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.

scholarly journals Inactivation of the Spirochete recA Gene Results in a Mutant with Low Viability and Irregular Nucleoid Morphology

2002 ◽  
Vol 184 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Ange-Patricia Tchamedeu Kameni ◽  
Evelyne Couture-Tosi ◽  
Isabelle Saint-Girons ◽  
Mathieu Picardeau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Uv Light ◽  
Fluorescent Microscopy ◽  
Kanamycin Resistance ◽  
Reca Gene ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Poor Growth ◽  
Kanamycin Resistance Cassette

ABSTRACT Recently, we have shown the first evidence for allelic exchange in Leptospira spp. By using the same methodology, the cloned recA of Leptospira biflexa was interrupted by a kanamycin resistance cassette, and the mutated allele was then introduced into the L. biflexa chromosome by homologous recombination. The recA double-crossover mutant showed poor growth in liquid media and was considerably more sensitive to DNA-damaging agents such as mitomycin C and UV light than the wild-type strain. The efficiency of plating of the recA mutant was about 10% of that of the parent strain. In addition, microscopic observation of the L. biflexa recA mutant showed cells that are more elongated than those of the wild-type strain. Fluorescent microscopy of stained cells of the L. biflexa wild-type strain revealed that chromosomal DNA is distributed throughout most of the length of the cell. In contrast, the recA mutant showed aberrant nucleoid morphologies, i.e., DNA is condensed at the midcell. Our data indicate that L. biflexa RecA plays a major role in ensuring cell viability via mechanisms such as DNA repair and, indirectly, active chromosome partitioning.

scholarly journals Relationship of plcR-Regulated Factors to BacillusEndophthalmitis Virulence

2003 ◽  
Vol 71 (6) ◽  
pp. 3116-3124 ◽  
Author(s):  
Michelle C. Callegan ◽  
Scott T. Kane ◽  
D. Clay Cochran ◽  
Michael S. Gilmore ◽  
Myriam Gominet ◽  
...  
Keyword(s):  
Inflammatory Cell ◽  
Wild Type Strain ◽  
Kanamycin Resistance ◽  
Retinal Function ◽  
Deficient Mutant ◽  
Wild Type ◽  
Global Regulator ◽  
Kanamycin Resistance Cassette ◽  
Insertional Inactivation ◽  
Relationship Of

ABSTRACT The explosive, destructive course of Bacillus endophthalmitis has been attributed to the production of toxins during infection. In this study we analyzed the contribution of toxins controlled by the global regulator plcR to the pathogenesis of experimental Bacillus endophthalmitis. Isogenic plcR-deficient mutants of Bacillus cereus and Bacillus thuringiensis were constructed by insertional inactivation of plcR by the kanamycin resistance cassette, aphA3. Rabbit eyes were injected intravitreally with approximately 100 CFU of wild-type B. cereus or B. thuringiensis or a plcR-deficient mutant. The evolution of endophthalmitis resulting from each plcR-deficient mutant was considerably slower than that caused by each wild-type strain. Retinal function was not eliminated until 42 h postinfection in rabbits with endophthalmitis caused by the plcR-deficient mutants, whereas wild-type infections resulted in a complete loss of retinal function within 18 h. The intraocular inflammatory cell influx and retinal destruction in plcR-deficient endophthalmitis approached the severity observed in wild-ype infections, but not until 36 h postinfection. Gross and histological examinations of eyes infected with plcR mutants demonstrated that the anterior and posterior segment changes were muted compared to the changes observed in eyes infected with the wild types. The loss of plcR-regulated factors significantly attenuated the severity of Bacillus endophthalmitis. The results therefore suggest that plcR may represent a target for which adjunct therapies could be designed for the prevention of blindness during Bacillus endophthalmitis.

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 Identification for mar mutants among quinolone-resistant clinical isolates of Escherichia coli.

1996 ◽  
Vol 40 (7) ◽  
pp. 1695-1698 ◽  
Author(s):  
K Maneewannakul ◽  
S B Levy
Keyword(s):  
Escherichia Coli ◽  
Constitutive Expression ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Transcriptional Fusion ◽  
E Coli ◽  
Kanamycin Resistance Cassette ◽  
Mutant Strains ◽  
Beta Galactosidase ◽  
Multiple Antibiotics

Quinolone-resistant clinical Escherichia coli isolates were examined for mutations in the marRAB operon of the multiple antibiotic resistance (mar) locus. Among 23 strains evaluated, 8 were chosen for further study: 3 that showed relatively high levels of uninduced, i.e., constitutive, expression of the operon and 5 with variable responses to induction by salicylate or tetracyclines. The marR genes, specifying the repressor of the operon, cloned from the three strains constitutively expressing the operon did not reduce the level of expression of beta-galactosidase from a marO::lacZ transcriptional fusion and were therefore mutant; however, marR genes cloned from the five other clinical strains repressed LacZ expression and were wild type. All three mutant marR genes contained more than one mutation: a deletion and a point mutation. Inactivation of the mar locus in the three known marR mutant strains with a kanamycin resistance cassette introduced by homologous recombination reduced resistance to quinolones and multiple antibiotics. These findings indicate that mar operon mutations exist in quinolone-resistant clinical E. coli isolates and contribute to quinolone and multidrug resistance.

scholarly journals Influence of the Alternative σ28 Factor on Virulence and Flagellum Expression of Legionella pneumophila

2002 ◽  
Vol 70 (3) ◽  
pp. 1604-1608 ◽  
Author(s):  
Klaus Heuner ◽  
Claudia Dietrich ◽  
Carina Skriwan ◽  
Michael Steinert ◽  
Jörg Hacker
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Blot Analysis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Kanamycin Resistance Cassette

ABSTRACT The fliA gene of Legionella pneumophila encoding the alternative σ28 factor was inactivated by introducing a kanamycin resistance cassette. Electron microscopy and Western blot analysis revealed that the fliA mutant strain is aflagellate and expresses no flagellin. Reporter gene assays indicated that the flaA promoter is not active in the fliA mutant strain. The fliA mutant strain multiplied less effectively in coculture with amoebae than the wild-type strain and was not able to replicate in coculture with Dictyostelium discoideum.

scholarly journals In Vivo Behavior of a Helicobacter pylori SS1 nixA Mutant with Reduced Urease Activity

2002 ◽  
Vol 70 (2) ◽  
pp. 685-691 ◽  
Author(s):  
Kylie J. Nolan ◽  
David J. McGee ◽  
Hazel M. Mitchell ◽  
Tassia Kolesnikow ◽  
Janette M. Harro ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Urease Activity ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Bacterial Colony ◽  
Nickel Ions ◽  
Kanamycin Resistance Cassette ◽  
Mutant Strains ◽  
H Pylori

ABSTRACT Helicobacter pylori mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The nixA gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A nixA mutant of the Sydney strain of H. pylori (SS1) was constructed by disruption of the nixA gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j (n = 75) and BALB/c (n = 75) mice were inoculated independently with the wild-type or the nixA strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the nixA strain in BALB/c mice were significantly lower compared with SS1 (P = 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the nixA strain in BALB/c mice. On the contrary, when C57BL/6j (n = 35) and BALB/c (n = 35) mice were coinfected with the wild-type and nixA strains simultaneously, the nixA mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the nixA gene for increasing the fitness of H. pylori for gastric colonization. Since nixA is required for full urease activity, the decreased fitness of the nixA mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.

scholarly journals Identification and Isolation of Genes Involved in Poly(β-Hydroxybutyrate) Biosynthesis in Azospirillum brasilense and Characterization of a phbC Mutant

2002 ◽  
Vol 68 (6) ◽  
pp. 2943-2949 ◽  
Author(s):  
Daniel Kadouri ◽  
Saul Burdman ◽  
Edouard Jurkevitch ◽  
Yaacov Okon
Keyword(s):  
Mutant Strain ◽  
Azospirillum Brasilense ◽  
Capsular Polysaccharide ◽  
Cell Aggregation ◽  
Growth Conditions ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Kanamycin Resistance Cassette ◽  
In The Wild ◽  
Phb Synthase

ABSTRACT Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(β-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (β-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.

scholarly journals areABC Genes Determine the Catabolism of Aryl Esters in Acinetobacter sp. Strain ADP1

1999 ◽  
Vol 181 (15) ◽  
pp. 4568-4575 ◽  
Author(s):  
Rheinallt M. Jones ◽  
Lauren S. Collier ◽  
Ellen L. Neidle ◽  
Peter A. Williams
Keyword(s):  
Benzyl Alcohol ◽  
Carboxylic Acids ◽  
Specific Activity ◽  
High Specific Activity ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Wild Type ◽  
Substrate Preferences ◽  
Kanamycin Resistance Cassette ◽  
Benzaldehyde Dehydrogenase

ABSTRACT Acinetobacter sp. strain ADP1 is able to grow on a range of esters of aromatic alcohols, converting them to the corresponding aromatic carboxylic acids by the sequential action of three inducible enzymes: an areA-encoded esterase, anareB-encoded benzyl alcohol dehydrogenase, and anareC-encoded benzaldehyde dehydrogenase. Theare genes, adjacent to each other on the chromosome and transcribed in the order areCBA, were located 3.5 kbp upstream of benK. benK, encoding a permease implicated in benzoate uptake, is at one end of the ben-cat supraoperonic cluster for benzoate catabolism by the β-ketoadipate pathway. Two open reading frames which may encode a transcriptional regulator,areR, and a porin, benP, separatebenK from areC. Each are gene was individually expressed to high specific activity in Escherichia coli. The relative activities against different substrates of the cloned enzymes were, within experimental error, identical to that of wild-type Acinetobacter sp. strain ADP1 grown on either benzyl acetate, benzyl alcohol, or 4-hydroxybenzyl alcohol as the carbon source. The substrate preferences of all three enzymes were broad, encompassing a range of substituted aromatic compounds and in the case of the AreA esterase, different carboxylic acids. TheareA, areB, and areC genes were individually disrupted on the chromosome by insertion of a kanamycin resistance cassette, and the rates at which the resultant strains utilized substrates of the aryl ester catabolic pathway were severely reduced as determined by growth competitions between the mutant and wild-type strains.

scholarly journals The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nicholas M. Negretti ◽  
Christopher R. Gourley ◽  
Prabhat K. Talukdar ◽  
Geremy Clair ◽  
Courtney M. Klappenbach ◽  
...  
Keyword(s):  
Host Cell ◽  
Campylobacter Jejuni ◽  
Foodborne Pathogen ◽  
Small Gtpase ◽  
Host Cells ◽  
Cell Protein ◽  
Host Cell Protein ◽  
Cell Binding ◽  
Actin Reorganization ◽  
Cell Cytosol

AbstractCampylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1.

scholarly journals Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting

2013 ◽  
Vol 304 (5) ◽  
pp. F522-F532 ◽  
Author(s):  
Luca Vedovelli ◽  
John T. Rothermel ◽  
Karin E. Finberg ◽  
Carsten A. Wagner ◽  
Anie Azroyan ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Collecting Duct ◽  
Egfp Expression ◽  
Intercalated Cells ◽  
Wild Type ◽  
Western Blots ◽  
Atpase Subunit ◽  
Protein Levels ◽  
Baseline Conditions ◽  
Deficient Mice

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1−/−) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1−/− and wild-type mice (Păunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915–F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1−/− mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1+/+ mice) with Atp6v1b1−/− mice to generate novel EGFP-B1−/− mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1+/+ and EGFP-B1−/− mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1−/− mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.

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