Phylogenomic analyses and comparative genomic studies of Thermus strains isolated from Tengchong and Tibet Hot Springs, China.

Genus Thermus is the main focus of researcher among the thermophiles. Members of this genus are the inhabitants of both natural and artificial thermal environments. We performed phylogenomic analyses and comparative genomic studies to unravel the genomic diversity among the strains belonging to the genus Thermus in geographically different thermal springs. Sixteen Thermus strains were isolated and sequenced from hot springs, Qucai hot springs in Tibet and Tengchong hot springs in Yunnan, China. 16S rRNA gene based phylogeny and phylogenomic analyses based on concatenated set of 971 Orthologous Protein Families (supermatrix and gene content methods) revealed a mixed distribution of the Thermus strains. Whole genome based phylogenetic analysis showed, all 16 Thermus strains belong to five species; Thermus oshimai (YIM QC-2-109, YIM 1640, YIM 1627, 77359, 77923, 77838), Thermus antranikianii (YIM 73052, 77412, 77311, 71206), Thermus brokianus (YIM 73518, 71318, 72351), Thermus hydrothermalis (YIM 730264 and 77927) and one potential novel species 77420 forming clade with Thermus thalpophilus SYSU G00506T. Although the genomes of different strains of Thermus of same species were highly similar in their metabolic pathways, but subtle differences were found. CRISPR loci were detected through genome-wide screening, which showed that Thermus isolates from two different thermal locations had well developed defense system against viruses and adopt similar strategy for survival. Additionally, comparative genome analysis screened competence loci across all the Thermus genomes which could be helpful to acquire DNA from environment. In the present study it was found that Thermus isolates use two mechanism of incomplete denitrification pathway, some Thermus strains produces nitric oxide while others nitrious oxide (dinitrogen oxide), which show the heterotrophic lifestyle of Thermus genus. All isolated organisms encoded complete pathways for glycolysis, tricarboxylic acid and pentose phosphate. Calvin Benson Bassham cycle genes were identified in genomes of T. oshimai and T. antranikianii strains, while genomes of all T. brokianus strains and organism 77420 were lacking. Arsenic, cadmium and cobalt-zinc-cadmium resistant genes were detected in genomes of all sequenced Thermus strains. Strains 77,420, 77,311, 73,518, 77,412 and 72,351 genomes were found harboring genes for siderophores production. Sox gene clusters were identified in all sequenced genomes, except strain YIM 730264, suggesting a mode of chemolithotrophy. Through the comparative genomic analysis, we also identified 77420 as the genome type species and its validity as novel organism was confirmed by whole genome sequences comparison. Although isolate 77420 had 99.0% 16S rRNA gene sequence similarity with T. thalpophilus SYSU G00506T but based on ANI 95.86% (Jspecies) and digital DDH 68.80% (GGDC) values differentiate it as a potential novel species. Similarly, in the phylogenomic tree, the novel isolate 77,420 forming a separate branch with their closest reference type strain T. thalpophilus SYSU G00506T.

Quorum Quenchers from Reynoutria japonica in the Battle against Methicillin-Resistant Staphylococcus aureus (MRSA).

Over the past decade, methicillin-resistant Staphylococcus aureus (MRSA) has become a major source of biofilm formation and a major contributor to antimicrobial resistance. The genes that govern biofilm formation are regulated by a signaling mechanism called the quorum-sensing system. There is a need for new molecules to treat the infections caused by dangerous pathogens like MRSA. The current study focused on an alternative approach using juglone derivatives from Reynoutria japonica as quorum quenchers. Ten bioactive compounds from this plant, i.e., 2-methoxy-6-acetyl-7-methyljuglone, emodin, emodin 8-o-b glucoside, polydatin, resveratrol, physcion, citreorosein, quercetin, hyperoside, and coumarin were taken as ligands and docked with accessory gene regulator proteins A, B, and C and the signal transduction protein TRAP. The best ligand was selected based on docking score, ADMET properties, and the Lipinski rule. Considering all these parameters, resveratrol displayed all required drug-like properties with a docking score of -8.9 against accessory gene regulator protein C. To further assess the effectiveness of resveratrol, it was compared with the commercially available antibiotic drug penicillin. A comparison of all drug-like characteristics showed that resveratrol was superior to penicillin in many aspects. Penicillin showed a binding affinity of -6.7 while resveratrol had a score of -8.9 during docking. This was followed by molecular dynamic simulations wherein inhibitors in complexes with target proteins showed stability inside the active site during the 100 ns simulations. Structural changes due to ligand movement inside the cavity were measured in the protein targets, but they remained static due to hydrogen bonds. The results showed acceptable pharmacokinetic properties for resveratrol as compared to penicillin. Thus, we concluded that resveratrol has protective effects against Staphylococcus aureus infections and that it suppresses the quorum-sensing ability of this bacterium by targeting its infectious proteins.

Noviherbaspirillum aridicola sp. nov., isolated from an arid soil in Pakistan.

Strain NCCP-691T was isolated from a soil sample collected from an arid soil in Karak, Khyber Pakhtunkhwa, Pakistan. Phenotypically, the cells were Gram-stain-negative, aerobic and motile rods. The organism was able to grow between 20-40 °C (optimum at 30-37 °C), at pH 5.5-8.0 (optimum at pH 7.0-7.2) and tolerated 0-1.5% NaCl (w/v) (optimum at 0-0.5). Based on 16S rRNA gene sequences, strain NCCP-691T formed a distinct phylogenetic clade with Noviherbaspirillum arenae, N. agri, N. denitrificans and N. autotrophicum (having sequence similarities of 99.0; 98.1; 98.0 and 97.7% respectively). Phylogenetic analyses based on the whole genome sequences confirmed that strain NCCP-691T should be affiliated to the genus Noviherbaspirillum. The average nucleotide identity values compared to other species of Noviherbaspirillum were below 95-96 % and digital DNA-DNA hybridization values were less than 70 %. Chemotaxonomic analyses showed that the strain had ubiquinone-8, as the only respiratory quinine. The major cellular fatty acids were summed feature 3 (C16 : 1 ω 7 c/C16 : 1 ω 6 c, 35.9 %), summed feature 8 (C18 : 1 ω 7 c/C18 : 1 ω 6 c, 26.9 %) and C16 : 0 (22.9 %) and the polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The genomic DNA G+C content was 65.5 mol% (from draft genome). Genome analyses showed that strain NCCP-691T had terpene and arylpolyene biosynthetic genes clusters and genes related to resistance against heavy metals. Based on phylogenetic analyses, phenotypic features and genomic comparison, it is proposed that strain NCCP-691T is a novel species of the genus Noviherbaspirillum and the name Noviherbaspirillum aridicola sp. nov. is proposed. Type strain is NCCP-691T (=KCTC 52721T=CGMCC 1.13600T).

Zafaria cholistanensis gen. nov. sp. nov., a moderately thermotolerant and halotolerant actinobacterium isolated from Cholistan desert soil of Pakistan.

A Gram-staining positive, non-spore forming, non-pigmented and non-motile bacterium, designated strain NCCP-1664T, was isolated from Cholistan desert, Pakistan. Cells of strain NCCP-1664T were strictly aerobic, catalase positive and oxidase negative with a rod to coccus growth cycle and can grow at pH 6.0-9.0 (optimum pH 7-8) at 28-45 °C (optimum 37 °C) and could tolerate 0-16% NaCl (optimum 2%). Phylogenetic analyses based on 16S rRNA gene sequence revealed that strain NCCP-1664T belongs to the family Micrococcaceae and was related to members of the genus Arthrobacter having highest sequence similarities with Arthrobacter ginkgonis (98.9%), A. halodurans (97.7%) and A. oryzae (97.1%) and less than 97% with other related taxa. DNA-DNA relatedness values of strain NCCP-1664T with above mentioned type strains were found to be less than 54%, whereas digital DDH and average nucleotide identity (ANI) values with A. oryzae were 20.9 and of 74.3%, respectively. DNA G + C content of strain NCCP-1664T was 70.0 mol%. Chemotaxonomic data of strain NCCP-1664T showed the peptidoglycan type as A3α L-Lys-L -Ala; menaquinones as MK-9(H2) (67%), MK-8(H2) (32%) and MK-7(H2) (1%), major fatty acids as anteiso -C15:0 (51.2%), anteiso-C17:0 (9.6%) and C18:1ω9c (6.9%) and polar lipids profile comprising of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, digalactosyldiacylglycerol, small amounts of monogalactosyldiacylglycerol, trimannosyldiacylglycerol and three unidentified lipids. The phylogenomic analyses along with chemotaxonomic data, physiological, biochemical characteristics allowed to describe it as representative of a novel genus, for which the name Zafaria cholistanensis gen. nov. sp. nov. is proposed with the type strain NCCP-1664T (= DSM 29936T = KCTC 39549T).

Nocardioides thalensis sp. nov., isolated from a desert.

A novel actinobacterial strain, designated NCCP-696T, was isolated from the Thal desert in Punjab, Pakistan, and characterized by using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain NCCP-696T belongs to the genus Nocardioides and showed the highest level of sequence similarity with respect to Nocardioides panacisoliGsoil 346T (98.2 %) and less than 96.4 % to the strains of other species of the genus Nocardioides. Cells of strain NCCP-696T were Gram-positive, aerobic, non-motile rods and formed cream-coloured colonies. The strain was positive for oxidase and catalase. Growth occurred at 20-42 °C (optimum 30-37 °C) at pH 5.5-9.0 (optimum pH 7.0) and with 0-4 % NaCl (optimum 0-2 %, w/v). Strain NCCP-696T contained Iso-C16 : 0, C18 : 1ω9c, C17 : 1ω8c and C17 : 0 as the predominant fatty acids and was found to have LL-2,6-diaminopimelic acid in the cell-wall peptidoglycan. The polar lipid profile consisted of phosphatidylinositol, phosphatidylglycerol and one unknown phospholipid. The major menaquinone was MK-8(H4) (98.7 %) while a minor amount (1.3 %) of MK-9(H2) was also detected. The DNA G+C content of the genomic DNA was 71.6 mol%. The DNA-DNA hybridization value of the isolate against the closely related type strain Nocardioides panacisoliGsoil 346T was 56.3±1.4. On the basis of the phylogenetic inference, chemotaxonomic characteristics and phenotypic data, strain NCCP-696T should be classified as a novel species, for which the name Nocardioides thalensis sp. nov. is proposed. The type strain is NCCP-696T (=DSM 103833T=CCTCC AB 2016296T).

Thermus caldifontis sp. nov., a thermophilic bacterium isolated from a hot spring.

A thermophilic bacterial strain, designated YIM 73026T was isolated from a sediment sample collected from a hot spring in Tibet, PR China. The taxonomic position of the novel isolate was investigated by a polyphasic approach. The novel isolate was Gram-stain-negative, aerobic and rod-shaped. Colonies were circular, convex, opaque and yellow. The strain grew at 50-70 °C (optimum, 60 °C), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of up to 1.0 % NaCl (w/v). Comparison of the 16S rRNA gene sequences of YIM 73026T and those of other members of the genus Thermus showed sequence similarities ranging from 91.2 to 97.5 %, with YIM 73026T showing closest sequence similarity to Thermus scotoductus SE-1T (97.5 %). DNA-DNA hybridization results, however, revealed that DNA-DNA reassociation values between YIM 73026T and T. scotoductus DSM 8553T (37.6 %), Thermusamyloliquefaciens YIM 77409T (34.5 %), Thermusantranikianii DSM 12462T (30.3 %), Thermuscaliditerrae YIM 77925T (28.6 %) and Thermustengchongensis YIM 77924T (27.3 %) were well below the 70 % limit for species identification. YIM 73026T contained MK-8 as the respiratory quinone, and iso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and anteiso-C15 : 0 as the major cellular fatty acids (>10 %). The polar lipids consisted of one aminophospholipid, one phospholipid and two glycolipids. The genomic DNA G+C content of YIM 73026T was 65.4 mol%. On the basis of morphological, chemotaxonomic and genotypic characteristics, it is proposed that the isolate represents a novel species of the genus Thermus, for which the name Thermus caldifontis sp. nov. is proposed. The type strain is YIM 73026T (=NBRC 112415T=CCTCC AB 2016305T).

Tibeticola sediminis gen. nov., sp. nov., a thermophilic bacterium isolated from a hot spring.

Two closely related thermophilic bacterial strains, designated YIM 73013T and YIM 73008, were isolated from a sediment sample collected from a hot spring in Tibet, western Tibet province, China. The taxonomic positions of the two isolates were investigated using a polyphasic approach. The novel isolates were Gram-stain-negative, aerobic, short-rod-shaped and motile by means of a polar flagellum. They were oxidase- and catalase-positive and were able to grow at 30-55 °C (optimum, 37-45 °C), at pH 6.0-8.0 (optimum, pH 7.0) and with NaCl tolerance up to 1 % (w/v). Phylogenetic analyses based on 16S rRNA gene sequences showed that strains YIM 73013T and YIM 73008 formed a distinct lineage with respect to closely related genera in the family Comamonadaceae and shared highest 16S rRNA gene sequences similarities with Acidovorax caeni R-24608T (96.3 and 96.4 %, respectively). The respiratory quinone was ubiquinone-8 (Q-8) and the major cellular fatty acids observed were C17 : 1ω6c, C16 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The genomic DNA G+C contents of strains YIM 73013T and YIM 73008 were 68.7 and 68.3 mol%, respectively. Based on the morphological, phylogenetic and chemotaxonomic results, the two isolates represent a novel species in a new genus, for which the name Tibeticola sediminis gen. nov., sp. nov. is proposed. The type strain of Tibeticola sediminis is YIM 73013T (=DSM 101684T=KCTC 42873T).

Diversity and Distribution of Thermophilic Bacteria in Hot Springs of Pakistan.

Chilas and Hunza areas, located in the Main Mantle Thrust and Main Karakoram Thrust of the Himalayas, host a range of geochemically diverse hot springs. This Himalayan geothermal region encompassed hot springs ranging in temperature from 60 to 95 °C, in pH from 6.2 to 9.4, and in mineralogy from bicarbonates (Tato Field), sulfates (Tatta Pani) to mixed type (Murtazaabad). Microbial community structures in these geothermal springs remained largely unexplored to date. In this study, we report a comprehensive, culture-independent survey of microbial communities in nine samples from these geothermal fields by employing a bar-coded pyrosequencing technique. The bacterial phyla Proteobacteria and Chloroflexi were dominant in all samples from Tato Field, Tatta Pani, and Murtazaabad. The community structures however depended on temperature, pH, and physicochemical parameters of the geothermal sites. The Murtazaabad hot springs with relatively higher temperature (90-95 °C) favored the growth of phylum Thermotogae, whereas the Tatta Pani thermal spring site TP-H3-b (60 °C) favored the phylum Proteobacteria. At sites with low silica and high temperature, OTUs belonging to phylum Chloroflexi were dominant. Deep water areas of the Murtazaabad hot springs favored the sulfur-reducing bacteria. About 40% of the total OTUs obtained from these samples were unclassified or uncharacterized, suggesting the presence of many undiscovered and unexplored microbiota. This study has provided novel insights into the nature of ecological interactions among important taxa in these communities, which in turn will help in determining future study courses in these sites.

Streptomyces caldifontis sp. nov., isolated from a hot water spring of Tatta Pani, Kotli, Pakistan.

A Gram-staining positive, non-motile, rod-shaped, catalase positive and oxidase negative bacterium, designated NCCP-1331T, was isolated from a hot water spring soil collected from Tatta Pani, Kotli, Azad Jammu and Kashmir, Pakistan. The isolate grew at a temperature range of 18-40 °C (optimum 30 °C), pH 6.0-9.0 (optimum 7.0) and with 0-6 % NaCl (optimum 2 % NaCl (w/v)). The phylogenetic analysis based on 16S rRNA gene sequence revealed that strain NCCP-1331T belonged to the genus Streptomyces and is closely related to Streptomyces brevispora BK160T with 97.9 % nucleotide similarity, followed by Streptomyces drosdowiczii NRRL B-24297T with 97.8 % nucleotide similarity. The DNA-DNA relatedness values of strain NCCP-1331T with S. brevispora KACC 21093T and S. drosdowiczii CBMAI 0498T were 42.7 and 34.7 %, respectively. LL-DAP was detected as diagnostic amino acid along with alanine, glycine, leucine and glutamic acid. The isolate contained MK-9(H8) as the predominant menaquinone. Major polar lipids detected in NCCP-1331T were phosphatidylethanolamine, phosphatidylinositol and unidentified phospholipids. Major fatty acids were iso-C16: 0, summed feature 8 (18:1 ω7c/18:1 ω6c), anteiso-C15:0 and C16:0. The genomic DNA G + C content was 69.8 mol %. On the basis of phylogenetic, phenotypic and chemotaxonomic analysis, it is concluded that strain NCCP-1331T represents a novel species of the genus Streptomyces, for which the name Streptomyces caldifontis sp. nov. is proposed. The type strain is NCCP-1331T (=KCTC 39537T = CPCC 204147T).

Microvirga pakistanensis sp. nov., a novel bacterium isolated from desert soil of Cholistan, Pakistan.

A Gram-negative, non-spore-forming, non-pigmented, strictly aerobic and non-motile short rod bacterium, designated NCCP-1258T, was isolated from Cholistan desert soil, Bahawalpur, Pakistan. Growth of strain NCCP-1258T was observed at pH range 6.5-9.5 (optimum 7.5-8.5) and temperature range 20-45 °C (optimum 40 °C), and it tolerated 0-2 % NaCl (optimum 0.5 %, w/v). Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain NCCP-1258T belongs to genus Microvirga and is most closely related to Microvirga lotononidis (98.0 %), Microvirga vignae (97.4 %), Microvirga lupini (97.2 %), Microvirga zambiensis (97.2 %) and Microvirga flocculans (97.1 %). Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA and rpoB) also confirmed the placement of strain NCCP-1258T within the genus Microvirga. DNA-DNA relatedness values of NCCP-1258T with above-mentioned type strains were less than 42 %. The DNA G+C content of strain NCCP-1258T was 64.3 mol%. Chemotaxonomic data (predominant menaquinone system was Q-10; major fatty acids were C16:0, C18:1 ω7c and C19:0 cyclo ω8c; the polar lipid profile contained diphosphatidylglycerol, phosphatidylcholine, phosphatidyl dimethyl ethanolamine and phosphatidyl ethanolamine) also supported the affiliation of strain NCCP-1258T to the genus Microvirga. On the basis of physiological and biochemical characteristics, phylogenetic analyses and DNA-DNA relatedness, strain NCCP-1258T can be distinguished from the closely related taxa and thus represents a novel species of the genus Microvirga, for which the name Microvirga pakistanensis sp. nov. is proposed with the type strain NCCP-1258T (=CGMCC 1.15074T = KCTC 42496T).

Nocardioides pakistanensis sp. nov., isolated from a hot water spring of Tatta Pani in Pakistan.

A Gram-staining positive, non-spore forming, non-pigmented and non-motile bacterium, designated as NCCP-1340(T), was isolated from a hot water spring, Tatta Pani, Pakistan. Cells of strain NCCP-1340(T) were observed to be aerobic, rod shaped, catalase and urease positive but H2S production and oxidase negative. Growth was observed at pH 6.0-8.0 (optimum pH 7.0) and at 20-40 °C (optimum 37 °C). The strain could tolerate 0-8 % NaCl (optimum 2 %, w/v). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain NCCP-1340(T) belongs to the genus Nocardioides and is closely related to Nocardioides iriomotensis JCM 17985(T) (96.8 %), Nocardioides daedukensis KCTC 19601(T) (96.6 %), Nocardioides jensenii KCTC 9134(T) (96.1 %) and Nocardioides daejeonensis KCTC 19772(T) (96.1 %). The DNA-DNA relatedness values of strain NCCP-1340(T) with N. iriomotensis JCM 17985(T), N. daedukensis KCTC 19601(T) and N. jensenii KCTC 9134(T) were found to be less than 53 %. The DNA G+C content of strain NCCP-1340(T) was determined to be 71.8 mol  %. The affiliation of strain NCCP-1340(T) to the genus Nocardioides was further supported by chemotaxonomic data which showed the presence of MK-8(H4) as major menaquinone system; iso-C16:0, C17:0, C16:0 10-methyl, iso-C15:0 and C 15:0 as major cellular fatty acids; and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and unidentified glycolipids and polar lipids in the polar lipids profile. The cell wall peptidoglycan contained LL-diaminopimelic acid as the diagnostic amino acid. On the basis of physiological and biochemical characteristics and the phylogenetic analyses, strain NCCP-1340(T) can be distinguished from the closely related taxa and thus represents a novel species of the genus Nocardioides, for which the name Nocardioides pakistanensis sp. nov. is proposed with the type strain NCCP-1340(T) (= DSM 29942(T) = JCM 30630(T)).

Untapped rich microbiota of mangroves of Pakistan: diversity and community compositions.

The mangrove ecosystem is the world's fourth most productive ecosystem in terms of service value and offering rich biological resources. Microorganisms play vital roles in these ecological processes, thus researching the mangroves-microbiota is crucial for a deeper comprehension of mangroves dynamics. Amplicon sequencing that targeted V4 region of 16S rRNA gene was employed to profile the microbial diversities and community compositions of 19 soil samples, which were collected from the rhizosphere of 3 plant species (i.e., Avicennia marina, Ceriops tagal, and Rhizophora mucronata) in the mangrove forests of Lasbela coast, Pakistan. A total of 67 bacterial phyla were observed from three mangroves species, and these taxa were classified into 188 classes, 453 orders, 759 families, and 1327 genera. We found that Proteobacteria (34.9-38.4%) and Desulfobacteria (7.6-10.0%) were the dominant phyla followed by Chloroflexi (6.6-7.3%), Gemmatimonadota (5.4-6.8%), Bacteroidota (4.3-5.5%), Planctomycetota (4.4-4.9%) and Acidobacteriota (2.7-3.4%), Actinobacteriota (2.5-3.3%), and Crenarchaeota (2.5-3.3%). After considering the distribution of taxonomic groups, we prescribe that the distinctions in bacterial community composition and diversity are ascribed to the changes in physicochemical attributes of the soil samples (i.e., electrical conductivity (ECe), pH, total organic matter (OM), total organic carbon (OC), available phosphorus (P), and extractable potassium (CaCO3). The findings of this study indicated a high-level species diversity in Pakistani mangroves. The outcomes may also aid in the development of effective conservation policies for mangrove ecosystems, which have been hotspots for anthropogenic impacts in Pakistan. To our knowledge, this is the first microbial research from a Pakistani mangrove forest.

Insights on comparative bacterial diversity between different arid zones of Cholistan Desert, Pakistan.

The present study was conducted to analyze bacterial diversity profile of Cholistan desert located in Pakistan. The study investigates the influence of physicochemical parameters of soil on distribution of different bacteria at all taxonomic levels and also study the distribution pattern between different desert environments, particularly rhizospheric and bulk desert sands. Species richness showed phyla Proteobacteria and Chloroflexi as the dominant OTUs in all the samples. Besides the two phyla, the rhizospheric soils with root remnants were dominated by Firmicutes, Deinococcus-Thermus, Actinobacteria and Acidobacteri, while phylum Thermotogae was present in significant quantity in rhizosheaths devoid of roots. In non-rhizospheric desert soils, a considerable number of OTUs belonged to phyla Proteobacteria, Chloroflexi, Bacteroidetes and Acidobacteria. An important finding from this study is that a bulk portion of the OTUs were assigned to unclassified taxa, indicating a large repertoire of unexplored taxa in the desert ecology of Pakistan. Distribution of taxonomic groups among various regions of the desert was collaborating well with the physicochemical parameters of the sites. The findings of this study establish the fundamental relationships between desert ecosystem, specific native plant and the total bacterial flora. This is the first study of microbial community analysis of any desert in Pakistan and thus, will serve as a future platform to explore further on desert ecosystem functioning by employing the ever-changing biotechnological tools.