26 research outputs found
Ubiquitous late competence genes in Bacillus species indicate the presence of functional DNA uptake machineries
P>Natural competence for genetic transformation, i.e. the ability to take up DNA and stably integrate it in the genome, has so far only been observed in the bacterial kingdom (both in Gram-negative and Gram-positive species) and may contribute to survival under adverse growth conditions. Bacillus subtilis, the model organism for the Bacillus genus, possesses a well-characterized competence machinery. Phylogenetic analysis of several genome sequences of different Bacillus species reveals the presence of many, but not all genes potentially involved in competence and its regulation. The recent demonstration of functional DNA uptake by B. cereus supports the significance of our genome analyses and shows that the ability for functional DNA uptake might be widespread among Bacilli
Response of Bacillus cereus ATCC 14579 to challenges with sublethal concentrations of enterocin AS-48
Background: Enterocin AS-48 is produced by Enterococcus faecalis S48 to compete with other bacteria in their environment. Due to its activity against various Gram positive and some Gram negative bacteria it has clear potential for use as a food preservative. Here, we studied the effect of enterocin AS-48 challenges on vegetative cells of Bacillus cereus ATCC 14579 by use of transcriptome analysis.
Results: Of the 5200 genes analysed, expression of 24 genes was found to change significantly after a 30 min treatment with a subinhibitory bacteriocin concentration of 0.5 μg/ml. Most of upregulated genes encode membrane-associated or secreted proteins with putative transmembrane segments or signal sequences, respectively. One operon involved in arginine metabolism was significantly downregulated. The BC4206-BC4207 operon was found to be the most upregulated target in our experiments. BC4206 codes for a PadR type transcriptional regulator, while BC4207 codes for a hypothetical membrane protein. The operon structure and genes are conserved in B. cereus and B. thuringiensis species, but are not present in B. anthracis and B. subtilis. Using real-time qPCR, we show that these genes are upregulated when we treated the cells with AS-48, but not upon nisin treatment. Upon overexpression of BC4207 in B. cereus, we observed an increased resistance against AS-48. Expression of BC4207 in B. subtilis 168, which lacks this operon also showed increased resistance against AS-48.
Conclusion: BC4207 membrane protein is involved in the resistance mechanism of B. cereus cells against AS-48.
Lipid Production From Waste Materials in Seawater-Based Medium by the Yeast Yarrowia lipolytica
The global limitation of fossil fuels impels scientists to search for new energy sources. A good alternative is biodiesel produced from crop plants. However, its production requires huge quantities of farmland, fertilizers and fresh water, which is in conflict with the human demand for water for consumption and land for food production. Thus, production of single cell oil (SCO) by oleaginous microorganisms remains the best solution for the coming years. Whereas most microorganisms require fresh water for proper cell metabolism, in this study we demonstrate that the unconventional yeast Yarrowia lipolytica is able to produce huge quantities of fatty acid in seawater-based medium. Here we shown that Y. lipolytica is able to produce fatty acids in medium based on seawater and crude glycerol as the main carbon source, which allows for low-cost production of SCO, is beneficial for industrial application and is ecologically friendly
Distinct Roles of ComK1 and ComK2 in Gene Regulation in Bacillus cereus
The B. subtilis transcriptional factor ComK regulates a set of genes coding for DNA uptake from the environment and for its integration into the genome. In previous work we showed that Bacillus cereus expressing the B. subtilis ComK protein is able to take up DNA and integrate it into its own genome. To extend our knowledge on the effect of B. subtilis ComK overexpression in B. cereus we first determined which genes are significantly altered. Transcriptome analysis showed that only part of the competence gene cluster is significantly upregulated. Two ComK homologues can be identified in B. cereus that differ in their respective homologies to other ComK proteins. ComK1 is most similar, while ComK2 lacks the C-terminal region previously shown to be important for transcription activation by B. subtilis ComK. comK1 and comK2 overexpression and deletion studies using transcriptomics techniques showed that ComK1 enhances and ComK2 decreases expression of the comG operon, when B. subtilis ComK was overexpressed simultaneously
Mutation in yl-HOG1 represses the filament-to-yeast transition in the dimorphic yeast Yarrowia lipolytica
Abstract Background Yarrowia lipolytica is a dimorphic fungus, which switches from yeast to filament form in response to environmental conditions. For industrial purposes it is important to lock cells in the yeast or filamentous form depending on the fermentation process. yl-Hog1 kinase is a key component of the HOG signaling pathway, responsible for activating the osmotic stress response. Additionally, deletion of yl-Hog1 leads to increased filamentation in Yarrowia lipolytica, but causes significant sensitivity to osmotic stress induced by a high concentration of a carbon source. Results In this study, we tested the effect of point mutations on the function of yl-Hog1 protein kinase. The targets of modification were the phosphorylation sites (T171A-Y173A) and the active center (K49R). Introduction of the variant HOG1-49 into the hog1∆ strain partially improved growth under osmotic stress, but did not recover the yeast-like shape of the cells. The HOG1-171/173 variant was not functional, and its introduction further weakened the growth of hog1∆ strains in hyperosmotic conditions. To verify a genetic modification in filament form, we developed a new system based on green fluorescent protein (GFP) for easier screening of proper mutants. Conclusions These results provide new insights into the functions of yl-Hog1 protein in dimorphic transition and constitute a good starting point for further genetic modification of Y. lipolytica in filament form
The Direct Anti-Virulence but Not Bactericidal Activity of Human Neutrophil Elastase against <i>Moraxella catarrhalis</i>
Neutrophil elastase (NE) contributes to innate antibacterial defense at both the intracellular (phagocytosis) and extracellular (degranulation, NETosis) levels. Moraxella catarrhalis, a human respiratory pathogen, can exist in an inflammatory milieu which contains NE. No data are available on the action of NE against M. catarrhalis or on the counteraction of NE-dependent host defenses by this pathogen. Using time-kill assays we found that bacteria are able to survive and replicate in the presence of NE. Transmission electron microscopy and flow cytometry studies with NE-treated bacteria revealed that while NE admittedly destabilizes the outer membrane leaflet, it does not cause cytoplasmic membrane rupture, suggesting that the enzyme does not target components that are essential for cell integrity. Using LC-MS/MS spectroscopy we determined that NE cleaved at least three virulent surface proteins in outer membrane vesicles (OMVs) of M. catarrhalis, including OMP CD, McaP, and TbpA. The cleavage of OMP CD contributes to the significant decrease in resistance to serum complement in the complement-resistant strain Mc6. The cleavage of McaP did not cause any sensitization to erythromycin nor did NE disturb its drug action. Identifying NE as a novel but subtle anti-virulence agent together with its extracellularly not-efficient bactericidal activity against M. catarrhalis may facilitate the pathogen’s existence in the airways under inflammation
Characterization of erythrose reductase from Yarrowia lipolytica and its influence on erythritol synthesis
Abstract Background Erythritol is a natural sweetener that is used in the food industry. It is produced as an osmoprotectant by bacteria and yeast. Due to its chemical properties, it does not change the insulin level in the blood, and therefore it can be safely used by diabetics. Previously, it has been shown that erythrose reductase (ER), which catalyzes the final step, plays a crucial role in erythritol synthesis. ER reduces erythrose to erythritol with NAD(P)H as a cofactor. Despite many studies on erythritol synthesis by Yarrowia lipolytica, the enzymes involved in this metabolic pathway have ever been described. Results The gene YALI0F18590g encoding the predicted erythrose reductase from Y. lipolytica was overexpressed, and its influence on erythritol synthesis was studied. The amino acid sequence of the Y. lipolytica ER showed a high degree of similarity to the previously described erythrose reductases from known erythritol producers, such as Candida magnoliae and Moniliella megachiliensis. Here, we found that the gene overexpression results in an enhanced titer of erythritol of 44.44 g/L (20% over the control), a yield of 0.44 g/g and productivity of 0.77 g/L/h. Moreover, on purification and characterization of the enzyme we found that it displays the highest activity at 37 °C and pH 3.0. The effects of various metal ions (Zn2+, Cu2+, Mn2+, Fe2+) on erythrose reductase were investigated. The addition of Zn2+ ions at 0.25 mM had a positive effect on the activity of erythrose reductase from Y. lipolytica, as well as on the erythritol production. Conclusions In this study we identified, overexpressed and characterized a native erythrose reductase in Y. lipolytica. Further optimizations of this strain via metabolic pathway engineering and media optimization strategies enabled 54 g/L to be produced in a shake-flask experiment. To date, this is the first reported study employing metabolic engineering of the native gene involved in the erythritol pathway to result in a high titer of the polyol. Moreover, it indicates the importance of environmental conditions for genetic targets in metabolic engineering
The Overexpression of YALI0B07117g Results in Enhanced Erythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica
The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis
In-depth analysis of erythrose reductase homologs in Yarrowia lipolytica
Abstract The unconventional yeast Yarrowia lipolytica produces erythritol as an osmoprotectant to adapt to osmotic stress. In this study, the array of putative erythrose reductases, responsible for the conversion of d-erythrose to erythritol, was analyzed. Single knockout and multiple knockout strains were tested for their ability to produce polyols in osmotic stress conditions. Lack of six of the reductase genes does not affect erythritol significantly, as the production of this polyol is comparable to the control strain. Deletion of eight of the homologous erythrose reductase genes resulted in a 91% decrease in erythritol synthesis, a 53% increase in mannitol synthesis, and an almost 8-fold increase in arabitol synthesis as compared to the control strain. Additionally, the utilization of glycerol was impaired in the media with induced higher osmotic pressure. The results of this research may shed new light on the production of arabitol and mannitol from glycerol by Y. lipolytica and help to develop strategies for further modification in polyol pathways in these microorganisms