Occurrence of F-Specific Bacteriophages in Untreated and Treated Wastewaters in Mumbai.

F + coliphages are considered as potential enteric viral indicators in water systems as a tool for on-site validation of wastewater treatment processes. The present study evaluated the occurrence of F + coliphages in wastewaters collected from three wastewater treatment plants (WWTPs) in Mumbai city, to assess this potential. The detection and enumeration of F + coliphages was carried out from WWTPs Z1, Z3 and Z5 using the ISO 10705-1 and U.S EPA 1601 methods. F + coliphages were majorly detected in untreated wastewater samples followed by a few secondary treated samples in WWTP-Z1 and Z3 and one tertiary treated sample from Z1, these differences were found to be statistically significant. The difference in F + coliphage levels between the treatment stages highlight their potential as indicators for monitoring the efficiency of wastewater treatment. The overall positivity of F + coliphage was 35.09% for Salmonella. typhimurium WG49 host (as per ISO 10705-1), was higher by 10.52% for Escherichia coli Famp HS host (as per U.S EPA 1601) (45.61%), highlighting the efficiency of the latter host over the former in F + coliphage detection. Significant difference in F + coliphage counts using the two bacterial hosts were observed in WWTP-Z3 (p = 0.001) and WWTP-Z1 (p = 0.047) but not in WWTP-Z5 (p = 0.332). Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01181-7.

Wastewater-Based Epidemiology of SARS-CoV-2: Assessing Prevalence and Correlation with Clinical Cases.

Wastewater-based epidemiology has been recognized as a tool to monitor the progress of COVID-19 pandemic worldwide. The study presented herein aimed at quantitating the SARS-CoV-2 RNA in the wastewaters, predicting the number of infected individuals in the catchment areas, and correlating it with the clinically reported COVID-19 cases. Wastewater samples (n = 162) from different treatment stages were collected from three wastewater treatment plants (WWTPs) from Mumbai city during the 2nd surge of COVID-19 (April 2021 to June 2021). SARS-CoV-2 causing COVID-19, was detected in 76.2% and 4.8% of raw and secondary treated (n = 63 each) wastewater samples respectively while all tertiary treated samples (n = 36) were negative. The quantity of SARS-CoV-2 RNA determined as gene copies/100 mL varied among all the three WWTPs under study. The gene copy numbers thus obtained were further used to estimate the number of infected individuals within the population served by these WWTPs using two published methods. A positive correlation (p < 0.05) was observed between the estimated number of infected individuals and clinically confirmed COVID-19 cases reported during the sampling period in two WWTPs. Predicted infected individuals calculated in this study were 100 times higher than the reported COVID-19 cases in all the WWTPs assessed. The study findings demonstrated that the present wastewater treatment technologies at the three WWTPs studied were adequate to remove the virus. However, SARS-CoV-2 genome surveillance with emphasis on monitoring its variants should be implemented as a routine practice to prepare for any future surge in infections.

Response Regulator CD1688 Is a Negative Modulator of Sporulation in Clostridioides difficile.

Two-component signal transduction systems (TCSs), consisting of a sensor histidine kinase (HK) and a response regulator (RR), sense environmental stimuli and then modulate cellular responses, typically through changes in gene expression. Our previous work identified the DNA binding motif of CD1586, an RR implicated in Clostridioides difficile strain R20291 sporulation. To determine the role of this RR in the sporulation pathway in C. difficile, we generated a deletion strain of cd1688 in the historical 630 strain, the homolog of cd1586. The C. difficile Δcd1688 strain exhibited a hypersporulation phenotype, suggesting that CD1688 negatively regulates sporulation. Complementation of the C. difficile Δcd1688 strain restored sporulation. In contrast, a nonphosphorylatable copy of cd1688 did not restore sporulation to wild-type (WT) levels, indicating that CD1688 must be phosphorylated to properly modulate sporulation. Expression of the master regulator spo0A, the sporulation-specific sigma factors sigF, sigE, sigG, and sigK, and a signaling protein encoded by spoIIR was increased in the C. difficile Δcd1688 strain compared to WT. In line with the increased spoIIR expression, we detected an increase in mature SigE at an earlier time point, which arises from SpoIIR-mediated processing of pro-SigE. Taken together, our data suggest that CD1688 is a novel negative modulator of sporulation in C. difficile and contributes to mediating progression through the spore developmental pathway. These results add to our growing understanding of the complex regulatory events involved in C. difficile sporulation, insight that could be exploited for novel therapeutic development. IMPORTANCE Clostridioides difficile causes severe gastrointestinal illness and is a leading cause of nosocomial infections in the United States. This pathogen produces metabolically dormant spores that are the major vehicle of transmission between hosts. The sporulation pathway involves an intricate regulatory network that controls a succession of morphological changes necessary to produce spores. The environmental signals inducing the sporulation pathway are not well understood in C. difficile. This work identified a response regulator, CD1688, that, when deleted, led to a hypersporulation phenotype, indicating that it typically acts to repress sporulation. Improving our understanding of the regulatory mechanisms modulating sporulation in C. difficile could provide novel strategies to eliminate or reduce spore production, thus decreasing transmission and disease relapse.

Insights revealed by the co-crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1.

Two-component signaling systems are the primary means by which bacteria, archaea, and certain plants and fungi react to their environments. The model yeast, Saccharomyces cerevisiae, uses the Sln1 signaling pathway to respond to hyperosmotic stress. This pathway contains a hybrid histidine kinase (Sln1) that autophosphorylates and transfers a phosphoryl group to its own receiver domain (R1). The phosphoryl group is then transferred to a histidine phosphotransfer protein (Ypd1) that finally passes it to the receiver domain (R2) of a downstream response regulator (Ssk1). Under normal conditions, Ssk1 is constitutively and preferentially phosphorylated in the phosphorelay. Upon detecting hyperosmotic stress, Ssk1 rapidly dephosphorylates and activates the high-osmolarity glycerol (HOG) pathway, initiating a response. Despite their distinct physiological roles, both Sln1 and Ssk1 bind to Ypd1 at a common docking site. Co-crystal structures of response regulators in complex with their phosphorelay partners are scarce, leaving many mechanistic and structural details uncharacterized for systems like the Sln1 pathway. In this work, we present the co-crystal structure of Ypd1 and a near wild-type variant of the receiver domain of Ssk1 (Ssk1-R2-W638A) at a resolution of 2.80 Å. Our structural analyses of Ypd1-receiver domain complexes, biochemical determination of binding affinities for Ssk1-R2 variants, in silico free energy estimates, and sequence comparisons reveal distinctive electrostatic properties of the Ypd1/Ssk1-R2-W638A complex that may provide insight into the regulation of the Sln1 pathway as a function of dynamic osmolyte concentration.

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins.

BACKGROUND: Many bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners. RESULTS: We report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1. CONCLUSIONS: Phosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.

Regulatory Targets of the Response Regulator RR_1586 from Clostridioides difficile Identified Using a Bacterial One-Hybrid Screen.

The Clostridioides difficile R20291 genome encodes 57 response regulator proteins that, as part of two-component signaling pathways, regulate adaptation to environmental conditions. Genomic and transcriptomic studies in C. difficile have been limited, due to technical challenges, to the analysis of either high-throughput screens or high-priority targets, such as primary regulators of toxins or spore biology. We present the use of several technically accessible and generally applicable techniques to elucidate the putative regulatory targets of a response regulator, RR_1586, involved in sporulation of the hypervirulent C. difficile strain R20291. A DNA-binding specificity motif for RR_1586 was determined using a bacterial one-hybrid assay originally developed for Drosophila transcription factors. Comparative bioinformatics approaches identified and in vitro experiments confirmed RR_1586 binding sites upstream of putative target genes, including those that encode phosphate ion transporters, spermidine/putrescine biosynthesis and transport pathways, ABC type transport systems, known regulators of sporulation, and genes encoding spore structural proteins. Representative examples of these regulatory interactions have been tested and confirmed in Escherichia coli-based reporter assays. Finally, evidence of possible regulatory mechanisms is also presented. A working model includes self-regulation by RR_1586 and phosphorylation-dependent and -independent DNA binding at low- and high-fidelity binding sites, respectively. Broad application of this and similar approaches is anticipated to be an important catalyst for the study of gene regulation by two-component systems from pathogenic or technically challenging bacteria.IMPORTANCEClostridioides difficile spores survive under harsh conditions and can germinate into actively dividing cells capable of causing disease. An understanding of the regulatory networks controlling sporulation and germination in C. difficile could be exploited for therapeutic advantage. However, such studies are hindered by the challenges of working with an anaerobic pathogen recalcitrant to genetic manipulation. Although two-component response regulators can be identified from genetic sequences, identification of their downstream regulatory networks requires further development. This work integrates experimental and bioinformatic approaches, which provide practical advantages over traditional transcriptomic analyses, to identify the putative regulon of the C. difficile response regulator RR_1586 by first screening for protein-DNA interactions in E. coli and then predicting regulatory outputs in C. difficile.

Crystal structure and DNA binding activity of a PadR family transcription regulator from hypervirulent Clostridium difficile R20291.

BACKGROUND: Clostridium difficile is a spore-forming obligate anaerobe that can remain viable for extended periods, even in the presence of antibiotics, which contributes to the persistence of this bacterium as a human pathogen during host-to-host transmission and in hospital environments. We examined the structure and function of a gene product with the locus tag CDR20291_0991 (cdPadR1) as part of our broader goal aimed at elucidating transcription regulatory mechanisms involved in virulence and antibiotic resistance of the recently emergent hypervirulent C. difficile strain R20291. cdPadR1 is genomically positioned near genes that are involved in stress response and virulence. In addition, it was previously reported that cdPadR1 and a homologue from the historical C. difficile strain 630 (CD630_1154) were differentially expressed when exposed to stressors, including antibiotics. RESULTS: The crystal structure of cdPadR1 was determined to 1.9 Å resolution, which revealed that it belongs to the PadR-s2 subfamily of PadR transcriptional regulators. cdPadR1 binds its own promoter and other promoter regions from within the C. difficile R20291 genome. DNA binding experiments demonstrated that cdPadR1 binds a region comprised of inverted repeats and an AT-rich core with the predicted specific binding motif, GTACTAT(N2)ATTATA(N)AGTA, within its own promoter that is also present in 200 other regions in the C. difficile R20291 genome. Mutation of the highly conserved W in α4 of the effector binding/oligomerization domain, which is predicted to be involved in multi-drug recognition and dimerization in other PadR-s2 proteins, resulted in alterations of cdPadR1 binding to the predicted binding motif, potentially due to loss of higher order oligomerization. CONCLUSIONS: Our results indicate that cdPadR1 binds a region within its own promoter consisting of the binding motif GTACTAT(N2)ATTATA(N)AGTA and seems to associate non-specifically with longer DNA fragments in vitro, which may facilitate promoter and motif searching. This suggests that cdPadR1 acts as a transcriptional auto-regulator, binding specific sites within its own promoter, and is part of a broad gene regulatory network involved, in part, with environmental stress response, antibiotic resistance and virulence.

Extended N-terminal region of the essential phosphorelay signaling protein Ypd1 from Cryptococcus neoformans contributes to structural stability, phosphostability and binding of calcium ions.

Rapid response to external stimuli is crucial for survival and proliferation of microorganisms. Pathogenic fungi employ histidine-to-aspartate multistep phosphorelay systems to respond to environmental stress, progress through developmental stages and to produce virulence factors. Because these His-to-Asp phosphorelay systems are not found in humans, they are potential targets for the development of new antifungal therapies. Here we report the characterization of the histidine phosphotransfer (HPt) protein Ypd1 from the human fungal pathogen Cryptococcus neoformans Results from this study demonstrate that CnYpd1 indeed functions as a phosphorelay protein in vitro, and that H138 is confirmed as the site of phosphorylation. We found that CnYpd1 exhibits unique characteristics in comparison to other histidine phosphotransfer proteins, such as an extended N-terminal amino acid sequence, which we find contributes to structural integrity, a longer phosphorylated life time and the ability to bind calcium ions.

Structural studies of E73 from a hyperthermophilic archaeal virus identify the "RH3" domain, an elaborated ribbon-helix-helix motif involved in DNA recognition.

Hyperthermophilic archaeal viruses, including Sulfolobus spindle-shaped viruses (SSVs) such as SSV-1 and SSV-Ragged Hills, exhibit remarkable morphology and genetic diversity. However, they remain poorly understood, in part because their genomes exhibit limited or unrecognizable sequence similarity to genes with known function. Here we report structural and functional studies of E73, a 73-residue homodimeric protein encoded within the SSV-Ragged Hills genome. Despite lacking significant sequence similarity, the nuclear magnetic resonance (NMR) structure reveals clear similarity to ribbon-helix-helix (RHH) domains present in numerous proteins involved in transcriptional regulation. In vitro double-stranded DNA (dsDNA) binding experiments confirm the ability of E73 to bind dsDNA in a nonspecific manner with micromolar affinity, and characterization of the K11E variant confirms the location of the predicted DNA binding surface. E73 is distinct, however, from known RHH domains. The RHH motif is elaborated upon by the insertion of a third helix that is tightly integrated into the structural domain, giving rise to the "RH3" fold. Within the homodimer, this helix results in the formation of a conserved, symmetric cleft distal to the DNA binding surface, where it may mediate protein-protein interactions or contribute to the high thermal stability of E73. Analysis of backbone amide dynamics by NMR provides evidence of a rigid core, fast picosecond to nanosecond time scale NH bond vector motions for residues located within the antiparallel ß-sheet region of the proposed DNA-binding surface, and slower microsecond to millisecond time scale motions for residues in the α1-α2 loop. The roles of E73 and its SSV homologues in the viral life cycle are discussed.

Structure of the response regulator PhoP from Mycobacterium tuberculosis reveals a dimer through the receiver domain.

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving α4-ß5-α5, a common interface for activated receiver domain dimers. However, the switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.

The crystal structure of D212 from sulfolobus spindle-shaped virus ragged hills reveals a new member of the PD-(D/E)XK nuclease superfamily.

Structural studies have made significant contributions to our understanding of Sulfolobus spindle-shaped viruses (Fuselloviridae), an important model system for archaeal viruses. Continuing these efforts, we report the structure of D212 from Sulfolobus spindle-shaped virus Ragged Hills. The overall fold and conservation of active site residues place D212 in the PD-(D/E)XK nuclease superfamily. The greatest structural similarity is found to the archaeal Holliday junction cleavage enzymes, strongly suggesting a role in DNA replication, repair, or recombination. Other roles associated with nuclease activity are also considered.

(1)H, (13)C, (15)N backbone and side chain NMR resonance assignments for E73 from Sulfolobus spindle-shaped virus ragged hills, a hyperthermophilic crenarchaeal virus from Yellowstone National Park.

Crenarchaeal viruses are commonly found in hyperthermal acidic environments such as those of Yellowstone National Park. These remarkable viruses not only exhibit unusual morphologies, but also display extreme genetic diversity. However, little is known about crenarchaeal viral life cycles, virus-host interactions, and their adaptation to hyperthermophilic environments. In an effort to better understand the functions of crenarchaeal viruses and the proteins encoded by their genomes, we have undertaken detailed structural and functional studies of gene products encoded in the open reading frames of Sulfolobus spindle-shaped virus ragged hills. Herein, we report ((15)N, (13)C, (1)H) resonance assignments of backbone and side chain atoms of a 19.1 kDa homodimeric E73 protein of SSVRH.

Structural and functional studies of archaeal viruses.

Viruses populate virtually every ecosystem on the planet, including the extreme acidic, thermal, and saline environments where archaeal organisms can dominate. For example, recent studies have identified crenarchaeal viruses in the hot springs of Yellowstone National Park and other high temperature environments worldwide. These viruses are often morphologically and genetically unique, with genomes that show little similarity to genes of known function, complicating efforts to understand their viral life cycles. Here, we review progress in understanding these fascinating viruses at the molecular level and the evolutionary insights coming from these studies.

Cysteine usage in Sulfolobus spindle-shaped virus 1 and extension to hyperthermophilic viruses in general.

Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs worldwide. The type virus, Sulfolobus spindle-shaped virus 1 (SSV1), has a double-stranded DNA genome that contains 34 open reading frames (ORFs). Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, tertiary protein structure can provide insight into protein function. We have thus undertaken a systematic investigation of the SSV1 proteome and report here on the F112 gene product. Biochemical, proteomic and structural studies reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Notably, the structure contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses, and reveals decreased cysteine content in the membrane proteins of hyperthermophilic viruses infecting Sulfolobales. The evolutionary implications of the SSV1 distribution are discussed.

A winged-helix protein from Sulfolobus turreted icosahedral virus points toward stabilizing disulfide bonds in the intracellular proteins of a hyperthermophilic virus.

Sulfolobus turreted icosahedral virus (STIV) was the first non-tailed icosahedral virus to be isolated from an archaeal host. Like other archaeal viruses, its 37 open reading frames generally lack sequence similarity to genes with known function. The roles of the gene products in this and other archaeal viruses are thus largely unknown. However, a protein's three-dimensional structure may provide functional and evolutionary insight in cases of minimal sequence similarity. In this vein, the structure of STIV F93 reveals a homodimer with strong similarity to the winged-helix family of DNA-binding proteins. Importantly, an interchain disulfide bond is found at the dimer interface, prompting analysis of the cysteine distribution in the putative intracellular proteins of the viral proteome. The analysis suggests that intracellular disulfide bonds are common in cellular STIV proteins, where they enhance the thermostability of the viral proteome.

Cystic schwannoma of the orbit-a case series.

A schwannoma is an uncommon benign orbital tumor that arises from Schwann cells in the peripheral nervous system. Schwannoma with cystic degeneration is an even more rarely reported entity. Clinical examination alone is inadequate for the diagnosis. Radiological examination, like computed tomography (CT) scans, can help in the diagnosis; however, the diagnosis can only be confirmed by histopathological examination (HPE) after excision biopsy. Here, the authors report four cases of orbital schwannoma with cystic degeneration that presented with proptosis and decreased vision. CT scans showed a well-defined non-enhancing intraconal mass with cystic spaces. The histopathological examination was diagnostic for orbital schwannoma with cystic degeneration. Schwannoma should be included in the differential diagnosis of cystic orbital lesions.

Thrombosed orbital varix -- a correlation between imaging studies and histopathology.

A thrombosed varix in the orbit is comparatively rare. Clinical examination alone is often inadequate for diagnosis. Radio-logical examination, such as a computed tomography (CT) scan of the orbit, is extremely important. Histopathological examination (HPE) after excision biopsy can confirm the diagnosis. The present authors describe a case of proptosis in the left eye of a 45-year-old man. CT-scan and HPE supported the diagnosis of a thrombosed orbital varix. This paper discusses the use of radiological investigations to supplement a clinical suspicion and make the diagnosis. This entity needs to be included in the differential diagnosis of proptosis and requires a coordinated approach for establishment of the diagnosis.