Ramón Cervantes-Rivera - Academia.edu (original) (raw)

Papers by Ramón Cervantes-Rivera

Research paper thumbnail of Infection-induced membrane ruffling initiates danger and immune signaling via the mechanosensor PIEZO1

Research paper thumbnail of RpuS/R Is a Novel Two-Component Signal Transduction System That Regulates the Expression of the Pyruvate Symporter MctP in Sinorhizobium fredii NGR234

Frontiers in Microbiology

The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-c... more The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-component signal transduction systems (TCSTS), in which the signaling domain is fused to an SLC5 solute symporter domain through a STAC domain. Only two members of this family have been characterized experimentally, the CrbS/R system that regulates acetate utilization in Vibrio and Pseudomonas, and the CbrA/B system that regulates the utilization of histidine in Pseudomonas and glucose in Azotobacter. In an attempt to expand the characterized members of this family beyond the Gammaproteobacteria, we identified two putative TCSTS in the Alphaproteobacterium Sinorhizobium fredii NGR234 whose sensor histidine kinases belong to the SLC5/STAC family. Using reverse genetics, we were able to identify the first TCSTS as a CrbS/R homolog that is also needed for growth on acetate, while the second TCSTS, RpuS/R, is a novel system required for optimal growth on pyruvate. Using RNAseq and transcripti...

Research paper thumbnail of Additional file 9 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 9: File: S2.1. General features of the S. flexneri 5a M90T virulence plasmid (pWR... more Additional file 9: File: S2.1. General features of the S. flexneri 5a M90T virulence plasmid (pWR100) annotated with RAST [58].

Research paper thumbnail of Additional file 8 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 8: File: S2. General features of the S. flexneri 5a M90T chromosome annotated wit... more Additional file 8: File: S2. General features of the S. flexneri 5a M90T chromosome annotated with RAST [58].

Research paper thumbnail of Additional file 7 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 7: File S1.1. General features of the S. flexneri 5a M90T virulence plasmid (pWR1... more Additional file 7: File S1.1. General features of the S. flexneri 5a M90T virulence plasmid (pWR100) annotated with Prokka [59].

Research paper thumbnail of Additional file 6 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 6: File S1. General features of the S. flexneri 5a M90T chromosome annotated with... more Additional file 6: File S1. General features of the S. flexneri 5a M90T chromosome annotated with Prokka [59].

Research paper thumbnail of Additional file 4 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 4: Table S3. Transcriptional start sites determined in S. flexneri 5a M90T virule... more Additional file 4: Table S3. Transcriptional start sites determined in S. flexneri 5a M90T virulence plasmid (pWR100) with ReadExplorer [64, 113].

Research paper thumbnail of Additional file 5 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 5: Table S4. Pseudogenes transcription abundance level. Reads counting per pseudo... more Additional file 5: Table S4. Pseudogenes transcription abundance level. Reads counting per pseudogene was performed with htseq/0.9.1 [113] using stranded mode and pseudogene as a feature type. The total expression of pseudogenes is the average of the six libraries used for TSS determination.

Research paper thumbnail of Additional file 3 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 3: Table S2. Transcriptional start sites determined in S. flexneri 5a M90T chromo... more Additional file 3: Table S2. Transcriptional start sites determined in S. flexneri 5a M90T chromosome with ReadExplorer [64, 113].

Research paper thumbnail of Additional file 2 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 2: Table S1. Comparison of general features of the S. flexneri 5a M90T predicted ... more Additional file 2: Table S1. Comparison of general features of the S. flexneri 5a M90T predicted with three different pipelines: Prokka [59], RAST [58] and PGAP/NCBI [60].

Research paper thumbnail of Additional file 1 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 1: Figure S1. Genome coverage plot of S. flexneri 5a M90T with aligned DNA or RNA... more Additional file 1: Figure S1. Genome coverage plot of S. flexneri 5a M90T with aligned DNA or RNA short-read sequences. a) Genome coverage of small DNA Illumina sequences. The percentage of genome covered with a depth of ≥5 is 99.98% with a mean coverage of 126X. b) Genome coverage of small RNA sequences. The percentage of genome covered with a depth of of ≥5 is 98.77% with a mean coverage of 989.9X. Coverage calculation was performed with Samtools depth [54, 55] using sorted alignments in BAM format as input. The figure was generated with ggplot2.3.2.1 in the R. 3.6.1 environment.

Research paper thumbnail of RESEARCH ARTICLE Open Access The

replication origin of a repABC plasmid

Research paper thumbnail of Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria

BIO-PROTOCOL, 2020

Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identif... more Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence. Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5′-monophosphatedependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis. Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.

Research paper thumbnail of A nalysis of the mechanism of action of the antisense RNA 2 that controls the replication of the repABC plasmid p 42 d 3 4 5 6 Running title : analysis of the antisense RNA of p 42 d plasmid

14 15 16 17 18 19 20 1. Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad... more 14 15 16 17 18 19 20 1. Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad 21 Nacional Autónoma de México, Apartado Postal 565-A, Cuernavaca, Morelos, 22 México. 23 24 2. Instituto de Parasitología y Biomedicina "López-Neyra", CSIC, Parque 25 Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla, 18100 26 Granada, España. 27 28 29 30 31 32 33 *Corresponding author: Miguel A. Cevallos. Tel 7773 11 46 63, Fax: 7773 17 55 81 34 E-mail: mac@ccg.unam.mx 35 Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. J. Bacteriol. doi:10.1128/JB.00118-10 JB Accepts, published online ahead of print on 30 April 2010

Research paper thumbnail of Complete genome sequence and annotation of the laboratory reference strainShigella flexneriserotype 5a M90T and genome-wide transcriptional start site determination

BackgroundShigellais a Gram-negative facultative intracellular bacterium that causes bacillary dy... more BackgroundShigellais a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans.Shigellainvades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strainS. flexneriserotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches.ResultsWe have sequenced, assembled, annotated and manually curated the full genome ofS. flexneri5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new pipeline to prepare gapless, highly accurate genome sequences. Furthermore,...

Research paper thumbnail of The Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy

Autophagy plays an essential role in the defence against many microbial pathogens as a regulator ... more Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy subversion mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy s...

Research paper thumbnail of The repABC plasmid family

Plasmid, 2008

repABC plasmids are widely distributed among alpha-proteobacteria. They are especially common in ... more repABC plasmids are widely distributed among alpha-proteobacteria. They are especially common in Rhizobiales. Some strains of this bacterial order can contain multiple repABC replicons indicating that this plasmid family includes several incompatibility groups. The replication and stable maintenance of these replicons depend on the presence of a repABC operon. The repABC operons sequenced to date share some general characteristics. All of them contain at least three protein-encoding genes: repA, repB and repC. The first two genes encode proteins involved in plasmid segregation, whereas repC encodes a protein crucial for replication. The origin of replication maps within the repC gene. In contrast, the centromere-like sequence (parS) can be located at various positions in the operon. In this review we will summarize current knowledge about this plasmid family, with special emphasis on their structural diversity and their complex genetic regulation. Finally, we will examine some ideas about their evolutionary origin and trends.

Research paper thumbnail of Analysis of the Mechanism of Action of the Antisense RNA That Controls the Replication of the repABC Plasmid p42d

Journal of Bacteriology, 2010

Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the pr... more Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the presence of a repABC operon, which carries all the plasmid-encoded elements required for these functions. All repABC operons share three protein-encoding genes (repA, repB, and repC), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (parS). The products of repA and repB, in conjunction with the parS region, make up the segregation system, and they negatively regulate operon transcription. The last gene of the operon, repC, encodes the initiator protein. The ctRNA is a negative posttranscriptional regulator of repC. In this work, we analyzed the secondary structures of the ctRNA and its target and mapped the motifs involved in the complex formed between them. Essential residues for the effective interaction localize at the unpaired 5 end of the antisense molecule and the loop of the target mRNA. In light of our results, we propose a model explaining the mechanism of action of this ctRNA in the regulation of plasmid replication in R. etli. . † Supplemental material for this article may be found at http://jb .asm.org/. ᰔ Published ahead of print on 30 April 2010. 3272 CERVANTES-RIVERA ET AL. J. BACTERIOL. FIG. 4. Secondary structure of the target mInc␣. (a) Autoradiogram of a polyacrylamide gel used to resolve 5Ј-labeled mInc␣ after treatment with RNase T1, lead acetate (Pb 2ϩ ), and RNase V1. Lane 1 (C), undigested probe; lane 2 (OHL), alkaline ladder; lane 3 (T1L), RNase T1 partial digestion of denatured mInc␣ used as a ladder; lane 4, RNase T1 partial digestion of mInc␣; lane 5, Pb 2ϩ partial degradation of mInc␣; lane 6, RNase V1 partial digestion of mInc␣. Vertical bars indicate loop and helixes of the mInc␣. (b) mInc␣ secondary structure consistent with cleavage patterns. Black arrowheads indicate RNase T1 sites, white arrowheads indicate Pb 2ϩ sites, and open circles indicate RNase V1 cleavages. Major cuts are indicated by asterisks ( * ). Relevant loop and helix regions are marked with the letters L and H, followed by a number and the Greek letter, "␣." The mInc␣ region complementary to the ctRNA is marked with a black line. "SD" indicates the position of repC Shine-Dalgarno sequence. The repC initiation codon (AUG) is encircled. 3274 CERVANTES-RIVERA ET AL. J. BACTERIOL.

Research paper thumbnail of The replication origin of a repABC plasmid

BMC Microbiology, 2011

Background repABC operons are present on large, low copy-number plasmids and on some secondary ch... more Background repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. Results To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d. Conclusions RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The oriV of this plasmid resides within the repC gene and is located close to or inside of a large A+T region. RepC can act as an incompatibility factor, and the last 39 amino acid residues of the carboxy-terminal region of this protein are involved in promoting this phenotype.

Research paper thumbnail of Analysis of the Mechanism of Action of the Antisense RNA

Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the pr... more Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the presence of a repABC operon, which carries all the plasmid-encoded elements required for these functions. All repABC operons share three protein-encoding genes (repA, repB, and repC), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (parS). The products of repA and repB, in conjunction with the parS region, make up the segregation system, and they negatively regulate operon transcription. The last gene of the operon, repC, encodes the initiator protein. The ctRNA is a negative posttranscriptional regulator of repC. In this work, we analyzed the secondary structures of the ctRNA and its target and mapped the motifs involved in the complex formed between them. Essential residues for the effective interaction localize at the unpaired 5 end of the antisense molecule and the loop of the target mRNA. In light of our results, we propose a model explaining the mechanism of action of this ctRNA in the regulation of plasmid replication in R. etli.

Research paper thumbnail of Infection-induced membrane ruffling initiates danger and immune signaling via the mechanosensor PIEZO1

Research paper thumbnail of RpuS/R Is a Novel Two-Component Signal Transduction System That Regulates the Expression of the Pyruvate Symporter MctP in Sinorhizobium fredii NGR234

Frontiers in Microbiology

The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-c... more The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-component signal transduction systems (TCSTS), in which the signaling domain is fused to an SLC5 solute symporter domain through a STAC domain. Only two members of this family have been characterized experimentally, the CrbS/R system that regulates acetate utilization in Vibrio and Pseudomonas, and the CbrA/B system that regulates the utilization of histidine in Pseudomonas and glucose in Azotobacter. In an attempt to expand the characterized members of this family beyond the Gammaproteobacteria, we identified two putative TCSTS in the Alphaproteobacterium Sinorhizobium fredii NGR234 whose sensor histidine kinases belong to the SLC5/STAC family. Using reverse genetics, we were able to identify the first TCSTS as a CrbS/R homolog that is also needed for growth on acetate, while the second TCSTS, RpuS/R, is a novel system required for optimal growth on pyruvate. Using RNAseq and transcripti...

Research paper thumbnail of Additional file 9 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 9: File: S2.1. General features of the S. flexneri 5a M90T virulence plasmid (pWR... more Additional file 9: File: S2.1. General features of the S. flexneri 5a M90T virulence plasmid (pWR100) annotated with RAST [58].

Research paper thumbnail of Additional file 8 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 8: File: S2. General features of the S. flexneri 5a M90T chromosome annotated wit... more Additional file 8: File: S2. General features of the S. flexneri 5a M90T chromosome annotated with RAST [58].

Research paper thumbnail of Additional file 7 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 7: File S1.1. General features of the S. flexneri 5a M90T virulence plasmid (pWR1... more Additional file 7: File S1.1. General features of the S. flexneri 5a M90T virulence plasmid (pWR100) annotated with Prokka [59].

Research paper thumbnail of Additional file 6 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 6: File S1. General features of the S. flexneri 5a M90T chromosome annotated with... more Additional file 6: File S1. General features of the S. flexneri 5a M90T chromosome annotated with Prokka [59].

Research paper thumbnail of Additional file 4 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 4: Table S3. Transcriptional start sites determined in S. flexneri 5a M90T virule... more Additional file 4: Table S3. Transcriptional start sites determined in S. flexneri 5a M90T virulence plasmid (pWR100) with ReadExplorer [64, 113].

Research paper thumbnail of Additional file 5 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 5: Table S4. Pseudogenes transcription abundance level. Reads counting per pseudo... more Additional file 5: Table S4. Pseudogenes transcription abundance level. Reads counting per pseudogene was performed with htseq/0.9.1 [113] using stranded mode and pseudogene as a feature type. The total expression of pseudogenes is the average of the six libraries used for TSS determination.

Research paper thumbnail of Additional file 3 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 3: Table S2. Transcriptional start sites determined in S. flexneri 5a M90T chromo... more Additional file 3: Table S2. Transcriptional start sites determined in S. flexneri 5a M90T chromosome with ReadExplorer [64, 113].

Research paper thumbnail of Additional file 2 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 2: Table S1. Comparison of general features of the S. flexneri 5a M90T predicted ... more Additional file 2: Table S1. Comparison of general features of the S. flexneri 5a M90T predicted with three different pipelines: Prokka [59], RAST [58] and PGAP/NCBI [60].

Research paper thumbnail of Additional file 1 of Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Additional file 1: Figure S1. Genome coverage plot of S. flexneri 5a M90T with aligned DNA or RNA... more Additional file 1: Figure S1. Genome coverage plot of S. flexneri 5a M90T with aligned DNA or RNA short-read sequences. a) Genome coverage of small DNA Illumina sequences. The percentage of genome covered with a depth of ≥5 is 99.98% with a mean coverage of 126X. b) Genome coverage of small RNA sequences. The percentage of genome covered with a depth of of ≥5 is 98.77% with a mean coverage of 989.9X. Coverage calculation was performed with Samtools depth [54, 55] using sorted alignments in BAM format as input. The figure was generated with ggplot2.3.2.1 in the R. 3.6.1 environment.

Research paper thumbnail of RESEARCH ARTICLE Open Access The

replication origin of a repABC plasmid

Research paper thumbnail of Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria

BIO-PROTOCOL, 2020

Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identif... more Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence. Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5′-monophosphatedependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis. Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.

Research paper thumbnail of A nalysis of the mechanism of action of the antisense RNA 2 that controls the replication of the repABC plasmid p 42 d 3 4 5 6 Running title : analysis of the antisense RNA of p 42 d plasmid

14 15 16 17 18 19 20 1. Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad... more 14 15 16 17 18 19 20 1. Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad 21 Nacional Autónoma de México, Apartado Postal 565-A, Cuernavaca, Morelos, 22 México. 23 24 2. Instituto de Parasitología y Biomedicina "López-Neyra", CSIC, Parque 25 Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla, 18100 26 Granada, España. 27 28 29 30 31 32 33 *Corresponding author: Miguel A. Cevallos. Tel 7773 11 46 63, Fax: 7773 17 55 81 34 E-mail: mac@ccg.unam.mx 35 Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. J. Bacteriol. doi:10.1128/JB.00118-10 JB Accepts, published online ahead of print on 30 April 2010

Research paper thumbnail of Complete genome sequence and annotation of the laboratory reference strainShigella flexneriserotype 5a M90T and genome-wide transcriptional start site determination

BackgroundShigellais a Gram-negative facultative intracellular bacterium that causes bacillary dy... more BackgroundShigellais a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans.Shigellainvades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strainS. flexneriserotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches.ResultsWe have sequenced, assembled, annotated and manually curated the full genome ofS. flexneri5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new pipeline to prepare gapless, highly accurate genome sequences. Furthermore,...

Research paper thumbnail of The Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy

Autophagy plays an essential role in the defence against many microbial pathogens as a regulator ... more Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy subversion mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy s...

Research paper thumbnail of The repABC plasmid family

Plasmid, 2008

repABC plasmids are widely distributed among alpha-proteobacteria. They are especially common in ... more repABC plasmids are widely distributed among alpha-proteobacteria. They are especially common in Rhizobiales. Some strains of this bacterial order can contain multiple repABC replicons indicating that this plasmid family includes several incompatibility groups. The replication and stable maintenance of these replicons depend on the presence of a repABC operon. The repABC operons sequenced to date share some general characteristics. All of them contain at least three protein-encoding genes: repA, repB and repC. The first two genes encode proteins involved in plasmid segregation, whereas repC encodes a protein crucial for replication. The origin of replication maps within the repC gene. In contrast, the centromere-like sequence (parS) can be located at various positions in the operon. In this review we will summarize current knowledge about this plasmid family, with special emphasis on their structural diversity and their complex genetic regulation. Finally, we will examine some ideas about their evolutionary origin and trends.

Research paper thumbnail of Analysis of the Mechanism of Action of the Antisense RNA That Controls the Replication of the repABC Plasmid p42d

Journal of Bacteriology, 2010

Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the pr... more Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the presence of a repABC operon, which carries all the plasmid-encoded elements required for these functions. All repABC operons share three protein-encoding genes (repA, repB, and repC), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (parS). The products of repA and repB, in conjunction with the parS region, make up the segregation system, and they negatively regulate operon transcription. The last gene of the operon, repC, encodes the initiator protein. The ctRNA is a negative posttranscriptional regulator of repC. In this work, we analyzed the secondary structures of the ctRNA and its target and mapped the motifs involved in the complex formed between them. Essential residues for the effective interaction localize at the unpaired 5 end of the antisense molecule and the loop of the target mRNA. In light of our results, we propose a model explaining the mechanism of action of this ctRNA in the regulation of plasmid replication in R. etli. . † Supplemental material for this article may be found at http://jb .asm.org/. ᰔ Published ahead of print on 30 April 2010. 3272 CERVANTES-RIVERA ET AL. J. BACTERIOL. FIG. 4. Secondary structure of the target mInc␣. (a) Autoradiogram of a polyacrylamide gel used to resolve 5Ј-labeled mInc␣ after treatment with RNase T1, lead acetate (Pb 2ϩ ), and RNase V1. Lane 1 (C), undigested probe; lane 2 (OHL), alkaline ladder; lane 3 (T1L), RNase T1 partial digestion of denatured mInc␣ used as a ladder; lane 4, RNase T1 partial digestion of mInc␣; lane 5, Pb 2ϩ partial degradation of mInc␣; lane 6, RNase V1 partial digestion of mInc␣. Vertical bars indicate loop and helixes of the mInc␣. (b) mInc␣ secondary structure consistent with cleavage patterns. Black arrowheads indicate RNase T1 sites, white arrowheads indicate Pb 2ϩ sites, and open circles indicate RNase V1 cleavages. Major cuts are indicated by asterisks ( * ). Relevant loop and helix regions are marked with the letters L and H, followed by a number and the Greek letter, "␣." The mInc␣ region complementary to the ctRNA is marked with a black line. "SD" indicates the position of repC Shine-Dalgarno sequence. The repC initiation codon (AUG) is encircled. 3274 CERVANTES-RIVERA ET AL. J. BACTERIOL.

Research paper thumbnail of The replication origin of a repABC plasmid

BMC Microbiology, 2011

Background repABC operons are present on large, low copy-number plasmids and on some secondary ch... more Background repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. Results To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d. Conclusions RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The oriV of this plasmid resides within the repC gene and is located close to or inside of a large A+T region. RepC can act as an incompatibility factor, and the last 39 amino acid residues of the carboxy-terminal region of this protein are involved in promoting this phenotype.

Research paper thumbnail of Analysis of the Mechanism of Action of the Antisense RNA

Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the pr... more Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the presence of a repABC operon, which carries all the plasmid-encoded elements required for these functions. All repABC operons share three protein-encoding genes (repA, repB, and repC), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (parS). The products of repA and repB, in conjunction with the parS region, make up the segregation system, and they negatively regulate operon transcription. The last gene of the operon, repC, encodes the initiator protein. The ctRNA is a negative posttranscriptional regulator of repC. In this work, we analyzed the secondary structures of the ctRNA and its target and mapped the motifs involved in the complex formed between them. Essential residues for the effective interaction localize at the unpaired 5 end of the antisense molecule and the loop of the target mRNA. In light of our results, we propose a model explaining the mechanism of action of this ctRNA in the regulation of plasmid replication in R. etli.