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 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 Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

2014 ◽  
Vol 82 (12) ◽  
pp. 5293-5307 ◽  
Author(s):  
Marina Falaleeva ◽  
Oliwia W. Zurek ◽  
Robert L. Watkins ◽  
Robert W. Reed ◽  
Hadeel Ali ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Streptococcus Pyogenes ◽  
Regulatory Region ◽  
Regulatory System ◽  
Invasive Disease ◽  
Negative Regulator ◽  
Mobility Shift ◽  
Putative Promoter ◽  
Transcriptional Terminator ◽  
Content Type

ABSTRACTThe important human pathogenStreptococcus pyogenes(group AStreptococcus[GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that thehasABCoperon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription ofhasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion ofhasSor of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through,hasAtranscription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.

scholarly journals ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

2012 ◽  
Vol 442 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Corinne Appia-Ayme ◽  
Andrea Hall ◽  
Elaine Patrick ◽  
Shiny Rajadurai ◽  
Thomas A. Clarke ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Regulatory System ◽  
Enteric Bacteria ◽  
Polymyxin B ◽  
Regulatory Function ◽  
Cationic Antimicrobial Peptide ◽  
Envelope Stress ◽  
Two Component ◽  
Periplasmic Proteins ◽  
Zinc Concentrations

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.

scholarly journals Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Brett M. Barney ◽  
Mary H. Plunkett ◽  
Velmurugan Natarajan ◽  
Florence Mus ◽  
Carolann M. Knutson ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Stationary Phase ◽  
Gene Transcription ◽  
Biological Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Content Type ◽  
Ammonium Production ◽  
Biological Nitrogen

ABSTRACT Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

scholarly journals Diazotrophic Growth Allows Azotobacter vinelandii To Overcome the Deleterious Effects of a glnE Deletion

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Florence Mus ◽  
Alex Tseng ◽  
Ray Dixon ◽  
John W. Peters
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Ammonium Assimilation ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Posttranslational Regulation ◽  
Knockout Mutant ◽  
Content Type ◽  
Carbon And Nitrogen ◽  
Ammonium Production

ABSTRACT Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of Azotobacter vinelandii, attempts to obtain deletion mutants in the gene encoding GS (glnA) have been unsuccessful. We have generated a glnE deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance. IMPORTANCE In this study, the characterization of the glnE knockout mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.

scholarly journals The Haemophilus ducreyi Fis Protein Is Involved in Controlling Expression of thelspB-lspA2Operon and Other Virulence Factors

2013 ◽  
Vol 81 (11) ◽  
pp. 4160-4170 ◽  
Author(s):  
Maria Labandeira-Rey ◽  
Dana A. Dodd ◽  
Chad A. Brautigam ◽  
Kate R. Fortney ◽  
Stanley M. Spinola ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Regulatory System ◽  
Structural Similarity ◽  
Enteric Bacteria ◽  
Secretion System ◽  
Haemophilus Ducreyi ◽  
Altered Expression ◽  
Content Type ◽  
Genes Encoding ◽  
Regulatory Effects

ABSTRACTExpression of thelspB-lspA2operon encoding a virulence-related two-partner secretion system inHaemophilus ducreyi35000HP is directly regulated by the CpxRA regulatory system (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402–3411, 2009). In the present study, we show that this secretion system is also regulated by the small nucleoid-associated protein Fis. Inactivation of theH. ducreyi fisgene resulted in a reduction in expression of both theH. ducreyiLspB and LspA2 proteins. DNA microarray experiments showed that aH. ducreyi fisdeletion mutant exhibited altered expression levels of genes encoding other importantH. ducreyivirulence factors, including DsrA and Flp1, suggesting a possible global role for Fis in the control of virulence in this obligate human pathogen. While theH. ducreyiFis protein has a high degree of sequence and structural similarity to the Fis proteins of other bacteria, its temporal pattern of expression was very different from that of enterobacterial Fis proteins. The use of alacZ-based transcriptional reporter provided evidence which indicated that theH. ducreyiFis homolog is a positive regulator ofgyrB, a gene that is negatively regulated by Fis in enteric bacteria. Taken together, the Fis protein expression data and the observed regulatory effects of Fis inH. ducreyisuggest that this small DNA binding protein has a regulatory role inH. ducreyiwhich may differ in substantial ways from that of other Fis proteins.

scholarly journals The Phosphorolytic Exoribonucleases Polynucleotide Phosphorylase and RNase PH Stabilize sRNAs and Facilitate Regulation of Their mRNA Targets

2016 ◽  
Vol 198 (24) ◽  
pp. 3309-3317 ◽  
Author(s):  
Todd A. Cameron ◽  
Nicholas R. De Lay
Keyword(s):  
Rna Binding ◽  
Environmental Changes ◽  
Rna Binding Proteins ◽  
Growth Conditions ◽  
Regulatory Function ◽  
Polynucleotide Phosphorylase ◽  
Base Pairing ◽  
Stationary Growth ◽  
Content Type ◽  
Mrna Targets

ABSTRACT Gene regulation by base pairing between small noncoding RNAs (sRNAs) and their mRNA targets is an important mechanism that allows bacteria to maintain homeostasis and respond to dynamic environments. In Gram-negative bacteria, sRNA pairing and regulation are mediated by several RNA-binding proteins, including the sRNA chaperone Hfq and polynucleotide phosphorylase (PNPase). PNPase and its homolog RNase PH together represent the two 3′ to 5′ phosphorolytic exoribonucleases found in Escherichia coli ; however, the role of RNase PH in sRNA regulation has not yet been explored and reported. Here, we have examined in detail how PNPase and RNase PH interact to support sRNA stability, activity, and base pairing in exponential and stationary growth conditions. Our results indicate that these proteins facilitate the stability and regulatory function of the sRNAs RyhB, CyaR, and MicA during exponential growth. PNPase further appears to contribute to pairing between RyhB and its mRNA targets. During stationary growth, each sRNA responded differently to the absence or presence of PNPase and RNase PH. Finally, our results suggest that PNPase and RNase PH stabilize only Hfq-bound sRNAs. Taken together, these results confirm and extend previous findings that PNPase participates in sRNA regulation and reveal that RNase PH serves a similar, albeit more limited, role as well. These proteins may, therefore, act to protect sRNAs from spurious degradation while also facilitating regulatory pairing with their targets. IMPORTANCE In many bacteria, Hfq-dependent base-pairing sRNAs facilitate rapid changes in gene expression that are critical for maintaining homeostasis and responding to stress and environmental changes. While a role for Hfq in this process was identified more than 2 decades ago, the identity and function of the other proteins required for Hfq-dependent regulation by sRNAs have not been resolved. Here, we demonstrate that PNPase and RNase PH, the two phosphorolytic RNases in E. coli , stabilize sRNAs against premature degradation and, in the case of PNPase, also accelerate regulation by sRNA-mRNA pairings for certain sRNAs. These findings are the first to demonstrate that RNase PH influences and supports sRNA regulation and suggest shared and distinct roles for these phosphorolytic RNases in this process.

scholarly journals ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation byBacillus amyloliquefaciensSQR9

2018 ◽  
Vol 84 (8) ◽  
pp. e02744-17 ◽  
Author(s):  
Xuan Zhou ◽  
Nan Zhang ◽  
Liming Xia ◽  
Qing Li ◽  
Jiahui Shao ◽  
...  
Keyword(s):  
Plant Growth ◽  
Biofilm Formation ◽  
Root Colonization ◽  
Oxygen Deficiency ◽  
Regulatory System ◽  
Regulatory Function ◽  
Content Type ◽  
Environmental Signals ◽  
Link Type ◽  
Two Component

ABSTRACTEfficient biofilm formation and root colonization capabilities facilitate the ability of beneficial plant rhizobacteria to promote plant growth and antagonize soilborne pathogens. Biofilm formation by plant-beneficialBacillusstrains is triggered by environmental cues, including oxygen deficiency, but the pathways that sense these environmental signals and regulate biofilm formation have not been thoroughly elucidated. In this study, we showed that the ResDE two-component regulatory system in the plant growth-promoting rhizobacteriumBacillus amyloliquefaciensstrain SQR9 senses the oxygen deficiency signal and regulates biofilm formation. ResE is activated by sensing the oxygen limitation-induced reduction of the NAD+/NADH pool through its PAS domain, stimulating its kinase activity, and resulting in the transfer of a phosphoryl group to ResD. The phosphorylated ResD directly binds to the promoter regions of theqoxABCDandctaCDEFoperons to improve the biosynthesis of terminal oxidases, which can interact with KinB to activate biofilm formation. These results not only revealed the novel regulatory function of the ResDE two-component system but also contributed to the understanding of the complicated regulatory network governingBacillusbiofilm formation. This research may help to enhance the root colonization and the plant-beneficial efficiency of SQR9 and otherBacillusrhizobacteria used in agriculture.IMPORTANCEBacillusspp. are widely used as bioinoculants for plant growth promotion and disease suppression. The exertion of their plant-beneficial functions is largely dependent on their root colonization, which is closely related to their biofilm formation capabilities. On the other hand,Bacillusis the model bacterium for biofilm study, and the process and molecular network of biofilm formation are well characterized (B. Mielich-Süss and D. Lopez, Environ Microbiol 17:555–565, 2015,https://doi.org/10.1111/1462-2920.12527; L. S. Cairns, L. Hobley, and N. R. Stanley-Wall, Mol Microbiol 93:587–598, 2014,https://doi.org/10.1111/mmi.12697; H. Vlamakis, C. Aguilar, R. Losick, and R. Kolter, Genes Dev 22:945–953, 2008,https://doi.org/10.1101/gad.1645008; S. S. Branda, A. Vik, L. Friedman, and R. Kolter, Trends Microbiol 13:20–26, 2005,https://doi.org/10.1016/j.tim.2004.11.006; C. Aguilar, H. Vlamakis, R. Losick, and R. Kolter, Curr Opin Microbiol 10:638–643, 2007,https://doi.org/10.1016/j.mib.2007.09.006; S. S. Branda, J. E. González-Pastor, S. Ben-Yehuda, R. Losick, and R. Kolter, Proc Natl Acad Sci U S A 98:11621–11626, 2001,https://doi.org/10.1073/pnas.191384198). However, the identification and sensing of environmental signals triggeringBacillusbiofilm formation need further research. Here, we report that the oxygen deficiency signal inducingBacillusbiofilm formation is sensed by the ResDE two-component regulatory system. Our results not only revealed the novel regulatory function of the ResDE two-component regulatory system but also identified the sensing system of a biofilm-triggering signal. This knowledge can help to enhance the biofilm formation and root colonization of plant-beneficialBacillusstrains and also provide new insights of bacterial biofilm formation regulation.

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