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Biodesulfurization poses as an ideal replacement to the high cost hydrodesulfurization of the recalcitrant heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and its derivatives. The increasingly stringent limits on fuel sulfur content intensify the need for improved desulfurization biocatalysts, without sacrificing the calorific value of the fuel. Selective sulfur removal in a wide range of biodesulfurization strains, as well as in the model biocatalyst Rhodococcus qingshengii IGTS8, occurs via the 4S metabolic pathway that involves the dszABC operon, which encodes enzymes that catalyze the generation of 2-hydroxybiphenyl and sulfite from DBT. Here, using a homologous recombination process, we generate two recombinant IGTS8 biocatalysts, harboring native or rearranged, nonrepressible desulfurization operons, within the native dsz locus. The alleviation of sulfate-, methionine-, and cysteine-mediated dsz repression is achieved through the exchange of the native promoter P dsz , with the nonrepressible P kap1 promoter. The Dsz-mediated desulfurization from DBT was monitored at three growth phases, through HPLC analysis of end product levels. Notably, an 86-fold enhancement of desulfurization activity was documented in the presence of selected repressive sulfur sources for the recombinant biocatalyst harboring a combination of three targeted genetic modifications, namely, a dsz operon rearrangement, a native promoter exchange, and a dszA-dszB overlap removal. In addition, transcript level comparison highlighted the diverse effects of our genetic engineering approaches on dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. IMPORTANCE Rhodococcus is perhaps the most promising biodesulfurization genus and is able to withstand the harsh process conditions of a biphasic biodesulfurization process. In the present work, we constructed an advanced biocatalyst harboring a combination of three genetic modifications, namely, an operon rearrangement, a promoter exchange, and a gene overlap removal. Our homologous recombination approach generated stable biocatalysts that do not require antibiotic addition, while harboring nonrepressible desulfurization operons that present very high biodesulfurization activities and are produced in simple and low-cost media. In addition, transcript level quantification validated the effects of our genetic engineering approaches on recombinant strains' dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. Based on these findings, the present work can pave the way for further strain and process optimization studies that could eventually lead to an economically viable biodesulfurization process.

During alcoholic fermentation of grape sugars, wine yeasts produce a range of secondary metabolites that play an important role in the aroma profile of wines. In this study, we have explored the ability of a large number of wine yeast strains to modulate wine aroma composition, focusing on the release of the "fruity" thiols 3-mercaptohexan-1-ol (3-MH) and 4-mercapto-4-methylpentan-2-one (4-MMP) from their respective cysteinylated nonvolatile precursors. The role of the yeast gene IRC7 in thiol release has been well established, and it has been shown that a 38-bp deletion found in many wine strains cause them to express a truncated version of Irc7p that does not possess cysteine- S -conjugate β-lyase activity. In our data, we find that IRC7 allele length alone does not fully explain the capacity of a strain to release thiols. Screening of a large number of strains coupled with analysis of genomic sequence data allowed us to identify several previously undescribed single-nucleotide polymorphisms (SNPs) in IRC7 that, when coupled with allele length, more robustly explain the ability of a particular yeast strain to release thiols from their cysteinylated precursors. We also demonstrate that allelic variation of IRC7 not only affects the release of thiols but modulates the formation of negative volatile sulfur compounds from the amino acid cysteine. The results of this study provide winemakers with an improved understanding of the genetic determinants that affect wine aroma and flavor, which can be used to guide the choice of yeast strains that are fit for purpose. IMPORTANCE Volatile sulfur compounds contribute to wine aromas that may be considered pleasant, such as "tropical," "passionfruit," and "guava," as well as aromas that are considered undesirable, such as "rotten eggs," "onions," and "sewer." During fermentation, wine yeasts release some of these compounds from odorless precursor molecules, a process that is most efficient when performed by yeasts that express active forms of the protein Irc7p. We show that most wine yeasts carry mutations that reduce activity of this protein, affecting the formation of volatile sulfur compounds that impart both pleasant and unpleasant aromas. The results provide winemakers with guidance on the choice of yeasts that can emphasize or deemphasize this particular contribution to wine quality., (Copyright © 2019 American Society for Microbiology.)

Certain species from the extremely thermoacidophilic genus Metallosphaera directly oxidize Fe(II) to Fe(III), which in turn catalyzes abiotic solubilization of copper from chalcopyrite to facilitate recovery of this valuable metal. In this process, the redox status of copper does not change as it is mobilized. Metallosphaera species can also catalyze the release of metals from ores with a change in the metal's redox state. For example, Metallosphae ra sedula catalyzes the mobilization of uranium from the solid oxide U 3 O 8 , concomitant with the generation of soluble U(VI). Here, the mobilization of metals from solid oxides (V 2 O 3 , Cu 2 O, FeO, MnO, CoO, SnO, MoO 2 , Cr 2 O 3 , Ti 2 O 3 , and Rh 2 O 3 ) was examined for M. sedula and M. prunae at 70°C and pH 2.0. Of these oxides, only V and Mo were solubilized, a process accelerated in the presence of FeCl 3 However, it was not clear whether the solubilization and oxidation of these metals could be attributed entirely to an Fe-mediated indirect mechanism. Transcriptomic analysis for growth on molybdenum and vanadium oxides revealed transcriptional patterns not previously observed for growth on other energetic substrates (i.e., iron, chalcopyrite, organic compounds, reduced sulfur compounds, and molecular hydrogen). Of particular interest was the upregulation of Msed_1191, which encodes a Rieske cytochrome b 6 fusion protein (Rcbf, referred to here as V/MoxA) that was not transcriptomically responsive during iron biooxidation. These results suggest that direct oxidation of V and Mo occurs, in addition to Fe-mediated oxidation, such that both direct and indirect mechanisms are involved in the mobilization of redox-active metals by Metallosphaera species. IMPORTANCE In order to effectively leverage extremely thermoacidophilic archaea for the microbially based solubilization of solid-phase metal substrates (e.g., sulfides and oxides), understanding the mechanisms by which these archaea solubilize metals is important. Physiological analysis of Metallosphaera species growth in the presence of molybdenum and vanadium oxides revealed an indirect mode of metal mobilization, catalyzed by iron cycling. However, since the mobilized metals exist in more than one oxidation state, they could potentially serve directly as energetic substrates. Transcriptomic response to molybdenum and vanadium oxides provided evidence for new biomolecules participating in direct metal biooxidation. The findings expand the knowledge on the physiological versatility of these extremely thermoacidophilic archaea., (Copyright © 2019 American Society for Microbiology.)

Adaptation to environmental perturbations requires living systems to coordinately regulate signaling pathways, gene expression, and metabolism. To better understand the mechanisms underlying adaptation, the regulatory nodes within networks must be elucidated. Here, ARO8-2 (which encodes an aminotransferase), PDC (which encodes a decarboxylase), and STR3 (which encodes a demethiolase) were identified as key genes involved in the catabolism of methionine in the mycoparasitic fungus Clonostachys rosea , isolated from Tuber melanosporum ascocarps. Exogenous Met induced the transcription of ARO8-2 and PDC but repressed the transcription of STR3 , which is controlled by the putative MSN2 and GLN3 binding sites responding to nitrogen catabolite repression. Met and its structural derivatives function as glutamine synthetase inhibitors, resulting in the downregulation of STR3 expression. The putative GLN3 binding site was necessary for STR3 downregulation. In Saccharomyces cerevisiae , Met and its structural derivatives also triggered downregulation of demethiolase gene expression. Altogether, the results indicated that exogenous Met triggered nitrogen catabolite repression, which stimulated the Ehrlich pathway and negatively regulated the demethiolation pathway via the methionine sulfoximine-responsive regulatory pathway. This finding revealed the regulatory nodes within the networks controlling the catabolism of Met into volatile organic sulfur-containing compounds, thereby enhancing our understanding of adaptation. IMPORTANCE Methionine shuttles organic nitrogen and plays a central role in nitrogen metabolism. Exogenous Met strongly induces the expression of ARO8-2 and PDC , represses the expression of STR3 , and generates volatile organic sulfur-containing compounds via the Ehrlich and demethiolation pathways. In this study, we used genetic, bioinformatic, and metabolite-based analyses to confirm that transcriptional control of the aminotransferase gene ARO8-2 , the decarboxylase gene PDC , and the demethiolase gene STR3 modulates Met catabolism into volatile organic sulfur-containing compounds. Importantly, we found that, in addition to the Ehrlich pathway, the demethiolation pathway was regulated by a nitrogen catabolite repression-sensitive regulatory pathway that controlled the transcription of genes required to catabolize poor nitrogen sources. This work significantly advances our understanding of nitrogen catabolite repression-sensitive transcriptional regulation of sulfur-containing amino acid catabolism and provides a basis for engineering Met catabolism pathways for the production of fuel and valuable flavor alcohols., (Copyright © 2018 American Society for Microbiology.)

Volatile compounds produced by plant-associated microorganisms represent a diverse resource to promote plant growth and health. Here, we investigated the effect of volatiles from root-associated Microbacterium species on plant growth and development. Volatiles of eight strains induced significant increases in shoot and root biomass of Arabidopsis but differed in their effects on root architecture. Microbacterium strain EC8 also enhanced root and shoot biomass of lettuce and tomato. Biomass increases were also observed for plants exposed only briefly to volatiles from EC8 prior to transplantation of the seedlings to soil. These results indicate that volatiles from EC8 can prime plants for growth promotion without direct and prolonged contact. We further showed that the induction of plant growth promotion is tissue specific; that is, exposure of roots to volatiles from EC8 led to an increase in plant biomass, whereas shoot exposure resulted in no or less growth promotion. Gas chromatography-quadrupole time of flight mass spectometry (GC-QTOF-MS) analysis revealed that EC8 produces a wide array of sulfur-containing compounds, as well as ketones. Bioassays with synthetic sulfur volatile compounds revealed that the plant growth response to dimethyl trisulfide was concentration-dependent, with a significant increase in shoot weight at 1 μM and negative effects on plant biomass at concentrations higher than 1 mM. Genome-wide transcriptome analysis of volatile-exposed Arabidopsis seedlings showed upregulation of genes involved in assimilation and transport of sulfate and nitrate. Collectively, these results show that root-associated Microbacterium primes plants, via the roots, for growth promotion, most likely via modulation of sulfur and nitrogen metabolism. IMPORTANCE In the past decade, various studies have described the effects of microbial volatiles on other (micro)organisms in vitro , but their broad-spectrum activity in vivo and the mechanisms underlying volatile-mediated plant growth promotion have not been addressed in detail. Here, we revealed that volatiles from root-associated bacteria of the genus Microbacterium can enhance the growth of different plant species and can prime plants for growth promotion without direct and prolonged contact between the bacterium and the plant. Collectively, these results provide new opportunities for sustainable agriculture and horticulture by exposing roots of plants only briefly to a specific blend of microbial volatile compounds prior to transplantation of the seedlings to the greenhouse or field. This strategy has no need for large-scale introduction or root colonization and survival of the microbial inoculant., (Copyright © 2018 American Society for Microbiology.)

The green sulfur bacteria ( Chlorobiaceae ) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S 0 , and thiosulfate) as electron donors for photoautotrophic growth. Chlorobaculum tepidum , the model system for the Chlorobiaceae , both produces and consumes extracellular S 0 globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added to Cba. tepidum growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e., the specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA sequencing (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S 0 , and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor-dependent transcriptional regulation. These motifs were conserved across homologous Chlorobiaceae promoters. Two lines of evidence suggest that sulfide-mediated repression is the dominant regulatory mode in Cba. tepidum First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of the Cba. tepidum 1277 ( CT1277 ) gene, encoding a putative regulatory protein, leads to constitutive overexpression of the sulfide:quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism in Cba. tepidum and the Chlorobiaceae Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology in Cba. tepidum and perhaps other Chlorobiaceae IMPORTANCE Elemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacterium Chlorobaculum tepidum either produces or consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics of Chlorobaculum tepidum on elemental sulfur and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This report identifies genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this report paves the way for the development of synthetic biology in Chlorobaculum tepidum and other Chlorobiaceae by providing a comprehensive identification of promoter elements for control of gene expression, a key element of strain engineering., (Copyright © 2018 American Society for Microbiology.)

The traditional view of the dependency of subsurface environments on surface-derived allochthonous carbon inputs is challenged by increasing evidence for the role of lithoautotrophy in aquifer carbon flow. We linked information on autotrophy (Calvin-Benson-Bassham cycle) with that from total microbial community analysis in groundwater at two superimposed-upper and lower-limestone groundwater reservoirs (aquifers). Quantitative PCR revealed that up to 17% of the microbial population had the genetic potential to fix CO2 via the Calvin cycle, with abundances of cbbM and cbbL genes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 × 10(3) to 6 × 10(6) genes liter(-1) over a 2-year period. The structure of the active microbial communities based on 16S rRNA transcripts differed between the two aquifers, with a larger fraction of heterotrophic, facultative anaerobic, soil-related groups in the oxygen-deficient upper aquifer. Most identified CO2-assimilating phylogenetic groups appeared to be involved in the oxidation of sulfur or nitrogen compounds and harbored both RubisCO forms I and II, allowing efficient CO2 fixation in environments with strong oxygen and CO2 fluctuations. The genera Sulfuricella and Nitrosomonas were represented by read fractions of up to 78 and 33%, respectively, within the cbbM and cbbL transcript pool and accounted for 5.6 and 3.8% of 16S rRNA sequence reads, respectively, in the lower aquifer. Our results indicate that a large fraction of bacteria in pristine limestone aquifers has the genetic potential for autotrophic CO2 fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Three succinate coenzyme A (succinate-CoA) ligases (SucCD) from Escherichia coli, Advenella mimigardefordensis DPN7(T), and Alcanivorax borkumensis SK2 were characterized regarding their substrate specificity concerning succinate analogues. Previous studies had suggested that SucCD enzymes might be promiscuous toward succinate analogues, such as itaconate and 3-sulfinopropionate (3SP). The latter is an intermediate of the degradation pathway of 3,3'-dithiodipropionate (DTDP), a precursor for the biotechnical production of polythioesters (PTEs) in bacteria. The sucCD genes were expressed in E. coli BL21(DE3)/pLysS. The SucCD enzymes of E. coli and A. mimigardefordensis DPN7(T) were purified in the native state using stepwise purification protocols, while SucCD from A. borkumensis SK2 was equipped with a C-terminal hexahistidine tag at the SucD subunit. Besides the preference for the physiological substrates succinate, itaconate, ATP, and CoA, high enzyme activity was additionally determined for both enantiomeric forms of malate, amounting to 10 to 21% of the activity with succinate. Km values ranged from 2.5 to 3.6 mM for l-malate and from 3.6 to 4.2 mM for d-malate for the SucCD enzymes investigated in this study. As l-malate-CoA ligase is present in the serine cycle for assimilation of C1 compounds in methylotrophs, structural comparison of these two enzymes as members of the same subsubclass suggested a strong resemblance of SucCD to l-malate-CoA ligase and gave rise to the speculation that malate-CoA ligases and succinate-CoA ligases have the same evolutionary origin. Although enzyme activities were very low for the additional substrates investigated, liquid chromatography/electrospray ionization-mass spectrometry analyses proved the ability of SucCD enzymes to form CoA-thioesters of adipate, glutarate, and fumarate. Since all SucCD enzymes were able to activate 3SP to 3SP-CoA, we consequently demonstrated that the activation of 3SP is not a unique characteristic of the SucCD from A. mimigardefordensis DPN7(T). The essential role of sucCD in the activation of 3SP in vivo was proved by genetic complementation.

Except for several conspicuous cases, very little is known about sulfur oxidizers living in natural freshwater environments. Sulfuricella denitrificans skB26 is a psychrotolerant sulfur oxidizer recently isolated from a freshwater lake as a representative of a new genus in the class Betaproteobacteria. In this study, an approximately 3.2-Mb draft genome sequence of strain skB26 was obtained. In the draft genome, consisting of 23 contigs, a single rRNA operon, 43 tRNA genes, and 3,133 coding sequences were identified. The identified genes include those required for sulfur oxidation, denitrification, and carbon fixation. Comparative proteomic analysis was conducted to assess cold adaptation mechanisms of this organism. From cells grown at 22°C and 5°C, proteins were extracted for analysis by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. In the cells cultured at 5°C, relative abundances of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases were increased in comparison to those at 22°C. These results suggest that maintenance of proper translation is critical for growth under low-temperature conditions, similar to the case for other cold-adapted prokaryotes.

The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-gamma-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade L-methionine as well as other sulfur-containing compounds such as L-cysteine, L-cystathionine, and L-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.

The pseudocolonial coral Lophelia pertusa (Scleractinia, Caryophylliidae) is a eurybathic, stenothermal cosmopolitan cold-water species. It occurs in two color varieties, white and red. L. pertusa builds vast cold-water coral reefs along the continental margins, which are among the most diverse deep-sea habitats. Microbiology of L. pertusa has been in scientific focus for only a few years, but the question of whether the coral holds a host-specific bacterial community has not been finally answered. Bacteria on coral samples from the Trondheimsfjord (Norway) were characterized by the culture-independent 16S rRNA gene-based techniques terminal restriction fragment length polymorphism and sequence analysis. L. pertusa revealed a high microbial richness. Clone sequences were dominated by members of the Alpha- and Gammaproteobacteria. Other abundant taxa were Bacteroidetes, Actinobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes. The bacterial community of L. pertusa not only differed conspicuously from that of the environment but also varied with both the location and color variety of its host. Therefore, the microbial colonization cannot be termed "specific" sensu stricto. However, similarities to other coral-bacterium associations suggest the existence of "cold-water coral-specific" bacterial groups sensu lato. L. pertusa-associated bacteria appear to play a significant role in the nutrition of their host by degradation of sulfur compounds, cellulose, chitin, and end products of the coral's anaerobic metabolism. Some coral-associated microbes were regarded as opportunistic pathogens. Dominance of mixotrophic members of the Rhodobacteraceae in white L. pertusa could explain the wider dispersal of this phenotype by supplementary nutrition.

The hitherto unstudied microbial degradation of the organic disulfide 3,3'-dithiodipropionic acid (DTDP) was investigated with the recently described bacterium Tetrathiobacter mimigardefordensis strain DPN7(T) (DSM 17166(T); LMG 22922(T)), which is able to use DTDP as the sole carbon source for growth. 3-Mercaptopropionic acid (3MP) and 3-sulfinopropionic acid (3SP) were detected in the growth medium and occurred as intermediates during DTDP degradation. To identify genes coding for enzymes of DTDP catabolism, Tn5::mob-induced mutants of T. mimigardefordensis were generated. Screening of transposon mutant libraries yielded many mutants fully or partially impaired in utilizing DTDP as a carbon source. Mapping of the insertion loci in some mutants identified four disrupted open reading frames (ORFs) with putative metabolic functions. The ORFs were assigned function on the basis of homologies with lpdA (EC 1.8.1.4), cdo (EC 1.13.11.20), sucCD (EC 6.2.1.5), and acnB (EC 4.2.1.3). Tn5::mob insertions occurred additionally in the vicinity of heat shock protein-encoding genes. The predicted function of the LpdA homologue in T. mimigardefordensis is cleavage of the disulfide bond of DTDP to form two molecules of 3MP. Cdo catalyzes the conversion of the sulfhydryl group of 3MP, yielding the corresponding sulfinic acid, 3SP. SucCD exhibits thiokinase activity, ligating coenzyme A (CoA) with 3SP to form 3SP-CoA. Afterwards, an elimination of sulfite via a putative desulfinase is expected. acnB encodes a putative 2-methylisocitrate dehydratase. Therefore, a new pathway is proposed for the catabolism of DTDP via 3MP, 3SP, and 3SP-CoA toward propionyl-CoA, which is then further catabolized via the 2-methylcitric acid cycle in T. mimigardefordensis.

Yarrowia lipolytica is one of the yeasts most frequently isolated from the surface of ripened cheeses. In previous work, it has been shown that this yeast is able to convert L-methionine into various volatile sulfur compounds (VSCs) that may contribute to the typical flavors of several cheeses. In the present study, we show that Y. lipolytica does not assimilate lactate in the presence of L-methionine in a cheeselike medium. Nineteen presumptive genes associated with L-methionine catabolism or pyruvate metabolism in Y. lipolytica were transcriptionally studied in relation to L-methionine degradation. The expression levels of the YlARO8 (YALI0E20977g), YlBAT1 (YALI0D01265g), and YlBAT2 (YALI0F19910g) genes (confirmed by real-time PCR experiments) were found to be strongly up-regulated by L-methionine, and a greater variety and larger amounts of VSCs, such as methanethiol and its autooxidation products (dimethyl disulfide and dimethyl trisulfide), were released in the medium when Y. lipolytica was grown in the presence of a high concentration of L-methionine. In contrast, other genes related to pyruvate metabolism were found to be down-regulated in the presence of L-methionine; two exceptions were the YlPDB1 (YALI0E27005g) and YlPDC6 (YALI0D06930g) genes, which encode a pyruvate dehydrogenase and a pyruvate decarboxylase, respectively. Both transcriptional and biochemical results corroborate the view that transamination is the first step of the enzymatic conversion of L-methionine to VSCs in Y. lipolytica and that the YlARO8, YlBAT1, and YlBAT2 genes could play a key role in this process.

Acidithiobacillus caldus has been proposed to play a role in the oxidation of reduced inorganic sulfur compounds (RISCs) produced in industrial biomining of sulfidic minerals. Here, we describe the regulation of a new cluster containing the gene encoding tetrathionate hydrolase (tetH), a key enzyme in the RISC metabolism of this bacterium. The cluster contains five cotranscribed genes, ISac1, rsrR, rsrS, tetH, and doxD, coding for a transposase, a two-component response regulator (RsrR and RsrS), tetrathionate hydrolase, and DoxD, respectively. As shown by quantitative PCR, rsrR, tetH, and doxD are upregulated to different degrees in the presence of tetrathionate. Western blot analysis also indicates upregulation of TetH in the presence of tetrathionate, thiosulfate, and pyrite. The tetH cluster is predicted to have two promoters, both of which are functional in Escherichia coli and one of which was mapped by primer extension. A pyrrolo-quinoline quinone binding domain in TetH was predicted by bioinformatic analysis, and the presence of an o-quinone moiety was experimentally verified, suggesting a mechanism for tetrathionate oxidation.

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.

Biphasic systems can overcome the problem of low productivity in conventional media and have been exploited for biocatalysis. Solvent-tolerant microorganisms are useful in biotransformation with whole cells in biphasic reactions. A solvent-tolerant desulfurizing bacterium, Pseudomonas putida A4, was constructed by introducing the biodesulfurizing gene cluster dszABCD, which was from Rhodococcus erythropolis XP, into the solvent-tolerant strain P. putida Idaho. Biphasic reactions were performed to investigate the desulfurization of various sulfur-containing heterocyclic compounds in the presence of various organic solvents. P. putida A4 had the same substrate range as R. erythropolis XP and could degrade dibenzothiophene at a specific rate of 1.29 mM g (dry weight) of cells(-1) h(-1) for the first 2 h in the presence of 10% (vol/vol) p-xylene. P. putida A4 was also able to degrade dibenzothiophene in the presence of many other organic solvents at a concentration of 10% (vol/vol). This study is a significant step in the exploration of the biotechnological potential of novel biocatalysts for developing an efficient biodesulfurization process in biphasic reaction mixtures containing toxic organic solvents.

Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of L-methionine. L-methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, KlARO8.1 and KlARO8.2, were found to be responsible for L-methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced.

Tracing experiments were carried out to identify volatile and nonvolatile L-methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the L-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and alpha-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of L-methionine catabolism. However, the L-methionine degradation pathways differ in the first steps of L-methionine degradation. L-Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum. Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G.candidum could also be used as precursors for VSC production by B. linens.

The enzymatic degradation of L-methionine and the subsequent formation of volatile sulfur compounds (VSCs) are essential for the development of the typical flavor in cheese. In the yeast Yarrowia lipolytica, the degradation of L-methionine was accompanied by the formation of the transamination product 4-methylthio-2-oxobutyric acid. A branched-chain aminotransferase gene (YlBCA1) of Y. lipolytica was amplified, and the L-methionine-degrading activity and the aminotransferase activity were measured in a genetically modified strain and compared to those of the parental strain. Our work shows that L-methionine degradation via transamination is involved in formation of VSCs in Y. lipolytica.

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.

A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using two-dimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c(552), a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.

By proteomic analysis we found a 21-kDa protein (P21) from Acidithiobacillus ferrooxidans ATCC 19859 whose synthesis was greatly increased by growth of the bacteria in pyrite, thiosulfate, elemental sulfur, CuS, and ZnS and was almost completely repressed by growth in ferrous iron. After we determined the N-terminal amino acid sequence of P21, we used the available preliminary genomic sequence of A. ferrooxidans ATCC 23270 to isolate the DNA region containing the p21 gene. The nucleotide sequence of this DNA fragment contained a putative open reading frame (ORF) coding for a 23-kDa protein. This difference in size was due to the presence of a putative signal peptide in the ORF coding for P21. When p21 was cloned and overexpressed in Escherichia coli, the signal peptide was removed, resulting in a mature protein with a molecular mass of 21 kDa and a calculated isoelectric point of 9.18. P21 exhibited 27% identity and 42% similarity to the Deinococcus radiodurans thiosulfate-sulfur transferase (rhodanese; EC 2.8.1.1) and similar values in relation to other rhodaneses, conserving structural domains and an active site with a cysteine, both characteristic of this family of proteins. However, the purified recombinant P21 protein did not show rhodanese activity. Unlike cytoplasmic rhodaneses, P21 was located in the periphery of A. ferrooxidans cells, as determined by immunocytochemical analysis, and was regulated depending on the oxidizable substrate. The genomic context around gene p21 contained other ORFs corresponding to proteins such as thioredoxins and sulfate-thiosulfate binding proteins, clearly suggesting the involvement of P21 in inorganic sulfur metabolism in A. ferrooxidans.

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC. A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.

Members of a group of marine bacteria that is numerically important in coastal seawater and sediments were characterized with respect to their ability to transform organic and inorganic sulfur compounds. Fifteen strains representing the Roseobacter group (a phylogenetic cluster of marine bacteria in the alpha-subclass of the class Proteobacteria) were isolated from seawater, primarily from the southeastern United States. Although more than one-half of the isolates were obtained without any selection for sulfur metabolism, all of the isolates were able to degrade the sulfur-containing osmolyte dimethyl sulfoniopropionate (DMSP) with production of dimethyl sulfide (DMS). Five isolates also degraded DMSP with production of methanethiol, indicating that both cleavage and demethylation pathways for DMSP occurred in the same organism, which is unusual. Five isolates were able to reduce dimethyl sulfoxide to DMS, and several isolates also degraded DMS and methanethiol. Sulfite oxygenase activity and methanesulfonic acid oxygenase activity were also present in some of the isolates. The ability to incorporate the reduced sulfur in DMSP and methanethiol into cellular material was studied with one of the isolates. A group-specific 16S rRNA probe indicated that the relative abundance of uncultured bacteria in the Roseobacter group increased in seawater enriched with DMSP or DMS. Because this group typically accounts for >10% of the 16S ribosomal DNA pool in coastal seawater and sediments of the southern United States, clues about its potential biogeochemical role are of particular interest. Studies of culturable representatives suggested that the group could mediate a number of steps in the cycling of both organic and inorganic forms of sulfur in marine environments.