scholarly journals Comparative genomics of the genus Roseburia reveals divergent biosynthetic pathways that may influence colonic competition among species

2020 ◽  
Vol 6 (7) ◽  
Author(s):  
Ethan T. Hillman ◽  
Ariangela J. Kozik ◽  
Casey A. Hooker ◽  
John L. Burnett ◽  
Yoojung Heo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Comparative Genomics ◽  
Type Species ◽  
Gene Content ◽  
Interspecies Interactions ◽  
Content Type ◽  
Link Type ◽  
Folate Biosynthesis ◽  
B Vitamin

Roseburia species are important denizens of the human gut microbiome that ferment complex polysaccharides to butyrate as a terminal fermentation product, which influences human physiology and serves as an energy source for colonocytes. Previous comparative genomics analyses of the genus Roseburia have examined polysaccharide degradation genes. Here, we characterize the core and pangenomes of the genus Roseburia with respect to central carbon and energy metabolism, as well as biosynthesis of amino acids and B vitamins using orthology-based methods, uncovering significant differences among species in their biosynthetic capacities. Variation in gene content among Roseburia species and strains was most significant for cofactor biosynthesis. Unlike all other species of Roseburia that we analysed, Roseburia inulinivorans strains lacked biosynthetic genes for riboflavin or pantothenate but possessed folate biosynthesis genes. Differences in gene content for B vitamin synthesis were matched with differences in putative salvage and synthesis strategies among species. For example, we observed extended biotin salvage capabilities in R. intestinalis strains, which further suggest that B vitamin acquisition strategies may impact fitness in the gut ecosystem. As differences in the functional potential to synthesize components of biomass (e.g. amino acids, vitamins) can drive interspecies interactions, variation in auxotrophies of the Roseburia spp. genomes may influence in vivo gut ecology. This study serves to advance our understanding of the potential metabolic interactions that influence the ecology of Roseburia spp. and, ultimately, may provide a basis for rational strategies to manipulate the abundances of these species.

scholarly journals Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Microbiology ◽  
2020 ◽  
Vol 166 (5) ◽  
pp. 484-497 ◽  
Author(s):  
Alejandra Arteaga Ide ◽  
Victor M. Hernández ◽  
Liliana Medina-Aparicio ◽  
Edson Carcamo-Noriega ◽  
Lourdes Girard ◽  
...  
Keyword(s):  
Type Species ◽  
Sinorhizobium Meliloti ◽  
Arginase Activity ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Link Type ◽  
Arginine Catabolism

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Nirbhay Singh ◽  
Anu Chauhan ◽  
Ram Kumar ◽  
Sudheer Kumar Singh
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Low Ph ◽  
Stress Conditions ◽  
Starvation Stress ◽  
Content Type ◽  
E Coli ◽  
Link Type

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

scholarly journals SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions

Microbiology ◽  
2020 ◽  
Vol 166 (3) ◽  
pp. 296-305 ◽  
Author(s):  
Tsaone Tamuhla ◽  
Lydia Joubert ◽  
Danicke Willemse ◽  
Monique J. Williams
Keyword(s):  
Mycobacterium Smegmatis ◽  
Type Species ◽  
Model Organism ◽  
Culture Conditions ◽  
Growth Defect ◽  
Content Type ◽  
Link Type ◽  
Targeted Gene Deletion ◽  
Accessory Factor

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. Deletion of the sufT gene in M. smegmatis had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.

scholarly journals bla OXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands

2021 ◽  
Vol 7 (5) ◽  
Author(s):  
Antoni P. A. Hendrickx ◽  
Fabian Landman ◽  
Angela de Haan ◽  
Sandra Witteveen ◽  
Marga G. van Santen-Verheuvel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
The Netherlands ◽  
Resistance Genes ◽  
Type Species ◽  
Antibiotic Resistance Genes ◽  
Gene Content ◽  
Content Type ◽  
E Coli ◽  
Link Type

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by bla OXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the bla OXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete bla OXA-48-like plasmids for E. coli and K. pneumoniae , respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the bla OXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded bla OXA-48-like alleles. Chromosomally localized bla OXA-48 and bla OXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The bla OXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli . Lastly, K. pneumoniae isolates carrying bla OXA-48 or bla OXA-232 were mostly resistant for meropenem, whereas E. coli bla OXA-48, bla OXA-181 and chromosomal bla OXA-48 or bla OXA-244 isolates were mostly sensitive. In conclusion, the overall bla OXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of bla OXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

scholarly journals In vitro exploration of the Xanthomonas hortorum pv. vitians genome using transposon insertion sequencing and comparative genomics to discriminate between core and contextual essential genes

2021 ◽  
Author(s):  
Lucas Morinière ◽  
Solène Lecomte ◽  
Erwan Gueguen ◽  
Franck Bertolla
Keyword(s):  
Comparative Genomics ◽  
Plant Pathogen ◽  
Type Species ◽  
Essential Genes ◽  
Content Type ◽  
Link Type ◽  
Transposon Insertion ◽  
Ecological Features ◽  
Genomic Study ◽  
Transposon Insertion Sequencing

The essential genome of a bacterium encompasses core genes associated with basic cellular processes and conditionally essential genes dependent upon environmental conditions or the genetic context. Comprehensive knowledge of those gene sets allows for a better understanding of fundamental bacterial biology and offers new perspectives for antimicrobial drug research against detrimental bacteria such as pathogens. We investigated the essential genome ofXanthomonas hortorumpv.vitians, a gammaproteobacterial plant pathogen of lettuce (Lactuca sativaL.) which belongs to the plant-pathogen reservoir genusXanthomonasand is affiliated to the familyXanthomonadaceae. No practical means of disease control or prevention against this pathogen is currently available, and its molecular biology is virtually unknown. To reach a comprehensive overview of the essential genome ofX. hortorumpv.vitiansLM16734, we developed a mixed approach combining high-quality full genome sequencing, saturated transposon insertion sequencing (Tn-Seq) in optimal growth conditions, and coupled computational analyses such as comparative genomics, synteny assessment and phylogenomics. Among the 370 essential loci identified by Tn-Seq, a majority was bound to critical cell processes conserved across bacteria. The remaining genes were either related to specific ecological features ofXanthomonasorXanthomonadaceaespecies, or acquired through horizontal gene transfer of mobile genetic elements and associated with ancestral parasitic gene behaviour and bacterial defence systems. Our study sheds new light on our usual concepts about gene essentiality and is pioneering in the molecular and genomic study ofX. hortorumpv.vitians.

scholarly journals Delivery of antibacterial silver nanoclusters to Pseudomonas aeruginosa using species-specific DNA aptamers

2020 ◽  
Vol 69 (4) ◽  
pp. 640-652 ◽  
Author(s):  
Jennifer Soundy ◽  
Darren Day
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Type Species ◽  
Galleria Mellonella ◽  
Host Cells ◽  
Silver Nanoclusters ◽  
Bacterial Cells ◽  
Content Type ◽  
Link Type

Introduction. The use of silver as an antimicrobial therapeutic is limited by its toxicity to host cells compared with that required to kill bacterial pathogens. Aim. To use aptamer targeting of DNA scaffolded silver nanoclusters as an antimicrobial agent for treating Pseudomonas aeruginosa infections. Methodology. Antimicrobial activity was assessed in planktonic cultures and in vivo using an invertebrate model of infection. Results. The aptamer conjugates that we call aptabiotics have potent antimicrobial activity. Targeted silver nanoclusters were more effective at killing P. aeruginosa than the equivalent quantity of untargeted silver nanoclusters. The aptabiotics have an IC50 of 1.3–2.6 µM against planktonically grown bacteria. Propidium iodide staining showed that they rapidly depolarize bacterial cells to kill approximately 50 % of the population within 10 min following treatment. In vivo testing in the Galleria mellonella model of infection prolonged survival from an otherwise lethal infection. Conclusion. Using P. aeruginosa as a model, we show that targeting of DNA-scaffolded silver nanoclusters with an aptamer has effective fast-acting antimicrobial activity in vitro and in an in vivo animal model.

Mining indigenous honeybee gut microbiota for Lactobacillus with probiotic potential

Microbiology ◽  
2021 ◽  
Author(s):  
Ghazal Aziz ◽  
Muhammad Tariq ◽  
Arsalan Haseeb Zaidi
Keyword(s):  
Type Species ◽  
Amplicon Sequencing ◽  
Culturable Bacteria ◽  
Human Pathogens ◽  
Carbohydrate Utilization ◽  
Potential Health ◽  
Content Type ◽  
Link Type

The present study was done to explore the diversity of lactic acid bacteria (LAB) associated with the gastrointestinal tract (GIT) of honeybee species endemic to northeastern Pakistan. Healthy worker bees belonging to Apis mellifera, A. dorsata, A. cerana and A. florea were collected from hives and the surroundings of a major apiary in the region. The 16S rRNA amplicon sequencing revealed a microbial community in A. florea that was distinct from the others in having an abundance of Lactobacillus and Bifidobacteria. However, this was not reflected in the culturable bacteria obtained from these species. The isolates were characterized for safety parameters, and 20 LAB strains deemed safe were evaluated for resistance to human GIT stresses like acid and bile, adhesion and adhesiveness, and anti-pathogenicity. The five most robust strains, Enterococcus saigonensis NPL780a, Lactobacillus rapi NPL782a, Lactobacillus kunkeei NPL783a, and NPL784, and Lactobacillus paracasei NPL783b, were identified through normalized Pearson (n) principal components analysis (PCA). These strains were checked for inhibition of human pathogens, antibiotic resistance, osmotic tolerance, metabolic and enzymatic functions, and carbohydrate utilization, along with antioxidative and cholesterol-removing potential. The findings suggest at least three strains (NPL 783a, 784 and 782a) as candidates for further in vitro and in vivo investigations of their potential health benefits and application as novel probiotic adjuncts.

Nitrogeniibacter mangrovi gen. nov., sp. nov., a novel anaerobic and aerobic denitrifying betaproteobacterium and reclassification of Azoarcus pumilus as Aromatoleum pumilum comb. nov.

2021 ◽  
Vol 71 (8) ◽  
Author(s):  
Hu Liao ◽  
Mingming Qu ◽  
Xinyue Hou ◽  
Xiaolan Lin ◽  
Hu Li ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Type Species ◽  
Novel Species ◽  
Mangrove Sediment ◽  
Respiratory Quinone ◽  
Content Type ◽  
Denitrifying Bacterium ◽  
Taxonomic Analysis ◽  
Link Type ◽  
Predominant Respiratory Quinone

Denitrification is a vital link in the global bio-nitrogen cycle. Here, we isolated a strain (M9-3-2T) that is a novel benzo[a]pyrene (BaP)-tolerant, anaerobic and aerobic denitrifying bacterium from a continuous BaP-enrichment cultured mangrove sediment. In silico comparative genomics and taxonomic analysis clearly revealed that strain M9-3-2T (=MCCC 1K03313T=JCM 32045T) represents a novel species of a novel genus named as Nitrogeniibacter mangrovi gen. nov., sp. nov., belonging to family Zoogloeaceae , order Rhodocyclales . In addition, the species Azoarcus pumilus is transferred into genus Aromatoleum and named Aromatoleum pumilum comb. nov. The predominant respiratory quinone of strain M9-3-2T was ubiquinone-8 and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified phospholipids and three unidentified aminophospholipids. In this study, the capacity of strain M9-3-2T to eliminate nitrate was detected under anaerobic and aerobic conditions, and the removal rates of nitrate were 6.1×10−6 µg N/l/h/cell and 3×10−7 µg N/l/h/cell, respectively. Our results suggested that strain M9-3-2T could play an important role in the nitrogen removal regardless of the presence of oxygen in natural or/and man-made ecosystems.

scholarly journals Identification of Essential Residues for Ciprofloxacin Resistance of Ciprofloxacin-Modifying Enzyme (CrpP) of pUM505

Microbiology ◽  
2020 ◽  
Vol 166 (4) ◽  
pp. 367-374 ◽  
Author(s):  
Víctor M. Chávez-Jacobo ◽  
Juan Pablo García Merinos ◽  
Yliana López ◽  
Victor Meza-Carmen ◽  
Martha I. Ramírez-Díaz
Keyword(s):  
Amino Acids ◽  
Type Species ◽  
Terminal Region ◽  
Content Type ◽  
Extended Spectrum Beta Lactamase ◽  
Enzymatic Mechanism ◽  
Link Type ◽  
P Gene ◽  
Ciprofloxacin Resistance ◽  
Modified Proteins

The ciprofloxacin-resistance crpP gene, encoded by the pUM505 plasmid, isolated from a P. aeruginosa clinical isolate, confers an enzymatic mechanism of antibiotic phosphorylation, which is ATP-dependent, that decreases ciprofloxacin susceptibility. Homologous crpP genes are distributed across extended spectrum beta-lactamase (ESBL)-producing isolates obtained from Mexican hospitals and which confer decreased susceptibility to CIP. The analysis of sequences of the CrpP of proteins showed that the residues Gly7, Thr8, Asp9, Lys33 and Gly34 (located at the N-terminal region) and Cys40 (located at the C-terminal region) are conserved in all proteins, suggesting that these residues could be essential for CrpP function. The aim of this study was to investigate the amino acids essential to ciprofloxacin resistance, which is conferred by the CrpP protein of pUM505 plasmid. Mutations in the codons encoding Gly7, Asp9, Lys33 and Cys40 of CrpP protein from pUM505 were generated by PCR fusion. The results showed that all mutations generated in CrpP proteins increased ciprofloxacin susceptibility in Escherichia coli . In addition, the CrpP modified proteins were purified and their enzymatic activity on ciprofloxacin was assayed, showing that these modified proteins do not exert catalytic activity on ciprofloxacin. Moreover, by infrared assays it was determined that the modified proteins were are not able to modify the ciprofloxacin molecule. Our findings are the first report that indicate that the amino acids, namely Gly7, Asp9, Lys33 and Cys40, which are conserved in the CrpP proteins, possess an essential role for the enzymatic mechanism that confers ciprofloxacin resistance.

scholarly journals Host factors abolish the need for polysaccharides and extracellular matrix-binding protein in Staphylococcus epidermidis biofilm formation

2021 ◽  
Author(s):  
Sandra M. Skovdal ◽  
Liva Kjær Hansen ◽  
Diana Malskær Ivarsen ◽  
Guanghong Zeng ◽  
Henning Büttner ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Human Plasma ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Type Species ◽  
Host Factors ◽  
Content Type ◽  
Link Type ◽  
Matrix Components

Introduction. Staphylococcus epidermidis is predominant in implant-associated infections due to its capability to form biofilms. It can deploy several strategies for biofilm development using either polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA) and/or proteins, such as the extracellular matrix-binding protein (Embp). Hypothesis/Gap Statement. We hypothesize that the dichotomic regulation of S. epidermidis adhesins is linked to whether it is inside a host or not, and that in vitro biofilm investigations in laboratory media may not reflect actual biofilms in vivo. Aim. We address the importance of PIA and Embp in biofilm grown in ‘humanized’ media to understand if these components play different roles in biofilm formation under conditions where bacteria can incorporate host proteins in the biofilm matrix. Methodology. S. epidermidis 1585 WT (deficient in icaADBC), and derivative strains that either lack embp, express embp from an inducible promotor, or express icaADBC from a plasmid, were cultivated in standard laboratory media, or in media with human plasma or serum. The amount, structure, elasticity and antimicrobial penetration of biofilms was quantified to describe structural differences caused by the different matrix components and growth conditions. Finally, we quantified the initiation of biofilms as suspended aggregates in response to host factors to determine how quickly the cells aggregate in response to the host environment and reach a size that protects them from phagocytosis. Results. S. epidermidis 1585 required polysaccharides to form biofilm in laboratory media. However, these observations were not representative of the biofilm phenotype in the presence of human plasma. If human plasma were present, polysaccharides and Embp were redundant for biofilm formation. Biofilms formed in human plasma were loosely attached and existed mostly as suspended aggregates. Aggregation occurred after 2 h of exposing cells to plasma or serum. Despite stark differences in the amount and composition of biofilms formed by polysaccharide-producing and Embp-producing strains in different media, there were no differences in vancomycin penetration or susceptibility. Conclusion. We suggest that the assumed importance of polysaccharides for biofilm formation is an artefact from studying biofilms in laboratory media void of human matrix components. The cell–cell aggregation of S. epidermidis can be activated by host factors without relying on either of the major adhesins, PIA and Embp, indicating a need to revisit the basic question of how S. epidermidis deploys self-produced and host-derived matrix components to form antibiotic-tolerant biofilms in vivo.

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