Characterization of thecydAB-Encoded Cytochrome bd Oxidase fromMycobacterium smegmatis

ABSTRACT The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. ThecydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. ThecydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss ofd-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster fromMycobacterium tuberculosis. Inactivation ofcydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 102 Pa of pO2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 102 Pa of pO2 or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase ind-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase inM. smegmatis.

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Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Brucella abortus Deficient in Internalization and Intracellular Growth in HeLa Cells

Infection and Immunity ◽

10.1128/iai.71.6.3020-3027.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3020-3027 ◽

Cited By ~ 63

Author(s):

Suk Kim ◽

Masahisa Watarai ◽

Yuki Kondo ◽

Janchivdorj Erdenebaatar ◽

Sou-ichi Makino ◽

...

Keyword(s):

Hela Cells ◽

Brucella Abortus ◽

Kanamycin Resistance ◽

Host Cells ◽

Class Iii ◽

Wild Type ◽

Intracellular Growth ◽

Isolation And Characterization ◽

Bacterial Genes

ABSTRACT Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.

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Dissection of the Mechanisms of Growth Inhibition Resulting from Loss of the PII Protein in the CyanobacteriumSynechococcus elongatusPCC 7942

Plant and Cell Physiology ◽

10.1093/pcp/pcab030 ◽

2021 ◽

Author(s):

Takayuki Sakamoto ◽

Nobuyuki Takatani ◽

Kintake Sonoike ◽

Haruhiko Jimbo ◽

Yoshitaka Nishiyama ◽

...

Keyword(s):

Nitrogen Assimilation ◽

Growth Defect ◽

Oxygen Species ◽

Wild Type ◽

Pii Protein ◽

Regulator Protein ◽

Synechococcus Elongatus Pcc 7942 ◽

Pcc 7942 ◽

Mutant Cells

AbstractIn cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the coactivator of the global nitrogen regulator protein NtcA. In Synechococcus elongatus PCC 7942, construction of a PII-less mutant retaining the wild-type pipX gene is difficult because of the toxicity of uncontrolled action of PipX and the other defect(s) resulting from the loss of PII per se, but the nature of the PipX toxicity and the PipX-independent defect(s) remains unclear. Characterization of a PipX-less glnB mutant (PD4) in this study showed that the loss of PII increases the sensitivity of PSII to ammonium. Ammonium was shown to stimulate the formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by the addition of an antioxidant α-tocopherol, confirming that photo-oxidative damage was the major cause of the growth defect. A targeted PII mutant retaining wild-type pipX was successfully constructed from the wild-type S. elongatus strain (SPc) in the presence of α-tocopherol. The resulting mutant (PD1X) showed an unusual chlorophyll fluorescence profile, indicating extremely slow reduction and re-oxidation of QA, which was not observed in mutants defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in an impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.

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Symbiotic Deficiencies Associated with acoxWXYZ Mutant of Bradyrhizobium japonicum

Applied and Environmental Microbiology ◽

10.1128/aem.65.1.339-341.1999 ◽

1999 ◽

Vol 65 (1) ◽

pp. 339-341 ◽

Cited By ~ 4

Author(s):

Marci Ann Surpin ◽

Robert J. Maier

Keyword(s):

Bradyrhizobium Japonicum ◽

Double Mutant ◽

Viable Cell ◽

Cell Number ◽

Viable Cell Number ◽

Terminal Oxidase ◽

Wild Type ◽

Difference Spectra ◽

Cyanide Inhibition ◽

Binding Component

ABSTRACT The terminal oxidase complexes encoded by coxMNOP andcoxWXYZ were studied by analysis of mutations in each of the two oxidases. Carbon monoxide difference spectra obtained from membranes of coxMNOP mutant bacteroids were like those obtained for the wild type, whereas bacteroid membranes of acoxWXYZ mutant were deficient in CO-reactive cytochromeb. Experiments involving cyanide inhibition of oxidase activity were consistent with the conclusion that the coxXmutant is deficient in a membrane-associated O2-binding component. The viable cell number (bacteria that could be recultured from crushed nodules) was 20 to 29% lower for the coxXmutant than for the wild-type or the CoxN− strain. In three separate greenhouse studies, nodules of a coxXmutant had significantly lower (28 to 34% less) acetylene reduction rates than the wild-type nodules did, and plants inoculated with a double mutant (coxMNOP coxWZYZ) had rates 30% lower than those of wild-type-inoculated plants.

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Deletion of oxyR in Legionella pneumophila causes growth defect on agar

Canadian Journal of Microbiology ◽

10.1139/cjm-2018-0129 ◽

2018 ◽

Vol 64 (12) ◽

pp. 1030-1041 ◽

Cited By ~ 1

Author(s):

Nilmini Mendis ◽

Hana Trigui ◽

Mariam Saad ◽

Adrianna Tsang ◽

Sébastien P. Faucher

Keyword(s):

Legionella Pneumophila ◽

Water Environment ◽

Host Cells ◽

Growth Defect ◽

Intracellular Pathogen ◽

Wild Type ◽

Severe Growth Defect ◽

In Trans ◽

Severe Growth

The intracellular pathogen Legionella pneumophila (Lp) is a strict aerobe, surviving and replicating in environments where it frequently encounters reactive oxygen species (ROS), such as the nutrient-poor water environment and its replicative niche inside host cells. In many proteobacteria, the LysR-type regulator OxyR controls the oxidative stress response; however, the importance of the OxyR homologue in Lp is still unclear. Therefore, we undertook the characterization of phenotypes associated with the deletion of oxyR in Lp. Contrary to the wild type, the oxyR deletion mutant exhibits a severe growth defect on charcoal – yeast extract (CYE) agar lacking α-ketoglutarate supplementation. Growth in AYE broth (CYE without agar and charcoal), in amoeba and in human cultured macrophages, and survival in water is unaffected by the deletion. Supplementing CYE agar with antioxidants that neutralize ROS or introducing the oxyR gene in trans rescues the observed growth defect. Moreover, the mutant grows as well as the wild type on CYE plates made with agarose instead of agar, suggesting that a compound present in the latter is responsible for the growth defect phenotype.

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Glutamate-354 of the CP43 polypeptide interacts with the oxygen-evolving Mn 4 Ca cluster of photosystem II: a preliminary characterization of the Glu354Gln mutant

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2007.2213 ◽

2007 ◽

Vol 363 (1494) ◽

pp. 1179-1188 ◽

Cited By ~ 14

Author(s):

Melodie A Strickler ◽

Hong Jin Hwang ◽

Robert L Burnap ◽

Junko Yano ◽

Lee M Walker ◽

...

Keyword(s):

Photosystem Ii ◽

Release Kinetics ◽

Difference Spectrum ◽

Wild Type ◽

Difference Spectra ◽

Paramagnetic Resonance ◽

Preliminary Characterization ◽

Essentially Normal ◽

Normal Period

In the recent X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is assigned as a ligand of the O 2 -evolving Mn 4 Ca cluster. In this communication, a preliminary characterization of the CP43-Glu354Gln mutant of the cyanobacterium Synechocystis sp. PCC 6803 is presented. The steady-state rate of O 2 evolution in the mutant cells is only approximately 20% compared with the wild-type, but the kinetics of O 2 release are essentially unchanged and the O 2 -flash yields show normal period-four oscillations, albeit with lower overall intensity. Purified PSII particles exhibit an essentially normal S 2 state multiline electron paramagnetic resonance (EPR) signal, but exhibit a substantially altered S 2 - minus -S 1 Fourier transform infrared (FTIR) difference spectrum. The intensities of the mutant EPR and FTIR difference spectra (above 75% compared with wild-type) are much greater than the O 2 signals and suggest that CP43-Glu354Gln PSII reaction centres are heterogeneous, with a minority fraction able to evolve O 2 with normal O 2 release kinetics and a majority fraction unable to advance beyond the S 2 or S 3 states. The S 2 - minus -S 1 FTIR difference spectrum of CP43-Glu354Gln PSII particles is altered in both the symmetric and asymmetric carboxylate stretching regions, implying either that CP43-Glu354 is exquisitely sensitive to the increased charge that develops on the Mn 4 Ca cluster during the S 1 →S 2 transition or that the CP43-Glu354Gln mutation changes the distribution of Mn(III) and Mn(IV) oxidation states within the Mn 4 Ca cluster in the S 1 and/or S 2 states.

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