Engineering topics

Trotter, Valentine V.

Publications and source records attributed to Trotter, Valentine V..

5 records

DOE OSTI2024

Fitness factors impacting survival of a subsurface bacterium in contaminated groundwater

Many factors contribute to the ability of a microbial species to persist when encountering complexly contaminated environments, including time of exposure, the nature and concentration of contaminants, availability of nutritional resources, and possession of a combination of appropriate molecular mechanisms needed for survival. Herein we sought to identify genes that are most important for survival of Gram-negative Enterobacteriaceae in contaminated groundwater environments containing high concentrations of nitrate and metals using the metal-tolerant Oak Ridge Reservation isolate, Pantoea sp. MT58 (MT58). Survival fitness experiments in which a randomly barcoded transposon insertion (RB-TnSeq) library of MT58 was exposed directly to contaminated Oak Ridge Reservation groundwater samples from across a nitrate and mixed metal contamination plume were used to identify genes important for survival with increasing exposure times and concentrations of contaminants, and availability of a carbon source. Genes involved in controlling and using carbon, encoding transcriptional regulators, and related to Gram-negative outer membrane processes were among those found to be important for survival in contaminated Oak Ridge Reservation groundwater. A comparative genomics analysis of 75 Pantoea genus strains allowed us to further separate the survival determinants into core and non-core genes in the Pantoea pangenome, revealing insights into the survival of subsurface microorganisms during contaminant plume intrusion.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Molecular mechanisms and environmental adaptations of flagellar loss and biofilm growth of Rhodanobacter under environmental stress

Biofilms aid bacterial adhesion to surfaces via direct and indirect mechanisms, and formation of biofilms is considered as an important strategy for adaptation and survival in suboptimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, and nitrate or metal concentrations are underexplored. Here, we examined biofilm formation under different nutrient, pH, metal, and nitrate regimens of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse levels of pH (3.5 to 5) and nitrates (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptations for survival and growth at low pH. Biofilms were intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation, findings warranting further investigation. Through random barcode transposon-site sequencing (RB-TnSeq), proteomics, use of specific mutants, and transmission electron microscopy analysis, we discovered flagellar loss under aluminum stress, indicating a potential relationship between motility, metal tolerance, and biofilm growth. Comparative genomic analyses revealed the absence of flagella and chemotaxis genes and the presence of a putative type VI secretion system in the highly biofilm-forming strain FW021-MT20. In this study we identified genetic determinants associated with biofilm growth under metal stress in a predominant environmental genus, Rhodanobacter, and identified traits aiding survival and adaptation to contaminated subsurface environments.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Draft Genome Sequence of Pedococcus sp. Strain 5OH_020, Isolated from California Grassland Soil

The draft genome sequence of the soil bacterium Pedococcus sp. strain 5OH_020, isolated on a natural cobalamin analog, comprises 4.4 Mbp, with 4,108 protein-coding genes. Its genome encodes cobalamin-dependent enzymes, including methionine synthase and class II ribonucleotide reductase. Taxonomic analysis suggests that it is a novel species within the genus Pedococcus .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Large-scale genetic characterization of the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough

Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Deciphering Microbial Metal Toxicity Responses using RB-TnSeq and Activity-Based Metabolomics

To uncover metal toxicity targets and defense mechanisms of the facultative anaerobe Pantoea sp. strain MT58 (MT58), we used a multiomic strategy combining two global techniques, random bar code transposon site sequencing (RB-TnSeq) and activity-based metabolomics. MT58 is a metal-tolerant Oak Ridge Reservation (ORR) environmental isolate that was enriched in the presence of metals at concentrations measured in contaminated groundwater at an ORR nuclear waste site. The effects of three chemically different metals found at elevated concentrations in the ORR contaminated environment were investigated: the cation Al 3+ , the oxyanion CrO 4 2- , and the oxycation UO 2 2+ . Both global techniques were applied using all three metals under both aerobic and anaerobic conditions to elucidate metal interactions mediated through the activity of metabolites and key genes/proteins. These revealed that Al 3+ binds intracellular arginine, CrO 4 2- enters the cell through sulfate transporters and oxidizes intracellular reduced thiols, and membrane-bound lipopolysaccharides protect the cell from UO 2 2+ toxicity. In addition, the Tol outer membrane system contributed to the protection of cellular integrity from the toxic effects of all three metals. Likewise, we found evidence of regulation of lipid content in membranes under metal stress. Individually, RB-TnSeq and metabolomics are powerful tools to explore the impact various stresses have on biological systems. In this work, we show that together they can be used synergistically to identify the molecular actors and mechanisms of these pertubations to an organism, furthering our understanding of how living systems interact with their environment.

59 BASIC BIOLOGICAL SCIENCES↗