Characterization of the promoter region of the murine Catsper2 gene.

CATSPER2 (Cation channel sperm-associated protein 2) protein, which is part of the calcium CATSPER channel located in the membrane of the flagellar principal piece of the sperm cell, is only expressed in the testis during spermatogenesis. Deletions or mutations in the Catsper2 gene are associated with the deafness-infertility syndrome (DIS) and non-syndromic male infertility. However, the mechanisms by which Catsper2 is regulated are unknown. Here, we report the characterization of the promoter region of murine Catsper2 and the role of CTCF and CREMτ in its transcription. We report that the promoter region has transcriptional activity in both directions, as determined by observing luciferase activity in mouse Sertoli and GC-1 spg transfected cells. WGBS data analysis indicated that a CpG island identified in silico is non-methylated; Chromatin immunoprecipitation (ChIP)-seq data analysis revealed that histone marks H3K4me3 and H3K36me3 are present in the promoter and body of the Catsper2 gene respectively, indicating that Catsper2 is subject to epigenetic regulation. In addition, the murine Catsper2 core promoter was delimited to a region between -54/+189 relative to the transcription start site (TSS), where three CTCF and one CRE binding site were predicted. The functionality of these sites was determined by mutation of the CTCF sites and deletion of the CRE site. Finally, ChIP assays confirmed that CREMτ and CTCF bind to the Catsper2 minimal promoter region. This study represents the first functional analysis of the murine Catsper2 promoter region and the mechanisms that regulate its expression.

Down Regulation of Catsper1 Expression by Calmodulin Inhibitor (Calmidazolium): Possible Implications for Fertility.

The CatSper channel localizes exclusively in the flagella of sperm cells. The Catsper1 protein, together with three pore units, is essential for the CatSper Channel formation, which produces flagellum hyperactivation and confers sperm fertility. Catsper1 expression is dependent on Sox transcription factors, which can recognize in vitro at least three Sox binding sites on the promoter. Sox transcription factors have calmodulin-binding domains for nuclear importation. Calmodulin (CaM) is affected by the specific inhibitor calmidazolium (CMZ), which prevents the nuclear transport of Sox factors. In this work, we assess the regulation of the Catsper1 promoter in vivo by Sox factors in the murine testis and evaluate the effects of the inhibitor calmidazolium on the expression of the Casper genes, and the motility and fertility of the sperm. Catsper1 promoter has significant transcriptional activity in vivo; on the contrary, three Sox site mutants in the Catsper1 promoter reduced transcriptional activity in the testis. CaM inhibition affects Sox factor nuclear transport and has notable implications in the expression and production of Catsper1, as well as in the motility and fertility capability of sperm. The molecular mechanism described here might conform to the basis of a male contraceptive strategy acting at the transcriptional level by affecting the production of the CatSper channel, a fundamental piece of male fertility.

Acciones de vigilancia y control ante un caso de COVID-19 en el Policlínico José Luis Chaviano de Cienfuegos, Cuba / Surveillance and control actions in a COVID-19 case at the José Luis Chaviano Polyclinic in Cienfuegos, Cuba

RESUMEN La enfermedad COVID-19 constituye una amenaza para la salud pública mundial. La realidad observada en los últimos meses evidencia el aumento exponencial de los casos confirmados y la rápida propagación de la enfermedad con una alta letalidad. El objetivo de este trabajo es presentar la secuencia práctica epidemiológica de un caso de COVID-19 en un paciente del Área de salud I del municipio de Cienfuegos, confirmado al SARS-CoV-2, del sexo masculino, con 35 años de edad, de color de piel negra y sin antecedentes de enfermedades concomitantes, quien se encontraba sin sintomatología. El tiempo transcurrido entre el contacto directo y el diagnóstico de positividad fue entre tres y cinco días. Le fueron identificados y aislados 26 contactos directos. Se aislaron además dos casos sintomáticos clasificados como sospechosos y 15 contactos de estos casos. En cada momento se implementaron acciones de aislamiento y control. Las medidas adoptadas en el área de salud I del municipio de Cienfuegos estuvieron encaminadas a detener la transmisión y prevenir el contagio, reduciendo el riesgo de exposición al virus. Se ha demostrado que todas las medidas fueron inmediatas y escalables, en función de la situación epidemiológica concreta.

ABSTRACT COVID-19 disease constitutes a threat to global public health. The reality observed in recent months shows the exponential increase in confirmed cases and the rapid spread of the disease with high lethality. This work is aimed at presenting a case of COVID-19 practical epidemiological sequence, in a patient from Health Area I of the Cienfuegos municipality, confirmed to SARS-CoV-2, male, 35 years old, black and without a history of concomitant diseases, who was asymptomatic. The time elapsed between direct contact and positive diagnosis was between three and five days. A number of 26 direct contacts were identified and isolated. Two symptomatic cases classified as suspicious and 15 contacts of these cases were also isolated. Isolation and control actions were implemented at all times. The measures adopted in health area I of the Cienfuegos municipality were focused at stopping transmission and preventing contagion, reducing the risk of exposure to the virus. All measures have been shown to be immediate and scalable, depending on the specific epidemiological situation.

Domains two and three of Escherichia coli ribosomal S1 protein confers 30S subunits a high affinity for downstream A/U-rich mRNAs.

S1, a multi-domain ribosomal protein associated with the 30S subunit, is essential for translation initiation. S1 binds with high affinity to single-stranded mRNA containing A/U-rich patches upstream of the start codon. It was previously reported that domains 1-3 of S1 protein play a role in the docking and unfolding of structured mRNAs to the ribosome. Moreover, S1-deficient 30S subunits are still able to bind to low structured mRNAs. However, mRNAs containing A/U-rich patches in the early base positions after start codon enhance protein synthesis and mRNA binding to the ribosome, which suggests that S1 is also able to interact with these A/U-rich regions. To evaluate the essentiality of S1 domains in the binding to low structured mRNAs containing A/U/G nucleotides after the start codon as well as their role in translation and cell viability, S1 protein deletion variants were generated. We show that S1 domain 3 is necessary to discriminate these mRNAs according to the nucleotide nature since its absence abrogated S1 binding to A/U-rich mRNAs and allowed binding to G-rich mRNAs. Interestingly, domains 2 and 3 were required for the binding of mRNAs containing A/U-rich sequences after the start codon to 30S, in vitro translation and cell viability.

Regulation of CATSPER1 expression by the testis-determining gene SRY.

CATSPER1 gene encodes a pore-forming and pH-sensing subunit of the CatSper Ca2+- permeable channel, a protein in the flagellum essential for sperm hyperactivation. Previous studies have shown that the murine Catsper1 gene promoter is regulated by different Sox proteins. Likewise, it is acknowledged that the human CATSPER1 gene promoter sequence is enriched in potential interaction sites for the sex-determining region Y gene (SRY), which suggest a novel regulatory transcriptional mechanism for CatSper1 channel expression. Therefore, in this work, we sought to determine whether the human CATSPER1 gene expression is regulated by the SRY transcription factor. To this end, a series of deletions and mutations were introduced in the wild- type CATSPER1 gene promoter to eliminate the SRY sites, and the different constructs were tested for their ability to activate transcription in human embryonic kidney and murine spermatogonial germ cell lines (HEK-293 and GC1-spg, respectively) using luciferase assays. In addition, by using a strategy that combines electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) we investigated whether the CATSPER1 gene expression is regulated by the SRY transcription factor both in vitro and in vivo. Our results show that the transcriptional factor SRY specifically binds to different sites in the promoter sequence and has the ability to control CATSPER1 gene transcription.

Human CATSPER1 Promoter Is Regulated by CREB1 and CREMτ Transcriptional Factors In Vitro.

BACKGROUND: The CATSPER1 gene encodes a CATSPER channel protein that selectively permeates Ca2+ ions, and CATSPER expression in sperm is essential for flagellum hyperactivation and, thus, male fertility. Little is known regarding the transcriptional regulation of CATSPER1, but previous studies have performed in silico analyses of transcription factor binding sites, including three CRE sites designated 0-2, in which CRE0 is located near the transcription start site. OBJETIVES: We investigate if overexpression of CREB-A and CREMτ transcription factors might regulate CATSPER1 expression. MATERIAL AND METHODS: In this study, the transcriptional regulation of the CATSPER1 gene by CREB-A and CREMτ transcriptions factors was determined by dual-luciferase assays in HEK293 and GC1-spg cells, and important CRE sites were mutated and analyzed for transcriptional regulation. RESULTS: The deletion of the CRE1 site dramatically increased the transcriptional activity of the CATSPER1 promoter in HEK293 and GC1-spg cells. In HEK293 cells, the CREB-A transcription factor positively regulated CATSPER1 gene expression, while the presence of CREB-A and CREMτ factors synergistically enhanced promoter activity in these cells. In contrast, deletion of CRE0 prevented any transcriptional activity of the CATSPER1 promoter in GC1-spg spermatogonial cells, but expression of either CREB-A or CREMτ restored such transcriptional activity. CONCLUSIONS: The human CATSPER1 promoter is positively regulated in vitro by CREB-A in HEK293 and GC1-spg cells. Both lines showed differential transcriptional regulation, which was defined by the factors and coactivators present in each cell line as well as the context in which the CRE sites were found in the promoter.

Cor interacts with outer membrane proteins to exclude FhuA-dependent phages.

Superinfection exclusion (Sie) of FhuA-dependent phages is carried out by Cor in the Escherichia coli mEp167 prophage lysogenic strain. In this work, we present evidence that Cor is an outer membrane (OM) lipoprotein that requires the participation of additional outer membrane proteins (OMPs) to exclude FhuA-dependent phages. Two Cor species of ~13 and ~8.5 kDa, corresponding to the preprolipoprotein/prolipoprotein and lipoprotein, were observed by Western blot. Cell mutants for CorC17F, CorA18D and CorA57E lost the Sie phenotype for FhuA-dependent phages. A copurification affinity binding assay combined with LC_ESI_MS/MS showed that Cor bound to OMPs: OmpA, OmpC, OmpF, OmpW, LamB, and Slp. Interestingly, Sie for FhuA-dependent phages was reduced on Cor overexpressing FhuA+ mutant strains, where ompA, ompC, ompF, ompW, lamB, fhuE, genes were knocked out. The exclusion was restored when these strains were supplemented with plasmids expressing these genes. Sie was not lost in other Cor overexpressing FhuA+ null mutant strains JW3938(btuB-), JW5100(tolB-), JW3474(slp-). These results indicate that Cor interacts and requires some OMPs to exclude FhuA-dependent phages.

Trichinella spiralis muscle larvae excretory-secretory products induce changes in cytoskeletal and myogenic transcription factors in primary myoblast cultures.

Trichinella spiralis infection in skeletal muscle culminates with nurse cell formation. The participation of excretory-secretory products of the muscle larvae has been implicated in this process through different studies performed in infected muscle and the muscle cell line C2C12. In this work, we developed primary myoblast cultures to analyse the changes induced by excretory-secretory products of the muscle larvae in muscle cells. Microarray analyses revealed expression changes in muscle cell differentiation, proliferation, cytoskeleton organisation, cell motion, transcription, cell cycle, apoptosis and signalling pathways such as MAPK, Jak-STAT, Wnt and PI3K-Akt. Some of these changes were further evaluated by other methodologies such as quantitative real-time PCR (qRT-PCR) and western blot, confirming that excretory-secretory products of the muscle larvae treated primary mouse myoblasts undergo increased proliferation, decreased expression of MHC and up-regulation of α-actin. In addition, changes in relevant muscle transcription factors (Pax7, Myf5 and Mef2c) were observed. Taken together, these results provide new information about how T. spiralis could alter the normal process of skeletal muscle repair after ML invasion to accomplish nurse cell formation.

Catsper1 promoter is bidirectional and regulates the expression of a novel lncRNA.

The Catsper1 gene, whose expression is restricted to male germ cells, has great importance in reproductive biology because of its function in sperm motility and fertilization. We previously reported that the promoter of this gene has transcriptional activity in either direction in a heterologous system. In the present study, we found that the Catsper1 promoter has in vitro transcriptional activity in either orientation in GC-1 spg mouse spermatogonial cells. The results also showed that this promoter regulates the expression of a new divergent Catsper1 gene named Catsper1au (Catsper1 antisense upstream transcript). Catsper1au is expressed in adult male mouse testis and liver tissues but not in female mouse liver or ovary tissues. In the testis, Catsper1au is expressed in embryos at 11.5 days post-coitum and from newborns to adults. This gene is also expressed in 1- to 3-week postnatal hearts and in 1-week to adult stage livers. The analysis of the 1402 bp whole genome sequence revealed that Catsper1au is an intronless and polyadenylated lncRNA, located in the nuclei of Sertoli and spermatogenic cells from adult testis. These data indicate that Catsper1au is divergently expressed from the Catsper1 promoter and could regulate gene expression during spermatogenesis.

Macrophage Activation by Ursolic and Oleanolic Acids during Mycobacterial Infection.

Oleanolic (OA) and ursolic acids (UA) are triterpenes that are abundant in vegetables, fruits and medicinal plants. They have been described as active moieties in medicinal plants used for the treatment of tuberculosis. In this study, we analyzed the effects of these triterpenes on macrophages infected in vitro with Mycobacterium tuberculosis (MTB). We evaluated production of nitric oxide (NO), reactive oxygen species (ROS), and cytokines (TNF-α and TGF-ß) as well as expression of cell membrane receptors (TGR5 and CD36) in MTB-infected macrophages following treatment with OA and UA. Triterpenes caused reduced MTB growth in macrophages, stimulated production of NO and ROS in the early phase, stimulated TNF-α, suppressed TGF-ß and caused over-expression of CD36 and TGR5 receptors. Thus, our data suggest immunomodulatory properties of OA and UA on MTB infected macrophages. In conclusion, antimycobacterial effects induced by these triterpenes may be attributable to the conversion of macrophages from stage M2 (alternatively activated) to M1 (classically activated).

The transcription factors Sox5 and Sox9 regulate Catsper1 gene expression.

Catsper is a Ca(2+)permeable channel required for sperm hyperactivation. In spite of its central role in male fertility, the transcriptional mechanisms that regulate Catsper1 expression are ill defined. In this work, we describe the identification and characterization of important regulatory elements in the murine Catsper1 gene proximal promoter. Four transcription start sites and three functional Sox-binding sites were identified in the Catsper1 promoter. Interestingly, transcription factors Sox5 and Sox9 caused a significant increase in transactivation of the Catsper1 promoter in heterologous systems, and chromatin immunoprecipitation assays showed that both transcription factors interact with the Catsper1 promoter in vivo. These results provide new insights into the molecular mechanisms that control Catsper channel expression.

Molecular cloning and analysis of the Catsper1 gene promoter.

CatSper channels are essential for hyperactivity of sperm flagellum, progesterone-mediated chemotaxis and oocyte fertilization. Catsper genes are exclusively expressed in the testis during spermatogenesis, but the function and regulation of the corresponding promoter regions are unknown. Here, we report the cloning and characterization of the promoter regions in the human and murine Catsper1 genes. These promoter regions were identified and isolated from genomic DNA, and transcriptional activities were tested in vitro after transfection into human embryonic kidney 293, mouse Sertoli cells 1 and GC-1spg cell lines as well as by injecting plasmids directly into mouse testes. Although the human and murine Catsper1 promoters lacked a TATA box, a well-conserved CRE site was identified. Both sequences may be considered as TATAless promoters because their transcriptional activity was not affected after deletion of TATA box-like sites. Several transcription initiation sites were revealed by RNA ligase-mediated rapid amplification of the cDNA 5'-ends. We also found that the immediate upstream region and the first exon in the human CATSPER1 gene negatively regulate transcriptional activity. In the murine Catsper1 promoter, binding sites for transcription factors SRY, SOX9 and CREB were protected by the presence of nuclear testis proteins in DNAse degradation assays. Likewise, the mouse Catsper1 promoter exhibited transcriptional activity in both orientations and displayed significant expression levels in mouse testis in vivo, whereas the suppression of transcription signals in the promoter resulted in low expression levels. This study, thus, represents the first identification of the transcriptional control regions in the genes encoding the human and murine CatSper channels.

Retinoic acid receptor ß deficiency reduces splenic dendritic cell population in a conditional mouse line.

Different studies have shown that retinoids and their receptors [retinoic acid receptors (RARs) and retinoid X receptors (RXRs)] have crucial effects on the differentiation and function of myeloid cells such as Dendritic cells (DCs) and the development of lymphoid tissue. However, the relationship between RARß expression and DCs has not been previously studied in vivo. This work examined the effect of decreased RARß expression on the number (and probably on differentiation) of splenic DCs and the structure of spleen using a conditional mouse that partially ablates floxed RARß gene (RARß(L-/L-) mice). Our results showed that RARß is expressed mainly in cells of the splenic White Pulp (WP) zone of Wild type mice. As expected, low levels of RARß expression were detected in the spleen of RARß(L-/L-) conditional mice. These results were consistent with a decrease in the population of splenic CD11c(+)MHC-II(+) cells. Histopathological analyses of conditional mice spleen indicated defects in cell organization and structure. The expression of Toll-like receptor 2 was also down-regulated in the spleen of these mice. These results suggest that RARß is involved in splenic cell organization as well as in the maintenance of splenic DCs population, indicating that RARß expression is important in homeostasis of immune system components.

Cloning and identification of a gene coding for a 26-kDa hemoglobin-binding protein from Entamoeba histolytica.

The capability of Entamoeba histolytica to use hemoglobin (Hb) as an iron source has been documented. However, the underlying mechanism to acquire iron from this source is poorly understood. In the present work, an in silico analysis in the E. histolytica genome (Pathema database) allowed us to identify a gene coding for a putative 26-kDa protein (Ehhmbp26) which contains the motifs necessary for Hb-binding. The purified Ehhmbp26 protein was able to bind Hb. Albeit with less efficiency, trophozoites were able to grow using Hb as the only iron source. In addition, ehhmbp26 RNA and the Ehhmbp26 protein were only expressed under iron restrictive conditions and ehhmbp26 RNA was subsequently inhibited after iron supplementation indicating that ehhmbp26 gene is negatively regulated by iron. These results suggest that the Ehhmbp26 protein may be involved in a mechanism by which E. histolytica scavenges iron from Hb.

Analysis of some phenotypic traits of feces-borne temperate lambdoid bacteriophages from different immunity groups: a high incidence of cor+, FhuA-dependent phages.

A group of previously isolated heterogeneous mEp lambdoid phages (43) from 19 different immunity groups for phage infection was further characterized to gain insight into some phenotypic traits and to assess their relationship with phage lambda. Interestingly, the FhuA host receptor was required by the majority of mEp phages (37 out of 43; approximately 85%). The cor gene, which has been reported to be involved in FhuA-dependent exclusion of lambdoid phages, was also found in most of the FhuA-dependent phages. Accordingly, no cor amplification by PCR was obtained among the six FhuA-independent mEp lambdoid phages. In contrast, it was found that around 25% of the population (10 out of 43 phages) required the specific and essential lambda N antitermination function, and the lambda site-specific DNA recombination function was observed only in two members (4.6%). Thus, a larger proportion of phages require the FhuA receptor for infection, and this is frequently correlated with the cor gene.

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon.

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3-8 codon minigenes containing AGAAGA codons before the stop codon. Beta-galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, beta-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 microg/microl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of beta-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNA(Arg4). These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA.

Giardia duodenalis: adhesion-deficient clones have reduced ability to establish infection in Mongolian gerbils.

The role of Giardia duodenalis surface molecules in the attachment of trophozoites to epithelial cells has been established through the dual strategies of characterizing G. duodenalis clones with deficient adhesion and blocking experiments with surface-specific monoclonal antibodies. Also, the infectivity of the analyzed clones was tested using Mongolian gerbils as experimental model. Two adhesion-deficient G. duodenalis clones, C6 and C7, were isolated from the wild type C5 clone which in turn was obtained from the WB strain. The adhesion efficiencies of C6 and C7 clones (48.2+/-4.9 and 32.6+/-2.4, respectively) were significantly lower as compared with WB strain or C5 clone (82.8+/-6.4 and 79.9+/-7.9). Analysis of radiolabel surface proteins by 1D and 2D SDS-PAGE revealed prominently labelled 28 and 88 kDa components in C6 and C7 clones and a major 200 kDa protein in the C5 clone and the WB strain. The 88 and 200 kDa components are acidic proteins by two-dimensional electrophoretic analyses. The most striking difference between wild-type and adhesion-deficient Giardia trophozoites was the reduced expression of a 200 kDa surface protein in the latter. Significantly, a mAb (IG3) specific for the 200 kDa protein that reacted with more than 99% of WB and C5 trophozoites and less than 1% of C6 and C7 trophozoites as determined by indirect immunofluorescence inhibited the adhesion of trophozoites from WB and C5 clone to Madin Darby Canine Kidney cells by 52% and 40.9%, respectively, suggesting a participation of this antigen in adherence. Finally, the functional relevance of trophozoite adhesion to epithelial cells was indicated by the reduced capacity of the adhesion-deficient clones to establish the infection in Mongolian gerbils.

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats.

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal differences in the kinetics of anti-NBL and ML Ab responses. While anti-NBL Abs declined slowly from day 19 until the end of the experiment, Abs to ML antigen remained high in the same period. It is remarkable the optimal Ab response to NBL antigens with 2000 ML infective dose and the reduced number of NBL antigens identified throughout the experimental T. spiralis infection, standing out the immunodominant 49 kDa antigen. Interestingly, this antigen, which was prominently expressed in NBL somatic proteins, was also detected in NBL-ESP.

Codon-specific and general inhibition of protein synthesis by the tRNA-sequestering minigenes.

The expression of minigenes in bacteria inhibits protein synthesis and cell growth. Presumably, the translating ribosomes, harboring the peptides as peptidyl-tRNAs, pause at the last sense codon of the minigene directed mRNAs. Eventually, the peptidyl-tRNAs drop off and, under limiting activity of peptidyl-tRNA hydrolase, accumulate in the cells reducing the concentration of specific aminoacylable tRNA. Therefore, the extent of inhibition is associated with the rate of starvation for a specific tRNA. Here, we used minigenes harboring various last sense codons that sequester specific tRNAs with different efficiency, to inhibit the translation of reporter genes containing, or not, these codons. A prompt inhibition of the protein synthesis directed by genes containing the codons starved for their cognate tRNA (hungry codons) was observed. However, a non-specific in vitro inhibition of protein synthesis, irrespective of the codon composition of the gene, was also evident. The degree of inhibition correlated directly with the number of hungry codons in the gene. Furthermore, a tRNA(Arg4)-sequestering minigene promoted the production of an incomplete beta-galactosidase polypeptide interrupted, during bacterial polypeptide chain elongation at sites where AGA codons were inserted in the lacZ gene suggesting ribosome pausing at the hungry codons.

An orthologue of the cor gene is involved in the exclusion of temperate lambdoid phages. Evidence that Cor inactivates FhuA receptor functions.

A new set of lambdoid phages (mEp) classified into different immunity groups was previously described. Phages mEp213, mEp237, and mEp410 were unable to grow in mEp167 lysogenic cells, presumably due to an exclusion mechanism expressed constitutively by the mEp167 repressed prophage. In this work, to analyze the exclusion phenomenon, we constructed a genomic library from mEp167 phage in a pPROEX derivative plasmid. A DNA fragment containing an open reading frame for a 77 amino acid polypeptide was selected by its ability to confer resistance to heteroimmune phage infection. This ORF shows high amino acid sequence identity with putative Cor proteins of phages HK022, phi80 and N15. Cells expressing the mEp167 cor gene from a plasmid (Cor(+) phenotype) excluded 13 of 20 phages from different infection immunity groups. This exclusion was observed in both tonB(-) and tonB(+) cells. Lambdoid mEp phages that were excluded in these cells were unable to infect cells defective in the outer membrane FhuA receptor (fhuA(-)). Thus, Cor-mediated exclusion was only observed in fhuA(+) cells. Phage production after DNA transfection or the spontaneous induction of mEp prophage in Cor(+) cells was not blocked. In addition, ferrichrome uptake, which is mediated by FhuA, was inhibited in Cor(+) cells. Our results show that not only phage infection via FhuA but also a FhuA transport activity (ferrichrome uptake) are inhibited by Cor, presumably by inactivation of FhuA.