scholarly journals Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

2015 ◽  
Vol 81 (13) ◽  
pp. 4316-4328 ◽  
Author(s):  
Brett M. Barney ◽  
Lauren J. Eberhart ◽  
Janet M. Ohlert ◽  
Carolann M. Knutson ◽  
Mary H. Plunkett
Keyword(s):  
Insertional Mutagenesis ◽  
Azotobacter Vinelandii ◽  
Regulatory System ◽  
Ammonium Transporter ◽  
Gene Deletions ◽  
Content Type ◽  
Living Bacterium ◽  
Obligate Aerobe ◽  
Biosensor Strain ◽  
Alternative Approaches

ABSTRACTAzotobacter vinelandiiis a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium,A. vinelandiihas evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies ofA. vinelandiiproducing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability ofA. vinelandiito recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter geneamtBthat resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green algaChlorella sorokinianaconfirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of theA. vinelandiiin coculture. The nature and quantities of nitrogen released by urease andamtBdisruptions were further compared to strains reported in previous efforts that altered thenifLAregulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.

scholarly journals NasT-Mediated Antitermination Plays an Essential Role in the Regulation of the Assimilatory Nitrate Reductase Operon in Azotobacter vinelandii

2012 ◽  
Vol 78 (18) ◽  
pp. 6558-6567 ◽  
Author(s):  
Baomin Wang ◽  
Leland S. Pierson ◽  
Christopher Rensing ◽  
Malkanthi K. Gunatilaka ◽  
Christina Kennedy
Keyword(s):  
Nitrogen Fixation ◽  
Rna Binding ◽  
Azotobacter Vinelandii ◽  
Regulatory System ◽  
Enteric Bacteria ◽  
Regulatory Function ◽  
Transcriptional Terminator ◽  
Content Type ◽  
Sequence Elements ◽  
Nitrate And Nitrite

ABSTRACTAzotobacter vinelandiiis a well-studied model system for nitrogen fixation in bacteria. Regulation of nitrogen fixation inA. vinelandiiis independent of NtrB/NtrC, a conserved nitrogen regulatory system in proteobacteria. Previous work showed that anntrCmutation inA. vinelandiiresulted in a loss of induction of assimilatory nitrate and nitrite reductases encoded by thenasABoperon. In addition to NtrC, several other proteins, including NasT, a protein containing a potential RNA-binding domain ANTAR (AmiR andNasRtranscriptionantiterminationregulators), have been implicated innasABregulation. In this work, we characterize the sequence upstream ofnasAand identify several DNA sequence elements, including two potential NtrC binding sites and a putative intrinsic transcriptional terminator upstream ofnasAthat are potentially involved innasABregulation. Our analyses confirm that thenasABpromoter,PnasA, is under NtrC control. However, unlike NtrC-regulated promoters in enteric bacteria,PnasAshows high activity in the presence of ammonium; in addition, thePnasAactivity is altered in thenifAgene mutation background. We discuss the implication of these results on NtrC-mediated regulation inA. vinelandii. Our study provides direct evidence that induction ofnasABis regulated by NasT-mediated antitermination, which occurs within the leader region of the operon. The results also support the hypothesis that NasT binds the promoter proximal hairpin ofnasABfor its regulatory function, which contributes to the understanding of the regulatory mechanism of ANTAR-containing antiterminators.

scholarly journals Natural Disruption of Two Regulatory Networks in Serotype M3 Group A Streptococcus Isolates Contributes to the Virulence Factor Profile of This Hypervirulent Serotype

2014 ◽  
Vol 82 (5) ◽  
pp. 1744-1754 ◽  
Author(s):  
Tram N. Cao ◽  
Zhuyun Liu ◽  
Tran H. Cao ◽  
Kathryn J. Pflughoeft ◽  
Jeanette Treviño ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Group A Streptococcus ◽  
Regulatory System ◽  
Mrna Levels ◽  
Bacterial Strains ◽  
Content Type ◽  
Small Regulatory Rna ◽  
Group A

ABSTRACTDespite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogenStreptococcus pyogenes(the group AStreptococcus[GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in thefasCgene of thefasBCAXregulatory system and an inactivating polymorphism in therivRregulator-encoding gene. ThefasCandrivRmutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of thefasCmutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of therivRmutation in M3 GAS restored the regulation ofgrabmRNA abundance but did not alter capsule mRNA levels. While important, thefasCandrivRmutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.

scholarly journals A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

2014 ◽  
Vol 81 (5) ◽  
pp. 1708-1714 ◽  
Author(s):  
Min-Sik Kim ◽  
Ae Ran Choi ◽  
Seong Hyuk Lee ◽  
Hae-Chang Jung ◽  
Seung Seob Bae ◽  
...  
Keyword(s):  
Production Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory System ◽  
Transcriptional Regulator ◽  
Bioreactor Culture ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

scholarly journals Spontaneous Gac Mutants of Pseudomonas Biological Control Strains: Cheaters or Mutualists?

2011 ◽  
Vol 77 (20) ◽  
pp. 7227-7235 ◽  
Author(s):  
William W. Driscoll ◽  
John W. Pepper ◽  
Leland S. Pierson ◽  
Elizabeth A. Pierson
Keyword(s):  
Alternative Hypothesis ◽  
Natural Populations ◽  
Regulatory System ◽  
Wild Type ◽  
Content Type ◽  
The Public ◽  
Extracellular Metabolites ◽  
Mutualistic Interaction ◽  
Alternative Hypotheses

ABSTRACTBacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are “cheaters” that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacteriumPseudomonas chlororaphisstrain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.

The public-private partnership valuation paradox

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Stephen Gray ◽  
Jason Hall ◽  
Grant Pollard ◽  
Damien Cannavan
Keyword(s):  
Mathematical Proof ◽  
Systematic Risk ◽  
Cash Flows ◽  
Current Theory ◽  
Apparent Paradox ◽  
Content Type ◽  
The Public ◽  
The Past ◽  
Alternative Approaches ◽  
Practical Implications

Purpose In the context of public-private partnerships (PPPs), it has been argued that the standard valuation framework produces a paradox whereby government appears to be made better off by taking on more systematic risk. This has led to a range of approaches being applied in practice, none of which are consistent with the standard valuation approach. The purpose of this paper is to demonstrate that these approaches are flawed and unnecessary. Design/methodology/approach The authors step through the proposed alternative valuation approaches and demonstrate their inconsistencies and illogical outcomes, using theory, logic and mathematical proof. Findings In this paper, the authors demonstrate that the proposed (alternative) approaches suffer from internal inconsistencies and produce illogical outcomes in some cases. The authors also show that there is no problem with the current accepted theory and that the apparent paradox is not the result of a deficiency in the current theory but is rather caused by its misapplication in practice. In particular, the authors show that the systematic risk of cash flows is frequently mis-estimated, and the correction of this error solves the apparent paradox. Practical implications Over the past 20 years, PPP activity around the globe amounts to many billions of dollars. Decisions on major infrastructure funding are of enormous social and economic importance. Originality/value To the best of the authors’ knowledge, this study is the first to demonstrate the flaws and internal inconsistencies with proposed valuation framework alternatives for the purposes of evaluating PPPs.

The role of Akkermansia muciniphila in obesity, diabetes and atherosclerosis

2021 ◽  
Vol 70 (10) ◽  
Author(s):  
Alka Hasani ◽  
Saba Ebrahimzadeh ◽  
Fatemeh Hemmati ◽  
Aytak Khabbaz ◽  
Akbar Hasani ◽  
...  
Keyword(s):  
Type Species ◽  
Anaerobic Bacteria ◽  
Regulatory System ◽  
Content Type ◽  
Akkermansia Muciniphila ◽  
Link Type ◽  
Animal Trials ◽  
Obesity And Diabetes ◽  
Underlying Mechanisms

Alteration in the composition of the gut microbiota can lead to a number of chronic clinical diseases. Akkermansia muciniphila is an anaerobic bacteria constituting 3–5% of the gut microbial community in healthy adults. This bacterium is responsible for degenerating mucin in the gut; its scarcity leads to diverse clinical disorders. In this review, we focus on the role of A. muciniphila in diabetes, obesity and atherosclerosis, as well as the use of this bacterium as a next-generation probiotic. In regard to obesity and diabetes, human and animal trials have shown that A. muciniphila controls the essential regulatory system of glucose and energy metabolism. However, the underlying mechanisms by which A. muciniphila alleviates the complications of obesity, diabetes and atherosclerosis are unclear. At the same time, its abundance suggests improved metabolic disorders, such as metabolic endotoxemia, adiposity insulin resistance and glucose tolerance. The role of A. muciniphila is implicated in declining aortic lesions and atherosclerosis. Well-characterized virulence factors, antigens and cell wall extracts of A. muciniphila may act as effector molecules in these diseases. These molecules may provide novel mechanisms and strategies by which this bacterium could be used as a probiotic for the treatment of obesity, diabetes and atherosclerosis.

scholarly journals Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

2014 ◽  
Vol 197 (5) ◽  
pp. 861-871 ◽  
Author(s):  
Kumiko Kurabayashi ◽  
Yuko Hirakawa ◽  
Koichi Tanimoto ◽  
Haruyoshi Tomita ◽  
Hidetada Hirakawa
Keyword(s):  
Phosphate Uptake ◽  
Response Regulator ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Carbon Substrate ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Two Component ◽  
Biological Cost

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagicEscherichia coli(EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression oftorR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression ofglpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.

Combining goods and service-dominant logics in purchasing strategies

2018 ◽  
Vol 33 (8) ◽  
pp. 1087-1099
Author(s):  
Tale Skjølsvik
Keyword(s):  
Future Research ◽  
Management Consulting ◽  
Content Type ◽  
Industrial Marketing ◽  
Knowledge Based ◽  
Consulting Services ◽  
Alternative Approaches ◽  
Knowledge Intensive ◽  
Relational Strategies ◽  
Practical Implications

Purpose While goods- and service-dominant logics are separated in most research as alternative and often incompatible paradigms, this paper aims to show how these logics can be and are combined in purchasing strategies in organizations. The paper also illustrates that multiple logics exist in addition to purely goods- or service-based logics. Design/methodology/approach The paper is based on empirical data on the purchasing of management consulting services, which represent an extreme context for understanding the combination and intersection of goods- and service-dominant logics. In particular, four in-depth case studies and interviews with 51 sellers and 30 buyers of management consulting services are used to develop a typology of purchasing approaches that combines goods- and service-dominant logics. Findings The study shows that goods- and service-dominant logics are combined in two main purchasing phases: supplier set selection and assignment selection. In both these phases, parallel and knowledge-based, embedded and experience-based approaches were identified as ways of combining goods- and service-dominant logics in the purchasing context. Research limitations/implications The research presented in the following adds to our existing understanding of possible purchasing strategies under multiple logics in buying organizations. Future research should explore the conditions under which different strategies are and should be applied in organizations. Practical implications This paper gives practitioners alternative approaches to choose from in their purchasing and sales of knowledge-intensive services, in addition to transactional and relational strategies. Originality/value The research adds to existing research on business and industrial marketing by identifying particular purchasing strategies on a continuum between goods- and service-dominant logics.

scholarly journals Gene Cluster Responsible for Secretion of and Immunity to Multiple Bacteriocins, the NKR-5-3 Enterocins

2014 ◽  
Vol 80 (21) ◽  
pp. 6647-6655 ◽  
Author(s):  
Naoki Ishibashi ◽  
Kohei Himeno ◽  
Yoshimitsu Masuda ◽  
Rodney Honrada Perez ◽  
Shun Iwatani ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Abc Transporter ◽  
Response Regulator ◽  
Regulatory System ◽  
Content Type ◽  
Expression Studies ◽  
A Genome ◽  
Wide Range ◽  
Close Proximity ◽  
Immunity Genes

ABSTRACTEnterococcus faeciumNKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA[ent53A],enkC[ent53C],enkD[ent53D], andenkZ[ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIazandenkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies ofenkTand ΔenkTmutant strains showed thatenkTis responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRKmutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter.

scholarly journals RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

2013 ◽  
Vol 79 (20) ◽  
pp. 6447-6451 ◽  
Author(s):  
Jung-Hoon Lee ◽  
Marcha L. Gatewood ◽  
George H. Jones
Keyword(s):  
Parental Strain ◽  
Insertional Mutagenesis ◽  
Southern Blotting ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Production Medium ◽  
Wild Type ◽  
Rnase Iii ◽  
Content Type ◽  
Streptomyces Antibioticus

ABSTRACTUsing insertional mutagenesis, we have disrupted the RNase III gene,rnc, of the actinomycin-producing streptomycete,Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of therncdisruption with the wild-typerncgene fromS. antibioticusrestored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case inStreptomyces coelicolor, RNase III is required for antibiotic production inS. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of theS. antibioticus rncmutant and its parental strain.

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