Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality.

Understanding how to program biological functions into artificial DNA sequences remains a key challenge in synthetic genomics. Here, we report the chemical synthesis and testing of Caulobacter ethensis-2.0 (C. eth-2.0), a rewritten bacterial genome composed of the most fundamental functions of a bacterial cell. We rebuilt the essential genome of Caulobacter crescentus through the process of chemical synthesis rewriting and studied the genetic information content at the level of its essential genes. Within the 785,701-bp genome, we used sequence rewriting to reduce the number of encoded genetic features from 6,290 to 799. Overall, we introduced 133,313 base substitutions, resulting in the rewriting of 123,562 codons. We tested the biological functionality of the genome design in C. crescentus by transposon mutagenesis. Our analysis revealed that 432 essential genes of C. eth-2.0, corresponding to 81.5% of the design, are equal in functionality to natural genes. These findings suggest that neither changing mRNA structure nor changing the codon context have significant influence on biological functionality of synthetic genomes. Discovery of 98 genes that lost their function identified essential genes with incorrect annotation, including a limited set of 27 genes where we uncovered noncoding control features embedded within protein-coding sequences. In sum, our results highlight the promise of chemical synthesis rewriting to decode fundamental genome functions and its utility toward the design of improved organisms for industrial purposes and health benefits.

Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation.

Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant-provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism-two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCH-N cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation.

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan.

BACKGROUND: Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars. RESULTS: Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al(3+) specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1. CONCLUSIONS: High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Quantitative Selection Analysis of Bacteriophage φCbK Susceptibility in Caulobacter crescentus.

Classical molecular genetics uses stringent selective conditions to identify mutants with distinct phenotypic responses. Mutations giving rise to less pronounced phenotypes are often missed. However, to gain systems-level insights into complex genetic interaction networks requires genome-wide assignment of quantitative phenotypic traits. In this paper, we present a quantitative selection approach coupled with transposon sequencing (QS-TnSeq) to globally identify the cellular components that orchestrate susceptibility of the cell cycle model bacterium Caulobacter crescentus toward bacteriophage φCbK infection. We found that 135 genes representing 3.30% of the Caulobacter genome exhibit significant accumulation of transposon insertions upon φCbK selection. More than 85% thereof consist of new factors not previously associated with phage φCbK susceptibility. Using hierarchical clustering of dose-dependent TnSeq datasets, we grouped these genes into functional modules that correlate with different stages of the φCbK infection process. We assign φCbK susceptibility to eight new genes that represent novel components of the pilus secretion machinery. Further, we demonstrate that, from 86 motility genes, only seven genes encoding structural and regulatory components of the flagellar hook increase phage resistance when disrupted by transposons, suggesting a link between flagellar hook assembly and pili biogenesis. In addition, we observe high recovery of Tn5 insertions within regulatory sequences of the genes encoding the essential NADH:ubiquinone oxidoreductase complex indicating that intact proton motive force is crucial for effective phage propagation. In sum, QS-TnSeq is broadly applicable to perform quantitative and genome-wide systems-genetics analysis of complex phenotypic traits.

Translation initiation factor AteIF(iso)4E is involved in selective mRNA translation in Arabidopsis thaliana seedlings.

One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5'end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions.

[High blood pressure prevalence in Oporto sedentary and regular exercise practicing people]. / Prevalência do diagnóstico de hipertensão arterial em pessoas sedentárias e em praticantes de exercício físico, na cidade do Porto.

INTRODUCTION: The association between the lack of exercise and prevalence of high blood pressure (HBP) is a well known fact. The non-pharmacological treatment for HBP includes regular aerobic exercise practicing. METHODS: A total of 900 individuals, aged above 40, resident in Oporto, representing the 15 parishes of the city, were approached to answer a questionnaire about exercise habits and diagnosis of HBP. The exercise practitioner was defined as the one who practices aerobic exercise for a minimal period of 30 minutes at least 3 times a week. For data analysis non-parametric tests (Pearson's Chi-Square) were used through the Statistical Package for the Social Sciences (SPSS) software. RESULTS: The HBP prevalence in the sample was 39.67% (Confidence Interval (IC) 95%, 36,46% to 42,87%). The HBP prevalence in the exercise practitioners was inferior to the one of the non-practitioners (13,78% vs 25,89%, p < 0,001). This result is valid to the male 33,5% vs 51,6%, p < 0,001) and female population (27,1% vs 51,9%, p < 0,001), as well as to the population without the most frequent HBP-related comorbidities (21,8% vs 44,6%, p < 0,001). CONCLUSIONS: The HBP prevalence in the population resident in Oporto city is higher in the non-practitioners of regular exercise. The HBP prevalence, in both sexes, is higher in the non-practitioners of exercise. Finally, we can also conclude that the studied comorbidities don't bias the obtained results because the group of individuals who don't have them the HBP prevalence is still higher in the group of sedentary.

Uso de antibiótico para profilaxis quirúrgica en el servicio de cirugía general del HEODRA, en el período enero a diciembre 2003 / Use of antibiotic for surgical prevention in the general surgery service of the HEODRA, in the period January to December 2003

El objetivo de esta investigación es describir la experiencia en el uso de antibióticos para la profilaxis en los procedimientos quirurgicos en el departamento de Cirugia del Hospital Escuela Oscar Damio Rosales Arguello (H.E.O.D.RA.), determinando los criterios utilizados en su implementacion, si su uso esta basado en el protocolo de este centro asistencial y cuales son los resultados obtenidos. El presente es un estudio descriptivo prospectivo realízado en el departamento de Cirugía General del H.E.O.D.R.A. durante los meses de enero a diciembre del 2003, se incluyeron un total de 688 cirugias realizadas en este periodo, obteniendose los datos de los expedientes clinicos de estos pacientes y registrandose en una base de datos. Los resultados muestran que la practica de usar antibióticos profilácticos esta bien difundida, prescribiéndose antibióticos en un 92.5 porciento de los procedimientos quirúrgicos, aunque los criterios de indicación de esta profilaxis en base al típo de cirugía fueron adecuados solo en un 71 2 porciento. Los antibioticos más utilizados en profilaxis fueron cefoxitina seguido de cef momento de aplicación, la dosis administrada, fueron adecuadas en el 98.2 porciento de los casos. La duración del régimen de antibióticos fue adecuada en un 93 porciento de los casos. Este estudio reporta un 5.8 prociento de infecciones postquirúrgicas en las intervenciones realizadas en el periodo de estudio. Además de mostrar una incidencia de infecciones en los procedimientos limpios por encima de las cifras descritas en la literatura...