Nitrite reductase activity in F420-dependent sulphite reductase (Fsr) from Methanocaldococcus jannaschii.

Methanocaldococcus jannaschii (Mj), a hyperthermophilic and evolutionarily deeply rooted methanogenic archaeon from a deep-sea hydrothermal vent, produces F420-dependent sulphite reductase (Fsr) in response to exposure to sulphite. This enzyme allows Mj to detoxify sulphite, a potent inhibitor of methyl coenzyme-M reductase (Mcr), by reducing it to sulphide with reduced coenzyme F420 (F420H2) as an electron donor; Mcr is essential for energy production for a methanogen. Fsr allows Mj to utilize sulphite as a sulphur source. Nitrite is another potent inhibitor of Mcr and is toxic to methanogens. It is reduced by most sulphite reductases. In this study, we report that MjFsr reduced nitrite to ammonia with F420H2 with physiologically relevant K m values (nitrite, 8.9 µM; F420H2, 9.7 µM). The enzyme also reduced hydroxylamine with a K m value of 112.4 µM, indicating that it was an intermediate in the reduction of nitrite to ammonia. These results open the possibility that Mj could use nitrite as a nitrogen source if it is provided at a low concentration of the type that occurs in its habitat.

A Reduced F420-Dependent Nitrite Reductase in an Anaerobic Methanotrophic Archaeon.

Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme F420-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a hyperthermophilic methanogen from deep-sea hydrothermal vents. M. jannaschii Fsr (MjFsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. MjFsrI reduces sulfite to sulfide with reduced F420 (F420H2), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F420H2. Instead, it acted as an F420H2-dependent nitrite reductase (FNiR) with physiologically relevant Km values (nitrite, 5 µM; F420H2, 14 µM). From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F420H2-derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite (E0' [standard potential], +440 mV) but not sulfite (E0', -116 mV). These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies. IMPORTANCE Coenzyme F420-dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade. We show that a member of this clade from ANME-2c functions as F420-dependent nitrite reductase (FNiR) and lacks Fsr activity. This specialization arose from a distinct feature of the reductant processing system and not the substrate recognition element. We hypothesize FNiR may protect ANME Mcr from inactivation by nitrite. This is an example of functional specialization within a protein family that is induced by changes in electron transfer modules to fit an ecological need.

Coenzyme F420-Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F420 in Mycobacteria.

Coenzyme F420 plays a key role in the redox metabolisms of various archaea and bacteria, including Mycobacterium tuberculosis In M. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420 carries multiple glutamate residues in the side chain, forming F420-n species (n, number of glutamate residues), and the length of this side chain impacts cellular physiology. M. tuberculosis strains with F420 species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420 is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420 However, purified FbiB of M. tuberculosis (MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that, in vitro, MtbFbiB synthesized side chains containing up to seven glutamate residues if F420 was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis in Mycobacterium bovis BCG and Mycobacterium smegmatis and an analysis of literature data on M. tuberculosis revealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420 to F420H2 We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2 modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate.IMPORTANCE Coenzyme F420-dependent reactions of Mycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism of M. tuberculosis Mutations that eliminate the production of F420 with longer tails make M. tuberculosis resistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420 requires concerted actions of two enzymes, one of which reduces the coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.

Proline utilization system is required for infection by the pathogenic α-proteobacterium Brucella abortus.

Proline utilization (Put) systems have been described in a number of bacteria; however, the importance and functionality of the Put system in the intracellular pathogen Brucellaabortus has not been explored. Generally, bacterial Put systems are composed of the bifunctional enzyme proline dehydrogenase PutA and its transcriptional activator PutR. Here, we demonstrate that the genes putA (bab2_0518) and putR (bab2_0517) are critical for the chronic infection of mice by B. abortus, but putA and putR are not required for the survival and replication of the bacteria in naive macrophages. Additionally, in vitro experiments revealed that putR is necessary for the ability of the bacteria to withstand oxidative stress, as a ΔputR deletion strain is hypersensitive to hydrogen peroxide exposure. Quantitative reverse transcription-PCR and putA-lacZ transcriptional reporter studies revealed that PutR acts as a transcriptional activator of putA in Brucella, and electrophoretic mobility shift assays confirmed that PutR binds directly to the putA promoter region. Biochemical analyses demonstrated that a purified recombinant B. abortus PutA protein possesses quintessential proline dehydrogenase activity, as PutA is capable of catalysing the conversion of proline to glutamate. Altogether, these data are the first to reveal that the Put system plays a significant role in the ability of B. abortus to replicate and survive within its host, as well as to describe the genetic regulation and biochemical activity of the Put system in Brucella.

Complete Genome Sequence of Bordetella pertussis Pelita III, the Production Strain for an Indonesian Whole-Cell Pertussis Vaccine.

PT Bio Farma, the sole World Health Organization-approved Indonesian vaccine producer, manufactures a whole-cell whooping cough vaccine (wP) that, as part of a pentavalent diphtheria-tetanus-pertussis/hepatitis B/Haemophilus influenzae b (DTP/HB/Hib) vaccine, is used in Indonesia and many other countries. We report here the whole-genome sequence for Bordetella pertussis Pelita III, PT Bio Farma's wP production strain.

F420H2 Is Required for Phthiocerol Dimycocerosate Synthesis in Mycobacteria.

UNLABELLED: Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria. IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.

Lack of mycothiol and ergothioneine induces different protective mechanisms in Mycobacterium smegmatis.

Mycobacterium smegmatis contains the low molecular weight thiols, mycothiol (MSH) and ergothioneine (ESH). Examination of transposon mutants disrupted in mshC and egtA, involved in the biosynthesis of MSH and ESH respectively, demonstrated that both mutants were sensitive to oxidative, alkylating, and metal stress. However, the mshC mutant exhibited significantly more protein carbonylation and lipid peroxidation than wildtype, while the egtA mutant had less protein and lipid damage than wildtype. We further show that Ohr, KatN, and AhpC, involved in protection against oxidative stress, are upregulated in the egtA mutant. In the mshC mutant, an Usp and a putative thiol peroxidase are upregulated. In addition, mutants lacking MSH also contained higher levels of Coenzyme F420 as compared to wildtype and two Coenzyme F420 dependent enzymes were found to be upregulated. These results indicate that lack of MSH and ESH result in induction of different mechanisms for protecting against oxidative stress.

Genetic resources for methane production from biomass described with the Gene Ontology.

Methane (CH4) is a valuable fuel, constituting 70-95% of natural gas, and a potent greenhouse gas. Release of CH4 into the atmosphere contributes to climate change. Biological CH4 production or methanogenesis is mostly performed by methanogens, a group of strictly anaerobic archaea. The direct substrates for methanogenesis are H2 plus CO2, acetate, formate, methylamines, methanol, methyl sulfides, and ethanol or a secondary alcohol plus CO2. In numerous anaerobic niches in nature, methanogenesis facilitates mineralization of complex biopolymers such as carbohydrates, lipids and proteins generated by primary producers. Thus, methanogens are critical players in the global carbon cycle. The same process is used in anaerobic treatment of municipal, industrial and agricultural wastes, reducing the biological pollutants in the wastes and generating methane. It also holds potential for commercial production of natural gas from renewable resources. This process operates in digestive systems of many animals, including cattle, and humans. In contrast, in deep-sea hydrothermal vents methanogenesis is a primary production process, allowing chemosynthesis of biomaterials from H2 plus CO2. In this report we present Gene Ontology (GO) terms that can be used to describe processes, functions and cellular components involved in methanogenic biodegradation and biosynthesis of specialized coenzymes that methanogens use. Some of these GO terms were previously available and the rest were generated in our Microbial Energy Gene Ontology (MENGO) project. A recently discovered non-canonical CH4 production process is also described. We have performed manual GO annotation of selected methanogenesis genes, based on experimental evidence, providing "gold standards" for machine annotation and automated discovery of methanogenesis genes or systems in diverse genomes. Most of the GO-related information presented in this report is available at the MENGO website (http://www.mengo.biochem.vt.edu/).

Genetic resources for advanced biofuel production described with the Gene Ontology.

Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary. The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology () project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. Here we review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.

Rv0132c of Mycobacterium tuberculosis encodes a coenzyme F420-dependent hydroxymycolic acid dehydrogenase.

The ability of Mycobacterium tuberculosis to manipulate and evade human immune system is in part due to its extraordinarily complex cell wall. One of the key components of this cell wall is a family of lipids called mycolic acids. Oxygenation of mycolic acids generating methoxy- and ketomycolic acids enhances the pathogenic attributes of M. tuberculosis. Thus, the respective enzymes are of interest in the research on mycobacteria. The generation of methoxy- and ketomycolic acids proceeds through intermediary formation of hydroxymycolic acids. While the methyl transferase that generates methoxymycolic acids from hydroxymycolic acids is known, hydroxymycolic acids dehydrogenase that oxidizes hydroxymycolic acids to ketomycolic acids has been elusive. We found that hydroxymycolic acid dehydrogenase is encoded by the rv0132c gene and the enzyme utilizes F420, a deazaflavin coenzyme, as electron carrier, and accordingly we called it F420-dependent hydroxymycolic acid dehydrogenase. This is the first report on the involvement of F420 in the synthesis of a mycobacterial cell envelope. Also, F420-dependent hydroxymycolic acid dehydrogenase was inhibited by PA-824, and therefore, it is a previously unknown target for this new tuberculosis drug.

Glucose 6-phosphate accumulation in mycobacteria: implications for a novel F420-dependent anti-oxidant defense system.

Glucose 6-phosphate (G6P) is a metabolic intermediate with many possible cellular fates. In mycobacteria, G6P is a substrate for an enzyme, F(420)-dependent glucose-6-phosphate dehydrogenase (Fgd), found in few bacterial genera. Intracellular G6P levels in six Mycobacterium sp. were remarkably higher ( approximately 17-130-fold) than Escherichia coli and Bacillus megaterium. The high G6P level in Mycobacterium smegmatis may result from 10-25-fold higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase when grown on glucose, glycerol, or acetate compared with B. megaterium and E. coli. In M. smegmatis this coincided with up-regulation of the first gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, when acetate was the carbon source, suggesting a cellular program for maintaining high G6P levels. G6P was depleted in cells under oxidative stress induced by redox cycling agents plumbagin and menadione, whereas an fgd mutant of M. smegmatis used G6P less well under such conditions. The fgd mutant was more sensitive to these agents and, in contrast to wild type, was defective in its ability to reduce extracellular plumbagin and menadione. These data suggest that intracellular G6P in mycobacteria serves as a source of reducing power and, with the mycobacteria-specific Fgd-F(420) system, plays a protective role against oxidative stress.

Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage.

In mycobacteria, F(420), a deazaflavin derivative, acts as a hydride transfer coenzyme for an F(420)-specific glucose-6-phosphate dehydrogenase (Fgd). Physiologically relevant reactions in the mycobacteria that use Fgd-generated reduced F(420) (F(420)H(2)) are unknown. In this work, F(420)H(2) was found to be oxidized by NO only in the presence of oxygen. Further analysis demonstrated that NO(2), produced from NO and O(2), was the oxidant. UV-visible spectroscopic and NO-sensor-based analyses proved that F(420)H(2) reduced NO(2) to NO. This reaction could serve as a defense system for Mycobacterium tuberculosis, which is more sensitive to NO(2) than NO under aerobic conditions. Activated macrophages produce NO, which in acidified phagosomes is converted to NO(2). Hence, by converting NO(2) back to NO with F(420)H(2), M. tuberculosis could decrease the effectiveness of antibacterial action of macrophages; such defense would correspond to active tuberculosis conditions where the bacterium grows aerobically. This hypothesis was consistent with the observation that a mutant strain of Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, which either did not produce or could not reduce F(420), was approximately 4-fold more sensitive to NO(2) than the wild-type strain. The phenomenon is reminiscent of the anticancer activity of gamma-tocopherol, which reduces NO(2) to NO and protects human cells from NO(2)-induced carcinogenesis.

Brucella abortus strain RB51 leucine auxotroph as an environmentally safe vaccine for plasmid maintenance and antigen overexpression.

To avoid potentiating the spread of an antibiotic resistance marker, a plasmid expressing a leuB gene and a heterologous antigen, green fluorescent protein (GFP), was shown to complement a leucine auxotroph of cattle vaccine strain Brucella abortus RB51, which protected CD1 mice from virulent B. abortus 2308 and elicited GFP antibodies.

Identification of pyruvate carboxylase genes in Pseudomonas aeruginosa PAO1 and development of a P. aeruginosa-based overexpression system for alpha4- and alpha4beta4-type pyruvate carboxylases.

Pyruvate carboxylase (PYC) is an ecologically, medically, and industrially important enzyme. It is widespread in all three domains of life, the archaea, bacteria, and eukarya. PYC catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate. Detailed structure-function studies of this enzyme have been hampered due to the unavailability of a facile recombinant overexpression system. Except for the alpha4 enzyme from a thermophilic Bacillus species, Escherichia coli has been unsuitable for overexpression of PYCs. We show that a Pseudomonas aeruginosa strain carrying the T7 polymerase gene can serve as a host for the overexpression of Mycobacterium smegmatis alpha4 PYC and Pseudomonas aeruginosa alpha4beta4 PYC under the control of the T7 promoter from a broad-host-range conjugative plasmid. Overexpression occurred both in aerobic (LB medium) and nitrate-respiring anaerobic (LB medium plus glucose and nitrate) cultures. The latter system presented a simpler option because it involved room temperature cultures in stationary screw-cap bottles. We also developed a P. aeruginosa Deltapyc strain that allowed the expression of recombinant PYCs in the absence of the native enzyme. Since P. aeruginosa can be transformed genetically and lysed for cell extract preparation rather easily, our system will facilitate site-directed mutagenesis, kinetics, X-ray crystallographic, and nuclear magnetic resonance-based structure-function analysis of PYCs. During this work we also determined that, contrary to a previous report (C. K. Stover et al., Nature 406:959-964, 2000), the open reading frame (ORF) PA1400 does not encode a PYC in P. aeruginosa. The alpha4beta4 PYC of this organism was encoded by the ORFs PA5436 and PA5435.