Chloramphenicol Acetyltransferase Gene Research Articles

Development of Bioluminescent Virulent Aeromonas hydrophila for Understanding Pathogenicity.

Virulent Aeromonas hydrophila (vAh) strains that cause motile Aeromonas septicemia (MAS) in farmed channel catfish (Ictalurus punctatus) have been an important problem for more than a decade. However, the routes of infection of vAh in catfish are not well understood. Therefore, it is critical to study the pathogenicity of vAh in catfish. To this goal, a new bioluminescence expression plasmid (pAKgfplux3) with the chloramphenicol acetyltransferase (cat) gene was constructed and mobilized into vAh strain ML09-119, yielding bioluminescent vAh (BvAh). After determining optimal chloramphenicol concentration, plasmid stability, bacteria number-bioluminescence relationship, and growth kinetics, the catfish were challenged with BvAh, and bioluminescent imaging (BLI) was conducted. Results showed that 5 to 10 µg/mL chloramphenicol was suitable for stable bioluminescence expression in vAh, with some growth reduction. In the absence of chloramphenicol, vAh could not maintain pAKgfplux3 stably, with the half-life being 16 h. Intraperitoneal injection, immersion, and modified immersion (adipose fin clipping) challenges of catfish with BvAh and BLI showed that MAS progressed faster in the injection group, followed by the modified immersion and immersion groups. BvAh was detected around the anterior mouth, barbels, fin bases, fin epithelia, injured skin areas, and gills after experimental challenges. BLI revealed that skin breaks and gills are potential attachment and entry portals for vAh. Once vAh breaches the skin or epithelial surfaces, it can cause a systemic infection rapidly, spreading to all internal organs. To our best knowledge, this is the first study that reports the development of a bioluminescent vAh and provides visual evidence for catfish-vAh interactions. Findings are expected to provide a better understanding of vAh pathogenicity in catfish.

Analysis of endogenous plasmids in Lacticaseibacillus paracasei ZY-1 and development of expression vectors

The construction of efficient and stable Lactobacillus expression vector is critical for strain improvement and development of customized strains. In this study, four endogenous plasmids were isolated from Lacticaseibacillus paracasei ZY-1 and subjected to functional analysis. The Escherichia coli-Lactobacillus shuttle vectors pLPZ3N and pLPZ4N were constructed by combining the replicon rep from pLPZ3 or pLPZ4, the chloramphenicol acetyltransferase gene cat from pNZ5319 and the replicon ori from pUC19. Moreover, the expression vectors pLPZ3E and pLPZ4E with the promoter Pldh3 of lactic acid dehydrogenase and the mCherry red fluorescent protein as a reporter gene were obtained. The size of pLPZ3 and pLPZ4 were 6 289 bp and 5 087 bp, respectively, and its GC content, 40.94% and 39.51%, were similar. Both shuttle vectors were successfully transformed into Lacticaseibacillus, and the transformation efficiency of pLPZ4N (5.23×102-8.93×102 CFU/μg) was slightly higher than that of pLPZ3N. Furthermore, the mCherry fluorescent protein was successfully expressed after transforming the expression plasmids pLPZ3E and pLPZ4E into L. paracasei S-NB. The β-galactosidase activity of the recombinant strain obtained from the plasmid pLPZ4E-lacG constructed with Pldh3 as promoter was higher than that of the wild-type strain. The construction of shuttle vectors and expression vectors provide novel molecular tools for the genetic engineering of Lacticaseibacillus strains.

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 × 105-1.2 × 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of ≥0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (β-lactams), aadA, aac (6')-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, β-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, thechloramphenicol acetyltransferase gene catB10, andthe rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 × 105-1.2 × 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of ≥0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae, and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (β-lactams), aadA, aac (6')-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, β-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the chloramphenicol acetyltransferase gene catB10, and the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Clinical and Environmental Vibrio cholerae Non-O1, Non-O139 Strains from Australia Have Similar Virulence and Antimicrobial Resistance Gene Profiles.

Cholera caused by pathogenic Vibrio cholerae is still considered one of the major health problems in developing countries including those in Asia and Africa. Australia is known to have unique V. cholerae strains in Queensland waterways, resulting in sporadic cholera-like disease being reported in Queensland each year. We conducted virulence and antimicrobial genetic characterization of O1 and non-O1, non-O139 V. cholerae (NOVC) strains (1983 to 2020) from Queensland with clinical significance and compared these to environmental strains that were collected as part of a V. cholerae monitoring project in 2012 of Queensland waterways. In this study, 87 V. cholerae strains were analyzed where O1 (n = 5) and NOVC (n = 54) strains from Queensland and international travel-associated NOVC (n = 2) (61 in total) strains were sequenced, characterized, and compared with seven previously sequenced O1 strains and 18 other publicly available NOVC strains from Australia and overseas to visualize the genetic context among them. Of the 61 strains, three clinical and environmental NOVC serogroup strains had cholera toxin-producing genes, namely, the CTX phage (identified in previous outbreaks) and the complete Vibrio pathogenicity island 1. Phylogenetic analysis based on core genome analysis showed more than 10 distinct clusters and interrelatedness between clinical and environmental V. cholerae strains from Australia. Moreover, 30 (55%) NOVC strains had the cholix toxin gene (chxA) while only 11 (20%) strains had the mshA gene. In addition, 18 (34%) NOVC strains from Australia had the type three secretion system and discrete expression of type six secretion system genes. Interestingly, four NOVC strains from Australia and one NOVC strain from Indonesia had intSXT, a mobile genetic element. Several strains were found to have beta-lactamase (blaCARB-9) and chloramphenicol acetyltransferase (catB9) genes. Our study suggests that Queensland waterways can harbor highly divergent V. cholerae strains and serve as a reservoir for various V. cholerae-associated virulence genes which could be shared among O1 and NOVC V. cholerae strains via mobile genetic elements or horizontal gene transfer. IMPORTANCE Australia has its own V. cholerae strains, both toxigenic and nontoxigenic, that are associated with cholera disease. This study aimed to characterize a collection of clinical and environmental NOVC strains from Australia to understand their virulence and antimicrobial resistance profile and to place strains from Australia in the genetic context of international strains. The findings from this study suggest the toxigenic V. cholerae strains in the Queensland River water system are of public health concern. Therefore, ongoing monitoring and genomic characterization of V. cholerae strains from the Queensland environment are important and would assist public health departments to track the source of cholera infection early and implement prevention strategies for future outbreaks. Understanding the genomics of V. cholerae could also inform the natural ecology and evolution of this bacterium in natural environments.

A paradox of bacterial persistence and antibiotic resistance: chloramphenicol acetyl transferase as a double barrel shot gun.

The problematic microbial resistance to antibiotics has led to an increasing interest in bacterial persistence and its impact on infection. Nonetheless, these two mechanisms are often assessed in independent studies, and there is a lack of knowledge about their relation or possible interactions, both at cellular and population levels. This work shows evidence that the insertion of the resistance gene Chloramphenicol Acetyl Transferase (cat) together with its cognate antibiotic chloramphenicol (CAM), is capable to modulate Salmonella Typhimurium persistence to several antibiotics and decrease its survival. This effect is independent of the antibiotics' mechanisms of action or the locus of cat. RelA [p(ppGpp) syntetase] has been shown to be involved in persistence. It was recently proposed that RelA [(p)ppGpp synthetase], binds to uncharged tRNAs, forming RelA.tRNA complexes. These complexes bind to vacant A-sites in the ribosome, and this mechanism is essential for the activation of RelA. In this study, we propose that the antibiotic chloramphenicol blocks the A-site of the ribosome, hindering the binding of RelA.tRNA complexes to the ribosome thus preventing the activation of RelA and (p)ppGpp synthesis, with a consequent decrease in the level of persistence of the population. Our discovery that the concomitant use of chloramphenicol and other antibiotics in chloramphenicol resistant bacteria can decrease the persister levels can be the basis of novel therapeutics aiming to decrease the persisters and recalcitrant infections.

Evaluation of inducible promoter-riboswitch constructs for heterologous protein expression in the cyanobacterial species Anabaena sp. PCC 7120.

Cyanobacteria are promising chassis for synthetic biology applications due to the fact that they are photosynthetic organisms capable of growing in simple, inexpensive media. Given their slower growth rate than other model organisms such as Escherichia coli and Saccharomyces cerevisiae, there are fewer synthetic biology tools and promoters available for use in model cyanobacteria. Here, we compared a small library of promoter–riboswitch constructs for synthetic biology applications in Anabaena sp. PCC 7120, a model filamentous cyanobacterium. These constructs were designed from six cyanobacterial promoters of various strengths, each paired with one of two theophylline-responsive riboswitches. The promoter–riboswitch pairs were cloned upstream of a chloramphenicol acetyltransferase (cat) gene, and CAT activity was quantified using an in vitro assay. Addition of theophylline to cultures increased the CAT activity in almost all cases, allowing inducible protein production with natively constitutive promoters. We found that riboswitch F tended to have a lower induced and uninduced production compared to riboswitch E for the weak and medium promoters, although the difference was larger for the uninduced production, in accord with previous research. The strong promoters yielded a higher baseline CAT activity than medium strength and weak promoters. In addition, we observed no appreciable difference between CAT activity measured from strong promoters cultured in uninduced and induced conditions. The results of this study add to the genetic toolbox for cyanobacteria and allow future natural product and synthetic biology researchers to choose a construct that fits their needs.

Construction of a reporter system for bifidobacteria using chloramphenicol acetyltransferase and its application for evaluation of promoters and terminators

A reporter assay system is an essential tool for investigating gene expression mechanisms. In the case of bifidobacteria, several convenient and sensitive reporter systems have been developed. Here, we developed a new reporter system for bifidobacteria using the chloramphenicol acetyltransferase gene (cat) from Staphylococcus aureus. This enzyme stoichiometrically produced free CoA-SH, which was analyzed quantitatively with Ellman’s test using 2-nitrobenzoic acid (DTNB). The 2-nitro-5-thiobenzoate (TNB2-) produced showed a strong yellowish color with maximum absorbance at 412 nm. We also constructed a new pBCMAT plasmid series for CAT assays in bifidobacteria to evaluate promoters and terminators. Analyses using promoters from Bifidobacterium longum NCC2705 indicated that the CAT assay using these promoters is quantitative, has a wide measurement range, and is stable. In addition, this assay was useful for several bifidobacterial species, including B. longum, Bifidobacterium breve, and Bifidobacterium adolescentis. Compared with evoglow-Bs2, a fluorescent protein used under anaerobic conditions, the CAT assay showed about 0.25% background activity. In analyses using this CAT assay, we identified 11 promoters and 12 terminators of B. longum NCC2705. The genes encoding ribosomal proteins, elongation factors, and transfer RNAs possessed strong promoters, and terminators that include strong stem-loops and poly-U tails structures tended to show high activities. Although the abovementioned promoters made stronger contributions to expression activities than the terminators, the maximum fold difference in the activities among the tested terminators was approximately 17-fold. Modification of the -10 box and 5’-UTR in the promoters and the structure around the stem-loop in the terminators affected expression levels. These results suggest that the CAT assay is useful for various analyses of bifidobacterial gene expression.

Antibiotic-Resistant and Non-Resistant Bacteria Display Similar Susceptibility to Dielectric Barrier Discharge Plasma.

Here, we examined whether antibiotic-resistant and non-resistant bacteria show a differential susceptibility to plasma treatment. Escherichia coli DH5α were transformed with pPRO-EX-HT-CAT, which encodes an ampicillin resistance gene and chloramphenicol acetyltransferase (CAT) gene, and then treated with a dielectric barrier discharge (DBD) plasma torch. Plasma treatment reduced the viable cell count of E. coli after transformation/selection and further cultured in ampicillin-containing and ampicillin-free medium. However, there was no significant difference in viable cell count between the transformed and untransformed E. coli after 1 min- and 2 min-plasma treatment. Furthermore, the enzyme-linked immunosorbent assay (ELISA) and acetyltransferase activity assay showed that the CAT activity was reduced after plasma treatment in both transformed and selected E. coli grown in ampicillin-containing or ampicillin-free medium. Loss of lipopolysaccharide and DNA damage caused by plasma treatment were confirmed by a Limulus test and polymerase chain reaction, respectively. Taken together, these findings suggest the plasma acts to degrade components of the bacteria and is therefore unlikely to display a differential affect against antibiotic-resistant and non-resistant bacteria. Therefore, the plasma method may be useful in eliminating bacteria that are recalcitrant to conventional antibiotic therapy.

Antimicrobial Resistance in Bordetella bronchiseptica.

Bordetella bronchiseptica is involved in respiratory tract infections mainly in dogs and pigs but may also cause infections in humans. Valid and representative data on antimicrobial susceptibility of B. bronchiseptica is rare. Approved antimicrobial susceptibility testing methods have been published, but very few clinical breakpoints are available. The MIC values are low for most agents but high for β-lactam antibiotics and macrolides. Information on the genetic basis of resistance is scarce. For a small number of isolates that are resistant or show elevated MICs, the molecular basis of resistance was identified. Three tetracycline resistance genes, tet(A), tet(C), and tet(31), coding for major facilitator superfamily efflux pumps, were identified. Two other major facilitator superfamily exporter genes confer resistance to chloramphenicol (cmlB1) or to chloramphenicol and florfenicol (floR). Two class B chloramphenicol acetyltransferase genes (catB1 and catB3), which confer resistance to nonfluorinated phenicols by enzymatic inactivation, have been identified in B. bronchiseptica. Like the trimethoprim resistance genes dfrA1 and dfrB1, which code for trimethoprim-insensitive dihydrofolate reductases, the genes catB1 and catB3 were located on gene cassettes and found in class 1 integrons also harboring the sulfonamide resistance gene sul1. In addition, the gene sul2 has also been detected. Both sul1 and sul2 code for sulfonamide-insensitive dihydropteroate synthases. A gene cassette harboring the β-lactamase gene blaOXA-2 was also identified, whereas β-lactam resistance in B. bronchiseptica seems to be more likely due to reduced influx in combination with the species-specific β-lactamase encoded by blaBOR-1. The resistance genes were mostly located on conjugative plasmids.

Genetic Determinants of Tetracycline Resistance in Clinical Streptococcus pneumoniae Serotype 1 Isolates from Niger.

Streptococcus pneumoniae serotype 1 is the first cause of pneumococcal meningitis Niger. To determine the underlying mechanism of resistance to tetracycline in serotype 1 Streptococcus pneumoniae, a collection of 37 isolates recovered from meningitis patients over the period of 2002 to 2009 in Niger were analyzed for drug susceptibility, and whole genome sequencing (WGS) was performed for molecular analyses. MIC level was determined for 31/37 (83.8%) isolates and allowed detection of full resistance (MIC = 8 µg) in 24/31 (77.4%) isolates. No resistance was found to macrolides and quinolones. Sequence-types deduced from WGS were ST217 (54.1%), ST303 (35.1%), ST2206 (5.4%), ST2839 (2.7%) and one undetermined ST (2.7%). All tetracycline resistant isolates carried a Tn5253 like element, which was found to be an association of two smaller transposons of Tn916 and Tn5252 families. No tet(O) and tet(Q) genes were detected. However, a tet(M) like sequence was identified in all Tn5253 positive strains and was found associated to Tn916 composite. Only one isolate was phenotypically resistant to chloramphenicol, wherein a chloramphenicol acetyl transferase (cat) gene sequence homologous to catpC194 from the Staphylococcus aureus plasmid pC194 was detected. In conclusion, clinical Streptococcus pneumoniae type 1 isolated during 2002 to 2009 meningitis surveillance in Niger were fully susceptible to macrolides and quinolones but highly resistant to tetracycline (77.4%) through acquisition of a defective Tn5253 like element composed of Tn5252 and Tn916 transposons. Of the 31 tested isolates, only one was exceptionally resistant to chloramphenicol and carried a Tn5253 transposon that contained cat gene sequence.

Phylogenetic Analysis of the Antibiotic Resistance Genes in Salmonella Species in silico.

Antibiotic resistance is an emerging problem in both developed and developing countries. It has been responsible for 700,000 deaths worldwide. Some genotypes of bacteria are sensitive to certain antibiotics than others. Hence by conducting phylogenetic analysis of bacteria and detecting the presence of resistance genes in each genotype, we can select the antibiotic that would be most effective for the bacteria in that certain genotype. A total of forty-five Salmonella species were investigated for the presence of antibiotic resistance genes through in silico PCR (polymerase chain reaction) amplification and PFGE (pulsed-field gel electrophoresis) analysis was conducted to assess the phylogenetic relationship. Total twenty-eight antibiotic resistance genes were selected for screening the isolates and seventeen antibiotic resistance genes among the Salmonella strains were found. Almost all the isolates (n=43) exhibited PCR amplification product for gyrA genes while fluoroquinolone resistance gyrB (66.67%), parC (68.89%) and parE (15.56%) genes were also present. About 15.56% and 11.11% isolates were found to harbor adenylyltransferase gene, aadA1 and aadA2, respectively while phosphotransferase gene was detected in only one isolate. Two isolates expressed both chloramphenicol acetyltransferase genes, cat1 and cat2. Three isolates (6.67%) harbored chloramphenicol resistance gene cmlA gene while two isolates (4.44%) expressed florfenicol resistance gene, floR. Tetracycline resistance gene, tetA was more prevalent (8.89%) than tetG genes (2.22%). Salmonella harbored all three sulfonamide resistance genes while sulII was more prevalent (17.78%). Genotype 2 contained fifteen antibiotic resistance genes while genotype 3 contained only one antibiotic resistance genes. These investigations used a computer aided approach to genotype isolates and assess the difference in antibiotic resistance profile of Salmonella species based on genotype. This data helps to predict antibiotic resistance genes that might be present for an isolate of known genotype and select antibiotic for the treatment of Salmonella infections based on their phylogenetic group.

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 β-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.

RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants.

The physiological function of Arabidopsis thaliana universal stress protein (AtUSP) in plant has remained unclear. Thus, we report here the functional role of the Arabidopsis universal stress protein, AtUSP (At3g53990). To determine how AtUSP affects physiological responses towards cold stress, AtUSP overexpression (AtUSP OE) and T-DNA insertion knock-out (atusp, SALK_146059) mutant lines were used. The results indicated that AtUSP OE enhanced plant tolerance to cold stress, whereas atusp did not. AtUSP is localized in the nucleus and cytoplasm, and cold stress significantly affects RNA metabolism such as by misfolding and secondary structure changes of RNA. Therefore, we investigated the relationship of AtUSP with RNA metabolism. We found that AtUSP can bind nucleic acids, including single- and double-stranded DNA and luciferase mRNA. AtUSP also displayed strong nucleic acid-melting activity. We expressed AtUSP in RL211 Escherichia coli, which contains a hairpin-loop RNA structure upstream of chloramphenicol acetyltransferase (CAT), and observed that AtUSP exhibited anti-termination activity that enabled CAT gene expression. AtUSP expression in the cold-sensitive Escherichia coli (E. coli) mutant BX04 complemented the cold sensitivity of the mutant cells. As these properties are typical characteristics of RNA chaperones, we conclude that AtUSP functions as a RNA chaperone under cold-shock conditions. Thus, the enhanced tolerance of AtUSP OE lines to cold stress is mediated by the RNA chaperone function of AtUSP.

Construction of novel pJRD215-derived plasmids using chloramphenicol acetyltransferase (cat) gene as a selection marker for Acidithiobacillus caldus.

BackgroundAcidithiobacillus caldus, a Gram-negative, chemolithotrophic sulfur-oxidizing bacterium, is widely applied in bioleaching. The absence of an ideal selection marker has become a major obstacle to achieve high efficiency of the gene transfer system for A. caldus. Plasmid pJRD215, widely used in Acidithiobacillus spp., has severe drawbacks in molecular manipulations and potential biosafety issues due to its mobility. Therefore, finding a new selection marker and constructing new plasmids have become an urgent and fundamental work for A. caldus.ResultsEffective inhibitory effect of chloramphenicol on the growth of A. caldus was elucidated for the first time. The P2-cat gene cassette, including a chloramphenicol acetyltransferase gene (cat) from plasmid pACBSR and a promoter (P2) upstream of the tetracycline resistance gene on pBR322, was designed, chloramphenicol acetyltransferase was expressed in A. caldus, and the enzyme activity was assessed. A new vector pSDU1 carrying the replication and mobilization regions derived from pJRD215, the P2-cat gene cassette and a multiple cloning site from pUC19 was successfully constructed. Compared with pJRD215, pSDU1 had a 27-fold increase in electrotransformation efficiency (30.43±0.88×104 CFU/μg DNA for pSDU1 and 1.09±0.11×104 CFU/μg DNA for pJRD215), better carrying capacity and could offer more convenience for the restriction enzyme digestion. In addition, the generated plasmid pSDU1Δmob, a novel non-mobilizable derivative of pSDU1 lacking some DNA sequences involved in the mobilization process, had increased copy number in A. caldus and lost its mobility for biosafety considerations. Both pSDU1 and pSDU1Δmob exhibited stable maintenance in A. caldus within 50 passages. However, further deletion of orfEF region involved in regulating repAC operon resulted in a negative effect on transformation efficiency, copy number and stability of plasmid pSDU1ΔmobΔorfEF in A. caldus.ConclusionChloramphenicol was proved to be an ideal selection marker for A. caldus. Novel plasmids carrying cat gene were constructed. The utilization of these vectors will undoubtedly facilitate efficient genetic manipulations and accelerate the research progress in A. caldus.

Unusual acylation of chloramphenicol in Lysobacter enzymogenes, a biocontrol agent with intrinsic resistance to multiple antibiotics

BackgroundThe environmental gliding bacteria Lysobacter are emerging as a new group of biocontrol agents due to their prolific production of lytic enzymes and potent antibiotic natural products. These bacteria are intrinsically resistant to many antibiotics, but the mechanisms behind the antibiotic resistance have not been investigated.ResultsPreviously, we have used chloramphenicol acetyltransferase gene (cat) as a selection marker in genetic manipulation of natural product biosynthetic genes in Lysobacter, because chloramphenicol is one of the two common antibiotics that Lysobacter are susceptible to. Here, we found L. enzymogenes, the most studied species of this genus, could still grow in the presence of a low concentration of chloramphenicol. Three chloramphenicol derivatives (1–3) with an unusual acylation pattern were identified in a cat-containing mutant of L. enzymogenes and in the wild type. The compounds included chloramphenicol 3'-isobutyrate (1), a new compound chloramphenicol 1'-isobutyrate (2), and a rare chloramphenicol 3'-isovalerate (3). Furthermore, a mutation of a global regulator gene (clp) or a Gcn5-related N-acetyltransferase (GNAT) gene in L. enzymogenes led to nearly no growth in media containing chloramphenicol, whereas a complementation of clp restored the chloramphenicol acylation as well as antibiotic HSAF production in the clp mutant.ConclusionsThe results indicated that L. enzymogenes contains a pool of unusual acyl donors for enzymatic modification of chloramphenicol that confers the resistance, which may involve the Clp-GNAT regulatory system. Because Lysobacter are ubiquitous inhabitants of soil and water, the finding may have important implications in understanding microbial competitions and bioactive natural product regulation.

Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae.

The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae.

Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?

Bacteria from the family Flavobacteriaceae often show low susceptibility to antibiotics. With the exception of two Chryseobacterium spp. isolates that were positive for the florfenicol resistance gene floR, no clinical resistance genes were identified by microarray in 36 Flavobacteriaceae isolates from salmonid fish that could grow in ≥ 4 mg/L florfenicol. Whole genome sequence analysis of the floR positive isolates revealed the presence of a region that contained the antimicrobial resistance genes floR, a tet(X) tetracycline resistance gene, a streptothricin resistance gene and a chloramphenicol acetyltransferase gene. In silico analysis of 377 published genomes for Flavobacteriaceae isolates from a range of sources confirmed that well-characterised resistance gene cassettes were not widely distributed in bacteria from this group. Efflux pump-mediated decreased susceptibility to a range of antimicrobials was confirmed in both floR positive isolates using an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide) assay. The floR isolates possessed putative virulence factors, including production of siderophores and haemolysins, and were mildly pathogenic in rainbow trout. Results support the suggestion that, despite the detection of floR, susceptibility to antimicrobials in Flavobacteriaceae is mostly mediated via intrinsic mechanisms rather than the horizontally acquired resistance genes more normally associated with Gram-negative bacterial pathogens such as Enterobacteriaceae.

Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments

Key messageWe have successfully transformed an exthemophilic red alga with the chloramphenicol acetyltransferase gene, rendering this organism insensitive to its toxicity. Our work paves the way to further work with this new modelorganism.Here we report the first successful attempt to achieve a stable, under selectable pressure, chloroplast transformation in Cyanidioschizon merolae—an extremophilic red alga of increasing importance as a new model organism. The following protocol takes advantage of a double homologous recombination phenomenon in the chloroplast, allowing to introduce an exogenous, selectable gene. For that purpose, we decided to use chloramphenicol acetyltransferase (CAT), as chloroplasts are particularly vulnerable to chloramphenicol lethal effects (Zienkiewicz et al. in Protoplasma, 2015, doi:10.1007/s00709-015-0936-9). We adjusted two methods of DNA delivery: the PEG-mediated delivery and the biolistic bombardment based delivery, either of these methods work sufficiently with noticeable preference to the former. Application of a codon-optimized sequence of the cat gene and a single colony selection yielded C. merolae strains, capable of resisting up to 400 µg/mL of chloramphenicol. Our method opens new possibilities in production of site-directed mutants, recombinant proteins and exogenous protein overexpression in C. merolae—a new model organism.

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.