Role of Iron-Containing Alcohol Dehydrogenases in Acinetobacter baumannii ATCC 19606 Stress Resistance and Virulence

Most bacteria possess alcohol dehydrogenase (ADH) genes (Adh genes) to mitigate alcohol toxicity, but these genes have functions beyond alcohol degradation. Previous research has shown that ADH can modulate quorum sensing in Acinetobacter baumannii, a rising opportunistic pathogen. However, the number and nature of Adh genes in A. baumannii have not yet been fully characterized. We identified seven alcohol dehydrogenases (NAD+-ADHs) from A. baumannii ATCC 19606, and examined the roles of three iron-containing ADHs, ADH3, ADH4, and ADH6. Marker-less mutation was used to generate Adh3, Adh4, and Adh6 single, double, and triple mutants. Disrupted Adh4 mutants failed to grow in ethanol-, 1-butanol-, or 1-propanol-containing mediums, and recombinant ADH4 exhibited strongest activity against ethanol. Stress resistance assays with inorganic and organic hydroperoxides showed that Adh3 and Adh6 were key to oxidative stress resistance. Virulence assays performed on the Galleria mellonella model organism revealed that Adh4 mutants had comparable virulence to wild-type, while Adh3 and Adh6 mutants had reduced virulence. The results suggest that ADH4 is primarily involved in alcohol metabolism, while ADH3 and ADH6 are key to stress resistance and virulence. Further investigation into the roles of other ADHs in A. baumannii is warranted.

Identification and Functional Analysis of ThADH1 and ThADH4 Genes Involved in Tolerance to Waterlogging Stress in Taxodium hybrid ‘Zhongshanshan 406’

The Taxodium hybrid ‘Zhongshanshan 406’ (T. hybrid ‘Zhongshanshan 406’) [Taxodium mucronatum Tenore × Taxodium distichum (L.). Rich] has an outstanding advantage in flooding tolerance and thus has been widely used in wetland afforestation in China. Alcohol dehydrogenase genes (ADHs) played key roles in ethanol metabolism to maintain energy supply for plants in low-oxygen conditions. Two ADH genes were isolated and characterized—ThADH1 and ThADH4 (GenBank ID: AWL83216 and AWL83217—basing on the transcriptome data of T. hybrid ‘Zhongshanshan 406’ grown under waterlogging stress. Then the functions of these two genes were investigated through transient expression and overexpression. The results showed that the ThADH1 and ThADH4 proteins both fall under ADH III subfamily. ThADH1 was localized in the cytoplasm and nucleus, whereas ThADH4 was only localized in the cytoplasm. The expression of the two genes was stimulated by waterlogging and the expression level in roots was significantly higher than those in stems and leaves. The respective overexpression of ThADH1 and ThADH4 in Populus caused the opposite phenotype, while waterlogging tolerance of the two transgenic Populus significantly improved. Collectively, these results indicated that genes ThADH1 and ThADH4 were involved in the tolerance and adaptation to anaerobic conditions in T. hybrid ‘Zhongshanshan 406’.

Alcohol dehydrogenase (ADH) genes family in wheat (Triticum aestivum): Genome-wide identification, characterization, phylogenetic relationship and expression patterns

Background Alcohol dehydrogenase (ADH) plays important roles in plant survival under anaerobic conditions. Although some research has been carried out the functions of ADH in other plants, that of wheat TaADH family genes in response to abiotic stress are unclear. Results A total of 22 ADH genes were obtained from 14 chromosomes of the wheat genome by systematic screening. Multiple sequence alignment and evolutionary relationship show that these genes contain the characteristics of GroES-like domain and Zinc-binding domain, and these belong to Medium-chain -ADH type and can be divided into three subfamilies. There are 17 pairs of fragment replication genes among TaADH family members in the wheat genome, while there are 9 pairs of collinear gene pairs from ADH family members between wheat and rice genome. We speculate that these fragment repetition events may be the main reason for the amplification of TaADH family genes. Ka/Ks analysis indicated that there were 64 repetitive gene pairs, and the Ka/Ks value of these gene pairs was less than 1, which indicated that these sequences of TaADH gene were relatively conservative and did not change greatly in the process of evolution. Promoter element analysis showed that almost all of the upstream promoters of these genes contained the responsive anaerobic inducible element. Tissue localized expression and expression patterns also demonstrated that the TaADH genes responded to abiotic stress and may play an important role in waterlogging stress during the seed germination stage. Conclusions The results of this study may be helpful to further study the function of TaADH genes and determine the candidate gene for wheat stress resistance breeding.

Molecular Characterization and Enological Potential of A High Lactic Acid-Producing Lachancea thermotolerans Vineyard Strain

Lactic acid production is an important feature of the yeast Lachancea thermotolerans that has gained increasing interest in winemaking. In particular, in light of climate change, the biological acidification and ethanol reduction by the use of selected yeast strains may counteract the effect of global warming in wines. Here, the enological potential of a high lactate-producing L. thermotolerans strain (P-HO1) in mixed fermentations with S. cerevisiae was examined. Among the different inoculation schemes evaluated, the most successful implantation of L. thermotolerans was accomplished by sequential inoculation of S. cerevisiae, i.e., at 1% vol. ethanol. P-HO1produced the highest levels of lactic acid ever recorded in mixed fermentations (10.4 g/L), increasing thereby the acidity and reducing ethanol by 1.6% vol. L. thermotolerans was also associated with increases in ethyl isobutyrate (strawberry aroma), free SO2, organoleptically perceived citric nuances and aftertaste. To start uncovering the molecular mechanisms of lactate biosynthesis in L. thermotolerans, the relative expressions of the three lactate dehydrogenase (LDH) paralogous genes, which encode the key enzyme for lactate biosynthesis, along with the alcohol dehydrogenase paralogs (ADHs) were determined. Present results point to the possible implication of LDH2, but not of other LDH or ADH genes, in the high production of lactic acid in certain strains at the expense of ethanol. Taken together, the important enological features of P-HO1 highlighted here, and potentially of other L. thermotolerans strains, indicate its great importance in modern winemaking, particularly in the light of the upcoming climate change and its consequences in the grape/wine system.

Recurrent single-gene duplication drives the expansion and expression diversification of the ADH gene family in pear and other Rosaceae species

Background Alcohol dehydrogenases (ADHs) are essential to plant growth and the formation of aromatic compounds in fruits. However, the evolutionary history and characteristics of ADH gene expression remain largely unclear in Chinese white pear ( Pyrus bretschneideri ) and other fruit species from the family Rosaceae.Results In this study, 464 ADH genes were identified in eight Rosaceae fruit species and 68 of the genes were from pear. Based on the analyses of phylogeny and conserved motifs, the pear ADH genes were classified into four subgroups (I, II, III, and IV). The chromosomal distribution of the genes was found to be uneven and numerous clusters of physically linked ADH genes were detected. Frequent single-gene duplication events were found to have contributed to the formation of ADH gene clusters and the expansion of the ADH gene family in these eight Rosaceae species. Purifying selection was the major force in ADH gene evolution. The younger genes derived from tandem and proximal duplications had evolved faster than those that derived from other types of duplication. RNA-sequencing and quantitative-real time-PCR analysis revealed that the expression levels of three ADH genes were closely correlated with the content of aromatic compounds that are found during fruit development.Conclusion Comprehensive analyses were conducted in eight Rosaceae species and 464 ADH genes were identified. The results of this study provide new insights into the evolution and expression characteristics of ADH family genes in pear and other Rosaceae species.

AdRAP2.3, a Novel Ethylene Response Factor VII from Actinidia deliciosa, Enhances Waterlogging Resistance in Transgenic Tobacco through Improving Expression Levels of PDC and ADH Genes

APETALA2/ethylene-responsive factor superfamily (AP2/ERF) is a transcription factor involved in abiotic stresses, for instance, cold, drought, and low oxygen. In this study, a novel ethylene-responsive transcription factor named AdRAP2.3 was isolated from Actinidia deliciosa ‘Jinkui’. AdRAP2.3 transcription levels in other reproductive organs except for the pistil were higher than those in the vegetative organs (root, stem, and leaf) in kiwi fruit. Plant hormones (Salicylic acid (SA), Methyl-jasmonate acid (MeJA), 1-Aminocyclopropanecarboxylic Acid (ACC), Abscisic acid (ABA)), abiotic stresses (waterlogging, heat, 4 °C and NaCl) and biotic stress (Pseudomonas Syringae pv. Actinidiae, Psa) could induce the expression of AdRAP2.3 gene in kiwi fruit. Overexpression of the AdRAP2.3 gene conferred waterlogging stress tolerance in transgenic tobacco plants. When completely submerged, the survival rate, fresh weight, and dry weight of transgenic tobacco lines were significantly higher than those of wile type (WT). Upon the roots being submerged, transgenic tobacco lines grew aerial roots earlier. Overexpression of AdRAP2.3 in transgenic tobacco improved the pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzyme activities, and improved the expression levels of waterlogging mark genes NtPDC, NtADH, NtHB1, NtHB2, NtPCO1, and NtPCO2 in roots under waterlogging treatment. Overall, these results demonstrated that AdRAP2.3 might play an important role in resistance to waterlogging through regulation of PDC and ADH genes in kiwi fruit.