Biology subjects

Brito, I. L.

Publications and source records attributed to Brito, I. L..

23 records · Page 2Linked to original sources

bioRxiv2022

Run-on sequencing reveals nascent transcriptomics of the human microbiome

Precise regulation of transcription initiation and elongation enables bacteria to control cellular responses to environmental stimuli. RNAseq is the most common tool for measuring the transcriptional output of bacteria, comprising predominantly mature transcripts. To gain further insight into transcriptional dynamics, it is necessary to discriminate actively transcribed loci from those represented in the total RNA pool. One solution is to capture RNA polymerase (RNAP) in the act of transcription, but current methods are restricted to culturable and genetically tractable organisms. Here, we apply precision run-on sequencing (PRO-seq) to profile nascent transcription, a method amenable to diverse species. We find that PRO-seq is well-suited to profile small, structured, or post-transcriptionally modified RNAs, which are often excluded from RNAseq libraries. When PRO-seq is applied to the human microbiome, we identify taxon-specific RNAP pause motifs. We also uncover concurrent transcription and cleavage of guide RNAs and tRNA fragments at active CRISPR and tRNA loci. We demonstrate the specific utility of PRO-seq as a tool for exploring transcriptional dynamics in diverse microbial communities.

microbiology↗

bioRxiv2021

Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR

The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links target ARGs with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases in a neutropenic patient population who are particularly vulnerable multidrug-resistant infections. We detect novel associations of two low-abundance genera, Romboutsia and Agathobacter, with a multi-drug resistant plasmid harbored by Klebsiella pneumoniae. We put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes in complex microbial communities.

microbiology↗

bioRxiv2020

Pangenome sequence evolution within human gut microbiomes is explained by gene-specific rather than host-specific selective pressures

Pangenomes - the cumulative set of genes encoded by a species - arise from evolutionary forces including horizontal gene transfer (HGT), drift, and selection. The relative importance of drift and selection in shaping pangenome structure has been recently debated, and the role of sequence evolution (point mutations) within mobile genes has been largely ignored, with studies focusing mainly on patterns of gene presence or absence. The effects of drift, selection, and HGT on pangenome evolution likely depends on the time scale being studied, ranging from ancient (e.g., between distantly related species) to recent (e.g., within a single animal host), and the unit of selection being considered (e.g., the gene, whole genome, microbial species, or human host). To shed light on pangenome evolution within microbiomes on relatively recent time scales, we investigate the selective pressures acting on mobile genes using a dataset that previously identified such genes in the gut metagenomes of 176 Fiji islanders. We mapped the metagenomic reads to mobile genes to call single nucleotide variants (SNVs) and calculate population genetic metrics that allowed us to infer deviations from a neutral evolutionary model. We found that mobile gene sequence evolution varied more by gene family than by human social attributes, such as household or village membership, suggesting that selection at the level of gene function is most relevant on these short time scales. Patterns of mobile gene sequence evolution could be qualitatively recapitulated with a simple evolutionary simulation, without the need to invoke an adaptive advantage of mobile genes to their bacterial host genome. This suggests that, at least on short time scales, a majority of the pangenome need not be adaptive. On the other hand, a subset of gene functions including defense mechanisms and secondary metabolism showed an aberrant pattern of molecular evolution, consistent with species-specific selective pressures or negative frequency-dependent selection not seen in prophages, transposons, or other gene categories. That mobile genes of different functions behave so differently suggests stronger selection at the gene level, rather than at the genome level. While pangenomes may be largely adaptive to their bacterial hosts on longer evolution time scales, here we show that, on shorter "human" time scales, drift and gene-specific selection predominate.

microbiology↗

bioRxiv2020

Widespread transfer of mobile antibiotic resistance genes within individual gut microbiomes revealed through bacterial Hi-C

The gut microbiome harbors a silent reservoir of antibiotic resistance (AR) genes that is thought to contribute to the emergence of multidrug-resistant pathogens through the process of horizontal gene transfer (HGT). To counteract the spread of AR genes, it is paramount to know which organisms harbor mobile AR genes and with which organisms they engage in HGT. Despite methods to characterize the bulk presence1, abundance2 and function3 of AR genes in the gut, technological limitations of short-read sequencing have precluded linking bacterial taxa to specific mobile genetic elements (MGEs) and their concomitant AR genes. Here, we apply and evaluate a high-throughput, culture-independent method for surveilling the bacterial carriage of MGEs, based on bacterial Hi-C protocols. We compare two healthy individuals with a cohort of seven neutropenic patients undergoing hematopoietic stem cell transplantation, who receive multiple courses of antibiotics throughout their prolonged hospitalizations, and are thus acutely vulnerable to the threat of multidrug-resistant infections4. We find that the networks of HGT are surprisingly distinct between individuals, yet AR and mobile genes are more dispersed across taxa within the neutropenic patients than the healthy subjects. Our data further suggest that HGT is occurring throughout the course of treatment in the microbiomes of neutropenic patients and within the guts of healthy individuals over a similar timeframe. Whereas most efforts to understand the spread of AR genes have focused on pathogenic species, our findings shed light on the role of the human gut microbiome in this process.

systems biology↗

bioRxiv2019

Transmission of human-associated microbiota along family and social networks

The human microbiome, described as an accessory organ because of the crucial functions it provides, is composed of species that are uniquely found in humans1,2. Yet, surprisingly little is known about the impact of routine interpersonal contacts in shaping microbiome composition. In a relatively closed cohort of 287 people from the Fiji Islands, where common barriers to bacterial transmission are absent, we examine putative bacterial transmission in individuals gut and oral microbiomes using strain-level data from both core SNPs and flexible genomic regions. We find a weak signal of transmission, defined by the inferred sharing of genotypes, across many organisms that, in aggregate, reveals strong transmission patterns, most notably within households and between spouses. We find that women harbor strains more closely related to those harbored by their familial and social contacts than men; and that transmission patterns of oral- and gut-associated microbiota need not be the same. Using strain-level data alone, we are able to confidently predict a subset of spouses, highlighting the role of shared susceptibilities, behaviors or social interactions that distinguish specific links in the social network.

microbiology↗