The expression ofnifBgene fromHerbaspirillum seropedicaeis dependent upon the NifA and RpoN proteins

2006 ◽  
Vol 52 (12) ◽  
pp. 1199-1207 ◽  
Author(s):  
Fabiane G.M Rego ◽  
Fábio O Pedrosa ◽  
Leda S Chubatsu ◽  
M Geoffrey Yates ◽  
Roseli Wassem ◽  
...  
Keyword(s):  
Binding Sites ◽  
Promoter Region ◽  
Integration Host Factor ◽  
Lacz Fusion ◽  
Herbaspirillum Seropedicae ◽  
Band Shift ◽  
E Coli ◽  
Low Levels ◽  
Ihf Protein

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a –24/–12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.Key words: Herbaspirillum seropedicae, nif, nitrogen fixation, NifA, RpoN.

scholarly journals Evaluation of Immunocompetent Urinary Tract Infected Balb/C Mouse Model For the Study of Antibiotic Resistance Development Using Escherichia Coli CFT073 Infection

Antibiotics ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 170 ◽  
Author(s):  
Ashok Chockalingam ◽  
Sharron Stewart ◽  
Lin Xu ◽  
Adarsh Gandhi ◽  
Murali K. Matta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Urinary Tract ◽  
Bladder Tissue ◽  
Resistance Development ◽  
Once Daily ◽  
E Coli ◽  
Low Levels ◽  
Tract Infections

Urinary tract infections (UTI) are common worldwide and are becoming increasingly difficult to treat because of the development of antibiotic resistance. Immunocompetent murine models of human UTI have been used to study pathogenesis and treatment but not for investigating resistance development after treatment with antibiotics. In this study, intravesical inoculation of uropathogenic Escherichia coli CFT073 in immunocompetent Balb/c mice was used as a model of human UTI. The value of the model in investigating antibiotic exposure on in vivo emergence of antibiotic resistance was examined. Experimentally infected mice were treated with 20 or 200 mg/kg ampicillin, 5 or 50 mg/kg ciprofloxacin, or 100 or 1000 mg/kg of fosfomycin. Ampicillin and ciprofloxacin were given twice daily at 8 h intervals, and fosfomycin was given once daily. Antibiotic treatment began 24 h after bacterial inoculation and ended after 72 h following the initial treatment. Although minimum inhibitory concentrations (MIC) for the experimental strain of E. coli were exceeded at peak concentrations in tissues and consistently in urine, low levels of bacteria persisted in tissues in all experiments. E. coli from bladder tissue, kidney, and urine grew on plates containing 1× MIC of antibiotic, but none grew at 3× MIC. This model is not suitable for studying emergent resistance but might serve to examine bacterial persistence.

The interaction of E. coli IHF protein with its specific binding sites

Cell ◽  
1989 ◽  
Vol 57 (5) ◽  
pp. 869-880 ◽  
Author(s):  
Chien-Chin Yang ◽  
Howard A. Nash
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
E Coli ◽  
Specific Binding Sites ◽  
Ihf Protein

scholarly journals Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator FlhD2C2

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1779-1788 ◽  
Author(s):  
Graham P. Stafford ◽  
Tomoo Ogi ◽  
Colin Hughes
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Class Ii ◽  
Promoter Regions ◽  
E Coli ◽  
Flagellar Genes

The gene hierarchy directing biogenesis of peritrichous flagella on the surface of Escherichia coli and other enterobacteria is controlled by the heterotetrameric master transcriptional regulator FlhD2C2. To assess the extent to which FlhD2C2 directly activates promoters of a wider regulon, a computational screen of the E. coli genome was used to search for gene-proximal DNA sequences similar to the 42–44 bp inverted repeat FlhD2C2 binding consensus. This identified the binding sequences upstream of all eight flagella class II operons, and also putative novel FlhD2C2 binding sites in the promoter regions of 39 non-flagellar genes. Nine representative non-flagellar promoter regions were all bound in vitro by active reconstituted FlhD2C2 over the K D range 38–356 nM, and of the nine corresponding chromosomal promoter–lacZ fusions, those of the four genes b1904, b2446, wzz fepE and gltI showed up to 50-fold dependence on FlhD2C2 in vivo. In comparison, four representative flagella class II promoters bound FlhD2C2 in the K D range 12–43 nM and were upregulated in vivo 30- to 990-fold. The FlhD2C2-binding sites of the four regulated non-flagellar genes overlap by 1 or 2 bp the predicted −35 motif of the FlhD2C2-activated σ 70 promoters, as is the case with FlhD2C2-dependent class II flagellar promoters. The data indicate a wider FlhD2C2 regulon, in which non-flagellar genes are bound and activated directly, albeit less strongly, by the same mechanism as that regulating the flagella gene hierarchy.

scholarly journals HOMOLOGOUS RECOMBINATION IN ESCHERICHIA COLI: DEPENDENCE ON SUBSTRATE LENGTH AND HOMOLOGY

Genetics ◽  
1986 ◽  
Vol 112 (3) ◽  
pp. 441-457 ◽  
Author(s):  
Ping Shen ◽  
Henry V Huang
Keyword(s):  
Dna Sequences ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
In Vivo Recombination ◽  
Recombinant Frequency ◽  
Genetic Length ◽  
Efficient Processing ◽  
Low Levels

ABSTRACT We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC  - cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.

scholarly journals Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzTPromoter

2016 ◽  
Vol 82 (14) ◽  
pp. 4350-4362 ◽  
Author(s):  
Ana Isabel Platero ◽  
Aroa López-Sánchez ◽  
Laura Tomás-Gallardo ◽  
Eduardo Santero ◽  
Fernando Govantes
Keyword(s):  
Complex Formation ◽  
Promoter Region ◽  
Atp Hydrolysis ◽  
Cyanuric Acid ◽  
Regulatory Protein ◽  
Dna Interaction ◽  
Integration Host Factor ◽  
Open Complex Formation

ABSTRACTPatzTis an internal promoter of theatzRSTUVWoperon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. PatzTis σNdependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety ofin vivoandin vitrogene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of PatzT. Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the PatzTpromoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC. Furthermore, repression requires precise rotational orientation of the AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between the two proteins on the promoter region. Further exploration of the antiactivation mechanism showed that although AtzR-dependent repression occurs prior to open complex formation, AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when bound to the PatzTpromoter region. Taken together, these results strongly suggest that PatzT-bound AtzR interacts with NtrC to prevent the coupling of NtrC-mediated ATP hydrolysis with the remodeling of the interactions between E-σNand PatzTthat lead to open complex formation.IMPORTANCEHere, we describe a unique mechanism by which the regulatory protein AtzR prevents the activation of the σN-dependent promoter PatzT. Promoters of this family are always positively regulated, but there are a few examples of overlapping negative regulation. The mechanism described here is highly unconventional and involves an interaction between the repressor and activator proteins to prevent the action of the repressor protein on the RNA polymerase-promoter complex.

Two adjacent C/EBP-binding sequences that participate in the cell-specific expression of the mouse serum amyloid A3 gene

1990 ◽  
Vol 10 (12) ◽  
pp. 6624-6631
Author(s):  
X X Li ◽  
J H Huang ◽  
H Y Rienhoff ◽  
W S Liao
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Serum Amyloid ◽  
Mouse Serum ◽  
Band Shift ◽  
Independent Manner ◽  
Specific Expression ◽  
Amyloid A

Serum amyloid A (SAA) is a major acute-phase protein synthesized primarily in the liver. Its expression, very low in normal animals, is increased several hundredfold following acute inflammation. To examine DNA sequences involved in liver-specific expression, 5'-flanking regions of the mouse SAA3 gene were analyzed by transient transfection, band shift, and DNase I protection assays. We found that a 56-bp fragment immediately 5' to the TATA box spanning the region -93 to -38 relative to the transcription start site was sufficient to confer liver cell-specific transcriptional activation onto a heterologous promoter in a dose-dependent and orientation-independent manner. This DNA fragment could form DNA-protein complexes with heat-stable nuclear proteins, and the complexes formed could be specifically competed for by excess oligomers corresponding to the C/EBP- or DBP-binding sites but not by binding sites for three other liver-specific factors, HNF1, HNF3, and HNF4. Footprint analysis using Hep3B nuclear extracts revealed two adjacent footprint regions within this 56-bp fragment, the distal region having at least fivefold-greater affinity than the proximal region. Identical footprint patterns were observed when purified recombinant C/EBP protein was used. These results indicated that binding of C/EBP to this 56-bp fragment plays an important role in vivo in enhancing expression of the mouse SAA3 gene in hepatocytes.

scholarly journals Integration of Global Regulation of Two Aromatic-Responsive σ54-Dependent Systems: a Common Phenotype by Different Mechanisms

2002 ◽  
Vol 184 (3) ◽  
pp. 760-770 ◽  
Author(s):  
Chun Chau Sze ◽  
Lisandro M. D. Bernardo ◽  
Victoria Shingler
Keyword(s):  
Promoter Region ◽  
Integration Host Factor ◽  
Target Component ◽  
Phosphotransferase System ◽  
Global Regulation ◽  
Regulatory Circuit ◽  
Regulatory Circuits ◽  
Rich Media ◽  
Two Factors

ABSTRACT Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related σ54-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding σ54 RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of σ54-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.

scholarly journals Involvement of the Cra Global Regulatory Protein in the Expression of the iscRSUA Operon, Revealed during Studies of Tricarballylate Catabolism in Salmonella enterica

2009 ◽  
Vol 191 (7) ◽  
pp. 2069-2076 ◽  
Author(s):  
Jeffrey A. Lewis ◽  
Jeffrey M. Boyd ◽  
Diana M. Downs ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Binding Sites ◽  
Salmonella Enterica ◽  
Promoter Region ◽  
Flavin Adenine Dinucleotide ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Cluster Assembly ◽  
Regulation Of Expression ◽  
Membrane Bound ◽  
Low Levels

ABSTRACT In Salmonella enterica, tricarballylate (Tcb) catabolism requires function of TcuB, a membrane-bound protein that contains [4Fe-4S] clusters and heme. TcuB transfers electrons from reduced flavin adenine dinucleotide in the Tcb dehydrogenase (TcuA) to electron acceptors in the membrane. We recently showed that functions needed to assemble [Fe-S] clusters (i.e., the iscRSUA-hscBA-fdx operon) compensate for the lack of ApbC during growth of an apbC strain on Tcb. ApbC had been linked to [Fe-S] cluster metabolism, and we showed that an apbC strain had decreased TcuB activity. Here we report findings that expand our understanding of the regulation of expression of the iscRSUA genes in Salmonella enterica. We investigated why low levels of glucose or other saccharides restored growth of an apbC strain on Tcb. Here we report the following findings. (i) A ≤1 mM concentration of glucose, fructose, ribose, or glycerol restores growth of an apbC strain on Tcb. (ii) The saccharide effect results in increased levels of TcuB activity. (iii) The saccharide effect depends on the global regulatory protein Cra. (iv) Putative Cra binding sites are present in the regulatory region of the iscRSUA operon. (v) Cra protein binds to all three sites in the iscRSUA promoter region in a concentration-dependent fashion. To our knowledge, this is the first report of the involvement of Cra in [Fe-S] cluster assembly.

scholarly journals Characterization of cis-Acting Sites Controlling Arginine Deiminase Gene Expression in Streptococcus gordonii

2006 ◽  
Vol 188 (3) ◽  
pp. 941-949 ◽  
Author(s):  
Lin Zeng ◽  
Yiqian Dong ◽  
Robert A. Burne
Keyword(s):  
Binding Sites ◽  
Promoter Region ◽  
Sequence Similarity ◽  
Acid Tolerance ◽  
Arginine Deiminase ◽  
Streptococcus Gordonii ◽  
Oral Diseases ◽  
Mobility Shift ◽  
Gel Mobility Shift

ABSTRACT The arginine deiminase system (ADS) is responsible for the production of ornithine, CO2, ammonia, and ATP from arginine. The ADS of the oral bacterium Streptococcus gordonii plays major roles in physiologic homeostasis, acid tolerance, and oral biofilm ecology. To further our understanding of the transcriptional regulation of the ADS (arc) operon, the binding of the ArcR transcriptional activator, which governs expression of the ADS in response to arginine, was investigated by DNase I protection and gel mobility shift assays. An ArcR binding sequence was found that was 27 bp in length and had little sequence similarity to binding sites of other arginine metabolism regulators. The presence of arginine at physiologically relevant concentrations enhanced the binding of ArcR to its target. Using cat fusions, various deletion and substitution mutations within the putative ArcR footprint were shown to cause dramatic reductions in expression from the arcA promoter in vivo, confirming that the 27-bp sequence is required for optimal expression and induction of the ADS by arginine. Mutation of two putative catabolite response elements (CREs) within the arc promoter region showed that both CREs contribute to catabolite repression. A thorough understanding of the regulation of the ADS in S. gordonii and related organisms is needed to develop ways to exploit arginine catabolism for the control of oral diseases. Identification of the ArcR and CcpA binding sites lays the foundation for a more complete understanding of the complex interactions of multiple regulatory proteins with elements in the arc promoter region.

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