Project description:Many sulfur-oxidizing prokaryotes oxidize sulfur compounds through a combination of initial extracytoplasmic and downstream cytoplasmic reactions. Facultative sulfur oxidizers adjust transcription to sulfur availability. While sulfur-oxidizing enzymes and transcriptional repressors have been extensively studied, sulfur import into the cytoplasm and how regulators sense external sulfur are poorly understood. Addressing this gap, we show that SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters and are encoded alongside sulfur oxidation and transcriptional regulation genes, fulfill these roles in the Alphaproteobacterium Hyphomicrobium denitrificans. RT-qPCR provided initial evidence that SoxT1A and SoxT1B from H. denitrificans may be intricate components of the oxidation pathway and/or involved in its transcriptional regulation. Here, we extend these analyses with genome-wide mRNA-Seq data for the reference strain, comparing transcription in the absence and presence of 2 mM thiosulfate
Project description:To get further insights in the dimethyl sulfide metabolism of Hyphomicrobium denitrificans, a LC-MS proteomic approach was used. The total protein of three biological replicates of H. denitrificans grown on dimethyl sulfide was compared with three biological replicates grown on dimethylamine. This approach resulted in the identification of several new proteins with a potential involvement in dimethyl sulfide degradation.
Project description:Beller, H. R., T. E. Letain, A. Chakicherla, S. R. Kane, T. C. Legler, and M. A. Coleman. 2006. Whole-genome transcriptional analysis of chemolithoautotrophic thiosulfate oxidation by Thiobacillus denitrificans under aerobic vs. denitrifying conditions. Journal of Bacteriology 188:7005-7015. Thiobacillus denitrificans is one of the few known obligate chemolithoautotrophic bacteria capable of energetically coupling thiosulfate oxidation to denitrification as well as aerobic respiration. As very little is known about the differential expression of genes associated with key chemolithoautotrophic functions (such as sulfur-compound oxidation and CO2 fixation) under aerobic versus denitrifying conditions, we conducted whole-genome, cDNA microarray studies to explore this topic systematically. The microarrays identified 277 genes (approximately ten percent of the genome) as differentially expressed using Robust Multi-array Average statistical analysis and a 2-fold cutoff. Genes upregulated (ca. 6- to 150-fold) under aerobic conditions included a cluster of genes associated with iron acquisition (e.g., siderophore-related genes), a cluster of cytochrome cbb3 oxidase genes, cbbL and cbbS (encoding the large and small subunits of form I ribulose 1,5-bisphosphate carboxylase/oxygenase, or RubisCO), and multiple molecular chaperone genes. Genes upregulated (ca. 4- to 95-fold) under denitrifying conditions included nar, nir, and nor genes (associated respectively with nitrate reductase, nitrite reductase, and nitric oxide reductase, which catalyze successive steps of denitrification), cbbM (encoding form II RubisCO), and genes involved with sulfur-compound oxidation (including two physically separated but highly similar copies of sulfide:quinone oxidoreductase and of dsrC, associated with dissimilatory sulfite reductase). Among genes associated with denitrification, relative expression levels (i.e., degree of upregulation with nitrate) tended to decrease in the order nar > nir > nor > nos. Reverse transcription, quantitative PCR analysis was used to validate these trends. Keywords: bacterial metabolism
Project description:Chemoautotrophic bacteria belonging to the genus Sulfurimonas in the class Campylobacteria (formerly classified as Epsilonproteobacteria) play a key role in the sulfur cycle in a variety of oxygen-deficient or –limited and sulfide-rich marine and terrestrial environments. Previously, they were identified as key players in the turnover of zero-valence sulfur, a central intermediate in the marine sulfur cycle, and S. denitrificans was further shown to be able to oxidize cyclooctasulfur. However, at present the mechanism involved in the activation and metabolism of cyclooctasulfur is not known. To this end, we assessed the transcriptome and proteome of S. denitrificans grown with either thiosulfate or cyclooctasulfur as the electron donor. While the overall profiles under the two growth conditions were rather similar, distinct differences were observed that could be attributed to the utilization of cyclooctasulfur. This included a higher abundance of expressed genes and proteins related to attachment in the presence of cyclooctasulfur and the differential expression of the sulfur-oxidation multienzyme complex (SOX). S. denitrificans uses the SOX system for the oxidation of reduced sulfur compounds, including two copies of the sulfur-binding SoxYZ proteins, encoded in two gene clusters: soxABXYZ1 and soxCDYZ2. While the proteins of both operons of the SOX system were detected in the presence of thiosulfate, only proteins of the soxCDYZ2 operon were detected when grown with cylcooctasulfur. Based on these findings a model for the oxidation of cylcooctasulfur is being proposed that might also apply to other Campylobacteria that share the same arrangement of the SOX system. Our results have implications for interpreting metatranscriptomic and -proteomic data and for the observed high level of diversification of soxYZ2 among sulfur-oxidizing Campylobacteria.
Project description:Investigation of whole genome gene expression level changes in anaerobic, nitrate-dependent Fe(II) oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans Here we report on a study to identify genes associated with nitrate-dependent Fe(II) oxidation by whole-genome transcriptional (microarray) assays including the use of FeCO3, Fe2+, and U(IV) oxides as electron donors under denitrifying conditions. A 25 chip study using total RNA recovered from wild-type T. denitrificans was cultivated at 30oC under strictly anaerobic conditions with growth medium that contained 20 mM thiosulfate, 20 mM nitrate, and 30 mM bicarbonate (pH ~7) and exposed to 8 treatments. Each chip measures the expression level of 2832 ORFs with N 24-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.
Project description:Transcriptome profiles of an aerobic photosynthetic bacterium Roseobacter denitrificans OCh114 grown under different oxygen tension and light irradiation conditions were determined by NimbleGen Prokaryotic Expression array (12x135K).