The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.

Influence of RNase E deficiency on the production of stx2-bearing phages and Shiga toxin in an RNase E-inducible strain of enterohaemorrhagic Escherichia coli (EHEC) O157:H7.

PURPOSE: In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naïve bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated. METHODOLOGY: Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively. RESULTS: RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures. CONCLUSION: RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.

Effect of RNase E deficiency on translocon protein synthesis in an RNase E-inducible strain of enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that assembles a type III secretion system (T3SS) on its surface. The last portion of the T3SS, called the 'translocon', is composed of a filament and a pore complex that is inserted into the membrane of intestinal epithelial cells. The genes encoding the translocon (espADB) are part of the LEE4 operon. Their expression is regulated by a complex post-transcriptional mechanism that involves the processing of LEE4 mRNA by the essential endoribonuclease RNase E. Here, we report the construction of an EHEC strain (TEA028-rne) in which RNase E can be induced by adding IPTG to the culture medium. EHEC cells deficient in RNase E displayed an abnormal morphology and slower growth, in agreement with published observations in E. coli K-12. Under those conditions, EspA and EspB were produced at higher concentrations, and protein secretion still occurred. These results indicate that RNase E negatively regulates translocon protein synthesis and demonstrate the utility of E. coli strain TEA028-rne as a tool for investigating the influence of this ribonuclease on EHEC gene expression in vitro.

The ribosome binding site of a mini-ORF protects a T3SS mRNA from degradation by RNase E.

Enterohaemorrhagic Escherichia coli harbours a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU-rich leader region and monophosphorylated 5'-terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine-Dalgarno element (SD2) and a translatable mini-ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini-ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini-ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU-rich leader region.

Post-transcriptional processing of the LEE4 operon in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) employs a type III secretion system (T3SS) to export translocator and effector proteins required for mucosal colonization. The T3SS is encoded in a pathogenicity island called the locus of enterocyte effacement (LEE) that is organized in five major operons, LEE1 to LEE5. LEE4 encodes a regulator of secretion (SepL), translocators (EspA, D and B), two chaperones (CesD2 and L0017), a T3SS component (EscF) and an effector protein (EspF). It was originally proposed that the esp transcript is transcribed from a promoter located at the end of sepL but other authors suggested that this transcript is the result of a post-transcriptional processing event. In this study, we established that the espADB mRNA is generated by post-transcriptional processing at the end of the sepL coding sequence. RNase E is the endonuclease involved in the cleavage, but the interaction of this enzyme with other proteins through its C-terminal half is dispensable. A putative transcription termination event in the cesD2 coding region would generate the 3' end of the transcript. Similar to what has been described for other processed transcripts, the cleavage of LEE4 seems a mechanism to differentially regulate SepL and Esp protein production.

Isolation, characterization and long term preservation of mutant strains of Xanthophyllomyces dendrorhous.

The yeast Xanthophyllomyces dendrorhous is biotechnologically important due to its ability to produce the pigment astaxanthin, but is poorly understood at the genetic level. This is mainly because its preservation is difficult and many of the mutants obtained are unstable. The objectives of the present work were (i) the mutagenesis X. dendrorhous and, (ii) isolation of mutants with auxotrophic markers suitable for genetic studies of the carotenogenesis pathway and sexual cycle. Additionally, two kinds of preservation methods at the laboratory level were tested for the storage of strains. A collection of X. dendrorhous mutants affected in the production of carotenoid pigments or development of sexual structures and auxotrophic requirements were isolated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine and the antibiotic nystatin. From a detailed analysis about the requirements of auxotrophic mutants the ARG7, ARG3 and PRO3 loci can be defined in this yeast. Among the methods assayed for the long-term preservation of X. dendrorhous strains, the dehydrated gelatin drop method showed the highest recovery of viable yeast after storage for 65 months. No changes in auxotrophic properties and in macro or micro morphology were observed after applying the latter method.

Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous.

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+/idi-::hph), crtE (crtE+/crtE-::hph), crtYB (crtYB+/crtYB-::hph), crtI (crtI+/crtI-::hph) and crtS (crtS+/crtS-::hph) and homozygote mutants crtYB (crtYB-::hph/crtYB-::hph), crtI (crtI-::hph/crtI-::hph) and crtS (crtS-::hph/crtS-::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.

Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+ / idi-::hph), crtE (crtE+ / crtE -::hph), crtYB (crtYB + / crtYB -::hph), crtI (crtI+ / crtI-::hph) and crtS (crtS +/crtS -::hph) and homozygote mutants crtYB (crtYB -::hph/crtYB -::hph), crtI (crtI -::hph/crtI -::hph) and crtS (crtS -::hph / crtS -::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.

A variation of the translation attenuation model can explain the inducible regulation of the pBC16 tetracycline resistance gene in Bacillus subtilis.

Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.

Study of the expression of carotenoid biosynthesis genes in wild-type and deregulated strains of Xanthophyllomyces dendrorhous (Ex.: Phaffia rhodozyma).

The expression, at the mRNA level, of carotenoid biosynthetic genes from Xanthophyllomyces dendrorhous was studied by RT-PCR. The experimental conditions for the RT-PCR assay were standardized to quantify the relative transcript levels of idi, crtE, crtYB and crtI genes. This work attempted to correlate astaxanthin production with the transcript levels of carotenogenic genes in a wild-type strain (UCD 67-385) and two overproducer deregulated strains (atxS1 and atxS2). At 3 day cultures, the wild-type strain contained higher transcript levels from the crtE and crtYB genes on minimal medium than on rich medium. Similarly, carotenoid production was higher on minimal medium than on rich medium. However, carotenoid production in the atxS1 and atxS2 strains was not correlated with the transcript level of carotenogenic genes under the same experimental conditions. This result suggests that there is not a linear relationship between carotenogenic transcript levels and carotenoid biosynthesis.

Study of the expression of carotenoid biosynthesis genes in wild-type and deregulated strains of Xanthophyllomyces dendrorhous (Ex: Phaffia rhodozyma)

The expression, at the mRNA level, of carotenoid biosynthetic genes from Xanthophyllomyces dendrorhous was studied by RT-PCR. The experimental conditions for the RT-PCR assay were standardized to quantify the relative transcript levels of idi, crtE, crtYB and crtI genes. This work attempted to correlate astaxanthin production with the transcript levels of carotenogenic genes in a wild-type strain (UCD 67-385) and two overproducer deregulated strains (atxS1 and atxS2). At 3 day cultures, the wild-type strain contained higher transcript levels from the crtE and crtYB genes on minimal medium than on rich medium. Similarly, carotenoid production was higher on minimal medium than on rich medium. However, carotenoid production in the atxS1 and atxS2 strains was not correlated with the transcript level of carotenogenic genes under the same experimental conditions. This result suggests that there is not a linear relationship between carotenogenic transcript levels and carotenoid biosynthesis.

Estudio genético molecular de levaduras de diversos ambientes / Molecular genetic study of yeasts from different environments

Se describe la presencia de polimorfísmo genético en levaduras nativas aisladas de diferentes ambientes, tales como: agua de mar, suelos forestales con poca intervención antrópica, suelos de viñedos, otros ambientes naturales y desde pacientes con fungemia severa. Se detectó la presencia de elementos genéticos extracromosómicos en cuatro cepas diferentes de levaduras. El análisis de amplificación de una región ITS utilizando partidores específicos para el ITS2 (partidores ITS3 e ITS4), permite determinar una banda de amplificación de rDNA que varia de tamaño entre 450 y 560 pb. dependiendo del género de levadura analizada. En una cepa de Cryptococcus terreus se observó la presencia de tres bandas de amplificación ITS que sugieren una organización compleja de los genes de rDNA en esa cepa. Finalmente el análisis del cariotipo electroforético de cepas ambientales y clínicas de Pichia anomala mostró un marcado polimorfísmo cromosómico en esta levadura emergente.

Aislamiento de genes carotenogénesis de xanthophyllomyces dendrorhous (ex. phaffia rhodozyma) mediante PCR / Isolation of carotenogenesis genes of xanthophyllomyces dendrohous (ex. phaffia rhodozyma) through PCR

Xanthophyllomyces dendrorhous (ex.phaffia rhodozyma) es una levadura basidiomicetica carotenogénica, en la cual aspectos importantes de su biología como la organización general de su genoma, número de cromosomas y nivel de ploidia aún no son completamente entendidos. En atención a esto, se han orientado esfuerzos en estudios moleculares con el objetivo de aumentar el conocimiento de su genética. En el presente trabajo, se describe un eficiente procedimiento para seleccionar y clonar genes a partir de una genoteca de DNA genómico de x. dendrorhous mediante la amplificación de DNA por PCR, utilizándo parejas de partidores específicos para el gen crtl. Adicionalmente, se describe la síntesis de cDNA a partir de RNA total de la levadura mediante transcripción reversa acoplada a amplificación de DNA (rt-pcr)