Membranome-based identification of amino acid substitution in Haemophilus influenzae multidrug efflux pump HmrM for reduced chloramphenicol susceptibility.

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

Chloramphenicol Binding Sites of Acinetobacter baumannii Chloramphenicol Acetyltransferase CatB8.

Acinetobacter baumannii is a multidrug-resistant pathogen that has become one of the most challenging pathogens in global healthcare. Several antibiotic-resistant genes, including catB8, have been identified in the A. baumannii genome. CatB8 protein, one of the chloramphenicol acetyltransferases (Cats), is encoded by the catB8 gene. Cats can convert chloramphenicol (chl) to 3-acetyl-chl, leading to bacterial resistance to chl. Here, we present the high-resolution cocrystal structure of CatB8 with chl. The structure that we resolved showed that each monomer of CatB8 binds to four chl molecules, while its homologous protein only binds to one chl molecule. One of the newly discovered chl binding site overlaps with the site of another substrate, acetyl-CoA. Through structure-based biochemical analyses, we identified key residues for chl recruiting and acetylation of chl in CatB8. Our work is of significant importance for understanding the drug resistance of A. baumannii and the effectiveness of antibiotic treatment.

A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers.

BACKGROUND: The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. RESULTS: This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. CONCLUSIONS: The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

Structural characterization of a Type B chloramphenicol acetyltransferase from the emerging pathogen Elizabethkingia anophelis NUHP1.

Elizabethkingia anophelis is an emerging multidrug resistant pathogen that has caused several global outbreaks. E. anophelis belongs to the large family of Flavobacteriaceae, which contains many bacteria that are plant, bird, fish, and human pathogens. Several antibiotic resistance genes are found within the E. anophelis genome, including a chloramphenicol acetyltransferase (CAT). CATs play important roles in antibiotic resistance and can be transferred in genetic mobile elements. They catalyse the acetylation of the antibiotic chloramphenicol, thereby reducing its effectiveness as a viable drug for therapy. Here, we determined the high-resolution crystal structure of a CAT protein from the E. anophelis NUHP1 strain that caused a Singaporean outbreak. Its structure does not resemble that of the classical Type A CATs but rather exhibits significant similarity to other previously characterized Type B (CatB) proteins from Pseudomonas aeruginosa, Vibrio cholerae and Vibrio vulnificus, which adopt a hexapeptide repeat fold. Moreover, the CAT protein from E. anophelis displayed high sequence similarity to other clinically validated chloramphenicol resistance genes, indicating it may also play a role in resistance to this antibiotic. Our work expands the very limited structural and functional coverage of proteins from Flavobacteriaceae pathogens which are becoming increasingly more problematic.

Characterization of permissive and non-permissive peptide insertion sites in chloramphenicol acetyltransferase.

The growing prevalence of antibiotic resistance in numerous pathogenic bacteria is a major public health concern and urgently requires the development of new therapeutic approaches. Multidrug resistant species that remain sensitive to chloramphenicol (CAM) treatment have engendered renewed interest in using this drug as a modern day antimicrobial agent. High-level resistance to CAM commonly is mediated by chloramphenicol acetyltransferase (CAT) which catalyzes the acetylation of CAM and renders the drug inactive. Of the three main types (CATI, CATII and CATIII), CATI is of broad clinical significance. Despite this importance, understanding of the catalytic mechanism of CATI largely is extrapolated from studies of CATIII. Here, pentapeptide scanning mutagenesis was used to generate a library of random insertions in CATI to gain a better understanding of structure-function relationships in the enzyme. Pentapeptide insertions in secondary structure elements which contain residues that form part of the CATI active site abolished CAM resistance in Escherichia coli. Insertions in secondary structures that have key roles in protein folding and CAM binding led to a reduction in resistance. In contrast, insertions in loop regions between the major secondary structure features exerted modest, if any, effects on CAM resistance. The analysis pinpoints regions of CATI that may serve as targets for the design of novel inhibitors that prevent the spread of CAM-resistant pathogens thereby enabling the drug to be re-deployed as a broad range antimicrobial agent. Moreover, regions of CATI that are tolerant of insertions may be suitable for the construction of bifunctional enzymes in which peptides, mini-proteins or amino acid tags are introduced at the permissive sites.

Mixed-culture fermentation for enhanced C21-hydroxylation of glucocorticoids.

Synthetic glucocorticoids are generally preferred over their natural counterparts as these compounds exhibit improved anti-inflammatory potency and glucocorticoid receptor selectivity. However, the biotechnological production of these molecules is often subject to limitations inferred by restricted enzyme stability, selectivity or inhibition thereof. The latter is particularly important during 6α-methylprednisolone production, as the essential C21-hydroxylation of its precursor medrane appears to be hampered by product inhibition of the steroid-21-hydroxylase (CYP21A2). To circumvent this bottleneck, we established a two-step reaction for controlled mixed-culture fermentation, using recombinant E. coli. This process comprises the previously reported C21-hydroxylation of medrane by CYP21A2, followed by an instant derivatization of the hydroxylated product premedrol by chloramphenicol acetyl transferase 1 (CAT1). The CAT1-mediated C21-acetylation prevents the product from regaining access to the enzyme's active site which effectively shifts the chemical equilibrium toward premedrol formation. The successful circumvention of product inhibition at optimized conditions resulted in the formation of more than 1.5 g of product per liter which corresponds to an increase by more than 100 %. Taken together, we demonstrate an efficient system to enhance cytochrome P450-mediated biotransformations, holding great ecologic and economic potential to be applied in industrial processes.

The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators.

In trypanosomatids, posttranscriptional controls are very important in regulation of individual gene expression. These are achieved through combinatorial sets of RNA-binding proteins (RBPs) which recognize RNA regulatory motifs or regions of secondary structure within RNAs. To analyze the potential functional impact of an RBP on their mRNA targets, we have applied a robust technique called tethering assay. In this method, the protein under study is attached to an mRNA reporter through an artificial RNA-protein interaction. Therefore, the functional activity of a protein can be analyzed independently of its intrinsic ability to bind to RNA. By making use of a cell line expressing a chloramphenicol acetyltransferase (CAT) reporter mRNA, we have characterized dozens of novel mRNA-fate regulators in cultured Trypanosoma brucei. After induction of the candidate fusion protein, the effect on the reporter expression is determined by a rapid CAT assay. The protocol is simple and typically takes one working day for analysis of a single protein and controls. In this chapter, we provide a description of materials and methods for the tethering method and should allow the assay to be successfully deployed in any laboratory with minimal user training.

Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness.

In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species.

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid-mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.

A novel family of intrinsic chloramphenicol acetyltransferase CATC in Vibrio parahaemolyticus: Naturally occurring variants reveal diverse resistance levels against chloramphenicol.

Intrinsic resistance of bacteria to antibiotics plays an increasingly significant role in antibiotic resistance. However, the breadth of intrinsic resistance has not been fully elucidated. Here we identified a novel class of chloramphenicol acetyltransferase (type C CAT or CATC) in Vibrio parahaemolyticus and its closely related species V. alginolyticus, V. antiquarius, and V. diabolicus. The catC genes encoding the CATC clade are distributed among the four Vibrio species and are consistently found in the same conserved genomic regions. Based on their prevalence, these genes are considered to be intrinsic in V. parahaemolyticus, V. alginolyticus, V. antiquarius, and V. diabolicus. We also demonstrated that naturally occurring variants of CATC can confer diverse resistance levels against chloramphenicol in Escherichia coli. Furthermore, the enzyme kinetics of CATC variant proteins supported the diversity of their resistance phenotypes. This work provides insights into the distribution and resistance phenotypes of a novel class of intrinsic resistance genes in bacteria.

Tracking of single tRNAs for translation kinetics measurements in chloramphenicol treated bacteria.

Chloramphenicol is a broad-spectrum antibiotic targeting the protein synthesis machinery by binding to the bacterial ribosome. Chloramphenicol has been considered a classic general inhibitor of translation, blocking the accommodation of aa-tRNA into the A site of the large ribosomal subunit. However, recent studies suggest that this proposed mechanism is a simplification and that the effect of chloramphenicol on mRNA translation is much more dynamic. By tracking single dye-labelled elongator and initiator tRNAs in Escherichia coli cells treated with chloramphenicol, we observe the direct effect of chloramphenicol on translation kinetics. We find clear indications of slow but significant mRNA translation on drug bound ribosomes.

Conditional MitoTimer reporter mice for assessment of mitochondrial structure, oxidative stress, and mitophagy.

Assessment of structural and functional changes of mitochondria is vital for biomedical research as mitochondria are the power plants essential for biological processes and tissue/organ functions. Others and we have developed a novel reporter gene, pMitoTimer, which codes for a redox sensitive mitochondrial targeted protein that switches from green fluorescence protein (GFP) to red fluorescent protein (DsRed) when oxidized. It has been shown in transfected cells, transgenic C. elegans and Drosophila m., as well as somatically transfected adult skeletal muscle that this reporter gene allows quantifiable assessment of mitochondrial structure, oxidative stress, and lysosomal targeting of mitochondria-containing autophagosomes. Here, we generated CAG-CAT-MitoTimer transgenic mice using a transgene containing MitoTimer downstream of LoxP-flanked bacterial chloramphenicol acetyltransferase (CAT) gene with stop codon under the control of the cytomegalovirus (CMV) enhancer fused to the chicken ß-actin promoter (CAG). When CAG-CAT-MitoTimer mice were crossbred with various tissue-specific (muscle, adipose tissue, kidney, and pancreatic tumor) or global Cre transgenic mice, the double transgenic offspring showed MitoTimer expression in tissue-specific or global manner. Lastly, we show that hindlimb ischemia-reperfusion caused early, transient increases of mitochondrial oxidative stress, mitochondrial fragmentation and lysosomal targeting of autophagosomes containing mitochondria as well as a later reduction of mitochondrial content in skeletal muscle along with mitochondrial oxidative stress in sciatic nerve. Thus, we have generated conditional MitoTimer mice and provided proof of principle evidence of their utility to simultaneously assess mitochondrial structure, oxidative stress, and mitophagy in vivo in a tissue-specific, controllable fashion.

Egg Yolk Immunoglobulin Supplementation Prevents Rat Liver from Aflatoxin B1-Induced Oxidative Damage and Genotoxicity.

Egg yolk immunoglobulins (IgY), as nutraceutical supplement for therapeutic or prophylactic intervention, have been extensively studied. The effects of IgY on small molecular toxin-induced toxicity in animals are unclear. In the present study, the protection of highly purified and specific anti-AFB1 IgY against AFB1-induced genotoxicity and oxidative damage on the rat liver model were investigated. Our results revealed that AFB1 induced significant oxidative damage markers, as well as AFB1-induced protein expression in antioxidant, pro- and antiapoptosis processes in rat liver. These effects could be significantly inhibited by cogavage with anti-AFB1 IgY in a dose-dependent manner. However, anti-AFB1 IgY did not significantly induce hepatic CAT and SOD1. To explore mechanisms, metabolite experiments were established to evaluate the influence of anti-AFB1 IgY on the absorption of AFB1 in rats. Middle and high doses of anti-AFB1 IgY reduced hepatic AFB1-DNA adducts by 43.3% and 52.9%, AFB1- N7-guanine urinary adducts by 19.6% and 34.4%, and AFB1-albumin adducts by 10.5% and 21.1%, respectively. The feces of high dose anti-AFB1 IgY cogavaged rats contained approximately 2-fold higher AFB1 equivalents at 3-6 h after ingestion than AFB1 group feces, indicating IgY inhibited AFB1 uptake. These results had provided insight that anti-AFB1 IgY could prevent animal organs from damage caused by AFB1 and will be beneficial for the application of detoxification antibody as a supplement in food.

The Genetic Transformation of Chlamydia pneumoniae.

We demonstrate the genetic transformation of Chlamydia pneumoniae using a plasmid shuttle vector system which generates stable transformants. The equine C. pneumoniae N16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearing C. pneumoniae transformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolate C. pneumoniae LPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolate C. pneumoniae CV-6 and the human community-acquired pneumonia-associated C. pneumoniae IOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown that Chlamydia spp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-free C. trachomatis, C. muridarum, C. caviae, C. pecorum, and C. abortus However, contrary to our expectation, pRSGFPCAT-Cpn did transform C. felis Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid of C. felis Taken together, we provide for the first time an easy-to-handle transformation protocol for C. pneumoniae that results in stable transformants. In addition, the vector can cross the species barrier to C. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid.IMPORTANCE The absence of tools for the genetic manipulation of C. pneumoniae has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for C. pneumoniae transformation based on a plasmid shuttle vector system. We constructed a C. pneumoniae plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in C. pneumoniaeC. pneumoniae transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in C. pneumoniae using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in C. pneumoniae biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.

Development of Assay Systems for Amber Codon Decoding at the Steps of Initiation and Elongation in Mycobacteria.

Genetic analysis of the mechanism of protein synthesis in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriate in vivo assay systems. We developed chloramphenicol acetyltransferase (CAT)-based in vivo reporter systems to study translation initiation and elongation in Mycobacterium smegmatis The CAT reporters utilize specific decoding of amber codons by mutant initiator tRNA (i-tRNA, metU) molecules containing a CUA anticodon (metUCUA). The assay systems allow structure-function analyses of tRNAs without interfering with the cellular protein synthesis and function with or without the expression of heterologous GlnRS from Escherichia coli We show that despite their naturally occurring slow-growth phenotypes, the step of i-tRNA formylation is vital in translation initiation in mycobacteria and that formylation-deficient i-tRNA mutants (metUCUA/A1, metUCUA/G72, and metUCUA/G72G73) with a Watson-Crick base pair at the 1·72 position participate in elongation. In the absence of heterologous GlnRS expression, the mutant tRNAs are predominantly aminoacylated (glutamylated) by nondiscriminating GluRS. Acid urea gels show complete transamidation of the glutamylated metUCUA/G72G73 tRNA to its glutaminylated form (by GatCAB) in M. smegmatis In contrast, the glutamylated metUCUA/G72 tRNA did not show a detectable level of transamidation. Interestingly, the metUCUA/A1 mutant showed an intermediate activity of transamidation and accumulated in both glutamylated and glutaminylated forms. These observations suggest important roles for the discriminator base position and/or a weak Watson-Crick base pair at 1·72 for in vivo recognition of the glutamylated tRNAs by M. smegmatis GatCAB.IMPORTANCE Genetic analysis of the translational apparatus in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriate in vivo assay systems. We developed chloramphenicol acetyltransferase (CAT)-based reporters which utilize specific decoding of amber codons by mutant tRNAs at the steps of initiation and/or elongation to allow structure-function analysis of the translational machinery. We show that formylation of the initiator tRNA (i-tRNA) is crucial even for slow-growing bacteria and that i-tRNA mutants with a CUA anticodon are aminoacylated by nondiscriminating GluRS. The discriminator base position, and/or a weak Watson-Crick base pair at the top of the acceptor stem, provides important determinants for transamidation of the i-tRNA-attached Glu to Gln by the mycobacterial GatCAB.

Novel Genetic Tool to Study the Stability of Genomic Islands.

BACKGROUND: Genomic islands (GIs) are discrete segments of mobile DNA with defined boundaries according to recent patents, acquired in the bacterial genome from another organism by horizontal gene transfer during the course of evolution. GIs contribute significantly to virulence, disease development, antimicrobial resistance and metabolic process. OBJECTIVE: The present study focuses on the development of a vector based genetic tool carrying selectable and counter-selectable markers, in order to flag the GIs in the bacterial chromosome and monitor their stability under in vitro and in vivo conditions. METHOD: We engineered suicide vectors, pSB40 and pSB41, carrying single or tandem copies of chloramphenicol acetyltransferase (cat) and levansucrase (sacB) alleles, respectively. The sacB-cat allele in both the vectors is flanked by several restriction sites. To test the suitability of sacB-cat allele for monitoring GI loss, we introduced the allele in the Vibrio Pathogenicity Island-1 (VPI-1) in Vibrio cholerae genome. RESULTS: The V. cholerae strain carrying sacB-cat allele in VPI-1 element showed resistance to chloramphenicol and sensitivity to sucrose at optimal growth conditions. Loss of VPI-1 element from the V. cholerae genome was simply monitored by growing the cells on selection agar plates supplemented with sucrose. Our results showed that the genetic tool we developed is suitable for monitoring GI stability in the bacterial genome. CONCLUSION: The present study indicates that pSB40 and pSB41are efficient and sensitive genetic tool that can be used for reverse genetics experiments and monitoring stability of mobile genetic elements in the bacterial genome.

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis.

Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations - particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.

Deletion of psbQ' gene in Cyanidioschyzon merolae reveals the function of extrinsic PsbQ' in PSII.

KEY MESSAGE: We have successfully produced single-cell colonies of C. merolae mutants, lacking the PsbQ' subunit in its PSII complex by application of DTA-aided mutant selection. We have investigated the physiological changes in PSII function and structure and proposed a tentative explanation of the function of PsbQ' subunit in the PSII complex. We have improved the selectivity of the Cyanidioschyzon merolae nuclear transformation method by the introduction of diphtheria toxin genes into the transformation vector as an auxiliary selectable marker. The revised method allowed us to obtained single-cell colonies of C. merolae, lacking the gene of the PsbQ' extrinsic protein. The efficiency of gene replacement was extraordinarily high, allowing for a complete deletion of the gene of interest, without undesirable illegitimate integration events. We have confirmed the absence of PsbQ' protein at genetic and protein level. We have characterized the physiology of mutant cells and isolated PSII protein complex and concluded that PsbQ' is involved in nuclear regulation of PSII activity, by influencing several parameters of PSII function. Among these: oxygen evolving activity, partial dissociation of PsbV, regulation of dimerization, downsizing of phycobilisomes rods and regulation of zeaxanthin abundance. The adaptation of cellular physiology appeared to favorite upregulation of PSII and concurrent downregulation of PSI, resulting in an imbalance of energy distribution, decrease of photosynthesis and inhibition of cell proliferation.

Investigation of specific interactions between T7 promoter and T7 RNA polymerase by force spectroscopy using atomic force microscope.

The specific recognition and binding of promoter and RNA polymerase is the first step of transcription initiation in bacteria and largely determines transcription activity. Therefore, direct analysis of the interaction between promoter and RNA polymerase in vitro may be a new strategy for promoter characterization, to avoid interference due to the cell's biophysical condition and other regulatory elements. In the present study, the specific interaction between T7 promoter and T7 RNA polymerase was studied as a model system using force spectroscopy based on atomic force microscope (AFM). The specific interaction between T7 promoter and T7 RNA polymerase was verified by control experiments, and the rupture force in this system was measured as 307.2 ± 6.7 pN. The binding between T7 promoter mutants with various promoter activities and T7 RNA polymerase was analyzed. Interaction information including rupture force, rupture distance and binding percentage were obtained in vitro, and reporter gene expression regulated by these promoters was also measured according to a traditional promoter activity characterization method in vivo Using correlation analysis, it was found that the promoter strength characterized by reporter gene expression was closely correlated with rupture force and the binding percentage by force spectroscopy. These results indicated that the analysis of the interaction between promoter and RNA polymerase using AFM-based force spectroscopy was an effective and valid approach for the quantitative characterization of promoters.