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

Sílvia Arantes,Ana Teresa Caldeira,Patrícia Branco

doi:10.3390/app12052348

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.

Highlights

The genus Paenibacillus (Paenibacillaceae family), defined as belonging to the genusBacillus, was reclassified in 1993 by Ash et al [1], based on the sequence comparison of the 16S RNA gene, and is characterized as a Gram-positive, rod-shaped endosporeforming aerobic or facultative anaerobic bacteria [2,3,4]
Probe with a good performance, for it is required that the probe holds a low probability of hairpin and self-dimer formation and a high affinity and level of specificity and hybridization efficiency [34,35]
The affinity of the DNA-fluorescence in situ hybridization (FISH) is the propensity of the probe to bind to its target under given hybridization conditions

Summary

Introduction

The genus Paenibacillus (Paenibacillaceae family), defined as belonging to the genusBacillus, was reclassified in 1993 by Ash et al [1], based on the sequence comparison of the 16S RNA gene, and is characterized as a Gram-positive, rod-shaped endosporeforming aerobic or facultative anaerobic bacteria [2,3,4]. The species of the genus Paenibacillus colonize different environments [2,5,6,7,8,9,10,11,12,13,14,15] and most of them are found in the soil, often associated with plant roots, in a symbiotic relationship (promote plant growth), and can be exploited for use in agriculture. Many species of Paenibacillus produce antimicrobial compounds that are useful in medicine, and some have shown biodeteriogenic potential to cultural heritage (CH) assets (i.e., biodeterioration of murals) [5,8]. Due to its ability to colonize different environments, the genus Paenibacillus, often in association with Arthrobacter and Bacillus species, has been associated with the biodeterioration of wall paintings, with a variety of hygroscopic salts, including carbonates, chlorides, nitrates and sulfates, which form efflorescences on their surfaces [8,16,17]. Detection and identification of CH biodeteriogenic microorganisms, such as Paenibacillus spp., is crucial for the CH objects safeguarding [19,20]

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