Chloramphenicol Acetyltransferase Research Articles

New PAH gene promoter KLF1 and 3′-region C/EBPalpha motifs influence transcription in vitro

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50% in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60% of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay

TOXCAT is a widely used genetic assay to study interactions of transmembrane helices within the inner membrane of the bacterium Escherichia coli. TOXCAT is based on a fusion construct that links a transmembrane domain of interest with a cytoplasmic DNA-binding domain from the Vibrio cholerae ToxR protein. Interaction driven by the transmembrane domain results in dimerization of the ToxR domain, which, in turn, activates the expression of the reporter gene chloramphenicol acetyl transferase (CAT). Quantification of CAT is used as a measure of the ability of the transmembrane domain to self-associate. Because the quantification of CAT is relatively laborious, we developed a high-throughput variant of the assay, TOXGREEN, based on the expression of super-folded GFP and detection of fluorescence directly in unprocessed cell cultures. Careful side-by-side comparison of TOXCAT and TOXGREEN demonstrates that the methods have comparable response, dynamic range, sensitivity and intrinsic variability both in LB and minimal media. The greatly enhanced workflow makes TOXGREEN much more scalable and ideal for screening, since hundreds of constructs can be rapidly assessed in 96 well plates. Even for small scale investigations, TOXGREEN significantly reduces time, labor and cost associated with the procedure. We demonstrate applicability with a large screening for self-association among the transmembrane domains of bitopic proteins of the divisome (FtsL, FtsB, FtsQ, FtsI, FtsN, ZipA and EzrA) belonging to 11 bacterial species. The analysis confirms a previously reported tendency for FtsB to self-associate, and suggests that the transmembrane domains of ZipA, EzrA and FtsN may also possibly oligomerize.

Homo- and heterologous reporter proteins for evaluation of promoter activity in Methylomicrobium alcaliphilum 20Z

A number of vectors were constructed based on the plasmid from the broad range of pMHA200 hosts. Also, the expression of some key genes of the haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z was studied. The activities of the promoter regions of genes for hexulose phosphate synthase, glutamine synthetase, and glucokinase, as well as the promoter of the ectABC-ask operon, which encodes enzymes for osmoprotectant ectoine biosynthesis, were evaluated with the use of the gfp gene; the evaluation was proven to be ineffective. Conversely, glucokinase and a heterologous enzyme of chloramphenicol acetyltransferase were useful for the evaluation of promoter activity. In M. alcaliphilum 20Z cells, the expression level of chloramphenicol acetyltransferase transcribed from the methanol dehydrogenase promoter was higher as compared with that of glucokinase. This seems to be due to a regulatory mechanism for homologous protein expression. The introduction of a synthetic nucleotide sequence forming the secondary structure in the 5′ untranslated region of the glucokinase mRNA resulted in an increase of this enzyme level. This is the first attempt to use M. alcaliphilum 20Z for homo- and heterologous protein expression.

Whole genome sequencing for deciphering the resistome of Chryseobacterium indologenes, an emerging multidrug-resistant bacterium isolated from a cystic fibrosis patient in Marseille, France.

We decipher the resistome of Chryseobacterium indologenes MARS15, an emerging multidrug-resistant clinical strain, using the whole genome sequencing strategy. The bacterium was isolated from the sputum of a hospitalized patient with cystic fibrosis in the Timone Hospital in Marseille, France. Genome sequencing was done with Illumina MiSeq using a paired-end strategy. The in silico analysis was done by RAST, the resistome by the ARG-ANNOT database and detection of polyketide synthase (PKS) by ANTISMAH. The genome size of C. indologenes MARS15 is 4 972 580 bp with 36.4% GC content. This multidrug-resistant bacterium was resistant to all β-lactams, including imipenem, and also to colistin. The resistome of C. indologenes MARS15 includes Ambler class A and B β-lactams encoding blaCIA and blaIND-2 genes and MBL (metallo-β-lactamase) genes, the CAT (chloramphenicol acetyltransferase) gene and the multidrug efflux pump AcrB. Specific features include the presence of an urease operon, an intact prophage and a carotenoid biosynthesis pathway. Interestingly, we report for the first time in C. indologenes a PKS cluster that might be responsible for secondary metabolite biosynthesis, similar to erythromycin. The whole genome sequence analysis provides insight into the resistome and the discovery of new details, such as the PKS cluster.

Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli

BackgroundThe occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. MethodsTo understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV–vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. ResultsWe observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37μM. ConclusionsIt was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. General significanceUnderstanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in <em>E. coli</em>

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in <em>E. coli</em>

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Everolimus (RAD001) inhibits the proliferation of rat vascular smooth muscle cells by up-regulating the activity of the p27/kip1 gene promoter.

Objective:We investigated whether the inhibitory effect of the immunosuppressant everolimus (RAD001) on vascular smooth muscle cell (VSMC) proliferation is mediated by p27/kip1 gene promoter activity.Methods:In this experimental study, cultured rat VSMCs were transiently transfected with a recombinant plasmid (pXp27) containing p27/kip1 gene promoter sequence and a chloramphenicol acetyltransferase (CAT) reporter gene. After stimulation with the mitogen platelet-derived growth factor (PDGF-BB, 10 ng/mL) in the presence or absence of RAD001 (10 nM), the promoter activity, mRNA expression, and protein expression of p27/kip1 were examined by CAT assay, RT–PCR, and immunoblotting, respectively. Cell cycle–related changes were detected by flow cytometry. DNA synthesis was determined using 3H-TdR incorporation.Results:Compared with the non-stimulation group, PDGF-BB stimulation induced a significant proliferative response in the VSMCs as indicated by decreased p27/kip1 gene promoter activity, decreased p27/kip1 mRNA and protein expression, increased S-phase and G2/M-phase cells, and increased DNA synthesis. RAD001 intervention increased p27/kip1 gene promoter activity 3.5-fold, promoted p27/kip1 mRNA and protein expression, increased G0-phase cells, reduced DNA synthesis, and, overall, inhibited PDGF-BB–stimulated cell proliferation.Conclusion:RAD001 inhibits PDGF-BB–stimulated proliferation of cultured VSMCs by upregulating p27/kip1 gene promoter activity and increasing p27/kip1 mRNA and protein expression.

Chloramphenicol acetyltransferase-a new selectable marker in stable nuclear transformation of the red alga Cyanidioschyzon merolae.

In this study, we have shown the applicability of chloramphenicol acetyltransferase as a new and convenient selectable marker for stable nuclear transformation as well as potential chloroplast transformation of Cyanidioschyzon merolae-a new model organism, which offers unique opportunities for studding the mitochondrial and plastid physiology as well as various evolutionary, structural, and functional features of the photosynthetic apparatus.

Rapid intranasal delivery of chloramphenicol acetyltransferase in the active form to different brain regions as a model for enzyme therapy in the CNS

BackgroundThe blood brain barrier (BBB) is critical for maintaining central nervous system (CNS) homeostasis by restricting entry of potentially toxic substances. However, the BBB is a major obstacle in the treatment of neurotoxicity and neurological disorders due to the restrictive nature of the barrier to many medications. Intranasal delivery of active enzymes to the brain has therapeutic potential for the treatment of numerous CNS enzyme deficiency disorders and CNS toxicity caused by chemical threat agents. New methodThe aim of this work is to provide a sensitive model system for analyzing the rapid delivery of active enzymes into various regions of the brain with therapeutic bioavailability. ResultsWe tested intranasal delivery of chloramphenicol acetyltransferase (CAT), a relatively large (75kD) enzyme, in its active form into different regions of the brain. CAT was delivered intranasally to anaesthetized rats and enzyme activity was measured in different regions using a highly specific High Performance Thin Layer Chromatography (HP-TLC)-radiometry coupled assay. Active enzyme reached all examined areas of the brain within 15min (the earliest time point tested). In addition, the yield of enzyme activity in the brain was almost doubled in the brains of rats pre-treated with matrix metalloproteinase-9 (MMP-9). Comparison with existing method (s)Intranasal administration of active enzymes in conjunction with MMP-9 to the CNS is both rapid and effective. ConclusionThe present results suggest that intranasal enzyme therapy is a promising method for counteracting CNS chemical threat poisoning, as well as for treating CNS enzyme deficiency disorders.

Identification and characterization of transcriptional control region of the human beta 1,4-mannosyltransferase gene.

All asparagine-linked glycans (N-glycans) on the eukaryotic glycoproteins are primarily derived from dolichol-linked oligosaccharides (DLO), synthesized on the rough endoplasmic reticulum membrane. We have previously reported cloning and identification of the human gene, HMT-1, which encodes chitobiosyldiphosphodolichol beta-mannosyltransferase (β1,4-MT) involved in the early assembly of DLO. Considering that N-glycosylation is one of the most ubiquitous post-translational modifications for many eukaryotic proteins, the HMT-1 could be postulated as one of the housekeeping genes, but its transcriptional regulation remains to be investigated. Here we screened a 1kb region upstream from HMT-1 open reading frame (ORF) for transcriptionally regulatory sequences by using chloramphenicol acetyl transferase (CAT) assay, and found that the region from -33 to -1 positions might act in HMT-1 transcription at basal level and that the region from -200 to -42 should regulate its transcription either positively or negatively. In addition, results with CAT assays suggested the possibility that two GATA-1 motifs and an Sp1 motif within a 200bp region upstream from HMT-1 ORF might significantly upregulate HMT-1 transcription. On the contrary, the observations obtained from site-directed mutational analyses revealed that an NF-1/AP-2 overlapping motif located at -148 to -134 positions should serve as a strong silencer. The control of the HMT-1 transcription by these motifs resided within the 200bp region could partially explain the variation of expression level among various human tissues, suggesting availability and importance of this region for regulatory role in HMT-1 expression.

Persistence of Antibiotic Resistant Vibrio spp. in Shellfish Hatchery Environment.

The characterization of antibiotic-resistant vibrios isolated from shellfish aquaculture is necessary to elucidate the potential transfer of resistance and to establish effective strategies against vibriosis. With this aim, we analyzed a collection of bacterial isolates obtained from 15 failed hatchery larval cultures that, for the most part, had been treated experimentally with chloramphenicol to prevent vibriosis. Isolates were obtained during a 2-year study from experimental cultures of five different clam species. Among a total of 121 Vibrio isolates studied, 28 were found to be chloramphenicol resistant, suggesting that the shellfish hatchery had been using a sublethal concentration of the antibiotic. Interestingly, chloramphenicol-resistant vibrios showed also resistance to tetracycline and amoxicillin (group A; n = 19) or to streptomycin (group B; n = 9). Chloramphenicol-resistant vibrios were subjected to a PCR amplification and DNA sequencing of the chloramphenicol acetyltransferase genes (cat), and the same approach was followed to study the tetracycline resistance markers (tet). 16S ribosomal RNA (rRNA) gene sequencing revealed that chloramphenicol-resistant vibrios pertained mostly to the Splendidus clade. Conjugation assays demonstrated that various R-plasmids which harbored the cat II/tet(D) genes and cat III gene in groups A and B respectively, were transferred to E. coli and bivalve pathogenic vibrios. Most interestingly, transconjugants exhibited the antibiotic resistance patterns of the donors, despite having been selected only on the basis of chloramphenicol resistance. This is the first report carried out in a bivalve hatchery elucidating the persistence of resistant vibrios, the mechanisms of antibiotic resistance, and the transfer of different R-plasmids.

On-chip enzymatic assay for chloramphenicol acetyltransferase using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Herein, we report a chloramphenicol (CAP) acetyltransferase (CAT) activity assay based on self-assembled monolayers on gold as an alternative to conventional CAT reporter gene assay systems, which sometimes require toxic materials and complicated steps that limit their use. A CAP derivative presented on a monolayer was converted to the acetylated CAP by CAT in the presence of acetyl-CoA. The conversion was directly monitored by observing the molecular weight changes in CAP using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CAT activity was determined under various reaction conditions by changing reaction times, CAT and acetyl-CoA concentrations. As a practical application, we identified gene expression in bacteria that were transformed with pCAT plasmid DNA. Our strategy can provide a simple and rapid assay that eliminates some commonly used but potentially detrimental steps in enzymatic assays, such as radioactive labeling and complicated separation and purification of analytes prior to detection.

Chloramphenicol Selection of IS10 Transposition in the cat Promoter Region of Widely Used Cloning Vectors.

The widely used pSU8 family of cloning vectors is based on a p15A replicon and a chloramphenicol acetyltransferase (cat) gene conferring chloramphenicol resistance. We frequently observed an increase in the size of plasmids derived from these vectors. Analysis of the bigger molecular species shows that they have an IS10 copy inserted at a specific site between the promoter and the cat open reading frame. Promoter activity from both ends of IS10 has been reported, suggesting that the insertion events could lead to higher CAT production. Insertions were observed in certain constructions containing inserts that could lead to plasmid instability. To test the possibility that IS10 insertions were selected as a response to chloramphenicol selection, we have grown these constructs in the presence of different amounts of antibiotic and we observed that insertions arise promptly under higher chloramphenicol selective pressure. IS10 is present in many E. coli laboratory strains, so the possibility of insertion in constructions involving cat-containing vectors should be taken into account. Using lower chloramphenicol concentrations could solve this problem.

Post-transcriptional regulation by distal Shine-Dalgarno sequences in the grpE-dnaK intergenic region of Streptococcus mutans.

A unique 373 bp region (igr66) between grpE and dnaK of Streptococcus mutans lacks a promoter but is required for optimal production of DnaK. Northern blotting using probes specific to hrcA, igr66 or dnaK revealed multiple transcripts produced from the dnaK operon and 5'-RACE mapped 5' termini of multiple dnaK transcripts within igr66. One product mapped to a predicted 5'-SL (stem-loop) and two others mapped just 5' to Shine-Dalgarno (SD)-like sequences located immediately upstream to dnaK and to a predicted SL 120 bp upstream of the dnaK start codon (3'-SL). A collection of cat reporter-gene strains containing mutant derivatives of igr66 were engineered. Chloramphenicol acetyltransferase (CAT) activity varied greatly between strains, but there were no correlative changes in cat mRNA levels. Interestingly, mutations introduced into the SD-like sequences 5' to the 3'-SL resulted in an 83-98% decrease in CAT activity. Markerless point mutations introduced upstream of dnaK in the SD-like sequences impaired growth at elevated temperatures and resulted in up to a 40% decrease in DnaK protein after heat shock. Collectively, these results indicate processing within igr66 enhances translation in a temperature dependent manner via non-canonical ribosome binding sites positioned >120 bp upstream of dnaK.

Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid pGKE75(αβ)-catA138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75(αβ)-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75(αβ)-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75(αβ)-catA138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75(αβ)-catA138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli.

The Cellular Factor NXP2/MORC3 Is a Positive Regulator of Influenza Virus Multiplication.

Transcription and replication of influenza A virus are carried out in the nuclei of infected cells in the context of viral ribonucleoproteins (RNPs). The viral polymerase responsible for these processes is a protein complex composed of the PB1, PB2, and PA proteins. We previously identified a set of polymerase-associated cellular proteins by proteomic analysis of polymerase-containing intracellular complexes expressed and purified from human cells. Here we characterize the role of NXP2/MORC3 in the infection cycle. NXP2/MORC3 is a member of the Microrchidia (MORC) family that is associated with the nuclear matrix and has RNA-binding activity. Influenza virus infection led to a slight increase in NXP2/MORC3 expression and its partial relocalization to the cytoplasm. Coimmunoprecipitation and immunofluorescence experiments indicated an association of NXP2/MORC3 with the viral polymerase and RNPs during infection. Downregulation of NXP2/MORC3 by use of two independent short hairpin RNAs (shRNAs) reduced virus titers in low-multiplicity infections. Consistent with these findings, analysis of virus-specific RNA in high-multiplicity infections indicated a reduction of viral RNA (vRNA) and mRNA after NXP2/MORC3 downregulation. Silencing of NXP2/MORC3 in a recombinant minireplicon system in which virus transcription and replication are uncoupled showed reductions in cat mRNA and chloramphenicol acetyltransferase (CAT) protein accumulation but no alterations in cat vRNA levels, suggesting that NXP2/MORC3 is important for influenza virus transcription. Influenza virus infections appear as yearly epidemics and occasional pandemics of respiratory disease, with high morbidity and occasional mortality. Influenza viruses are intracellular parasites that replicate and transcribe their genomic ribonucleoproteins in the nuclei of infected cells, in a complex interplay with host cell factors. Here we characterized the role of the human NXP2/MORC3 protein, a member of the Microrchidia family that is associated with the nuclear matrix, during virus infection. NXP2/MORC3 associates with the viral ribonucleoproteins in infected cells. Downregulation of NXP2/MORC3 reduced virus titers and accumulations of viral genomic RNA and mRNAs. Silencing of NXP2/MORC3 in an influenza virus CAT minireplicon system diminished CAT protein and cat mRNA levels but not genomic RNA levels. We propose that NXP2/MORC3 plays a role in influenza virus transcription.

Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus.

BackgroundExotic genes, especially clustered multiple-genes for a complex pathway, are normally integrated into chromosome for heterologous expression. The influences of insertion sites on heterologous expression and allotropic expressions of exotic genes on host remain mostly unclear.ResultsWe compared the integration and expression efficiencies of single and multiple exotic genes that were inserted into Myxococcus xanthus genome by transposition and attB-site-directed recombination. While the site-directed integration had a rather stable chloramphenicol acetyl transferase (CAT) activity, the transposition produced varied CAT enzyme activities. We attempted to integrate the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M. xanthus genome by site-direction but failed, which was determined to be due to the insertion size limitation at the attB site. The transposition technique produced many recombinants with varied production capabilities of epothilones, which, however, were not paralleled to the transcriptional characteristics of the local sites where the genes were integrated. Comparative transcriptomics analysis demonstrated that the allopatric integrations caused selective changes of host transcriptomes, leading to varied expressions of epothilone genes in different mutants.ConclusionsWith the increase of insertion fragment size, transposition is a more practicable integration method for the expression of exotic genes. Allopatric integrations selectively change host transcriptomes, which lead to varied expression efficiencies of exotic genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0294-5) contains supplementary material, which is available to authorized users.

Over-expression of recombinant proteins with N-terminal His-tag via subcellular uneven distribution in <italic>Escherichia coli</italic>

Specific tags with defined amino acid residues are widely used to purify or probe target proteins. Interestingly, the tagging system occasionally results in an increase of the recombinant protein expression in vivo. Here, we systematically examined this phenomenon using a poly-histidine (His)-tag fused to N- or C-terminal region of green, red, and blue fluorescent proteins by quantification and uneven distribution in cytoplasm of Escherichia coli. This effect was further supported by the distinct over-expression of several unrelated proteins, such as esterase, neopullulanase, and chloramphenicol acetyltransferase, tagging with the same tag. These results suggest that a poly-His-tag placed at N-terminal region can induce over-expression of recombinant protein via subcellular uneven distribution in vivo.

Regioselective Acetylation of C21 Hydroxysteroids by the Bacterial Chloramphenicol Acetyltransferase I.

Chloramphenicol acetyltransferase I (CATI) detoxifies the antibiotic chloramphenicol and confers a corresponding resistance to bacteria. In this study we identified this enzyme as a steroid acetyltransferase and designed a new and efficient Escherichia-coli-based biocatalyst for the regioselective acetylation of C21 hydroxy groups in steroids of pharmaceutical interest. The cells carried a recombinant catI gene controlled by a constitutive promoter. The capacity of the whole-cell system to modify different hydroxysteroids was investigated, and NMR spectroscopy revealed that all substrates were selectively transformed into the corresponding 21-acetoxy derivatives. The biotransformation was optimized, and the reaction mechanism is discussed on the basis of a computationally modeled substrate docking into the crystal structure of CATI.