Janthinobacterium kumbetense sp. nov., a violacein-producing bacterium isolated from spring water in Turkey, and investigation of antimicrobial activity of violacein.

Strain GKT was isolated from the Kumbet plateu of Giresun in Turkey. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GKT belonged to genus Janthinobacterium and 16S rRNA gene sequence similarities with all type strains of the genus Janthinobacterium were 98.89%-99.78%. The calculated pairwise average nucleotide identity (ANI) values between strain GKT and all type strains of Janthinobacterium species were in the range of 79.8%-93.2%. In addition, digital DNA-DNA hybridization (dDDH) values were in the range of 23.0%-51.7%. Major fatty acids are C10:03OH, C12:0, C16:1ω7c, C16:0, and C18:1ω7c, and polar lipids included phosphatidylethanolamine, phosphatidylglycerol, also one unidentified phospholipid and one unidentified aminophospholipid. The respiratory quinone of strain GKT was determinated to be Q-8. The genome sizes of strain GKT was 6 197 538 bp with 63.16% G + C ratio. Strain GKT is Gram-stain-negative, aerobic, rod-shaped, and motile. A violet pigment was produced by strain GKT. The crude violacein pigments were separated into three diferent bands on a TLC sheet. Then violacein and deoxyviolacein were purifed by vacuum liquid column chromatography and identifed by NMR spectroscopy. The antimicrobial activities of purifed violacein and deoxyviolacein were screened for seven microorganisms. Based on the results of the morphological, biochemical, physiological, phylogenetic, and genomic characteristics, we propose classifying the strain GKT as representative of a novel species of the genus Janthinobacterium, for which the name Janthinobacterium kumbetense sp. nov. is proposed (GKT = LMG 32662T = DSM 11423T).

Investigation of potential inhibitor properties of violacein against HIV-1 RT and CoV-2 Spike RBD:ACE-2.

A violacein-producing bacterium was isolated from a mud sample collected near a hot spring on Kümbet Plateau in Giresun Province and named the GK strain. According to the phylogenetic tree constructed using 16S rRNA gene sequence analysis, the GK strain was identified and named Janthinobacterium sp. GK. The crude violacein pigments were separated into three different bands on a TLC sheet. Then violacein and deoxyviolacein were purified by vacuum liquid column chromatography and identified by NMR spectroscopy. According to the inhibition studies, the HIV-1 RT inhibition rate of 1 mM violacein from the GK strain was 94.28% and the CoV-2 spike RBD:ACE2 inhibition rate of 2 mM violacein was 53%. In silico studies were conducted to investigate the possible interactions between violacein and deoxyviolacein and three reference molecules with the target proteins: angiotensin-converting enzyme 2 (ACE2), HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain. Ligand violacein binds strongly to the receptor ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of -9.94 kcal/mol, -9.32 kcal/mol, and -8.27 kcal/mol, respectively. Deoxyviolacein strongly binds to the ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of -10.38 kcal/mol, -9.50 kcal/mol, and -8.06 kcal/mol, respectively. According to these data, violacein and deoxyviolacein bind to all the receptors quite effectively. SARS-CoV-2 spike protein and HIV-1-RT inhibition studies with violacein and deoxyviolacein were performed for the first time in the literature.

Arthrobacter pityocampae sp. nov., isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae).

A bacterium (strain Tp2(T)) was isolated from a caterpillar of the pine processionary moth, Thaumetopoea pityocampa (Den. & Schiff.) (Lepidoptera: Thaumetopoeidae), a destructive pine forest pest. The bacterium is a Gram-stain-positive, red-pigmented coccus, oxidase-negative, nitrate-reducing, non-motile and non-spore-forming. Strain Tp2(T) was subjected to a taxonomic study using polyphasic approach that included morphological and biochemical characterizations, 16S rRNA gene sequence analysis, DNA-DNA hybridization, DNA G+C content analysis, comparative fatty acid profiles, and analyses of quinones and polar lipids. The 16S rRNA gene sequence of strain Tp2(T) revealed that Arthrobacter agilis DSM 20550(T) was the closest known strain (98% 16S rRNA gene sequence similarity). DNA-DNA hybridization of A. agilis DSM 20550(T) and strain Tp2(T) resulted in a DNA-DNA relatedness value of 11.9% (20.2% reciprocal). The DNA base composition of strain Tp2(T) was 69.5 mol%, which is consistent with the other recognized members of Actinobacteria that have a high G+C content in their genome. The polar lipid pattern of strain Tp2(T) consisted of diphosphatidylglycerol (major), phosphatidylglycerol and phosphatidylinositol and unknown glycolipids. The cellular fatty acids were anteiso C15:0 and anteiso C17:0 and the major menaquinone was MK-9(II-H2). The peptidoglycan type was A3α with an L-Lys-L-Thr-L-Ala3 interpeptide bridge. The above-mentioned characterization qualifies strain Tp2(T) as genotypically and phenotypically distinct from closely related species of the genus Arthrobacter with validly published names. Strain Tp2(T) is therefore proposed to represent a novel species of the genus Arthrobacter, described as Arthrobacter pityocampae sp. nov. The type strain is Tp2(T) ( = DSM 21719(T) = NCCB 100254(T)).

Purification and characterization of the bacteriocin Thuricin Bn1 produced by Bacillus thuringiensis subsp. kurstaki Bn1 isolated from a hazelnut pest.

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.

Brevibacterium pityocampae sp. nov., isolated from caterpillars of Thaumetopoea pityocampa (Lepidoptera, Thaumetopoeidae).

This work deals with the taxonomic study of a bacterium, strain Tp12(T), isolated from caterpillars of the pine processionary moth (Thaumetopoea pityocampa Denis & Schiffermüller, 1775; Lepidoptera, Thaumetopoeidae). The isolate was assigned to the genus Brevibacterium on the basis of a polyphasic taxonomic study, including morphological and biochemical characteristics, 16S rRNA gene sequence analysis, fatty acid analysis and DNA G+C content. The highest 16S rRNA gene sequence similarity to this isolate was approximately 96 %, with the type strains of Brevibacterium album and Brevibacterium samyangense. Cellular fatty acids of the isolate are of the branched type, with the major components being anteiso-C(15 : 0) and anteiso-C(17 : 0). The DNA G+C content was 69.8 mol%. Although the strain was related to B. album and B. samyangense according to 16S rRNA gene sequence analysis, it differed from any known species of Brevibacterium. Based on this evidence, the novel species Brevibacterium pityocampae sp. nov. is proposed, with strain Tp12(T) (=DSM 21720(T) =NCCB 100255(T)) as the type strain.

Characterisation and toxicity of Bacillus thuringiensis strains from hazelnut pests and fields.

BACKGROUND: In order to find and identify more toxic insecticidal Bacillus thuringiensis Berliner (Bt) strains, a survey was carried out of B. thuringiensis isolate pests belonging to Coleoptera, Lepidoptera and Diptera and from soils in hazelnut fields. Of 16 isolates having Bacillus cereus-B. thuringiensis morphology, eight were classified as B. thuringiensis because of the production of parasporal delta-endotoxin crystals. RESULTS: In this study, eight isolates of B. thuringiensis from hazelnut pests (isolates Bn1, Mm2, Mnd and Xd3) and from hazelnut soils (isolates 6, 27, 40 and 46) have been characterised in detail. These isolates were compared with reference strains by electron microscopy, SDS-PAGE analysis, cry gene content, serological test and insecticidal activity. CONCLUSION: Results indicate that Bn1 and MnD are B. thuringiensis subsp. kurstaki, and Mm2 and Xd3 are B. thuringiensis subsp. tenebrionis. In addition, isolate 6 is B. thuringiensis subsp. israelensis, isolates 27 and 46 are B. thuringiensis subsp. kumamotoensis and isolate 40 is B. thuringiensis subsp. indiana. The four B. thuringiensis isolates from hazelnut pests may be valuable as biological control agents against coleopteran and lepidopteran insects.

A highly pathogenic strain of Bacillus thuringiensis serovar kurstaki in lepidopteran pests.

In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.

Investigations on bacteria as a potential biological control agent of summer chafer, Amphimallon solstitiale L. (Coleoptera: Scarabaeidae).

Studying the bacteria of hazardous insects allows the opportunity to find potentially better biological control agents. Therefore, in this study, bacteria from summer chafer (Amphimallon solstitiale L., Coleoptera: Scarabaeidae) we isolated and identified the insecticidal effects of bacteria isolated from A. solstitiale and Melolontha melolontha L. (common cockchafer, Coleoptera: Scarabaeidae) and the mixtures of these bacterial isolates were investigated on A. solstitiale larvae. Crystals from Bacillus sp. isolated from M. melolontha were also purified, and tested against the second and third-stage larvae of A. solstitiale. The bacterial isolates of A. solstitiale were identified as Pseudomonas sp., Pseudomonas sp., Bacillus cereus and Micrococcus luteus, based on their morphology, spore formation, nutritional features, and physiological and biochemical characteristics. The insecticidal effects of the bacterial isolates determined on the larvae of A. solstitiale were 90% with B. cereus isolated from A. solstitiale, and 75% with B. cereus, B. sphaericus and B. thuringiensis isolated from M. melolontha within ten days. The highest insecticidal effects of the mixed infections on the larvae of A. solstitiale were 100% both with B. cereus+B. sphaericus and with B. cereus+B. thuringiensis. In the crystal protein bioassays, the highest insecticidal effect was 65% with crystals of B. thuringiensis and B. sphaericus isolated from M. melolontha within seven days. Finally, our results showed that the mixed infections could be utilized as microbial control agents, as they have a 100% insecticidal effect on the larvae of A. solstitiale.