Genetic, physiological and biochemical characterization of multiple methanol methyltransferase isozymes in Methanosarcina acetivorans C2A

Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes.     

Comparison of mammalian cell entry operons of mycobacteria: in silico analysis and expression profiling

Mammalian cell entry (mce) operons, implicated in the entry of mycobacteria into host cells, are present in pathogenic and saprophytic species. It is likely that the genes in these operons have functions other than those required for entry into host cells. Using in silico analysis we have identified domains within the mce operons that might justify their occurrence in saprophytic species like Mycobacterium smegmatis. Our analysis identified in addition to the mce domain, the presence of the Ttg2B and Ttg2C domains, typical of proteins involved in transport. We have also analysed and compared the expression profile between mce operons of Mycobacterium tuberculosis, Mycobacterium bovis and M. smegmatis under different growth conditions. In case of M. smegmatis, each operon presented domain truncation for at least one gene. We observe differential expression among the operons in M. smegmatis growing under different culture conditions. Bacilli growing in nutritionally rich medium with aeration, only the mce4 operon was expressed while during stationary phase of a standing culture, all four mce operons were expressed. In M. bovis, in addition to the absence of the mce3 operon, several protein domains encoded by the other operons were truncated. We detected expression of the mce2 operon in the exponential and stationary growth phase, while the mce1 operon was only expressed in the stationary growth phase. Differential expression of mce operons and their redundancy in the genome of the majority members of mycobacteria are discussed in view of our results.     

Growth substrate effects on acetate and methanol catabolism in Methanosarcina sp. strain TM-1.

When Methanosarcina sp. strain TM-1 is grown in medium in which both methanol and acetate are present, growth is biphasic, with methanol used as the primary catabolic substrate during the first phase. To better understand this phenomenon, we grew cells on methanol or on acetate or on both and examined the abilities of anaerobically washed cells to catabolize these substrates. Washed acetate-grown cells incubated with 10 mM acetate, 10 mM methanol, or both substrates together produced methane at initial rates of 325, 3, and 315 nmol min-1 mg of protein-1, respectively. Although the initial rate of methanogenesis from both substrates was nearly identical to the rate for acetate alone, after several hours of incubation the rate was greater for cells provided with both substrates. Studies with 14C-labeled methanol indicated that methanol was catabolized to methane at increasing rates by acetate-grown cells in a manner reminiscent of an induction curve, but only when cells were provided with acetate as a cosubstrate. Acetate was presumably providing energy and carbon for induction of methanol-catabolic enzymes. Methanol-grown cells showed a pattern of substrate utilization significantly different from that of acetate-grown cells, producing methane from 10 mM acetate, 10 mM methanol, or both substrates at initial rates of 10, 280, and 450 nmol min-1 mg of protein-1, respectively. There was significant oxidation of the methyl group of acetate during metabolism of both substrates. Cells grown on methanol-acetate and harvested before methanol depletion (methanol phase) showed catabolic patterns nearly identical to those of methanol-grown cells, including a low rate of methanogenesis from acetate. Cells harvested from methanol-acetate cultures in the acetate phase were capable of significant methanogenesis from either methanol or acetate alone, and the rate from both substrates together was nearly equal to the sum of the rates for the single substrates. When both 10 mM methanol and 10 mM acetate were presented to the acetate-phase cells, there was a preference for the methanol. These results are consistent with a model for regulation in Methanosarcina sp. strain TM-1 in which methanol represses acetate catabolism while methanol catabolism is inducible.     

Development of a plasmid-mediated reporter system for in vivo monitoring of gene expression in the archaeon Methanosarcina acetivorans

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.     

Coordinate regulation of the Escherichia coli formate dehydrogenase fdnGHI and fdhF genes in response to nitrate,nitrite, and formate: roles for NarL and NarP

Escherichia coli possesses three distinct formate dehydrogenase enzymes encoded by the fdnGHI, fdhF, and fdoGHI operons. To examine how two of the formate dehyrogenase operons (fdnGHI and fdhF) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with fdnG-lacZ and fdhF-lacZ reporter fusions. Complementary patterns of gene expression were seen. Optimal fdhF-lacZ expression occurred only at low to intermediate levels of nitrate, while high nitrate levels caused up to 10-fold inhibition of gene expression. In contrast, fdnG-lacZ expression was induced 25-fold in the presence of intermediate to high nitrate concentrations. Consistent with prior reports, NarL was able to induce fdnG-lacZ expression. However, NarP could not induce expression; rather, it functioned as an antagonist of fdnG-lacZ expression under low-nitrate conditions (i.e., it was a negative regulator). Nitrite, a reported signal for the Nar sensory system, was unable to stimulate or suppress expression of either formate dehydrogenase operon via NarL and NarP. The different gene expression profiles of the alternative formate dehydrogenase operons suggest that the two enzymes have complementary physiological roles under environmental conditions when nitrate and formate levels are changing. Revised regulatory schemes for NarL- and NarP-dependent nitrate control are presented for each operon.     

Development of a Plasmid-Mediated Reporter System for In Vivo Monitoring of Gene Expression in the Archaeon Methanosarcina acetivorans

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.     

The htx and ptx operons of Pseudomonas stutzeri WM88 are new members of the pho regulon

The htx and ptx operons of Pseudomonas stutzeri WM88 allow for the use of the inorganic reduced phosphorus (P) compounds hypophosphite (P valence, +1) and phosphite (P valence, +3) as sole P sources. To support the proposed in vivo role for the htx and ptx operons, namely the use of phosphite and hypophosphite as alternative P sources, we used reporter gene fusions to examine their expression levels with respect to various P conditions. Expression of the htx and ptx operons was induced up to 17- and 22-fold, respectively, in cultures grown under phosphate starvation conditions relative to expression in medium with excess phosphate (Pi). However, the presence of the reduced P substrate hypophosphite, phosphite, or methylphosphonate, in addition to excess Pi, did not result in an increase in the expression of either operon. To provide further support for a role of the htx and ptx operons in Pi acquisition, we identified P. stutzeri phoBR homologs and constructed deletion mutants. Induction of the htx and ptx reporter gene fusions in response to growth on limiting Pi was abolished in DeltaphoB, DeltaphoR, and DeltaphoBR mutants, demonstrating that htx and ptx expression is phoBR dependent. The putative LysR-type regulator encoded by ptxE has no apparent role in the expression of the htx and ptx operons, as no effect was observed on the level of induction of either operon in a DeltaptxE mutant.     

Expression of MsPG3-GFP fusions in Medicago truncatula'hairy roots' reveals preferential tip localization of the protein in root hairs.

Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle-enriched zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our laboratory, specifically expressed during the interaction of this plant with its symbiotic partner Sinorhizobium meliloti and which might participate in tip growth processes during symbiosis. We have used MsPG3-GFP fusions to study in vivo protein transport processes and localization during root hair growth. Different MsPG3-GFP fusions were expressed in Medicago truncatula'hairy roots' following a protocol developed for this study and also tested by transient expression in onion epidermal cells. Preferential accumulation of an MsPG3-GFP fusion protein in the tip of the growing root hair at different developmental stages was found, confirming the delivery of MsPG3 to the newly synthesized cell wall. This indicates that this protein may participate in tip growth processes during symbiosis and, in addition, that this fusion could be a useful tool to study this process in plants.     

Growth and Methanogenesis by Methanosarcina Strain 227 on Acetate and Methanol

Methanosarcina strain 227 exhibited exponential growth on sodium acetate in the absence of added H₂. Under these conditions, rates of methanogenesis were limited by concentrations of acetate below 0.05 M. One mole of methane was formed per mole of acetate consumed. Additional evidence from radioactive labeling studies indicated that sufficient energy for growth was obtained by the decarboxylation of acetate. Diauxic growth and sequential methanogenesis from methanol followed by acetate occurred in the presence of mixtures of methanol and acetate. Detailed studies showed that methanol-grown cells did not metabolize acetate in the presence of methanol, although acetate-grown cells did metabolize methanol and acetate simultaneously before shifting to methanol. Acetate catabolism appeared to be regulated in response to the presence of better metabolizable substrates such as methanol or H₂-CO₂ by a mechanism resembling catabolite repression. Inhibition of methanogenesis from acetate by 2-bromoethanesulfonate, an analog of coenzyme M, was reversed by addition of coenzyme M. Labeling studies also showed that methanol may lie on the acetate pathway. These results suggested that methanogenesis from acetate, methanol, and H₂-CO₂ may have some steps in common, as originally proposed by Barker. Studies with various inhibitors, together with molar growth yield data, suggest a role for electron transport mechanisms in energy metabolism during methanogenesis from methanol, acetate, and H₂-CO₂.