Operon Fusion Research Articles

A triple-sugar regulated Salmonella vaccine protects against Clostridium perfringens-induced necrotic enteritis in broiler chickens

Gram-positive Clostridium perfringens type G, the causative agent of necrotic enteritis (NE), has gained more attention in the poultry industry due to governmental restrictions on the use of growth-promoting antibiotics in poultry feed. Our previous work has proved that regulated delayed lysis Salmonella vaccines delivering a plasmid encoding an operon fusion of the nontoxic C-terminal adhesive part of alpha toxin and a GST-NetB toxin fusion were able to elicit significant protective immunity in broilers against C. perfringens challenge. We recently improved our S. Typhimurium antigen delivery vaccine strain by integrating a rhamnose-regulated O-antigen synthesis gene enabling a triple-sugar regulation system to control virulence, antigen-synthesis and lysis in vivo traits. The strain also includes a ΔsifA mutation that was previously shown to increase the immunogenicity of and level of protective immunity induced by Salmonella vectored influenza and Eimeria antigens. The new antigen-delivery vaccine vector system confers on the vaccine strain a safe profile and improved protection against C. perfringens challenge. The strain with the triple-sugar regulation system delivering a regulated lysis plasmid pG8R220 encoding the PlcC and GST-NetB antigens protected chickens at a similar level observed in antibiotic-treated chickens. Feed conversion and growth performance were also similar to antibiotic-treated chickens. These studies made use of a severe C. perfringens challenge with lesion formation and mortality enhanced by pre-exposure to Eimeria maxima oocysts. The vaccine achieved effectiveness through three different immunization routes, oral, spray and in drinking water. The vaccine has a potential for application in commercial hatcher and broiler-rearing conditions.

Strain-specific transcriptional and posttranscriptional regulation of heat-labile toxin expression by enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) represents one of the most important etiological agents of diarrhea in developing countries and characteristically produces at least one of two enterotoxins: heat-labile toxin (LT) and heat-stable toxin (ST). It has been previously shown that the production and release of LT by human-derived ETEC strains are variable. Although the natural genetic polymorphisms of regulatory sequences of LT-encoding (eltAB) genes may explain the variable production of LT, the knowledge of the transcriptional and posttranscriptional aspects affecting LT expression among ETEC strains is not clear. To further understand the factors affecting LT expression, we evaluated the impact of the natural polymorphism in noncoding regulatory sequences of eltAB among clinically derived ETEC strains. Sequence analyses of seven clinically derived strains and the reference strain H10407 revealed polymorphic sites at both the promoter and upstream regions of the eltAB operon. Operon fusion assays with GFP revealed that specific nucleotide changes in the Pribnow box reduce eltAB transcription. Nonetheless, the total amounts of LT produced by the tested ETEC strains did not strictly correspond to the detected LT-specific mRNA levels. Indeed, the stability of LT varied according to the tested strain, indicating the presence of posttranscriptional mechanisms affecting LT expression. Taken together, our results indicate that the production of LT is a strain-specific process and involves transcriptional and posttranscriptional mechanisms that regulate the final amount of toxin produced and released by specific strains.Electronic supplementary materialThe online version of this article (10.1007/s42770-020-00231-2) contains supplementary material, which is available to authorized users.

Salmonella Enteritidis와 Salmonella Gallinarum의 세균막 스트레스를 인식하는 spy-gfp 오페론 융합체

Emergence of drug resistant strains of Salmonella enterica threatens milk processing and related dairy industries, thereby increasing the need for development of new anti-bacterials. Developments of antibacterial drugs are largely aimed to target the bacterial envelope, but screening their efficacy on bacterial envelope is laborious. This study presents a potential biosensor for envelope-specific stress in which a gfp reporter gene fused to spy gene encoding a periplasmic chaperone protein Spy (spheroplast protein y) that can sense envelope stress signals transduced by two major two-component signal transduction systems BaeSR and CpxAR in Salmonella enterica serovars Enteritidis and S. Gallinarum. Using spy-gfp operon fusions in S. Enterititis and S. Gallinarum, we found that spy transcription in both serovars was greatly induced when Salmonella cells were forming the spheroplast and were treated with ethanol or a membrane-disrupting antibiotic polymyxin B. These envelope stress-specific inductions of spy transcription were abrogated in mutant Salmonella lacking either BaeR or CpxR. Results illustrate that induction of Spy expression can be efficiently triggered by two-component signal transduction systems sensing envelope stress conditions, and thereby suggest that monitoring the spy transcription by spy-gfp operon fusions would be helpful to determine if developing antimicrobials can damage envelopes of S. Enteritidis and S. Gallinarum.

DksA involvement in transcription fidelity buffers stochastic epigenetic change.

DksA is an auxiliary transcription factor that interacts with RNA polymerase and influences gene expression. Depending on the promoter, DksA can be a positive or negative regulator of transcription initiation. Moreover, DksA has a substantial effect on transcription elongation where it prevents the collision of transcription and replication machineries, plays a key role in maintaining transcription elongation when translation and transcription are uncoupled and has been shown to be involved in transcription fidelity. Here, we assessed the role of DksA in transcription fidelity by monitoring stochastic epigenetic switching in the lac operon (with and without an error-prone transcription slippage sequence), partial phenotypic suppression of a lacZ nonsense allele, as well as monitoring the number of lacI mRNA transcripts produced in the presence and absence of DksA via an operon fusion and single molecule fluorescent in situ hybridization studies. We present data showing that DksA acts to maintain transcription fidelity in vivo and the role of DksA seems to be distinct from that of the GreA and GreB transcription fidelity factors.

Tn5/7-lux: a versatile tool for the identification and capture of promoters in gram-negative bacteria.

BackgroundThe combination of imaging technologies and luciferase-based bioluminescent bacterial reporter strains provide a sensitive and simple non-invasive detection method (photonic bioimaging) for the study of diverse biological processes, as well as efficacy of therapeutic interventions, in live animal models of disease. The engineering of bioluminescent bacteria required for photonic bioimaging is frequently hampered by lack of promoters suitable for strong, yet stable luciferase gene expression.ResultsWe devised a novel method for identification of constitutive native promoters in Gram-negative bacteria. The method is based on a Tn5/7 transposon that exploits the unique features of Tn5 (random transposition) and Tn7 (site-specific transposition). The transposons are designed such that Tn5 transposition will allow insertion of a promoter-less bacterial luxCDABE operon downstream of a bacterial gene promoter. Cloning of DNA fragments from luminescent isolates results in a plasmid that replicates in pir+ hosts. Sequencing of the lux-chromosomal DNA junctions on the plasmid reveals transposon insertion sites within genes or operons. The plasmid is also a mini-Tn7-lux delivery vector that can be used to introduce the promoter-lux operon fusion into other derivatives of the bacterium of interest in an isogenic fashion. Alternatively, promoter-containing sequences can be PCR-amplified from plasmid or chromosomal DNA and cloned into a series of accompanying mini-Tn7-lux vectors. The mini-Tn5/7-lux and mini-Tn7-lux vectors are equipped with diverse selection markers and thus applicable in numerous Gram-negative bacteria. Various mini-Tn5/7-lux vectors were successfully tested for transposition and promoter identification by imaging in Acinetobacter baumannii, Escherichia coli, and Burkholderia pseudomallei. Strong promoters were captured for lux expression in E. coli and A. baumannii. Some mini-Tn7-lux vectors are also equipped with attB sites for swapping of the lux operon with other reporter genes using Gateway technology.ConclusionsAlthough mini-Tn5-lux and mini-Tn7-lux elements have previously been developed and used for bacterial promoter identification and chromosomal insertion of promoter-lux gene fusions, respectively, the newly developed mini-Tn5/7-lux and accompanying accessory plasmids streamline and accelerate the promoter discovery and bioluminescent strain engineering processes. Availability of vectors with diverse selection markers greatly extend the host-range of promoter probe and lux gene fusion vectors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0354-3) contains supplementary material, which is available to authorized users.

The influence of CsgD on the expression of genes of folate metabolism and hmp in Escherichia coli K-12

The csgD gene codes for the regulatory protein CsgD. CsgD upregulates the synthesis of the adhesive fimbriae, curli, that are important for biofilm formation and downregulates flagellar synthesis. We compared the expression of genes involved in folate metabolism and a gene (hmp) in strains with an intact csgD gene and with a deletion in csgD. The hmp gene codes a flavohemoglobin that inactivates nitric oxide. Expression was monitored by measuring light production from single copy lux operon fusions. At late times of growth, expression of genes responsible for methylene tetrahydrofolate synthesis (glyA and gcvTHP) and formyltetrahydrofolate recycling (purU) was higher in cells with CsgD than those without. In contrast, expression of hmp was lower in the presence of CsgD throughout the period monitored. We used a novel defined medium which should assist in defining nutritional factors that contribute to curli formation.

Identification of PhoB binding sites of the yibD and ytfK promoter regions in Escherichia coli

By using a lacZ operon fusion genomic library of the Escherichia coli 0157:H7 Sakai, we identified phosphate-starvation-inducible (psi) promoters located upstream of the yibD and ytfK genes. They have been previously proposed to belong to the phosphate regulon (pho regulon) by Beak and Lee (2006), based on the DNA array and in vivo transcriptional experiments. However, the direct interaction of these promoters with the activator protein of the pho regulon, PhoB, has not been determined. We determined the binding regions of PhoB in these promoter regions by DNase I footprinting. Both regions contained two pho boxes similar to the consensus sequence for PhoB binding.

T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ-ProA-ProH route is responsible for the production of proline as an osmoprotectant, and the ProB-ProA-ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB-treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine.

Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment

The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 harbors a subset of genes that are expressed specifically on plant surfaces. The function of these genes is central to the ecological success of SBW25, but their study poses significant challenges because no phenotype is discernable in vitro. Here, we describe a genetic strategy with general utility that combines suppressor analysis with IVET (SPyVET) and provides a means of identifying regulators of niche-specific genes. Central to this strategy are strains carrying operon fusions between plant environment-induced loci (EIL) and promoterless 'dapB. These strains are prototrophic in the plant environment but auxotrophic on laboratory minimal medium. Regulatory elements were identified by transposon mutagenesis and selection for prototrophs on minimal medium. Approximately 10(6) mutants were screened for each of 27 strains carrying 'dapB fusions to plant EIL and the insertion point for the transposon determined in approximately 2,000 putative regulator mutants. Regulators were functionally characterized and used to provide insight into EIL phenotypes. For one strain carrying a fusion to the cellulose-encoding wss operon, five different regulators were identified including a diguanylate cyclase, the flagella activator, FleQ, and alginate activator, AmrZ (AlgZ). Further rounds of suppressor analysis, possible by virtue of the SPyVET strategy, revealed an additional two regulators including the activator AlgR, and allowed the regulatory connections to be determined.

Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of σs activity and levels

Upon environmental changes, bacteria reschedule gene expression by directing alternative sigma factors to core RNA polymerase (RNAP). This sigma factor switch is achieved by regulating relative amounts of alternative sigmas and by decreasing the competitiveness of the dominant housekeeping sigma(70). Here we report that during stationary phase, the unorthodox Crl regulator supports a specific sigma factor, sigma(S) (RpoS), in its competition with sigma(70) for core RNAP by increasing the formation of sigma(S)-containing RNAP holoenzyme, Esigma(S). Consistently, Crl has a global regulatory effect in stationary phase gene expression exclusively through sigma(S), that is, on sigma(S)-dependent genes only. Not a specific promoter motif, but sigma(S) availability determines the ability of Crl to exert its function, rendering it of major importance at low sigma(S) levels. By promoting the formation of Esigma(S), Crl also affects partitioning of sigma(S) between RNAP core and the proteolytic sigma(S)-targeting factor RssB, thereby playing a dual role in fine-tuning sigma(S) proteolysis. In conclusion, Crl has a key role in reorganising the Escherichia coli transcriptional machinery and global gene expression during entry into stationary phase.

YtxR, a Conserved LysR-Like Regulator That Induces Expression of Genes Encoding a Putative ADP-Ribosyltransferase Toxin Homologue inYersinia enterocolitica

Yersinia enterocolitica causes human gastroenteritis, and many isolates have been classified as either "American" or "non-American" strains based on their geographic prevalence and virulence properties. In this study we describe identification of a transcriptional regulator that controls expression of the Y. enterocolitica ytxAB genes. The ytxAB genes have the potential to encode an ADP-ribosylating toxin with similarity to pertussis toxin. However, a ytxAB null mutation did not affect virulence in mice. Nevertheless, the ytxAB genes are conserved in many Y. enterocolitica strains. Interestingly, American and non-American strains have different ytxAB alleles encoding proteins that are only 50 to 60% identical. To obtain further insight into the ytxAB locus, we investigated whether it is regulated as part of a known or novel regulon. Transposon mutagenesis identified a LysR-like regulator, which we designated YtxR. Expression of ytxR from a nonnative promoter increased Phi(ytxA-lacZ) operon fusion expression up to 35-fold. YtxR also activated expression of its own promoter. DNase I footprinting showed that a His(6)-YtxR fusion protein directly interacted with the ytxA and ytxR control regions at similar distances upstream of their probable transcription initiation sites, identified by primer extension. Deletion analysis demonstrated that removal of the regions protected by His(6)-YtxR in vitro eliminated YtxR-dependent induction in vivo. The ytxAB locus is not present in most Yersinia species. In contrast, ytxR is conserved in multiple Yersinia species, as well as in the closely related organisms Photorhabdus luminescens and Photorhabdus asymbiotica. These observations suggest that YtxR may play a conserved role involving regulation of other genes besides ytxAB.

Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable beta-galactosidase.

Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable beta-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3'- and 5'-end of the amyE gene (encoding alpha-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the beta-lactamase-encoding gene. Whereas one vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated.

Regulation of fumarase (fumB) gene expression in Escherichia coli in response to oxygen, iron and heme availability: role of the arcA, fur, and hemA gene products.

Three distinct fumarases, FumA, FumB and FumC, have been reported in Escherichia coli. While the fumA and fumC gene products are expressed under aerobic cell growth conditions, the FumB fumarase appears to be more abundant during anaerobic growth. To study the transcriptional regulation of the fumB gene, a fumB-lacZ operon fusion was constructed and analyzed in a single copy under a variety of cell culture conditions. Expression of fumB-lacZ was fourfold higher under anaerobic than aerobic growth conditions. This anaerobic response is modulated by the ArcA and Fnr proteins, which function independently as anaerobic activators of fumB gene expression. Cellular iron limitation in a fur mutant caused fumB-lacZ expression to decrease sevenfold while cellular heme limitation decreased fumB gene expression twofold. In addition, fumB-lacZ expression was shown to vary depending on the DNA superhelicity. This study further delineates the regulation of the fumB gene in cell growth.

Expression of the heme biosynthetic pathway genes hemCD, hemH, hemM, and hemA of Escherichia coli.

Little is known about the control of latter steps of heme biosynthesis in Escherichia coli. In this study we examined the transcriptional regulation of genes that encode two intermediate heme pathway enzymes, porphobilinogen deaminase (hemC) and uroporphyrinogen III cosynthase (hemD), and the final enzyme of the pathway, ferrochelatase (hemH). We also reexamined the regulation of hemA and the gene located immediately upstream of hemA, hemM. The regulatory regions of hemC, hemH, hemA and hemM were fused to lacZ. The resultant operon fusions were inserted into the E. coli chromosome in single copy and expression monitored under conditions of oxygen and heme limitation. Expression of hemM appeared constitutive under the conditions tested here. In contrast, expression of hemCD, hemH and hemA were shown to be mildly regulated in response to heme availability. Thus, transcription of four of the nine genes of the E. coli heme pathway appears to be only mildly regulated in response to heme limitation.

Identification of yebG as a DNA damage-inducible Escherichia coli gene.

The nucleotide sequence upstream of the Escherichia coli yebG gene presents features similar to those found in SOS system regulatory sites (putative SOS box, -10 and -35 promoter boxes and a ribosome binding site). Operon fusion assays demonstrate now that this region controls transcription in a recA-, lexA-dependent way and that the reporter gene expression is inducible by DNA damage consequent to mitomycin C treatment. Increased expression does not result from an increase in plasmid copy number. These results indicate that yebG is a novel SOS regulon gene. The yebG product is predicted to be a 96 amino acid residue, 10.7 kDa protein whose function is not yet known. Unlike other SOS genes, the construct carrying the yebG regulatory region is not stationary phase inducible.

Development of a Functional Genomics Platform for Sinorhizobium meliloti : Construction of an ORFeome

The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.

Improved System for Construction and Analysis of Single-Copy β-Galactosidase Operon Fusions in Yersinia enterocolitica

We report a significantly improved system for studying single-copy lacZ operon fusions in Yersinia enterocolitica: a simple procedure for the stable integration of lacZ operon fusions into the ara locus and a strain with a deletion mutation that abolishes the low level of endogenous beta-galactosidase activity.

Regulation of the Ubiquinone (Coenzyme Q) Biosynthetic Genes ubiCA in Escherichia coli

Ubiquinone (Coenzyme Q) is an essential component of bacterial respiratory chains. The first committed step in the biosynthetic pathway is the formation of 4-hydroxybenzoate from chorismate by the enzyme chorismate pyruvate-lyase encoded by the ubiC gene. The 4-hydroxybenzoate is prenylated by 4-hydroxybenzoate octaprenyltransferase encoded by the ubiA gene. The two genes are linked at 91.5 min in the Escherichia coli chromosome. To study the regulation, operon fusions were constructed between these two genes and the lacZ gene. The fusions were introduced into the chromosome as a single copy at the lambda attachment site. Expression of beta-galactosidase was determined in strains carrying the operon fusions ubiC'-lacZ(+) ubiCA'-lacZ(+), and ubiA'-lacZ(+). In glycerol media, the highest level of expression was observed with the operon fusion ubiC'-lacZ(+). Compared with the ubiC'-lacZ(+), the ubiCA'-lacZ(+) operon fusion showed 26% of the activity while the ubiA'-lacZ(+) operon fusion had an activity of 1%. Thus, the ubiC gene is regulated by the upstream promoter while the ubiA gene lacks its own promoter. The effect of fermentable and oxidizable carbon sources on the expression of ubiC'-lacZ(+) was determined. The expression was low in the case of a fermentable carbon source, glucose, while in the presence of oxidizable carbon sources the expression increased 2- to 3-fold. When the expression of ubiC'-lacZ(+) and ubiCA'-lacZ(+) operon fusions were compared under a wide variety of conditions, the levels of beta-galactosidase varied coordinately, suggesting that the ubiCA genes are organized into an operon. The variations in transcription of the operon under different nutritional conditions and in the regulatory mutants, arcA, fnr, and narXL are presented.

The Metal Permease ZupT from Escherichia coli Is a Transporter with a Broad Substrate Spectrum

The Escherichia coli zupT (formerly ygiE) gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An E. coli strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if zupT was expressed. Heterologous expression of Arabidopsis thaliana ZIP1 could also alleviate iron deficiency in this E. coli strain, as could expression of indigenous mntH or feoABC. Transport studies with intact cells showed that ZupT facilitates uptake of 55Fe2+ similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using 57Co2+. Expression of zupT rendered E. coli cells hypersensitive to Co2+ and sensitive to Mn2+. ZupT did not appear to be metal regulated: expression of a Phi(zupT-lacZ) operon fusion indicated that zupT is expressed constitutively at a low level.

The kgmB gene, encoding ribosomal RNA methylase from Streptomyces tenebrarius, is autogenously regulated

The KgmB methylase (the kanamycin-gentamicin resistance methylase from Streptomyces tenebrarius) acts at G-1405 of 16S rRNA within the sequence CGUCA that is also found 6 bp in front of ribosomal binding site of the kgmB gene. The kgmBColon, two colonslacZ gene and operon fusions were used in order to test for translational autoregulation of kgmB gene. Overexpression of kgmB either in cis or in trans drastically decreased the level of expression of the fusion protein. However, mutagenesis eliminated any role for the CGUCA sequence in translational autoregulation. Hence, the role of second putative regulatory sequence (CGCCC) that was shown to be involved in regulation of another methylase, Sgm (sisomicin-gentamicin methylase gene from Micromonospora zionensis) was examined. It was shown that the Sgm methylase can also decrease the level of expression of the kgmBColon, two colonslacZ fusion protein.