Expanding the toolbox for molecular microbial studies: from communities to single cells

Abstract

Recently, the molecular revolution and nanotechnologies have provided tools for expanding the knowledge of microorganisms. This thesis had its starting point in utilizing existing methods to identify specific microbes. Polymerase chain reaction (PCR)-based assays were employed for the detection of zoonotic virus (Orthopoxvirus) in capybara’s (Hydrochoerus hydrochaeris) faecal samples in an attempt to examine the spread of viruses and a possible route for viral outbreaks. Here, the existing method was sufficient to answer the question and find that viral genetic material is indeed present in these stool samples. The established PCR and sequencing-based methods were also applied to study bacteria responsible for the removal of highly chlorinated phenols from contaminated sites. Despite detecting bacterial species from samples in situ or from microcosm experiments, it became evident that the used method did not clarify which microbes specifically were responsible for bioremediation. The abundance of pentachlorophenol hydroxylase gene (pcpB), the major gene involved in the bioremediation process, was detected with quantitative PCR and the overall bacterial diversity with sequencing using the universal marker – the 16S rRNA gene. Yet, the real players of the process remained unknown. This led to the quest to specify the microorganisms that are carrying a specific gene. As a result, a novel method based on droplet microfluidics technology was developed to investigate microbes at single-cell resolution. Within these droplets, PCR is used to fuse 16S rRNA and a second gene of interest into concatemers. Through sequencing, the second gene can be associated with specific bacterial species. To make the method reliable, several individual steps in the procedure required both engineering and careful method validation. These included the elimination of errors stemming from the existence of DNA outside of the cells, the efficient breaking of the emulsion and hence the collection of DNA, the blocking of unwanted PCR amplification that was confounding the sequencing results, and ensuring that the sample was studied at single-cell resolution. The established methods may be applied to a variety of research where it is necessary to identify the microorganism carriers of specific genetically encoded functions, and therefore highlight the potential actors in the environmental processes. Keywords: Methods in microbiology; NGS; PCR; single-cell technology; methods in microbiology.