Design of a tunable bacterial gene expression system using engineered σ factors.

Extracytoplasmic function (ECF) σ factors selectively upregulate expression of specific genes in bacteria. These σ factors, belonging to the σ70 family, are much smaller than the primary, housekeeping σ factor with two helical domains that interact with the Pribnow box and the -35 element of the promoter DNA. Structural studies reveal that promoter specificity in a σ factor is determined by the interactions between a loop (L3) and the Pribnow box element. Similarly, the efficiency of transcription initiation is governed by the polypeptide linker between the two promoter-binding domains. Both these polypeptide segments are dynamic and poorly conserved among ECF σ factor homologs. This feature hitherto limited insights from protein-DNA interactions to be correlated with transcription initiation efficiency. Here, we describe an approach to characterize these features that govern the dynamic range of gene expression using chimeric Escherichia coli σE. The L3 loop and linker polypeptides in these σE chimeras were replaced by the corresponding segments from 10 annotated and functional Mycobacterium tuberculosis ECF σ's. In vitro and in vivo measurements to determine the effect of these polypeptide replacements provided an experimentally validated σE chimera- gene expression level data set. We illustrate the utility of this chimeric σE library in improving the efficiency of a biosynthetic pathway in E. coli. In a two-enzyme step, unaffected by feedback inhibition and substrate concentration, we show an increase in desired product levels by altering the relative intracellular levels of the target enzymes using this library of σ factors. The chimeric σE library thus demonstrates the feasibility of engineering σ factors to achieve bespoke expression levels of target genes for diverse applications in synthetic microbiology. IMPORTANCE: The synthesis of organic compounds involves the action of multiple enzymes in a biosynthetic pathway. Incorporating such biosynthetic pathways into microbes often leads to substantial cellular and metabolic stress resulting in low titers of the target compound. This limitation can be offset, in part, by optimizing enzyme efficiency and cellular enzyme concentration. The former involves significant efforts to achieve improvements in catalytic efficiency with the caveat that the metabolic load on a microbial cell imposed by the overexpression of the exogenous enzyme could result in reduced cell fitness. Here, we demonstrate the feasibility of engineered σ factors to modulate gene expression levels without significant genetic engineering. We note that changing the sequence of two flexible polypeptide loops without any changes to the structural scaffold of the transcription initiation factor σE could modulate the expression levels of the target genes. This ability provides a route to improve the efficiency of a biosynthetic pathway without altering the overall genomic makeup. The σE chimera library thus provides an avenue for pre-determined conditional gene expression of specific genes in Escherichia coli.

Characterization of RNase J.

RNase J is involved in RNA maturation as well as degradation of RNA to the level of mononucleotides. This enzyme plays a vital role in maintaining intracellular RNA levels and governs different steps of the cellular metabolism in bacteria. RNase J is the first ribonuclease that was shown to have both endonuclease and 5'-3' exonuclease activity. RNase J enzymes can be identified by their characteristic sequence features and domain architecture. The quaternary structure of RNase J plays a role in regulating enzyme activity. The structure of RNase J has been characterized from several homologs. These reveal extensive overall structural similarity alongside a distinct active site topology that coordinates a metal cofactor. The metal cofactor is essential for catalytic activity. The catalytic activity of RNase J is influenced by oligomerization, the choice and stoichiometry of metal cofactors, and the 5' phosphorylation state of the RNA substrate. Here we describe the sequence and structural features of RNase J alongside phylogenetic analysis and reported functional roles in diverse organisms. We also provide a detailed purification strategy to obtain an RNase J enzyme sample with or without a metal cofactor. Different methods to identify the nature of the bound metal cofactor, the binding affinity and stoichiometry are presented. Finally, we describe enzyme assays to characterize RNase J using radioactive and fluorescence-based strategies with diverse RNA substrates.

Crystal structures of non-uracil ring fragments in complex with Mycobacterium tuberculosis uracil DNA glycosylase (MtUng) as a starting point for novel inhibitor design: A case study with the barbituric acid fragment.

Uracil DNA glycosylase (UDG or Ung) is a key enzyme involved in uracil excision from the DNA as a repair mechanism. Designing Ung inhibitors is thus a promising strategy to treat different cancers and infectious diseases. The uracil ring and its derivatives have been shown to inhibit Mycobacterium tuberculosis Ung (MtUng), resulting from specific and strong binding with the uracil-binding pocket (UBP). To design novel MtUng inhibitors, we screened several non-uracil ring fragments hypothesised to occupy MtUng UBP due to their high similarity to the uracil structural motif. These efforts have resulted in the discovery of novel MtUng ring inhibitors. Here we report the co-crystallised poses of these fragments, confirming their binding within the UBP, thus providing a robust structural framework for the design of novel lead compounds. We selected the barbituric acid (BA) ring as a case study for further derivatisation and SAR analysis. The modelling studies predicted the BA ring of the designed analogues to interact with the MtUng UBP much like the uracil ring. The synthesised compounds were screened in vitro using radioactivity and a fluorescence-based assay. These studies led to a novel BA-based MtUng inhibitor 18a (IC50 = 300 µM) displaying ∼24-fold potency over the uracil ring.

Mutational and structural analyses of UdgX: insights into the active site pocket architecture and its evolution.

UdgX excises uracil from uracil-containing DNA to concurrently form a covalent bond with the resulting AP-DNA. Structurally, UdgX is highly similar to family-4 UDGs (F4-UDGs). However, UdgX is unique in possessing a flexible R-loop (105KRRIH109). Among the class-defining motifs, while its motif A (51GEQPG55) diverged to possess Q53 in place of A53/G53 in F4-UDGs, motif B [178HPS(S/A)(L/V)(L/V)R184] has remained unchanged. Previously, we proposed an SN1 mechanism resulting in a covalent bond between H109 and AP-DNA. In this study, we investigated several single/double mutants of UdgX. The H109A, H109S, H109G, H109Q, H109C and H109K mutants gain conventional UDG activity to varying levels. The crystal structures of UdgX mutants show topological changes in their active sites, rationalizing their UDG activities. The E52Q, E52N and E52A mutants reveal that E52 forms a catalytic dyad with H109 to enhance its nucleophilicity. The Q53A mutant supports that UdgX specific evolution of Q53 occurred essentially to stabilize the R-loop conformation. The R184A mutation (motif B) supports the role of R184 in substrate-binding. Taken together, the structural, bioinformatics, and mutational studies suggest that UdgX diverged from F4-UDGs, and the emergence of the characteristic R-loop in UdgX is functionally assisted by A53/G53 to Q53 changes in motif A.

Allosteric Determinants in High Temperature Requirement A Enzymes Are Conserved and Regulate the Population of Active Conformations.

High temperature requirement A (HtrA) are allosterically regulated enzymes wherein effector binding to the PDZ domain triggers proteolytic activity. Yet, it remains unclear if the inter-residue network governing allostery is conserved across HtrA enzymes. Here, we investigated and identified the inter-residue interaction networks by molecular dynamics simulations on representative HtrA proteases, Escherichia coli DegS and Mycobacterium tuberculosis PepD, in effector-bound and free forms. This information was used to engineer mutations that could potentially perturb allostery and conformational sampling in a different homologue, M. tuberculosis HtrA. Mutations in HtrA perturbed allosteric regulation─a finding consistent with the hypothesis that the inter-residue interaction network is conserved across HtrA enzymes. Electron density from data collected on cryo-protected HtrA crystals revealed that mutations altered the topology of the active site. Ensemble models fitted into electron density calculated from room-temperature diffraction data showed that only a fraction of these models had a catalytically competent active site conformation alongside a functional oxyanion hole thus providing experimental evidence that these mutations influenced conformational sampling. Mutations at analogous positions in the catalytic domain of DegS perturbed the coupling between effector binding and proteolytic activity, thus confirming the role of these residues in the allosteric response. The finding that a perturbation in the conserved inter-residue network alters conformational sampling and the allosteric response suggests that an ensemble allosteric model best describes regulated proteolysis in HtrA enzymes.

Paraneoplastic leukemoid syndrome in squamous cell carcinoma of the forearm.

The paraneoplastic leukemoid reaction is a rare haematological paraneoplastic syndrome, which is typically seen with solid tumours and squamous cell carcinomas. As an indication of bone marrow infiltration and malignancy involvement, it indicates a poor outcome and a grave prognosis. We report a woman in her 50s, who presented with an ulcer over the right forearm. Biopsy revealed squamous cell carcinoma. The patient underwent radiological investigations, which showed the presence of metastatic squamous cell carcinoma. Incidentally, the patient was found to have leucocytosis, which was attributed to a paraneoplastic leukemoid reaction, after ruling out all other causes of leukemoid reaction. Due to metastatic disease, the patient was planned for palliative radiotherapy and the best supportive care.

The unique N-terminal region of Mycobacterium tuberculosis sigma factor A plays a dominant role in the essential function of this protein.

SigA (σA) is an essential protein and the primary sigma factor in Mycobacterium tuberculosis (Mtb). However, due to the absence of genetic tools, our understanding of the role and regulation of σA activity and its molecular attributes that help modulate Mtb survival is scant. Here, we generated a conditional gene replacement of σA in Mtb and showed that its depletion results in a severe survival defect in vitro, ex vivo, and in vivo in a murine infection model. Our RNA-seq analysis suggests that σA either directly or indirectly regulates ∼57% of the Mtb transcriptome, including ∼28% of essential genes. Surprisingly, we note that despite having ∼64% similarity with σA, overexpression of the primary-like σ factor SigB (σB) fails to compensate for the absence of σA, suggesting minimal functional redundancy. RNA-seq analysis of the Mtb σB deletion mutant revealed that 433 genes are regulated by σB, of which 283 overlap with the σA transcriptome. Additionally, surface plasmon resonance, in vitro transcription, and functional complementation experiments reveal that σA residues between 132-179 that are disordered and missing from all experimentally determined σA-RNAP structural models are imperative for σA function. Moreover, phosphorylation of σA in the intrinsically disordered N-terminal region plays a regulatory role in modulating its activity. Collectively, these observations and analysis provide a rationale for the centrality of σA for the survival and pathogenicity of this bacillus.

Neuromucormycosis of Posterior Tibial Nerve: A Rare Presentation of Mucormycosis.

Opportunistic fungal infections are known to occur in immunocompromised patients. Mucormycosis is one of the most common opportunistic fungal infections with significant mortality rates. In this article, we present a case of an adult female, a known diabetic who presented with fever and pus discharge from the amputation site of toes in the left foot with blackening of the foot. Examination revealed gangrenous changes of the left foot with no distal pulses palpable. Computed tomography angiogram revealed no flow of blood in distal vessels of the left lower limb. Left below knee guillotine amputation was done. Intraoperative biopsy of the neurovascular bundle revealed invasive neuromucormycosis. She was started on liposomal amphotericin B. The wound started granulating after a few days with serial dressings and the patient was planned for split skin grafting.

Curation and cheminformatics analysis of a Ugi-reaction derived library (URDL) of synthetically tractable small molecules for virtual screening application.

Virtual screening (VS) is an important approach in drug discovery and relies on the availability of a virtual library of synthetically tractable molecules. Ugi reaction (UR) represents an important multi-component reaction (MCR) that reliably produces a peptidomimetic scaffold. Recent literature shows that a tactically assembled Ugi adduct can be subjected to further chemical modifications to yield a variety of rings and scaffolds, thus, renewing the interest in this old reaction. Given the reliability and efficiency of UR, we collated an UR derived library (URDL) of small molecules (total = 5773) for VS. The synthesis of the majority of URDL molecules may be carried out in 1-2 pots in a time and cost-effective manner. The detailed analysis of the average property and chemical space of URDL was also carried out using the open-source Datawarrior program. The comparison with FDA-approved oral drugs and inhibitors of protein-protein interactions (iPPIs) suggests URDL molecules are 'clean', drug-like, and conform to a structurally distinct space from the other two categories. The average physicochemical properties of compounds in the URDL library lie closer to iPPI molecules than oral drugs thus suggesting that the URDL resource can be applied to discover novel iPPI molecules. The URDL molecules consist of diverse ring systems, many of which have not been exploited yet for drug design. Thus, URDL represents a small virtual library of drug-like molecules with unexplored chemical space designed for VS. The structures of all molecules of URDL, oral drugs, and iPPI compounds are being made freely accessible as supplementary information for broader application.

Identification of a new and diverse set of Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) inhibitors using structure-based virtual screening: Experimental validation and molecular dynamics studies.

Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng), a key DNA repair enzyme, represents an attractive target for the design of new antimycobacterial agents. However, only a limited number of weak MtUng inhibitors are reported, primarily based on the uracil ring, and hence, lack diversity. We report the first structure-based virtual screening (SBVS) using three separate libraries consisting of uracil and non-uracil small molecules, together with the FDA-approved drugs. Twenty diverse virtual hits with the highest predicted binding were procured and screened using a fluorescence-based assay to evaluate their potential to inhibit MtUng. Several of these molecules were found to inhibit MtUng activity at low mM and µM levels, comparable to or better than several other reported Ung inhibitors. Thus, these molecules represent a diverse set of scaffolds for developing next-generation MtUng inhibitors. The most active uracil-based compound 5 (IC50 = 0.14 mM) was found to be âˆ¼ 15-fold more potent than the positive control, uracil. The binding stability and conformation of compound 5 in complex with the enzyme were further confirmed using molecular dynamics simulation.

Mycobacterium tuberculosis Transcription Factor EmbR Regulates the Expression of Key Virulence Factors That Aid in Ex Vivo and In Vivo Survival.

Mycobacterium tuberculosis encodes ~200 transcription factors that modulate gene expression under different microenvironments in the host. Even though high-throughput chromatin immunoprecipitation sequencing and transcriptome sequencing (RNA-seq) studies have identified the regulatory network for ~80% of transcription factors, many transcription factors remain uncharacterized. EmbR is one such transcription factor whose in vivo regulon and biological function are yet to be elucidated. Previous in vitro studies suggested that phosphorylation of EmbR by PknH upregulates the embCAB operon. Using a gene replacement mutant of embR, we investigated its role in modulating cellular morphology, antibiotic resistance, and survival in the host. Contrary to the prevailing hypothesis, under normal growth conditions, EmbR is neither phosphorylated nor impacted by ethambutol resistance through the regulation of the embCAB operon. The embR deletion mutant displayed attenuated M. tuberculosis survival in vivo. RNA-seq analysis suggested that EmbR regulates operons involved in the secretion pathway, lipid metabolism, virulence, and hypoxia, including well-known hypoxia-inducible genes devS and hspX. Lipidome analysis revealed that EmbR modulates levels of all lysophospholipids, several phospholipids, and M. tuberculosis-specific lipids, which is more pronounced under hypoxic conditions. We found that the EmbR mutant is hypersusceptible to hypoxic stress, and RNA sequencing performed under hypoxic conditions indicated that EmbR majorly regulates genes involved in response to acidic pH, hypoxia, and fatty acid metabolism. We observed condition-specific phosphorylation of EmbR, which contributes to EmbR-mediated transcription of several essential genes, ensuring enhanced survival. Collectively, the study establishes EmbR as a key modulator of hypoxic response that facilitates mycobacterial survival in the host. IMPORTANCE Mycobacterium tuberculosis modulates its transcriptional machinery in response to dynamic microenvironments encountered within the host. In this study, we identified that EmbR, a transcription factor, plays important roles in modulating cellular morphology, antibiotic resistance, and survival in the host. We found that EmbR undergoes condition-specific phosphorylation for its activation. Together, the study establishes a key role of EmbR as a transcriptional activator of genes belonging to multiple pathways, viz., virulence, secretion, or polyketide synthesis, that aid in mycobacterial survival during hypoxia and within the host.

Hepatic haemangioma causing isolated right-sided pleural effusion and bilateral pedal oedema.

Hepatic haemangioma (HH) is a common benign tumour of the liver and is usually asymptomatic. HH causing isolated right-sided pleural effusion and bilateral pedal oedema due to inferior vena cava (IVC) compression have never been reported in the literature. We report a 35-year-old male patient who presented with breathlessness and mass per abdomen. On examination, the patient was found to have right-sided pleural effusion, bilateral pedal oedema, hepatomegaly. Contrast-enhanced CT showed compression of the IVC by the HH. The patient was managed with right-sided intercostal drain insertion for pleural effusion and hepatic artery embolisation. The patient improved gradually with reduced pleural effusion and resolving pedal oedema.

Particle uptake driven phagocytosis in macrophages and neutrophils enhances bacterial clearance.

Humans are exposed to numerous synthetic foreign particles in the form of drug delivery systems and diagnostic agents. Specialized immune cells (phagocytes) clear these particles by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particles may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize Escherichia coli (E. coli) faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and Staphylococcus epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable abiotic and biotic material.

Mycobacterium tuberculosis SufR responds to nitric oxide via its 4Fe-4S cluster and regulates Fe-S cluster biogenesis for persistence in mice.

The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtb's physiology are not fully understood. Since NO damages iron-sulfur (Fe-S) clusters of essential enzymes, the mechanism(s) involved in regulating Fe-S cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O2 and H2O2 gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA-sequencing of MtbΔsufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of MtbΔsufR. Lastly, MtbΔsufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics.

Redox Regulation of the Quorum-sensing Transcription Factor AgrA by Coenzyme A.

Staphylococcus aureus (S. aureus) is an aggressive opportunistic pathogen of prominent virulence and antibiotic resistance. These characteristics are due in part to the accessory gene regulator (agr) quorum-sensing system, which allows for the rapid adaptation of S. aureus to environmental changes and thus promotes virulence and the development of pathogenesis. AgrA is the agr system response regulator that binds to the P2 and P3 promoters and upregulates agr expression. In this study, we reveal that S. aureus AgrA is modified by covalent binding of CoA (CoAlation) in response to oxidative or metabolic stress. The sites of CoAlation were mapped by liquid chromatography tandem mass spectrometry (LC-MS/MS) and revealed that oxidation-sensing Cys199 is modified by CoA. Surface plasmon resonance (SPR) analysis showed an inhibitory effect of CoAlation on the DNA-binding activity, as CoAlated AgrA had significantly lower affinity towards the P2 and P3 promoters than non-CoAlated AgrA. Overall, this study provides novel insights into the mode of transcriptional regulation in S. aureus and further elucidates the link between the quorum-sensing and oxidation-sensing roles of the agr system.

Novel insights into ATP-Stimulated Cleavage of branched DNA and RNA Substrates through Structure-Guided Studies of the Holliday Junction Resolvase RuvX.

Much of our understanding of the homologous recombination (HR) machinery hinges on studies using Escherichia coli as a model organism. Interestingly enough, studies on the HR machinery in different bacterial species casts doubt on the universality of the E. coli paradigm. The human pathogen Mycobacterium tuberculosis encodes two Holliday junction (HJ)-resolvase paralogues, namely RuvC and RuvX; however, insights into their structural features and functional relevance is still limited. Here, we report on structure-guided functional studies of the M. tuberculosis RuvX HJ resolvase (MtRuvX). The crystalline MtRuvX is a dimer in the asymmetric unit, and each monomer has a RNAse H fold vis-à-vis RuvC-like nucleases. Interestingly, MtRuvX also contains some unique features, including the residues essential for ATP binding/coordination of Mg2+ ions. Indeed, MtRuvX exhibited an intrinsic, robust ATPase activity, which was further accentuated by DNA cofactors. Structure-guided substitutions of single residues at the ATP binding/Mg2+coordination sites while markedly attenuating the ATPase activity completely abrogated HJ cleavage, indicating an unanticipated relationship between ATP hydrolysis and DNA cleavage. However, the affinity of ATPase-deficient mutants for the HJ was not impaired. Contrary to RuvC, MtRuvX exhibits relaxed substrate specificity, cleaving a variety of branched DNA/RNA substrates. Notably, ATP hydrolysis plays a regulatory role, rendering MtRuvX from a canonical HJ resolvase to a DNA/RNA non-sequence specific endonuclease, indicating a link between HJ resolvase and nucleic acid metabolism. These findings provide novel insights into the structure and dual-functional activities of MtRuvX, and suggest that it may play an important role in DNA/RNA metabolism.

Use of a molecular beacon based fluorescent method for assaying uracil DNA glycosylase (Ung) activity and inhibitor screening.

Uracil DNA glycosylases are an important class of enzymes that hydrolyze the N-glycosidic bond between the uracil base and the deoxyribose sugar to initiate uracil excision repair. Uracil may arise in DNA either because of its direct incorporation (against A in the template) or because of cytosine deamination. Mycobacteria with G, C rich genomes are inherently at high risk of cytosine deamination. Uracil DNA glycosylase activity is thus important for the survival of mycobacteria. A limitation in evaluating the druggability of this enzyme, however, is the absence of a rapid assay to evaluate catalytic activity that can be scaled for medium to high-throughput screening of inhibitors. Here we report a fluorescence-based method to assay uracil DNA glycosylase activity. A hairpin DNA oligomer with a fluorophore at its 5' end and a quencher at its 3' ends was designed incorporating five consecutive U:A base pairs immediately after the first base pair (5' C:G 3') at the top of the hairpin stem. Enzyme assays performed using this fluorescent substrate were seen to be highly sensitive thus enabling investigation of the real time kinetics of uracil excision. Here we present data that demonstrate the feasibility of using this assay to screen for inhibitors of Mycobacterium tuberculosis uracil DNA glycosylase. We note that this assay is suitable for high-throughput screening of compound libraries for uracil DNA glycosylase inhibitors.

Draft Genome Sequence of the Community-Associated Staphylococcus aureus Sequence Type 88 Strain LVP-7, Isolated from an Ocular Infection.

We report a de novo-assembled draft genome sequence of the Indian Staphylococcus aureus sequence type 88 (ST88) strain LVP-7, isolated from an ocular infection. The genome harbors a Panton-Valentine leukocidin phage, a type V staphylococcal cassette chromosome mec element, the delta-hemolysin-converting Newman phage ΦNM3, and the pathogenicity island SaPI3, encoding the superantigen enterotoxin B.

A non-resolving splenic 'abscess' in an elderly male - An unusual manifestation of splenic lymphoma.

Splenic abscess is a relatively uncommon condition, posing a diagnostic and therapeutic challenge for the treating physician. It occurs due to haematogenous spread from endocarditis or other septic foci, especially in immune-compromised individuals and diabetics. We describe an elderly male who presented with splenomegaly and low-grade fever with no predisposing factors. Examination revealed a tender splenomegaly. Ultrasonography (US) showed a hypoechoic area within the spleen from which guided aspiration of pus grew Staphylococcus aureus and Klebsiella pneumoniae. Percutaneous drainage and culture-based antibiotics failed to resolve the abscess, obligating surgical drainage. Intraoperative biopsy from the abscess wall was reported as splenic marginal lymphoma. This unusual presentation of lymphoma needs to be considered in splenic abscess without known risk factors.

Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2.

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.