for these organisms. Proteus actively moved in the control, but in presence of 10 μg/mL of batumin was almost no growth. The biofilm formation by P. batumici UCM B-321 was stimulated by supplementing batumin into the medium. The stimulation effect by batumin on the biofilm formation was equally strong when the compound was applied in the concentrations of 1 and 10 μg/mL. Batumin was not an attractant of the producer strain. However, in one of our experiments batumin has shown the properties of positive effector (attractant) for B. subtilis UCM B-7023 strain. Conclusion. The discovered features allow to consider the antibiotic batumin synthesized by P. batumici UCM B-321 as the essential tool for survival and competition of the producer strain in a natural habitat.

THE BIOLOGICAL PECULIARITIES OF PRODUCER STRAIN OF MICROBIOPREPARATION 11-1 BACILLUS SP. – THE ANATAGONIST OF SUNFLOWER PHOMA ROT PATHOGEN

We studied the cultural and physiological characteristics of the producer strain 11-1 Bacillus sp. – the antagonist of sunflower Phoma rot pathogen to develop the technological regulations for the production of a microbiological preparation in a «wettable powder» form. We studied the cultural characteristics of the producer strain on three agar media: potato sucrose agar (PSA), Czapek’s agar and Tylon-3; the characteristics varied significantly depending on the nutrient medium. The maximum diameter of colonies on the tenth day of incubation developed on the Tylon-3 medium – 66×99 mm. We established the optimal conditions for the cultivation of the strain on liquid nutrient media: temperature – 30–35 °С, medium pH from 6 to 10. We found that molasses is an optimal source of carbon nutrition, while peptone is the most favorable source of nitrogen nutrition. We established that the Tylon-3 medium is the optimal complex liquid nutrient medium for the cultivation of the bacterial strain 11-1 Bacillus sp.