Discovery of a novel glucuronan lyase system in Trichoderma parareesei

Glucuronan lyases (EC 4.2.2.14) catalyze depolymerization of linear β-(1, 4)-polyglucuronic acid (glucuronan). Only a few glucuronan lyases have been characterized until now, most of them originating from bacteria. Here we report the discovery, recombinant production, and functional characterization of the full complement of six glucuronan specific polysaccharide lyases in the necrotic mycoparasite Trichoderma parareesei . The enzymes belong to four different polysaccharide lyase families and have different reaction optima and glucuronan degradation profiles. Four of them showed endo-lytic action and two, TpPL8A and TpPL38A, displayed exo-lytic action. NMR revealed that the monomeric end-product from TpPL8A and TpPL38A underwent spontaneous rearrangements to tautomeric forms. Proteomic analysis of the secretomes from T. parareesei growing on pure glucuronan and lyophilized A. bisporus fruiting bodies, respectively, showed secretion of five of the glucuronan lyases and HPAEC-PAD analysis confirmed the presence of glucuronic acid in the A. bisporus fruiting bodies. By systematic genome annotation of more than 100 fungal genomes and subsequent phylogenetic analysis of the putative glucuronan lyases, we show that glucuronan lyases occur in several ecological and taxonomic groups in the fungal kingdom. Our findings suggest that a diverse repertoire of glucuronan lyases is a common trait among Hypocreales spp. with mycoparasitic and entomopathogenic lifestyles. IMPORTANCE This paper reports the discovery of a set of six complementary glucuronan lyase enzymes in the mycoparasite Tricoderma parareseei . Apart from the novelty of the discovery of these enzymes in T. parareesei, the key importance of the study is the finding that the majority of these lyases are induced when T. parareseei is inoculated on Basidiomycete cell walls that contain glucuronan. The study also reveals putative glucuronan lyase encoding genes in a wealth of other fungi that furthermore points at fungal cell wall glucuronan being a target C-source for many types of fungi. In a technical context, the findings may lead to controlled production of glucuronan oligomers for advanced pharmaceutical applications and pave the way for development of new fungal biocontrol agents.

Genomics and Phenotypical Characterization of Two New Lytic Bacteriophages for Biocontrol of Salmonella enterica

Aims: To perform the isolation, characterization and sequencing of the bacteriophages. To demonstrate that the bacteriophages can be used for biocontrol of different Salmonella enterica serovars. Study Design: This study was an experimental study. Place and Duration of Study: Bacteriology and Mycology Laboratory in the Veterinary Hospital at the Faculty of Agronomy and Veterinary Medicine of the University of Passo Fundo (FAMV/UPF), Biotechnology Center (CBiotec) of the Federal University of Paraíba (UFPB), Center for Microscopy and Microanalysis at the Faculty of Veterinary of the Federal University of Rio Grande do Sul (UFRGS), between January – September 2016. Methodology: Twelve Salmonella enterica serovars (S. Anatum, S. Agona, S. Brandenburg, S. Bredeney, S. Infantis, S. Lexington, S. Panama, S. Rissen, S. Schwarzengrund, S. Tennessee, S. Enteritidis ATCC 13076 and S. Typhimurium ATCC 14028) were selected to be the hosts. We isolate, purify, produce and determine the bacteriophage titers to verify the potential for lysis of these phages against the hosts. Having determined the action of the phages against the hosts, we performed the sequencing of the bacteriophages on the Illumina Mi-Seq platform and the morphology was performed by transmission electron microscopy (TEM). Results: We isolated, characterized and sequenced the genome of two new bacteriophages, Salmonella phage UPF_BP1, belonging to the family Podoviridae and Salmonella phage UPF_BP2, family Myoviridae. UPF_BP1 has lytic action against seven tested Salmonella enterica serovars, while UPF_BP2 has action against the twelve tested serovars. Conclusion: The two new bacteriophages have a lytic action against different Salmonella enterica serovars, feeding our expectations for the development of alternatives for the use of antimicrobials, being possible candidates for use as a biocontrol of Salmonella enterica in food, animals and the environment.

Characteristics of bacteriophages of the Staphylococcus aureus variant bovis

Bacteriophages may be an alternative method of treatment for antibiotic-resistant bacteria, including mastitis in cows. Our study describes the initial isolation and bacteriological activity of bacteriophages, circulating on dairy farms, the against S. aureus var. bovis. Samples of cow’s milk secretions with signs of mastitis and sewage water were used as the study material. The isolation and production of pure bacteriophage lines were performed according to the double agar method. The method of studying a single cycle of phage reproduction was used to determine the duration of the latency period. Determination of the spectrum of the lytic activity of bacteriophages against the clinical isolates of the microorganisms was carried out by the drop method. As a result of the research, four phages, specific for S. aureus var. bovis were isolated: Phage SAvB07, Phage SAvB08, Phage SAvB12 and Phage SAvB14. The negative colonies of the isolated phages were 1–2 mm in size, rounded with clear edges, with varying degrees of transparency. The latency period of Phage SAvB14 was 35 min, with the number of active virions increasing by 8 orders. In the study on growth curves of other bacteriophages, taken in the experiment, the latency period was more than 35 min, and their titre increased by only two orders. Phage SAvB07, Phage SAvB08 and Phage SAvB12 were able to lyse the bacterial strains of S. aureus var. bovis in 25–45.6% of the cases (low lytic activity), whereas Phage SAvB14 lysed 94.1% of S. aureus strains were isolated from the cows. Studies have shown that among the bacteriophages we have studied, Phage SAvB14 with a short latency period has the best lytic action on the culture S. aureus var. bovis. The resulting bacteriophage strain can be used to create a bacteriophage-based drug for the treatment of mastitis in cows.

Lytic Activity of Staphylococcal Bacteriophage on Different Biotypes of Staphylococcus aureus

Bacteriophage is a virus that infects a bacterium by injecting a phage genome into a bacterial cytoplasm and uses a host cell as a propagation mechanism. The studied models of phages show a narrow range of hosts. Previous studies on the investigation of lytic activity of staphylococcal bacteriophages were focused on determining the sensitivity of S. aureus isolated from patients from different clinical material and clinically healthy people. However, there is no information as to how refractory are the already described agents against S. aureus ecovars, isolated from animals. The purpose of the work is to study the lytic activity of the agent “Staphylococcal Bacteriophage” in relation to different biotypes of Staphylococcus aureus. Microbiological treatment of samples for isolation of S. aureus was performed using BD Baird-Parker Agar according to standard techniques. To confirm the presence of S. aureus, tests were used for catalase, coagulase, oxidase, for D-mannitol fermentation, DNase production and acetoin. In cultures belonging to S. aureus, the biotype was determined using the scheme: determining the colour of pigment, the presence of beta hemolysis, the activity of coagulase in the bovine plasma, the formation of colonies in a medium with crystal violet. Determination of the range of action of bacteriophages in relation to clinical isolates of microorganisms was carried out by droplet method. The results of determining the lytic activity of staphylococcal bacteriophage in relation to S. aureus isolates of various biological origin showed that the lytic activity of staphylococcal bacteriophage is most active against S. aureus var. hominis. From the studied cultures S. aureus var. hominis full lysis in the course of dripping were found only in 4.8%, and in 42.8% of cultures showed a semiconfluent lysis. 14.3% of cultures S. aureus var. hominis were subjected to weak lytic activity of the bacteriophage. Also, there were detected 4,8% of cultures of this biotype, which were resistant to staphylococcal bacteriophage. In the study of lytic activity of staphylococcal bacteriophage up to 35 cultures S. aureus var. bovis, the manifestation of the lytic action of only one culture is established. Moreover, the level of lysis was estimated at “+/–”, that is, they showed less than 20 phage colonies. At the same time, the studied by us staphylococcal bacteriophage did not show the lytic action on S. aureus var. avium and S. aureus var. canis. Therefore, conducted studies have shown that the lytic activity of the agent “Staphylococcal Bacteriophage” is directed mainly to S. aureus var. hominis, and practically does not work on other biotypes. In consideration of the apparent lack of activity of phage agents in relation to the studied biotypes, attention should be focused on the specificity of phages, not only within the species of bacteria, but also within their biotypes.

INTERACTION BETWEEN PHAGES AND BACTERIA AS A TOOL FOR THE OBTAINING OF IMAGES

The obtaining of images by lytic action of bacteriophage T4 on the Escherichia coli bacterial lawn is considered. Methodical aspects of the approach are discussed, namely, use of different stencil types, total and partial staining of obtained image by different dyes. The perspectives of the practical use are proposed namely restriction of the action of microorganisms in out-of-theway places etc.