New Insights into Molecular Basis Identification of Three Novel Strains of the Bacillus Subtilis Group Produce Cry Proteins Isolated from Soil Samples in Adana, Turkey

Aims: This study aimed to analyze the evolutionary relationship between Bacillus species isolated from agricultural soil using in-silico tools. Methods and Results: A cross-sectional study was conducted in Adana province, in Turkey. A total of 120 Bacillus species were isolated from 80 soil samples. However, the phylogenetic tree diverged into two lineages; one belongs to B. subtilis group while the other belongs to B. cereus group. Interestingly, three native strains (SY27.1A, SY35.3A, and SY58.5A), which produce Cry proteins, shared high similarity with B. subtilis group (over 99%) and less than 95% similarity with known B. thuringiensis and other species of B. cereus group. Furthermore, 11 canonical SNPs (canSNPs) were identified in strains that belong to B. pumilus group when compared with B. subtilis reference sequences. Conclusions: Phylogenetic analysis of 16S rRNA sequences was found valuable for differentiation between Bacillus species isolated from soil samples. In addition, SNPs analysis provided more intra-specific information in the cases of B. subtilis group. Significance and Impact of Study: A detailed analysis was provided for the SNPs present in a conserved region of 16S rRNA gene of Bacillus species. Also, we proposed three novel Bacillus strains that produce Cry proteins and belong to B. subtilis group.

Micellar Antibiotics of Bacillus

Members of the Bacillus genus, particularly the “Bacillus subtilis group”, are known to produce amphipathic lipopeptides with biosurfactant activity. This includes the surfactins, fengycins and iturins that have been associated with antibacterial, antifungal, and anti-viral properties. We have screened a large collection of Bacillus, isolated from human, animal, estuarine water and soil samples and found that the most potent lipopeptide producers are members of the species Bacillus velezensis. B. velezensis lipopeptides exhibited anti-bacterial activity which was localised on the surface of both vegetative cells and spores. Interestingly, lipopeptide micelles (6–10 nm diameter) were detectable in strains exhibiting the highest levels of activity. Micelles were stable (heat and gastric stable) and shown to entrap other antimicrobials produced by the host bacterium (exampled here was the dipeptide antibiotic chlorotetaine). Commercially acquired lipopeptides did not exhibit similar levels of inhibitory activity and we suspect that micelle formation may relate to the particular isomeric forms produced by individual bacteria. Using naturally produced micelle formulations we demonstrated that they could entrap antimicrobial compounds (e.g., clindamycin, vancomycin and resveratrol). Micellar incorporation of antibiotics increased activity. Bacillus is a prolific producer of antimicrobials, and this phenomenon could be exploited naturally to augment antimicrobial activity. From an applied perspective, the ability to readily produce Bacillus micelles and formulate with drugs enables a possible strategy for enhanced drug delivery.

The Change of Bacterial Spectrum after Storage of X. campestris pv. campestris Inoculated Cabbage Heads (Brassica oleracea var. capitata L.)

Changes in the bacterial spectrum of cabbage heads after storage under commonly used storage conditions were examined in this study. Cabbage seeds (Brassica oleracea var. capitata L.) were artificially inoculated with X. campestris pv. campestris (Xcc), a serious pathogen of cruciferous plants causing black rot. Isolation of bacterial cultures from Xcc-inoculated and non-inoculated cabbage heads were carried out in two time points—at the day of harvest and after four months of storage. According to our previous research and literature reports, the most frequent genera of bacteria were chosen for PCR testing, i.e., Bacillus cereus group, Bacillus subtilis group, Pseudomonas sp., and X. campestris pv. campestris. A few of the obtained bacterial cultures were negative for the four above-mentioned species. In those, other bacteria were identified by 16S rRNA sequencing. In both Xcc-inoculated and non-inoculated cabbage heads, changes of the bacterial spectrum over time were observed. The severity of Xcc infection of heads increased after four months of storage. Bacillus species represented the most frequently occurring bacterial genus. The presence of the Bacillus subtilis group increased significantly after storage in non-inoculated cabbage heads. The minor part of the other genera identified by sequencing in the first sampling were not detected in the stored cabbage heads. This was associated with a possible antagonistic behavior of Pseudomonas sp. and Bacillus sp.

Antibiotic susceptibilty of bacteria from the Bacillus subtilis group

The ability of bacteria to quickly acquire antibiotic resistance and transfer it among different groups are created a global problem, since antibiotics are used not only in medicine, but also in agriculture. That is why to study of the antibiotic sensitivity of not only pathogenic bacteria, but also of beneficial microbes like Bacillus spp. is extremely important. Our results demonstrated that all the studied strains of the B. subtilis group were highly sensitive to cephalexin and enrofloxacin, while resistant to oxacillin. The B. subtilis B-5449 strain was the least resistant to the studied antibiotics, and the B. subtilis B-2896 strain was the most resistant.

Endophytic Detection in Selected European Herbal Plants

A total of 181 cultivable endophytic bacterial isolates were collected from stems of 13 species of herbs inhabiting Europe (Poland): Chelidonium majus L., Elymus repens L., Erigeron annuus L., Euphrasia rostkoviana Hayne, Foeniculum vulgare L., Geranium pratense L., Humulus lupulus L., Matricaria chamomilla L., Mentha arvensis L., Papaver rhoeas L., Rosmarinus officinalis L., Solidago gigantea L. and Vinca minor L. The isolates were screened for their antifungal activity and fifty three were found to inhibit fungal growth. Of these, five had strong antifungal properties. These selected isolates were identified as: Pseudomonas azotoformans, P. cedrina, Bacillus subtilis group and Erwinia persicina.