scholarly journals Modification of the 1-Phosphate Group during Biosynthesis of Capnocytophaga canimorsus Lipid A

2015 ◽  
Vol 84 (2) ◽  
pp. 550-561 ◽  
Author(s):  
Francesco Renzi ◽  
Ulrich Zähringer ◽  
Courtney E. Chandler ◽  
Robert K. Ernst ◽  
Guy R. Cornelis ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Lipid A ◽  
Sequence Similarity ◽  
Phosphate Group ◽  
Capnocytophaga Canimorsus ◽  
Content Type ◽  
Double Deletion ◽  
Severe Infections ◽  
Single Operon ◽  
Subsequent Addition

Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that theC. canimorsusgenome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of anP-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion oflpxEoreptAled to mutants lacking theP-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant oflpxE-eptAwas similar to that of a singlelpxEmutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains.

scholarly journals Na+/H+Antiport Is Essential for Yersinia pestis Virulence

2013 ◽  
Vol 81 (9) ◽  
pp. 3163-3172 ◽  
Author(s):  
Yusuke Minato ◽  
Amit Ghosh ◽  
Wyatt J. Faulkner ◽  
Erin J. Lind ◽  
Sara Schesser Bartra ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Drug Target ◽  
Deletion Mutant ◽  
Ex Vivo ◽  
Ion Homeostasis ◽  
Content Type ◽  
Double Deletion ◽  
Derivative Strain

ABSTRACTNa+/H+antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na+/H+antiport inYersinia pestisvirulence and found thatY. pestisstrains lacking the major Na+/H+antiporters, NhaA and NhaB, are completely attenuated in anin vivomodel of plague. TheY. pestisderivative strain lacking thenhaAandnhaBgenes showed markedly decreased survival in blood and blood serumex vivo. Complementation of eithernhaAornhaBintransrestored the survival of theY. pestis nhaA nhaBdouble deletion mutant in blood. ThenhaA nhaBdouble deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na+levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na+/H+antiport is indispensable for the survival ofY. pestisin the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused byY. pestisand possibly for those caused by other blood-borne bacterial pathogens.

scholarly journals Lack of Lipid A Pyrophosphorylation and FunctionallptAReduces Inflammation by Neisseria Commensals

2012 ◽  
Vol 80 (11) ◽  
pp. 4014-4026 ◽  
Author(s):  
Constance M. John ◽  
Mingfeng Liu ◽  
Nancy J. Phillips ◽  
Zhijie Yang ◽  
Courtney R. Funk ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Lipid A ◽  
Polymyxin B ◽  
Toll Like Receptor ◽  
Inflammatory Signaling ◽  
Toll Like Receptor 4 ◽  
Bioinformatics Analyses ◽  
Monocytic Cells ◽  
Content Type ◽  
High Level

ABSTRACTThe interaction of the immune system withNeisseriacommensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenicNeisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded bylptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensalNeisseriaspecies confirmed thatlptAwas absent in 15 of 17 strains examined but was present inN. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lackinglptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensalNeisseriastrains that lackedlptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of twoN. lactamicacommensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcallptAdeletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensalNeisseriastrains by reducing the inflammatory potential of LOS.

scholarly journals The rbmBCDEF Gene Cluster Modulates Development of Rugose Colony Morphology and Biofilm Formation in Vibrio cholerae

2007 ◽  
Vol 189 (6) ◽  
pp. 2319-2330 ◽  
Author(s):  
Jiunn C. N. Fong ◽  
Fitnat H. Yildiz
Keyword(s):  
Vibrio Cholerae ◽  
Intergenic Region ◽  
Biofilm Formation ◽  
Deletion Mutant ◽  
Sequence Similarity ◽  
Colony Morphology ◽  
Colony Development ◽  
Carbohydrate Binding ◽  
Double Deletion ◽  
Biofilm Structure

ABSTRACT Vibrio cholerae, the causative agent of cholera, can undergo phenotypic variation generating rugose and smooth variants. The rugose variant forms corrugated colonies and well-developed biofilms and exhibits increased levels of resistance to several environmental stresses. Many of these phenotypes are mediated in part by increased expression of the vps genes, which are organized into vps-I and vps-II coding regions, separated by an intergenic region. In this study, we generated in-frame deletions of the five genes located in the vps intergenic region, termed rbmB to -F (rugosity and biofilm structure modulators B to F) in the rugose genetic background, and characterized the mutants for rugose colony development and biofilm formation. Deletion of rbmB, which encodes a protein with low sequence similarity to polysaccharide hydrolases, resulted in an increase in colony corrugation and accumulation of exopolysaccharides relative to the rugose variant. RbmC and its homolog Bap1 are predicted to encode proteins with carbohydrate-binding domains. The colonies of the rbmC bap1 double deletion mutant and bap1 single deletion mutant exhibited a decrease in colony corrugation. Furthermore, the rbmC bap1 double deletion mutant was unable to form biofilms at the air-liquid interface after 2 days, while the biofilms formed on solid surfaces detached readily. Although the colony morphology of rbmDEF mutants was similar to that of the rugose variant, their biofilm structure and cell aggregation phenotypes were different than those of the rugose variant. Taken together, these results indicate that vps intergenic region genes encode proteins that are involved in biofilm matrix production and maintenance of biofilm structure and stability.

scholarly journals Identification of Virulent Capnocytophaga canimorsus Isolates by Capsular Typing

2017 ◽  
Vol 55 (6) ◽  
pp. 1902-1914 ◽  
Author(s):  
Estelle Hess ◽  
Francesco Renzi ◽  
Dunia Koudad ◽  
Mélanie Dol ◽  
Guy R. Cornelis
Keyword(s):  
Innate Immune System ◽  
Capsular Polysaccharide ◽  
Innate Immune ◽  
Capnocytophaga Canimorsus ◽  
Typing Method ◽  
Content Type ◽  
Severe Infections ◽  
Geographical Bias ◽  
Human Infections ◽  
Capsular Typing

ABSTRACTCapnocytophaga canimorsusis a dog oral commensal that causes rare but severe infections in humans.C. canimorsuswas recently shown to be endowed with a capsular polysaccharide implicated in resistance to the innate immune system of the host. Here, we developed the firstC. canimorsuscapsular serotyping scheme. We describe nine different serovars (A to I), and this serotyping scheme allowed typing of 25/25 isolates from human infections but only 18/52 isolates from dog mouths, indicating that the repertoire of capsules in the species is vast. However, while only three serovars (A, B, and C) covered 88% of the human isolates tested (22/25), they covered only 7.7% of the dog isolates (4/52). Serovars A, B, and C were found 22.9-, 14.6-, and 4.2-fold more often, respectively, among human isolates than among dog isolates, with no geographical bias, implying that isolates endowed with these three capsular types are more virulent for humans than other isolates. Capsular serotyping would thus allow identification of virulent isolates in dogs, which could contribute to the prevention of these infections. To this end, we developed a PCR typing method based on the amplification of specific capsular genes.

scholarly journals Deletion Mutants of Schmallenberg Virus Are Avirulent and Protect from Virus Challenge

2014 ◽  
Vol 89 (3) ◽  
pp. 1825-1837 ◽  
Author(s):  
Franziska Kraatz ◽  
Kerstin Wernike ◽  
Silke Hechinger ◽  
Patricia König ◽  
Harald Granzow ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Schmallenberg Virus ◽  
Wild Type Virus ◽  
Growth Defect ◽  
Type I ◽  
Nonstructural Proteins ◽  
Content Type ◽  
Live Vaccines ◽  
Double Deletion

ABSTRACTSince its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, has caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still not available. Here, the properties of novel recombinant SBV mutants lacking the nonstructural protein NSs (rSBVΔNSs) or NSm (rSBVΔNSm) or both of these proteins (rSBVΔNSs/ΔNSm) were testedin vitroandin vivoin type I interferon receptor knockout mice (IFNAR−/−) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both nonstructural proteins of SBV are not essential for viral growthin vitro. In interferon-defective BHK-21 cells, rSBVΔNSs and rSBVΔNSm replicated to levels comparable to that of the parental rSBV; the double mutant virus, however, showed a mild growth defect, resulting in lower final virus titers. Additionally, both mutants with an NSs deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR−/−mice, all mutants were virulent, with the highest mortality rate for rSBVΔNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viremia and seroconversion comparable to those caused by the wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both nonstructural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV vaccines which could be also a model for other viruses of the Simbu serogroup and related orthobunyaviruses.IMPORTANCESBV induces only mild clinical signs in adult ruminants but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genusOrthobunyavirus, and in addition, such a vaccine type has never been tested in cattle for any virus of the entire familyBunyaviridae. Therefore, the described vaccine also represents the first model for a broad range of related viruses and is of high importance to the field.

scholarly journals Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 679
Author(s):  
Benedict-Uy Fabia ◽  
Joshua Bingwa ◽  
Jiyeon Park ◽  
Nguyen-Mihn Hieu ◽  
Jung-Hoon Ahn
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Pseudomonas Fluorescens ◽  
Abc Transporter ◽  
Transforming Growth Factor Beta ◽  
Deletion Mutant ◽  
Transforming Growth Factor ◽  
Gene Knockout ◽  
Type I ◽  
Double Deletion

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

scholarly journals Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 781-786 ◽  
Author(s):  
Maximo Sánchez ◽  
Martha-Helena Ramírez-Bahena ◽  
Alvaro Peix ◽  
María J. Lorite ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Lotus Corniculatus ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

scholarly journals Resistance and Heteroresistance to Colistin in Pseudomonas aeruginosa Isolates from Wenzhou, China

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Jie Lin ◽  
Chunquan Xu ◽  
Renchi Fang ◽  
Jianming Cao ◽  
Xiucai Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Population Analysis ◽  
Colistin Resistance ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Expression Levels ◽  
Maldi Tof ◽  
Broth Microdilution Method ◽  
Tof Ms

ABSTRACT The goal was to investigate the mechanisms of colistin resistance and heteroresistance in Pseudomonas aeruginosa clinical isolates. Colistin resistance was determined by the broth microdilution method. Colistin heteroresistance was evaluated by population analysis profiling. Time-kill assays were also conducted. PCR sequencing was performed to detect the resistance genes among (hetero)resistant isolates, and quantitative real-time PCR assays were performed to determine their expression levels. Pulsed-field gel electrophoresis and multilocus sequence typing were performed. Lipid A characteristics were determined via matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS). Two resistant isolates and 9 heteroresistant isolates were selected in this study. Substitutions in PmrB were detected in 2 resistant isolates. Among heteroresistant isolates, 8 of 9 heteroresistant isolates had nonsynonymous PmrB substitutions, and 2 isolates, including 1 with a PmrB substitution, had PhoQ alterations. Correspondingly, the expression levels of pmrA or phoP were upregulated in PmrB- or PhoQ-substituted isolates. One isolate also found alterations in ParRS and CprRS. The transcript levels of the pmrH gene were observed to increase across all investigated isolates. MALDI-TOF MS showed additional 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in lipid A profiles in (hetero)resistant isolates. In conclusion, both colistin resistance and heteroresistance in P. aeruginosa in this study mainly involved alterations of the PmrAB regulatory system. There were strong associations between mutations in specific genetic loci for lipid A synthesis and regulation of modifications to lipid A. The transition of colistin heteroresistance to resistance should be addressed in future clinical surveillance.

scholarly journals Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae

2014 ◽  
Vol 64 (Pt_11) ◽  
pp. 3877-3884 ◽  
Author(s):  
Celine De Maesschalck ◽  
Filip Van Immerseel ◽  
Venessa Eeckhaut ◽  
Siegrid De Baere ◽  
Margo Cnockaert ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Link Type

Strains LMG 27428T and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428T and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734T. Strain LMG 27428T could be distinguished from S. pleomorphus ATCC 29734T based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734T. The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428T and LMG 27427. Strain LMG 27428T ( = DSM 26963T) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( = DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756T = ATCC 29734T = DSM 20574T). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983T (94.4 % 16S rRNA gene sequence similarity to strain LMG 27428T) and Eubacterium biforme DSM 3989T (92.7 % 16S rRNA gene sequence similarity to strain LMG 27428T). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983T = ATCC 27803T = JCM 10261T and that of Holdemanella biformis is DSM 3989T = ATCC 27806T = CCUG 28091T.

scholarly journals Amphritea ceti sp. nov., isolated from faeces of Beluga whale (Delphinapterus leucas)

2014 ◽  
Vol 64 (Pt_12) ◽  
pp. 4068-4072 ◽  
Author(s):  
Young-Ok Kim ◽  
Sooyeon Park ◽  
Doo Nam Kim ◽  
Bo-Hye Nam ◽  
Sung-Min Won ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Sequence Similarity ◽  
Beluga Whale ◽  
Delphinapterus Leucas ◽  
Rrna Gene ◽  
Phenotypic Properties ◽  
Content Type ◽  
Link Type ◽  
Beluga Whale Delphinapterus Leucas ◽  
Type Strains

A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated and rod-shaped or ovoid bacterial strain, designated RA1T, was isolated from faeces collected from Beluga whale (Delphinapterus leucas) in Yeosu aquarium, South Korea. Strain RA1T grew optimally at 25 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain RA1T joins the cluster comprising the type strains of three species of the genus Amphritea , with which it exhibited 95.8–96.0 % sequence similarity. Sequence similarities to the type strains of other recognized species were less than 94.3 %. Strain RA1T contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω7c and C16 : 0 as the major fatty acids. The major polar lipids of strain RA1T were phosphatidylethanolamine, phosphatidylglycerol, two unidentified lipids and one unidentified aminolipid. The DNA G+C content of strain RA1T was 47.4 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain RA1T is separated from other species of the genus Amphritea . On the basis of the data presented, strain RA1T is considered to represent a novel species of the genus Amphritea , for which the name Amphritea ceti sp. nov. is proposed. The type strain is RA1T ( = KCTC 42154T = NBRC 110551T).

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