EnsembleSeq: a workflow towards real-time, rapid, and simultaneous multi-kingdom-amplicon sequencing for holistic and resource-effective microbiome research at scale.

Bacterial communities are often concomitantly present with numerous microorganisms in the human body and other natural environments. Amplicon-based microbiome studies have generally paid skewed attention, that too at a rather shallow genus level resolution, to the highly abundant bacteriome, with interest now forking toward the other microorganisms, particularly fungi. Given the generally sparse abundance of other microbes in the total microbiome, simultaneous sequencing of amplicons targeting multiple microbial kingdoms could be possible even with full multiplexing. Guiding studies are currently needed for performing and monitoring multi-kingdom-amplicon sequencing and data capture at scale. Aiming to address these gaps, amplification of full-length bacterial 16S rRNA gene and entire fungal internal-transcribed spacer (ITS) region was performed for human saliva samples (n = 96, including negative and positive controls). Combined amplicon DNA libraries were prepared for nanopore sequencing using a major fraction of 16S molecules and a minor fraction of ITS amplicons. Sequencing was performed in a single run of an R10.4.1 flow cell employing the latest V14 chemistry. An approach for real-time monitoring of the species saturation using dynamic rarefaction was designed as a guiding determinant of optimal run time. Real-time saturation monitoring for both bacterial and fungal species enabled the completion of sequencing within 30 hours, utilizing less than 60% of the total nanopores. Approximately 5 million high quality (HQ) taxonomically assigned reads were generated (~4.2 million bacterial and 0.7 million fungal), providing a wider (beyond bacteriome) snapshot of human oral microbiota at species-level resolution. Among the more than 400 bacterial and 240 fungal species identified in the studied samples, the species of Streptococcus (e.g., Streptococcus mitis and Streptococcus oralis) and Candida (e.g., Candida albicans and Candida tropicalis) were observed to be the dominating microbes in the oral cavity, respectively. This conformed well with the previous reports of the human oral microbiota. EnsembleSeq provides a proof-of-concept toward the identification of both fungal and bacterial species simultaneously in a single fully multiplexed nanopore sequencing run in a time- and resource-effective manner. Details of this workflow, along with the associated codebase, are provided to enable large-scale application for a holistic species-level microbiome study. IMPORTANCE: Human microbiome is a sum total of a variety of microbial genomes (including bacteria, fungi, protists, viruses, etc.) present in and on the human body. Yet, a majority of amplicon-based microbiome studies have largely remained skewed toward bacteriome as an assumed proxy of the total microbiome, primarily at a shallow genus level. Cost, time, effort, data quality/management, and importantly lack of guiding studies often limit progress in the direction of moving beyond bacteriome. Here, EnsembleSeq presents a proof-of-concept toward concomitantly capturing multiple-kingdoms of microorganisms (bacteriome and mycobiome) in a fully multiplexed (96-sample) single run of long-read amplicon sequencing. In addition, the workflow captures dynamic tracking of species-level saturation in a time- and resource-effective manner.

Insights into Changing Dermatophyte Spectrum in India Through Analysis of Cumulative 161,245 Cases Between 1939 and 2021.

Dermatophytosis is one of the most common superficial infections of the skin affecting nearly one-fifth of the world population at any given time. With nearly 30% of worldwide terbinafine-resistance cases in Trichophyton mentagrophytes/Trichophyton interdigitale and Trichophyton rubrum reported from India in recent years, there is a significant burden of the emerging drug resistance epidemic on India. Here, we carry out a comprehensive retrospective analysis of dermatophytosis in India using 1038 research articles pertaining to 161,245 cases reported from 1939 to 2021. We find that dermatophytosis is prevalent in all parts of the country despite variable climatic conditions in different regions. Our results show T. rubrum as the most prevalent until 2015, with a sudden change in dermatophyte spectrum towards T. mentagrophytes/T. interdigitale complex since then. We also carried out an 18S rRNA-based phylogenetics and an average nucleotide identity-and single nucleotide polymorphism-based analysis of available whole genomes and find very high relatedness among the prevalent dermatophytes, suggesting geographic specificity. The comprehensive epidemiological and phylogenomics analysis of dermatophytosis in India over the last 80 years, presented here, would help in region-specific prevention, control and treatment of dermatophyte infections, especially considering the large number of emerging resistance cases.

Biophysical Characterization of the C-Terminal Tail of T. rubrum PacC Reveals an Inherent Intrinsically Disordered Structure with pH-Induced Structural Plasticity.

PacC is a key transcriptional regulator of human pathogenic fungus Trichophyton rubrum with pivotal roles in pH homeostasis and virulence. We report the first biophysical characterization of the C-terminal inhibitory tail of PacC, pertinent to its physiological role in maintaining the inactive state of PacC at acidic pH which undergoes conformational changes for its proteolytic removal and activation, at alkaline pH. To gain insights into the structural features of PacC that enable the required conformational flexibility, we performed gel filtration chromatography, dynamic light scattering, circular dichroism, and 1-anilino-8-naphthalenesulfonate binding and showed that the tail exhibits properties similar to intrinsically disordered proteins, as also predicted by bioinformatics tools. We demonstrate that the C-terminal tail is conformationally flexible and attains a molten globule-like state at extremely acidic pH and undergoes biphasic GdmCl-induced unfolding in a noncooperative manner with an intermediate X state. We hypothesize that the conformational plasticity of the C-terminal tail of PacC may play a significant role in modulating its pH-dependent transcriptional activation.

A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene pacC in a Clinical Isolate of Trichophyton rubrum.

Trichophyton rubrum is the most prevalent causative agent responsible for 80-90% of all known superficial fungal infections in humans, worldwide. Limited available methods for genetic manipulations have been one of the major bottlenecks in understanding relevant molecular mechanisms of disease pathogenesis in T. rubrum. Here, a dual-plasmid-based CRISPR/Cas9 strategy to edit pH regulatory transcription factor, pacC, of a clinical isolate of T. rubrum by non-homologous end joining (NHEJ) repair is presented. A cas9-eGFP fusion that aids pre-screening of primary transformants through detection of GFP fluorescence is expressed from one plasmid while target-specific sgRNA from the other brings about mutagenesis of pacC with an overall efficiency of 33.8-37.3%. The mutants had reduced transcript levels of pacC at both acidic and alkaline pH with several morphological abnormalities. We believe this dual-plasmid-based CRISPR/Cas9 strategy will aid functional genomics studies, especially in non-lab-adapted clinical strains of T. rubrum.

Structural investigation and gene deletion studies of mycobacterial oligoribonuclease reveal modulation of c-di-GMP-mediated phenotypes.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger required for normal physiology as well as survival under hypoxic and reductive stress conditions of mycobacterial cells. Complete degradation of c-di-GMP is necessary for signal termination and maintaining its homeostasis inside the cells. Homeostasis of c-di-GMP in mycobacteria is brought about by the bifunctional diguanylate cyclase (DGC) that synthesizes c-di-GMP from two molecules of GTP and also catalyses the asymmetric cleavage of c-di-GMP to linear pGpG through its phosphodiesterase activity. However, the mycobacterial enzyme for the last step of degradation from pGpG to GMP has not been characterized thus far. Here, we present the identification of oligoribonuclease (Orn) as the most likely phosphodiesterase to degrade pGpG to GMP through AlphaFold-empowered structural homology that exhibited in vitro phosphodiesterase activity on pGpG substrates. In order to understand the physiological role of Orn in mycobacteria, we created a deletion mutant of orn in M. smegmatis and analysed the phenotypes that are associated with c-di-GMP signaling. We find that orn plays important roles in vivo and is required not only for proper growth of M. smegmatis in normal and stress conditions but also for biofilm formation.

Three-dimensional structure of a mycobacterial oligoribonuclease reveals a unique C-terminal tail that stabilizes the homodimer.

Oligoribonucleases (Orns) are highly conserved DnaQ-fold 3'-5' exoribonucleases that have been found to carry out the last step of cyclic-di-GMP (c-di-GMP) degradation, that is, pGpG to GMP in several bacteria. Removal of pGpG is critical for c-di-GMP homeostasis, as excess uncleaved pGpG can have feedback inhibition on phosphodiesterases, thereby perturbing cellular signaling pathways regulated by c-di-GMP. Perturbation of c-di-GMP levels not only affects survival under hypoxic, reductive stress, or nutrient-limiting conditions but also affects pathogenicity in infection models as well as antibiotic response in mycobacteria. Here, we have determined the crystal structure of MSMEG_4724, the Orn of Mycobacterium smegmatis (Ms_orn) to 1.87 Å resolution to investigate the function of its extended C-terminal tail that is unique among bacterial Orns. Ms_orn is a homodimer with the canonical RNase-H fold of exoribonucleases and conserved catalytic residues in the active site. Further examination of the substrate-binding site with a modeled pGpG emphasized the role of a phosphate cap and "3'OH cap" in constricting a 2-mer substrate in the active site. The unique C-terminal tail of Ms_orn aids dimerization by forming a handshake-like flap over the second protomer of the dimer. Our thermal and denaturant-induced unfolding experiments suggest that it helps in higher stability of Ms_orn as compared with Escherichia coli Orn or a C-terminal deletion mutant. We also show that the C-terminal tail is required for modulating response to stress agents in vivo. These results will help in further evaluating the role of signaling and regulation by c-di-GMP in mycobacteria.

MarkerML - Marker Feature Identification in Metagenomic Datasets Using Interpretable Machine Learning.

Identification of environment specific marker-features is one of the key objectives of many metagenomic studies. It aims to identify such features in microbiome datasets that may serve as markers of the contrasting or comparable states. Hypothesis testing and black-box machine learnt models which are conventionally used for identification of these features are generally not exhaustive, especially because they generally do-not provide any quantifiable relevance (context) of/between the identified features. We present MarkerML web-server, that seeks to leverage the emergence of interpretable machine learning for facilitating the contextual discovery of metagenomic features of interest. It does so through a comprehensive and automated application of the concept of Shapley Additive Explanations in companionship to the compositionality accounted hypothesis testing for the multi-variate microbiome datasets. MarkerML not only helps in identification of marker-features, but also enables insights into the role and inter-dependence of the identified features in driving the decision making of the supervised machine learnt model. Generation of high quality and intuitive visualizations spanning prediction effect plots, model performance reports, feature dependency plots, Shapley and abundance informed cladograms (Sungrams), hypothesis tested violin plots along-with necessary provisions for excluding the participant bias and ensuring reproducibility of results, further seek to make the platform a useful asset for the scientists in the field of microbiome (and even beyond). The MarkerML web-server is freely available for the academic community at https://microbiome.igib.res.in/markerml/.

Correction to: Whole genome sequences of two Trichophyton indotineae clinical isolates from India emerging as threats during therapeutic treatment of dermatophytosis.

[This corrects the article DOI: 10.1007/s13205-021-02950-1.].

Whole genome sequences of two Trichophyton indotineae clinical isolates from India emerging as threats during therapeutic treatment of dermatophytosis.

In the current study, we report the genome sequence of two different clinical isolates from India, Trichophyton indotineae UCMS-IGIB-CI12 and Trichophyton indotineae UCMS-IGIB-CI14. The resulting genome assembly achieved a 143-fold coverage in 824 contigs for T. indotineae UCMS-IGIB-CI12 and a 136-fold coverage in 904 contigs for T. indotineae UCMS-IGIB-CI14. Both the clinical isolates contain a c.1342G>A mutation corresponding to Ala448Thr amino acid substitution in erg1 and exhibit an intermittent drug response to terbinafine. Comparative genomics analysis with available genomes of Trichophyton interdigitale/Trichophyton mentagrophytes species complex revealed a similar genome architecture and identified large number of genes associated with virulence and pathogenicity, namely, lipases, proteases, LysM domain-containing factors, carbon metabolism enzymes and cytochrome P450 enzymes, in all the genomes. An analysis of single amino acid polymorphisms (SAPs) in the protein sequences of subtilisin and lipase enzyme families identified a higher frequency of SAPs in functionally important proteins, Sub3 and Sub6 and their possible use in multilocus phylogenetic analysis of T. interdigitale/T. mentagrophytes species complex. The whole genome sequences of T. indotineae clinical isolates provided in this report will, hence, serve as a key reference point for investigation of clinical strains and emerging drug resistance among dermatophytes originating from different parts of the world.

Sputum Protein Biomarkers in Airway Diseases: A Pilot Study.

Background: Chronic mucous hypersecretion (CMH or chronic bronchitis) per se or when associated with chronic inflammatory airway diseases such as asthma or chronic obstructive pulmonary disease (COPD) has several adverse clinical consequences. The sputum fluid phase has several candidate proteins including mucins which have the potential of being therapeutic targets, but has not yet been explored in-depth. This study aimed at exploring the profile of sputum proteins in various airway diseases. Methods: Sputum from thirty-one patients with various airway diseases was collected and the fluid phase analyzed by LC-MS/MS and subsequently by sequential window acquisition of all theoretical fragments ion spectra (SWATH) (n = 15) for protein quantitation. Hierarchical clustering and functional grouping were performed. Results: A total of 185 proteins were quantitated by SWATH of which 21 proteins were identified which could distinguish between the clinical phenotypes by hierarchical clustering. Functional protein clustering revealed 4 groups: those that are inflammation related, oxidative stress related, mucin related and a cytoskeletal and calcium related group. The levels of eight proteins (Azurocidin1, Neutrophil defensin 3, Lactotransferrin, Calmodulin 3, Coronin1A, Mucin 5B, Mucin 5AC and BPI fold containing family B1) were significantly altered (relative to mean) in exacerbator prone subjects compared to nonexacerbators. Another simple but useful metric which emerged from this study was total protein concentration in sputum which was significantly higher in frequent exacerbators. Conclusion: Sputum proteins can detect the various airway disease clinical phenotypes. Total protein concentration and eight other proteins are biomarkers for frequent exacerbators. The clinical and therapeutic implications of the functional groups of proteins need further evaluation.

Loss of U1498 methylation in 16S rRNA by RsmE methyltransferase associates its role with aminoglycoside resistance in mycobacteria.

OBJECTIVES: Modulation of methylation pattern through mutations in ribosomal methyltransferases is a key mechanism of bacterial drug resistance. However, RsmG (GidB), which specifically methylates G527 in 16S rRNA, remains the only conserved methyltransferase known to be associated with low-level drug resistance in mycobacterial isolates. The mycobacterial RsmE homologue methylates U1498 in 16S rRNA in a highly specific manner. U1498 lies in the vicinity of the binding site for various aminoglycosides in the ribosome. However, the association of methylation at U1498 with altered drug response remains poorly understood. METHODS: A deletion mutant of the RsmE homologue in Mycobacterium smegmatis was generated by a suicidal vector strategy and drug susceptibility assays were performed on wild-type, knockout and complemented strains with varying concentrations of ribosomal- and non-ribosomal-targeting drugs. RESULTS: Deletion of the RsmE homologue of M. smegmatis led to an at least two-fold increase in the minimum inhibitory concentrations (MICs) of aminoglycosides that bind in the decoding centre proximal to U1498 in the 30S subunit. The change in MICs was highly specific and reproducible and did not show any cross-resistance to other drug classes. Surprisingly, Rv2372c, the RsmE homologue of Mycobacterium tuberculosis, has the largest number of mutations among conserved ribosomal methyltransferases, after gidB, highlighting the role of mutations in RsmE methyltransferase as a key emerging mechanism of resistance in clinical strains. CONCLUSION: We present the first evidence of an association of methylation of U1498 in 16S rRNA with development of low-level resistance in mycobacteria that must be tackled in a timely manner.

Structural and thermodynamic characterization of a highly stable conformation of Rv2966c, a 16S rRNA methyltransferase, at low pH.

Rv2966c is a highly specific methyltransferase that methylates G966 at the N2 position in 16S rRNA of mycobacterial ribosome and can be secreted inside the host cell to methylate host DNA. However, how the secreted protein retains its structure and function in the harsh environment of host cell, remains unclear. In this work, we investigate structural features of Rv2966c at pH 4.0 and pH 7.5 by far-UV- and near-UV-circular dichroism (CD) and fluorescence spectroscopy, to gain insights into its folding and stability at the acidic pH, that it is likely to encounter inside the macrophage. We show that Rv2966c exists in a compact, folded state at both pH 7.5 and pH 4.0, a result corroborated by molecular dynamics simulations as a function of pH. In fact, Rv2966c was found to be more stable at pH 4.0 than pH 7.5, as evidenced by thermal-induced CD and nanodifferential scanning fluorimetry, and urea-induced denaturation measurements. Interestingly, unlike pH 7.5 (two-state unfolding), denaturation of Rv2966c at pH 4.0 occurs in a biphasic (N ↔ X ↔ U) manner. Further spectroscopic characterization of 'X' state, identifies characteristics of a molten globule-like intermediate. We finally conclude that Rv2966c maintains a compact folded state at pH 4.0 akin to that at pH 7.5 but with higher stability.

A novel mutation alters the stability of PapA2 resulting in the complete abrogation of sulfolipids in clinical mycobacterial strains.

The analysis of whole genomes has revealed specific geographical distribution of Mycobacterium tuberculosis (Mtb) strains across the globe suggestive of unique niche dependent adaptive mechanisms. We provide an important correlation of a genome-based mutation to a molecular phenotype across two predominant clinical Mtb lineages of the Indian subcontinent. We have identified a distinct lineage specific mutation-G247C, translating into an alanine-proline conversion in the papA2 gene of Indo-oceanic lineage 1 (L1) Mtb strains, and restoration of cell wall sulfolipids by simple genetic complementation of papA2 from lineage 3 (L3) or from H37Rv (lineage 4-L4) attributed the loss of this glycolipid to this specific mutation in Indo-Oceanic L1 Mtb. The investigation of structure of Mtb PapA2 revealed a distinct nonribosomal peptide synthetase (NRPS) C domain conformation with an unconventional presence of a zinc binding motif. Surprisingly, the A83P mutation did not map to either the catalytic center in the N-terminal subdomain or any of the substrate-binding region of the protein. On the contrary, the inherent ability of mutant PapA2 to form insoluble aggregates and molecular simulations with the wild-type/mutant (Wt/mut) PapA2 purports an important role for the surface associated 83rd residue in protein conformation. This study demonstrates the importance of a critical structural residue in the papA2 protein of Mtb and helps establish a link between observed genomic alteration and its molecular consequence in the successful human pathogen Mtb. Significance We demonstrate the effect of a unique SNP in PapA2 gene of Indo-oceanic Mycobacterium tuberculosis (Mtb) strains leading to the loss of sulfolipid from these strains. By X-ray crystallographic analysis and molecular dynamics (MD) simulations, we show the importance of this residue in the global PapA2 structure. The presence of a Zn atom has not been reported before for this class of proteins. Here, we provide an important link between genomic alteration and its molecular consequence in Mtb highlighting one of the many adaptive mechanisms that have contributed to its success as a human pathogen. A high degree of identity with PapA1, 3, or 4 would help in interpreting the structure of these PapA proteins and other acyl transferases of other biological systems.

Improved regression model to predict an impact of SOD1 mutations on ALS patients survival time based on analysis of hydrogen bond stability.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterised by the inevitable degeneration of central and peripheral motor neurons. Aggregation of mutant SOD1 is one of the molecular mechanisms underlying the onset of the disease. There are a number of regression models designed to predict the survival of patients based on an analysis of experimental data on thermostability, heterodimerisation energy, and changes in the hydrophobicity of SOD1 mutants. Previously, we proposed regression models linking the change in the stability of hydrogen bonds in mutant SOD1 calculated using molecular dynamics and elastic networks with patients survival time. In this study, these models were improved in terms of accuracy of survival time prediction by taking into account the variance of survival time values relative to the mean, the number of patients carrying each specific mutation, and the use of random forest regression as a regression method. The accuracy of the previous models was roughly 5.2 years while the accuracy of the new ones are up to 4 years. The model is also superior to those published by other authors. It was found that the hydrogen bonds important for prediction of survival time are formed by residues at positions located in the regions of the protein responsible for aggregation as well as in structural and functionally important sites.

Genome Analysis of Staphylococcus capitis TE8 Reveals Repertoire of Antimicrobial Peptides and Adaptation Strategies for Growth on Human Skin.

Staphylococcus capitis TE8 was isolated from skin surface of a healthy human foot, and exhibited a strong antibacterial activity against Gram-positive bacteria, including Staphylococcus aureus. Whole genome sequence of S. capitis TE8 was obtained by shotgun and paired-end pyrosequencing with a coverage of 109-fold. The draft genome contains 2,516,639 bp in 8 scaffolds with 209 total contigs. The genome contains 2319 protein coding sequences, 58 tRNA and 3 rRNA. Genome sequence analysis revealed 4 distinct gene loci with the ability to encode antimicrobial peptides: (i) an epidermicin gene cluster; (ii) a gallidermin gene cluster; (iii) a gene cluster encoding six phenol soluble modulin (PSM) ß-type peptides (PSMß1-ß6) and (iv) an additional gene that belonged to PSMß family and encoded a 44 residues long peptide, HTP2388. Synthetic peptides with sequence identical to seven PSMß-like peptides i.e. PSMß1-ß6 and peptide HTP2388 showed antibacterial activity. Genome sequence also revealed genes for adhesins, intracellular adhesins, osmoadaptation, oxidative and acid stress tolerance possibly responsible for initial attachment, colonization and survival of S. capitis TE8 on human skin. Comparative genome analysis revealed presence of a gamut of genes in S. capitis strains in comparison to Staphylococcus epidermidis and Staphylococcus caprae indicating towards their possible role in better adaptation and survival on human skin.

Structural and thermodynamic characterisation of L94F mutant of horse cytochrome c.

Mammalian mitochondrial cytochromes c (cyts c) has a conserved Leu94 which is replaced by valine/isoleucine in some lower eukaryotes and prokaryotes. It is expected that nature substituted Leu94 with Val/Ile, for they have similar van der Waals volume and hydrophobicity with a difference in side chain branching only. Reports also suggested the presence of phenylalanine at position 94, which leads to questions: (i) How bulky aromatic amino acid residue fitted at position 94 in cyt c family proteins? (ii) What is the effect of L94F mutation on protein stability and folding? Here, we selected horse cyt-c as a model to answer the second question. We generated L94F mutant of horse cytochrome c and subsequently characterised using far-UV, near-UV and Soret circular dichroism, absorbance, intrinsic and extrinsic ANS (8-anilino-1-napthalenesulfonic acid) fluorescence and dynamic light scattering measurements. We observed that this mutation affects the native state and arrests the protein folding at the molten globule state. Thermal stability of L94F mutant is also measured by spectroscopic techniques and differential scanning calorimetry. The midpoint of thermal denaturation of L94F mutant is 17°C less than wild type. Molecular dynamics simulation study also supports our in vitro observation that this mutant has stable backbone conformation.

Identification of L84F mutation with a novel nucleotide change c.255G > T in the superoxide dismutase gene in a North Indian family with amyotrophic lateral sclerosis.

Mutations in the superoxide dismutase (SOD1) gene account for ∼15% and in the transactive response DNA binding protein (TARDBP) gene for ∼5% of familial amyotrophic lateral sclerosis (FALS) cases. These two genes were analysed in two siblings from North India with ALS and a positive family history. The coding region of SOD1 and TARDBP genes was sequenced in both siblings. Genetic variation identified in SOD1 was typed in unaffected family members (n = 11), sporadic ALS patients (n = 48) and healthy controls (n = 35). Molecular dynamic (MD) simulations were performed on wild-type (WT) and mutant monomers of SOD1 to determine structural changes due to the identified mutation. A novel heterozygous nucleotide variation (c.255G > T) was identified in exon 4 of SOD1 in the two siblings and two asymptomatic family members but not in SALS patients and healthy controls. This variation results in a known non-synonymous substitution from leucine to phenylalanine at position 84 (L84F), making it a triallelic variation. Large conformational changes were observed in the zinc loop and electrostatic loop in an L84F mutant compared to WT SOD1 in MD simulations. In conclusion, this is the first report of mutation in SOD1 associated with FALS in India. Structural perturbations in L84F SOD1 may cause dimer destabilization, with decreased metal affinity leading to oligomerization.

Genome sequence of a clinical isolate of dermatophyte, Trichophyton rubrum from India.

Trichophyton rubrum is one of the major causative agents of dermatophytosis in humans worldwide. We report the draft genome sequence of T. rubrum var. raubitschekii from Delhi, India, isolated from a patient presenting symptoms of onychomycosis. The total estimated genome size of the clinical isolate is 25.2 MB containing 8265 predicted protein-coding sequences, 91 tRNA and 15 rRNA genes. Sequence analysis of the secreted subtilases, one of the major virulence factors in dermatophytes, clusters them into three subfamilies with distinct sequence features. The genome sequence is a step in understanding diversity of dermatophytes worldwide and will aid in identification of virulence factors and dissecting mechanisms of pathogenesis among them.

Unique subunit packing in mycobacterial nanoRNase leads to alternate substrate recognitions in DHH phosphodiesterases.

DHH superfamily includes RecJ, nanoRNases (NrnA), cyclic nucleotide phosphodiesterases and pyrophosphatases. In this study, we have carried out in vitro and in vivo investigations on the bifunctional NrnA-homolog from Mycobacterium smegmatis, MSMEG_2630. The crystal structure of MSMEG_2630 was determined to 2.2-Å resolution and reveals a dimer consisting of two identical subunits with each subunit folding into an N-terminal DHH domain and a C-terminal DHHA1 domain. The overall structure and fold of the individual domains is similar to other members of DHH superfamily. However, MSMEG_2630 exhibits a distinct quaternary structure in contrast to other DHH phosphodiesterases. This novel mode of subunit packing and variations in the linker region that enlarge the domain interface are responsible for alternate recognitions of substrates in the bifunctional nanoRNases. MSMEG_2630 exhibits bifunctional 3'-5' exonuclease [on both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) substrates] as well as CysQ-like phosphatase activity (on pAp) in vitro with a preference for nanoRNA substrates over single-stranded DNA of equivalent lengths. A transposon disruption of MSMEG_2630 in M. smegmatis causes growth impairment in the presence of various DNA-damaging agents. Further phylogenetic analysis and genome organization reveals clustering of bacterial nanoRNases into two distinct subfamilies with possible role in transcriptional and translational events during stress.

The structure of Rv2372c identifies an RsmE-like methyltransferase from Mycobacterium tuberculosis.

U1498 of 16S rRNA plays an important role in translation fidelity as well as in antibiotic response. U1498 is present in a methylated form in the decoding centre of the ribosome. In this study, Rv2372c from Mycobacterium tuberculosis has been identified as an RsmE-like methyltransferase which specifically methylates U1498 of 16S rRNA at the N3 position and can complement RsmE-deleted Escherichia coli. The crystal structure of Rv2372c has been determined, and reveals that the protein belongs to a distinct class in the SPOUT superfamily and exists as a dimer. The deletion of critical residues at the C-terminus of Rv2372c leads to an inability of the protein to form stable dimers and to abolition of the methyltransferase activity. A ternary model of Rv2372c with its cofactor S-adenosylmethionine (SAM) and the 16S rRNA fragment (1487)16S rRNA(1510) helps to identify binding pockets for SAM (in the deep trefoil knot) and substrate RNA (at the dimer interface) and suggests an S(N)2 mechanism for the methylation of N3 of U1498 in 16S rRNA.