Bacterial droplet-based single-cell RNA-seq reveals antibiotic-associated heterogeneous cellular states.

We introduce BacDrop, a highly scalable technology for bacterial single-cell RNA sequencing that has overcome many challenges hindering the development of scRNA-seq in bacteria. BacDrop can be applied to thousands to millions of cells from both gram-negative and gram-positive species. It features universal ribosomal RNA depletion and combinatorial barcodes that enable multiplexing and massively parallel sequencing. We applied BacDrop to study Klebsiella pneumoniae clinical isolates and to elucidate their heterogeneous responses to antibiotic stress. In an unperturbed population presumed to be homogeneous, we found within-population heterogeneity largely driven by the expression of mobile genetic elements that promote the evolution of antibiotic resistance. Under antibiotic perturbation, BacDrop revealed transcriptionally distinct subpopulations associated with different phenotypic outcomes including antibiotic persistence. BacDrop thus can capture cellular states that cannot be detected by bulk RNA-seq, which will unlock new microbiological insights into bacterial responses to perturbations and larger bacterial communities such as the microbiome.

Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria.

There is increasing evidence that prokaryotes maintain chromosome structure, which in turn impacts gene expression. We recently characterized densely occupied, multi-kilobase regions in the E. coli genome that are transcriptionally silent, similar to eukaryotic heterochromatin. These extended protein occupancy domains (EPODs) span genomic regions containing genes encoding metabolic pathways as well as parasitic elements such as prophages. Here, we investigate the contributions of nucleoid-associated proteins (NAPs) to the structuring of these domains, by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and B. subtilis. We identify key NAPs contributing to the silencing of specific EPODs, whose deletion opens a chromosomal region for RNA polymerase binding at genes contained within that region. We show that changes in E. coli EPODs facilitate an extra layer of transcriptional regulation, which prepares cells for exposure to exotic carbon sources. Furthermore, we distinguish novel xenogeneic silencing roles for the NAPs Fis and Hfq, with the presence of at least one being essential for cell viability in the presence of domesticated prophages. Our findings reveal previously unrecognized mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements.

Correction: Dynamic landscape of protein occupancy across the Escherichia coli chromosome.

[This corrects the article DOI: 10.1371/journal.pbio.3001306.].

Dynamic landscape of protein occupancy across the Escherichia coli chromosome.

Free-living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein-DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using an optimized version of in vivo protein occupancy display technology (in vivo protein occupancy display-high resolution, IPOD-HR) and present a pilot application to Escherichia coli. We observe that the E. coli protein-DNA interactome organizes into 2 distinct prototypic features: (1) highly dynamic condition-dependent transcription factor (TF) occupancy; and (2) robust kilobase scale occupancy by nucleoid factors, forming silencing domains analogous to eukaryotic heterochromatin. We show that occupancy dynamics across a range of conditions can rapidly reveal the global transcriptional regulatory organization of a bacterium. Beyond discovery of previously hidden regulatory logic, we show that these observations can be utilized to computationally determine sequence specificity models for the majority of active TFs. Our study demonstrates that global observations of protein occupancy combined with statistical inference can rapidly and systematically reveal the transcriptional regulatory and structural features of a bacterial genome. This capacity is particularly crucial for non-model bacteria that are not amenable to routine genetic manipulation.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast.

Timely completion of genome replication is a prerequisite for mitosis, genome integrity, and cell survival. A challenge to this timely completion comes from the need to replicate the hundreds of untranscribed copies of rDNA that organisms maintain in addition to the copies required for ribosome biogenesis. Replication of these rDNA arrays is relegated to late S phase despite their large size, repetitive nature, and essentiality. Here, we show that, in Saccharomyces cerevisiae, reducing the number of rDNA repeats leads to early rDNA replication, which results in delaying replication elsewhere in the genome. Moreover, cells with early-replicating rDNA arrays and delayed genome-wide replication aberrantly release the mitotic phosphatase Cdc14 from the nucleolus and enter anaphase prematurely. We propose that rDNA copy number determines the replication time of the rDNA locus and that the release of Cdc14 upon completion of rDNA replication is a signal for cell cycle progression.

Nucleoid-associated proteins shape chromatin structure and transcriptional regulation across the bacterial kingdom.

Genome architecture has proven to be critical in determining gene regulation across almost all domains of life. While many of the key components and mechanisms of eukaryotic genome organization have been described, the interplay between bacterial DNA organization and gene regulation is only now being fully appreciated. An increasing pool of evidence has demonstrated that the bacterial chromosome can reasonably be thought of as chromatin, and that bacterial chromosomes contain transcriptionally silent and transcriptionally active regions analogous to heterochromatin and euchromatin, respectively. The roles played by histones in eukaryotic systems appear to be shared across a range of nucleoid-associated proteins (NAPs) in bacteria, which function to compact, structure, and regulate large portions of bacterial chromosomes. The broad range of extant NAPs, and the extent to which they differ from species to species, has raised additional challenges in identifying and characterizing their roles in all but a handful of model bacteria. Here we review the regulatory roles played by NAPs in several well-studied bacteria and use the resulting state of knowledge to provide a working definition for NAPs, based on their function, binding pattern, and expression levels. We present a screening procedure which can be applied to any species for which transcriptomic data are available. Finally, we note that NAPs tend to play two major regulatory roles - xenogeneic silencers and developmental regulators - and that many unrecognized potential NAPs exist in each bacterial species examined.

Polyphosphate drives bacterial heterochromatin formation.

Heterochromatin is most often associated with eukaryotic organisms. Yet, bacteria also contain areas with densely protein-occupied chromatin that appear to silence gene expression. One nucleoid-associated silencing factor is the conserved protein Hfq. Although seemingly nonspecific in its DNA binding properties, Hfq is strongly enriched at AT-rich DNA regions, characteristic of prophages and mobile genetic elements. Here, we demonstrate that polyphosphate (polyP), an ancient and highly conserved polyanion, is essential for the site-specific DNA binding properties of Hfq in bacteria. Absence of polyP markedly alters the DNA binding profile of Hfq, causes unsolicited prophage and transposon mobilization, and increases mutagenesis rates and DNA damage­induced cell death. In vitro reconstitution of the system revealed that Hfq and polyP interact with AT-rich DNA sequences and form phase-separated condensates, a process that is mediated by the intrinsically disordered C-terminal extensions of Hfq. We propose that polyP serves as a newly identified driver of heterochromatin formation in bacteria.

The ENCODE Blacklist: Identification of Problematic Regions of the Genome.

Functional genomics assays based on high-throughput sequencing greatly expand our ability to understand the genome. Here, we define the ENCODE blacklist- a comprehensive set of regions in the human, mouse, worm, and fly genomes that have anomalous, unstructured, or high signal in next-generation sequencing experiments independent of cell line or experiment. The removal of the ENCODE blacklist is an essential quality measure when analyzing functional genomics data.

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants.

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae.