RESUMO
Many different protein domains are conserved among numerous species, but their function remains obscure. Proteins with DUF1127 domains number >17 000 in current databases, but a biological function has not yet been assigned to any of them. They are mostly found in alpha- and gammaproteobacteria, some of them plant and animal pathogens, symbionts or species used in industrial applications. Bioinformatic analyses revealed similarity of the DUF1127 domain of bacterial proteins to the RNA binding domain of eukaryotic Smaug proteins that are involved in RNA turnover and have a role in development from Drosophila to mammals. This study demonstrates that the 71 amino acid DUF1127 protein CcaF1 from the alphaproteobacterium Rhodobacter sphaeroides participates in maturation of the CcsR sRNAs that are processed from the 3' UTR of the ccaF mRNA and have a role in the oxidative stress defense. CcaF1 binds to many cellular RNAs of different type, several mRNAs with a function in cysteine / methionine / sulfur metabolism. It affects the stability of the CcsR RNAs and other non-coding RNAs and mRNAs. Thus, the widely distributed DUF1127 domain can mediate RNA-binding, affect stability of its binding partners and consequently modulate the bacterial transcriptome, thereby influencing different physiological processes.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Processamento Pós-Transcricional do RNA , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Rhodobacter sphaeroides/genética , Alphaproteobacteria/genética , Proteínas de Bactérias/fisiologia , Simulação por Computador , Endorribonucleases/fisiologia , Estabilidade de RNA , Proteínas de Ligação a RNA/fisiologia , Rhodobacter sphaeroides/metabolismo , Estresse Fisiológico , TranscriptomaRESUMO
UNLABELLED: In bacteria, regulatory RNAs play an important role in the regulation and balancing of many cellular processes and stress responses. Among these regulatory RNAs, trans-encoded small RNAs (sRNAs) are of particular interest since one sRNA can lead to the regulation of multiple target mRNAs. In the purple bacterium Rhodobacter sphaeroides, several sRNAs are induced by oxidative stress. In this study, we focused on the functional characterization of four homologous sRNAs that are cotranscribed with the gene for the conserved hypothetical protein RSP_6037, a genetic arrangement described for only a few sRNAs until now. Each of the four sRNAs is characterized by two stem-loops that carry CCUCCUCCC motifs in their loops. They are induced under oxidative stress, as well as by various other stress conditions, and were therefore renamed here sRNAs CcsR1 to CcsR4 (CcsR1-4) for conserved CCUCCUCCC motif stress-induced RNAs 1 to 4. Increased CcsR1-4 expression decreases the expression of genes involved in C1 metabolism or encoding components of the pyruvate dehydrogenase complex either directly by binding to their target mRNAs or indirectly. One of the CcsR1-4 target mRNAs encodes the transcriptional regulator FlhR, an activator of glutathione-dependent methanol/formaldehyde metabolism. Downregulation of this glutathione-dependent pathway increases the pool of glutathione, which helps to counteract oxidative stress. The FlhR-dependent downregulation of the pyruvate dehydrogenase complex reduces a primary target of reactive oxygen species and reduces aerobic electron transport, a main source of reactive oxygen species. Our findings reveal a previously unknown strategy used by bacteria to counteract oxidative stress. IMPORTANCE: Phototrophic organisms have to cope with photo-oxidative stress due to the function of chlorophylls as photosensitizers for the formation of singlet oxygen. Our study assigns an important role in photo-oxidative stress resistance to a cluster of four homologous sRNAs in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides. We reveal a function of these regulatory RNAs in the fine-tuning of C1 metabolism. A model that relates oxidative stress defense to C1 metabolism is presented.
Assuntos
Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexo Piruvato Desidrogenase/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Sequência de Bases , Transporte de Elétrons , Glutationa/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estresse Oxidativo , Homologia de SequênciaRESUMO
Maternal exposure to various stimuli can influence pre- and postnatal development of the offspring. This potential has been discussed for glyphosate (GLY), active substance in some non-selective herbicides. Accordingly, present study investigated putative effects of GLY residues in rations on cows and their offspring. Dams received either GLY-contaminated (GLY groups) or control (CON groups) rations combined with low (LC groups) or high (HC groups) concentrate feed proportions (CFP) for 16 weeks during mid- and late lactation and early gestation (59±4 days at beginning of GLY exposure; mean±SE). During this feeding trial, average daily GLY exposures of dams were 1.2 (CONLC), 1.1 (CONHC), 112.5 (GLYLC) and 130.3 (GLYHC) µg/kg body weight/d. After a depletion period (107±4 days; mean±SE) and calving, blood samples of dams and their calves were collected (5-345 min after birth) before calves were fed colostrum and analyzed for hematological and clinical-chemical traits, redox parameters, functional properties of leukocytes and DNA damage in leukocytes. No evidence for malformations of newborn calves could be collected. At parturition, most analyzed blood parameters were not affected by dietary treatment of dams during gestation. Significant GLY effects were observed for some traits, e.g. blood non-esterified fatty acids (NEFA) in calves. These deviations of GLY groups from CON groups likely resulted from strong time-dependent responses of NEFA levels within the first 105 minutes after birth and before colostrum intake (Spearman´s rank correlation R = 0.76, p<0.001). Additionally, significant GLY effects did not result in differences in measures that were beyond normally observed ranges questioning a pathological relevance. In summary, no evidence for teratogenic or other clear effects of GLY or CFP on analyzed parameters of dams and their newborn calves could be collected under applied conditions. However, detailed studies including GLY exposure during late and complete gestation period would be needed to rule out teratogenic effects.
Assuntos
Dieta , Ácidos Graxos não Esterificados , Animais , Bovinos , Feminino , Gravidez , Ração Animal/análise , Animais Recém-Nascidos , Células Sanguíneas , Dieta/veterinária , Dano ao DNA , Ácidos Graxos não Esterificados/análise , Leite/química , GlifosatoRESUMO
Glyphosate (GLY), the active substance in non-selective herbicides, is often found in ruminant feed. The present feeding study aimed to investigate the effects of GLY-contaminated rations and different concentrate feed proportions (CFP) on the health of fattening German Holstein bulls. Bulls were grouped by low (LC) or high (HC) CFP with (GLYLC, GLYHC) or without GLY-contaminations (CONLC, CONHC) in their rations. Intakes (dry matter, water) and body weight were documented continuously lasting over an average range from 392.2 ± 60.4 kg to 541.2 ± 67.4 kg (mean ± SD). Blood samples collected at the trial's beginning, and after 7 and 15 weeks, were analyzed for hematological and clinical-chemical traits, functional properties of leukocytes, redox parameters and DNA damage. The average GLY exposures of 128.6 (GLYHC), 213.7 (GLYLC), 1.3 (CONHC) and 2.0 µg/kg body weight/d (CONLC) did not lead to GLY effects for most of the assessed parameters relating to animal health and performance. CFP and time displayed marked influences on most of the experimental parameters such as higher dry matter intake and average daily gain in HC compared with the LC groups. GLY effects were rather weak. However, the observed interactive effects between GLY and CFP and/or time occurring in an inconsistent manner are likely not reproducible. Finally, all animals remained clinically inconspicuous, which brings into question the physiological relevance of putative GLY effects.
RESUMO
The sensitivity of pigs to deoxynivalenol (DON) might be increased by systemic inflammation (SI), which also has consequences for hepatic integrity. Liver lesions and a dys-regulated gene network might hamper hepatic handling and elimination of DON whereby the way of initiation of hepatic inflammation might play an additional role. First and second-pass exposure of the liver with LPS for triggering a SI was achieved by LPS infusion via pre- or post-hepatic venous route, respectively. Each infusion group was pre-conditioned either with a control diet (0.12 mg DON/kg diet) or with a DON-contaminated diet (4.59 mg DON/kg diet) for 4 wk. Liver transcriptome was evaluated at 195 min after starting infusions. DON exposure alone failed to modulate the mRNA expression significantly. However, pre- and post-hepatic LPS challenges prompted transcriptional responses in immune and metabolic levels. The mRNAs for B-cell lymphoma 2-like protein 11 as a key factor in apoptosis and IFN-γ released by T cells were clearly up-regulated in DON-fed group infused with LPS post-hepatically. On the other hand, mRNAs for nucleotide binding oligomerization domain containing 2, IFN-α and eukaryotic translation initiation factor 2α kinase 3 as ribosomal stress sensors were exclusively up-regulated in control pigs with pre-hepatic LPS infusion. These diverse effects were traced back to differences in TLR4 signalling.
Assuntos
Reação de Fase Aguda/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Fígado/fisiologia , Tricotecenos/toxicidade , Reação de Fase Aguda/metabolismo , Ração Animal , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Dieta/efeitos adversos , Exposição Dietética , Contaminação de Alimentos , Lipopolissacarídeos/metabolismo , Micotoxinas , Suínos , TranscriptomaRESUMO
Glyphosate (GLY) is worldwide one of the most used active substances in non-selective herbicides. Although livestock might be orally exposed via GLY-contaminated feedstuffs, not much is known about possible hepatotoxic effects of GLY. As hepatic xenobiotic and nutrient metabolism are interlinked, toxic effects of GLY residues might be influenced by hepatic nutrient supply. Therefore, a feeding trial with lactating dairy cows was conducted to investigate effects of GLY-contaminated feedstuffs and different concentrate feed proportions (CFP) in the diets as tool for varying nutrient supply to the liver. For this, 61 German Holstein cows (207 ± 49 days in milk; mean ± standard deviation) were either fed a GLY-contaminated total mixed ration (TMR, GLY groups, mean GLY intake 122.7 µg/kg body weight/day) or control TMR (CON groups, mean GLY intake 1.2 µg/kg body weight/day) for 16 weeks. Additionally, both groups were further split into subgroups fed a lower (LC, 30% on dry matter basis) or higher (HC, 60% on dry matter basis) CFP resulting in groups CONHC (n = 16), CONLC (n = 16), GLYHC (n = 15), GLYLC (n = 14). Blood parameters aspartate aminotransferase, γ-glutamyltransferase, glutamate dehydrogenase, cholesterol, triglyceride, total protein, calcium, phosphorus, acetic acid and urea and histopathological evaluation were not influenced by GLY, whereas all mentioned parameters were at least affected by time, CFP or an interactive manner between time and CFP. Total bilirubin blood concentration was significantly influenced by an interaction between GLY and CFP with temporarily elevated concentrations in GLYHC, whereas the biological relevance remained unclear. Gene expression analysis indicated 167 CFP-responsive genes, while seven genes showed altered expression in GLY groups compared to CON groups. Since expression changes of GLY-responsive genes were low and liver-related blood parameters changed either not at all or only slightly, the tested GLY formulation was considered to have no toxic effects on the liver of dairy cows.
Assuntos
Ração Animal/análise , Indústria de Laticínios , Regulação da Expressão Gênica , Glicina/análogos & derivados , Fígado/metabolismo , Fígado/patologia , Animais , Bovinos , Regulação da Expressão Gênica/efeitos dos fármacos , Glicina/toxicidade , Fígado/efeitos dos fármacos , Reprodutibilidade dos Testes , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , GlifosatoRESUMO
Glyphosate-based herbicides are among the most used non-selective herbicides worldwide and inhibit synthesis of aromatic amino acids in plants, bacteria, and fungi. Given the broad usage, controversies concerning potential effects of glyphosate on health and especially on gut microbiomes arose. For cattle, it has been proposed based on in vitro data that glyphosate has detrimental effects on the ruminal microbiome, which manifest as a specific inhibition of bacteria involved in fiber degradation and as an enrichment of specific pathogens. In the present study, glyphosate effects on the ruminal microbiome were analyzed in vivo using glyphosate contaminated feedstuffs with strong differences in dietary fiber and dietary energy content in order to reproduce the proposed detrimental glyphosate effects on the rumen microbiome. While significant impact of dietary factors on the ruminal microbiome and its products are pointed out, no adverse glyphosate effects on ruminal microbiome composition, diversity, and microbial metabolites are observed.