Biology subjects

Laub, M. T.

Publications and source records attributed to Laub, M. T..

25 records · Page 2Linked to original sources

bioRxiv2021

High-resolution, genome-wide mapping of positive supercoiling in chromosomes

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the "twin-domain" model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

molecular biology↗

bioRxiv2020

Host adaptations in the fixLJ pathway of the Burkholderia cepacia complex increase virulence

The Burkholderia cepacia complex (BCC) is composed of multiple species, including B. multivorans and B. dolosa, that are significant pathogens for people with cystic fibrosis (CF) and are extensively resistant to many antibiotics. The fixL gene of the fixLJ 2-component system (TCS) in these BCC species shows evidence of positive selection for nonsynonymous mutations during chronic lung infection in CF. Previous work showed that the B. dolosa fixLJ system regulates 11% of the genome and modulates biofilm formation, motility, persistence within macrophages, and virulence in a murine pneumonia model. Here, we assess the impacts of clinically observed FixL evolved variants in fixLJ pathway-mediated phenotypes in B. dolosa and B. multivorans. BCC carrying the ancestral fixL sequence are less pathogenic than constructs carrying evolved variants in both a macrophage infection model and a murine pneumonia model. In vitro phospho-transfer experiments demonstrate that the evolved B. dolosa FixL variants are able to reduce fixLJ pathway activity by either having lower levels of kinase activity or increased phosphatase activity. Notably, the ancestral fixL genotype has increased ability to survive within the soil compared to isogenic constructs with evolved fixL genotypes, demonstrating that increased virulence comes at an expense. Modulation of the FixLJ system has profound effects on many BCC phenotypes including full pathogenicity, and this modulation is critical for BCC adaptation to the host.

microbiology↗

bioRxiv2020

Mucin glycans signal through the sensor kinase RetS to inhibit virulence-associated traits in Pseudomonas aeruginosa

Mucus is a densely populated ecological niche that coats all epithelia, and plays a critical role in protecting the human body from infections. Although traditionally viewed as a physical barrier, emerging evidence suggests that mucus can directly suppress virulence traits in opportunistic pathogens like Pseudomonas aeruginosa. However, the molecular mechanisms by which mucus affords this protection are unclear. Here, we show that mucins, and particularly their associated glycans, activate the sensor kinase RetS via its Dismed2 domain in P. aeruginosa. We find that this RetS-dependent signaling leads to the direct inhibition of the GacS-GacA two-component system, the activity of which is associated with a chronic infection state. This signaling includes the downregulation of the type VI secretion system (T6SS), and prevents T6SS-dependent bacterial killing by P. aeruginosa. Overall, these results shed light on how mucus impacts P. aeruginosa behavior in the human host, and may inspire novel approaches for controlling P. aeruginosa infections.

microbiology↗

bioRxiv2020

Stress induces the transcription of toxin-antitoxin systems but does not activate toxin

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking 10 TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA-sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems likely do not strongly promote survival following stress.

microbiology↗

bioRxiv2020

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

The profiling of gene expression by RNA-sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is ribosomal RNA (rRNA), it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued. Here, we report the development a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin beads then depletes rRNA from a complex, total RNA sample such that ~75-80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold-change measurements in RNA-seq experiments between our method and the previously available Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or metagenomic sample of interest. ImportanceThe ability to examine global patterns of gene expression in microbes through RNA-sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of ribosomal RNA from total RNA samples. Otherwise, rRNA would comprise upwards of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from messenger RNA or requiring high total read depth. A commonly used, kit for rRNA subtraction from Illumina was recently discontinued. Here, we report the development of a do-it-yourself kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligos enable sufficient rRNA depletion to produce RNA-seq data with 75-80% of reads comming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

microbiology↗

bioRxiv2018

Constriction rate modulation can drive cell size control and homeostasis in C. crescentus

Rod-shaped bacteria typically grow first via sporadic and dispersed elongation along their lateral walls, then via a combination of zonal elongation and constriction at the division site to form the poles of daughter cells. Although constriction comprises up to half of the cell cycle, its impact on cell size control and homeostasis has rarely been considered. To reveal the roles of cell elongation and constriction in bacterial size regulation during cell division, we captured the shape dynamics of Caulobacter crescentus with time-lapse structured illumination microscopy and used molecular markers as cell-cycle landmarks. We perturbed constriction rate using a hyperconstriction mutant or fosfomycin inhibition. We report that constriction rate contributes to both size control and homeostasis, by determining elongation during constriction, and by compensating for variation in pre-constriction elongation on a single-cell basis.

biophysics↗

bioRxiv2017

SMC Progressively Aligns Chromosomal Arms In Caulobacter crescentus But Is Antagonized By Convergent Transcription

The Structural Maintenance of Chromosomes (SMC) complex plays an important role in chromosome organization and segregation in most living organisms. In Caulobacter crescentus, SMC is required to align the left and the right arms of the chromosome that run in parallel down the long axis of the cell. However, the mechanism of SMC-mediated alignment of chromosomal arms remains elusive. Here, using a genome-wide chromosome conformation capture assay (Hi-C), chromatin immunoprecipitation with deep sequencing (ChIP-seq) and microscopy of single cells, we show that Caulobacter SMC is recruited to the centromeric parS site and that SMC-mediated arm alignment depends on the chromosome partitioning protein ParB. We provide evidence that SMC likely tethers the parS-proximal regions of the chromosomal arms together, promoting arm alignment. Strikingly, the co-orientation of DNA replication and the transcription of highly-expressed genes is crucial for chromosome-wide alignment. Highly-transcribed genes near parS that are oriented against DNA replication disrupt arm alignment suggesting that head-on transcription interferes with SMC translocation and arm alignment. Our results demonstrate a tight interdependence of bacterial chromosome organization and global patterns of transcription.

microbiology↗