scholarly journals Mutagenesis of the rpoS gene involved in alteration of outer membrane composition

2019 ◽  
Author(s):  
Sayyed Shahryar Rahpeyma ◽  
Jamshid Raheb
Keyword(s):  
Outer Membrane ◽  
Sigma Factor ◽  
Critical Role ◽  
Membrane Composition ◽  
Wild Type ◽  
Rpos Gene ◽  
Suicide Vector ◽  
Transmission Electron ◽  
Bacterial Morphology ◽  
Suitable Vector

Background and Objectives: rpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of the genes which control regulons and play a critical role in survival against stresses. Few suitable vectors are available which could be maintained successfully in Flexibacter chinesis cells and could in particular be used as a suicide vector to make mutation in the rpoS gene. The aim of this study was to investigate if rpoS mutagenesis has impact on bacterial morphology in addition to cell division. Materials and Methods: A 0.603 kb BamHI-PstI fragment subclone of pICRPOS38Ω was cloned into linearized pLYLO3. The final construct, pLRPOS38 suicide vector, was introduced into Flexibacter chinesis. Then the cytoplasm of mutant strain and wild-type were investigated by transmission electron microscopy. Results: After successful subcloning of suicide vector into F. chinesis, based on TEM study, it was demonstrated that muta- tion in rpoS gene leads to decomposition of outer membrane of F. chinesis. Conclusion: A suitable vector to make suicide mutation in rpoS was constructed for F. chinesi.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals The Quorum-Sensing Molecule Autoinducer 2 Regulates Motility and Flagellar Morphogenesis in Helicobacter pylori

2007 ◽  
Vol 189 (17) ◽  
pp. 6109-6117 ◽  
Author(s):  
Bethany A. Rader ◽  
Shawn R. Campagna ◽  
Martin F. Semmelhack ◽  
Bonnie L. Bassler ◽  
Karen Guillemin
Keyword(s):  
Helicobacter Pylori ◽  
Quorum Sensing ◽  
Sigma Factor ◽  
Critical Role ◽  
Soft Agar ◽  
Dependent Manner ◽  
Flagellar Gene ◽  
Wild Type ◽  
Autoinducer 2 ◽  
Mutant Phenotypes

ABSTRACT The genome of the gastric pathogen Helicobacter pylori contains a homologue of the gene luxS, which has been shown to be responsible for production of the quorum-sensing signal autoinducer 2 (AI-2). We report here that deletion of the luxS gene in strain G27 resulted in decreased motility on soft agar plates, a defect that was complemented by a wild-type copy of the luxS gene and by the addition of cell-free supernatant containing AI-2. The flagella of the luxS mutant appeared normal; however, in genetic backgrounds lacking any of three flagellar regulators—the two-component sensor kinase flgS, the sigma factor σ28 (also called fliA), and the anti-sigma factor flgM—loss of luxS altered flagellar morphology. In all cases, the double mutant phenotypes were restored to the luxS + phenotype by the addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Furthermore, in all mutant backgrounds loss of luxS caused a decrease in transcript levels of the flagellar regulator flhA. Addition of DPD to luxS cells induced flhA transcription in a dose-dependent manner. Deletion of flhA in a wild-type or luxS mutant background resulted in identical loss of motility, flagella, and flagellar gene expression. These data demonstrate that AI-2 functions as a secreted signaling molecule upstream of FlhA and plays a critical role in global regulation of flagellar gene transcription in H. pylori.

scholarly journals Isolation and Characterization of Outer Membrane Vesicles of Pectobacterium brasiliense 1692

2021 ◽  
Vol 9 (9) ◽  
pp. 1918
Author(s):  
Silindile Maphosa ◽  
Lucy Novungayo Moleleki
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Outer Membrane ◽  
Critical Role ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Broad Host Range ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Animal Pathogens

Pectobacterium brasiliense (Pbr) 1692 is an aggressive phytopathogen affecting a broad host range of crops and ornamental plants, including potatoes. Previous research on animal pathogens, and a few plant pathogens, revealed that Outer Membrane Vesicles (OMVs) are part of Gram-negative bacteria’s (GNB) adaptive toolkit. For this reason, OMV production and subsequent release from bacteria is a conserved process. Therefore, we hypothesized that OMVs might transport proteins that play a critical role in causing soft rot disease and in the survival and fitness of Pbr1692. Here, we show that the potato pathogen, Pbr1692, releases OMVs of various morphologies in Luria Bertani media at 31 °C. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) confirmed the production of OMVs by Pbr1692 cells. Transmission Electron Microscopy showed that these exist as chain-, single-, and double-membrane morphologies. Mass spectrometry followed by Gene Ontology, Clusters of Orthologous Groups, Virulence Factor, CAZymes, Antibiotic Resistance Ontology, and Bastion6 T6SE annotations identified 129 OMV-associated proteins with diverse annotated roles, including antibiotic stress response, virulence, and competition. Pbr1692 OMVs contributed to virulence in potato tubers and elicited a hypersensitive response in Nicotiana benthamiana leaves. Furthermore, Pbr1692 OMVs demonstrated antibacterial activity against Dickeya dadantii.

scholarly journals Control of the Ferric Citrate Transport System ofEscherichia coli: Mutations in Region 2.1 of the FecI Extracytoplasmic-Function Sigma Factor Suppress Mutations in the FecR Transmembrane Regulatory Protein

2001 ◽  
Vol 183 (1) ◽  
pp. 162-170 ◽  
Author(s):  
Alfred Stiefel ◽  
Susanne Mahren ◽  
Martina Ochs ◽  
Petra T. Schindler ◽  
Sabine Enz ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Gene Transcription ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Caulobacter Crescentus ◽  
Ferric Citrate ◽  
Missense Mutations ◽  
Wild Type ◽  
Citrate Transport ◽  
K 12

ABSTRACT Transcription of the ferric citrate transport genes is initiated by binding of ferric citrate to the FecA protein in the outer membrane ofEscherichia coli K-12. Bound ferric citrate does not have to be transported but initiates a signal that is transmitted by FecA across the outer membrane and by FecR across the cytoplasmic membrane into the cytoplasm, where the FecI extracytoplasmic-function (ECF) sigma factor becomes active. In this study, we isolated transcription initiation-negative missense mutants in the cytoplasmic region of FecR that were located at four sites, L13Q, W19R, W39R, and W50R, which are highly conserved in FecR-like open reading frames of thePseudomonas aeruginosa, Pseudomonas putida,Bordetella pertussis, Bordetella bronchiseptica, and Caulobacter crescentus genomes. The cytoplasmic portion of the FecR mutant proteins, FecR1–85, did not interact with wild-type FecI, in contrast to wild-type FecR1–85, which induced FecI-mediated fecB transport gene transcription. Two missense mutations in region 2.1 of FecI, S15A and H20E, partially restored induction of ferric citrate transport gene induction of thefecR mutants by ferric citrate. Region 2.1 of ς70 is thought to bind RNA polymerase core enzyme; the residual activity of mutated FecI in the absence of FecR, however, was not higher than that of wild-type FecI. In addition, missense mutations in the fecI promoter region resulted in a twofold increased transcription in fecR wild-type cells and a partial restoration of fec transport gene transcription in thefecR mutants. The mutations reduced binding of the Fe2+ Fur repressor and as a consequence enhancedfecI transcription. The data reveal properties of the FecI ECF factor distinct from those of ς70 and further support the novel transcription initiation model in which the cytoplasmic portion of FecR is important for FecI activity.

Interaction of mutants of Xenorhabdus nematophilus (Enterobacteriaceae) with antibacterial systems of Galleria mellonella larvae (Insecta: Pyralidae)

1994 ◽  
Vol 40 (3) ◽  
pp. 161-168 ◽  
Author(s):  
Gary B. Dunphy
Keyword(s):  
Outer Membrane ◽  
Galleria Mellonella ◽  
Outer Membrane Proteins ◽  
Bacterial Attachment ◽  
Membrane Composition ◽  
Wild Type ◽  
Insect Haemocytes ◽  
Increased Sensitivity ◽  
Xenorhabdus Nematophilus ◽  
Bacterial Hydrophobicity

Xenorhabdus nematophilus mutants that took longer to kill insects than did the wild type were used to determine the relationship of the physicochemical properties and outer membrane composition to bacterial interaction with the antibacterial systems of Galleria mellonella larvae and to bacterial virulence. Insect serum slowed the growth of the wild-type and mutant bacteria. This was attributed to increased spheroplast formation for the mutants. Spheroplast formation was associated with an increased sensitivity to insect lysozyme and a reduction in overall bacterial cationic charge. Increasing bacterial hydrophobicity was correlated with both increased bacterial attachment to the insect's haemocytes and the accelerated removal of the bacteria from the haemolymph. Attachment of the mutants to the insect haemocytes also increased as the bacterial lipopolysaccharide content increased, the level of prophenoloxidase activation increased, and cationic charge declined. Bacterial emergence into the haemolymph occurred in parallel with haemocyte damage but neither the total lipopolysaccharide levels in the bacteria nor the rate of bacterial emergence were associated with virulence. The rate of lipopolysaccharide release into the haemolymph influenced the rate of haemocyte damage. The contribution of outer membrane proteins to lipopolysaccharide release, bacterial adhesion to haemocytes, and virulence is discussed. Virulence reflects bacterial tolerance to the host's antibacterial defences, favouring an increase in bacteria and toxic lipopolysaccharides.Key words: Xenorhabdus, antibacterial systems, insects.

scholarly journals Extracellular Reduction of Hexavalent Chromium by Cytochromes MtrC and OmcA of Shewanella oneidensis MR-1

2011 ◽  
Vol 77 (12) ◽  
pp. 4035-4041 ◽  
Author(s):  
Sara M. Belchik ◽  
David W. Kennedy ◽  
Alice C. Dohnalkova ◽  
Yuanmin Wang ◽  
Papatya C. Sevinc ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Outer Membrane ◽  
Initial Rate ◽  
Mutant Cell ◽  
Shewanella Oneidensis ◽  
Wild Type ◽  
Content Type ◽  
Transmission Electron ◽  
Mutant Cells

ABSTRACTTo characterize the roles of cytochromes MtrC and OmcA ofShewanella oneidensisMR-1 in Cr(VI) reduction, the effects of deleting themtrCand/oromcAgene on Cr(VI) reduction and the cellular locations of reduced Cr(III) precipitates were investigated. Compared to the rate of reduction of Cr(VI) by the wild type (wt), the deletion ofmtrCdecreased the initial rate of Cr(VI) reduction by 43.5%, while the deletion ofomcAor bothmtrCandomcAlowered the rate by 53.4% and 68.9%, respectively. In wt cells, Cr(III) precipitates were detected by transmission electron microscopy in the extracellular matrix between the cells, in association with the outer membrane, and inside the cytoplasm. No extracellular matrix-associated Cr(III) precipitates, however, were found in the cytochrome mutant cell suspension. In mutant cells without either MtrC or OmcA, most Cr(III) precipitates were found in association with the outer membrane, while in mutant cells lacking both MtrC and OmcA, most Cr(III) precipitates were found inside the cytoplasm. Cr(III) precipitates were also detected by scanning election microscopy on the surfaces of the wt and mutants without MtrC or OmcA but not on the mutant cells lacking both MtrC and OmcA, demonstrating that the deletion ofmtrCandomcAdiminishes the extracellular formation of Cr(III) precipitates. Furthermore, purified MtrC and OmcA reduced Cr(VI) with apparentkcatvalues of 1.2 ± 0.2 (mean ± standard deviation) and 10.2 ± 1 s−1andKmvalues of 34.1 ± 4.5 and 41.3 ± 7.9 μM, respectively. Together, these results consistently demonstrate that MtrC and OmcA are the terminal reductases used byS. oneidensisMR-1 for extracellular Cr(VI) reduction where OmcA is a predominant Cr(VI) reductase.

scholarly journals Altered Envelope Structure and Nanomechanical Properties of a C-Terminal Protease A-Deficient Rhizobium leguminosarum

2020 ◽  
Vol 8 (9) ◽  
pp. 1421
Author(s):  
Dong Jun ◽  
Ubong Idem ◽  
Tanya E. S. Dahms
Keyword(s):  
Outer Membrane ◽  
Creep Deformation ◽  
High Performance ◽  
Rhizobium Leguminosarum ◽  
Cell Envelope ◽  
Time Dependent ◽  
Wild Type ◽  
Force Microscopy ◽  
Viscoelastic Parameters ◽  
Transmission Electron

(1) Background: Many factors can impact bacterial mechanical properties, which play an important role in survival and adaptation. This study characterizes the ultrastructural phenotype, elastic and viscoelastic properties of Rhizobium leguminosarum bv. viciae 3841 and the C-terminal protease A (ctpA) null mutant strain predicted to have a compromised cell envelope; (2) Methods: To probe the cell envelope, we used transmission electron microscopy (TEM), high performance liquid chromatography (HPLC), mass spectrometry (MS), atomic force microscopy (AFM) force spectroscopy, and time-dependent AFM creep deformation; (3) Results: TEM images show a compromised and often detached outer membrane for the ctpA mutant. Muropeptide characterization by HPLC and MS showed an increase in peptidoglycan dimeric peptide (GlcNAc-MurNAc-Ala-Glu-meso-DAP-Ala-meso-DAP-Glu-Ala-MurNAc-GlcNAc) for the ctpA mutant, indicative of increased crosslinking. The ctpA mutant had significantly larger spring constants than wild type under all hydrated conditions, attributable to more highly crosslinked peptidoglycan. Time-dependent AFM creep deformation for both the wild type and ctpA mutant was indicative of a viscoelastic cell envelope, with best fit to the four-element Burgers model and generating values for viscoelastic parameters k1, k2, η1, and η2; (4) Conclusions: The viscoelastic response of the ctpA mutant is consistent with both its compromised outer membrane (TEM) and fortified peptidoglycan layer (HPLC/MS).

scholarly journals OmcF, a Putative c-Type Monoheme Outer Membrane Cytochrome Required for the Expression of Other Outer Membrane Cytochromes in Geobacter sulfurreducens

2005 ◽  
Vol 187 (13) ◽  
pp. 4505-4513 ◽  
Author(s):  
Byoung-Chan Kim ◽  
Ching Leang ◽  
Yan-Huai R. Ding ◽  
Richard H. Glaven ◽  
Maddalena V. Coppi ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Critical Role ◽  
Electron Acceptors ◽  
Geobacter Sulfurreducens ◽  
Lag Phase ◽  
Membrane Association ◽  
Deficient Mutant ◽  
Wild Type ◽  
Cytochrome Content ◽  
Doubling Times

ABSTRACT Outer membrane cytochromes are often proposed as likely agents for electron transfer to extracellular electron acceptors, such as Fe(III). The omcF gene in the dissimilatory Fe(III)-reducing microorganism Geobacter sulfurreducens is predicted to code for a small outer membrane monoheme c-type cytochrome. An OmcF-deficient strain was constructed, and its ability to reduce and grow on Fe(III) citrate was found to be impaired. Following a prolonged lag phase (150 h), the OmcF-deficient strain developed the ability to grow in Fe(III) citrate medium with doubling times and yields that were ca. 145% and 70% of those of the wild type, respectively. Comparison of the c-type cytochrome contents of outer membrane-enriched fractions prepared from wild-type and OmcF-deficient cultures confirmed the outer membrane association of OmcF and revealed multiple changes in the cytochrome content of the OmcF-deficient strain. These changes included loss of expression of two previously characterized outer membrane cytochromes, OmcB and OmcC, and overexpression of a third previously characterized outer membrane cytochrome, OmcS, during growth on Fe(III) citrate. The omcB and omcC transcripts could not be detected in the OmcF-deficient mutant by either reverse transcriptase PCR or Northern blot analyses. Expression of the omcF gene in trans restored both the capacity of the OmcF-deficient mutant to reduce Fe(III) and wild-type levels of omcB and omcC mRNA and protein. Thus, elimination of OmcF may impair Fe(III) reduction by influencing expression of OmcB, which has previously been demonstrated to play a critical role in Fe(III) reduction.

scholarly journals Role of the Extracytoplasmic Function Protein Family Sigma Factor RpoE in Metal Resistance of Escherichia coli

2005 ◽  
Vol 187 (7) ◽  
pp. 2297-2307 ◽  
Author(s):  
Monique Egler ◽  
Cornelia Grosse ◽  
Gregor Grass ◽  
Dietrich H. Nies
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Sigma Factor ◽  
Double Mutant ◽  
Expression Profiles ◽  
Protein Family ◽  
Wild Type ◽  
Qrt Pcr ◽  
Metal Treatment

ABSTRACT RpoE of Escherichia coli is a sigma factor of the extracytoplasmic function protein family and is required for the expression of proteins involved in maintaining the integrity of periplasmic and outer membrane components. RpoE of E. coli was needed for full resistance to Zn(II), Cd(II), and Cu(II). Promoter gene fusion and quantitative real time reverse transcription (RT)-PCR (qRT-PCR) assays demonstrated that expression of RpoE was induced by metals. Global gene expression profiles upon metal treatment of a ΔrpoE mutant strain and its wild-type strain were analyzed with microarrays, and selected genes were confirmed by qRT-PCR. The absolute number of genes that were changed in their expression upon metal stress was similar in both strains, but the increase or decrease in transcript levels upon metal treatment was smaller in the ΔrpoE mutant strain than in the wild type. Genes showing increased expression in the ΔrpoE mutant strain encoded proteins that belong to general defense systems against protein-denaturing agents. Genes showing decreased expression were part of the RpoE modulon itself plus the ompC gene, encoding a major outer membrane protein. A ΔompC deletion strain was as sensitive to Cu(II) and Cd(II) as the ΔrpoE mutant or a ΔrpoE ΔompC double mutant strain. In the case of Zn(II), the double mutant was more sensitive than either single mutant. This indicates that increased expression of OmpC contributes to the RpoE modulon-mediated response to metals.

scholarly journals Outer Membrane Vesicles (OMVs) of Pseudomonas aeruginosa Provide Passive Resistance but Not Sensitization to LPS-Specific Phages

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 121
Author(s):  
Daria Augustyniak ◽  
Tomasz Olszak ◽  
Zuzanna Drulis-Kawa
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Passive Resistance ◽  
Bacterial Physiology ◽  
Transmission Electron ◽  
Growth Assay ◽  
One Step

Outer membrane vesicles (OMVs) released from gram-negative bacteria are key elements in bacterial physiology, pathogenesis, and defence. In this study, we investigated the role of Pseudomonas aeruginosa OMVs in the anti-phage defence as well as in the potential sensitization to LPS-specific phages. Using transmission electron microscopy, virion infectivity, and neutralization assays, we have shown that both phages efficiently absorb on free vesicles and are unable to infect P. aeruginosa host. Nevertheless, the accompanying decrease in PFU titre (neutralization) was only observed for myovirus KT28 but not podovirus LUZ7. Next, we verified whether OMVs derived from wild-type PAO1 strain can sensitize the LPS-deficient mutant (Δwbpl PAO1) resistant to tested phages. The flow cytometry experiments proved a quite effective and comparable association of OMVs to Δwbpl PAO1 and wild-type PAO1; however, the growth kinetic curves and one-step growth assay revealed no sensitization event of the OMV-associated phage-resistant P. aeruginosa deletant to LPS-specific phages. Our findings for the first time identify naturally formed OMVs as important players in passive resistance (protection) of P. aeruginosa population to phages, but we disproved the hypothesis of transferring phage receptors to make resistant strains susceptible to LPS-dependent phages.

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