Structural Evolution of the Ancient Enzyme, Dissimilatory Sulfite Reductase

Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme diversified into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving sulfate/sulfite reducing organismal (SRO) lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.

Expression of Cytochrome c3 from Desulfovibrio vulgaris in Plant Leaves Enhances Uranium Uptake and Tolerance of Tobacco

Cytochrome c3 (uranyl reductase) from Desulfovibrio vulgaris can reduce uranium in bacterial cells and in cell-free systems. This gene was introduced in tobacco under control of the RbcS promoter, and the resulting transgenic plants accumulated uranium when grown on a uranyl ion containing medium. The uptaken uranium was detected by EM in chloroplasts. In the presence of uranyl ions in sublethal concentration, the transgenic plants grew phenotypically normal while the control plants’ development was impaired. The data on uranium oxidation state in the transgenic plants and the possible uses of uranium hyperaccumulation by plants for environmental cleanup are discussed.

Assessing microbial growth monitoring methods for challenging strains and cultures

This is a paper focusing on the comparison of growth curves using field relevant testing methods and moving away from colony counts. Challenges exist to explore antimicrobial growth of fastidious strains, poorly culturable bacterial and bacterial communities of environmental interest. Thus, various approaches have been explored to follow bacteria growth that can be an efficient surrogate for classical optical density or colony forming unit measurements.Here we tested optical density, ATP assays, DNA concentrations and 16S rRNA qPCR as means to monitor pure culture growth of six different species including Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica. Optical density is and excellent, rapid monitoring method of pure culture planktonic cells but cannot be applied to environmental or complex samples. ATP assays provide rapid results but conversions to cell counts may be misleading for different species. DNA concentration is a very reliable technique which can be used for any sample type and provides genetic materials for downstream applications. qPCR of the 16S rRNA gene is a widely applicable technique for monitoring microbial cell concentrations but is susceptible to variation between replicates. DNA concentrations were found to correlate the best with the other three assays and provides the advantages of rapid extraction, consistency between replicates and potential for downstream analysis, DNA concentrations is determined to be the best universal monitoring method for complex environmental samples.

MICROEVOLUTION OF DESULFOVIBRIO VULGARIS CO-CULTURED WITH METHANOSARCINA BARKERI REVEALED BY GENOME RE-SEQUENCING AND SINGLE-CELL RT-QPCR ANALYSIS

An integrative approach of adaptive laboratory evolution, whole-genome sequencing and single-cell analysis was used to explore mechanisms related to establishment and maintenance of syntrophic interaction between sulfate-reducing Desulfovibrio vulgaris and methanogen Methanosarcina barkeri. Adaptive laboratory evolution of the D. vulgaris and M. barkeri dual-cultures under two different concentrations of electron donor lactate (38 mM and 50 mM) was conducted by propagating continuously for 50 transfers (~200 generations). Physiological analysis showed that, compared with the initial dual-cultures, the adapted dual-cultures (E38 and E50) have increased growth rates (1.1-fold and 1.2 -fold) and higher biomass yields (3.0-fold and 3.8-fold) on 38 mM and 50 mM lactate, respectively. Whole-genome re-sequencing of D. vulgaris in the adapted dual-cultures revealed 11 and 12 mutations in the D. vulgaris genomes of E38 and E50 dual-cultures, respectively, among which 4 mutations were found in both adapted dual-cultures. RT-qPCR analysis showed that the expression levels of 8 mutated genes were gradually up-regulated in D. vulgaris along with the evolution process. In addition, their heterogeneity was found decreased along with the evolution, as revealed by single-cell RT-qPCR analysis, reflecting adjustments of both gene expression and gene heterogeneity to the gradually established syntrophic relationship.

Genetic Basis of Chromate Adaptation and the Role of the Pre-existing Genetic Divergence during an Experimental Evolution Study with Desulfovibrio vulgaris Populations

Chromium is one of the most common heavy metal pollutants of soil and groundwater. The potential of Desulfovibrio vulgaris Hildenborough in heavy metal bioremediation such as Cr(VI) reduction was reported previously; however, experimental evidence of key functional genes involved in Cr(VI) resistance are largely unknown.

Microbial corrosion of DSS 2205 in an acidic chloride environment under continuous flow

Corrosion under flow conditions is a major problem in the transportation industry. Various studies have shown the direct impact of different flow rates on bacteria biofilm formation, mass transfer and resulting different corrosion behaviour of materials in neutral environments. However, little is understood on corrosion under acidic flow conditions. This study investigated the impact of an acidic artificial seawater environment containing Desulfovibrio vulgaris on DSS 2205 microbial corrosion under different velocities (0.25 m.s-1 and 0.61 m.s-1). Experiments containing no bacteria were performed as controls. Bacterial attachment was observed by optical and scanning electron microscope (SEM). Materials corrosion was assessed using open circuit potential (OCP), electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization. Pits formed after potentiodynamic test were observed under SEM. The largest area of bacterial attachment was found on coupons immersed at a velocity of 0.25 m.s-1; however, the corrosion rate was lower than at higher velocity. Shallow pits occurred in the metal coupons when bacteria were present, while deep pits occurred in the controls. The study indicates the positive impact of biofilm formation in corrosion prevention of materials under acidic condition. The nature of corrosion behaviour of duplex stainless is discussed.