scholarly journals The TOL Plasmid pWW0 xylN Gene Product fromPseudomonas putida Is Involved inm-Xylene Uptake

2001 ◽  
Vol 183 (22) ◽  
pp. 6662-6666 ◽  
Author(s):  
Yuki Kasai ◽  
Jun Inoue ◽  
Shigeaki Harayama
Keyword(s):  
Outer Membrane ◽  
Gene Product ◽  
Sequence Similarity ◽  
Kanamycin Resistance ◽  
Fatty Acid Transport ◽  
Wild Type ◽  
High Concentration ◽  
Tol Plasmid ◽  
Intact Cells ◽  
Cell Extracts

ABSTRACT The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport inEscherichia coli. To analyze the role of thexylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type andxylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylNmutant TOL plasmid was not. The apparentK s value for the oxidation ofm-xylene in intact cells of the xylNmutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that thexylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.

scholarly journals Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit).

1996 ◽  
Vol 16 (9) ◽  
pp. 5194-5209 ◽  
Author(s):  
H G Dohlman ◽  
J Song ◽  
D Ma ◽  
W E Courchesne ◽  
J Thorner
Keyword(s):  
G Protein ◽  
Gene Product ◽  
Genetic Interaction ◽  
Sequence Similarity ◽  
Negative Regulator ◽  
Rgs Proteins ◽  
Pheromone Response ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Cell Extracts

Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.

scholarly journals Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity.

1992 ◽  
Vol 119 (5) ◽  
pp. 1297-1307 ◽  
Author(s):  
M J Levis ◽  
H R Bourne
Keyword(s):  
Adenylyl Cyclase ◽  
Cholera Toxin ◽  
Soluble Fraction ◽  
Covalent Modification ◽  
Alpha Subunit ◽  
Wild Type ◽  
Hormonal Stimulation ◽  
Intact Cells ◽  
Cell Extracts ◽  
Membrane Bound

Binding of GTP induces alpha subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the alpha subunit (alpha s) of Gs in genetically alpha s-deficient S49 cyc- cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type alpha s to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-alpha s) quantitatively from cell extracts. We activated the epitope-tagged alpha s in intact cells by: (a) exposure of cells to cholera toxin, which activates alpha s by covalent modification; (b) mutational replacement of arginine-201 in HA-alpha s by a cysteine residue, to create HA-alpha s-R201C; like the cholera toxin-catalyzed modification, this mutation activates alpha s by slowing its intrinsic GTPase activity; and (c) treatment of cells with the beta-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to alpha s, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of alpha s (0.03 h-1) by three- to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-alpha s- R201C was found in the soluble fraction, as compared to 10% of wild type HA-alpha s. Isoproterenol rapidly (in < 2 min) caused 20% of HA-alpha s to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-alpha s-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of alpha s in response to activation. Taken together, these findings show that activation of alpha s induces a conformational change that loosens its attachment to membranes and increases its degradation rate.

scholarly journals Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli.

1981 ◽  
Vol 77 (2) ◽  
pp. 121-135 ◽  
Author(s):  
H Nikaido ◽  
E Y Rosenberg
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Specific Class ◽  
Wild Type ◽  
Intact Cells ◽  
E Coli ◽  
Low Concentrations ◽  
Transmembrane Pores ◽  
Diffusion Rates ◽  
Type Strains

Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.

scholarly journals Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE, a Homolog of the pulE (gspE) Type II Protein Secretion Gene

2002 ◽  
Vol 184 (1) ◽  
pp. 142-151 ◽  
Author(s):  
Thomas J. DiChristina ◽  
Charles M. Moore ◽  
Carolyn A. Haller
Keyword(s):  
Outer Membrane ◽  
Protein Secretion ◽  
Sequence Similarity ◽  
Shewanella Putrefaciens ◽  
Metal Reduction ◽  
Nucleotide Sequence Analysis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Secretion Systems

ABSTRACT Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468–1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer−) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).

A sulphite respiration system in the chemoheterotrophic human pathogen Campylobacter jejuni

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 233-242 ◽  
Author(s):  
Jonathan D. Myers ◽  
David J. Kelly
Keyword(s):  
Campylobacter Jejuni ◽  
Kanamycin Resistance ◽  
Exchange Chromatography ◽  
Cell Protein ◽  
Human Pathogen ◽  
Wild Type ◽  
Western Blots ◽  
Intact Cells ◽  
Kanamycin Resistance Cassette ◽  
Inhibitory Compound

The ability to use sulphite as a respiratory electron donor is usually associated with free-living chemolithotrophic sulphur-oxidizing bacteria. However, this paper shows that the chemoheterotrophic human pathogen Campylobacter jejuni has the ability to respire sulphite, with oxygen uptake rates of 23±8 and 28±15 nmol O2 min−1 (mg cell protein)−1 after the addition of 0·5 mM sodium sulphite or metabisulphite, respectively, to intact cells. The C. jejuni NCTC 11168 Cj0004c and Cj0005c genes encode a monohaem cytochrome c and molybdopterin oxidoreductase, respectively, homologous to the sulphite : cytochrome c oxidoreductase (SOR) of Starkeya novella. Western blots of C. jejuni periplasm probed with a SorA antibody demonstrated cross-reaction of a 45 kDa band, consistent with the size of Cj0005. The Cj0004c gene was inactivated by insertion of a kanamycin-resistance cassette. The resulting mutant showed wild-type rates of formate-dependent respiration but was unable to respire with sulphite or metabisulphite as electron donors. 2-Heptyl-4-hydroxyquinoline-N-oxide (HQNO), a cytochrome bc 1 complex inhibitor, did not affect sulphite respiration at concentrations up to 25 μM, whereas formate respiration (which occurs partly via a bc 1 dependent route) was inhibited 50 %, thus suggesting that electrons from sulphite enter the respiratory chain after the bc 1 complex at the level of cytochrome c. Periplasmic extracts of wild-type C. jejuni 11168 showed a symmetrical absorption peak at 552 nm after the addition of sulphite, demonstrating the reduction of cytochrome c. No cytochrome c reduction was observed after addition of sulphite to periplasmic extracts of the Cj0004c mutant. A fractionation study confirmed that the majority of the SOR activity is located in the periplasm in C. jejuni, and this activity was partially purified by ion-exchange chromatography. The presence of a sulphite respiration system in C. jejuni is another example of the surprising diversity of the electron-transport chain in this small-genome pathogen. Sulphite respiration may be of importance for survival in environmental microaerobic niches and some foods, and may also provide a detoxification mechanism for this normally growth-inhibitory compound.

Proteolytic digestion and labelling studies of the organization of the proteins in the outer membrane of Escherichia coli

1977 ◽  
Vol 55 (10) ◽  
pp. 1082-1090 ◽  
Author(s):  
Reinhart A. F. Reithmeier ◽  
Philip D. Bragg
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Hydrophobic Interactions ◽  
Proteolytic Digestion ◽  
Wild Type ◽  
Terminal Portion ◽  
Intact Cells ◽  
Protein Components ◽  
Pronase Treatment ◽  
Protein B

The arrangement of the proteins in the outer membrane of Escherichia coli was examined by treating intact cells and isolated membrane preparations with fluorescamine and with pronase. Intact wild-type cells, or those of a mutant in which the core region of the lipopolysaccharide was absent, were equally resistant to pronase treatment. The protein components of isolated outer membrane preparations varied in their rate of digestion and labelling with fluorescamine. The N-terminal portion of protein B was removed by pronase to yield a fragment (protein Bp) still embedded in the membrane. Protein Bp was not significantly enriched in nonpolar amino acids, suggesting that protein B may not be held in the membrane primarily by hydrophobic interactions. This was confirmed by reconstitution experiments in which protein B could be reassociated with itself, without lipopolysaccharide or phospholipid, in the presence of a divalent cation such that pronase digestion of the reassociated material gave protein Bp.

scholarly journals Functional Analysis of a Rickettsial OmpA Homology Domain of Shigella flexneri IcsA

1999 ◽  
Vol 181 (3) ◽  
pp. 869-878 ◽  
Author(s):  
Macarthur Charles ◽  
Juana Magdalena ◽  
Julie A. Theriot ◽  
Marcia B. Goldberg
Keyword(s):  
Outer Membrane ◽  
Sequence Similarity ◽  
Shigella Flexneri ◽  
Cytoskeletal Proteins ◽  
Site Directed Mutagenesis ◽  
Actin Assembly ◽  
Wild Type ◽  
Amino Terminal ◽  
Carboxy Terminal

ABSTRACT Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri andEscherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathioneS-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.

Molecular and genetic characterization of natural variants of HIV-1 Nef gene from North India and its functional implication in down-regulation of MHC-I and CD-4

2020 ◽  
Vol 18 ◽  
Author(s):  
J. Singh ◽  
L. Ronsard ◽  
M. Pandey ◽  
R. Kapoor ◽  
V.G. Ramachandran ◽  
...  
Keyword(s):  
Biological Activities ◽  
Sequence Similarity ◽  
Eukaryotic Expression ◽  
North India ◽  
Cell Surface Expression ◽  
Functional Domains ◽  
Wild Type ◽  
Down Regulation ◽  
Subtype B ◽  
Hiv 1

Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of HIV-1 Nef gene shows extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North-India and to find out their functional implications. Methods: The unique representative variants were sub-cloned in eukaryotic expression vector and further characterized with respect to their ability to down regulate cell surface expression of CD4 and MHC-1molecules. Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 down regulation activity. Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising out of amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

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.

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