Comprehensive quantitative modeling of translation efficiency in a genome-reduced bacterium.

Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half-lives, proteins, and protein half-lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae (Mpn). We found that, although the initiation rate varied over 160-fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100-fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn.

SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome.

The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field.

FASTQINS and ANUBIS: two bioinformatic tools to explore facts and artifacts in transposon sequencing and essentiality studies.

Transposon sequencing is commonly applied for identifying the minimal set of genes required for cellular life; a major challenge in fields such as evolutionary or synthetic biology. However, the scientific community has no standards at the level of processing, treatment, curation and analysis of this kind data. In addition, we lack knowledge about artifactual signals and the requirements a dataset has to satisfy to allow accurate prediction. Here, we have developed FASTQINS, a pipeline for the detection of transposon insertions, and ANUBIS, a library of functions to evaluate and correct deviating factors known and uncharacterized until now. ANUBIS implements previously defined essentiality estimate models in addition to new approaches with advantages like not requiring a training set of genes to predict general essentiality. To highlight the applicability of these tools, and provide a set of recommendations on how to analyze transposon sequencing data, we performed a comprehensive study on artifacts corrections and essentiality estimation at a 1.5-bp resolution, in the genome-reduced bacterium Mycoplasma pneumoniae. We envision FASTQINS and ANUBIS to aid in the analysis of Tn-seq procedures and lead to the development of accurate genome essentiality estimates to guide applications such as designing live vaccines or growth optimization.

Protein quality control and regulated proteolysis in the genome-reduced organism Mycoplasma pneumoniae.

Protein degradation is a crucial cellular process in all-living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP-dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA-seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half-life measurements also suggest a role for Lon-modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation.

Impact of C-terminal amino acid composition on protein expression in bacteria.

The C-terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C-terminal sequence and levels of protein expression remains unknown. Here, we identified C-terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C-terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels.

Mycoplasma genitalium Nonadherent Phase Variants Arise by Multiple Mechanisms and Escape Antibody-Dependent Growth Inhibition.

Antigenic variation of the immunodominant MgpB and MgpC proteins has been suggested to be a mechanism of immune evasion of the human pathogen Mycoplasma genitalium, a cause of several reproductive tract disease syndromes. Phase variation resulting in the loss of adherence has also been documented, but the molecular mechanisms underlying this process and its role in pathogenesis are still poorly understood. In this study, we isolated and characterized 40 spontaneous, nonadherent phase variants from in vitro-passaged M. genitalium cultures. In all cases, nonadherence was associated with the loss of MgpBC protein expression, attributable to sequence changes in the mgpBC expression site. Phase variants were grouped into seven classes on the basis of the nature of the mutation. Consistent with the established role of RecA in phase variation, 31 (79.5%) variants arose via recombination with MgPa repeat regions that contain mgpBC variable sequences. The remaining mutants arose via nonsense or frameshift mutations. As expected, revertants were obtained for phase variants that were predicted to be reversible but not for those that arose via an irreversible mechanism. Furthermore, phase variants were enriched in M. genitalium cultures exposed to antibodies reacting to the extracellular, conserved C terminus of MgpB but not in cultures exposed to antibodies reacting to an intracellular domain of MgpB or the cytoplasmic HU protein. Genetic characterization of the antibody-selected phase variants confirmed that they arose via reversible and irreversible recombination and point mutations within mgpBC These phase variants resisted antibody-mediated growth inhibition, suggesting that phase variation promotes immune evasion.

MG428 is a novel positive regulator of recombination that triggers mgpB and mgpC gene variation in Mycoplasma genitalium.

The human pathogen Mycoplasma genitalium employs homologous recombination to generate antigenic diversity in the immunodominant MgpB and MgpC proteins. Only recently, some of the molecular factors involved in this process have been characterized, but nothing is known about its regulation. Here, we show that M. genitalium expresses N-terminally truncated RecA isoforms via alternative translation initiation, but only the full-length protein is essential for gene variation. We also demonstrate that overexpression of MG428 positively regulates the expression of recombination genes, including recA, ruvA, ruvB and ORF2, a gene of unknown function co-transcribed with ruvAB. The co-ordinated induction of these genes correlated with an increase of mgpBC gene variation. In contrast, cells lacking MG428 were unable to generate variants despite expressing normal levels of RecA. Similarly, deletion analyses of the recA upstream region defined sequences required for gene variation without abolishing RecA expression. The requirement of these sequences is consistent with the presence of promoter elements associated with MG428-dependent recA induction. Sequences upstream of recA also influence the relative abundance of RecA isoforms, possibly through translational regulation. Overall, these results suggest that MG428 is a positive regulator of recombination and that precise control of recA expression is required to initiate mgpBC variation.

Characterization of the operon encoding the Holliday junction helicase RuvAB from Mycoplasma genitalium and its role in mgpB and mgpC gene variation.

Mycoplasma genitalium is an emerging sexually transmitted pathogen associated with reproductive tract disease in men and women, and it can persist for months to years despite the development of a robust antibody response. Mechanisms that may contribute to persistence in vivo include phase and antigenic variation of the MgpB and MgpC adhesins. These processes occur by segmental recombination between discrete variable regions within mgpB and mgpC and multiple archived donor sequences termed MgPa repeats (MgPars). The molecular factors governing mgpB and mgpC variation are poorly understood and obscured by the paucity of recombination genes conserved in the M. genitalium genome. Recently, we demonstrated the requirement for RecA using a quantitative PCR (qPCR) assay developed to measure recombination between the mgpB and mgpC genes and MgPars. Here, we expand these studies by examining the roles of M. genitalium ruvA and ruvB homologs. Deletion of ruvA and ruvB impaired the ability to generate mgpB and mgpC phase and sequence variants, and these deficiencies could be complemented with wild-type copies, including the ruvA gene from Mycoplasma pneumoniae. In contrast, ruvA and ruvB deletions did not affect the sensitivity to UV irradiation, reinforcing our previous findings that the recombinational repair pathway plays a minor role in M. genitalium. Reverse transcription-PCR (RT-PCR) and primer extension analyses also revealed a complex transcriptional organization of the RuvAB system of M. genitalium, which is cotranscribed with two novel open reading frames (ORFs) (termed ORF1 and ORF2 herein) conserved only in M. pneumoniae. These findings suggest that these novel ORFs may play a role in recombination in these two closely related bacteria.

Engineering Mycoplasma pneumoniae to bypass the association with Guillain-Barré syndrome.

A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis.

RecA mediates MgpB and MgpC phase and antigenic variation in Mycoplasma genitalium, but plays a minor role in DNA repair.

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived 'MgPar' donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10(-4) events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption-deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C-MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.

Development of a Serum-Free Medium To Aid Large-Scale Production of Mycoplasma-Based Therapies.

To assist in the advancement of the large-scale production of safe Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin (in the animal component-free medium) were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available. IMPORTANCE Mycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies.

Widespread ribosome stalling in a genome-reduced bacterium and the need for translational quality control.

Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, Mycoplasma pneumoniae (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show in vivo that in MPN the sole quality control cytoplasmic protease (Lon) degrades efficiently tmRNA-tagged proteins. Analysis of tmRNA-mutants encoding a tag resistant to proteolysis reveals extensive tagging activity under normal growth. Unlike knockout strains, these mutants are viable demonstrating the requirement of tmRNA-mediated ribosome recycling. Chaperone and Lon steady-state levels maintain proteostasis in these mutants suggesting a model in which co-evolution of Lon and their substrates offer simple mechanisms of regulation without specialized degradation machineries. Finally, comparative analysis shows relative increase in Lon/Chaperone levels in slow-growing bacteria suggesting physiological adaptation to growth demand.

Model-driven design allows growth of Mycoplasma pneumoniae on serum-free media.

Mycoplasma pneumoniae is a slow-growing, human pathogen that causes atypical pneumonia. Because it lacks a cell wall, many antibiotics are ineffective. Due to its reduced genome and dearth of many biosynthetic pathways, this fastidious bacterium depends on rich, undefined medium for growth, which makes large-scale cultivation challenging and expensive. To understand factors limiting growth, we developed a genome-scale, constraint-based model of M. pneumoniae called iEG158_mpn to describe the metabolic potential of this bacterium. We have put special emphasis on cell membrane formation to identify key lipid components to maximize bacterial growth. We have used this knowledge to predict essential components validated with in vitro serum-free media able to sustain growth. Our findings also show that glycolysis and lipid metabolism are much less efficient under hypoxia; these findings suggest that factors other than metabolism and membrane formation alone affect the growth of M. pneumoniae. Altogether, our modelling approach allowed us to optimize medium composition, enabled growth in defined media and streamlined operational requirements, thereby providing the basis for stable, reproducible and less expensive production.

Deletion of the Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by altering terminal organelle curvature.

Motility is often a virulence factor of pathogenic bacteria. Although recent works have identified genes involved in gliding motility of mycoplasmas, little is known about the mechanisms governing the cell gliding behaviour. Here, we report that Mycoplasma genitalium MG217 is a novel protein involved in the gliding apparatus of this organism and it is, at least, one of the genes that are directing cells to move in narrow circles when they glide. In the absence of MG_217 gene, cells are still able to glide but they mainly move drawing erratic or wide circular paths. This change in the gliding behaviour correlates with a rearrangement in the terminal organelle disposition, suggesting that the terminal organelle operates as a guide to steer the mycoplasma cell in a specific direction. Immunogold labelling reveals that MG217 protein is located intracellular at the distal end of the terminal organelle, between the cell membrane and the terminal button. Such location is consistent with the idea that MG217 could act as a modulator of the terminal organelle curvature, allowing cells to move in specific directions.

Post-Hurricane Distress Scale (PHDS): A Novel Tool for First Responders and Disaster Researchers.

OBJECTIVE: The aim of this study was the construction and validation of a novel research instrument to quantify the degree of post-hurricane trauma and distress in an affected population. The Post-Hurricane Distress Scale (PHDS) has quantitative measures of both acute and prolonged distress, attributable to meteorological and hydrological disasters. METHODS: A careful evaluation of existing questionnaires, as well as extensive canvasing of the post-Maria population of Puerto Rico, availed the construction of the PHDS. The PHDS consists of 20 items, organized into 4 subscales. The PHDS was pre-validated (n=79), revised, and then distributed to a broad sampling of the post-Hurricane Maria Puerto Rican population (n=597). Validation, including factor analysis, analyses of concurrent validity, discriminant validity, and internal reliability, was performed. RESULTS: After comparing various scales, factor loading profiles, concurrent validities, and models of fit, we show that the PHDS is best scored as a single 0-6 distress scale. When compared with the Traumatic Exposure Severity Scale, the PHDS shows superior concurrent validity, more accurately predicting scores for the Peritraumatic Distress Inventory, Impact of Event Scale - Revised, and Generalized Anxiety Disorder 7 Scale. The PHDS shows good internal reliability and discriminant validity. CONCLUSIONS: The PHDS represents a novel, useful instrument for disaster first-responders and researchers. The prompt identification of high-risk populations is possible using this instrument. (Disaster Med Public Health Preparedness. 2019;13:82-89).

Mechanical resistance of a new biomaterial, ostrich pericardium, and a new method of joining tissues combining suturing and a biological adhesive.

We studied the mechanical behavior in response to tensile stress of samples of ostrich pericardium bonded with a cyanoacrylate glue or sewn with a rectangular, overlapping suture that was subsequently sealed with the same bioadhesive. Seventy-two trials were performed in three series of 24 samples each: series AG, glued with an overlap of 1 cm2; series ASG, sewn with a rectangular, overlapping suture and sealed; and series AC, control samples that were left intact. The mean stress at rupture in series AG (glued) was 0.1 MPa, much lower than the working stress of a human valve leaflet, which is approximately 0.25 MPa. In the control series, this stress was 26.28 MPa. At rupture in series ASG (sutured/glued), the suture material was being subjected to a stress of 64.91 MPa, thus confirming the existence of an interaction between the suture and the shear stress exerted by the suture on the samples of pericardium. In series ASG, the mean value for the resistance to rupture when measured in machine kg was 8.83 kg, lower than but similar to that recorded in the control series AC (10.26 kg). The percentages of reversible deformation, or elongation, once the samples were torn were similar in series AC (19.15%) and ASG (21.93%). This phenomenon can only be explained by the damage to the collagen fibers in the area around the rupture, while other more distant regions work at a lower load within the elastic limit. We conclude that cyanocrylate adhesives alone are not suitable as bonding materials in cardiac bioprostheses. The results with the rectangular, overlapping suture, when subsequently sealed with an adhesive, can be considered good because, although this approach does not impede shear stress, it does maintain an excellent degree of resistance to rupture of the samples thus joined. We stress the need to take into account the concentration of the load in the design of bioprostheses.

[Valve repair for chronic mitral regurgitation]. / Reparación valvular en la insuficiencia mitral crónica.

Valve repair is the best surgical treatment for mitral regurgitation. In the present article we describe the results of mitral valve repair in patients with chronic mitral regurgitation treated at our center during the last eight years. The degree of correction of valve insufficiency, functional benefit, in-hospital morbidity and mortality, postoperative outcome of ventricular function, and middle-term overall and reoperation-free survival are analyzed.

Edge-to-Edge tricuspid repair for redeveloped valve incompetence after DeVega's annuloplasty.

"Edge-to-edge" technique is a well-accepted procedure with excellent results for correction of mitral insufficiency. We describe a simple edge-to-edge combined with bicuspidalization repair method that was successfully applied in 2 patients for the treatment of redeveloped functional tricuspid regurgitation after previous annuloplasty. Significant improvement in symptoms and echocardiographic results were achieved.

[Surgical treatment for hypertrophic obstructive cardiomyopathy]. / Tratamiento quirúrgico de la miocardiopatía hipertrófica obstructiva.

INTRODUCTION AND OBJECTIVES: Five percent of the patients with hypertrophic obstructive cardiomyopathy (HOCM) have symptoms unresponsive to medical treatment and are candidates for invasive therapy. The objective of this study was to analyze our results with surgical treatment of HOCM during the last 10 years. PATIENTS AND METHOD: Between July 1993 and January 2004 26 patients with HOCM refractory to drug therapy were operated on. An extended septal myectomy was performed, in combination with anterior mitral leaflet plication in 19 cases (73%) and with mitral valve replacement in 5 (19%). Evolution of the grade of dyspnea, left ventricle outflow tract gradient (LVOTG), mitral regurgitation, and systolic anterior motion after surgery was analyzed. RESULTS: Mean follow-up was 63 (37) months. After surgery, a significant reduction in LVOTG (from 96.5 to 19.5 mmHg; P<.001), grade of mitral regurgitation (from 2.54 to 0.69; P<.001) and systolic anterior motion (from 2.92 to 0.23; P<.001) was achieved, which led to improvement in functional class. Hospital mortality and need for pacemaker implantation due to complete heart block after surgery was 3.8% (n=1). There were no cases of iatrogenic ventricular septal defect or mitro-aortic valve injury. Actuarial survival at 5 years was 96% (4%). CONCLUSIONS: Surgery in patients with HOCM yields great clinical improvements with low morbidity and mortality. Simultaneous intervention for both myocardial and valvular components of the disease allows not only reduction in the LVOTG but also correction of mitral regurgitation and abolition of systolic anterior motion.

Characteristics of patients with survival longer than 20 years following heart transplantation.

INTRODUCTION AND OBJECTIVES: The number of heart-transplant recipients exceeding 20 years of follow-up is steadily increasing. However, little is known about their functional status, comorbidities, and mortality. Identifying the predictors of prolonged survival could guide the selection of candidates for the low number of available donors. METHODS: Functional status, morbidities, and mortality of heart-transplant patients between 1984 and 1992 were analyzed. To identify predictors of 20-year survival, a logistic regression model was constructed using the covariates associated with survival in the univariate analysis. RESULTS: A total of 39 patients who survived 20 years (26% of patients transplanted before 1992) were compared to 90 recipients from the same period who died between 1 and 20 years post-transplantation. Major complications were hypertension, renal dysfunction, infections, and cancer. After a mean follow-up of 30 months, 6 survivors had died, yielding a mortality rate of 6% per year (vs 2.5%-3% in years 1-19). Causes of mortality were infection (50%), malignancy (33%), and allograft vasculopathy (17%). Long-term survivors were younger and leaner, and had nonischemic cardiomyopathy and lower ischemic time. Logistic regression identified recipient age <45 years (odds ratio=3.9; 95% confidence interval, 1.6-9.7; P=.002) and idiopathic cardiomyopathy (odds ratio=3; 95% confidence interval, 1.4-7.8; P=.012) as independent predictors for 20-year survival. CONCLUSIONS: One fourth of all heart-transplant patients in our series survived >20 years with the same graft, and most enjoy independent lives despite significant comorbidities. Recipient age <45 years and idiopathic cardiomyopathy were associated with survival beyond 2 decades. These data may help decide donor allocation.