Coyne, M. J. | bioRxiv Science

bioRxiv2021

Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species sweeps and geographical clustering

The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on integrative and conjugative elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population sweeps of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 177 MGE that sweep through multiple Bacteroidales species within individual gut microbiomes. We further show that only eight MGE demonstrate multi-species population sweeps in as many human gut microbiomes as the GA1 and GA2 ICE. These data underscore the ubiquity and rapid dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.

microbiology↗

bioRxiv2020

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen (1), with oxygen available to bacteria growing at the mucus layer. Using a combination of analyses, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 - 0.14% oxygen, defined as nanaerobic conditions (2). Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields high fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic Type VI secretion system processes in metabolically active B. fragilis. The ability to visualize fluorescently-labeled Bacteroides and fluorescently-linked proteins in actively growing nanaerobic gut symbionts ushers in a new age of imaging analyses in these bacteria. SignificanceDespite many recent technological advances to study the human gut microbiota, we still lack a facile system to image dynamic cellular processes in most abundant gut species due to the requirement of oxygen for chromophore maturation of commonly used fluorescent proteins. Here, we took advantage of the ability of anaerobes of the gut microbiota to respire aerobically and grow robustly at 0.10- 0.14% oxygen. This physiologic concentration of oxygen is sufficient for fluorescent proteins to mature, allowing for visualization of biological processes never before imaged in these bacteria. This advance will allow for numerous types of analyses in actively-growing "nanaerobic" gut bacteria including subcellular protein localizations, single-cell analyses, biofilm imaging, and protein interactions with other microbes and the host.

microbiology↗