Desenvolvimento de sistema automatizado para controle de dosagem de ração animal

O presente trabalho propõe o desenvolvimento de um sistema automatizado para fábricas de ração animal, com o intuito de realizar melhorias no processo de dosagem, com enfoque na conformidade e a eficiência no processo. O trabalho iniciou-se a partir de análise de dados fornecidos por duas empresas, onde identificou-se a necessidade de um sistema que melhore a conformidade das dosagens, visando reduzir os erros e desperdícios do processo atual, sem afetar a eficiência do sistema. O sistema irá atuar no controle de roscas dosadoras comandadas por inversor de frequência, com base em informações de uma balança industrial. Atualmente, as empresas estudadas possuem um sistema automatizado do processo, porém diversos parâmetros são informados manualmente pelo operador, gerando oscilações e erros de dosagem esporádicos. No intuito de amenizar estas oscilações, para que o erro tenda a zero, identificou-se a necessidade de alterar o software de dosagem atual, substituindo os parâmetros informados manualmente pelo operador, por uma lógica automatizada que calcule as velocidades das roscas dosadoras, para que o sistema seja preciso e eficiente. O software desenvolvido segue os padrões da IEC 61131, podendo ser aplicado em qualquer Controlador Lógico Programável que siga esta norma.This paper proposes the development of an automated system for animal ration industries, aiming improvements on the dosing process, focusing on efficiency and conformity. The paper is based on an analysis of data provided by two industries, where there's the need of a system that improves the conformity of dosing, aiming on reduction of error and wasting of the actual process, without affecting the efficiency of the system. the system will control the dosing thread commanded by Frequency Inverters, based on information of an industrial balance. Actually, the studied companies have automation, but several parameters are informed manually by the operator, causing oscillations and sporadic dosing errors. So, attending to reduce the error to zero, this paper proposes a new dosing software, replacing the manual parameters by an automated logic that calculates the speed of dosing, aiming precision and efficiency. The developed software follows the IEC 61131 standard, being able to apply it on any PLC that follows this normative

Electron Cryotomography of Bacterial Secretion Systems

In biology, function arises from form. For bacterial secretion systems, which often span two membranes, avidly bind to the cell wall, and contain hundreds of individual proteins, studying form is a daunting task, made possible by electron cryotomography (ECT). ECT is the highest-resolution imaging technique currently available to visualize unique objects inside cells, providing a three-dimensional view of the shapes and locations of large macromolecular complexes in their native environment. Over the past 15 years, ECT has contributed to the study of bacterial secretion systems in two main ways: by revealing intact forms for the first time and by mapping components into these forms. Here we highlight some of these contributions, revealing structural convergence in type II secretion systems, structural divergence in type III secretion systems, unexpected structures in type IV secretion systems, and unexpected mechanisms in types V and VI secretion systems. Together, they offer a glimpse into a world of fantastic forms—nanoscale rotors, needles, pumps, and dart guns—much of which remains to be explored

TssA from Aeromonas hydrophila: expression, purification and crystallographic studies

TssA is a core subunit of the type VI secretion system, which is a major player in interspecies competition in Gram-negative bacteria. Previous studies on enteroaggregative Escherichia coli TssA suggested that it is comprised of three putative domains: a conserved N-terminal domain, a middle domain and a ring-forming C-terminal domain. X-ray studies of the latter two domains have identified their respective structures. Here, the results of the expression and purification of full-length and domain constructs of TssA from Aeromonas hydrophila are reported, resulting in diffraction-quality crystals for the middle domain (Nt2) and a construct including the middle and C-terminal domains (Nt2-CTD)

TssA: The cap protein of the Type VI secretion system tail

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Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in <em>Serratia marcescens</em>

Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions

Genomic Characterization of the Taylorella Genus

The Taylorella genus comprises two species: Taylorella equigenitalis, which causes contagious equine metritis, and Taylorella asinigenitalis, a closely-related species mainly found in donkeys. We herein report on the first genome sequence of T. asinigenitalis, analyzing and comparing it with the recently-sequenced T. equigenitalis genome. The T. asinigenitalis genome contains a single circular chromosome of 1,638,559 bp with a 38.3% GC content and 1,534 coding sequences (CDS). While 212 CDSs were T. asinigenitalis-specific, 1,322 had orthologs in T. equigenitalis. Two hundred and thirty-four T. equigenitalis CDSs had no orthologs in T. asinigenitalis. Analysis of the basic nutrition metabolism of both Taylorella species showed that malate, glutamate and alpha-ketoglutarate may be their main carbon and energy sources. For both species, we identified four different secretion systems and several proteins potentially involved in binding and colonization of host cells, suggesting a strong potential for interaction with their host. T. equigenitalis seems better-equipped than T. asinigenitalis in terms of virulence since we identified numerous proteins potentially involved in pathogenicity, including hemagluttinin-related proteins, a type IV secretion system, TonB-dependent lactoferrin and transferrin receptors, and YadA and Hep_Hag domains containing proteins. This is the first molecular characterization of Taylorella genus members, and the first molecular identification of factors potentially involved in T. asinigenitalis and T. equigenitalis pathogenicity and host colonization. This study facilitates a genetic understanding of growth phenotypes, animal host preference and pathogenic capacity, paving the way for future functional investigations into this largely unknown genus

Mobilizable IncQ-Related Plasmid Carrying a New Quinolone Resistance Gene, qnrS2, Isolated from the Bacterial Community of a Wastewater Treatment Plant

Plasmid-encoded quinolone resistance was previously reported for different bacteria isolated from patients not only in the United States and Asia but also in Europe. Here we describe the isolation, by applying a new selection strategy, of the quinolone resistance plasmid pGNB2 from an activated sludge bacterial community of a wastewater treatment plant in Germany. The hypersensitive Escherichia coli strain KAM3 carrying a mutation in the multidrug efflux system genes acrAB was transformed with total plasmid DNA preparations isolated from activated sludge bacteria and subsequently selected on medium containing the fluoroquinolone norfloxacin. This approach resulted in the isolation of plasmid pGNB2 conferring decreased susceptibility to nalidixic acid and to different fluoroquinolones. Analysis of the pGNB2 nucleotide sequence revealed that it is 8,469 bp in size and has a G+C content of 58.2%. The plasmid backbone is composed of a replication initiation module (repA-repC) belonging to the IncQ-family and a two-component mobilization module that confers horizontal mobility to the plasmid. In addition, plasmid pGNB2 carries an accessory module consisting of a transposon Tn1721 remnant and the quinolone resistance gene, qnrS2, that is 92% identical to the qnrS gene located on plasmid pAH0376 from Shigella flexneri 2b. QnrS2 belongs to the pentapeptide repeat protein family and is predicted to protect DNA-gyrase activity against quinolones. This is not only the first report on a completely sequenced plasmid mediating quinolone resistance isolated from an environmental sample but also on the first qnrS-like gene detected in Europe

IncP-1β Plasmid pGNB1 Isolated from a Bacterial Community from a Wastewater Treatment Plant Mediates Decolorization of Triphenylmethane Dyes▿

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1β plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the α-proteobacterium Sinorhizobium meliloti and the γ-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1β plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes

Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion

The recently identified type VI secretion systems (T6SS) have a crucial function in the virulence of various proteobacteria, including the human pathogen Vibrio cholerae. T6SS are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of Hcp and VgrG proteins in cell culture supernatants. Here, we analysed the function of the V. cholerae T6SS member ClpV, a specialized AAA+ protein. ClpV is crucial for a functional T6SS and interacts through its N-terminal domain with the VipA/VipB complex that is composed of two conserved and essential members of T6SS. Transferring ClpV substrate specificity to a distinct AAA+ protein involved in proteolysis caused degradation of VipA but not Hcp or VgrG2, suggesting that VipA rather than Hcp/VgrG2 functions as a primary ClpV substrate. Strikingly, VipA/VipB form tubular, cogwheel-like structures that are converted by a threading activity of ClpV into small complexes. ClpV-mediated remodelling of VipA/VipB tubules represents a crucial step in T6S, illuminating an unexpected role of an ATPase component in protein secretion