Biology subjects

Pogliano, J.

Publications and source records attributed to Pogliano, J..

34 records · Page 2Linked to original sources

bioRxiv2023

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were unknown. By studying phages that encode the major phage nucleus protein chimallin, including previously sequenced yet uncharacterized phages, we discovered that chimallin-encoding phages share a set of 72 highly conserved genes encoded within seven distinct gene blocks. Of these, 21 core genes are unique to this group, and all but one of these unique genes encode proteins of unknown function. We propose that phages with this core genome comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryo-electron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication encoded in the core genome are conserved among diverse chimalliviruses, and reveal that non-core components can confer intriguing variations on this replication mechanism. For instance, unlike previously studied nucleus-forming phages, RAY doesnt degrade the host genome, and its PhuZ homolog appears to form a five-stranded filament with a lumen. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

microbiology↗

bioRxiv2022

Advanced transcriptomic analysis reveals the role of efflux pumps and media composition in antibiotic responses of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen and major cause of hospital acquired infections. The pathogenicity and virulence of P. aeruginosa is largely determined by its transcriptional regulatory network (TRN). We used 411 transcription profiles of P. aeruginosa from diverse growth conditions to construct a quantitative TRN by identifying independently modulated sets of genes (called iModulons) and their condition-specific activity levels. The current study focused on the use of iModulons to analyze pathogenicity and antibiotic resistance of P. aeruginosa. Our analysis revealed: 1) 116 iModulons, 81 of which show strong association with known regulators; 2) novel roles of two-component systems in regulating antibiotics efflux pumps; 3) substrate-efflux pump associations; 4) differential iModulon activity in response to beta-lactam antibiotics in bacteriological and physiological media; 5) differential activation of Cell Division iModulon resulting from exposure to different beta-lactam antibiotics; and 6) a role of the PprB iModulon in the stress-induced transition from planktonic to biofilm lifestyle. In light of these results, the construction of an iModulon-based TRN provides a transcriptional regulatory basis for key aspects of P. aeruginosa infection, such as antibiotic stress responses and biofilm formation. Taken together, our results offer a novel mechanistic understanding of P. aeruginosa pathogenicity. SignificanceLarge data sets and machine learning are impacting a growing number of areas of research in the life sciences. Once the compendia of bacterial transcriptomes reached a critical size, we could use source signal extraction algorithms to find lists of co-regulated genes (called iModulons) associated with a transcription factor (TF) to them. The gene composition of iModulons and their condition-dependent activity levels constitute a quantitative description of the composition of bacterial transcriptomes. This study shows how this approach can be used to reveal the responses of P. aeruginosa to antibiotics and thus yield a deep regulatory understanding of pathogenicity properties. This study motivates the execution of similar studies for the other ESKAPEEs to yield a broad understanding of the role of TRNs in antibiotic responses to these urgent threat bacterial pathogens.

systems biology↗

bioRxiv2022

Architecture and self-assembly of the jumbo bacteriophage nuclear shell

Bacteria encode myriad defenses that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR/Cas systems. In response, one family of large bacteriophage employs a nucleus-like compartment to protect their replicating genomes by excluding host defense factors. However, the principle composition and structure of this compartment remain unknown. Here, we find that the bacteriophage nuclear shell assembles primarily from one protein, termed chimallin. Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin cooperatively self-assembles as a flexible sheet into closed micron-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

biophysics↗

bioRxiv2021

A Cytoskeletal Vortex Drives Phage Nucleus Rotation During Jumbo Phage Replication inE. coli

Vortex-like arrays of cytoskeletal filaments that drive cytoplasmic streaming and nucleus rotation have been identified in eukaryotes, but similar structures have not been described in prokaryotes. The only known example of a rotating intracellular body in prokaryotic cells occurs when nucleus-forming jumbo phages infect Pseudomonas. During infection, a bipolar spindle of PhuZ filaments drives intracellular rotation of the phage nucleus, a key aspect of the replication cycle. Here we show the E. coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of mutant PhuZ strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes, this work identifies the first known example of a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.

microbiology↗

bioRxiv2021

Machine Learning of Pseudomonas aeruginosa transcriptomes identifies independently modulated sets of genes associated with known transcriptional regulators

The transcriptional regulatory network (TRN) of Pseudomonas aeruginosa plays a critical role in coordinating numerous cellular processes. We extracted and quality controlled all publicly available RNA-sequencing datasets for P. aeruginosa to find 281 high-quality transcriptomes. We produced 83 new RNAseq data sets under critical conditions to generate a comprehensive compendium of 364 transcriptomes. We used this compendium to reconstruct the TRN of P. aeruginosa using independent component analysis (ICA). We identified 104 independently modulated sets of genes (called iModulons), among which 81 (78%) reflect the effects of known transcriptional regulators. We show that iModulons: 1) play an important role in defining the genomic boundaries of biosynthetic gene clusters (BGCs); 2) show increased expression of the BGCs and associated secretion systems in conditions that emulate cystic fibrosis (CF); 3) show the presence of a novel BGC named RiPP (bacteriocin producer) which might have a role in worsening CF outcomes; 4) exhibit the interplay of amino acid metabolism regulation and central metabolism across carbon sources, and 5) clustered according to their activity changes to define iron and sulfur stimulons. Finally, we compare the iModulons of P. aeruginosa with those of E. coli to observe conserved regulons across two gram negative species. This comprehensive TRN framework covers almost every aspect of the transcriptional regulatory machinery in P. aeruginosa, and thus could prove foundational for future research of its physiological functions.

bioinformatics↗

bioRxiv2021

Subcellular Organization of Viral Particles During Maturation of Nucleus-Forming Jumbo Phage

Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages were long assumed to assemble randomly throughout the host cell cytoplasm. Here we visualized the subcellular location of viral particles formed during replication of Pseudomonas nucleus-forming jumbo phages and discovered that they assemble a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to the capsids, and these particles accumulate to form bouquets at specific subcellular locations. In these bouquets, the viral particles are arranged in a spherical pattern with tails oriented inward and the heads outwards. Localized at fixed distances on either side of the phage nucleus, bouquets grow in size and number over time as new phage particles are added. In the presence of mutations that cause the phage nucleus to be mispositioned away from its typical position at the midcell, bouquets still localize at the same fixed distance from the nucleus, suggesting an active mechanism for their formation and positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phage, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.

microbiology↗

bioRxiv2021

Bacterial Cytological Profiling Identifies Rhodanine-containing PAINS Analogs as Specific Inhibitors of E. coli Thymidylate Kinase in Vivo

In this study, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules inhibiting the growth of E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite their structural similarity to related PAINS. A genetic analysis of resistant mutants revealed that thymidylate kinase (an enzyme essential for DNA synthesis) is an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by assays of in vitro activity as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC50 in vitro and MIC in vivo displayed the greatest specificity for the inhibition of DNA replication in E. coli, despite containing a rhodamine moiety. While its generally thought that PAINS cannot be developed as antibiotics, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics, and it showcases novel inhibitors of E. coli thymidylate kinase. ImportanceWe demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotics in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

microbiology↗

bioRxiv2020

Selective transport of fluorescent proteins into the phage nucleus

Upon infection of Pseudomonas cells, jumbo phages 201{Phi}2-1, {Phi}PA3, and {Phi}KZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with {Phi}PA3 or 201{Phi}2-1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the {Phi}KZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the {Phi}KZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the {Phi}KZ phage nucleus.

microbiology↗

bioRxiv2020

Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcares most potent antibiotics are becoming obsolete. Approximately two-thirds of the worlds antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, in order to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 101 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

microbiology↗

bioRxiv2020

Characterizing the response of Acinetobacter baumannii ATCC 17978 to azithromycin in multiple in vitro growth conditions

Multi-drug resistant (MDR) Acinetobacter baumannii is one of the most concerning pathogens in hospital infections. A. baumannii is categorized as an "Urgent Threat" by the U.S. Centers for Disease Control and the highest priority pathogen by the World Health Organization due to its propensity for broad antibiotic resistance and its associated high mortality rates. New treatment options are urgently needed for MDR A. baumannii infections. Our prior studies have demonstrated an unappreciated utility of the macrolide azithromycin (AZM) against MDR A. baumannii in tissue-culture medium. This finding is all the more surprising since AZM has no appreciable activity against A. baumannii in standard bacteriological media. The basis for this media-dependent activity of AZM against A. baumannii is not fully defined. In this study, we utilize a variety of techniques (growth dynamics, bacterial cytological profiling, RNA sequencing, and LC/MS) to profile the response of MDR A. baumannii to AZM in both standard bacteriological and more physiological relevant mammalian tissue-culture medium. [Table 1]

microbiology↗

bioRxiv2020

Identifying the effect of vancomycin on HA-MRSA strains using bacteriological and physiological media

Healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern due to their evolving antibiotic resistant. They are resistant to a broad class of antibiotics like macrolides, aminoglycosides, fluoroquinolones, and many more. The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (CA-MHB) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behaviour of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments. The study reveals the vancomycin resistance behavior of USA100 strains in dual media conditions using wide-ranging experiments.

microbiology↗

bioRxiv2020

Profiling the effect of nafcillin on HA-MRSA D592 using bacteriological and physiological media

Staphylococcus aureus is a leading human pathogen associated with both hospital-acquired and community-acquired infections. The bacterium has steadily gained resistance to {beta}-lactams and other important first-line antibiotics culminating in its categorization as an urgent threat by the U.S. Centers for Disease Control and Prevention. Observations of a varying response to antimicrobial exposure as a function of media type has revealed that clinical susceptibility testing performed in standard bacteriological media might not adequately represent pharmacological responses in the patient. Such observations have encouraged research designed to identify media types that more closely mimic the in vivo environment. In this study, we examine the response of a hospital-acquired USA100 lineage methicillin-resistant, vancomycin-intermediate S. aureus (MRSA/VISA) strain (D592) to nafcillin in a bacteriological compared to a more physiological tissue culture-based medium. We performed multi-dimensional analysis including growth and bacterial cytological profiling, RNA sequencing, and exo-metabolomics measurements (both HPLC and LC/MS) to shed light on the media-dependent activity of the commonly prescribed {beta}-lactam antibiotic nafcillin. O_TBL View this table: org.highwire.dtl.DTLVardef@85b95borg.highwire.dtl.DTLVardef@14c1504org.highwire.dtl.DTLVardef@1f7e976org.highwire.dtl.DTLVardef@100598dorg.highwire.dtl.DTLVardef@1f770e9_HPS_FORMAT_FIGEXP M_TBL C_TBL

microbiology↗

bioRxiv2020

Revealing 29 sets of independently modulated genes in Staphylococcus aureus, their regulators and role in key physiological responses

The ability of Staphylococcus aureus to infect many different tissue sites is enabled, in part, by its Transcriptional Regulatory Network (TRN) that coordinates its gene expression to respond to different environments. We elucidated the organization and activity of this TRN by applying Independent Component Analysis (ICA) to a compendium of 108 RNAseq expression profiles from two S. aureus clinical strains (TCH1516 and LAC). ICA decomposed the S. aureus transcriptome into 29 independently modulated sets of genes (i-modulons) that revealed (1) high confidence associations between 21 i-modulons and known regulators; (2) an association between an i-modulon and {sigma}S, whose regulatory role was previously undefined; (3) the regulatory organization of 65 virulence factors in the form of three i-modulons associated with AgrR, SaeR and Vim-3, (4) the roles of three key transcription factors (codY, Fur and ccpA) in coordinating the metabolic and regulatory networks; and (5) a low dimensional representation, involving the function of few transcription factors, of changes in gene expression between two laboratory media (RPMI, CAMHB) and two physiological media (blood and serum). This representation of the TRN covers 842 genes representing 76% of the variance in gene expression that provides a quantitative reconstruction of transcriptional modules in S. aureus, and a platform enabling its full elucidation. Significance StatementStaphylococcus aureus infections impose an immense burden on the healthcare system. To establish a successful infection in a hostile host environment, S. aureus must coordinate its gene expression to respond to a wide array of challenges. This balancing act is largely orchestrated by the Transcriptional Regulatory Network (TRN). Here, we present a model of 29 independently modulated sets of genes that form the basis for a segment of the TRN in clinical USA300 strains of S. aureus. Using this model, we demonstrate the concerted role of various cellular systems (e.g. metabolism, virulence and stress response) underlying key physiological responses, including response during blood infection.

microbiology↗

bioRxiv2018

Mechanistic Origins of Dynamic Instability in Filaments from the Phage Tubulin, PhuZ

A bacteriophage-encoded tubulin homologue, PhuZ, harnesses dynamic instability to position genomes of KZ-like bacteriophage at the midline of their Pseudomonas hosts, facilitating phage infectivity. While much has been learned about molecular origins of microtubule dynamics, how GTP binding and hydrolysis control dynamics in the divergent 3-stranded PhuZ filaments is not understood. Here we present cryo-EM reconstructions of the PhuZ filamentin a pre-hydrolysis (3.5[A]) and three post-hydrolysis states (4.2 [A], 7.3 [A] and 8.1 [A] resolutions), likely representing distinct depolymerization stages. Core polymerization-induced structural changes reveal similarities to {beta}-tubulin, suggesting broad conservation within the tubulin family. By contrast, GTP hydrolysis is sensed quite differently and is communicated by the divergent PhuZ C-terminus to the lateral interface, leading to PhuZ polymer destabilization. This provides a contrasting molecular description of how nucleotide state can be harnessed by the tubulin fold to regulate filament assembly, metastability and disassembly.

cell biology↗

bioRxiv2017

Phenotypic consequences of RNA polymerase dysregulation in Escherichia coli

Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the adapted bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits.

microbiology↗

bioRxiv2017

Polymer dynamics of Alp7A reveals two ‘critical’ concentrations that govern dynamically unstable actin-like proteins

ABSTRACTDynamically unstable polymers capture and move cellular cargos in both bacteria and eukaryotes, but the regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial Actin-Like Protein (ALP) that segregates the low copy-number plasmid pLS20 in Bacillus subtilis. Purified Alp7A forms dynamically unstable polymers with two critical points: an intrinsic critical concentration (0.6 M), observed when ATP hydrolysis is blocked, and a dynamic critical concentration (10.3 M), observed when ATP hydrolysis occurs. From biochemical and kinetic analysis, the intrinsic critical concentration reflects a balance between filament elongation and shortening, while the dynamic critical concentration reflects a balance between filament nucleation and catastrophic disassembly. Although Alp7A does not form stable polymers at physiological concentrations, rapid nucleation by an accessory factor, Alp7R, decreases the dynamic critical concentration into the physiological range. Intrinsic and dynamic critical concentrations are fundamental parameters that can be used to describe the behavior of all dynamically unstable polymers.

biochemistry↗