A Culture-Independent Approach to Unravel Uncultured Bacteria and Functional Genes in a Complex Microbial Community

Abstract

Most microorganisms in nature are uncultured with unknown functionality. Sequence-based metagenomics alone answers ‘who/what are there?’ but not ‘what are they doing and who is doing it and how?’. Function-based metagenomics reveals gene function but is usually limited by the specificity and sensitivity of screening strategies, especially the identification of clones whose functional gene expression has no distinguishable activity or phenotypes. A ‘biosensor-based genetic transducer’ (BGT) technique, which employs a whole-cell biosensor to quantitatively detect expression of inserted genes encoding designated functions, is able to screen for functionality of unknown genes from uncultured microorganisms. In this study, BGT was integrated with Stable isotope probing (SIP)-enabled Metagenomics to form a culture-independent SMB toolbox. The utility of this approach was demonstrated in the discovery of a novel functional gene cluster in naphthalene contaminated groundwater. Specifically, metagenomic sequencing of the 13C-DNA fraction obtained by SIP indicated that an uncultured Acidovorax sp. was the dominant key naphthalene degrader in-situ, although three culturable Pseudomonas sp. degraders were also present in the same groundwater. BGT verified the functionality of a new nag2 operon which co-existed with two other nag and two nah operons for naphthalene biodegradation in the same microbial community. Pyrosequencing analysis showed that the nag2 operon was the key functional operon in naphthalene degradation in-situ, and shared homology with both nag operons in Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2. The SMB toolbox will be useful in providing deep insights into uncultured microorganisms and unravelling their ecological roles in natural environments.

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community

“…It is increasingly evident that as yet uncultured bacteria can have key roles in the biodegradation and bioremediation of environmental pollutants (Huang et al, 2009;Chen and Murrell, 2010;Wang et al, 2012). In this study, we demonstrated that MMI can be used to recover live cells of key pollutant degraders from a complex microbial community, such as biosludge, and that MRCs can be used for further eco-physiological studies.…”

“…Stable isotope probing (SIP) links uncultured microbial cells with the metabolism of specific stable isotope ( 13 C or 15 N)-labelled substrates (Radajewski et al, 2000;Manefield et al, 2002). SIP combined with metagenomics is able to establish a connection between bacterial identity and ecological function (Chen and Murrell, 2010;Wang et al, 2012). SIP requires that stable isotopes such as 13 C and 15 N be incorporated into biomass (DNA, RNA or protein) and therefore has limited success in processes that have no incorporation of stable isotopes into biomass, such as nitrification, denitrification, sulphate reduction, iron reduction, methanogenesis, co-metabolism in consortia or mixed organic carbon anabolism (Bombach et al, 2010;Nelson and Carlson, 2012).…”

Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil

“…To overcome the problem associated with the low recovery of labeled DNA inherent to SIP, amplification methods such as multiple displacement amplification may be used. Employing this method, Wang et al were able to assemble a nag gene cluster responsible for the conversion of naphthalene to salicylate (17). Presumably, this gene cluster was part of the genome of an uncultured Acidovorax sp., which was identified as a prevalent naphthalene degrader in situ.…”

“…Although this combination has been rec-ognized as a promising new approach in soil bioremediation studies (16), it has not been frequently implemented thus far (17). In the present study, we investigated the soil bacterial community of a facility collecting the road runoffs of a highway.…”

Fingerprinting microbiomes towards screening for microbial antibiotic resistance

“…Biofilm formation offers microbes an entirely different lifestyle weighing against the planktonic state providing protection from external exposures (e.g., antibiotics) for the community [45][46][47][48][49] . Recently, the field of single cell study has developed some breakthroughs allowing characterisation and interrogation of particular microbes at reasonable levels [50][51][52][53][54][55][56][57][58][59][60] . However, these remain insufficient to fulfil the need to study microbial interactions within the microbiome and their surrounding environment.…”