Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

Background

Methylobacterium extorquens AM1 is an aerobic facultative methylotrophic α-proteobacterium that can use reduced one-carbon compounds such as methanol, but also multi-carbon substrates like acetate (C₂) or succinate (C₄) as sole carbon and energy source. The organism has gained interest as future biotechnological production platform based on methanol as feedstock.

Methodology/Principal Findings

We present a comprehensive study of all postulated enzymes for the assimilation of methanol and their regulation in response to the carbon source. Formaldehyde, which is derived from methanol oxidation, is assimilated via the serine cycle, which starts with glyoxylate and forms acetyl-CoA. Acetyl-CoA is assimilated via the proposed ethylmalonyl-CoA pathway, which thereby regenerates glyoxylate. To further the understanding of the central carbon metabolism we identified and quantified all enzymes of the pathways involved in methanol assimilation. We observed a strict differential regulation of their activity level depending on whether C₁, C₂ or C₄ compounds are used. The enzymes, which are specifically required for the utilization of the individual substrates, were several-fold up-regulated and those not required were down-regulated. The enzymes of the ethylmalonyl-CoA pathway showed specific activities, which were higher than the calculated minimal values that can account for the observed growth rate. Yet, some enzymes of the serine cycle, notably its first and last enzymes serine hydroxymethyl transferase and malate thiokinase, exhibit much lower values and probably are rate limiting during methylotrophic growth. We identified the natural C₁ carrying coenzyme as tetrahydropteroyl-tetraglutamate rather than tetrahydrofolate.

Conclusion/Significance

This study provides the first complete picture of the enzymes required for methanol assimilation, the regulation of their activity levels in response to the growth substrate, and the identification of potential growth limiting steps.     

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C₁ and C₂ compounds, showing that both modes of growth have this pathway in common. However, growth on C₂ compounds via the ethylmalonyl-CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/β-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C₂ compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and ¹³C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/β-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C₂ compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H₄F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C₂ compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.Methylobacterium extorquens AM1 grows on one-carbon (C₁) compounds using the serine cycle for assimilation (25). This metabolism requires the conversion of acetyl-coenzyme A (CoA) to glyoxylate, which occurs via a novel pathway in which acetyl-CoA is converted to methylsuccinyl-CoA via acetoacetyl-CoA, ß-hydroxybutyryl-CoA, and ethylmalonyl-CoA (30-33). Recently, the steps involved in the conversion of methylsuccinyl-CoA to glyoxylate have been elucidated, and the pathway has been termed the ethylmalonyl-CoA (EMC) pathway (1, 19, 20, 40). Careful labeling measurements coupled to measurements of intermediates has confirmed that, during the growth of M. extorquens AM1 on methanol, methylsuccinyl-CoA is converted to glyoxylate and propionyl-CoA via mesaconyl-CoA and ß-methylmalyl-CoA (40).This finding has raised questions regarding how M. extorquens AM1 grows on two-carbon (C₂) compounds. The pathway involved in the conversion of acetyl-CoA to glyoxylate is known to operate during growth on both C₁ and C₂ compounds, as mutants in genes involved in this conversion are unable to grow on either C₁ or C₂ compounds, and in both cases they are rescued by glyoxylate (11, 15-17, 44). If glyoxylate is produced as an end product of this pathway during C₂ growth, then it must be converted to an intermediate of central metabolism, which has been proposed to involve a malate synthase activity (2, 14-17) (Fig. ​(Fig.1).1). In M. extorquens AM1, the apparent malate synthase activity is carried out in two steps, first by converting acetyl-CoA and glyoxylate to malyl-CoA by malyl-CoA lyase and then by converting malyl-CoA to malate by malyl-CoA hydrolase (Fig. ​(Fig.1)1) (14). However, a mutant (PCT57) defective in malyl-CoA lyase (MclA1) (22), which contains no detectable malate synthase activity during growth on methanol, is able to grow on C₂ compounds (43).Open in a separate windowFIG. 1.Enzymes and genes involved in the ethylmalonyol-CoA pathway. The colors of gene names denote a change in gene expression from microarray results comparing wild-type cells grown on ethylamine to those grown on succinate: dark red, >3-fold increase; light red, 1.5- to 3-fold increase; black, no significant change (1.49-fold increase to 1.49-fold decrease); light green, 1.5- to 3-fold decrease; dark green, >3-fold decrease. Parentheses denote a predicted function not confirmed by the mutant phenotype. See Table ​Table11 for enzyme names.Clearly, the finding that glyoxylate is generated as a direct product of the EMC pathway presents a conundrum. Apparently acetyl-CoA is converted to glyoxylate via this pathway, but M. extorquens AM1 lacking malate synthase is able to grow on C₂ compounds. Another apparent conundrum involving the malyl-CoA lyase (mclA1) mutant is that the EMC pathway requires an enzyme that carries out ß-methylmalyl-CoA cleavage, a reaction that homologs of MclA1 are known to carry out (38). The MclA1 enzyme has been purified from M. extorquens AM1 and shown to have activity with glyoxylate and propionyl-CoA (27), which would produce ß-methylmalyl-CoA. These results have led to the suggestion that MclA homologs actually are malyl-CoA/ß-methylmalyl-CoA lyases (38). Since the mclA1 mutant does not contain detectable malyl-CoA lyase activity, and by inference has correspondingly low ß-methylmalyl-CoA lyase activity, it was not clear how M. extorquens AM1 could convert acetyl-CoA to propionyl-CoA and glyoxylate via the EMC pathway in the mclA1 mutant.The purpose of this study was to solve these conundrums and determine how mutants of M. extorquens AM1 grow on C₂ compounds in the absence of malyl-CoA/ß-methylmalyl-CoA lyase or malate synthase activity. Our results show (i) that the known homolog of MclA1 (MclA2) appears to be capable of supporting both ß-methylmalyl-CoA cleavage and condensation between glyoxylate and acetyl-CoA in the mclA1 mutant, and (ii) that an alternative route for glyoxylate consumption occurs in this bacterium, in which it is converted to intermediates of central metabolism via a part of the serine cycle coupled with the glycine cleavage system.     

Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targeted metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)–meso-diaminopimelic acid (mDAP) and Ala–mDAP–Ala, each over 45-fold higher in the ecm over-expressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. This research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.     

MarkerSet: a marker selection tool based on markers location and informativity in experimental designs

Background

Although genome sequences are available for an ever-increasing number of bacterial species, the availability of facile genetic tools for physiological analysis have generally lagged substantially behind traditional genetic models.

Results

Here I describe the development of an improved, broad-host-range "in-out" allelic exchange vector, pCM433, which permits the generation of clean, marker-free genetic manipulations. Wild-type and mutant alleles were reciprocally exchanged at three loci in Methylobacterium extorquens AM1 in order to demonstrate the utility of pCM433.

Conclusion

The broad-host-range vector for marker-free allelic exchange described here, pCM433, has the advantages of a high copy, general Escherichia coli replicon for easy cloning, an IncP oriT enabling conjugal transfer, an extensive set of restriction sites in its polylinker, three antibiotic markers, and sacB (encoding levansucrase) for negative selection upon sucrose plates. These traits should permit pCM433 to be broadly applied across many bacterial taxa for marker-free allelic exchange, which is particularly important if multiple manipulations or more subtle genetic manipulations such as point mutations are desired.     

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

Background

Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared.

Methodology/Principal Findings

The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).

Conclusion/Significance

These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.     

Inoculation with arbuscular mycorrhizal fungi suppresses initiation of haustoria in the root hemiparasite Pedicularis tricolor

Background and Aims

Plant parasitism and arbuscular mycorrhizal (AM) associations have many parallels and share a number of regulatory pathways. Despite a rapid increase in investigations addressing the roles of AM fungi in regulating interactions between parasitic plants and their hosts, few studies have tested the effect of AM fungi on the initiation and differentiation of haustoria, the parasite-specific structures exclusively responsible for host attachment and nutrient transfer. In this study, we tested the influence of AM fungi on haustorium formation in a root hemiparasitic plant.

Methods

Using a facultative root hemiparasitic species (Pedicularis tricolor) with the potential to form AM associations, the effects of inoculation were tested with two AM fungal species, Glomus mosseae and Glomus intraradices, on haustorium initiation in P. tricolor grown alone or with Hordeum vulgare ‘Fleet’ (barley) as the host plant. This study consisted of two greenhouse pot experiments.

Key Results

Both AM fungal species dramatically suppressed intraspecific haustorium initiation in P. tricolor at a very low colonization level. The suppression over-rode inductive effects of the parasite''s host plant on haustoria production and caused significant growth depression of P. tricolor.

Conclusions

AM fungi had strong and direct suppressive effects on haustorium formation in the root hemiparasite. The significant role of AM fungi in haustorium initiation of parasitic plants was demonstrated for the first time. This study provides new clues for the regulation of haustorium formation and a route to development of new biocontrol strategies in management of parasitic weeds.     

Pre-Diagnostic Leukocyte Genomic DNA Methylation and the Risk of Colorectal Cancer in Women

Background

Abnormal one-carbon metabolism may lead to general genomic (global) hypomethylation, which may predispose an individual to the development of colorectal neoplasia.

Methods

We evaluated the association between pre-diagnostic leukocyte genomic DNA methylation level and the risk of colorectal cancer in a nested case-control study of 358 colorectal cancer cases and 661 matched controls within the all-female cohort of the Nurses’ Health Study (NHS). Among control subjects, we further examined major plasma components in the one-carbon metabolism pathway in relation to genomic DNA methylation level. Liquid chromatography/tandem mass spectrometry was used to examine leukocyte genomic DNA methylation level. We calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) using logistic regression.

Results

Overall genomic DNA methylation level was not associated with the risk of colorectal cancer (p for trend, 0.45). Compared with women in the lowest quintile of methylation, the multivariate OR of colorectal cancer risk was 1.32 (95% CI, 0.82–2.13) for those in the highest quintile. We did not find significant associations between major plasma components of one-carbon metabolism or risk factors for colorectal cancer and genomic DNA methylation level (all p for trend >0.05). Also, neither one-carbon metabolism-related plasma components nor well-known risk factors for colorectal cancer modified the association between genomic DNA methylation level and the risk of colorectal cancer (all p for interaction >0.05).

Conclusions

We found no evidence that hypomethylation of leukocyte genomic DNA increases risk of colorectal cancer among women. Additional studies are needed to investigate the association between pre-diagnostic genomic DNA methylation level and colorectal cancer risk among diverse populations.     

Direct and indirect influences of arbuscular mycorrhizal fungi on phosphorus uptake by two root hemiparasitic Pedicularis species: do the fungal partners matter at low colonization levels?

Background and Aims

Because most parasitic plants do not form mycorrhizal associations, the nutritional roles of arbuscular mycorrhizal (AM) fungi in them have hardly been tested. Some facultative root hemiparasitic Pedicularis species form AM associations and hence are ideal for testing both direct and indirect effects of AM fungi on their nutrient acquisition. The aim of this study was to test the influence of AM inoculation on phosphorus (P) uptake by Pedicularis rex and P. tricolor.

Methods

³²P labelling was used in compartmented pots to assess the contribution of the AM pathway and the influence of AM inoculation on P uptake from a host plant into the root hemiparasites. Laboratory isolates of fungal species (Glomus mosseae and G. intraradices) and the host species (Hordeum vulgare ‘Fleet’) to which the two Pedicularis species showed obvious responses in haustorium formation and growth in previous studies were used.

Key Results

The AM colonization of both Pedicularis spp. was low (<15 % root length) and only a very small proportion of total plant P (<1 %) was delivered from the soil via the AM fungus. In a separate experiment, inoculation with AM fungi strongly interfered with P acquisition by both Pedicularis species from their host barley, almost certainly because the numbers of haustoria formed by the parasite were significantly reduced in AM plants.

Conclusions

Roles of AM fungi in nutrient acquisition by root parasitic plants were quantitatively demonstrated for the first time. Evidence was obtained for a novel mechanism of preventing root parasitic plants from overexploiting host resources through AM fungal-induced suppression of the absorptive structures in the parasites.     

Alcohol Consumption,One-Carbon Metabolites,Liver Cancer and Liver Disease Mortality

Background

Excess alcohol consumption adversely affects one-carbon metabolism and increases the risk of liver disease and liver cancer. Conversely, higher folate levels have been inversely associated with liver damage. The current study investigated the effects of alcohol and one-carbon metabolite intake on liver cancer incidence and liver disease mortality within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study.

Methods

Cox proportional hazards modeling was used to calculate hazard ratios and 95% confidence intervals (CIs) in a population of 27,086 Finnish males with 194 incident liver cancers and 213 liver disease deaths. In a nested case-control subset (95 liver cancers, 103 controls), logistic regression was used to calculate odds ratios and 95% CIs for serum one-carbon metabolites in relation to liver cancer risk.

Results

Daily alcohol consumption of more than 20.44 g was associated with an increased risk of both liver cancer incidence (Hazard Ratio (HR) 1.52, 95%CI 1.06–2.18) and liver disease mortality (HR 6.68, 95%CI 4.16–10.71). These risks were unaffected by one-carbon metabolite intake. Similarly, in the case-control study, none of the serum one-carbon metabolites were associated with liver cancer.

Conclusions

The current study provided no convincing evidence for a protective association of one-carbon metabolite intake or serum level on the risk of liver cancer or liver disease mortality.     

The Apparent Malate Synthase Activity of Rhodobacter sphaeroides Is Due to Two Paralogous Enzymes, (3S)-Malyl-Coenzyme A (CoA)/β-Methylmalyl-CoA Lyase and (3S)- Malyl-CoA Thioesterase

Assimilation of acetyl coenzyme A (acetyl-CoA) is an essential process in many bacteria that proceeds via the glyoxylate cycle or the ethylmalonyl-CoA pathway. In both assimilation strategies, one of the final products is malate that is formed by the condensation of acetyl-CoA with glyoxylate. In the glyoxylate cycle this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway the reaction is separated into two proteins: malyl-CoA lyase, a well-known enzyme catalyzing the Claisen condensation of acetyl-CoA with glyoxylate and yielding malyl-CoA, and an unidentified malyl-CoA thioesterase that hydrolyzes malyl-CoA into malate and CoA. In this study the roles of Mcl1 and Mcl2, two malyl-CoA lyase homologs in Rhodobacter sphaeroides, were investigated by gene inactivation and biochemical studies. Mcl1 is a true (3S)-malyl-CoA lyase operating in the ethylmalonyl-CoA pathway. Notably, Mcl1 is a promiscuous enzyme and catalyzes not only the condensation of acetyl-CoA and glyoxylate but also the cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA during acetyl-CoA assimilation. In contrast, Mcl2 was shown to be the sought (3S)-malyl-CoA thioesterase in the ethylmalonyl-CoA pathway, which specifically hydrolyzes (3S)-malyl-CoA but does not use β-methylmalyl-CoA or catalyze a lyase or condensation reaction. The identification of Mcl2 as thioesterase extends the enzyme functions of malyl-CoA lyase homologs that have been known only as “Claisen condensation” enzymes so far. Mcl1 and Mcl2 are both related to malate synthase, an enzyme which catalyzes both a Claisen condensation and thioester hydrolysis reaction.Many organic compounds are initially metabolized to acetyl coenzyme A (acetyl-CoA), at which point they enter the central carbon metabolism. Examples of such growth substrates are C₁ and C₂ compounds (e.g., methanol and ethanol), fatty acids, waxes, esters, alkenes, or (poly)hydroxyalkanoates. The synthesis of all cell constituents from acetyl-CoA requires a specialized pathway for the conversion of this central C₂ unit into other biosynthetic precursor metabolites. This (anaplerotic) process is referred to as acetyl-CoA assimilation, and two very different strategies have been described, i.e., the glyoxylate cycle and the ethylmalonyl-CoA pathway (12, 21) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Pathways for acetyl-CoA assimilation. (A) Glyoxylate cycle. The key enzymes are isocitrate lyase and malate synthase. (B) Ethylmalonyl-CoA pathway. The unique enzymes of the pathway are crotonyl-CoA carboxylase/reductase, ethylmalonyl-CoA/methylmalonyl-CoA epimerase, (2R)-ethylmalonyl-CoA mutase, (2S)-methylsuccinyl-CoA dehydrogenase, mesaconyl-CoA hydratase, (3S)-malyl-CoA/β-methylmalonyl-CoA lyase, and (3S)-malyl-CoA thioesterase. The enzymes involved in the (apparent) malate synthase reaction(s) are boxed for each pathway.The glyoxylate cycle for acetyl-CoA assimilation is in fact a modified citric acid cycle that converts two molecules of acetyl-CoA to the citric acid cycle intermediate malate (Fig. ​(Fig.1A)1A) (21). In a first reaction sequence, one molecule of acetyl-CoA is converted into glyoxylate due to the combined action of the initial enzymes of the citric acid cycle and isocitrate lyase, the key enzyme of this assimilation strategy. Isocitrate lyase cleaves the citric cycle intermediate isocitrate into succinate and glyoxylate (22). The glyoxylate formed is then condensed in a second step with another molecule of acetyl-CoA to yield malate and free CoA. Because the two decarboxylation reactions of the citric acid cycle are circumvented by this acetyl-CoA assimilation strategy, the glyoxylate cycle is also referred to as the “glyoxylate bypass” or “glyoxylate shunt.”The ethylmalonyl-CoA pathway for acetyl-CoA assimilation replaces the glyoxylate cycle in bacteria that lack isocitrate lyase (1, 12). In this linear pathway, three molecules of acetyl-CoA, one molecule of CO₂, and one molecule of bicarbonate are converted to the citric acid cycle intermediates succinyl-CoA and malate (Fig. ​(Fig.1B).1B). The ethylmalonyl-CoA pathway requires at least seven unique enzymes. Crotonyl-CoA carboxylase/reductase, ethylmalonyl-CoA mutase, and methylsuccinyl-CoA dehydrogenase are considered key enzymes of the pathway, and all three enzymes have been characterized from Rhodobacter sphaeroides (12-14).Although these two acetyl-CoA strategies differ with respect to their reaction sequence, intermediates and overall balance, the glyoxylate cycle and the ethylmalonyl-CoA pathway both require the condensation of acetyl-CoA and glyoxylate to form malate (Fig. ​(Fig.1,1, boxed). In the glyoxylate cycle, this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway malate synthase is catalyzed by two separate enzymes, malyl-CoA lyase and malyl-CoA thioesterase (7, 26).Malyl-CoA lyases catalyze the reversible condensation of acetyl-CoA and glyoxylate into malyl-CoA and have been purified from Methylobacterium extorquens, Chloroflexus aurantiacus, Aminobacter aminovorans, and Rhodobacter capsulatus; the corresponding genes were identified as mclA (M. extorquens), mcl (C. aurantiacus), and mcl1 (R. capsulatus) (5, 16, 17, 19, 26). Remarkably, these proteins are promiscuous enzymes that also catalyze the (reversible) cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA, and it has been suggested that these enzymes catalyze both reactions in vivo (16, 19, 26). However, in contrast to malyl-CoA lyase, the malyl-CoA thioesterase catalyzing the highly exergonic hydrolysis of the CoA-thioester into malate and free CoA has not been identified so far, and the nature of the enzyme has remained enigmatic (7, 26).For R. sphaeroides, a malyl-CoA lyase homolog has been shown to be upregulated during growth on acetate, and it was proposed that this protein (Mcl1) catalyzes the cleavage of β-methylmalyl-CoA, as well as the condensation of acetyl-CoA and glyoxylate in the ethylmalonyl-CoA pathway (1). Interestingly, R. sphaeroides encodes a second malyl-CoA lyase homolog with 34% amino acid sequence identity to Mcl1. This protein, named Mcl2, was also shown to be upregulated during growth of R. sphaeroides on acetate, but a function could not be assigned so far (1). We therefore addressed the function of both malyl-CoA lyase homologs by gene inactivation and biochemical studies of recombinant Mcl1 and Mcl2. Based on our findings, we confirm here the function of Mcl1 in R. sphaeroides as (3S)-malyl-CoA/β-methylmalyl-CoA lyase and identify its paralog Mcl2 as the long-sought (3S)-malyl-CoA thioesterase.     

Comprehensive evaluation of one-carbon metabolism pathway gene variants and renal cell cancer risk

Introduction

Folate and one-carbon metabolism are linked to cancer risk through their integral role in DNA synthesis and methylation. Variation in one-carbon metabolism genes, particularly MTHFR, has been associated with risk of a number of cancers in epidemiologic studies, but little is known regarding renal cancer.

Methods

Tag single nucleotide polymorphisms (SNPs) selected to produce high genomic coverage of 13 gene regions of one-carbon metabolism (ALDH1L1, BHMT, CBS, FOLR1, MTHFR, MTR, MTRR, SHMT1, SLC19A1, TYMS) and the closely associated glutathione synthesis pathway (CTH, GGH, GSS) were genotyped for 777 renal cell carcinoma (RCC) cases and 1,035 controls in the Central and Eastern European Renal Cancer case-control study. Associations of individual SNPs (n = 163) with RCC risk were calculated using unconditional logistic regression adjusted for age, sex and study center. Minimum p-value permutation (Min-P) tests were used to identify gene regions associated with risk, and haplotypes were evaluated within these genes.

Results

The strongest associations with RCC risk were observed for SLC19A1 (P_min-P = 0.03) and MTHFR (P_min-P = 0.13). A haplotype consisting of four SNPs in SLC19A1 (rs12483553, rs2838950, rs2838951, and rs17004785) was associated with a 37% increased risk (p = 0.02), and exploratory stratified analysis suggested the association was only significant among those in the lowest tertile of vegetable intake.

Conclusions

To our knowledge, this is the first study to comprehensively examine variation in one-carbon metabolism genes in relation to RCC risk. We identified a novel association with SLC19A1, which is important for transport of folate into cells. Replication in other populations is required to confirm these findings.     

Identification of a novel gene product that promotes survival of Mycobacterium smegmatis in macrophages

Background

Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs.

Methodology/Principal Findings

We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate.

Conclusions/Significance

MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.     

The ADMR Receptor Mediates the Effects of Adrenomedullin on Pancreatic Cancer Cells and on Cells of the Tumor Microenvironment

Background

Adrenomedullin (AM) is highly expressed in pancreatic cancer and stimulates pancreatic cancer cells leading to increased tumor growth and metastasis. The current study examines the role of specific AM receptors on tumor and cells resembling the tumor microenvironment (human pancreatic stellate - HPSC, human umbilical vein – HUVEC and mouse lung endothelial cells - MLEC).

Methods and Findings

AM receptors ADMR and CRLR were present in HPSC, HUVEC and MLECs while PDAC cells possessed only ADMR receptors as assessed by RT-PCR and western blotting. All cell lines expressed and secreted AM as indicated by ELISA. The growth of each of the cell lines was stimulated by exogenous AM and inhibited by the antagonist AMA. AM also stimulated in vitro angiogenesis assessed by polygon formation of endothelial cell lines. SiRNA-mediated silencing of ADMR, but not CRLR, reduced basal growth of all cells examined and reduced polygon formation of endothelial cells in vitro. Orthotopic tumors developed with shADMR bearing cancer cells had dramatically reduced primary tumor volume (>90%) and lung and liver metastasis compared to shControl bearing cells. To validate ADMR as a potential therapeutic target, in vivo studies were conducted using neutral nanoliposomes to systemically deliver human siRNA to ADMR to silence human cancer cells and mouse siRNA to ADMR to silence mouse tumor stromal cells. Systemic silencing of both human and mouse ADMR had no obvious adverse effects but strongly reduced tumor development.

Conclusion

ADMR mediates the stimulatory effects of AM on cancer cells and on endothelial and stellate cells within the tumor microenvironment. These data support the further development of ADMR as a useful target treatment of pancreatic cancer.     

Comparative genome analysis of pathogenic and non-pathogenic Clavibacter strains reveals adaptations to their lifestyle

Background

The genus Clavibacter harbors economically important plant pathogens infecting agricultural crops such as potato and tomato. Although the vast majority of Clavibacter strains are pathogenic, there is an increasing number of non-pathogenic isolates reported. Non-pathogenic Clavibacter strains isolated from tomato seeds are particularly problematic because they affect the current detection and identification tests for Clavibacter michiganensis subsp. michiganensis (Cmm), which is regulated with a zero tolerance in tomato seed. Their misidentification as pathogenic Cmm hampers a clear judgment on the seed quality and health.

Results

To get more insight in the genetic features linked to the lifestyle of these bacteria, a whole-genome sequence of the tomato seed-borne non-pathogenic Clavibacter LMG 26808 was determined. To gain a better understanding of the molecular determinants of pathogenicity, the genome sequence of LMG 26808 was compared with that of the pathogenic Cmm strain (NCPPB 382). The comparative analysis revealed that LMG 26808 does not contain plasmids pCM1 and pCM2 and also lacks the majority of important virulence factors described so far for pathogenic Cmm. This explains its apparent non-pathogenic nature in tomato plants. Moreover, the genome analysis of LMG 26808 detected sequences from a plasmid originating from a member of Enterobacteriaceae/Klebsiella relative. Genes received that way and coding for antibiotic resistance may provide a competitive advantage for survival of LMG 26808 in its ecological niche. Genetically, LMG 26808 was the most similar to the pathogenic Cmm NCPPB 382 but contained more mobile genetic elements. The genome of this non-pathogenic Clavibacter strain contained also a high number of transporters and regulatory genes.

Conclusions

The genome sequence of the non-pathogenic Clavibacter strain LMG 26808 and the comparative analyses with other pathogenic Clavibacter strains provided a better understanding of the genetic bases of virulence and adaptation mechanisms present in the genus Clavibacter.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-392) contains supplementary material, which is available to authorized users.     

Vitamins A & D inhibit the growth of mycobacteria in radiometric culture

Background

The role of vitamins in the combat of disease is usually conceptualized as acting by modulating the immune response of an infected, eukaryotic host. We hypothesized that some vitamins may directly influence the growth of prokaryotes, particularly mycobacteria.

Methods

The effect of four fat-soluble vitamins was studied in radiometric Bactec® culture. The vitamins were A (including a precursor and three metabolites,) D, E and K. We evaluated eight strains of three mycobacterial species (four of M. avium subspecies paratuberculosis (MAP), two of M. avium and two of M. tb. complex).

Principal Findings

Vitamins A and D cause dose-dependent inhibition of all three mycobacterial species studied. Vitamin A is consistently more inhibitory than vitamin D. The vitamin A precursor, β-carotene, is not inhibitory, whereas three vitamin A metabolites cause inhibition. Vitamin K has no effect. Vitamin E causes negligible inhibition in a single strain.

Significance

We show that vitamin A, its metabolites Retinyl acetate, Retinoic acid and 13-cis Retinoic acid and vitamin D directly inhibit mycobacterial growth in culture. These data are compatible with the hypothesis that complementing the immune response of multicellular organisms, vitamins A and D may have heretofore unproven, unrecognized, independent and probable synergistic, direct antimycobacterial inhibitory activity.     

Combined Inhibition of IGF-1R/IR and Src Family Kinases Enhances Antitumor Effects in Prostate Cancer by Decreasing Activated Survival Pathways

Background

Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways.

Materials and Methods

Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively).

Results

In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1-induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS-754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers.

Conclusions

Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth.     

Inhaled fluticasone propionate impairs pulmonary clearance of Klebsiella Pneumoniae in mice

Background

Recent trials demonstrate increased pneumonia risk in chronic obstructive pulmonary disease patients treated with the inhaled corticosteroid (ICS) fluticasone propionate (FP). There is limited work describing FP effects on host defenses against bacterial pneumonia.

Methods

C57BL/6 mice received daily, nose-only exposure to nebulized FP or vehicle for 8 days, followed by pulmonary challenge with Klebsiella pneumoniae. Bacterial burden, phagocytosis, leukocyte recruitment, cytokine expression, nitric oxide release, and survival were measured.

Results

Inhaled FP increased bacterial burden in lungs and blood 48 h after infection but affected neither in vivo phagocytosis of bacteria by alveolar macrophages (AM) nor alveolar neutrophil recruitment. AM from FP-treated mice showed impaired expression of infection induced TNF-alpha, IP-10 (CXCL-10), and interleukin 6 (IL-6), and AM also showed a trend towards impaired intracellular pathogen control following in vivo infection. In vitro FP treatment resulted in a dose-dependent impairment of cytokine expression by AM. Furthermore, infection-induced nitric oxide (but not hydrogen peroxide) production was impaired by FP in vivo and in vitro. FP decreased survival in this model.

Conclusions

Exposure to inhaled FP impairs pulmonary clearance of K. pneumoniae in mice, an effect associated with greater systemic bacteremia and death. Decreased AM cytokine and nitric oxide expression parallel the failure to control infection. These results support the study of ICS effects on human pulmonary host defenses.     

Low-Dose Dextromethorphan,a NADPH Oxidase Inhibitor,Reduces Blood Pressure and Enhances Vascular Protection in Experimental Hypertension

Background

Vascular oxidative stress may be increased with age and aggravate endothelial dysfunction and vascular injury in hypertension. This study aimed to investigate the effects of dextromethorphan (DM), a NADPH oxidase inhibitor, either alone or in combination treatment, on blood pressure (BP) and vascular protection in aged spontaneous hypertensive rats (SHRs).

Methodology/Principal Findings

Eighteen-week-old WKY rats and SHRs were housed for 2 weeks. SHRs were randomly assigned to one of the 12 groups: untreated; DM monotherapy with 1, 5 or 25 mg/kg/day; amlodipine (AM, a calcium channel blocker) monotherapy with 1 or 5 mg/kg/day; and combination therapy of DM 1, 5 or 25 mg/kg/day with AM 1 or 5 mg/kg/day individually for 4 weeks. The in vitro effects of DM were also examined. In SHRs, AM monotherapy dose-dependently reduced arterial systolic BP. DM in various doses significantly and similarly reduced arterial systolic BP. Combination of DM with AM gave additive effects on BP reduction. DM, either alone or in combination with AM, improved aortic endothelial function indicated by ex vivo acetylcholine-induced relaxation. The combination of low-dose DM with AM gave most significant inhibition on aortic wall thickness in SHRs. Plasma total antioxidant status was significantly increased by all the therapies except for the combination of high-dose DM with high-dose AM. Serum nitrite and nitrate level was significantly reduced by AM but not by DM or the combination of DM with AM. Furthermore, in vitro treatment with DM reduced angiotensin II-induced reactive oxygen species and NADPH oxidase activation in human aortic endothelial cells.

Conclusions/Significance

Treatment of DM reduced BP and enhanced vascular protection probably by inhibiting vascular NADPH oxidase in aged hypertensive animals with or without AM treatment. It provides the potential rationale to a novel combination treatment with low-dose DM and AM in clinical hypertension.