Ligilactobacillus agilis BKN88 possesses thermo-/acid-stable heteropolymeric flagellar filaments

Microbiology ◽  
2021 ◽  
Author(s):  
Naoto Eguchi ◽  
Shunya Suzuki ◽  
Kenji Yokota ◽  
Shizunobu Igimi ◽  
Akinobu Kajikawa
Keyword(s):  
Type Species ◽  
Wild Type ◽  
Flagellin Gene ◽  
Specific Staining ◽  
Content Type ◽  
Link Type ◽  
Mutant Strains ◽  
In The Wild ◽  
Toll Like Receptor 5 ◽  
Acid Stable

Many flagellated bacteria possess multiple flagellins, but the roles and the compositions of each flagellin are diverse and poorly understood. In Ligilactobacillus agilis BKN88, there are two active flagellin gene paralogues but their function and composition in its flagellar filaments have not been described. The aim of this study is to find the function and composition of the flagellins by employing mutant strains each of which expresses a single flagellin or a modified flagellin. Two single flagellin-expressing strains were both flagellated while the number of flagella per cell in the single flagellin-expressing derivatives was lower than that in the wild type. Nonetheless, these derivative strains were apparently equally motile as the wild type. This indicates that either flagellin is sufficient for cell motility. The immunological activity via Toll-like receptor 5 of the single flagellin-expressing strains or purified single flagellins was readily detectable but mostly variably weaker than that of the wild type. The flagellar filaments of wild type L. agilis BKN88 were more acid-/thermo-stable than those of single flagellin-expressing derivatives. Using a combination of immunoprecipitation and flagellin-specific staining, wild type BKN88 appeared to possess heteropolymeric flagellar filaments consisting of both flagellins and each flagellin appeared to be equally distributed throughout the filaments. The results of this study suggest that the two flagellins together form a more robust filament than either alone and are thus functionally complementary.

scholarly journals Comparative genomics of wild-type and laboratory-evolved biofilm-overproducing Deinococcus metallilatus strains

2020 ◽  
Vol 6 (12) ◽  
Author(s):  
Chulwoo Park ◽  
Bora Shin ◽  
Wonjae Kim ◽  
Hoon Cheong ◽  
Soyoon Park ◽  
...  
Keyword(s):  
Cell Death ◽  
Biofilm Formation ◽  
Type Species ◽  
High Rate ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Extracellular Matrix Components ◽  
Mutant Strains ◽  
Mutant Cells

Deinococcus metallilatus MA1002 was exposed to ultraviolet radiation to generate mutants with enhanced biofilm production. Two strains (nos 5 and 6) were then selected based on their high biofilm formation, as well as their possession of higher concentrations of extracellular matrix components (eDNA, protein and saccharides) than the wild-type (WT). Genomic sequencing revealed the presence of large genome deletions in a secondary chromosome in the mutants. Expression analyses of the WT and mutant strains indicated the upregulation of genes associated with exopolysaccharide synthesis and stress response. The mutant strains showed high mortality in glucose-supplemented (TYG) medium; however, cell death and biofilm formation were not increased in mutant cells grown under acetate- or glyoxylate-added media, suggesting that metabolic toxicity during glucose metabolism induced a high rate of cell death but improved biofilm formation in mutant strains. In damaged cells, eDNAs contributed to the enhanced biofilm formation of D. metallilatus .

Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice

Microbiology ◽  
2021 ◽  
Vol 167 (10) ◽  
Author(s):  
Mengting Shi ◽  
Yue Zheng ◽  
Xianghong Wang ◽  
Zhengjia Wang ◽  
Menghua Yang
Keyword(s):  
Mouse Model ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Type Species ◽  
Wild Type ◽  
Expression Library ◽  
Content Type ◽  
Genomic Expression ◽  
Infant Mouse ◽  
Link Type

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae . In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

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.

scholarly journals ClpP is required for proteolytic regulation of type II toxin–antitoxin systems and persister cell formation in Streptococcus mutans

2019 ◽  
Vol 1 (8) ◽  
Author(s):  
Xiao-Lin Tian ◽  
Miao Li ◽  
Zachariah Scinocca ◽  
Heather Rutherford ◽  
Yung-Hua Li
Keyword(s):  
Streptococcus Mutans ◽  
Type Species ◽  
Critical Role ◽  
Cell Formation ◽  
Type Ii ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Viability Assay ◽  
Persister Cell

The type II toxin–antitoxin (TA) modules, mazEF and relBE, in Streptococcus mutans have been implicated in stress response, antibiotic tolerance and persister cell formation. However, how S. mutans regulates these systems to prevent unwanted toxin activation and persister cell formation is unclear. In this study, we provide evidence that ClpP is required for the proteolytic regulation of these TA systems and persister cell formation in S. mutans following antibiotic challenge. A persister viability assay showed that S. mutans UA159 (WT) formed a larger quantity of persister cells than its isogenic mutant ΔclpP following antibiotic challenge. However, the lux reporter assay revealed that clpP deletion did not affect the transcriptional levels of mazEF and relBE, since no significant differences (P>0.05) in the reporter activities were detected between the wild-type and ΔclpP background. Instead, all antibiotics tested at a sub-minimum inhibitory concentration (sub-MIC) induced transcriptional levels of mazEF and relBE operons. We then examined the protein profiles of His-tagged MazE and RelB proteins in the UA159 and ΔclpP backgrounds by Western blotting analysis. The results showed that S. mutans strains grown under non-stress conditions expressed very low but detectable levels of MazE and RelB antitoxin proteins. Antibiotics at sub-MICs induced the levels of the MazE and RelB proteins, but the protein levels decreased rapidly in the wild-type background. In contrast, a stable level of MazE and RelB proteins could be detected in the ΔclpP mutant background, suggesting that both proteins accumulated in the ΔclpP mutant. We conclude that ClpP is required for the proteolytic regulation of cellular levels of the MazE and RelB antitoxins in S. mutans , which may play a critical role in modulating the TA activities and persister cell formation of this organism following antibiotic challenge.

scholarly journals Differential susceptibility of airway and ocular surface cell lines to FlhDC-mediated virulence factors PhlA and ShlA from Serratia marcescens

2020 ◽  
Author(s):  
Nicholas A. Stella ◽  
Kimberly M. Brothers ◽  
Robert M. Q. Shanks
Keyword(s):  
Epithelial Cells ◽  
Serratia Marcescens ◽  
Cell Lines ◽  
Ocular Surface ◽  
Type Species ◽  
Wild Type ◽  
Bacterial Killing ◽  
Content Type ◽  
Link Type ◽  
Surface Cell

Introduction. Serratia marcescens is a bacterial pathogen that causes ventilator-associated pneumonia and ocular infections. The FlhD and FlhC proteins complex to form a heteromeric transcription factor whose regulon, in S. marcescens , regulates genes for the production of flagellum, phospholipase A and the cytolysin ShlA. The previously identified mutation, scrp-31, resulted in highly elevated expression of the flhDC operon. The scrp-31 mutant was observed to be more cytotoxic to human airway and ocular surface epithelial cells than the wild-type bacteria and the present study sought to identify the mechanism underlying the increased cytotoxicity phenotype. Hypothesis/Gap Statement. Although FlhC and FlhD have been implicated as virulence determinants, the mechanisms by which these proteins regulate bacterial cytotoxicity to different cell types remains unclear. Aim. This study aimed to evaluate the mechanisms of FlhDC-mediated cytotoxicity to human epithelial cells by S. marcescens . Methodology. Wild-type and mutant bacteria and bacterial secretomes were used to challenge airway and ocular surface cell lines as evaluated by resazurin and calcein AM staining. Pathogenesis was further tested using a Galleria mellonella infection model. Results. The increased cytotoxicity of scrp-31 bacteria and secretomes to both cell lines was eliminated by mutation of flhD and shlA. Mutation of the flagellin gene had no impact on cytotoxicity under any tested condition. Elimination of the phospholipase gene, phlA, had no effect on bacteria-induced cytotoxicity to either cell line, but reduced cytotoxicity caused by secretomes to airway epithelial cells. Mutation of flhD and shlA, but not phlA, reduced bacterial killing of G. mellonella larvae. Conclusion. This study indicates that the S. marcescens FlhDC-regulated secreted proteins PhlA and ShlA, but not flagellin, are cytotoxic to airway and ocular surface cells and demonstrates differences in human epithelial cell susceptibility to PhlA.

scholarly journals The putative phosphate transporter PitB (PP1373) is involved in tellurite uptake in Pseudomonas putida KT2440

Microbiology ◽  
2020 ◽  
Author(s):  
Rafael Montenegro ◽  
Sofía Vieto ◽  
Daniela Wicki-Emmenegger ◽  
Felipe Vásquez-Castro ◽  
Carolina Coronado-Ruiz ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Type Strain ◽  
Type Species ◽  
Wild Type Strain ◽  
Phosphate Transporter ◽  
Transport Systems ◽  
Wild Type ◽  
Pseudomonas Putida Kt2440 ◽  
Content Type ◽  
Link Type

Tellurium oxyanions are chemical species of great toxicity and their presence in the environment has increased because of mining industries and photovoltaic and electronic waste. Recovery strategies for this metalloid that are based on micro-organisms are of interest, but further studies of the transport systems and enzymes responsible for implementing tellurium transformations are required because many mechanisms remain unknown. Here, we investigated the involvement in tellurite uptake of the putative phosphate transporter PitB (PP1373) in soil bacterium Pseudomonas putida KT2440. For this purpose, through a method based on the CRISPR/Cas9 system, we generated a strain deficient in the pitB gene and characterized its phenotype on exposing it to varied concentrations of tellurite. Growth curves and transmission electronic microscopy experiments for the wild-type and ΔpitB strains showed that both were able to internalize tellurite into the cytoplasm and reduce the oxyanion to black nano-sized and rod-shaped tellurium particles, although the ΔpitB strain showed an increased resistance to the tellurite toxic effects. At a concentration of 100 μM tellurite, where the biomass formation of the wild-type strain decreased by half, we observed a greater ability of ΔpitB to reduce this oxyanion with respect to the wild-type strain (~38 vs ~16 %), which is related to the greater biomass production of ΔpitB and not to a greater consumption of tellurite per cell. The phenotype of the mutant was restored on over-expressing pitB in trans. In summary, our results indicate that PitB is one of several transporters responsible for tellurite uptake in P. putida KT2440.

scholarly journals Investigation of LuxS-mediated quorum sensing in Klebsiella pneumoniae

2020 ◽  
Vol 69 (3) ◽  
pp. 402-413 ◽  
Author(s):  
Lijiang Chen ◽  
Jonathan J. Wilksch ◽  
Haiyang Liu ◽  
Xiaoxiao Zhang ◽  
Von V. L. Torres ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Type Species ◽  
Microtiter Plate ◽  
Resistant Isolate ◽  
Wild Type ◽  
Content Type ◽  
Link Type

Introduction. Autoinducer-2 (AI-2) quorum sensing is a bacterial communication system that responds to cell density. The system requires luxS activity to produce AI-2, which can regulate gene expression and processes such as biofilm formation. Aim. To investigate the role of luxS in biofilm formation and gene expression in the nosocomial pathogen Klebsiella pneumoniae . Methodology. A ΔluxS gene deletion was made in K. pneumoniae KP563, an extensively drug-resistant isolate. AI-2 production was assessed in wild-type and ΔluxS strains grown in media supplemented with different carbohydrates. Potential roles of luxS in biofilm formation were investigated using a microtiter plate biofilm assay and scanning electron microscopy. Quantitative RT-PCR evaluated the expression of lipopolysaccharide (wzm and wbbM), polysaccharide (pgaA), and type 3 fimbriae (mrkA) synthesis genes in wild-type and ΔluxS mutant biofilm extracts. Results. AI-2 production was dependent on the presence of luxS. AI-2 accumulation was highest during early stationary phase in media supplemented with glucose, sucrose or glycerol. Changes in biofilm architecture were observed in the ΔluxS mutant, with less surface coverage and reduced macrocolony formation; however, no differences in biofilm formation between the wild-type and ΔluxS mutant using a microtiter plate assay were observed. In ΔluxS mutant biofilm extracts, the expression of wzm was down-regulated, and the expression of pgaA, which encodes a porin for poly-β−1,6-N-acetyl-d-glucosamine (PNAG) polysaccharide secretion, was upregulated. Conclusion. Relationships among AI-2-mediated quorum sensing, biofilm formation and gene expression of outer-membrane components were identified in K. pneumoniae . These inter-connected processes could be important for bacterial group behaviour and persistence.

scholarly journals A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum

2020 ◽  
Vol 69 (12) ◽  
pp. 1339-1345
Author(s):  
Norihito Tarumoto ◽  
Kazuo Imai ◽  
Shu-ichi Nakayama ◽  
Ichiro Itoda ◽  
Jun Sakai ◽  
...  
Keyword(s):  
Type Species ◽  
Treponema Pallidum ◽  
Isothermal Amplification ◽  
23S Rrna ◽  
Wild Type ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Link Type ◽  
Wild Type Sequence ◽  
Type Sequence

Introduction. Macrolides could be a potential alternative treatment for Treponema pallidum infections in patients; however, macrolide-resistant T. pallidum is spreading rapidly worldwide. Hypothesis/Gap Statement. There are presently no alternatives to serological tests for syphilis that can be used to evaluate therapeutic effects due to the fact that T. pallidum cannot be cultured in vitro. Aim. In this study, we constructed a method for rapidly identifying T. pallidum and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs). Methodology. A set of LAMP primers was designed to span nucleotide positions 2058 and 2059 in 23S rRNA. A PNA clamping probe was also designed to be complementary to the wild-type sequence (A2058/A2059) and positioned to interfere with both the annealing of the 3′ end of the backward inner primer and the concomitant extension. Prior to the LAMP assay, swab samples from suspected syphilitic lesions were boiled for DNA extraction. Results. The assay had an equivalent detection limit of 1.0×101 copies/reaction and showed specificity against 38 pathogens. In the presence of a 4 µM PNA probe, LAMP amplified up to 1.0×101 copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild-type sequence up to a concentration of 1.0×103 copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays. Conclusion. We developed a PNA-mediated LAMP method that enabled us to rapidly identify T. pallidum and determine its macrolide susceptibility via a culture-independent protocol.

scholarly journals Biochemical and functional characterization of the SMC holocomplex from Mycobacterium smegmatis

Microbiology ◽  
2020 ◽  
Author(s):  
Suchitra Pradhan ◽  
Shwetha K. ◽  
Pratibha Kumari ◽  
Ravi Kumar
Keyword(s):  
Mycobacterium Smegmatis ◽  
Type Species ◽  
Functional Characterization ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
Link Type ◽  
Growth Difference ◽  
Smc Proteins

Multi-subunit SMC complexes are required to perform essential functions, such as chromosome compaction, segregation and DNA repair, from bacteria to humans. Prokaryotic SMC proteins form complexes with two non-SMC subunits, ScpA and ScpB, to condense the chromosome. The mutants of both scpa and scpb genes in Bacillus subtilis have been shown to display characteristic phenotypes such as growth defects and increased frequency of anucleate cells. Here, we studied the function of the Smc-ScpAB complex from Mycobacterium smegmatis . We observed no significant growth difference between the scpb null mutant and wild-type M. smegmatis under both standard and stress conditions. Furthermore, we characterized the Smc-ScpAB holocomplex from M. smegmatis . The MsSMC consists of the dimerization hinge and ATPase head domains connected by long coiled-coils. The MsSMC interacts with two non-SMC proteins, ScpA and ScpB, and the resulting holocomplex binds to different DNA substrates independent of ATP. The Smc-ScpAB complex showed DNA-stimulated ATPase activity in the presence of ssDNA. A cytological profiling assay revealed that upon overexpression the Smc-ScpAB ternary complex compacts the decondensed nucleoid of rifampicin-treated wild-type and null mukb mutant of Escherichia coli in vivo. Together, our study suggests that M. smegmatis has a functional Smc-ScpAB complex capable of DNA binding and condensation. Based on our observations, we speculate that the presence of alternative SMCs such as MksB or other SMC homologues might have rescued the scpb mutant phenotype in M. smegmatis .

scholarly journals Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marianna Iorio ◽  
Sahar Davatgarbenam ◽  
Stefania Serina ◽  
Paolo Criscenzo ◽  
Mitja M. Zdouc ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Systematic Investigation ◽  
Biosynthetic Gene ◽  
Wild Type ◽  
Bacterial Rna ◽  
Soil Isolate ◽  
Mutant Strains ◽  
In The Wild ◽  
Polymerase Inhibitor

AbstractWe report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640.

Sign in / Sign up

Export Citation Format

Share Document