@article {8674, title = {Cryo-EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH [National Cryo-EM Facility, BLiSC]}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 Jan 20}, pages = {638}, abstract = {

Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome. Mycobacterium tuberculosis encounters hypoxia (low oxygen) as a major stress in the host macrophages, and upregulates the expression of RafH protein, which is crucial for its survival. The RafH, a dual domain HPF, an orthologue of bacterial long HPF (HPF), hibernates ribosome in 70S monosome form, whereas in other bacteria, the HPF induces 70S ribosome dimerization and hibernates its ribosome in 100S disome form. Here, we report the cryo- EM structure of M. smegmatis, a close homolog of M. tuberculosis, 70S ribosome in complex with the RafH factor at an overall 2.8 {\r A} resolution. The N- terminus domain (NTD) of RafH binds to the decoding center, similarly to HPF NTD. In contrast, the C- terminus domain (CTD) of RafH, which is larger than the HPF CTD, binds to a distinct site at the platform binding center of the ribosomal small subunit. The two domain-connecting linker regions, which remain mostly disordered in earlier reported HPF structures, interact mainly with the anti-Shine Dalgarno sequence of the 16S rRNA.

}, keywords = {Bacterial Proteins, Mycobacterium tuberculosis, Ribosomal Proteins, Ribosomes, RNA, Ribosomal, 16S}, issn = {2041-1723}, doi = {10.1038/s41467-024-44879-y}, author = {Kumar, Niraj and Sharma, Shivani and Kaushal, Prem S} } @article {8318, title = {High-quality single amplicon sequencing method for illumina MiSeq platform using pool of {\textquoteright}N{\textquoteright} (0-10) spacer-linked target specific primers without PhiX spike-in [Next Gen Genomics Facility (INT)]}, journal = {BMC Genomics}, volume = {24}, year = {2023}, month = {2023 Mar 23}, pages = {141}, abstract = {

BACKGROUND: Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing {\textquoteright}N{\textquoteright} (0-10) spacers in target gene amplification primers that are pooled for simple handling.

RESULT: We evaluated the efficiency of {\textquoteright}N{\textquoteright} (0-10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of {\textquoteright}N{\textquoteright} (0-10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,"MetReTrim", to trim the {\textquoteright}N{\textquoteright} (0-10) spacers from the raw reads ( https://github.com/Mohak91/MetReTrim ). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS{\texttrademark} microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method.

CONCLUSION: This method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using {\textquoteright}N{\textquoteright} (0-10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.

}, keywords = {Bacteria, Gene Library, High-Throughput Nucleotide Sequencing, Microbiota, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1471-2164}, doi = {10.1186/s12864-023-09233-4}, author = {Naik, Tejali and Sharda, Mohak and C P, Lakshminarayanan and Virbhadra, Kumar and Pandit, Awadhesh} } @article {8472, title = {Marinobacterium lacunae sp. nov. isolated from estuarine sediment [Next Gen Genomics Facility]}, journal = {Arch Microbiol}, volume = {205}, year = {2023}, month = {2023 Jul 22}, pages = {294}, abstract = {

A novel motile bacterium was isolated from a sediment sample collected in Kochi backwaters, Kerala, India. This bacterium is Gram negative, rod shaped, 1.0-1.5\ {\textmu}m wide, and 2.0-3.0\ {\textmu}m long. It was designated as strain AK27. Colonies were grown on marine agar displayed circular, off-white, shiny, moist, translucent, flat, margin entire, 1-2\ mm in diameter. The major fatty acids identified in this strain were C ω7c, C, and summed in feature 3. The composition of polar lipids in the strain AK27 included phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified amino lipid, two unidentified aminophospholipids, two unidentified phospholipids, and six unidentified lipids. The genomic DNA of strain AK27 exhibited a G+C content of 56.4\ mol\%. Based on the analysis of 16S rRNA gene sequence, strain AK27 showed sequence similarity to M. ramblicola D7 and M. zhoushanense WM3 as 98.99\% and 98.58\%, respectively. Compared to other type strains of the Marinobacterium genus, strain AK27 exhibited sequence similarities ranging from 91.7\% to 96.4\%. When compared to Marinobacterium zhoushanense WM3 and Marinobacterium ramblicola D7, strain AK27 exhibited average nucleotide identity values of 80.25\% and 79.97\%, and dDDH values of 22.9\% and 22.6\%, respectively. The genome size of the strain AK27 was 4.55\ Mb, with 4,229 coding sequences. Based on the observed phenotypic and chemotaxonomic features, and the results of phylogenetic and phylogenomic analysis, this study proposes the classification of strain AK27 as a novel species within the genus Marinobacterium. The proposed name for this novel species is Marinobacterium lacunae sp. nov.

}, keywords = {Agar, Alteromonadaceae, Cardiolipins, Phylogeny, RNA, Ribosomal, 16S}, issn = {1432-072X}, doi = {10.1007/s00203-023-03627-4}, author = {Tanuku, Srinivas Naga Radha and Pinnaka, Anil Kumar and Behera, Swarnaprava and Singh, Aditya and Pydi, Sudharani and Vasudeva, Gunjan and Vaidya, Bhumika and Sharma, Gunjan and Ganta, Sampath Kumar and Garbhapu, Naveen Sagar} } @article {8356, title = {Lunatimonas lonarensis gen. nov., sp. nov., a haloalkaline bacterium of the family Cyclobacteriaceae with nitrate reducing activity [Next Gen Genomics Facility]}, journal = {Syst Appl Microbiol}, volume = {37}, year = {2014}, month = {2014 Feb}, pages = {10-6}, abstract = {

Novel pinkish-orange pigmented, Gram-negative staining, half-moon shaped, non-motile, strictly aerobic strains designated AK24(T) and AK26 were isolated from water and sediment samples of Lonar Lake, Buldhana district, Maharahstra, India. Both strains were positive for oxidase, catalase and β-galactosidase activities. The predominant fatty acids were iso-C15:0 (41.5\%), anteiso-C15:0 (9.7\%), iso-C17:0 3OH (9.6\%), iso-C17:1 ω9c (10.2\%) and C16:1 ω7c/C16:1 ω6c/iso-C15:0 2OH (summed feature 3) (14.4\%). The strains contained MK-7 as the major respiratory quinone, and phosphatidylethanolamine and five unidentified lipids as the polar lipids. Blast analysis of the 16S rRNA gene sequence of strain AK24(T) showed that it was closely related to Aquiflexum balticum, with a pair-wise sequence similarity of 91.6\%, as well as to Fontibacter ferrireducens, Belliella baltica and Indibacter alkaliphilus (91.3, 91.2 and 91.2\% pair-wise sequence similarity, respectively), but it only had between 88.6 and 91.0\% pair-wise sequence similarity to the rest of the family members. The MALDI-TOF assay reported no significant similarities for AK24(T) and AK26, since they potentially represented a new species. A MALDI MSP dendrogram showed close similarity between the two strains, but they maintained a distance from their phylogenetic neighbors. The genome of AK24(T) showed the presence of heavy metal tolerance genes, including the genes providing resistance to arsenic, cadmium, cobalt and zinc. A cluster of heat shock resistance genes was also found in the genome. Two lantibiotic producing genes, LanR and LasB, were also found in the genome of AK24(T). Strains AK24(T) and AK26 were very closely related to each other with 99.5\% pair-wise sequence similarity. Phylogenetic analysis indicated that the strains were members of the family Cyclobacteriaceae and they clustered with the genus Mariniradius, as well as with the genera Aquiflexum, Cecembia, Fontibacter, Indibacter, and Shivajiella. DNA-DNA hybridization between strains AK24(T) and AK26 showed a relatedness of 82\% and their rep-PCR banding patterns were very similar. Based on data from the current polyphasic study, it is proposed that the isolates be placed in a new genus and species with the name Lunatimonas lonarensis gen. nov., sp. nov. The type strain of Lunatimonas lonarensis is AK24(T) (=JCM 18822(T)=MTCC 11627(T)).

}, keywords = {Bacterial Typing Techniques, Bacteroidetes, Cluster Analysis, DNA, Bacterial, DNA, Ribosomal, Fatty Acids, Fresh Water, Genome, Bacterial, Geologic Sediments, India, Molecular Sequence Data, Nitrates, Oxidation-Reduction, Phospholipids, Phylogeny, Quinones, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1618-0984}, doi = {10.1016/j.syapm.2013.10.003}, author = {Srinivas, T N R and Aditya, S and Bhumika, V and Kumar, P Anil} }