Expression Of Cassettes Research Articles

The expression of integron arrays is shaped by the translation rate of cassettes

Integrons are key elements in the rise and spread of multidrug resistance in Gram-negative bacteria. These genetic platforms capture cassettes containing promoterless genes and stockpile them in arrays of variable length. In the current integron model, expression of cassettes is granted by the Pc promoter in the platform and is assumed to decrease as a function of its distance. Here we explored this model using a large collection of 136 antibiotic resistance cassettes and show the effect of distance is in fact negligible. Instead, cassettes have a strong impact in the expression of downstream genes because their translation rate affects the stability of the whole polycistronic mRNA molecule. Hence, cassettes with reduced translation rates decrease the expression and resistance phenotype of cassettes downstream. Our data puts forward an integron model in which expression is contingent on the translation of cassettes upstream, rather than on the distance to the Pc.

Hif-1α expression targets the TMA/Fmo3/TMAO axis to participate in gallbladder cholesterol stone formation in individuals living in plateau regions

The incidence of gallbladder cholesterol stones (GCS) increases rapidly among people living in high-altitude hypoxic environments compared to those in normoxic areas. Upregulation of hepatic hypoxia inducible factor 1α (Hif-1α) plays a key role in the formation of GCS. High plasma trimethylamine-N-oxide (TMAO) levels are positively correlated with the occurrence of GCS. We hypothesized that HIF-1α may upregulate TMAO levels by promoting the transcription of flavin-containing monooxygenase 3 (Fmo3), which eventually leads to GCS formation. Our study shows that in women, high plasma total cholesterol and apolipoprotein B were positively correlated with cholecystolithiasis and hypoxia. Hif-1α binds to the Fmo3 promoter and promotes Fmo3 expression. Hypoxia and lithogenic diet induce the expression of Hif-1α, Fmo3, TMAO and cholesterol tube transporters in the livers of mice, disturb the proportion of bile and plasma components, and induce the formation of GCS. In cell experiments, silencing Hif-1α downregulates the expression of Fmo3, TMAO and cholesterol tube transporters. In a mouse model of hypoxic cholecystolithiasis, silencing Hif-1α downregulates the expression of related genes, restores the proportion of bile and plasma lipid components, and reduces the formation of GCS. Our study shows that Hif-1α binds to the promoter region of Fmo3 and promotes Fmo3 transcription. Thus, it mediates the transcriptional activation of the TMA/Fmo3/TMAO pathway, upregulates the expression of ATP-binding cassettes (Abc) g5 and g8, and participates in the regulation of the occurrence of GCS in the plateau region.

Identification of promoter activity in gene-less cassettes fromVibrionaceae superintegrons.

Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions. ICs generally encode promoterless genes, whose expression relies on the platform-associated Pc promoter, with the cassette array functioning as an operon-like structure regulated by the distance to the Pc. This is relevant in large sedentary chromosomal integrons (SCIs) carrying hundreds of ICs, like those in Vibrio species. We selected 29 gene-less cassettes in four Vibrio SCIs, and explored whether their function could be related to the transcription regulation of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Additionally, we identified the transcription start sites of gene-less ICs through 5'-RACE. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 cassette downstream, increasing trimethoprim resistance>512-fold in V. cholerae and Escherichia coli. Furthermore, one cassette with an antisense promoter can reduce trimethoprim resistance when cloned downstream. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in SCIs and their clinical importance if captured by mobile integrons.

Distribution diversity and expression regulation of class 1 integron promoters in clinical isolates of Morganella morganii.

Morganella morganii is an emerging nosocomial opportunistic pathogen with increasing multidrug resistance. Antibiotic resistance, driven primarily by the horizontal transfer of resistance genes, has become a global health crisis. Integrons, mobile genetic elements, are now understood to facilitate the transfer of these genes, contributing to the rapid proliferation of resistant strains. Understanding the regulatory role of integrons in drug resistance gene expression is crucial for developing novel strategies to combat this pressing public health issue. To investigate the distribution of promoter types in the variable regions of class 1 integrons isolated from clinical isolates of M. morganii and their regulatory role in the expression of downstream drug resistance gene cassettes. Ninety seven clinical isolates of M. morganii were screened for the presence of class 1 integrons (intI1) using polymerase chain reaction (PCR). Gene cassettes within the variable regions of positive isolates were characterized, and the gene cassette promoter Pc variants and downstream auxiliary promoter P2 were identified. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was employed for homology analysis. Recombinant plasmids containing different variable region promoters and gene cassettes were constructed to evaluate drug resistance genes and integrase (intI1) expression levels using reverse transcription-quantitative PCR (RT-qPCR) and antimicrobial susceptibility testing. Of the clinical isolates, 28.9% (n = 28/97) were positive for class 1 integrons. 24.7% (n = 24/97) of these isolates carried gene cassettes encoding resistance to aminoglycosides and trimethoprim. Three Pc promoter types (PcH1, PcS, and PcW) were identified, while all P2 promoters were inactive with a 14-base pair spacing between the -35 and -10 regions. ERIC-PCR analysis classified the integron-positive strains into 6 genotypes, with high consistency in promoter types and gene cassettes within each genotype. RT-qPCR and antimicrobial susceptibility testing demonstrated that strong promoters significantly enhanced the expression of downstream drug resistance gene cassettes compared to weak promoters. Additionally, RT-qPCR revealed a negative correlation between intI1 expression and Pc promoter strength. Class 1 integrons are prevalent in M. morganii. The promoter types within these integrons are diverse, and promoter strength is closely linked to downstream gene cassette expression. Integron-positive strains exhibit high homology, suggesting horizontal gene transfer and dissemination in clinical settings.

NFR2/ABC transporter axis in drug resistance of breast cancer cells.

Breast cancer is one of the most serious malignancies among women, accounting for about 12% of all cancers. The inherent complexity and heterogeneity of breast cancer results in failure to respond to treatment in the advanced stages of the disease. Breast cancer is caused by several genetic and environmental factors. One of the significant factors involved in the development of breast cancer is oxidative stress, which is generally regulated by nuclear factor erythroid 2-related factor 2 (NRF2). The level of NRF2 expression is low in healthy cells, which maintains the balance of the antioxidant system; however, its expression is higher in cancer cells, which have correlation characteristics such as angiogenesis, stem cell formation, drug resistance, and metastasis. Drug resistance increases with the upregulation of NRF2 expression, which contributes to cell protection. NRF2 controls this mechanism by increasing the expression of ATP-binding cassettes (ABCs). Considering the growing number of studies in this field, we aimed to investigate the relationship between NRF2 and ABCs, as well as their role in the development of drug resistance in breast cancer.

Characterization and application of several lysis cassettes

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S105, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD600, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S105, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.

Biochar and Manure Applications Differentially Altered the Class 1 Integrons, Antimicrobial Resistance, and Gene Cassettes Diversity in Paddy Soils.

Integrons are genetic components that are critically involved in bacterial evolution and antimicrobial resistance by assisting in the propagation and expression of gene cassettes. In recent decades, biochar has been introduced as a fertilizer to enhance physiochemical properties and crop yield of soil, while manure has been used as a fertilizer for centuries. The current study aimed to investigate the impact of biochar, manure, and a combination of biochar and manure on integrons, their gene cassettes, and relative antimicrobial resistance in paddy soil. Field experiments revealed class 1 (CL1) integrons were prevalent in all samples, with higher concentration and abundance in manure-treated plots than in biochar-treated ones. The gene cassette arrays in the paddy featured a broad pool of cassettes with a total of 35% novel gene cassettes. A majority of gene cassettes encoded resistance to aminoglycosides, heat shock protein, heavy metals, pilus secretory proteins, and twin-arginine translocases (Tat), TatA, TatB, and TatC. Both in combination and solo treatments, the diversity of gene cassettes was increased in the manure-enriched soil, however, biochar reduced the gene cassettes’ diversity and their cassettes array. Manure considerably enhanced CL1 integrons abundance and antimicrobial resistance, whereas biochar amendments significantly reduced integrons and antimicrobial resistance. The results highlighted the differential effects of biochar and manure on integrons and its gene cassette arrays, showing increased abundance of integrons and antibiotic resistance upon manure application and decrease of the same with biochar. The use of biochar alone or in combination with manure could be a beneficial alternative to mitigate the spread of antimicrobial resistance and bacterial evolution in the environment, specifically in paddy soils.

Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato.

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

Promoter activity of ORF-less gene cassettes isolated from the oral metagenome

Integrons are genetic elements consisting of a functional platform for recombination and expression of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA mediated gene regulation. In this study, the promoter activity of ORF-less GCs, previously recovered from the oral metagenome, was verified by cloning them upstream of a gusA reporter, proving they can function as a promoter, presumably allowing bacteria to adapt to multiple stresses within the complex physico-chemical environment of the human oral cavity. A bi-directional promoter detection system was also developed allowing direct identification of clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified from the human oral metagenomic DNA using this construct, called pBiDiPD. This is the first demonstration and detection of promoter activity of ORF-less GCs from Treponema bacteria and the development of an agar plate-based detection system will enable similar studies in other environments.

Chlamydomonas BAC vectörlerinin RED/ET yöntemiyle tek basamakta modifikasyonu

With the availability of the complete genome, Chlamydomonas BACs are being used extensively in functional genomics analysis. The following aspects of their construction, however, make them less than optimal for some types of analysis. (1) These BACs do not contain a gene to allow direct selection of transformants in complementation analysis. (2) Co-transformation using the BAC and an independent vector with a selectable marker has a low efficiency. (3) Most BACs have more than one gene, necessitating sub-cloning of each gene into a different vector (relying on the use of restriction enzymes). To simplify this process, we modified Chlamydomonas BACs by inserting 2 selectable marker cassettes, using the RED-ET system. We ligated a eukaryotic and a prokaryotic selectable marker cassette and used it in a one-step modification instead of a two-step counter selection protocol and showed the expression of both cassettes. This method will decrease the time needed for use of BACs in functional genomics analysis in Chlamydomonas.

Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and CAR NK-cell lines

BackgroundCytotoxic activity of T- and NK-cells can be efficiently retargeted against cancer cells using chimeric antigen receptors (CARs) and rTCRs. In the context of solid cancers, use of armored CAR T- and NK cells secreting additional anti-cancer molecules such as cytokines, chemokines, antibodies, BiTEs, inverted cytokine receptors, and checkpoint inhibitors, appears particularly promising, as this may help overcome immunosuppressive tumor microenvironment, attract bystander immune cells, and boost CAR T/NK-cell persistence. Placing the expression of such molecules under the transcriptional control downstream of CAR-mediated T/NK-cell activation offers the advantage of targeted delivery, high local concentration, and reduced toxicity. Several canonic DNA sequences that are known to function as activation-inducible promoters in human T and B cells have been described to date and typically encompass the multimers of NFkB and NFAT binding sites. However, relatively little is known about the DNA sequences that may function as activation-driven switches in the context of NK cells. We set out to compare the functionality of several activation-inducible promoters in primary human T cells, as well as in NK cell lines NK-92 and YT.MethodsLentiviral constructs were engineered to express two fluorescent reporters: mCherry under 4xNFAT, 2xNFkB, 5xNFkB, 10xNFkB, 30xNFkB promoters, as well as two variants of the CD69 promoter, and copGFP under the strong constitutive promoter of the human EF1a gene. Pseudotyped lentiviral particles obtained using these constructs were transduced into primary human T cells and NK-92 and YT cell lines expressing a CAR specific for PSMA. The transgenic cells obtained were activated by CD3/CD28 beads (T cells) or via a CAR (CAR-NK cell lines). Promoter activity before and after activation was assayed using FACS analysis.ResultsIn T cells, the CD69 promoter encompassing CNS1 and CNS2 regions displayed the highest signal/noise ratio. Intriguingly, in the context of CAR-YT cell line neither of the seven promoters tested displayed acceptable activation profile. In CAR-NK-92 cells, the largest fold activation (which was modest) was achieved with the 10xNFkB and 30xNFkB promoters, however its expression was clearly leaky in “resting” non-activated cells.ConclusionsUnlike in T cells, the robust activation-driven inducible expression of genetic cassettes in NK cells requires unbiased genome-wide identification of promoter sequences.

Class 1 and 2 Integrons in Hospital Strains of Gram-Negative Bacteria Isolated in Moscow and in Regions of the Russian Federation

Natural systems of cloning and expression of mobile gene cassettes caught by site-specific recombination, class 1 and 2 integrons, play an important role in mobilization and spread of genetic determinants of antibiotic resistance in gram-negative bacterial human pathogens, especially in a hospital environment. The gene cassettes localized in variable parts of integrons determine resistance to antibacterial drugs (AD) of different functional classes. The aim of the work is the detection and characteristic of class 1 and 2 integrons in gram-negative bacteria isolated in multidisciplinary hospitals of Moscow and other regions of the Russian Federation in 2003–2015. Clinical strains of gram-negative bacteria (n = 1248) mainly had multidrug resistance phenotype (94%). An amount of 10% of strains were resistant to AD of three functional groups; 19%, four; 42%, five; 17%, six; and 7%, seven. A high level of resistance of the studied strains to beta-lactams is associated with the presence of beta-lactamase genes of blaTEM (35% strains), blaSHV (25%), blaCTX-M (38%), blaOXA (31%), blaVIM (3%), and blaNDM (2%) types; to AD of other functional groups, with the presence of class 1 integrons (59%) and class 2 integrons (8%). Most class 1 integrons (54%) and class 2 integrons (88%) contained in its variable part 22 variants of gene cassette arrays in class 1 integrons and 4 variants in class 2 integrons. During the study, 31 types of gene cassettes were identified (including the most widespread, aadB, aacA4, aacC1, aadA1, aadA2, aadA5, blaVIM-2, dfrA1, dfrA7, dfrA12, orfC, orfE, orfY, and sat1) associated with the resistance of strains to aminoglycosides, chloramphenicol, sulfonamides, and beta-lactams, as well as orf cassettes encoding the proteins with unknown functions. New gene cassette arrays were identified: dfrA12s-orfF-aadA2 (In1249) and dfrA1-IS911-sat1-aadA1 (not numbered).

Class 1 integrons in Acinetobacter baumannii: a weak expression of gene cassettes to counterbalance the lack of LexA-driven integrase repression

Integrons recruit resistance genes through integrase-driven recombination events that are regulated by the bacterial SOS response and require the repressor LexA. Class 1 integrons genes are expressed from a common promoter, Pc, of which at least 5 predominant variants, classified from weak to strong, have been described. In Escherichia coli, there is an intertwined regulation between gene cassette expression and integrase activity: the stronger the promoter, the weaker the integrase. Class 1 integrons have been frequently described in Acinetobacter baumannii. However, Acinetobacter spp. lack the LexA repressor, suggesting that the integrase is constitutively expressed. We characterized the integron content of 83 clinical and environmental A. baumannii strains. We found a predominance of Pc variants described as strong in E. coli. The Pc expression level was 2- to 4-fold lower in A. baumannii than in E. coli, and the diversity of the gene cassette array was low. In A. baumannii, integrons with a PcS promoter might have been selected to enable sufficient resistance while avoiding the toxicity of a highly active integrase. Furthermore, a transcriptional interference between PcS and PintI1 (as shown in E. coli) may limit the expression of the integrase and thus counterbalance the lack of LexA-driven integrase repression to prevent the cost of the integrase.

Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco

<p>Reporter gene assays are commonly used to study the expression pattern of a gene and the promoter activity. The purpose of this study was to assemble the chimeric gene constructs consisting of CaMV 35S promoter orOsAER1 gene promoter connected to the β-glucuronidase (GUS) reporter gene encoding the β-glucuronidase enzyme and to obtain an efficient method for Agrobacterium tumefaciens-mediated transient transformation of tobacco sprouts. The CaMV 35S promoter fragment reamplified from pCAMBIA1301 binary vector and the OsAER1 gene promoter fragment amplified from rice cv. Awan Kuning were ligated into pCAMBIA1300int::gus::tNOS to produce binary vectors pCAMBIA1300int::p35S::gus::tNOS and pCAMBIA1300int::prOsAER1::gus::tNOS. The vectors were used for transient transformation of 5–day old tobacco seedlings. The transformation was carried out using two bacterial cultures with densities of OD600 = 0.5 or OD600 = 1.0 combined with a vacuum for 15 or 30 minutes. Tobacco seedlings transformed with pCAMBIA1300int::p35S::gus::tNOS showed higher transformation efficiency as compared tothe ones transformed with pCAMBIA1300int::prOsAER1::gus::tNOS. A higher efficiency was obtained from transformation using bacterial culture with density of OD600 = 0.5 in combination with a vacuum for 30 minutes. Expression of GUS gene in the tobacco sprouts transformed with CaMV 35S promoter construct was observed through out the sprouts area (root, hypocotyl, cotyledon, and leaf), where as expression of GUS gene was observed in root, hypocotyl, and cotyledon, but not in leaf on tobacco sprouts transformed with OsAER1 promoter construct. These results indicate that the transient transformation is a quick and simple method for testing a chimeric gene construct.</p>

Abstract 447: Lipid Droplet Associated Hydrolase (LDAH) Impacts Oxysterol Metabolism and Prevents Atherosclerosis

Macrophage/foam cells in the arterial wall engulf apolipoprotein B-containing lipoproteins and store the surplus of lipids derived from these particles in lipid droplets (LDs). Even though cholesterol is abundant in the LDs of atheroma, the lipidome of LDs displays a variety of lipid species, including other sterols that have a deep impact on foam cell and plaque biology. While the LD is increasingly seen as a reservoir of signaling precursors, how the bioactive lipids are mobilized from the LDs remains elusive. Previously, we identified LDAH as a novel LD protein that contains a lipase/esterase sequence and reported it as a CE hydrolase. Our in vivo atherosclerosis studies on LDAH knock-out (KO) and transgenic (Tg) mice in apoE KO background revealed an athero-protective role of LDAH driven by its ability to reduce lipid accumulation in the lesional foam cells. LDAH increases expression of ATP-binding cassettes (ABC) A1 and G1, two cholesterol transporters that are regulated by liver X receptor (LXR). Interestingly, we also found that LDAH promotes favorable tissue remodeling, evidenced by a remarkable increase in lesional collagen content, which is known to contribute to plaque stability. To determine molecular mechanism behind these phenotypes we performed combinatory “omics” studies of LDAH WT, KO, and Tg foam cells: lipidomics (targeted and untargeted) and transcriptomics. Lipidomics analyses confirmed that LDAH inversely regulates total cholesterol levels, and revealed that several other lipid species were also altered by LDAH. Interestingly, among sterol lipids, LDAH preferentially targets esterified oxysterols whose free forms are LXR ligands. RNA-seq analysis identified several collagen synthesis genes regulated by LDAH, supporting the phenotype seen in the atheroma. Overall, our studies suggest that LDAH might play a central role in the production of messengers from ester precursors stored in LDs of foam cells. Therefore, unraveling the mechanisms of trafficking through LDs may be key to exploit the atheroprotective potential of endogenous lipid mediators.

Increased IFRD1 Expression in Human Colon Cancers Predicts Reduced Patient Survival.

Colon cancer (CRC) is the third most common cancer worldwide. CRC develops through combinations of genetic and epigenetic changes. However, there is marked heterogeneity in the "driver gene" mutational profiles within and among colon cancers from individual patients, and these are not sufficient to explain differences in colon cancer behavior and treatment response. Global modulation of the tumor landscape may play a role in cancer behavior. Interferon-related developmental regulator 1 (IFRD1) is a transcriptional co-regulator that modulates expression of large gene cassettes and plays a role in gut epithelial proliferation following massive intestinal resection. We address the hypothesis that increased IFRD1 expression in colon cancers is associated with poorer patient survival. Tumor and normal tissue from colon cancer patient cohorts from the USA, Spain, and China were used for this study. Cancers were scored for the intensity of IFRD1 immunostaining. The primary clinical outcome was overall survival defined as time from diagnosis to death due to cancer. Kaplan-Meier method and log-rank analysis were used to assess the association between IFRD1 expression and survival. Almost all (98.7%) colon cancers showed readily detectable IFRD1 expression, with immunoreactivity primarily in the tumor cytoplasm. High IFRD1 colon cancer expression was significantly associated with decreased 5-year patient survival. Patients in the American cohort with high IFRD1 expression had a poorer prognosis. We have demonstrated that high IFRD1 protein expression in colon cancer is associated with poorer patient prognosis, suggesting a potential role for IFRD1 in modulating tumor behavior.

Gene Expression in Class 2 Integrons Is SOS-Independent and Involves Two Pc Promoters.

Integrons are powerful bacterial genetic elements that permit the expression and dissemination of antibiotic-resistance gene cassettes. They contain a promoter Pc that allows the expression of gene cassettes captured through site-specific recombination catalyzed by IntI, the integron-encoded integrase. Class 1 and 2 integrons are found in both clinical and environmental settings. The regulation of intI and of Pc promoters has been extensively studied in class 1 integrons and the regulatory role of the SOS response on intI expression has been shown. Here we investigated class 2 integrons. We characterized the PintI2 promoter and showed that intI2 expression is not regulated via the SOS response. We also showed that, unlike class 1 integrons, class 2 integrons possess not one but two active Pc promoters that are located within the attI2 region that seem to contribute equally to gene cassette expression. Class 2 integrons mostly encode an inactive truncated integrase, but the rare class 2 integrons that encode an active integrase are associated with less efficient Pc2 promoter variants. We propose an evolutionary model for class 2 integrons in which the absence of repression of the integrase gene expression led to mutations resulting in either inactive integrase or Pc variants of weaker activity, thereby reducing the potential fitness cost of these integrons.

Preferential expression of domain cassettes 4, 8 and 13 of Plasmodium falciparum erythrocyte membrane protein 1 in severe malaria imported in France

ObjectivesSevere Plasmodium falciparum malaria (SM) involves cytoadhesion of parasitized red blood cells, mediated by P. falciparum erythrocyte membrane protein 1, which is encoded by var genes. Expression of var gene group A and B or encoding domain cassettes DC4, DC5, DC8 and DC13 has been implicated in SM in African children, but no data exist in the context of imported malaria. The aim of this study was to investigate var gene expression linked to clinical presentation and host factors in SM imported into France. MethodsExpression level of var gene groups A, B, C, var1, var2csa, var3 and var genes encoding DC4, DC5, DC8 and DC13 was measured by quantitative RT-PCR and expressed in transcript units. Seventy SM and 48 uncomplicated malaria (UM) P. falciparum cases were analysed according to disease severity, epidemiological characteristics (migrants or travellers) and anti-P. falciparum antibodies. Cluster analysis was performed to identify gene expression profiles. ResultsVar1 and B/C expression were higher in UM than SM (0.66 (0–1.1) and 1.88 (1.3–2.4); p <0.04, respectively). Group C expression differed between migrants and travellers (0.21 (0–0.75) versus 0 (0–0); p 0.002). Group A differed in naive and pre-exposed patients (1.1 (0.7–1.5) versus 0.4 (0–1.1); p 0.01). Population clusters revealed increased expression from group A and B var genes, and DC4, DC8 and DC13 in SM. ConclusionsThese results corroborate the implication of DC4, DC8 and DC13 in severe imported malaria cases as African children, and their expression depends of host factors.

Efficiency of integron cassette insertion in correct orientation is ensured by the interplay of the three unpaired features of attC recombination sites.

The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of cassettes carrying protein-coding sequences, and is responsible for the emergence and rise of multiresistance in Gram-negative bacteria. The functionality of this system depends on the insertion of promoterless cassettes in correct orientation, allowing their expression from the promoter located upstream of the cassette array. Correct orientation is ensured by strand selectivity of integron integrases for the bottom strand of cassette recombination sites (attC), recombined in form of folded single-stranded hairpins. Here, we investigated the basis of such strand selectivity by comparing recombination of wild-type and mutated attC sites with different lengths, sequences and structures. We show that all three unpaired structural features that distinguish the bottom and top strands contribute to strand selectivity. The localization of Extra-Helical Bases (EHBs) directly favors integrase binding to the bottom strand. The Unpaired Central Spacer (UCS) and the Variable Terminal Structure (VTS) influence strand selectivity indirectly, probably through the stabilization of the bottom strand and the resulting synapse due to the nucleotide skew between the two strands. These results underscore the importance of the single-stranded nature of the attC site that allows such tight control over integron cassette orientation.

DNA Secondary Structure Formation in Bacterial Gene Capture Systems at Single-Molecule Resolution

Bacteria with multiple antibiotic resistances are a threat to human health. These resistances spread faster than could be expected from mutations alone. It was shown that bacteria exhibit a mechanism of exchanging and collecting genetic sections - coding for antibiotic resistances or other adaptive traits - between individuals or even across species boundaries.This mechanism relies on genetic elements termed integrons. They allow the incorporation and expression of exogenous gene cassettes through a site-specific recombination process. The process involves an enzyme, integrase, which mediates recombination between a double stranded integron recombination site (attI site) and a single-stranded cassette recombination site (attC site). The attC site is supposably recognized by integrase through specificity to the secondary structure of the DNA hairpin formed by the single-stranded attC site (1).This poses the question of how the DNA hairpin forms inside the living cell. It was shown in vivo that negative superhelicity promotes integron recombination, most likely through cruciform extrusion from double-stranded DNA (2).Here, we use single-molecule FRET and magnetic tweezers to study the formation of the postulated DNA hairpin in the presence of various proteins. We present data on the competition between SSB and integrase and on the extrusion of E. coli attC sites from dsDNA and their interaction with integrase hinting at possible molecular mechanisms underlying the function of this system in bacteria.1. Mazel, D. 2006. Integrons: agents of bacterial evolution. Nat Rev Micro. 4: 608-620.2. Loot, C., D. Bikard, A. Rachlin, and D. Mazel. 2010. Cellular pathways controlling integron cassette site folding. EMBO J. 29: 3745-3745.