Prophage-encoded methyltransferase drives adaptation of community-acquired methicillin-resistant Staphylococcus aureus.

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a novel mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.

Targeting Borrelia burgdorferi HtpG with a berserker molecule, a strategy for anti-microbial development.

Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization.

Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric ß-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.

Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete.

Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete's length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.

A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi.

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen.IMPORTANCE Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

Long-Distance Cooperative and Antagonistic RNA Polymerase Dynamics via DNA Supercoiling.

Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.

The purine biosynthesis regulator PurR moonlights as a virulence regulator in Staphylococcus aureus.

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli.

Cell size, cell growth, and cell cycle events are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. Here, we describe an image-based quantitative screen of the single-gene knockout collection of Escherichia coli and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics, and cell division. Functional analyses, together with high-dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.

Crosstalk between the tricarboxylic acid cycle and peptidoglycan synthesis in Caulobacter crescentus through the homeostatic control of α-ketoglutarate.

To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in Caulobacter crescentus alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the hfq deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis. In addition to serving as a key intermediate of the tricarboxylic acid (TCA) cycle, KG is a by-product of an enzymatic reaction required for the synthesis of peptidoglycan, a major component of the bacterial cell wall. Accumulation of KG in the hfq deletion mutant interferes with peptidoglycan synthesis, resulting in cell morphology defects and increased susceptibility to peptidoglycan-targeting antibiotics. This work thus reveals a direct crosstalk between the TCA cycle and cell wall morphogenesis. This crosstalk highlights the importance of metabolic homeostasis in not only ensuring adequate availability of biosynthetic precursors, but also in preventing interference with cellular processes in which these intermediates arise as by-products.

Ultra-High Resolution 3D Imaging of Whole Cells.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills.

Association of RNAs with Bacillus subtilis Hfq.

The prevalence and characteristics of small regulatory RNAs (sRNAs) have not been well characterized for Bacillus subtilis, an important model system for Gram-positive bacteria. However, B. subtilis was recently found to synthesize many candidate sRNAs during stationary phase. In the current study, we performed deep sequencing on Hfq-associated RNAs and found that a small subset of sRNAs associates with Hfq, an enigmatic RNA-binding protein that stabilizes sRNAs in Gram-negatives, but whose role is largely unknown in Gram-positive bacteria. We also found that Hfq associated with antisense RNAs, antitoxin transcripts, and many mRNA leaders. Several new candidate sRNAs and mRNA leader regions were also discovered by this analysis. Additionally, mRNA fragments overlapping with start or stop codons associated with Hfq, while, in contrast, relatively few full-length mRNAs were recovered. Deletion of hfq reduced the intracellular abundance of several representative sRNAs, suggesting that B. subtilis Hfq-sRNA interactions may be functionally significant in vivo. In general, we anticipate this catalog of Hfq-associated RNAs to serve as a resource in the functional characterization of Hfq in B. subtilis.

Identification of regulatory RNAs in Bacillus subtilis.

Post-transcriptional regulatory mechanisms are widespread in bacteria. Interestingly, current published data hint that some of these mechanisms may be non-random with respect to their phylogenetic distribution. Although small, trans-acting regulatory RNAs commonly occur in bacterial genomes, they have been better characterized in Gram-negative bacteria, leaving the impression that they may be less important for Firmicutes. It has been presumed that Gram-positive bacteria, in particular the Firmicutes, are likely to utilize cis-acting regulatory RNAs located within the 5' mRNA leader region more often than trans-acting regulatory RNAs. In this analysis we catalog, by a deep sequencing-based approach, both classes of regulatory RNA candidates for Bacillus subtilis, the model microorganism for Firmicutes. We successfully recover most of the known small RNA regulators while also identifying a greater number of new candidate RNAs. We anticipate these data to be a broadly useful resource for analysis of post-transcriptional regulatory strategies in B. subtilis and other Firmicutes.

A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales.

Formation of a Bacillus subtilis biofilm community requires an abundant matrix protein, TasA and an exopolysaccharide. The transcriptional regulatory pathways that control synthesis of these structural features are complex and responsive to multiple physiological and population signals. We report herein that an additional layer of co-transcriptional regulation is required for exopolysaccharide (eps) expression. This mechanism is mediated by a novel cis-acting RNA element, coined 'EAR', located between the second and third gene of the eps operon. The presence of the EAR element within the eps operon is required for readthrough of distally located termination signals. We also find that the EAR element promotes readthrough of heterologous termination sites. Based upon these observations, we hypothesize that the EAR element associates with RNA polymerase to promote processive antitermination, a process wherein the transcription elongation complex is altered by accessory factors to become resistant to pausing and termination signals. It is likely that this mechanism is required for eps expression to ensure full synthesis of the unusually long transcript (16 kb). We also identify the EAR element in other species within the order Bacillales, suggesting that a similar mechanism is required for synthesis of biofilm and capsular polysaccharide operons in other microorganisms.

Mechanism of mRNA destabilization by the glmS ribozyme.

An array of highly structured domains that function as metabolite-responsive genetic switches has been found to reside within noncoding regions of certain bacterial mRNAs. In response to intracellular fluctuations of their target metabolite ligands, these RNA elements exert control over transcription termination or translation initiation. However, for a particular RNA class within the 5' untranslated region (UTR) of the glmS gene, binding of glucosamine-6-phosphate stimulates autocatalytic site-specific cleavage near the 5' of the transcript in vitro, resulting in products with 2'-3' cyclic phosphate and 5' hydroxyl termini. The sequence corresponding to this unique natural ribozyme has been subjected to biochemical and structural scrutiny; however, the mechanism by which self-cleavage imparts control over gene expression has yet to be examined. We demonstrate herein that metabolite-induced self-cleavage specifically targets the downstream transcript for intracellular degradation. This degradation pathway relies on action of RNase J1, a widespread ribonuclease that has been proposed to be a functional homolog to the well-studied Escherichia coli RNase E protein. Whereas RNase E only poorly degrades RNA transcripts containing a 5' hydroxyl group, RNase J1 specifically degrades such transcripts in vivo. These findings elucidate key features of the mechanism for genetic control by a natural ribozyme and suggest that there may be fundamental biochemical differences in RNA degradation machinery between E. coli and other bacteria.

Structure and mechanism of a metal-sensing regulatory RNA.

Organisms maintain the correct balance of intracellular metals primarily through metal-sensing proteins that control transport and storage of the target ion(s). Here, we reveal the basis of metal sensing and genetic control by a metalloregulatory RNA. Our data demonstrate that a previously uncharacterized orphan riboswitch, renamed the "M-box," is a divalent metal-sensing RNA involved in Mg(2+) homeostasis. A combination of genetic, biochemical, and biophysical techniques demonstrate that Mg(2+) induces a compacted tertiary architecture for M-box RNAs that regulates the accessibility of nucleotides involved in genetic control. Molecular details are provided by crystallographic structure determination of a Mg(2+)-bound M-box RNA. Given the distribution of this RNA element, it may constitute a common mode for bacterial metal ion regulation, and its discovery suggests the possibility of additional RNA-based metal sensors in modern and primordial organisms.