Association of the Chromosome Replication Initiator DnaA with the Escherichia coli Inner Membrane In Vivo: Quantity and Mode of Binding

DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein’s domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication

Structural insight into Helicobacter pylori DNA replication initiation

While increasing knowledge is accumulating about the molecular mechanisms allowing the human pathogen Helicobacter pylori to survive and to subvert host defenses, much less is known about fundamental aspects of its biology, including DNA replication. We have studied the initiation step of chromosome replication of H. pylori and particularly the interaction between the initiator protein DnaA and its recently identified regulator HobA. This work has recently culminated in the determination of the crystal structure of the domains I and II of DnaA (DnaAI−II) in complex with HobA. By combining the structure with a variety of biochemical experiments we show that a tetramer of HobA can accommodate up to four DnaA molecules organized in a particular conformation within the complex. Mutations of the HobA interface that impaired the binding with DnaA were designed and proved to be lethal once introduced into H. pylori. These features suggest that HobA provides a molecular scaffold onto which regular oligomers of DnaA can assemble. The HobA-promoted oligomerization of DnaA could have a determinant role in the formation of the open complex. We propose a speculative model of HobA-dependent DnaA oligomerization leading to DNA unwinding. More generally, the parallel we draw with Escherichia coli DnaA and DiaA (HobA-like E. coli protein) will direct new studies that will contribute to the understanding of bacterial DNA replication

Efeitos do consumo agudo de cafeína sobre parâmetros metabólicos e de desempenho em indivíduos do sexo masculino Effects of caffeine acute consumption on the metabolic and performance parameters in male individuals

O objetivo deste estudo foi avaliar o efeito do consumo agudo de cafeína sobre a oxidação de lipídeos e desempenho durante o exercício aeróbico. Foram avaliados 15 indivíduos do sexo masculino, com idade média de 22,3 ± 2,7 anos, que realizaram teste de cargas progressivas em esteira rolante para determinação do consumo máximo de oxigênio (VO2máx) e limiares ventilatórios (LV). Cada voluntário realizou três testes submáximos na intensidade de 10% abaixo do segundo LV, sendo orientados a permanecer em exercício até a exaustão. Trinta minutos antes de cada teste submáximo, foram ingeridos 250ml de uma das bebidas compostas por: café com adoçante (CAD), café com açúcar (CA) e café descafeinado com adoçante (CD). Durante o exercício, os indivíduos foram monitorados pelo ergoespirômetro e frequencímetro. A oxidação de lipídeos foi predita pelo quociente respiratório (QR) durante o teste, e o desempenho foi verificado pelo tempo de exercício. Para comparar os resultados de QR e tempo de exercício entre os grupos, foi utilizado Anova fatorial, e considerou-se significância estatística um valor de p < 0,05. A média de VO2máx foi de 50,18 ± 9,9ml/kg/min. Com a ingestão de CAD, a média do QR foi de 0,98 ± 0,18 e o tempo médio em exercício foi de 24,1 ± 17,04 minutos. Com a ingestão de CA, a média do QR foi de 0,96 ± 0,2 e o tempo médio em exercício foi de 24,4 ± 17,8min. No teste com ingestão de CD, a média do QR foi de 1,01 ± 0,24 e a média do tempo em exercício foi de 20,6 ± 9,7min. Não houve diferença significativa entre os testes nos valores do QR e nem tempo de exercício (p = 0,697 e p = 0,598, respectivamente). A cafeína não aumentou a oxidação de lipídeos nem o desempenho de indivíduos jovens do sexo masculino.<br>The purpose of this study was to evaluate the effect of acute caffeine consumption on lipid oxidation and performance during aerobic exercise. Fifteen healthy male individuals, 22.3 ± 2.7 years old, performed a progressive test on treadmill for determination of maximal oxygen uptake (VO2max) and ventilatory thresholds. Each volunteer performed three submaximal tests at the intensity of 10% below the second ventilatory threshold, being guided to remain on exercise until exhaustion. Thirty minutes before each submaximal test, the subjects ingested 250ml of one of following drinks: coffee with sweetener (CSW), coffee with sugar (CS) or decaffeinated coffee with sweetener (CD). During the exercise, the individuals's heart rate was monitored and respiratory gases analyses were done. The lipid oxidation was predicted by the respiratory quotient (RQ) during the test and performance was verified by exercise duration. In order to compare the RQ results and time of exercise among the three groups, factorial Anova was used, and a value of p < 0.05 was considered as statistically significant. The individuals had VO2max of 50.18 ± 9.9 ml/kg/min. CAD ingestion caused RQ average of 0.98 ± 0.18, and the average exercise duration was of 24.1 ± 17.04 min; CA ingestion caused RQ average of 0.96 ± 0.2 and the average exercise duration was 24.4 ± 17.8 min. Finally, CD ingestion caused the RQ average of 1.01 ± 0.24, and the average exercise duration was of 20.6 ± 9.7 min. There were no significant differences in the RQ values or exercise duration among the three interventions (p = 0.697 and p = 0.598, respectively). Caffeine did not increase lipid oxidation or performance of young male individuals

When signalling goes wrong: pathogenic variants in structural and signalling proteins causing cardiomyopathies

Cardiomyopathies are a diverse group of cardiac disorders with distinct phenotypes, depending on the proteins and pathways affected. A substantial proportion of cardiomyopathies are inherited and those will be the focus of this review article. With the wide application of high-throughput sequencing in the practice of clinical genetics, the roles of novel genes in cardiomyopathies are recognised. Here, we focus on a subgroup of cardiomyopathy genes [TTN, FHL1, CSRP3, FLNC and PLN, coding for Titin, Four and a Half LIM domain 1, Muscle LIM Protein, Filamin C and Phospholamban, respectively], which, despite their diverse biological functions, all have important signalling functions in the heart, suggesting that disturbances in signalling networks can contribute to cardiomyopathies.</p

Advances in Genetic Engineering of Microalgae

Hallmann A. Advances in Genetic Engineering of Microalgae. In: Grand Challenges in Algae Biotechnology. Grand Challenges in Biology and Biotechnology. Cham: Springer International Publishing; 2020: 159-221