Overview of physiological, biochemical, and regulatory aspects of nitrogen fixation in Azotobacter vinelandii.

Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged scientists for more than a century. Such an understanding is a key aspect toward accomplishing the transfer of the genetic determinants of biological nitrogen fixation to crop plants as well as for the development of improved synthetic catalysts based on the biological mechanism. Over the past 30 years, the free-living nitrogen-fixing bacterium Azotobacter vinelandii emerged as a preferred model organism for mechanistic, structural, genetic, and physiological studies aimed at understanding biological nitrogen fixation. This review provides a contemporary overview of these studies and places them within the context of their historical development.

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNAGlu, Gln, Asp contains a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm5s2U to mnm5s2U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Sulfur Availability Impacts Accumulation of the 2-Thiouridine tRNA Modification in Bacillus subtilis.

Posttranscriptional modifications to tRNA are critical elements for the folding and functionality of these adaptor molecules. Sulfur modifications in tRNA are installed by specialized enzymes that act on cognate tRNA substrates at specific locations. Most studied organisms contain a general cysteine desulfurase to mobilize sulfur for the synthesis of S-tRNA and other thio-cofactors. Bacillus subtilis and other Gram-positive bacteria encode multiple cysteine desulfurases that partner with specific sulfur acceptors in the biosynthesis of thio-cofactors. This metabolic layout suggests an alternate mode of regulation in these biosynthetic pathways. In this study, tRNA modifications were exploited as a readout for the functionality of pathways involving cysteine desulfurases. These analyses showed that the relative abundance of 2-thiouridine-modified tRNA (s2U) responds to sulfur availability in the growth medium in a dose-dependent manner. This study found that low sulfur concentrations lead to decreased levels of the s2U cysteine desulfurase YrvO and thiouridylase MnmA, without altering the levels of other cysteine desulfurases, SufS, NifS, and NifZ. Analysis of pathway metabolites that depend on the activity of cysteine desulfurases indicates that sulfur nutrient availability specifically impacts s2U accumulation while having no effect on the levels of other S-modified tRNA or activity levels of Fe-S enzymes. Collectively, these results support a model in which s2U tRNA serves as a marker for sulfur availability in B. subtilis. IMPORTANCE The 2-thiouridine (s2U) tRNA modification is found ubiquitously across all domains of life. YrvO and MnmA, the enzymes involved in this modification, are essential in B. subtilis, confirming the well-established role of s2U in maintaining translational efficiency and, consequently, cellular viability. Herein, we show that in the model Gram-positive organism Bacillus subtilis, the levels of s2U are responsive to sulfur availability. Downregulation of the s2U biosynthetic components leads to lower s2U levels, which may serve as a signal for the slowing of the translational apparatus during cellular nutrient insufficiency. Our findings provide the basis for the identification of a potential bacterial mode of regulation during S-metabolite depletion that may use s2U as a marker of suboptimal metabolic status.

Distribution of Nitrogen-Fixation Genes in Prokaryotes Containing Alternative Nitrogenases.

Biological nitrogen fixation is an inherent trait exclusive to a select number of prokaryotes. Although molybdenum nitrogenase is the dominant catalyst for dinitrogen reduction, some diazotrophs also contain one or two additional types of nitrogenase that use alternative metal content as the active-site cofactor. The occurrence of alternative nitrogenases has not been well studied due to the discriminatory expression of the molybdenum nitrogenase and lack of comprehensive genomic data. This study reports on the genomic analysis of 87 unique species containing alternative nitrogenase sequences. The distribution of nitrogen-fixing genes within these species from distinct taxonomic groups shows the presence of the minimum gene set required for nitrogen fixation, including catalytic and biosynthetic enzymes of the Mo-dependent system (NifHDKENB) and the varying occurrence of additional Nif-dedicated components. These include NifS and NifU, found primarily in aerobic species, thus suggesting that these genes are necessary to accommodate the high demand for Fe-S clusters during aerobic nitrogen fixation.

The Thioredoxin System Reduces Protein Persulfide Intermediates Formed during the Synthesis of Thio-Cofactors in Bacillus subtilis.

The biosynthesis of Fe-S clusters and other thio-cofactors requires the participation of redox agents. A shared feature in these pathways is the formation of transient protein persulfides, which are susceptible to reduction by artificial reducing agents commonly used in reactions in vitro. These agents modulate the reactivity and catalytic efficiency of biosynthetic reactions and, in some cases, skew the enzymes' kinetic behavior, bypassing sulfur acceptors known to be critical for the functionality of these pathways in vivo. Here, we provide kinetic evidence for the selective reactivity of the Bacillus subtilis Trx (thioredoxin) system toward protein-bound persulfide intermediates. Our results demonstrate that the redox flux of the Trx system modulates the rate of sulfide production in cysteine desulfurase assays. Likewise, the activity of the Trx system is dependent on the rate of persulfide formation, suggesting the occurrence of coupled reaction schemes between both enzymatic systems in vitro. Inactivation of TrxA (thioredoxin) or TrxR (thioredoxin reductase) impairs the activity of Fe-S enzymes in B. subtilis, indicating the involvement of the Trx system in Fe-S cluster metabolism. Surprisingly, biochemical characterization of TrxA reveals that this enzyme is able to coordinate Fe-S species, resulting in the loss of its reductase activity. The inactivation of TrxA through the coordination of a labile cluster, combined with its proposed role as a physiological reducing agent in sulfur transfer pathways, suggests a model for redox regulation. These findings provide a potential link between redox regulation and Fe-S metabolism.

Metallocluster transactions: dynamic protein interactions guide the biosynthesis of Fe-S clusters in bacteria.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors present in all domains of life. The chemistries catalyzed by these inorganic cofactors are diverse and their associated enzymes are involved in many cellular processes. Despite the wide range of structures reported for Fe-S clusters inserted into proteins, the biological synthesis of all Fe-S clusters starts with the assembly of simple units of 2Fe-2S and 4Fe-4S clusters. Several systems have been associated with the formation of Fe-S clusters in bacteria with varying phylogenetic origins and number of biosynthetic and regulatory components. All systems, however, construct Fe-S clusters through a similar biosynthetic scheme involving three main steps: (1) sulfur activation by a cysteine desulfurase, (2) cluster assembly by a scaffold protein, and (3) guided delivery of Fe-S units to either final acceptors or biosynthetic enzymes involved in the formation of complex metalloclusters. Another unifying feature on the biological formation of Fe-S clusters in bacteria is that these systems are tightly regulated by a network of protein interactions. Thus, the formation of transient protein complexes among biosynthetic components allows for the direct transfer of reactive sulfur and Fe-S intermediates preventing oxygen damage and reactions with non-physiological targets. Recent studies revealed the importance of reciprocal signature sequence motifs that enable specific protein-protein interactions and consequently guide the transactions between physiological donors and acceptors. Such findings provide insights into strategies used by bacteria to regulate the flow of reactive intermediates and provide protein barcodes to uncover yet-unidentified cellular components involved in Fe-S metabolism.

Effects of supervised exercise and dietary nitrate in older adults with controlled hypertension and/or heart failure with preserved ejection fraction.

Aerobic exercise training is an effective therapy to improve peak aerobic power (peak VO2) in individuals with hypertension (HTN, AHA/ACC class A) and heart failure patients with preserved ejection fraction (HFpEF). High nitrate containing beetroot juice (BRJ) also improves sub-maximal endurance and decreases blood pressure in both HTN and HFpEF. We hypothesized that combining an aerobic exercise and dietary nitrate intervention would result in additive or even synergistic positive effects on exercise tolerance and blood pressure in HTN or HFpEF. We report results from two pilot studies examining the effects of supervised aerobic exercise combined with dietary nitrate in patients with controlled HTN (n = 26, average age 65 ± 5 years) and in patients with HFpEF (n = 20, average age 69 ± 7 years). All patients underwent an aerobic exercise training regimen; half were randomly assigned to consume a high nitrate-containing beet juice beverage (BRJ containing 6.1 mmol nitrate for the HFpEF study consumed three times a week and 8 mmol nitrate for the HTN study consumed daily) while the other half consumed a beet juice beverage with the nitrate removed (placebo). The main result was that there was no added benefit observed for any outcomes when comparing BRJ to placebo in either HTN or HFpEF patients undergoing exercise training (p ≥ 0.14). There were within-group benefits. In the pilot study in patients with HFpEF, aerobic endurance (primary outcome), defined as the exercise time to volitional exhaustion during submaximal cycling at 75% of maximal power output, improved during exercise training within each group from baseline to end of study, 369 ± 149 s vs 520 ± 257 s (p = 0.04) for the placebo group and 384 ± 129 s vs 483 ± 258 s for the BRJ group (p = 0.15). Resting systolic blood pressure in patients with HFpEF also improved during exercise training in both groups, 136 ± 16 mm Hg vs 122 ± 3 mm Hg for the placebo group (p < 0.05) and 132 ± 12 mm Hg vs 119 ± 9 mm Hg for the BRJ group (p < 0.05). In the HTN pilot study, during a treadmill graded exercise test, peak oxygen consumption (primary outcome) did not change significantly, but time to exhaustion (also a primary outcome) improved in both groups, 504 ± 32 s vs 601 ± 38 s (p < 0.05) for the placebo group and 690 ± 38 s vs 772 ± 95 s for the BRJ group (p < 0.05) which was associated with a reduction in supine resting systolic blood pressure in BRJ group. Arterial compliance also improved during aerobic exercise training in both the HFpEF and the HTN patients for both BRJ and placebo groups. Future work is needed to determine if larger nitrate doses would provide an added benefit to supervised aerobic exercise in HTN and HFpEF patients.

Shared-intermediates in the biosynthesis of thio-cofactors: Mechanism and functions of cysteine desulfurases and sulfur acceptors.

Cysteine desulfurases utilize a PLP-dependent mechanism to catalyze the first step of sulfur mobilization in the biosynthesis of sulfur-containing cofactors. Sulfur activation and integration into thiocofactors involve complex mechanisms and intricate biosynthetic schemes. Cysteine desulfurases catalyze sulfur-transfer reactions from l-cysteine to sulfur acceptor molecules participating in the biosynthesis of thio-cofactors, including Fe-S clusters, thionucleosides, thiamin, biotin, and molybdenum cofactor. The proposed mechanism of cysteine desulfurases involves the PLP-dependent cleavage of the C-S bond from l-cysteine via the formation of a persulfide enzyme intermediate, which is considered the hallmark step in sulfur mobilization. The subsequent sulfur transfer reaction varies with the class of cysteine desulfurase and sulfur acceptor. IscS serves as a mecca for sulfur incorporation into a network of intertwined pathways for the biosynthesis of thio-cofactors. The involvement of a single enzyme interacting with multiple acceptors, the recruitment of shared-intermediates partaking roles in multiple pathways, and the participation of Fe-S enzymes denote the interconnectivity of pathways involving sulfur trafficking. In Bacillus subtilis, the occurrence of multiple cysteine desulfurases partnering with dedicated sulfur acceptors partially deconvolutes the routes of sulfur trafficking and assigns specific roles for these enzymes. Understanding the roles of promiscuous vs. dedicated cysteine desulfurases and their partnership with shared-intermediates in the biosynthesis of thio-cofactors will help to map sulfur transfer events across interconnected pathways and to provide insight into the hierarchy of sulfur incorporation into biomolecules. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis.

UNLABELLED: The 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s(2)U in Escherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). The E. coli MnmA adenylates and subsequently thiolates tRNA to form the s(2)U modification. Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene, yrvO, directly adjacent to mnmA. The genomic synteny of yrvO and mnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s(2)U formation. Here, we provide evidence that the B. subtilis YrvO and MnmA are sufficient for s(2)U biosynthesis. A conditional B. subtilis knockout strain showed that s(2)U abundance correlates with MnmA expression, and in vivo complementation studies in E. coli IscS- or MnmA-deficient strains revealed the competency of these proteins in s(2)U biosynthesis. In vitro experiments demonstrated s(2)U formation by YrvO and MnmA, and kinetic analysis established a partnership between the B. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype of E. coli ΔiscS and ΔmnmA strains associated with s(2)U depletion is recovered by B. subtilis yrvO and mnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s(2)U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA. IMPORTANCE: The 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s(2)U in Escherichia coli requires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE). Bacillus subtilis and most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, the mnmA coding sequence is located adjacent to yrvO, encoding a cysteine desulfurase. In this work, we provide evidence that the B. subtilis YrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s(2)U biosynthesis in a pathway independent of the one used in E. coli.

The relationship between plasma and salivary NOx.

Several studies have shown that fasting plasma nitrite (NO2(-)) is an indicator of endothelial nitric oxide synthase (NOS) activity while plasma nitrate (NO3(-)) or the sum of NO2(-) and NO3(-) (NOx) does not reflect NOS function. Plasma NO2(-) can also be elevated through dietary NO3(-) where the NO3(-) is partially reduced to NO2(-) by oral bacteria and enters the plasma through the digestive system. NO3(-) is taken up from plasma by salivary glands and the cycle repeats itself. Thus, one may propose that salivary NO2(-) is an indicator of plasma NO2(-) and consequently of NO production. Many brands of nitric oxide (NO) saliva test strips have been developed that suggest that their product is indicative of circulatory NO availability. However, data supporting a relationship between salivary and plasma NO2(-) or NO bioavailability are lacking. Here we have measured basal salivary and plasma NO2(-) and NO3(-) to determine if any correlation exists between these in 13 adult volunteers. We found no significant correlation between basal salivary and plasma NO2(-). Also no correlation exists between salivary NO3(-) and plasma NO2(-). However, we did see a correlation between salivary NO3(-) and plasma NO3(-), and between salivary NO2(-) and plasma NO3(-). In a separate study, we compared the efficiency of salivary NO3(-) reduction with the efficacy of increasing plasma NO3(-) and NO2(-) after drinking beet juice, a high NO3(-)-containing beverage, in 10 adult volunteers. No significant correlation was observed between the ex vivo salivary reduction of NO3(-) to NO2(-) and plasma increases in NO3(-) or NO2(-). These results suggest that measures of salivary NO3(-), NO2(-) or NOx are not good indicators of endothelial function. In addition, the efficiency of saliva to reduce NO3(-) to NO2(-)ex-vivo does not demonstrate one's ability to increase plasma NO2(-) following consumption of dietary NO3(-).

Fe-S cluster biogenesis in Gram-positive bacteria: SufU is a zinc-dependent sulfur transfer protein.

The biosynthesis of Fe-S clusters in Bacillus subtilis and other Gram-positive bacteria is catalyzed by the SufCDSUB system. The first step in this pathway involves the sulfur mobilization from the free amino acid cysteine to a sulfur acceptor protein SufU via a PLP-dependent cysteine desulfurase SufS. In this reaction scheme, the formation of an enzyme S-covalent intermediate is followed by the binding of SufU. This event leads to the second half of the reaction where a deprotonated thiol of SufU promotes the nucleophilic attack onto the persulfide intermediate of SufS. Kinetic analysis combined with spectroscopic methods identified that the presence of a zinc atom tightly bound to SufU (Ka = 10(17) M(-1)) is crucial for its structural and catalytic competency. Fe-S cluster assembly experiments showed that despite the high degree of sequence and structural similarity to the ortholog enzyme IscU, the B. subtilis SufU does not act as a standard Fe-S cluster scaffold protein. The involvement of SufU as a dedicated agent of sulfur transfer, rather than as an assembly scaffold, in the biogenesis of Fe-S clusters in Gram-positive microbes indicates distinct strategies used by bacterial systems to assemble Fe-S clusters.

Cross-functionalities of Bacillus deacetylases involved in bacillithiol biosynthesis and bacillithiol-S-conjugate detoxification pathways.

BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N-acetylglucosamine malate to generate glucosamine malate. In Bacillus anthracis, BA1557 has been identified as the N-acetylglucosamine malate deacetylase (BshB); however, a high content of bacillithiol (~70%) was still observed in the B. anthracis ∆BA1557 strain. Genomic analysis led to the proposal that another deacetylase could exhibit cross-functionality in bacillithiol biosynthesis. In the present study, BA1557, its paralogue BA3888 and orthologous Bacillus cereus enzymes BC1534 and BC3461 have been characterized for their deacetylase activity towards N-acetylglucosamine malate, thus providing biochemical evidence for this proposal. In addition, the involvement of deacetylase enzymes is also expected in bacillithiol-detoxifying pathways through formation of S-mercapturic adducts. The kinetic analysis of bacillithiol-S-bimane conjugate favours the involvement of BA3888 as the B. anthracis bacillithiol-S-conjugate amidase (Bca). The high degree of specificity of this group of enzymes for its physiological substrate, along with their similar pH-activity profile and Zn²âº-dependent catalytic acid-base reaction provides further evidence for their cross-functionalities.

Protected sulfur transfer reactions by the Escherichia coli Suf system.

The first step in sulfur mobilization for the biosynthesis of Fe-S clusters under oxidative stress and iron starvation in Escherichia coli involves a cysteine desulfurase SufS. Its catalytic reactivity is dependent on the presence of a sulfur acceptor protein, SufE, which acts as the preferred substrate for this enzyme. Kinetic analysis of the cysteine:SufE sulfurtransferase reaction of the E. coli SufS that is partially protected from reducing agents, such as dithiothreitol and glutathione, was conducted. Under these conditions, the reaction displays a biphasic profile in which the first phase involves a fast sulfur transfer reaction from SufS to SufE. The accumulation of persulfurated/polysulfurated forms of SufE accounts for a second phase of the slow catalytic turnover rate. The presence of the SufBCD complex enhances the activity associated with the second phase, while modestly inhibiting the activity associated with the initial sulfur transfer from SufS to SufE. Thus, the rate of sulfur transfer from SufS to the final proposed SufBCD Fe-S cluster scaffold appears to be dependent on the availability of the final sulfur acceptor. The use of a stronger reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] elicited the maximal activity of the SufS-SufE reaction and surpassed the stimulatory effect of SufBCD. This concerted sulfur trafficking path involving sequential transfer from SufS to SufE to SufBCD guarantees the protection of intermediates at a controlled flux to meet cellular demands encountered under conditions detrimental to thiol chemistry and Fe-S cluster metabolism.

Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide.

Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.

Sulfinamide Formation from the Reaction of Bacillithiol and Nitroxyl.

Bacillithiol (BSH) replaces glutathione (GSH) as the most prominent low-molecular-weight thiol in many low G + C gram-positive bacteria. BSH plays roles in metal binding, protein/enzyme regulation, detoxification, redox buffering, and bacterial virulence. Given the small amounts of BSH isolated from natural sources and relatively lengthy chemical syntheses, the reactions of BSH with pertinent reactive oxygen, nitrogen, and sulfur species remain largely unexplored. We prepared BSH and exposed it to nitroxyl (HNO), a reactive nitrogen species that influences bacterial sulfur metabolism. The profile of this reaction was distinct from HNO oxidation of GSH, which yielded mixtures of disulfide and sulfinamide. The reaction of BSH and HNO (generated from Angeli's salt) gives only sulfinamide products, including a newly proposed cyclic sulfinamide. Treatment of a glucosamine-cysteine conjugate, which lacks the malic acid group, with HNO forms disulfide, implicating the malic acid group in sulfinamide formation. This finding supports a mechanism involving the formation of an N-hydroxysulfenamide intermediate that dehydrates to a sulfenium ion that can be trapped by water or internally trapped by an amide nitrogen to give the cyclic sulfinamide. The biological relevance of BSH reactivity toward HNO is provided through in vivo experiments demonstrating that Bacillus subtilis exposed to HNO shows a growth phenotype, and a strain unable to produce BSH shows hypersensitivity toward HNO in minimal medium cultures. Thiol analysis of HNO-exposed cultures shows an overall decrease in reduced BSH levels, which is not accompanied by increased levels of BSSB, supporting a model involving the formation of an oxidized sulfinamide derivative, identified in vivo by high-pressure liquid chromatography/mass spectrometry. Collectively, these findings reveal the unique chemistry and biology of HNO with BSH in bacteria that produce this biothiol.

Alternate routes to mnm 5 s 2 U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In Bacteria tRNA Glu, Gln, Asp contain a variety of xnm 5 s 2 U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm 5 s 2 U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the installation of this modification. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the Radical Sam superfamily was found to be involved in the synthesis of mnm 5 s 2 U in both Bacillus subtilis and Streptococcus mutans . This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm 5 s 2 U into mnm 5 s 2 U in B. subtilis . Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathways intermediates in regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. The occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in nature. Importance: The xnm 5 s 2 U modifications found in several tRNAs at the wobble base position are widespread in Bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile Radical SAM superfamily and is involved in the synthesis of mnm 5 s 2 U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA.

ThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the tRNA thionucleoside modification, 4-thiouridine. In Escherichia coli and Salmonella enterica, ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS and transferring it to the target molecule or additional sulfur carrier proteins. However, in Bacillus subtilis and most species from the Firmicutes phylum, ThiI lacks the rhodanese domain that contains the site responsible for the sulfurtransferase activity. The lack of the gene encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in both biosynthetic pathways. Here, we have carried out functional analysis of B. subtilis thiI and the adjacent gene, nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments in B. subtilis indicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not thiamine. In vitro synthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments, suggests the occurrence of an alternate transient site for sulfur transfer, thus obviating the need for a rhodanese domain. In vivo complementation studies in E. coli IscS- or ThiI-deficient strains provide further support for specific interactions between NifZ and ThiI. These results are compatible with the proposal that B. subtilis NifZ and ThiI utilize mechanistically distinct and mutually specific sulfur transfer reactions.

Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes.

BACKGROUND: The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. RESULTS: Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. CONCLUSIONS: Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.

Co-ordination and fine-tuning of nitrogen fixation in Azotobacter vinelandii.

Nitrogen fixation by the free-living organism Azotobacter vinelandii can occur through the activity of three different systems that are genetically distinct but mechanistically related. A combination of bioinformatic and biochemical-genetic studies has revealed that at least 82 different genes are likely to be associated with the formation and regulation of these systems. Studies performed over many years have established that cross-talk occurs between the various nitrogen fixation systems, and that expression and fine-tuning of their activities are integrated with overall cellular physiology. Martinez-Noel and co-workers now report another newly discovered aspect of the process. Evidence is presented to suggest that a nitrogen fixation-specific paralogue of ClpX is used to control the accumulation of proteins involved in formation of a metal-sulphur cluster that provides a nitrogenase active site. The intriguing aspect of this work is that it indicates that the nitrogen fixation-associated ClpX must recruit ClpP, for which a paralogue is not duplicated within any of the nitrogen fixation regions of the genome, to achieve its function related to nitrogen fixation. Inspection of the A. vinelandii genome indicates that such recruitment of cellular housekeeping components is a common feature used to integrate nitrogen fixation with global cellular physiology.