Design and Experimental Evaluation of a New RNA-FISH Probe to Detect and Identify Paenibacillus sp.

Abstract

Paenibacillus, rod-saped gram-positive endospores forming aerobic or facultative anaerobic bacteria, colonize diverse ecosystems and are involved in the biodegradation of cultural heritage assets. Biodeteriogenic microorganisms can be easily detected/identified by ribonucleic acid- fluorescent in situ hybridization RNA-FISH with specific probes. In this work, probes designed in silico were analyzed to calculate hybridization efficiency and specificity by varying the formamide concentration in the hybridization. The Pab489 probe showed excellent in silico performance with high theoretical maximum efficiency hybridization (99.99%) and specificity and was selected for experimental assays with target Paenibacillus sp. and non-target biodeteriogenic microorganisms. Results assessed by epifluorescence microscopy and flow cytometry revealed that, regardless of the formamide concentration, it was possible to observe that the Pab489-Cy3 probe had a similar signal intensity to the EUB338-Cy3 probe (positive control), so the presence of formamide, a highly toxic and carcinogenic compound used to aid the hybridization process, is not necessary. The designed probe used in FISH assays allows specific in situ identification of Paenibacillus spp. in microbial communities in a culture-independent way. This approach can be employed for screening Paenibacillus spp., showing great potential for future application in biodeterioration of heritage assets, in the search for Paenibacillus strains that produce compounds with biotechnological or medical potential.