α-Ketoglutarate Accumulation Is Not Dependent on Isocitrate Dehydrogenase Activity during Tellurite Detoxification inEscherichia coli

Tellurite is toxic to most microorganisms because of its ability to generate oxidative stress. However, the way in which tellurite interferes with cellular processes is not fully understood to date. In this line, it was previously shown that tellurite-exposed cells displayed reduced activity of theα-ketoglutarate dehydrogenase complex (α-KGDH), which resulted inα-ketoglutarate (α-KG) accumulation. In this work, we assessed ifα-KG accumulation in tellurite-exposedE. colicould also result from increased isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) activities, both enzymes involved inα-KG synthesis. Unexpectedly both activities were found to decrease in the presence of the toxicant, an observation that seems to result from the decreased transcription oficdAandgdhAgenes (encoding ICDH and GDH, resp.). Accordingly, isocitrate levels were found to increase in tellurite-exposedE. coli. In the presence of the toxicant, cells lackingicdAorgdhAexhibited decreased reactive oxygen species (ROS) levels and higher tellurite sensitivity as compared to the wild type strain. Finally, a novel branch activity of ICDH as tellurite reductase is presented.

Download Full-text

The Gene yggE Functions in Restoring Physiological Defects of Escherichia coli Cultivated under Oxidative Stress Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.71.5.2762-2765.2005 ◽

2005 ◽

Vol 71 (5) ◽

pp. 2762-2765 ◽

Cited By ~ 17

Author(s):

SunYoung Kim ◽

Motomu Nishioka ◽

Shuhei Hayashi ◽

Hiroyuki Honda ◽

Takeshi Kobayashi ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Type Strain ◽

Wild Type Strain ◽

Specific Growth ◽

Maximum Specific Growth Rate ◽

Oxygen Species ◽

Wild Type ◽

Amino Acid Requirement ◽

E Coli

ABSTRACT DNA microarray analysis showed that yfiD, yggB, and yggE genes were up-regulated when superoxide dismutase (SOD)-deficient Escherichia coli IM303 (I4) was cultivated under the oxidative stress generated by photoexcited TiO2, and pYFD, pYGB, and pYGE were constructed by inserting the respective genes into a pUC 19 vector. The content of reactive oxygen species (ROS) in IM303 (I4) cells carrying pYGE was reduced to 31% of ROS content in the control cells with pUC 19. In the culture of wild-type strain, E. coli MM294, in the medium with paraquat (10 μmol/l), maximum specific growth rate of the cells with pYGE was about five times higher than that of the control cells, with a decreased ROS content in the former cells. The introduction of pYGE also suppressed the occurrence of the cells with altered amino acid requirement in the culture of MM294 cells with paraquat.

Download Full-text

Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

Scientific Reports ◽

10.1038/s41598-020-80125-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

José Francisco Cruz-Pérez ◽

Roxana Lara-Oueilhe ◽

Cynthia Marcos-Jiménez ◽

Ricardo Cuatlayotl-Olarte ◽

María Luisa Xiqui-Vázquez ◽

...

Keyword(s):

Azospirillum Brasilense ◽

Type Strain ◽

Wild Type Strain ◽

Soil Conditions ◽

Wild Type ◽

Gene Encoding ◽

Wheat Roots ◽

Genes Encoding ◽

In The Wild ◽

Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

Download Full-text

Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

Journal of Bacteriology ◽

10.1128/jb.183.17.5187-5197.2001 ◽

2001 ◽

Vol 183 (17) ◽

pp. 5187-5197 ◽

Cited By ~ 302

Author(s):

Vanessa Sperandio ◽

Alfredo G. Torres ◽

Jorge A. Girón ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Quorum Sensing ◽

Type Strain ◽

Wild Type Strain ◽

Regulatory Mechanism ◽

Sos Response ◽

Enterohemorrhagic Escherichia Coli ◽

Trna Genes ◽

Wild Type ◽

E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

Download Full-text

Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.2.445-452.2006 ◽

2006 ◽

Vol 50 (2) ◽

pp. 445-452 ◽

Cited By ~ 39

Author(s):

Daniel Criswell ◽

Virginia L. Tobiason ◽

J. Stephen Lodmell ◽

D. Scott Samuels

Keyword(s):

16S Rrna ◽

Borrelia Burgdorferi ◽

Type Strain ◽

Wild Type Strain ◽

Small Subunit ◽

Wild Type ◽

Spectinomycin Resistance ◽

E Coli ◽

Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

Download Full-text

The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.184.10.2850-2853.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2850-2853 ◽

Cited By ~ 39

Author(s):

Annie Conter ◽

Rachel Sturny ◽

Claude Gutierrez ◽

Kaymeuang Cam

Keyword(s):

Escherichia Coli ◽

Type Strain ◽

Wild Type Strain ◽

Cell Envelope ◽

Wild Type ◽

E Coli ◽

Induced Stress ◽

Mutant Strains ◽

Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

Download Full-text

Expression of Different-Size Transcripts from theclpP-clpX Operon of Escherichia coli during Carbon Deprivation

Journal of Bacteriology ◽

10.1128/jb.182.23.6630-6637.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6630-6637 ◽

Cited By ~ 13

Author(s):

Chin Li ◽

Yi Ping Tao ◽

Lee D. Simon

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Type Strain ◽

Wild Type Strain ◽

Transcription Termination ◽

Carbon Starvation ◽

Lower Percentage ◽

Wild Type ◽

Lower Efficiency ◽

E Coli

ABSTRACT Transcription of the clpP-clpX operon ofEscherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entireclpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpXcoding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς32-dependent promoter of the clpP-clpXoperon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of theclpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoBmutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpXtranscript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

Download Full-text

DOSEc, a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.19.6678-6682.2005 ◽

2005 ◽

Vol 187 (19) ◽

pp. 6678-6682 ◽

Cited By ~ 16

Author(s):

Tokiko Yoshimura-Suzuki ◽

Ikuko Sagami ◽

Nao Yokota ◽

Hirofumi Kurokawa ◽

Toru Shimizu

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Type Strain ◽

Redox State ◽

Wild Type Strain ◽

Functional Domain ◽

Wild Type ◽

Aerobic Conditions ◽

E Coli ◽

Knockout Strain

ABSTRACT Heme-regulated phosphodiesterase from Escherichia coli (DOSEc) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because DOSEc was originally cloned from E. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of DOSEc using cAMP as a substrate is only 0.15 min−1, which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the DOSEc gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOSEc gene knockout strain (Δdos). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher than in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOSEc is regulated according to environmental O2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOSEc expression.

Download Full-text

Comparative analysis and mutation effects of fpp2–fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum

Microbiology ◽

10.1099/mic.0.046938-0 ◽

2011 ◽

Vol 157 (4) ◽

pp. 1196-1204 ◽

Cited By ~ 16

Author(s):

David Pérez-Pascual ◽

Esther Gómez ◽

Beatriz Álvarez ◽

Jessica Méndez ◽

Pilar Reimundo ◽

...

Keyword(s):

Gene Function ◽

Type Strain ◽

Wild Type Strain ◽

Proteolytic Enzymes ◽

Function Analysis ◽

Phenotypic Characterization ◽

Pcr Analysis ◽

Wild Type ◽

Flavobacterium Psychrophilum ◽

Genes Encoding

Flavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2–fpp1 tandem in the genome of strain F. psychrophilum THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F. psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the fpp2 gene. Phenotypic characterization of the fpp2 mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD50 experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of F. psychrophilum, open new perspectives for the study of gene function in this bacterium.

Download Full-text

Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism

Journal of Bacteriology ◽

10.1128/jb.184.3.679-686.2002 ◽

2002 ◽

Vol 184 (3) ◽

pp. 679-686 ◽

Cited By ~ 59

Author(s):

Brant K. J. Pohorelic ◽

Johanna K. Voordouw ◽

Elisabeth Lojou ◽

Alain Dolla ◽

Jens Harder ◽

...

Keyword(s):

Sulfate Reduction ◽

Electron Donor ◽

Type Strain ◽

Wild Type Strain ◽

Physiological Role ◽

Hydrogen Uptake ◽

Desulfovibrio Vulgaris ◽

Lactate Metabolism ◽

Wild Type ◽

Genes Encoding

ABSTRACT The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H2 as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H2 was also present, growth in lactate- and sulfate-containing media lacking H2 was less efficient. The hyd mutant produced, transiently, significant amounts of H2 under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688–3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D. vulgaris genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.

Download Full-text

Insights into an alternative pathway for glycerol metabolism in a glycerol kinase deficientPseudomonas putidaKT2440

10.1101/567230 ◽

2019 ◽

Cited By ~ 2

Author(s):

Meg Walsh ◽

William Casey ◽

Shane T. Kenny ◽

Tanja Narancic ◽

Lars M. Blank ◽

...

Keyword(s):

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Alternative Pathway ◽

Glycerol Kinase ◽

Glycerol Metabolism ◽

Wild Type ◽

Short Chain Length ◽

Genes Encoding ◽

In The Wild

AbstractPseudomonas putidaKT2440 is known to metabolise glycerol via glycerol-3-phosphate using glycerol kinase an enzyme previously described as critical for glycerol metabolism (1). However, when glycerol kinase was knocked out inP. putidaKT2440 it retained the ability to use glycerol as the sole carbon source, albeit with a much-extended lag period and 2 fold lower final biomass compared to the wild type strain. A metabolomic study identified glycerate as a major and the most abundant intermediate in glycerol metabolism in this mutated strain with levels 21-fold higher than wild type. Erythrose-4-phosphate was detected in the mutant strain, but not in the wild type strain. Glyceraldehyde and glycraldehyde-3-phosphate were detected at similar levels in the mutant strain and the wild type. Transcriptomic studies identified 191 genes that were more than 2-fold upregulated in the mutant compared to the wild type and 175 that were down regulated. The genes involved in short chain length fatty acid metabolism were highly upregulated in the mutant strain. The genes encoding 3-hydroxybutyrate dehydrogenase were 5.8-fold upregulated and thus the gene was cloned, expressed and purified to reveal it can act on glyceraldehyde but not glycerol as a substrate.

Download Full-text