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Background: Escherichia coli JU15 is a metabolically engineered strain capable to metabolize C5 and C6 sugars with a high yield of D-lactic acid production at its optimal growth temperature (37 °C). The simultaneous saccharification and fermentation process allow to use lignocellulosic biomass as a cost-effective and high-yield strategy. However, this process requires microorganisms capable of growth at a temperature close to 50 °C, at which the activity of cellulolytic enzymes works efficiently., Results: The thermotolerant strain GT48 was generated by adaptive laboratory evolution in batch and chemostat cultures under temperature increments until 48 °C. The strain GT48 was able to grow and ferment glucose to D-lactate at 47 °C. It was found that a pH of 6.3 conciliated with GT48 growth and cellulase activity of a commercial cocktail. Hence, this pH was used for the SSF of a diluted acid-pretreated corn stover (DAPCS) at a solid load of 15% (w/w), 15 FPU/g- DAPCS , and 47 °C. Under such conditions, the strain GT48 exhibited remarkable performance, producing D-lactate at a level of 1.41, 1.42, and 1.48-fold higher in titer, productivity, and yield, respectively, compared to parental strain at 45 °C., Conclusions: In general, our results show for the first time that a thermal-adapted strain of E. coli is capable of being used in the simultaneous saccharification and fermentation process without pre-saccharification stage at high temperatures., (© 2024. The Author(s).)

Acinetobacter pittii has increasingly been associated with several types of hospital-acquired severe infections. Genes implicated in carbapenem resistance, tigecycline resistance, or genes encoding extended spectrum cephalosporinases, such as bla ADC, are commonly found in isolates implicated in these infections. A. pittii strains that are pandrug resistant have occasionally been identified. Food for human consumption, animals and plants are environmental sources of this pathogen. An alarming situation is that A. pitti has been identified as responsible for outbreaks in different regions worldwide. In this study, 384 genomes of A. pittii were analyzed, comprising sequences from clinical and non-clinical origins from 32 countries. The objective was to investigate if clinical strains possess genetic traits facilitating hospital adaptation. Results indicate significant genomic variability in terms of size and gene content among A. pittii isolates. The core genome represents a small portion (25-36%) of each isolate's genome, while genes associated with antibiotic resistance and virulence predominantly belong to the accessory genome. Notably, antibiotic resistance genes are encoded by a diverse array of plasmids. As the core genome between environmental and hospital isolates is the same, we can assume that hospital isolates acquired ARGs due to a high selective pressure in these settings. The strain's phylogeographic distribution indicates that there is no geographical bias in the isolate distribution; isolates from different geographic regions are dispersed throughout a core genome phylogenetic tree. A single clade may include isolates from extremely distant geographical areas. Furthermore, strains isolated from the environment or animal, or plant sources frequently share the same clade as hospital isolates. Our analysis showed that the clinical isolates do not already possess specific genes, other than antibiotic-resistant genes, to thrive in the hospital setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Bello-López, Escobedo-Muñoz, Guerrero, Cruz-Córdova, Garza-González, Hernández-Castro, Zarain, Morfín-Otero, Volkow, Xicohtencatl-Cortes and Cevallos.)

Introduction: The Acinetobacter calcoaceticus-Acinetobacter baumannii complex, or Acb complex, consists of six species: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii , and Acinetobacter lactucae . A. baumannii is the most clinically significant of these species and is frequently related to healthcare-associated infections (HCAIs). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and associated genes ( cas ) constitute bacterial adaptive immune systems and function as variable genetic elements. This study aimed to conduct a genomic analysis of Acb complex genomes available in databases to describe and characterize CRISPR systems and cas genes., Methods: Acb complex genomes available in the NCBI and BV-BRC databases, the identification and characterization of CRISPR-Cas systems were performed using CRISPRCasFinder, CRISPRminer, and CRISPRDetect. Sequence types (STs) were determined using the Oxford scheme and ribosomal multilocus sequence typing (rMLST). Prophages were identified using PHASTER and Prophage Hunter., Results: A total of 293 genomes representing six Acb species exhibited CRISPR-related sequences. These genomes originate from various sources, including clinical specimens, animals, medical devices, and environmental samples. Sequence typing identified 145 ribosomal multilocus sequence types (rSTs). CRISPR-Cas systems were confirmed in 26.3% of the genomes, classified as subtypes I-Fa, I-Fb and I-Fv. Probable CRISPR arrays and cas genes associated with CRISPR-Cas subtypes III-A, I-B, and III-B were also detected. Some of the CRISPR-Cas systems are associated with genomic regions related to Cap4 proteins, and toxin-antitoxin systems. Moreover, prophage sequences were prevalent in 68.9% of the genomes. Analysis revealed a connection between these prophages and CRISPR-Cas systems, indicating an ongoing arms race between the bacteria and their bacteriophages. Furthermore, proteins associated with anti-CRISPR systems, such as AcrF11 and AcrF7, were identified in the A. baumannii and A. pittii genomes., Discussion: This study elucidates CRISPR-Cas systems and defense mechanisms within the Acb complex, highlighting their diverse distribution and interactions with prophages and other genetic elements. This study also provides valuable insights into the evolution and adaptation of these microorganisms in various environments and clinical settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mancilla-Rojano, Flores, Cevallos, Ochoa, Parra-Flores, Arellano-Galindo, Xicohtencatl-Cortes and Cruz-Córdova.)

We report the draft genomes of four Kluyveromyces marxianus isolates obtained from the elaboration process of henequen ( Agave fourcroydes ) mezcal, a Mexican alcoholic beverage. The average nucleotide identity analysis revealed that isolates derived from agave plants are distinct from those from other environments, including agave fermentations., Competing Interests: The authors declare no conflict of interest.

Acinetobacter pittii 978-A_19 was obtained from a parrot with pneumonia. It is resistant to ampicillin, carbenicillin, cephalosporins, clindamycin, and trimethoprim + sulfamethoxazole. The genome encodes a new bla ADC allele, a bla OXA-502 gene, possesses several virulence genes related to adherence and biofilm formation, and has types I, II, and IV secretion systems., Competing Interests: The authors declare no conflict of interest.

In 1989, Bouvet and Jeanjean delineated five proteolytic genomic species (GS) of Acinetobacter , each with two to four human isolates. Three were later validly named, whereas the remaining two (GS15 and GS16) have been awaiting nomenclatural clarification. Here we present the results of the genus-wide taxonomic study of 13 human strains classified as GS16 ( n =10) or GS15 ( n =3). Based on core genome phylogenetic analysis, the strains formed two respective but closely related phylogroups within the Acinetobacter haemolytic clade. The intraspecies genomic average nucleotide identity based on blast (ANIb) values for GS16 and GS15 reached ≥94.9 % and ≥98.7, respectively, whereas ANIb values between them were 92.5-93.5% and those between them and the known species were ≤91.5 %. GS16 and GS15 could be differentiated from the other Acinetobacter species by their ability to lyse gelatin and sheep blood and to assimilate d,l-lactate, along with their inability to acidify d-glucose and assimilate glutarate. In contrast, GS16 and GS15 were indistinguishable from one another by metabolic/physiological features or whole-cell MALDI-TOF mass spectra. All the GS15/GS16 genomes contained genes encoding a class D β-lactamase, Acinetobacter -derived cephalosporinase and aminoglycoside 6'- N -acetyltransferase. Searching NCBI databases revealed genome sequences of three additional isolates of GS16, but none of GS15. We conclude that our data support GS16 as representing a novel species, but leave the question of the taxonomic status of GS15 open, given its close relatedness to GS16 and the small number of available strains. We propose the name Acinetobacter higginsii sp. nov. for GS16, with the type strain NIPH 1872 T (CCM 9243 T =CIP 70.18 T =ATCC 17988 T ).

The objective of this research is to identify and systematize the medical conditions generated by SARS-CoV-2 on the optic nerve and retina of young, adult, and elderly adults who suffered from COVID-19 in the period 2019-2022. A theoretical documentary review (TDR) was conducted within the framework of an investigation to determine the current state of knowledge of the subject under study. The TDR includes the analysis of publications in the scientific databases PubMed/Medline, Ebsco, Scielo and Google. A total of 167 articles were found, of which 56 were studied in depth, and these evidence the impact of COVID-19 infection on the retina and optic nerve of infected patients, both during the acute phase and in subsequent recovery. Among the reported findings, the following stand out: anterior and posterior non-arteritic ischemic optic neuropathy, optic neuritis, central or branch vascular occlusion, paracentral acute medial maculopathy, neuroretinitis, as well as concomitant diagnoses such as possible Vogt-Koyanagi-Harada disease, multiple evanescent white dot syndrome (MEWDS), Purtscher-like retinopathy, among others., (Copyright © 2023 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.)

The Pseudomonas aeruginosa genome can change to adapt to different ecological niches. We compared four genomes from a Mexican hospital and 59 genomes from GenBank from different niches, such as urine, sputum, and environmental. The ST analysis showed that high-risk STs (ST235, ST773, and ST27) were present in the genomes of the three niches from GenBank, and the STs of Mexican genomes (ST167, ST2731, and ST549) differed from the GenBank genomes. Phylogenetic analysis showed that the genomes were clustering according to their ST and not their niche. When analyzing the genomic content, we observed that environmental genomes had genes involved in adapting to the environment not found in the clinics and that their mechanisms of resistance were mutations in antibiotic resistance-related genes. In contrast, clinical genomes from GenBank had resistance genes, in mobile/mobilizable genetic elements in the chromosome, except for the Mexican genomes that carried them mostly in plasmids. This was related to the presence of CRISPR-Cas and anti-CRISPR; however, Mexican strains only had plasmids and CRISPR-Cas. bla OXA-488 (a variant of bla OXA50 ) with higher activity against carbapenems was more prevalent in sputum genomes. The virulome analysis showed that exoS was most prevalent in the genomes of urinary samples and exoU and pldA in sputum samples. This study provides evidence regarding the genetic variability among P. aeruginosa isolated from different niches.

The CS21 pilus produced by enterotoxigenic Escherichia coli (ETEC) is involved in adherence to HT-29 intestinal cells. The CS21 pilus assembles proteins encoded by 14 genes clustered into the lng operon., Aim: This study aimed to determine whether E . coli BL21 (ECBL) transformed with the lng operon lacking the lngA gene (pE9034AΔ lngA ) and complemented in trans with lngA variants of ETEC clinical strains, as well as point substitutions, exhibited modified adherence to HT-29 cells., Methods: A kanamycin cassette was used to replace the lngA gene in the lng operon of the E9034A strain, and the construct was transformed into the ECBL strain. The pJET1.2 vector carrying lngA genes with allelic variants was transformed into ECBLpE9034AΔ lngA (ECBLΔ lngA ). The point substitutions were performed in the pJET lngA FMU073332 vector., Results: Bioinformatic alignment analysis of the LngA proteins showed hypervariable regions and clustered the clinical ETEC strains into three groups. Variations in amino acid residues affect the adherence percentages of recombinant ECBL strains with lngA variants and site-specific mutations with HT-29 cells., Conclusion: In this study, ECBL carrying the lng operon harboring lngA variants of six clinical ETEC strains, as well as point substitutions, exerted an effect on the adherence of ECBL to HT-29 cells, thereby confirming the importance of the CS21 pilus in adherence.

Chytridiomycosis, a lethal fungal disease caused by Batrachochytrium dendrobatidis (Bd), is responsible for population declines and extinctions of amphibians worldwide. However, not all amphibian species are equally susceptible to the disease; some species persist in Bd enzootic regions with no population reductions. Recently, it has been shown that the amphibian skin microbiome plays a crucial role in the defense against Bd. Numerous bacterial isolates with the capacity to inhibit the growth of Batrachochytrium fungi have been isolated from the skin of amphibians. Here, we characterized eight Acinetobacter bacteria isolated from the frogs Agalychnis callidryas and Craugastor fitzingeri at the genomic level. A total of five isolates belonged to Acinetobacter pittii,Acinetobacter radioresistens, or Acinetobactermodestus, and three were not identified as any of the known species, suggesting they are members of new species. We showed that seven isolates inhibited the growth of Bd and that all eight isolates inhibited the growth of the phytopathogen fungus Botrytis cinerea. Finally, we identified the biosynthetic gene clusters that could be involved in the antifungal activity of these isolates. Our results suggest that the frog skin microbiome includes Acinetobacter isolates that are new to science and have broad antifungal functions, perhaps driven by distinct genetic mechanisms., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

bla IMP and bla VIM are the most detected plasmid-encoded carbapenemase genes in Pseudomonas aeruginosa . Previous studies have reported plasmid sequences carrying bla IMP variants, except bla IMP-56 . In this study, we aimed to characterize a plasmid carrying bla IMP-56 in a P. aeruginosa strain isolated from a Mexican hospital. The whole genome of P. aeruginosa strain PE52 was sequenced using Illumina Miseq 2 × 150 bp, with 5 million paired-end reads. We characterized a 27 kb plasmid (pPE52IMP) that carried bla IMP-56 . The phylogenetic analysis of RepA in pPE52IMP and 33 P. aeruginosa plasmids carrying resistance genes reported in the GenBank revealed that pPE52IMP and four plasmids (pMATVIM-7, unnamed (FDAARGOS_570), pD5170990, and pMRVIM0713) were in the same clade. These closely related plasmids belonged to the MOB P11 subfamily and had similar backbones. Another plasmid (p4130-KPC) had a similar backbone to pPE52IMP; however, its RepA was truncated. In these plasmids, the resistance genes bla KPC-2 , bla VIM variants, aac(6')-Ib4 , bla OXA variants, and bla IMP-56 were inserted between phd and resolvase genes. This study describes a new family of plasmids carrying resistance genes, with a similar backbone, the same RepA, and belonging to the MOB P11 subfamily in P. aeruginosa . In addition, our characterized plasmid harboring bla IMP-56 (pPE52IMP) belongs to this family.

Background: Given the chemical richness of medicinal plants ( Bidens pilosa L. and Croton floccosus ) in Ecuador, they are considered the natural source of numerous medicines. Methods: The leaves were dried at 40°C and 50°C and the extracts were characterized by means of phytochemical screening, verifying the presence of secondary metabolites such as alkaloids, reducing sugars, phenols, flavonoids, tannins and saponins. Three extraction processes were carried out, with two solvents of different polarities: hexane and ethanol. The extraction methods that were applied to the leaves of the plants were Soxhlet, ultrasonic bath and maceration, the latter two at room temperature and Soxhlet at the boiling temperature of the solvent. Determination of the total content of phenols and flavonoids is carried out using the Follin-Ciocalteau colorimetric reaction, Quercetin standard, Aluminum Chloride solution measured with a UV-Vis spectrophotometer. The antioxidant activity was performed with the DPPH radical and measured with the same equipment. Results : The highest content of total phenols obtained by employing the Soxhlet method for extraction when the material was dried at 50°C was 48.609 ± 0.370 mg GAE/g of dry sample for Bidens pilosa L. while in the case of Croton floccosus it was 128.212 ± 0.601 mg GAE/g of dry sample obtained from the extraction by means of maceration. Finally, the antioxidant activity against the 1.1-diphenyl-2-picryl-hydrazyl radical was determined, and it was found that the Bidens pilosa L. species performed better and responded better to the test, with an IC 50 value of 239.33 µg/mL, than Croton floccosus (IC 50 of 644.125 µg/mL). Conclusions: The following preliminary phytochemical study of the Bidens pilosa L. and Croton floccosus plants provided important information on the content of secondary metabolites and response to the DPPH radical reported for the first time in Ecuador, which may be future use for medicinal application., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 Ruiz-Reyes E et al.)

We survey here the Alphaproteobacteria, a large class encompassing physiologically diverse bacteria which are divided in several orders established since 2007. Currently, there is considerable uncertainty regarding the classification of an increasing number of marine metagenome-assembled genomes (MAGs) that remain poorly defined in their taxonomic position within Alphaproteobacteria. The traditional classification of NCBI taxonomy is increasingly complemented by the Genome Taxonomy Database (GTDB), but the two taxonomies differ considerably in the classification of several Alphaproteobacteria, especially from ocean metagenomes. We analyzed the classification of Alphaproteobacteria lineages that are most common in marine environments, using integrated approaches of phylogenomics and functional profiling of metabolic features that define their aerobic metabolism. Using protein markers such as NuoL, the largest membrane subunit of complex I, we have identified new clades of Alphaproteobacteria that are specific to marine niches with steep oxygen gradients (oxycline). These bacteria have relatives among MAGs found in anoxic strata of Lake Tanganyika and together define a lineage that is distinct from either Rhodospirillales or Sneathiellales. We characterized in particular the new 'oxycline' clade. Our analysis of Alphaproteobacteria also reveals new clues regarding the ancestry of mitochondria, which likely evolved in oxycline marine environments.

The bioinformatic analysis that we made of 492 Acinetobacter baumannii plasmid sequences identified 418 genes encoding Replication Initiator (Rep) proteins that fell into at least fourteen groups according to the protein domains that they contained. The most abundant group of Rep proteins contained a Rep_3 superfamily domain, followed by Rep proteins containing Replicase/PriCT_1 superfamily domains, and then by Reps possessing only an HTH_MerR-SF superfamily domain. The remaining eleven groups contain only a few members. To evaluate the diversity of these Rep proteins, we classify them using the current scheme of GR homology groups, which contains 34 groups. However, we needed to create 22 additional GR homology groups to capture all the Rep protein diversity of the plasmid collection. Finally, our bioinformatic analysis suggests that a large fraction of the plasmids seem to have a restricted host range limited to Acinetobacter species, except for those belonging to GR38 that have a very wide host range. To facilitate the future analysis of the Rep proteins, we included a list of the DNA and protein sequences, in fasta format, of the representatives of each one of the GR homology groups., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bacteriophages are considered the most abundant biological entities on earth, and they are able to modulate the populations of their bacterial hosts. Although the potential of bacteriophages has been accepted as an alternative strategy to combat multidrug-resistant pathogenic bacteria, there still exists a considerable knowledge gap regarding their genetic diversity, which hinders their use as antimicrobial agents. In this study, we undertook a genomic and phylogenetic characterization of the phage Ab11510-phi, which was isolated from a multidrug-resistant Acinetobacter baumannii strain (Ab11510). We found that Ab11510-phi has a narrow host range and belongs to a small group of transposable phages of the genus Vieuvirus that have only been reported to infect Acinetobacter bacteria. Finally, we showed that Ab11510-phi (as well as other vieuvirus phages) has a high level of mosaicism. On a broader level, we demonstrate that comparative genomics and phylogenetic analysis are necessary tools for the proper characterization of phage diversity.

The Acinetobacter calcoaceticus - baumannii ( Acb ) complex is regarded as a group of phenotypically indistinguishable opportunistic pathogens responsible for mainly causing hospital-acquired pneumonia and bacteremia. The aim of this study was to determine the frequency of isolation of the species that constitute the Acb complex, as well as their susceptibility to antibiotics, and their distribution at the Hospital Infantil de Mexico Federico Gomez (HIMFG). A total of 88 strains previously identified by Vitek 2®, 40 as Acinetobacter baumannii and 48 as Acb complex were isolated from 52 children from 07, January 2015 to 28, September 2017. A. baumannii accounted for 89.77% (79/88) of the strains; Acinetobacter pittii , 6.82% (6/88); and Acinetobacter nosocomialis , 3.40% (3/88). Most strains were recovered mainly from patients in the intensive care unit (ICU) and emergency wards. Blood cultures (BC) provided 44.32% (39/88) of strains. The 13.63% (12/88) of strains were associated with primary bacteremia, 3.4% (3/88) with secondary bacteremia, and 2.3% (2/88) with pneumonia. In addition, 44.32% (39/88) were multidrug-resistant (MDR) strains and, 11.36% (10/88) were extensively drug-resistant (XDR). All strains amplified the bla OXA-51 gene; 51.13% (45/88), the bla OXA-23 gene; 4.54% (4/88), the bla OXA-24 gene; and 2.27% (2/88), the bla OXA-58 gene. Plasmid profiles showed that the strains had 1-6 plasmids. The strains were distributed in 52 pulsotypes, and 24 showed identical restriction patterns, with a correlation coefficient of 1.0. Notably, some strains with the same pulsotype were isolated from different patients, wards, or years, suggesting the persistence of more than one clone. Twenty-seven sequence types (STs) were determined for the strains based on a Pasteur multilocus sequence typing (MLST) scheme using massive sequencing; the most prevalent was ST 156 (27.27%, 24/88). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas I-Fb system provided amplification in A. baumannii and A. pittii strains (22.73%, 20/88). This study identified an increased number of MDR strains and the relationship among strains through molecular typing. The data suggest that more than one strain could be causing an infection in some patient. The implementation of molecular epidemiology allowed the characterization of a set of strains and identification of different attributes associated with its distribution in a specific environment., (Copyright © 2020 Mancilla-Rojano, Ochoa, Reyes-Grajeda, Flores, Medina-Contreras, Espinosa-Mazariego, Parra-Ortega, Rosa-Zamboni, Castellanos-Cruz, Arellano-Galindo, Cevallos, Hernández-Castro, Xicohtencatl-Cortes and Cruz-Córdova.)

Objective: To describe the clinical features, outcomes, and molecular epidemiology of an outbreak of multidrug resistant (MDR) A. baumannii., Methods: We performed a retrospective analysis of all MDR A. baumannii isolates recovered during an outbreak from 2011 to 2015 in a tertiary care cancer hospital. Cases were classified as colonized or infected. We determined sequence types following the Bartual scheme and plasmid profiles., Results: There were 106 strains of A. baumannii isolated during the study period. Sixty-six (62.3%) were considered as infection and 40 (37.7%) as colonization. The index case, identified by molecular epidemiology, was a patient with a drain transferred from a hospital outside Mexico City. Ninety-eight additional cases had the same MultiLocus Sequence Typing (MLST) 758, of which 94 also had the same plasmid profile, two had an extra plasmid, and two had a different plasmid. The remaining seven isolates belonged to different MLSTs. Fifty-three patients (50%) died within 30 days of A. baumanniii isolation: 28 (20%) in colonized and 45 (68.2%) in those classified as infection (p<0.001). In multivariate regression analysis, clinical infection and patients with hematologic neoplasm, predicted 30-day mortality. The molecular epidemiology of this outbreak showed the threat posed by the introduction of MDR strains from other institutions in a hospital of immunosuppressed patients and highlights the importance of adhering to preventive measures, including contact isolation, when admitting patients with draining wounds who have been hospitalized in other institutions., Competing Interests: The authors have declared that no competing interests exist.

Acinetobacter baumannii is an emergent bacterial pathogen that provokes many types of infections in hospitals around the world. The genome of this organism consists of a chromosome and plasmids. These plasmids vary over a wide size range and many of them have been linked to the acquisition of antibiotic-resistance genes. Our bioinformatic analyses indicate that A. baumannii plasmids belong to a small number of plasmid lineages. The general structure of these lineages seems to be very stable and consists not only of genes involved in plasmid maintenance functions but of gene sets encoding poorly characterized proteins, not obviously linked to survival in the hospital setting, and opening the possibility that they improve the parasitic properties of plasmids. An analysis of genes involved in replication, suggests that members of the same plasmid lineage are part of the same plasmid incompatibility group. The same analysis showed the necessity of classifying the Rep proteins in ten new groups, under the scheme proposed by Bertini et al. (2010). Also, we show that some plasmid lineages have the potential capacity to replicate in many bacterial genera including those embracing human pathogen species, while others seem to replicate only within the limits of the Acinetobacter genus. Moreover, some plasmid lineages are widely distributed along the A. baumannii phylogenetic tree. Despite this, a number of them lack genes involved in conjugation or mobilization functions. Interestingly, only 34.6% of the plasmids analyzed here possess antibiotic resistance genes and most of them belong to fourteen plasmid lineages of the twenty one described here. Gene flux between plasmid lineages appears primarily limited to transposable elements, which sometimes carry antibiotic resistance genes. In most plasmid lineages transposable elements and antibiotic resistance genes are secondary acquisitions. Finally, broad host-range plasmids appear to have played a crucial role., (Copyright © 2020 Salgado-Camargo, Castro-Jaimes, Gutierrez-Rios, Lozano, Altamirano-Pacheco, Silva-Sanchez, Pérez-Oseguera, Volkow, Castillo-Ramírez and Cevallos.)

Bryophytes are the earliest plant group on Earth. They are a fundamental component of many ecosystems around the World. Some of their main roles are related to soil development, water retention, and biogeochemical cycling. Bryophytes include liverworts, hornworts, and mosses. The sequencing of chloroplast and mitochondria genomes has been useful to elucidate the taxonomy of this heterogeneous plant group. To date, despite their ecological importance only 41 mosses mitogenomes have been deposited in the GenBank. Here, the complete mitochondria genome sequence of Pseudocrossidium replicatum , a moss of the Pottiaceae family isolated in Tlaxcala, Mexico, is reported. The mitochondrial genome size of P. replicatum comprises 105,495 bp and contains the groups of genes described for other bryophytes mitogenomes. Our phylogenetic analysis shows that during the evolution of the mosses' mitogenome, nad7 , rps4 , rpl16 , and rpl10 genes were lost independently in several lineages. The complete mitogenome sequence reported here would be a useful tool for our comprehension of the evolutionary and population genetics of this group of plants., Competing Interests: No potential conflict of interest was reported by the author(s), (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Acinetobacter haemolyticus is a Gammaproteobacterium that has been involved in serious diseases frequently linked to the nosocomial environment. Most of the strains causing such infections are sensitive to a wide variety of antibiotics, but recent reports indicate that this pathogen is acquiring very efficiently carbapenem-resistance determinants like the bla NDM-1 gene, all over the world. With this work we contribute with a collection set of 31 newly sequenced nosocomial A. haemolyticus isolates. Genome analysis of these sequences and others collected from RefSeq indicates that their chromosomes are organized in 12 syntenic blocks that contain most of the core genome genes. These blocks are separated by hypervariable regions that are rich in unique gene families, but also have signals of horizontal gene transfer. Genes involved in virulence or encoding different secretion systems are located inside syntenic regions and have recombination signals. The relative order of the synthetic blocks along the A. haemolyticus chromosome can change, indicating that they have been subject to several kinds of inversions. Genomes of this microorganism show large differences in gene content even if they are in the same clade. Here we also show that A. haemolyticus has an open pan-genome., (Copyright © 2020 Castro-Jaimes, Bello-López, Velázquez-Acosta, Volkow-Fernández, Lozano-Zarain, Castillo-Ramírez and Cevallos.)

Irritable bowel syndrome (IBS) is the most frequent functional gastrointestinal disorder, worldwide, with a high prevalence among Mestizo Latin Americans. Because several inflammatory disorders appear to affect this population, a further understanding of host genomic background variants, in conjunction with colonic mucosa dysbiosis, is necessary to determine IBS physiopathology and the effects of environmental pressures. Using a simple polygenic model, host single nucleotide polymorphisms (SNPs) and the taxonomic compositions of microbiota were compared between IBS patients and healthy subjects. As proof of concept, five IBS-Rome III patients and five healthy controls (HCs) were systematically studied. The human and bacterial intestinal metagenome of each subject was taxonomically annotated and screened for previously annotated IBS, ulcerative colitis, and Crohn's disease-associated SNPs or taxon abundance. Dietary data and fecal markers were collected and associated with the intestinal microbiome. However, more than 1,000 variants were found, and at least 76 SNPs differentiated IBS patients from HCs, as did associations with 4 phyla and 10 bacterial genera. In this study, we found elements supporting a polygenic background, with frequent variants, among the Mestizo population, and the colonic mucosal enrichment of Bacteroides, Alteromonas, Neisseria, Streptococcus , and Microbacterium , may serve as a hallmark for IBS., (Copyright © 2020 Arredondo-Hernández, Schmulson, Orduña, López-Leal, Zarate, Alanis-Funes, Alcaraz, Santiago-Cruz, Cevallos, Villa, Ponce-de-León Rosales and Lopez-Vidal.)

There is increased evidence demonstrating the association between Crohn's Disease (CD), a type of Inflammatory Bowel Disease (IBD), and non-diarrheagenic Adherent/Invasive Escherichia coli (AIEC) isolates. AIEC strains are phenotypically characterized by their adhesion, invasion and intra-macrophage survival capabilities. In the present study, the genomes of five AIEC strains isolated from individuals without IBD (four from healthy donors and one from peritoneal liquid) were sequenced and compared with AIEC prototype strains (LF82 and NRG857c), and with extra-intestinal uropathogenic strain (UPEC CFT073). Non-IBD-AIEC strains showed an Average Nucleotide Identity up to 98% compared with control strains. Blast identities of the five non-IBD-AIEC strains were higher when compared to AIEC and UPEC reference strains than with another E. coli pathotypes, suggesting a relationship between them. The SNPs phylogeny grouped the five non-IBD-AIEC strains in one separated cluster, which indicates the emergence of these strains apart from the AIEC group. Additionally, four genomic islands not previously reported in AIEC strains were identified. An incomplete Type VI secretion system was found in non-IBD-AIEC strains; however, the Type II secretion system was complete. Several groups of genes reported in AIEC strains were searched in the five non-IBD-AIEC strains, and the presence of fimA, fliC, fuhD, chuA, irp2 and cvaC were confirmed. Other virulence factors were detected in non-IBD-AIEC strains, which were absent in AIEC reference strains, including EhaG, non-fimbrial adhesin 1, PapG, F17D-G, YehA/D, FeuC, IucD, CbtA, VgrG-1, Cnf1 and HlyE. Based on the differences in virulence determinants and SNPs, it is plausible to suggest that non-IBD AIEC strains belong to a different pathotype., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The use of antimicrobials and myeloablative chemotherapy regimens has promoted multiresistant microorganisms to emerge as nosocomial pathogens, such as vancomycin-resistant Enterococcus faecium (VREfm). We described a polyclonal outbreak of bloodstream infection caused by Efm in a hemato-oncological ward in Mexico. Our aim was to describe the clonal complex (CC) of the Efm strains isolated in the outbreak in comparison with commensal and environmental isolates. Methodology: Sixty Efm clinical, environmental, and commensal strains were included. We constructed a cladogram and a phylogenetic tree using Vitek and Multilocus sequence typing data, respectively. Results: We reported 20 new sequence types (ST), among which 17/43 clinical isolates belonged to CC17. The predominant ST in the clinical strains were ST757, ST1304, ST412, and ST770. Neither environmental nor commensal isolates belonged to CC17. The phylogeny of our collection shows that the majority of the clinical isolates were different from the environmental and commensal isolates, and only a small group of clinical isolates was closely related with environmental and commensal isolates. The cladogram revealed a similar segregation to that of the phylogeny. Conclusions: We found a high diversity among clinical, environmental, and commensal strains in a group of samples in a single hospital. Highest diversity was found between commensal and environmental isolates.

Over the last few decades, carbapenemase-producing Acinetobacter baumannii has become a major cause of nosocomial infections all over the world. However, the genome identity of lineages of this species in Latin America has not been studied as much as in developed countries. Here, through a population genomics approach considering the whole genomes of 148 isolates (almost 40 from Mexico and Honduras), we describe the recent emergence of the lineage sequence type 758 (ST758), which belongs to the international clone V and has spread out to Canada, Mexico, Honduras, and Colombia. Notably, this lineage was found to coexist with other A. baumannii lineages in hospitals in Mexico and Honduras. Isolates from this lineage show considerable variation in antibiotic resistance profiles, but most of them are resistant to carbapenems. Moreover, we found a variety of acquired oxacillinase (OXA) families within this lineage and tracked the very recent inception, and subsequent horizontal transmission, of the OXA-239 carbapenemase. This work highlights the urgent need to investigate recently emerged lineages of this species in Latin America and elsewhere, as these might harbor novel antibiotic resistance genes. IMPORTANCE A. baumannii is a major cause of nosocomial infections all over the world. Although many isolates from developed countries have been studied in terms of their genome sequence, isolates from Latin America have been much less studied. In this study, using a population genomics approach considering the whole genomes of 148 isolates, we describe the recent emergence of the lineage ST758 endemic to Latin America and the inception of the OXA-239 carbapenemase. Our study highlights the urgent need to investigate recently emerged lineages of this species in Latin America and elsewhere, as these might harbor novel antibiotic resistance genes., (Copyright © 2020 Graña-Miraglia et al.)

Although Acinetobacter baumannii has become one of the most important nosocomial pathogens worldwide, very little is known about the genetic identity of isolates from less developed countries in Latin America. To alleviate this, we sequenced the genomes of 16 A. baumannii isolates from Honduras. Whole-genome sequencing was conducted on 16 isolates from five Honduran Hospitals. With the sequences of these Honduran isolates and other 42 publically available genomes, a maximum likelihood phylogeny was constructed to establish the relationship between the Honduran isolates and those belonging to the International Clones (ICs). In addition, sequence type (ST) assignation was conducted by the PubMLST, and antibiotic resistance genes were identified using ResFinder. The Honduran isolates are highly diverse and contain new allele combinations under the Bartual multilocus sequence typing scheme. The most common STs were ST B 447/ST P 10 and ST B 758/ST P 156. Furthermore, none of these isolates belongs to clonal complexes related to the ICs. Antibiotic susceptibility profiles of these isolates showed that they are multidrug resistant (MDR) or extensively drug resistant (XDR). In addition, the Honduran isolates had genes involved in resistance to seven antibiotic families. For instance, several bla OXA alleles were found, including bla OXA-23 and a gene encoding the metallo-beta-lactamase NDM-1. Notably, nine of the Honduran isolates have antibiotic resistance genes to three or more antibiotic families. In summary, in this study, we unveiled an untapped source of genetic diversity of MDR and XDR isolates; notably, these isolates did not belong to the well-known ICs.

Mosses in conjunction with hornworts and liverworts are collectively referred to as bryophytes. These seedless, nonvascular plants are the closest extant relatives of early terrestrial plants and their study is essential to understand the evolutionary first steps of land plants. Here we report the complete chloroplast (cp) genome sequence of Pseudocrossidium replicatum, a moss belonging to the Pottiaceae family that is common in the central highlands of Mexico, in South America, in southern USA, and in Kenia. The cp genome (plastome) of P. replicatum is 123,512 bp in size, comprising inverted repeats of 9,886 bp and single-copy regions of 85,146 bp (LSC) and 18,594 bp (SSC). The plastome encodes 82 different proteins, 31 different tRNAs, and 4 different rRNAs. Phylogenetic analysis using 16 cp protein-coding genes demonstrated that P. replicatum is closely related to Syntrichia ruralis, and the most basal mosses are Takakia lepidozioides followed by Sphagnum palustre. Our analysis indicates that during the evolution of the mosses' plastome, eight genes were lost. The complete plastome sequence reported here can be useful in evolutionary and population genetics.

Zymomonas mobilis ZM4 is an ethanol-producing microbe that is constitutively tolerant to this solvent. For a better understanding of the ethanol tolerance phenomenon we obtained and characterized two ZM4 mutants (ER79ap and ER79ag) with higher ethanol tolerance than the wild-type. Mutants were evaluated in different ethanol concentrations and this analysis showed that mutant ER79ap was more tolerant and had a better performance in terms of cell viability, than the wild-type strain and ER79ag mutant. Genotyping of the mutant strains showed that both carry non-synonymous mutations in clpP and spoT/relA genes. A third non-synonymous mutation was found only in strain ER79ap, in the clpB gene. Considering that ER79ap has the best tolerance to added ethanol, the mutant alleles of this strain were evaluated in ZM4 and here we show that while all of them contribute to ethanol tolerance, mutation within spo T/ rel A gene seems to be the most important.

Acinetobacter baumannii is an opportunistic pathogen and is one of the primary etiological agents of healthcare-associated infections (HAIs). A. baumannii infections are difficult to treat due to the intrinsic and acquired antibiotic resistance of strains of this bacterium, which frequently limits therapeutic options. In this study, five A. baumannii strains (810CP, 433H, 434H, 483H, and A-2), all of which were isolated from a child with leukemia M2, were characterized through antibiotic susceptibility profiling, the detection of genes encoding carbapenem hydrolyzing oxacillinases, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), adherence and invasion assays toward the A549 cell line, and the whole-genome sequence (WGS). The five strains showed Multidrug resistant (MDR) profiles and amplification of the bla OXA-23 gene, belonging to ST758 and grouped into two PFGE clusters. WGS of 810CP revealed the presence of a circular chromosome and two small plasmids, pAba810CPa and pAba810CPb. Both plasmids carried genes encoding the Sp1TA system, although resistance genes were not identified. A gene-by-gene comparison analysis was performed among the A. baumannii strains isolated in this study and others A . baumannii ST758 strains (HIMFG and INCan), showing that 86% of genes were present in all analyzed strains. Interestingly, the 433H, 434H, and 483H strains varied by 8-10 single-nucleotide variants (SNVs), while the A2 and 810CP strains varied by 46 SNVs. Subsequently, an analysis using BacWGSTdb showed that all of our strains had the same resistance genes and were ST758. However, some variations were observed in relation to virulence genes, mainly in the 810CP strain. The genes involved in the synthesis of hepta-acylated lipooligosaccharides, the pgaABCD locus encoding poly-β-1-6- N -acetylglucosamine, the ompA gene, Csu pili, bap , the two-component system bfms / bfmR , a member of the phospholipase D family, and two iron-uptake systems were identified in our A . baumannii strains genome. The five A. baumannii strains isolated from the child were genetically different and showed important characteristics that promote survival in a hospital environment. The elucidation of their genomic sequences provides important information for understanding their epidemiology, antibiotic resistance, and putative virulence factors.

This communication reports a further examination of venom gland transcripts and venom composition of the Mexican scorpion Thorellius atrox using RNA-seq and tandem mass spectrometry. The RNA-seq, which was performed with the Illumina protocol, yielded more than 20,000 assembled transcripts. Following a database search and annotation strategy, 160 transcripts were identified, potentially coding for venom components. A novel sequence was identified that potentially codes for a peptide with similarity to spider ω-agatoxins, which act on voltage-gated calcium channels, not known before to exist in scorpion venoms. Analogous transcripts were found in other scorpion species. They could represent members of a new scorpion toxin family, here named omegascorpins. The mass fingerprint by LC-MS identified 135 individual venom components, five of which matched with the theoretical masses of putative peptides translated from the transcriptome. The LC-MS/MS de novo sequencing allowed to reconstruct and identify 42 proteins encoded by assembled transcripts, thus validating the transcriptome analysis. Earlier studies conducted with this scorpion venom permitted the identification of only twenty putative venom components. The present work performed with more powerful and modern omic technologies demonstrates the capacity of accomplishing a deeper characterization of scorpion venom components and the identification of novel molecules with potential applications in biomedicine and the study of ion channel physiology., Competing Interests: The authors declare no conflict of interest.

Genome sequencing has been useful to gain an understanding of bacterial evolution. It has been used for studying the phylogeography and/or the impact of mutation and recombination on bacterial populations. However, it has rarely been used to study gene turnover at microevolutionary scales. Here, we sequenced Mexican strains of the human pathogen Acinetobacter baumannii sampled from the same locale over a 3 year period to obtain insights into the microevolutionary dynamics of gene content variability. We found that the Mexican A. baumannii population was recently founded and has been emerging due to a rapid clonal expansion. Furthermore, we noticed that on average the Mexican strains differed from each other by over 300 genes and, notably, this gene content variation has accrued more frequently and faster than the accumulation of mutations. Moreover, due to its rapid pace, gene content variation reflects the phylogeny only at very short periods of time. Additionally, we found that the external branches of the phylogeny had almost 100 more genes than the internal branches. All in all, these results show that rapid gene turnover has been of paramount importance in producing genetic variation within this population and demonstrate the utility of genome sequencing to study alternative forms of genetic variation.

Bacterial cell division is a complex process that relies on a multiprotein complex composed of a core of widely conserved and generally essential proteins and on accessory proteins that vary in number and identity in different bacteria. The assembly of this complex and, particularly, the initiation of constriction are regulated processes that have come under intensive study. In this work, we characterize the function of DipI, a protein conserved in Alphaproteobacteria and Betaproteobacteria that is essential in Caulobacter crescentus Our results show that DipI is a periplasmic protein that is recruited late to the division site and that it is required for the initiation of constriction. The recruitment of the conserved cell division proteins is not affected by the absence of DipI, but localization of DipI to the division site occurs only after a mature divisome has formed. Yeast two-hybrid analysis showed that DipI strongly interacts with the FtsQLB complex, which has been recently implicated in regulating constriction initiation. A possible role of DipI in this process is discussed. IMPORTANCE Bacterial cell division is a complex process for which most bacterial cells assemble a multiprotein complex that consists of conserved proteins and of accessory proteins that differ among bacterial groups. In this work, we describe a new cell division protein (DipI) present only in a group of bacteria but essential in Caulobacter crescentus Cells devoid of DipI cannot constrict. Although a mature divisome is required for DipI recruitment, DipI is not needed for recruiting other division proteins. These results, together with the interaction of DipI with a protein complex that has been suggested to regulate cell wall synthesis during division, suggest that DipI may be part of the regulatory mechanism that controls constriction initiation., (Copyright © 2017 American Society for Microbiology.)

Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México., (Copyright © 2017 Saldaña-Ahuactzi et al.)

A clinical isolate of extended-spectrum-β-lactamase-producing Klebsiella quasipneumoniae subsp. similipneumoniae 06-219 with hypermucoviscosity phenotypes obtained from a urine culture of an adult patient was used for whole-genome sequencing. Here, we report the draft genome sequences of this strain, consisting of 53 contigs with an ~5.6-Mb genome size and an average G+C content of 57.36%. The annotation revealed 6,622 coding DNA sequences and 77 tRNA genes., (Copyright © 2016 Garza-Ramos et al.)

Acinetobacter baumannii has emerged as a dangerous nosocomial pathogen, particularly for severely ill patients in intensive care units and patients with hematologic malignancies. Here, we present the complete genome sequence of a multidrug-resistant A. baumannii isolate, recovered from a Mexican hospital and classified as sequence type 422 according to the multilocus sequence typing Pasteur scheme., (Copyright © 2016 Castro-Jaimes et al.)

Sigma factors are an essential part of bacterial gene regulation and have been extensively studied as far as their molecular mechanisms and protein structure are concerned. However, their molecular evolution, especially for the alternative sigma factors, is poorly understood. Here, we analyze the evolutionary forces that have shaped the rpoH sigma factors within the alphaproteobacteria. We found that an ancient duplication gave rise to two major groups of rpoH sigma factors and that after this event horizontal gene transfer (HGT) occurred in rpoH 1 group. We also noted that purifying selection has differentially affected distinct parts of the gene; singularly, the gene segment that encodes the region 4.2, which interacts with the -35 motif of the RpoH-dependent genes, has been under relaxed purifying selection. Furthermore, these two major groups are clearly differentiated from one another regarding their promoter selectivity, as rpoH 1 is under the transcriptional control of σ(70) and σ(32), whereas rpoH 2 is under the transcriptional control of σ(24). Our results suggest a scenario in which HGT, gene loss, variable purifying selection and clear promoter specialization occurred after the ancestral duplication event. More generally, our study offers insights into the molecular evolution of alternative sigma factors and highlights the importance of analyzing not only the coding regions but also the promoter regions.

Acinetobacter baumanniihas emerged as an important nosocomial pathogen worldwide. Here, we present the draft genome of the first multidrug-resistantA. baumanniiisolate, sampled from a tertiary hospital in Mexico City. This genome will provide a starting point for studying the genomic diversity of this species in Mexico., (Copyright © 2016 Graña-Miraglia et al.)

We characterized an outbreak of imipenem-resistant Acinetobacter baumannii with clinical and environmental isolates from a tertiary care hospital in San Luis Potosi, Mexico. During a 4-month period, a total of 32 nonrepetitive imipenem-resistant clinical isolates of A. baumannii were collected. All isolates were susceptible to colistin and tigecycline and resistant to cefepime, ceftazidime, ceftriaxone, imipenem, and meropenem. Genotyping by pulsed-field gel electrophoresis showed a major clone (A). Multilocus sequence type (MLST) analysis was performed, revealing sequence type (ST) 417 (ST417) and 208 (ST208). The blaIMP-, blaVIM-, blaGIM-, blaSIM-, blaNDM-type, and blaOXA-type (blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, and blaOXA-58-like) genes were screened and showed that the blaOXA-51-like and blaOXA-24-like genes were present in all isolates. Sequencing and southern hybridization were performed, confirming the presence of the blaOXA-72 gene and its plasmid-borne nature. In addition, the blaOXA-72-XerC/XerD-like association was identified. These findings indicate that a clonal spread of blaOXA-72-producing A. baumannii ST417 had occurred throughout the hospital. The ST417 corresponded with a previous ST described in the United States.

Helicobacter pylori infection is a risk factor for the development of gastric cancer and other gastroduodenal diseases. We report here the complete genome sequence of H. pylori strain 29CaP, isolated from a Mexican patient with gastric cancer. The genomic data analysis revealed a cag-negative H. pylori strain that contains a prophage sequence., (Copyright © 2016 Mucito-Varela et al.)

Helicobacter pylori-induced gastritis is a risk factor for developing gastric pathologies. Here, we report the complete genome sequence of a multidrug-resistant H. pylori strain isolated from a chronic gastritis patient in Mexico City, Mexico. Nonvirulent VacA and cag-pathogenicity island (PAI) genotypes were found, but the presence of a potential mobilizable plasmid carrying an IS605 element is of outstanding interest., (Copyright © 2016 Mucito-Varela et al.)

The maintenance of large plasmid in a wide variety of alpha-proteobacteria depends on the repABC replication/segregation unit. The intergenic repB-repC region of these plasmids encodes a countertranscribed RNA (ctRNA) that modulates the transcription/translation rate of RepC, the initiator protein. The ctRNA acts as a strong incompatibility factor when expressed in trans. We followed a site directed mutagenesis approach to map those sequences of the ctRNA that are required for plasmid incompatibility and for plasmid replication control. We found that the first three nucleotides of the 5'-end of the ctRNA are essential for interactions with its target RNA. We also found that stretches of 4-5 nucleotides of non-complementarity within the first 10 nucleotides of the left arm of the ctRNA and the target RNA are sufficient to avoid plasmid incompatibility. Additionally, miniplasmid derivatives expressing ctRNAs with mutations in the 5' end or small deletions in the ctRNA are capable of controlling their own replication and coexisting with the parental plasmid. We suggest that a mechanism that could have a crucial role in the speciation process of repABC plasmids is to accumulate enough changes in this small region of the ctRNA gene to disrupt heteroduplex formation between the target RNA of one plasmid and the ctRNA of the other. Plasmids carrying these changes will not have defects in their maintenance., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C., Results: YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the "genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid., Conclusions: We showed that the transfer of the YU39 blaCMY-2 gene harbored on a non- conjugative pA/C requires the machinery of a highly conjugative pX1 plasmid. Our experiments demonstrate the complex interactions a single strain can exploit to contend with the challenge of horizontal transfer and antibiotic selective pressure.

Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The repABC replication/partitioning systems are commonly found in alpha-proteobacteria plasmids and in secondary chromosomes. All of the elements required for their replication and stable maintenance are encoded within a single transcription unit: the repABC operon. The repC gene encodes an initiator protein, while RepA, RepB and centromere-like sequence (parS) direct plasmid segregation. Strains containing two or more repABC plasmids are a common feature in some alpha proteobacteria groups, indicating that the repABC plasmid family embraces several incompatibility groups. Genes encoded within repABC operons are highly dynamic: each one possess its own distinctive phylogeny and homologous recombination events are common within these operons. Additionally, alpha-proteobacterial genomes contain repAB genes not associated with the ctRNA or with repC as well as plasmids whose replication depends on a ctRNA-repC module without the participation of repAB genes. Some alphaproteobacteria have repC genes clustered with other genes that are not involved in replication/partitioning functions. These atypical associations of genes could have an important role in the origin and diversification of new plasmids. Here we evaluated the functionality and possible evolutionary consequences of one of these atypical gene associations: the repAC genes present in the Rhizobium leguminosarum plasmid pRL7. The repAC genes are organized in an operon and they are capable of sustaining replication but in an unstable manner. RepC was essential for replication, and the origin of replication resides within its coding region. In contrast, RepA plays a minor role in the negative regulation of its own transcription., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Studies of Mycobacterium bovis BCG strains used in different countries and vaccination programs show clear variations in the genomes and immune protective properties of BCG strains. The aim of this study was to characterise the genomic and immune proteomic profile of the BCG 1931 strain used in Mexico., Results: BCG Mexico 1931 has a circular chromosome of 4,350,386 bp with a G+C content and numbers of genes and pseudogenes similar to those of BCG Tokyo and BCG Pasteur. BCG Mexico 1931 lacks Region of Difference 1 (RD1), RD2 and N-RD18 and one copy of IS6110, indicating that BCG Mexico 1931 belongs to DU2 group IV within the BCG vaccine genealogy. In addition, this strain contains three new RDs, which are 53 (RDMex01), 655 (RDMex02) and 2,847 bp (REDMex03) long, and 55 single-nucleotide polymorphisms representing non-synonymous mutations compared to BCG Pasteur and BCG Tokyo. In a comparative proteomic analysis, the BCG Mexico 1931, Danish, Phipps and Tokyo strains showed 812, 794, 791 and 701 protein spots, respectively. The same analysis showed that BCG Mexico 1931 shares 62% of its protein spots with the BCG Danish strain, 61% with the BCG Phipps strain and only 48% with the BCG Tokyo strain. Thirty-nine reactive spots were detected in BCG Mexico 1931 using sera from subjects with active tuberculosis infections and positive tuberculin skin tests., Conclusions: BCG Mexico 1931 has a smaller genome than the BCG Pasteur and BCG Tokyo strains. Two specific deletions in BCG Mexico 1931 are described (RDMex02 and RDMex03). The loss of RDMex02 (fadD23) is associated with enhanced macrophage binding and RDMex03 contains genes that may be involved in regulatory pathways. We also describe new antigenic proteins for the first time.

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.

Background: Salmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids., Results: The blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations., Conclusions: The ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the insertion and deletion of DNA stretches have shaped their evolutionary histories.

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.

Background: The repABC plasmid family, which is extensively present within Alphaproteobacteria, and some secondary chromosomes of the Rhizobiales have the particular feature that all the elements involved in replication and partitioning reside within one transcriptional unit, the repABC operon. Given the functional interactions among the elements of the repABC operon, and the fact that they all reside in the same operon, a common evolutionary history would be expected if the entire operon had been horizontally transferred. Here, we tested whether there is a common evolutionary history within the repABC operon. We further examined different incompatibility groups in terms of their differentiation and degree of adaptation to their host., Results: We did not find a single evolutionary history within the repABC operon. Each protein had a particular phylogeny, horizontal gene transfer events of the individual genes within the operon were detected, and different functional constraints were found within and between the Rep proteins. When different repABC operons coexisted in the same genome, they were well differentiated from one another. Finally, we found different levels of adaptation to the host genome within and between repABC operons coexisting in the same species., Conclusion: Horizontal gene transfer with conservation of the repABC operon structure provides a highly dynamic operon in which each member of this operon has its own evolutionary dynamics. In addition, it seems that different incompatibility groups present in the same species have different degrees of adaptation to their host genomes, in proportion to the amount of time the incompatibility group has coexisted with the host genome.