scholarly journals The FixM Flavoprotein Modulates Inhibition by AICAR or 5′AMP of Respiratory and Nitrogen Fixation Gene Expression in Sinorhizobium meliloti

2002 ◽  
Vol 15 (6) ◽  
pp. 598-607 ◽  
Author(s):  
Céline Cosseau ◽  
Anne Marie Garnerone ◽  
Jacques Batut
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Regulatory System ◽  
Wild Type ◽  
Meliloti Strain ◽  
Related Metabolite ◽  
Nitrogen Fixation Gene ◽  
Nifa Gene

AICAR, a purine-related metabolite, was recently shown to inhibit respiratory and nifA gene expression in Sinorhizobium meliloti. Here, we demonstrate that AICAR has essentially no or little effect in a wild-type S. meliloti strain and inhibits respiratory and nitrogen fixation gene expression only in specific mutant backgrounds. We have analyzed in detail a mutant in which addition of AICAR inhibited fixK, fixN, fixT, and nifA expression. The corresponding gene, fixM, is located just downstream of fixK1 on pSymA megaplasmid and encodes a flavoprotein oxidore-ductase. 5′AMP, a structural analogue of AICAR, mimicked AICAR effect as well as the nucleoside precursors AICAriboside and adenosine. The mode of action of AICAR and 5′AMP in vivo was investigated. We demonstrate that AICAR does not affect FixK transcriptional activity and instead regulates fixK and nifA gene expression. We hypothesize that AICAR and 5′AMP may modulate, possibly indirectly, the activity of the FixLJ two-component regulatory system. The possible physiological roles of AICAR, 5′AMP, and fixM in the context of symbiosis are discussed.

scholarly journals Quorum Sensing: a Transcriptional Regulatory System Involved in the Pathogenicity of Burkholderia mallei

2004 ◽  
Vol 72 (11) ◽  
pp. 6589-6596 ◽  
Author(s):  
Ricky L. Ulrich ◽  
David DeShazer ◽  
Harry B. Hines ◽  
Jeffrey A. Jeddeloh
Keyword(s):  
Quorum Sensing ◽  
Virulence Factor ◽  
Regulatory System ◽  
In Silico Analysis ◽  
Homoserine Lactone ◽  
Burkholderia Mallei ◽  
Wild Type ◽  
Transcriptional Regulatory ◽  
A Cell

ABSTRACT Numerous gram-negative bacterial pathogens regulate virulence factor expression by using a cell density mechanism termed quorum sensing (QS). An in silico analysis of the Burkholderia mallei ATCC 23344 genome revealed that it encodes at least two luxI and four luxR homologues. Using mass spectrometry, we showed that wild-type B. mallei produces the signaling molecules N-octanoyl-homoserine lactone and N-decanoyl-homoserine lactone. To determine if QS is involved in the virulence of B. mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models. Disruption of the B. mallei QS alleles, especially in RJ16 (bmaII) and RJ17 (bmaI3), which are luxI mutants, significantly reduced virulence, as indicated by the survival of mice who were aerosolized with 104 CFU (10 50% lethal doses [LD50s]). For the B. mallei transcriptional regulator mutants (luxR homologues), mutation of the bmaR5 allele resulted in the most pronounced decrease in virulence, with 100% of the challenged animals surviving a dose of 10 LD50s. Using a Syrian hamster intraperitoneal model of infection, we determined the LD50s for wild-type B. mallei and each QS mutant. An increase in the relative LD50 was found for RJ16 (bmaI1) (>967 CFU), RJ17 (bmaI3) (115 CFU), and RJ20 (bmaR5) (151 CFU) compared to wild-type B. mallei (<13 CFU). These findings demonstrate that B. mallei carries multiple luxIR homologues that either directly or indirectly regulate the biosynthesis of an essential virulence factor(s) that contributes to the pathogenicity of B. mallei in vivo.

scholarly journals Effects of Saline, an Ambient Acidic Environment, and Sodium Salicylate on OXA-Mediated Carbapenem Resistance in Acinetobacter baumannii: TABLE 1

2016 ◽  
Vol 60 (6) ◽  
pp. 3415-3418 ◽  
Author(s):  
Esther Zander ◽  
Harald Seifert ◽  
Paul G. Higgins
Keyword(s):  
Gene Expression ◽  
Acinetobacter Baumannii ◽  
Sodium Salicylate ◽  
Carbapenem Resistance ◽  
Low Ph ◽  
Wild Type ◽  
Experimental Conditions ◽  
Content Type ◽  
Reference Strains

Different physiological conditions, such as NaCl, low pH, and sodium salicylate, have been shown to affect antibiotic resistance determinants inAcinetobacter baumanniiisolates. Therefore, the aim of this study was to investigate the effects of NaCl, sodium salicylate, and low pH on the susceptibility ofA. baumanniito carbapenem. We cloned genes encoding oxacillinases (OXA) of different subclasses, with their associated promoters, from carbapenem-resistantA. baumanniiisolates into the same vector and transferred them to theA. baumanniireference strains ATCC 19606 and ATCC 17978. Carbapenem MICs were determined at least in triplicate by agar dilution under standard conditions, as well as in the presence of 200 mM NaCl or 16 mM sodium salicylate, or at pH 5.8. OXA-58-like gene expression was determined by reverse transcription-quantitative PCR (qRT-PCR). Under some experimental conditions, significant MIC reductions were shown for some transformants but not for others. Only in one instance were all transformants harboring the same OXA affected by the same condition: at pH 5.8, the imipenem and meropenem MICs for strains expressing OXA-58-like enzymes decreased from a resistant level (32 to 64 mg/liter) to an intermediate-susceptible level (8 mg/liter). However,blaOXA-58-likegene expression remained the same. MICs for both wild-type reference strains were not affected by the conditions tested. Our results indicate that the effects of the experimental conditions tested on OXAin vivoare mostly strain dependent. MICs were not reduced to wild-type levels, suggesting that the conditions tested do not lead to complete OXA inhibition in the bacterial cell.

scholarly journals Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Microbiology ◽  
2020 ◽  
Vol 166 (5) ◽  
pp. 484-497 ◽  
Author(s):  
Alejandra Arteaga Ide ◽  
Victor M. Hernández ◽  
Liliana Medina-Aparicio ◽  
Edson Carcamo-Noriega ◽  
Lourdes Girard ◽  
...  
Keyword(s):  
Type Species ◽  
Sinorhizobium Meliloti ◽  
Arginase Activity ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Link Type ◽  
Arginine Catabolism

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

scholarly journals Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice

2018 ◽  
Vol 92 (16) ◽  
Author(s):  
Hui Cai ◽  
Meisui Liu ◽  
Charles J. Russell
Keyword(s):  
Gene Expression ◽  
Influenza Virus ◽  
Virus Infection ◽  
Directed Evolution ◽  
Influenza Virus Infection ◽  
Foreign Gene ◽  
Reporter Virus ◽  
Wild Type ◽  
Bioluminescence Signal

ABSTRACTReporter viruses provide a powerful tool to study infection, yet incorporating a nonessential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus withNanoLuc luciferaseinserted into the 5′ end of the PA gene segment grew to titers 10-fold less than those of the wild type in MDCK cells and in DBA/2 mice and was less virulent. For 12 rounds, we propagated DBA/2 lung samples with the highest bioluminescence-to-titer ratios. Every three rounds, we comparedin vivoreplication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino acid mutations andPB2noncoding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate that directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-likein vivofitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCEInfluenza viruses contribute to 290,000 to 650,000 deaths globally each year. Infection is studied in mice to learn how the virus causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene that causes infected cells to light up, scientists could see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza virus infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.

The zygotic control of Drosophila pair-rule gene expression. I. A search for new pair-rule regulatory loci

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 663-672 ◽  
Author(s):  
S.H. Vavra ◽  
S.B. Carroll
Keyword(s):  
Gene Expression ◽  
Regulatory System ◽  
Wild Type ◽  
Periodic Patterns ◽  
Pair Rule Gene ◽  
New Type ◽  
Regulatory Loci ◽  
Direct Inspection ◽  
Pair Rule ◽  
Zygotic Control

The examination of pair-rule gene expression in wild-type and segmentation mutant embryos has identified many, but not necessarily all, of the elements of the regulatory system that establish their periodic patterns. Here we have conducted a new type of search for previously unknown regulators of these genes by examining pair-rule gene expression in blastoderm embryos lacking parts of or entire chromosomes. This method has the advantage of direct inspection of abnormal pair-rule gene patterns without relying upon mutagenesis or interpretation of larval phenotypes for the identification of segmentation genes. From these experiments we conclude that: (i) most zygotically required regulators of the fushi tarazu (ftz), even-skipped (eve) and hairy (h) pair-rule genes have been identified, except for one or more loci we have uncovered on chromosome arm 2L; (ii) the repression of the ftz and eve genes in the anterior third of the embryo is under maternal, not zygotic control; and (iii) there are no general zygotically required activators of pair-rule gene expression. The results suggest that the molecular basis of pair-rule gene regulation can be pursued with greater confidence now that most key trans-acting factors are already in hand.

scholarly journals Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression

2003 ◽  
Vol 285 (5) ◽  
pp. H2240-H2247 ◽  
Author(s):  
Elizabeth A. Nunamaker ◽  
Hai-Ying Zhang ◽  
Yuichi Shirasawa ◽  
Joseph N. Benoit ◽  
David A. Dean
Keyword(s):  
Gene Expression ◽  
Experimental Studies ◽  
Wild Type ◽  
Protein Levels ◽  
Dna Oligonucleotides ◽  
Pkc Isoforms ◽  
Cell Layers ◽  
Cultured Smooth Muscle Cells ◽  
Decrease Gene Expression

The development of inexpensive and effective approaches to transiently decrease gene expression in vivo would be useful for the study of physiological processes in living animals. DNAzymes are a novel class of DNA oligonucleotides that can catalytically cleave target mRNAs and thereby reduce protein production. However, current methods for their delivery in vivo are limited and inefficient. In this study, we show that electroporation can be used to deliver DNAzymes to the intact mesenteric vasculature of rats. With the use of PKC-ϵ as a target, a set of wild-type and mutant control DNAzymes was designed and shown to reduce both PKC-ϵ mRNA and protein levels in cultured smooth muscle cells in a specific manner. The wild-type DNAzyme reduced PKC-ϵ protein levels by 70% at 24 h in two different cell lines without decreasing the levels of the five other PKC isoforms tested. When delivered to the intact vasculature using electroporation, the DNAzyme reduced PKC-ϵ protein levels by >60% without affecting these other PKC isoforms. Electroporation was required for oligonucleotide transfer and was able to deliver the DNAzymes to multiple cell layers in the vessel wall. Protein levels were reduced maximally by 24 h postelectroporation and returned to normal by 48 h. These results suggest that electroporation can be used to deliver DNAzymes and other DNA oligonucleotides to the vasculature in vivo and can decrease gene expression for a window of time that can be used for experimental studies.

scholarly journals Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Michael J. Mitsch ◽  
George C. diCenzo ◽  
Alison Cowie ◽  
Turlough M. Finan
Keyword(s):  
Nitrogen Fixation ◽  
Carbon Source ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Symbiotic Nitrogen Fixation ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Changes ◽  
Symbiotic Properties

ABSTRACTSymbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) inSinorhizobium melilotiRm1021dctmutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF withMedicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-typeS. meliloti.Rhizobium leguminosarumbv. viciae 3841dctmutants unable to transport C4-dicarboxylates but expressing themaePtransporter had strong symbiotic properties, withPisum sativumplants inoculated with these strains appearing similar to plants inoculated with wild-typeR. leguminosarum. This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combinedP. sativum-R. leguminosarumnodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses,l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCESymbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

scholarly journals Interrelations between Glycine Betaine Catabolism and Methionine Biosynthesis in Sinorhizobium meliloti Strain 102F34

2006 ◽  
Vol 188 (20) ◽  
pp. 7195-7204 ◽  
Author(s):  
Lise Barra ◽  
Catherine Fontenelle ◽  
Gwennola Ermel ◽  
Annie Trautwetter ◽  
Graham C. Walker ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Methionine Synthase ◽  
Vitamin B ◽  
Wild Type ◽  
Methionine Biosynthesis ◽  
Catabolic Pathway ◽  
Methyl Transferase ◽  
Meliloti Strain

ABSTRACT Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B12-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experiment.

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