lacZ Promoter Research Articles

Cloning of cellulase gene using metagenomic approach of soils collected from Wadi El Natrun, an extremophilic desert valley in Egypt

BackgroundWadi El Natrun microorganisms have been considered as a new resource for natural products due to its extreme condition of salinity and alkalinity. Therefore, this study was devoted to generate metagemic library from soils collected from such an extreme environment in order to clone a novel cellulase for physique industrial applications. ResultsTotal soil-DNA was successfully extracted, and then digested by different restriction enzymes. Purified fragments ranged ~ 200–6500 bp were ligated and were cloned into plasmid cloning vector (pUC19) by using Escherichia coli DH5α (E. coli) host cells. A constructed metagenomic library composed of 270 clones was screened on carboxymethylcellulose (CMC) agar plate where the active clones had been characterized by the formation of the yellowish halo zone. Thereafter, clone 1 was selected as the most active as being based on cellulase activity quantification (19 μ/ml). Plasmid related to clone 1 encoded cellSNSY gene of approximately 1.5 kb was subjected to molecular characterization; the obtained partial sequence of 861 bps encoded 287 amino acids showing 76% similarity to the endoglucanase gene of Bacillus amyloliquefaciens. The recombinant cellSNSY was expressed under lacz promoter at 1 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG), giving 21 μ/ml cellulase after ~ 27 h. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and an activity staining of the recombinant cellSNSY which revealed an active band with a molecular mass ~ 59 kDa appeared in the induced sample. The maximum enzyme activity of crude cellSNSY was observed at 45 °C and for a pH of 8.5. Interestingly, the enzyme activity was slightly inhibited by ethylenediamine tetraacetic acid (EDTA) and methanol. It showed high resistance to the tested heavy metals and the surfactant which ordered Zn> (SDS,Fe)>Mn>Cu. ConclusionsThis study established an easy and a skillful way to clone/express a new found cellulase gene(s) under lacZ promoter. The isolated recombinant cellSNSY showed 76% similarity to endoglucanase gene, and the enzyme showed tolerance to the mostly tested agents including heavy metals, surfactant, solvents, and EDTA. Additionally, the studied recombinant showed a high stability up to 55 °C and for alkaline pH 8.5. These features make it an ample and viable for many applications.

Sinorhizobium medicae WSM419 Genes That Improve Symbiosis between Sinorhizobium meliloti Rm1021 and Medicago truncatula Jemalong A17 and in Other Symbiosis Systems.

Some soil bacteria, called rhizobia, can interact symbiotically with legumes, in which they form nodules on the plant roots, where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobium-plant combinations can differ in how successful this symbiosis is: for example, Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17, but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. coli lacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA (increased symbiotic effectiveness), had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split-root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant-induced resistance to rhizobial infection, suggesting an interaction with the plant's regulation of nodule formation. IMPORTANCE Legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle. Symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach identifies new genes that may more generally contribute to symbiotic productivity.

Construction and application of a Xanthomonas campestris CGMCC15155 strain that produces white xanthan gum

In the industrial production of xanthan gum using Xanthomonas campestris CGMCC15155, large amounts of ethanol are required to extract xanthan gum from the fermentation broth and remove xanthomonadin impurities. To reduce the amount of ethanol and the overall production cost of xanthan gum, a xanthomonadin‐deficient strain of CGMCC15155 was constructed by inserting the Vitreoscilla globin (vgb) gene, under the control of the LacZ promoter, into the region of the pigA gene, which is involved in xanthomonadin synthesis. The insertion of vgb inactivated pigA, resulting in the production of white xanthan gum. The lack of xanthomonadins resulted in a decreased yield of xanthan gum. However, the expression product of vgb gene, VHb, could increase the metabolism of X. campestris, which allowed the production of xanthan gum to reach wild‐type levels in the engineered strain. The yield, molecular weight, and rheological properties of the xanthan gum synthesized by the engineered and wild‐type bacteria were essentially the same. When the same volume of ethanol was used, the whiteness values of the xanthan gum extracted from engineered and wild‐type bacteria were 65.20 and 38.17, respectively. To extract xanthan gum with the same whiteness, three and seven times the fermentation volume of ethanol was required for the engineered and wild‐type strains, respectively. Thus, the engineered train reduced the requirement for ethanol in xanthan gum production by 133.3%. The results demonstrated that the engineered bacteria used less ethanol, thus reducing the downstream processing cost in xanthan gum production.

Improved heterologous expression of the membrane-bound quinoprotein quinate dehydrogenase from Gluconobacter oxydans

Gluconobacter oxydans produces 3-dehydroquinate by oxidation of quinate through a reaction catalyzed by the quinate dehydrogenase (QDH), membrane-bound, pyrroloquinoline quinone (PQQ)-dependent dehydrogenase. We previously reported the nucleotide and deduced amino acid sequence of QDH and constructed a heterologous expression system of QDH in Pseudomonas sp. (A.S. Vangnai, W. Promden, W. De-Eknamkul, K. Matsushita, H. Toyama, Biochemistry (Moscow) 75:452–459, 2010). Through this study, we aim to update the sequences of QDH and improve the heterologous expression of QDH in Gluconobacter strains using a broad-host-range plasmid. Expression of QDH using a plasmid containing a long 5′-UTR was higher than that using a plasmid with a short 5′-UTR. In addition, the usage of the putative promoter region of the membrane-bound, alcohol dehydrogenase (ADH) of Gluconobacter resulted in higher expression levels compared to the usage of the lacZ promoter. Base substitution experiments allowed to identify the correct TTG initiation codon between two possibilities, and the result of these experiments were consistent with the N-terminal amino acid sequence of the expressed QDH. However, change of the TTG codon to ATG did not increase QDH expression. Therefore, the optimal plasmid for QDH expression included the structural gene with a long 5′-UTR and the ADH promoter. Cell membrane of the recombinant Gluconobacter strain presented approximately 10-times higher specific QDH activity than that observed in the wild-type strain.

Expression of the arsenite oxidation regulatory operon in Rhizobium sp. str. NT-26 is under the control of two promoters that respond to different environmental cues.

Rhizobium sp. str. NT‐26 is a Gram‐negative facultative chemolithoautotrophic arsenite oxidizer that has been used as a model organism to study various aspects of arsenite oxidation including the regulation of arsenite oxidation. The three regulatory genes, aioX, aioS, and aioR, are cotranscribed when NT‐26 was grown in the presence or absence of arsenite. The aioXSR operon is upregulated in stationary phase but not by the presence of arsenite in the growth medium. The two transcription start sites upstream of aioX were determined which led to the identification of two promoters, the housekeeping promoter RpoD and the growth‐phase‐dependent promoter RpoE2. Promoter–lacZ fusions confirmed their constitutive and stationary phase expressions. The involvement of the NT‐26 sigma factor RpoE2 in acting on the NT‐26 RpoE2 promoter was confirmed in vivo in Escherichia coli, which lacks a rpoE2 homolog, using a strain carrying both the promoter–lacZ fusion and the NT‐26 rpoE2 gene. An in silico approach was used to search for other RpoE2 promoters and AioR‐binding motifs and led to the identification of other genes that could be regulated by these proteins including those involved in quorum sensing, chemotaxis, and motility expanding the signaling networks important for the microbial metabolism of arsenite.

Functional annotation of a novel toxin-antitoxin system Xn-RelT of Xenorhabdus nematophila; a combined in silico and in vitro approach.

Toxin-antitoxin (TA) complexes play an important role in stress responses and programmed cell death in bacteria. The RelB-RelE toxin antitoxin system is well studied in Escherichia coli. In this study, we used combined in silico and in vitro approaches to study a novel Xn-RelT toxin from Xenorhabdus nematophila bearing its own antitoxin Xn-RelAT-a RelB homolog of E. coli. The structure for this toxin-antitoxin pair is yet unknown. We generated homology-based models of X. nematophila RelT toxin and antitoxin. The deduced models were further characterized for protein-nucleic acid, protein-protein interactions and gene ontology. A detrimental effect of recombinant Xn-RelT on host E. coli was determined through endogenous toxicity assay. When expressed from a isopropyl β-D-1-thiogalactopyranoside-regulated LacZ promoter, Xn-RelT toxin showed a toxic effect on E. coli cells. These observations imply that the conditional cooperativity governing the Xn-RelT TA operon in X. nematophila plays an important role in stress management and programmed cell death.

Transcriptional and post-transcriptional regulation of pst2 operon expression in Vibrio cholerae O1

One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem–loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter–lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR.The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3′ untranslated regions that are known to block 3′-5′ exonucleolytic attacks.

Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli.

BackgroundThe bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system.ResultsTo fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a “landing pad” (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %.ConclusionsIntegration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.

Improved production of carotenoid-free welan gum in a genetic-engineered Alcaligenes sp. ATCC31555

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs. The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5l fermentor, welan gum was produced at 24±0.4gl(-1) compared to 21g±0.4gl(-1) in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties. Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

Activating C4-dicarboxylate transporters DcuB and DcuC for improving succinate production

Although many efforts had been performed to engineer Escherichia coli for succinate production, succinate efflux system had not been investigated as an engineering target for improving succinate production. In this work, four Dcu transporters, which had been reported to be responsible for C4-dicarboxylates transportation of E. coli, were investigated for their succinate efflux capabilities. These four dcu genes were deleted individually in a previously constructed succinate-producing strain to study their effects on succinate production. Deleting dcuA and dcuD genes had nearly no influence, while deleting dcuB and dcuC genes led to 15 and 11% decrease of succinate titer, respectively. Deleting both dcuB and dcuC genes resulted in 90% decrease of succinate titer, suggesting that DcuB and DcuC were the main transporters for succinate efflux and they functioned as independent and mutually redundant succinate efflux transporters. Furthermore, RBS library having strengths varied from 0.17 to 8.6 times of induced E. coli lacZ promoter was used to modulate dcuB and dcuC genes for improving succinate production. Modulating these two genes in combination led to 34% increase of succinate titer. To the best of knowledge, this was the first report about improving succinate production through engineering succinate efflux system.

Functional characterization of an ACCase subunit from the diatom Phaeodactylum tricornutum expressed in Escherichia coli

The marine diatom Phaeodactylum tricornutum, a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase (PtACC) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene (PtACC-1) was cloned. PtACC-1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli. The expression of the PtACC-1-Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72-fold. The fatty acid composition, analyzed by GC-MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC-1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC-1, providing an additional genetic resource with the potential for biodiesel development.

The Heme Chaperone ApoCcmE Forms a Ternary Complex with CcmI and Apocytochrome c

Cytochrome c maturation (Ccm) is a post-translational process that occurs after translocation of apocytochromes c to the positive (p) side of energy-transducing membranes. Ccm is responsible for the formation of covalent bonds between the thiol groups of two cysteines residues at the heme-binding sites of the apocytochromes and the vinyl groups of heme b (protoporphyrin IX-Fe). Among the proteins (CcmABCDEFGHI and CcdA) required for this process, CcmABCD are involved in loading heme b to apoCcmE. The holoCcmE thus formed provides heme b to the apocytochromes. Catalysis of the thioether bonds between the apocytochromes c and heme b is mediated by the heme ligation core complex, which in Rhodobacter capsulatus contains at least the CcmF, CcmH, and CcmI components. In this work we show that the heme chaperone apoCcmE binds to the apocytochrome c and the apocytochrome c chaperone CcmI to yield stable binary and ternary complexes in the absence of heme in vitro. We found that during these protein-protein interactions, apoCcmE favors the presence of a disulfide bond at the apocytochrome c heme-binding site. We also establish using detergent-dispersed membranes that apoCcmE interacts directly with CcmI and CcmH of the heme ligation core complex CcmFHI. Implications of these findings are discussed with respect to heme transfer from CcmE to the apocytochromes c during heme ligation assisted by the core complex CcmFHI.

Enhanced 2,3-Butanediol Production in Recombinant Klebsiella pneumoniae via Overexpression of Synthesis-Related Genes

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fedbatch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesisrelated genes can enhance 2,3-BD production in K. pneumoniae by fermentation.

A comprehensive toolbox for the rapid construction of lacZ fusion reporters

β-Galactosidase encoded by lacZ remains a popular reporter enzyme. Here, we present three fast and convenient tools that facilitate rapid construction of reporter lacZ fusions. The first enables the simple generation of lacZ (slacZ)-based chromosomally encoded reporter fusions within the lac operon in Escherichia coli using Red®/ET® recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the λ Red recombinase, and blue/white screening. This permits construction of transcriptional and translational reporter lacZ fusions within a day. The second tool allows the introduction of lacZ reporter fusions into the chromosome by a single-crossover method. The strategy relies on the γ-origin-based suicide vector pNPTS138-R6KT, which can only replicate in λpir E. coli strains. The third tool comprises four pBBR1-based broad-host-range vectors for transcriptional and translational lacZ fusions. The functionality of our toolbox was confirmed by the K+-dependent activation of kdp promoter–lacZ fusions in vivo.

The fimbriae activator MatA switches off motility in Escherichia coli by repression of the flagellar master operon flhDC

Flagella provide advantages to Escherichia coli by facilitating taxis towards nutrients and away from unfavourable niches. On the other hand, flagellation is an energy sink to the bacterial cell, and flagella also stimulate host innate inflammatory responses against infecting bacteria. The flagellar assembly pathway is ordered and under a complex regulatory circuit that involves three classes of temporally regulated promoters as well as the flagellar master regulator FlhD(4)C(2). We report here that transcription of the flhDC operon from the class 1 promoter is under negative regulation by MatA, a key activator of the common mat (or ecp) fimbria operon that enhances biofilm formation by E. coli. Ectopic expression of MatA completely precluded motility and flagellar synthesis in the meningitis-associated E. coli isolate IHE 3034. Northern blotting, analysis of chromosomal promoter-lacZ fusions and electrophoretic mobility shift assays revealed an interaction between MatA and the flhDC promoter region that apparently repressed flagellum biosynthesis. However, inactivation of matA in the chromosome of IHE 3034 had only a minor effect on flagellation, which underlines the complexity of regulatory signals that promote flagellation in E. coli. We propose that the opposite regulatory actions of MatA on mat and on flhDC promoters advance the adaptation of E. coli from a planktonic to an adhesive lifestyle.

A cloning vector for creation of Escherichia colilacZ translational fusions and generation of linear template for chromosomal integration

A novel cloning vector to aid in the construction of single copy β-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. colilacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5′ to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown.

The β-propeller gene Rv1057 of Mycobacterium tuberculosis has a complex promoter directly regulated by both the MprAB and TrcRS two-component systems

The β-propeller gene Rv1057 of Mycobacterium tuberculosis is activated by envelope stress and was first characterized as a regulatory target of the TrcRS two-component system (TCS). Rv1057 expression is repressed by TrcRS, and the Rv1057 proximal promoter contains a TrcR binding site. In this study, we determined that Rv1057 is also directly regulated by MprAB, a TCS associated with envelope stress. Multiple potential MprA binding sites (MprA boxes) were identified in the 1kb intergenic region upstream of Rv1057, and four sites were shown to bind MprA. Although MprA boxes were found in the proximal promoter, analyses suggest that MprA and TrcR do not compete for binding in this region. An MprAB-dependent, detergent-inducible transcriptional start point for Rv1057 was identified downstream of the MprA boxes, and a second TrcR binding site and small ORF of the 13E12 family were discovered in the distal promoter. MprAB was required for activation of Rv1057 during growth in macrophages and under detergent stress, and lacZ promoter constructs suggest the entire intergenic region is utilized during MprAB-dependent activation of Rv1057. These findings indicate that Rv1057 has an extensive and complex promoter, and provide evidence for coordinated regulation of stress response genes by TCSs.

Purification and characterization of an arginine regulatory protein, ArgR, in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive bacterium, has been widely used for industrial amino acid production. We previously showed that, in C. glutamicum, argCJBDFRGH arginine biosynthesis genes are clustered but independently transcribed from argC and argG promoters, leading to the generation of two transcripts corresponding to argCJBDFR and argGH. In this report, we show the effect of the C. glutamicum ArgR repressor on argC and argG promoters by overexpressing or disrupting the argR gene. Gel filtration assay results indicate that native ArgR is a hexamer of equal subunits with molecular mass of 110 kDa. Protein sequence analysis revealed the presence of an "SR" (Ser₅₇-Arg₅₈) motif for the DNA binding site at the N-terminal region and the "GTIAGDDTV" motif for arginine binding and its oligomerization at the C-terminal region. An argC or argG promoter-lacZ fusion reporter assay and argR mutational analysis showed that transcription of the argCJBDFR arginine biosynthesis genes is regulated from the argC promoter by ArgR in cooperation with L-arginine in C. glutamicum. This finding was supported by the gel mobility-shift assay showing direct binding of hexameric ArgR to the argC promoter in the presence of L-arginine. Unexpectedly, argGH transcription was not responsive to the level of ArgR repressor and/or arginine. In a further study, a C. glutamicum argR mutant was constructed by disrupting the chromosomal argR gene to manufacture an improved arginine-producing strain. Arginine productivity was increased in the C. glutamicum argR mutant strain under conditions of both limited and excessive arginine.

Transcription of AAT•ATT Triplet Repeats in Escherichia coli Is Silenced by H-NS and IS1E Transposition

BackgroundThe trinucleotide repeats AAT•ATT are simple DNA sequences that potentially form different types of non-B DNA secondary structures and cause genomic instabilities in vivo.Methodology and Principal FindingsThe molecular mechanism underlying the maintenance of a 24-triplet AAT•ATT repeat was examined in E.coli by cloning the repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E.coli genome. Either the AAT or the ATT strand acted as lagging strand template in a replication fork. Propagations of the repeats in either orientation on plasmids did not affect colony morphology when triplet repeat transcription using the lacZ promoter was repressed either by supplementing LacIQ in trans or by adding glucose into the medium. In contrast, transparent colonies were formed by inducing transcription of the repeats, suggesting that transcription of AAT•ATT repeats was toxic to cell growth. Meanwhile, significant IS1E transposition events were observed both into the triplet repeats region proximal to the promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region itself. Transposition reversed the transparent colony phenotype back into healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT repeat in either orientation on plasmids did not produce significant changes in cell morphology and did not promote IS1E transposition events. We further found that a role of IS1E transposition into plasmids was to inhibit transcription through the repeats, which was influenced by the presence of the H-NS protein, but not of its paralogue StpA.Conclusions and SignificanceOur findings thus suggest that the longer AAT•ATT triplet repeats in E.coli become vulnerable after transcription. H-NS and its facilitated IS1E transposition can silence long triplet repeats transcription and preserve cell growth and survival.

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (P(C1), P(Z), P(C2), P(F) and P(I) respectively). P(C1) and P(I) are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of P(F) are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the P(C1) and P(I) promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at P(C1) promoter (named OPRc1) and a 29 bp region at P(I) promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid beta-oxidation.