Biology subjects

Eltis, L. D.

Publications and source records attributed to Eltis, L. D..

6 recordsLinked to original sources

bioRxiv2026

Burkholderia cenocepacia physiology and molecular adaptations to the acidic pH of the CF nutritional environment

Burkholderia cenocepacia is an opportunistic pathogen associated with increased disease severity and mortality in cystic fibrosis (CF) patients. We have previously shown that elevated iron and acidic pH in the CF nutritional environment increases B. cenocepacia growth rate and decreases its susceptibility to some of the antimicrobials used clinically to treat CF infections. Here, we aimed to characterize B. cenocepacia physiology and its molecular response under acidic pH and increased zinc and iron concentrations using a modified synthetic CF sputum media (SCFM-FeZn). By investigating B. cenocepacia internal pH homeostasis, we found that it maintains a neutral internal pH when exposed to mildly acidic media at pH 5.5. We also assessed the effect of B. cenocepacia growth on the pH of SCFM-FeZn. When cultured at pH 6.8, B. cenocepacia maintained a media pH of [~]6.5. In contrast, when the culture pH value was initially 5.5, it increased to 6.5 during growth. Using comparative transcriptomics and metabolomics analysis, we identified 990 differentially expressed genes, and 23 differentially abundant metabolites in supernatants at acidic compared to neutral pH. Some of these genes and metabolites were involved in aromatic amino acid metabolism including the upregulated trpE gene, encoding a tryptophan biosynthetic enzyme. A tryptophan auxotrophic trpE deletion strain grew slower in SCFM-FeZn. Overall, this work identifies mechanisms involved in B. cenocepacia adaptation to acidic pH under conditions to model the CF nutritional environment. Some of these mechanisms are also associated with pathogenicity and virulence. ImportancePathogenic bacteria can be exposed to acidic pH inside and outside the host. Their ability to adapt to pH fluctuations contributes to success in host colonization. B. cenocepacia can grow at acidic pH ([~]3.5) and has been recovered from intracellular acidic compartments of amoebas and macrophages. Adaptation to acidic pH depends on molecular mechanisms that maintain a near optimal pH inside the cell for the function of vital processes. A few mechanisms that contribute to its adaptation to acidic pH have been described, but not in conditions reflecting the CF nutritional environment. Here, we identified multiple differentially-regulated systems that are associated with bacterial susceptibility to antimicrobials and pathogenesis. This research provides a better understanding of the role of acidic pH on B. cenocepacia physiology in the CF nutritional context and highlights possible systems that should be further characterized.

microbiology↗

bioRxiv2026

Engineering a cytochrome P450 O-demethylase for the bioconversion of hardwood lignin

Lignin is a promising alternative to petroleum as a feedstock for the chemical industry. Emergent strategies for lignin valorization involve tandem processes in which biomass is chemo-catalytically fractionated followed by biotransformation of the depolymerized lignin by microbial cell factories. A rate-limiting step in this biotransformation is O-demethylation of the lignin-derived monomers. The reductive catalytic fractionation of hardwood biomass generates high yields of two classes of monomers: 4-alkylguaiacols and 4-alkylsyringols. To better understand the biotransformation of these monomers, we studied AgcA, a cytochrome P450, and AgcB, the cognate reductase, that together catalyze the O-demethylation of 4-alkylguaiacols. A 1.82 [A] resolution crystal structure of AgcAEP4 from Rhodococcus rhodochrous EP4 in complex with 4-ethylguaiacol identified residues Leu78, Ala293 and Phe166 as potential specificity determinants. Substitution of Ala293 and Leu78 decreased the specificity of AgcAEP4 for alkylguaiacols. Substitution of Phe166 yielded a variant that bound 4-propylsyringol but did not transform it. In contrast, the corresponding variant in the Rhodococcus aromaticivorans RHA1 homolog, AgcARHA1 Y166A, catalyzed the O-demethylated of both methoxy groups of 4-propylsyringol with a kcat/Km of 8500 M-1 s-1 for the first O-demethylation, nearly 7-fold higher than WT AgcARHA1. A strain of RHA1 harboring the variant did not grow on 4-propylsyringol but consumed it at approximately the same rate as 4-propylguaiacol and transformed some of it to pentanoyl-CoA, consistent with metabolism via the meta-cleavage pathway that catabolizes 4-alkylguaiacols. These studies improve our understanding of a critical lignin-degrading enzyme system and facilitate its efficient implementation into biocatalysts. SignificanceLignin is a highly abundant source of aromatic carbon and a promising alternative to petroleum to generate materials. Fulfilling this promise depends on technological advances in areas such as catalytic fractionation and biocatalysis. Catalytic fractionation of hardwood biomass generates mixtures of aromatics enriched in 4-propylguaiacol and 4-propylsyringol. Here, we biochemically and structurally characterized a cytochrome P450 that initiates 4-propylguaiacol catabolism. Informed by the structure, we engineered the enzyme to have dual activity on both 4-propylguaiacol and 4-propylsyringol, and implemented this enzyme into a bacterial biocatalyst. Metabolomic analysis of this strain provided insights into the catabolism of both aromatics. Overall, these findings greatly facilitate the engineering of P450s and bacteria to biocatalytically upgrade lignin.

biochemistry↗

bioRxiv2020

A nanocompartment containing the peroxidase DypB contributes to defense against oxidative stress in M. tuberculosis

Encapsulin nanocompartments are an emerging class of prokaryotic protein-based organelles consisting of an encapsulin protein shell that encloses a protein cargo1. Genes encoding nanocompartments are widespread in bacteria and archaea, and recent works have characterized the biochemical function of several cargo enzymes2. However, the importance of these organelles to host physiology is poorly understood. Here, we report that the human pathogen Mycobacterium tuberculosis (Mtb) produces a nanocompartment that contains the dye-decolorizing peroxidase DypB. We show that this nanocompartment is important for the ability of Mtb to resist oxidative stress in low pH environments, including during infection of host cells and upon treatment with a clinically relevant antibiotic. Our findings are the first to implicate a nanocompartment in bacterial pathogenesis and reveal a new mechanism that Mtb uses to combat oxidative stress.

microbiology↗

bioRxiv2019

Steryl ester formation and accumulation in steroid-degrading bacteria

Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl- and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole cell systems in the biotechnological production of SEs for use in functional foods and cosmetics.\n\nIMPORTANCEOleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

microbiology↗

bioRxiv2019

Catabolism of alkylphenols in Rhodococcus via a meta-cleavage pathway associated with genomic islands

The bacterial catabolism of aromatic compounds has considerable promise to convert lignin depolymerization products to commercial chemicals. Alkylphenols are a key class of depolymerization products whose catabolism is not well elucidated. We isolated Rhodococcus rhodochrous EP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 and Rhodococcus jostii RHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by the aphABCDEFGHIQRS genes that degrades 4-ethylphenol via the meta-cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by the aphAB genes, which were up-regulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2000 U/mg) but did not detectably transform phenol. Nevertheless, a {Delta}aphA mutant in RHA1 grew on 4-ethylphenol by compensatory up-regulation of phenol hydroxylase genes (pheA1-3). Deletion of aphC, encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption of pcaL in the {beta}-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-ethylphenol and 4-propylphenol are catabolized exclusively via meta-cleavage in rhodococci while phenol is subject to ortho-cleavage. Putative genomic islands encoding aph genes were identified in EP4 and several other rhodococci. Overall, this study identifies a 4-alkylphenol pathway in rhodococci, demonstrates key enzymes involved, and presents evidence that the pathway is encoded in a genomic island. These advances are of particular importance for wide-ranging industrial applications of rhodococci, including upgrading of lignocellulose biomass.\n\nImportanceElucidation of bacterial alkylphenol catabolism is important for the development of biotechnologies to upgrade the lignin component of plant biomass. We isolated a new strain, Rhodococcus rhodochrous EP4, on 4-ethylphenol, an alkylphenol that occurs in lignin-derived streams, including reductive catalytic fractionation products of corn stover. We further demonstrated its degradation via a meta-cleavage pathway (Aph) with transcriptomics. A new class of Actinobacterial hydroxylase, AphAB, acts specifically on alkylphenols. Phylogenomic analysis indicated that the aph genes occur on putative genomic islands in several rhodococcal strains. These genes were identified in the genetically-tractable strain Rhodococcus jostii RHA1. Strains missing this element cannot metabolise 4-ethylphenol and 4-propylphenol. Overall, we advanced the understanding of how aromatic compounds are degraded by environmental bacteria and identified enzymes that can be employed in the transition away from petro-chemicals towards renewable alternatives.

microbiology↗

bioRxiv2018

Bacterial and fungal contributions to delignification and lignocellulose degradation in forest soils with metagenomic and quantitative stable isotope probing

Delignification, or lignin-modification, facilitates the decomposition of lignocellulose in woody plant biomass. The extant diversity of lignin-degrading bacteria and fungi is underestimated by culture-dependent methods, limiting our understanding of the functional and ecological traits of decomposers populations. Here, we describe the use of stable isotope probing (SIP) coupled with amplicon and shotgun metagenomics to identify and characterize the functional attributes of lignin-, cellulose-and hemicellulose-degrading fungi and bacteria in coniferous forest soils from across North America. We tested the extent to which catabolic genes partitioned among different decomposer taxa; the relative roles of bacteria and fungi, and whether taxa or catabolic genes correlated with variation in lignocellulolytic activity, measured as the total assimilation of 13C-label into DNA and phospholipid fatty acids. We found high overall bacterial degradation of our model lignin substrate, particularly by gram-negative bacteria (Comamonadaceae and Caulobacteraceae), while fungi were more prominent in cellulose-degradation. Very few taxa incorporated 13C-label from more than one lignocellulosic polymer, suggesting specialization among decomposers. Collectively, members of Caulobacteraceae could degrade all three lignocellulosic polymers, providing new evidence for their importance in lignocellulose degradation. Variation in lignin-degrading activity was better explained by microbial community properties, such as catabolic gene content and community structure, than cellulose-degrading activity. SIP significantly improved shotgun metagenome assembly resulting in the recovery of several high-quality draft metagenome-assembled genomes and over 7,500 contigs containing unique clusters of carbohydrate-active genes. These results improve understanding of which organisms, conditions and corresponding functional genes contribute to lignocellulose decomposition.

molecular biology↗