Identification of Chlamydia trachomatis Genomic Sequences Recognized by Chlamydial Divalent Cation-Dependent Regulator A (DcrA)

ABSTRACT The Chlamydia trachomatis divalent cation-dependent regulator (DcrA), encoded by open reading frame CT296, is a distant relative of the ferric uptake regulator (Fur) family of iron-responsive regulators. Chlamydial DcrA specifically binds to a consensus Escherichia coli Fur box and is able to complement an E. coli Fur mutant. In this report, the E. coli Fur titration assay (FURTA) was used to locate chlamydial genomic sequences that are recognized by E. coli Fur. The predictive regulatory regions of 28 C. trachomatis open reading frames contained sequences functionally recognized by E. coli Fur; targets include components of the type III secretion pathway, elements involved in envelope and cell wall biogenesis, predicted transport proteins, oxidative defense enzymes, and components of metabolic pathways. Selected FURTA-positive sequences were subsequently examined for recognition by C. trachomatis DcrA using an electrophoretic mobility shift assay. The resultant data show that C. trachomatis DcrA binds to native chlamydial genomic sequences and, overall, substantiate a functional relationship between chlamydial DcrA and the Fur family of regulators.

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Salmonella enteritidis: AmpC Plasmid-Mediated Inducible β-Lactamase (DHA-1) with anampR Gene from Morganella morganii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.9.2352 ◽

1998 ◽

Vol 42 (9) ◽

pp. 2352-2358 ◽

Cited By ~ 89

Author(s):

Guilene Barnaud ◽

Guillaume Arlet ◽

Charlotte Verdet ◽

Olivier Gaillot ◽

Philippe H. Lagrange ◽

...

Keyword(s):

Dna Sequence ◽

Salmonella Enteritidis ◽

Diffusion Method ◽

Morganella Morganii ◽

Mobility Shift ◽

Reading Frame ◽

E Coli ◽

Amino Terminal ◽

Mobility Shift Assay ◽

Gel Mobility Shift

ABSTRACT DHA-1, a plasmid-mediated cephalosporinase from a single clinicalSalmonella enteritidis isolate, conferred resistance to oxyimino-cephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and this resistance was transferable toEscherichia coli HB101. An antagonism was observed between cefoxitin and aztreonam by the diffusion method. Transformation of the transconjugant E. coli strain with plasmid pNH5 carrying the ampD gene (whose product decreases the level of expression of ampC) resulted in an eightfold decrease in the MIC of cefoxitin. A clone with the same AmpC susceptibility pattern with antagonism was obtained, clone E. coliJM101(pSAL2-ind), and its nucleotide sequence was determined. It contained an open reading frame with 98.7% DNA sequence identity with the ampC gene of Morganella morganii. DNA sequence analysis also identified a gene upstream of ampCwhose sequence was 97% identical to the partial sequence of the ampR gene (435 bp) from M. morganii. The gene encoded a protein with an amino-terminal DNA-binding domain typical of transcriptional activators of the LysR family. Moreover, the intercistronic region between the ampC andampR genes was 98% identical to the corresponding region from M. morganii DNA. AmpR was shown to be functional by enzyme induction and a gel mobility-shift assay. An ampGgene was also detected in a Southern blot of DNA from the S. enteritidis isolate. These findings suggest that this inducible plasmid-mediated AmpC type β-lactamase, DHA-1, probably originated from M. morganii.

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Identification and characterization of the regulatory elements of the inducible acetamidase operon from Mycobacterium smegmatis

Canadian Journal of Microbiology ◽

10.1139/w06-147 ◽

2007 ◽

Vol 53 (5) ◽

pp. 599-606 ◽

Cited By ~ 2

Author(s):

Selvakumar Subbian ◽

Sujatha Narayanan

Keyword(s):

Mycobacterium Smegmatis ◽

Regulatory Elements ◽

Growth Conditions ◽

Open Reading Frames ◽

Reporter System ◽

Mobility Shift ◽

Crude Cell Lysate ◽

Multiple Promoters ◽

Promoter Probe ◽

Mobility Shift Assay

The highly inducible acetamidase promoter from Mycobacterium smegmatis has been used as a tool in the study of mycobacterial genetics. The 4.2 kb acetamidase operon contains four putative open reading frames (ORFs) (amiC, amiA, amiD, and amiS) upstream of the 1.2 kb acetamidase ORF (amiE). In this article, using electrophoretic mobility shift assay and promoter probe analyses with a lacZ reporter system, we show the position of three putative operators within the acetamidase operon in M. smegmatis. Results from these studies reinforce previous findings about the involvement of multiple promoters in the regulation of acetamidase gene expression. Each of the identified operators are positioned upstream of the respective promoter reported in previous studies. We also found that the crude cell lysate of M. smegmatis containing potential regulators, obtained from bacteria grown under inducing or noninducing conditions, binds to specific operators. The binding affinity of each operator with its cognate regulator is significantly different from the other. This supports not only the previous model of acetamidase gene regulation in M. smegmatis but also explains the role of these operators in controlling the expression of respective promoters under different growth conditions.

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Genetic Composition of the Bacillus subtilis SOS System

Journal of Bacteriology ◽

10.1128/jb.187.22.7655-7666.2005 ◽

2005 ◽

Vol 187 (22) ◽

pp. 7655-7666 ◽

Cited By ~ 111

Author(s):

Nora Au ◽

Elke Kuester-Schoeck ◽

Veena Mandava ◽

Laura E. Bothwell ◽

Susan P. Canny ◽

...

Keyword(s):

Dna Damage ◽

Bacillus Subtilis ◽

Binding Sites ◽

Consensus Sequence ◽

Transcriptional Response ◽

Open Reading Frames ◽

Mobility Shift ◽

E Coli ◽

Recombination Protein ◽

Lexa Binding

ABSTRACT The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5′-CGAACATATGTTCG-3′. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.

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Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon

Journal of Bacteriology ◽

10.1128/jb.00741-09 ◽

2009 ◽

Vol 192 (1) ◽

pp. 77-85 ◽

Cited By ~ 39

Author(s):

Lydgia A. Jackson ◽

Thomas F. Ducey ◽

Michael W. Day ◽

Jeremy B. Zaitshik ◽

Joshua Orvis ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Transcriptional Control ◽

Cell Communication ◽

Essential Element ◽

Open Reading Frames ◽

Promoter Regions ◽

Mobility Shift ◽

Sequence Logos ◽

Mobility Shift Assay

ABSTRACT To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies—temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)—to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.

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Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R

International Journal of Molecular Sciences ◽

10.3390/ijms22189805 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9805

Author(s):

Dharmender K. Gahlot ◽

Gyles Ifill ◽

Sheila MacIntyre

Keyword(s):

Yersinia Pestis ◽

Vaccine Development ◽

Recombinant Expression ◽

Bacterial Pathogen ◽

Clinical Value ◽

Mobility Shift ◽

E Coli ◽

Capsular Antigen ◽

Mobility Shift Assay

The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis.

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Moraxella catarrhalis Expresses an Unusual Hfq Protein

Infection and Immunity ◽

10.1128/iai.01652-07 ◽

2008 ◽

Vol 76 (6) ◽

pp. 2520-2530 ◽

Cited By ~ 24

Author(s):

Ahmed S. Attia ◽

Jennifer L. Sedillo ◽

Wei Wang ◽

Wei Liu ◽

Chad A. Brautigam ◽

...

Keyword(s):

Moraxella Catarrhalis ◽

Cell Envelope ◽

Stress Sensitivity ◽

Wild Type ◽

Mobility Shift ◽

Altered Expression ◽

Reading Frame ◽

E Coli ◽

Different Types

ABSTRACT The Hfq protein is recognized as a global regulatory molecule that facilitates certain RNA-RNA interactions in bacteria. BLAST analysis identified a 630-nucleotide open reading frame in the genome of Moraxella catarrhalis ATCC 43617 that was highly conserved among M. catarrhalis strains and which encoded a predicted protein with significant homology to the Hfq protein of Escherichia coli. This protein, containing 210 amino acids, was more than twice as large as the Hfq proteins previously described for other bacteria. The C-terminal half of the M. catarrhalis Hfq protein was very hydrophilic and contained two different types of amino acid repeats. A mutation in the M. catarrhalis hfq gene affected both the growth rate of this organism and its sensitivity to at least two different types of stress in vitro. Provision of the wild-type M. catarrhalis hfq gene in trans eliminated these phenotypic differences in the hfq mutant. This M. catarrhalis hfq mutant exhibited altered expression of some cell envelope proteins relative to the wild-type parent strain and also had a growth advantage in a continuous flow biofilm system. The presence of the wild-type M. catarrhalis hfq gene in trans in an E. coli hfq mutant fully reversed the modest growth deficiency of this E. coli mutant and partially reversed the stress sensitivity of this E. coli mutant to methyl viologen. The use of an electrophoretic mobility shift assay showed that this M. catarrhalis Hfq protein could bind RNA derived from a gene whose expression was altered in the M. catarrhalis hfq mutant.

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Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli

Biochemistry and Cell Biology ◽

10.1139/o90-017 ◽

1990 ◽

Vol 68 (1) ◽

pp. 123-137 ◽

Cited By ~ 37

Author(s):

Krishna G. Peri ◽

Hughes Goldie ◽

E. Bruce Waygood

Keyword(s):

Escherichia Coli ◽

Genomic Library ◽

Sodium Dodecyl ◽

Open Reading Frames ◽

Open Reading Frame ◽

Gene Products ◽

Reading Frame ◽

E Coli ◽

Palindromic Sequences ◽

Repetitive Extragenic Palindromic

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate – polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62 000; nagA, 45 000; nagB, 29 000. In addition, another product of Mr 44 000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weights (MWs) of the gene products derived from the sequence are as follows: nagA, 40 954; nagB, 29 657; nagC, 44 664; nagE, 68 356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27 181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: [Formula: see text]Key words: N-acetylglucosamine, N-acetylglucosamine-6-P deacetylase, glucosamine-6-P isomerase, repetitive extragenic palindromic sequences, catabolite repression.

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A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in Pseudomonas sp. Strain U2

Journal of Bacteriology ◽

10.1128/jb.180.9.2522-2530.1998 ◽

1998 ◽

Vol 180 (9) ◽

pp. 2522-2530 ◽

Cited By ~ 157

Author(s):

Sergio L. Fuenmayor ◽

Mark Wild ◽

Alastair L. Boyes ◽

Peter A. Williams

Keyword(s):

Single Gene ◽

Open Reading Frames ◽

Open Reading Frame ◽

Gas Chromatography Mass Spectrometry ◽

Nucleotide Sequencing ◽

Naphthalene Dioxygenase ◽

Sequence Comparisons ◽

Reading Frame ◽

Selective Enrichment ◽

E Coli

ABSTRACT Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kbBamHI fragment. The genes for the naphthalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb(for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for nagB (for naphthalenecis-dihydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAbwere two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant Escherichia coli strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated nagG, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designatednagH, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together inE. coli, nagG, nagH, andnagAb, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts ofnagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although E. coli appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene ordernagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.

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Chlamydia trachomatis Serovar L2 Can Utilize Exogenous Lipoic Acid through the Action of the Lipoic Acid Ligase LplA1

Journal of Bacteriology ◽

10.1128/jb.00717-10 ◽

2010 ◽

Vol 192 (23) ◽

pp. 6172-6181 ◽

Cited By ~ 8

Author(s):

Aishwarya V. Ramaswamy ◽

Anthony T. Maurelli

Keyword(s):

Chlamydia Trachomatis ◽

Lipoic Acid ◽

Open Reading Frames ◽

Sequence Motifs ◽

Gene Encoding ◽

E Coli ◽

Genes Encoding ◽

Surrogate Host ◽

Lipoate Synthase

ABSTRACT Lipoic acid is an essential protein bound cofactor that is vital for the functioning of several important enzymes involved in central metabolism. Genomes of all sequenced chlamydiae show the presence of two genes encoding lipoic acid ligases and one gene encoding a lipoate synthase. However, the roles of these proteins in lipoic acid utilization or biosynthesis have not yet been characterized. The two distinct lipoic acid ligases in Chlamydia trachomatis serovar L2, LplA1Ct and LplA2Ct (encoded by the open reading frames ctl0537 and ctl0761) display moderate identity with Escherichia coli LplA (30 and 27%, respectively) but possess amino acid sequence motifs that are well conserved among all lipoyl protein ligases. The putative lipoic acid synthase LipACt, encoded by ctl0815, is ca. 43% identical to the E. coli LipA homolog. We demonstrate here the presence of lipoylated proteins in C. trachomatis serovar L2 and show that the lipoic acid ligase LplA1Ct is capable of utilizing exogenous lipoic acid for the lipoylation Therefore, host-derived lipoic acid may be important for intracellular growth and development. Based on genetic complementation in a surrogate host, our study also suggests that the C. trachomatis serovar L2 LipA homolog may not be functional in vivo.

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1-Deoxy-d-Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 inRhodobacter capsulatus

Journal of Bacteriology ◽

10.1128/jb.183.1.1-11.2001 ◽

2001 ◽

Vol 183 (1) ◽

pp. 1-11 ◽

Cited By ~ 59

Author(s):

Frederick M. Hahn ◽

Lisa M. Eubanks ◽

Charles A. Testa ◽

Brian S. J. Blagg ◽

Jonathan A. Baker ◽

...

Keyword(s):

Rhodobacter Capsulatus ◽

Open Reading Frames ◽

Reading Frame ◽

Methylerythritol Phosphate Pathway ◽

E Coli ◽

Methylerythritol Phosphate ◽

Steady State Kinetic ◽

Photosynthetic Machinery ◽

State Kinetic ◽

Phosphate Synthase

ABSTRACT In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and d-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase. InRhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-d-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-d-xylulose 5-phosphate synthase A and B, containing a His6 tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-d-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-d-xylulose 5-phosphate synthase A and B: Km pyruvate = 0.61 and 3.0 mM, Km d-glyceraldehyde 3-phosphate = 150 and 120 μM, andV max = 1.9 and 1.4 μmol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-d-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.

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