scholarly journals Sialylation of Lipooligosaccharide Cores Affects Immunogenicity and Serum Resistance of Campylobacter jejuni

2000 ◽  
Vol 68 (12) ◽  
pp. 6656-6662 ◽  
Author(s):  
Patricia Guerry ◽  
Cheryl P. Ewing ◽  
Thomas E. Hickey ◽  
Martina M. Prendergast ◽  
Anthony P. Moran
Keyword(s):  
Electrophoretic Mobility ◽  
Campylobacter Jejuni ◽  
Polyclonal Antiserum ◽  
Wild Type ◽  
Gene Encoding ◽  
Whole Cells ◽  
Acetylneuraminic Acid ◽  
Genes Encoding ◽  
Hyperimmune Antiserum ◽  
Human Sera

ABSTRACT Three genes involved in biosynthesis of the lipooligosaccharide (LOS) core of Campylobacter jejuni MSC57360, the type strain of the HS:1 serotype, whose structure mimics GM2ganglioside, have been cloned and characterized. Mutation of genes encoding proteins with homology to a sialyl transferase (cstII) and a putative N-acetylmannosamine synthetase (neuC1), part of the biosynthetic pathway ofN-acetylneuraminic acid (NeuNAc), have identical phenotypes. The LOS cores of these mutants display identical changes in electrophoretic mobility, loss of reactivity with cholera toxin (CT), and enhanced immunoreactivity with a hyperimmune polyclonal antiserum generated against whole cells of C. jejuni MSC57360. Loss of sialic acid in the core of the neuC1 mutant was confirmed by fast atom bombardment mass spectrometry. Mutation of a gene encoding a putative β-1,4-N-acetylgalactosaminyltransferase (Cgt) resulted in LOS cores intermediate in electrophoretic mobility between that of wild type and the mutants lacking NeuNAc, loss of reactivity with CT, and a reduced immunoreactivity with hyperimmune antiserum. Chemical analyses confirmed the loss of N-acetylgalactosamine (GalNAc) and the presence of NeuNAc in the cgt mutant. These data suggest that the Cgt enzyme is capable of transferring GalNAc to an acceptor with or without NeuNAc and that the Cst enzyme is capable of transferring NeuNAc to an acceptor with or without GalNAc. A mutant with a nonsialylated LOS core is more sensitive to the bactericidal effects of human sera than the wild type or the mutant lacking GalNAc.

scholarly journals KatG Is the Primary Detoxifier of Hydrogen Peroxide Produced by Aerobic Metabolism in Bradyrhizobium japonicum

2004 ◽  
Vol 186 (23) ◽  
pp. 7874-7880 ◽  
Author(s):  
Heather R. Panek ◽  
Mark R. O'Brian
Keyword(s):  
Catalase Activity ◽  
Bradyrhizobium Japonicum ◽  
Aerobic Metabolism ◽  
Short Exposure ◽  
Wild Type ◽  
Gene Encoding ◽  
Whole Cells ◽  
Cell Extracts ◽  
Genes Encoding ◽  
High Concentrations

ABSTRACT Bacteria are exposed to reactive oxygen species from the environment and from those generated by aerobic metabolism. Catalases are heme proteins that detoxify H2O2, and many bacteria contain more than one catalase enzyme. Also, the nonheme peroxidase alkyl hydroperoxide reductase (Ahp) is the major scavenger of endogenous H2O2 in Escherichia coli. Here, we show that aerobically grown Bradyrhizobium japonicum cells express a single catalase activity. Four genes encoding putative catalases in the B. japonicum genome were identified, including a katG homolog encoding a catalase-peroxidase. Deletion of the katG gene resulted in loss of catalase activity in cell extracts and of exogenous H2O2 consumption by whole cells. The katG strain had a severe aerobic growth phenotype but showed improved growth in the absence of O2. By contrast, a B. japonicum ahpCD mutant grew well aerobically and consumed H2O2 at wild-type rates. A heme-deficient hemA mutant expressed about one-third of the KatG activity as the wild type but grew well aerobically and scavenged low concentrations of exogenous H2O2. However, cells of the hemA strain were deficient in consumption of high concentrations of H2O2 and were very sensitive to killing by short exposure to H2O2. In addition, KatG activity did not decrease as a result of mutation of the gene encoding the transcriptional activator OxyR. We conclude that aerobic metabolism produces toxic levels of H2O2 in B. japonicum, which is detoxified primarily by KatG. Furthermore, the katG level sufficient for detoxification does not require OxyR.

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

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.

scholarly journals Analysis of Cytadherence-Deficient, GapA-NegativeMycoplasma gallisepticum Strain R

2000 ◽  
Vol 68 (12) ◽  
pp. 6643-6649 ◽  
Author(s):  
L. Papazisi ◽  
K. E. Troy ◽  
T. S. Gorton ◽  
X. Liao ◽  
S. J. Geary
Keyword(s):  
Shuttle Vector ◽  
Phenotypic Expression ◽  
Mycoplasma Genitalium ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Wild Type ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Low Passage ◽  
Translational Start

ABSTRACT Comparison of the phenotypic expression of Mycoplasma gallisepticum strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the gapAtranslational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type gapAin R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by gapA complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream ofgapA in the same operon and was designatedcrmA. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in bothMycoplasma pneumoniae and Mycoplasma genitalium. Transcriptional analysis revealed thatcrmA is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-typegapA and crmA for transformation into R high to assess the role of CrmA in the cytadherence process.

scholarly journals Mmp10 is required for post-translational methylation of arginine at the active site of methyl-coenzyme M reductase

2017 ◽  
Author(s):  
Zhe Lyu ◽  
Chau-wen Chou ◽  
Hao Shi ◽  
Ricky Patel ◽  
Evert C. Duin ◽  
...  
Keyword(s):  
Active Site ◽  
Methane Formation ◽  
Wild Type ◽  
Coenzyme M ◽  
Methane Cycle ◽  
Gene Encoding ◽  
Post Translational Modifications ◽  
Whole Cells ◽  
Type Gene ◽  
Methyl Coenzyme M Reductase

AbstractCatalyzing the key step for anaerobic methane production and oxidation, methyl-coenzyme M reductase or Mcr plays a key role in the global methane cycle. The McrA subunit possesses up to five post-translational modifications (PTM) at its active site. Bioinformatic analyses had previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To examine its role, MMP1554, the gene encoding Mmp10 inMethanococcus maripaludis, was deleted with a new genetic tool, resulting in the specific loss of the 5-(S)-methylarginine PTM of residue 275 in the McrA subunit and a 40~60 % reduction in the maximal rates of methane formation by whole cells. Methylation was restored by complementations with the wild-type gene. However, the rates of methane formation of the complemented strains were not always restored to the wild type level. This study demonstrates the importance of Mmp10 and the methyl-Arg PTM on Mcr activity.

scholarly journals A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

2016 ◽  
Vol 82 (6) ◽  
pp. 1917-1923 ◽  
Author(s):  
Wenyu Gu ◽  
Muhammad Farhan Ul Haque ◽  
Bipin S. Baral ◽  
Erick A. Turpin ◽  
Nathan L. Bandow ◽  
...  
Keyword(s):  
Methane Monooxygenase ◽  
Copper Uptake ◽  
Wild Type ◽  
Particulate Methane Monooxygenase ◽  
Methylosinus Trichosporium Ob3b ◽  
Gene Encoding ◽  
Methylosinus Trichosporium ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Genes Encoding

ABSTRACTMethanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin,mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well asmbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine ifmbnTis truly responsible for methanobactin uptake, a knockout was constructed inMethylosinus trichosporiumOB3b using marker exchange mutagenesis. The resultingM. trichosporiummbnT::Gmrmutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression ofmmoXandpmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-typeM. trichosporiumOB3b, indicating that the TonB-dependent transporter encoded bymbnTis responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When thembnT::Gmrmutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-typeM. trichosporiumOB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.

scholarly journals Disruption of sucA, Which Encodes the E1 Subunit of α-Ketoglutarate Dehydrogenase, Affects the Survival of Nitrosomonas europaea in Stationary Phase

2006 ◽  
Vol 188 (1) ◽  
pp. 343-347 ◽  
Author(s):  
Norman G. Hommes ◽  
Elizabeth G. Kurth ◽  
Luis A. Sayavedra-Soto ◽  
Daniel J. Arp
Keyword(s):  
Stationary Phase ◽  
Mutant Strain ◽  
Nitrosomonas Europaea ◽  
Wild Type ◽  
Late Stationary Phase ◽  
Gene Encoding ◽  
Hydroxylamine Oxidoreductase ◽  
Genes Encoding ◽  
Knockout Mutation ◽  
Ketoglutarate Dehydrogenase

ABSTRACT Although Nitrosomonas europaea lacks measurable α-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the sucA gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of ammonia monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.

scholarly journals Increased Production of Zeaxanthin and Other Pigments by Application of Genetic Engineering Techniques toSynechocystis sp. Strain PCC 6803

2000 ◽  
Vol 66 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Delphine Lagarde ◽  
Laurent Beuf ◽  
Wim Vermaas
Keyword(s):  
Mutant Strain ◽  
Fold Increase ◽  
Carotenoid Biosynthesis ◽  
Carotenoid Content ◽  
Wild Type ◽  
Gene Encoding ◽  
Genes Encoding ◽  
In The Wild ◽  
Β Carotene ◽  
Pcc 6803

ABSTRACT The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis inSynechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and theSynechocystis β-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP andcrtB, respectively) were introduced intoSynechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, andcrtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the β-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the β-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtOgene, encoding β-carotene ketolase, which converts β-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

scholarly journals The N-Acetylglucosamine Kinase from Yarrowia lipolytica Is a Moonlighting Protein

2021 ◽  
Vol 22 (23) ◽  
pp. 13109
Author(s):  
Carmen-Lisset Flores ◽  
Joaquín Ariño ◽  
Carlos Gancedo
Keyword(s):  
Yarrowia Lipolytica ◽  
Kinase Activity ◽  
Rna Seq ◽  
Wild Type ◽  
Gene Encoding ◽  
Moonlighting Protein ◽  
Genes Expression ◽  
Genes Encoding ◽  
Utilization Pathway

In Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes.

scholarly journals A truncated haemoglobin implicated in oxygen metabolism by the microaerophilic food-borne pathogen Campylobacter jejuni

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 4079-4091 ◽  
Author(s):  
Laura M. Wainwright ◽  
Karen T. Elvers ◽  
Simon F. Park ◽  
Robert K. Poole
Keyword(s):  
Campylobacter Jejuni ◽  
Nitrosative Stress ◽  
Bacterial Pathogen ◽  
Oxygen Metabolism ◽  
Wild Type ◽  
Gene Encoding ◽  
Food Borne ◽  
Micro Organisms ◽  
Mutant Cells ◽  
Kinetics Of

Of the three groups of haemoglobins identified in micro-organisms (single-domain globins, flavohaemoglobins and truncated globins), the last group is the least well understood. The function of the truncated haemoglobin (Ctb) encoded by Cj0465c in the microaerophilic food-borne bacterial pathogen Campylobacter jejuni was investigated by constructing a ctb mutant and characterizing its phenotype. The effects of the ctb mutation on the kinetics of terminal oxidase function in C. jejuni were investigated using oxyleghaemoglobin and oxymyoglobin as sensitive reporters of O2 consumption. The V max of ctb mutant cells for O2, calculated using either globin, was greater than that of wild-type cells at extracellular O2 concentrations up to ∼1 μM, suggesting a role for Ctb in moderating O2 supply for reduction by high-affinity terminal oxidases. However, cells mutated in ctb were disadvantaged when grown under conditions of high aeration, as revealed by measurements of growth yields and rates in batch culture. Furthermore, the rate at which ctb mutant cells consumed O2 in an O2 electrode (10–200 μM O2) was approximately half the rate displayed by wild-type cells, reflecting a role for Ctb in respiration at physiologically relevant external O2 concentrations. However, a lack of sensitivity of the mutant to paraquat or H2O2 indicated that increased oxidative stress under such conditions was not the cause of these phenotypes. O2 affinities of cells (K m values of approximately 40 nM and 1 μM) were unaffected by mutation of either Ctb or the full-length C. jejuni globin, Cgb. Although the gene encoding Ctb was found to be upregulated by S-nitrosoglutathione (GSNO) and the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP), a ctb mutant did not display sensitivity to a number of nitrosative stress-generating compounds. The authors conclude that Ctb is involved in moderating O2 flux within C. jejuni.

scholarly journals The Role of PerR in O2-Affected Gene Expression of Clostridium acetobutylicum

2009 ◽  
Vol 191 (19) ◽  
pp. 6082-6093 ◽  
Author(s):  
Falk Hillmann ◽  
Christina Döring ◽  
Oliver Riebe ◽  
Armin Ehrenreich ◽  
Ralf-Jörg Fischer ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Clostridium Acetobutylicum ◽  
Wild Type ◽  
Gene Encoding ◽  
Homologous Protein ◽  
Strict Anaerobe ◽  
Genes Encoding ◽  
Highly Expressed Genes

ABSTRACT In the strict anaerobe Clostridium acetobutylicum, a PerR-homologous protein has recently been identified as being a key repressor of a reductive machinery for the scavenging of reactive oxygen species and molecular O2. In the absence of PerR, the full derepression of its regulon resulted in increased resistance to oxidative stress and nearly full tolerance of an aerobic environment. In the present study, the complementation of a Bacillus subtilis PerR mutant confirmed that the homologous protein from C. acetobutylicum acts as a functional peroxide sensor in vivo. Furthermore, we used a transcriptomic approach to analyze gene expression in the aerotolerant PerR mutant strain and compared it to the O2 stimulon of wild-type C. acetobutylicum. The genes encoding the components of the alternative detoxification system were PerR regulated. Only few other targets of direct PerR regulation were identified, including two highly expressed genes encoding enzymes that are putatively involved in the central energy metabolism. All of them were highly induced when wild-type cells were exposed to sublethal levels of O2. Under these conditions, C. acetobutylicum also activated the repair and biogenesis of DNA and Fe-S clusters as well as the transcription of a gene encoding an unknown CO dehydrogenase-like enzyme. Surprisingly few genes were downregulated when exposed to O2, including those involved in butyrate formation. In summary, these results show that the defense of this strict anaerobe against oxidative stress is robust and by far not limited to the removal of O2 and its reactive derivatives.

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