Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase

Biochemical studies have revealed two distinct classes of Coenzyme B‐Coenzyme M heterodisulfide (CoB‐S‐S‐CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methane‐producing archaea. A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane‐bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic methanogenesis, based on reduced growth and methanogenesis rates of an hdrA1C1B1 mutant on methylotrophic substrates and downregulation of the genes during growth on acetate. This conclusion is further supported by phylogenetic analysis showing that the presence of hdrA1 in an organism is specifically correlated with the presence of genes for methylotrophic methanogenesis. Examination of mRNA abundance in methanol‐grown ΔhdrA1C1B1 strains relative to wild‐type revealed upregulation of genes required for synthesis of (di)methylsulfide and for transport and biosynthesis of CoB‐SH and CoM‐SH, suggesting that the mutant has a defect in electron transfer from ferredoxin to CoB‐S‐S‐CoM that causes cofactor limitation.     

Function and Regulation of Isoforms of Carbon Monoxide Dehydrogenase/Acetyl Coenzyme A Synthase in Methanosarcina acetivorans

Conversion of acetate to methane (aceticlastic methanogenesis) is an ecologically important process carried out exclusively by methanogenic archaea. An important enzyme for this process as well as for methanogenic growth on carbon monoxide is the five-subunit archaeal CO dehydrogenase/acetyl coenzyme A (CoA) synthase multienzyme complex (CODH/ACS) catalyzing both CO oxidation/CO(2) reduction and cleavage/synthesis of acetyl-CoA. Methanosarcina acetivorans C2A contains two very similar copies of a six-gene operon (cdh genes) encoding two isoforms of CODH/ACS (Cdh1 and Cdh2) and a single CdhA subunit, CdhA3. To address the role of the CODH/ACS system in M. acetivorans, mutational as well as promoter/reporter gene fusion analyses were conducted. Phenotypic characterization of cdh disruption mutants (three single and double mutants, as well as the triple mutant) revealed a strict requirement of either Cdh1 or Cdh2 for acetotrophic or carboxidotrophic growth, as well as for autotrophy, which demonstrated that both isoforms are bona fide CODH/ACS. While expression of the Cdh2-encoding genes was generally higher than that of genes encoding Cdh1, both appeared to be regulated differentially in response to growth phase and to changing substrate conditions. While dispensable for growth, CdhA3 clearly affected expression of cdh1, suggesting that it functions in signal perception and transduction rather than in catabolism. The data obtained argue for a functional hierarchy and regulatory cross talk of the CODH/ACS isoforms.     

Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin-dependent C-1 oxidation/reduction pathway and differences in H(2) metabolism between closely related species

A mutation in the mch gene, encoding the enzyme 5,10-methenyl tetrahydromethanopterin (H(4)MPT) cyclohydrolase, was constructed in vitro and recombined onto the chromosome of the methanogenic archaeon Methanosarcina barkeri. The resulting mutant does not grow in media using H(2)/CO(2), methanol, or acetate as carbon and energy sources, but does grow in media with methanol/H(2)/CO(2), demonstrating its ability to utilize H(2) as a source of electrons for reduction of methyl groups. Cell suspension experiments showed that methanogenesis from methanol or from H(2)/CO(2) is blocked in the mutant, explaining the lack of growth on these substrates. The corresponding mutation in Methanosarcina acetivorans C2A, which cannot grow on H(2)/CO(2), could not be made in wild-type strains, but could be made in strains carrying a second copy of mch, suggesting that M. acetivorans is incapable of methyl group reduction using H(2). M. acetivorans mch mutants could also be constructed in strains carrying the M. barkeri ech hydrogenase operon, suggesting that the block in the methyl reduction pathway is at the level of H(2) oxidation. Interestingly, the ech-dependent methyl reduction pathway of M. acetivorans involves an electron transport chain distinct from that used by M. barkeri, because M. barkeri ech mutants remain capable of H(2)-dependent methyl reduction.     

Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA

Mutants of Salmonella enterica carrying the igaA1 allele, selected as able to overgrow within fibroblast cells in culture, are mucoid and show reduced motility. Mucoidy is caused by derepression of wca genes (necessary for capsule synthesis); these genes are regulated by the RcsC/YojN/RcsB phosphorelay system and by the RcsA coregulator. The induction of wca expression in an igaA1 mutant is suppressed by mutations in rcsA and rcsC. Reduced motility is caused by lowered expression of the flagellar master operon, flhDC, and is suppressed by mutations in rcsB or rcsC, suggesting that mutations in the igaA gene reduce motility by activating the RcsB/C system. A null igaA allele can be maintained only in an igaA(+)/igaA merodiploid, indicating that igaA is an essential gene. Lethality is suppressed by mutations in rcsB, rcsC, and yojN, but not in rcsA, suggesting that the viability defect of an igaA null mutant is mediated by the RcsB/RcsC system, independently of RcsA (and therefore of the wca genes). Because all the defects associated with igaA mutations are suppressed by mutations that block the RcsB/RcsC system, we propose a functional interaction between the igaA gene product and either the Rcs regulatory network or one of its regulated products.     

Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments

We have worked out a system to obtain mutations that map in the promoter region of the Escherichia coli galactose operon. In order to easily detect small changes in gal promoter activity, we constructed a plasmid containing an operon fusion in which the lactose operon structural genes were controlled by the galactose operon promoter region. In cells harbouring this plasmid, even modest variations in the expression of the lac genes could be detected on MacConkey lactose indicator plates.Enrichment for mutations that map in the promoter segment of the galactose operon was achieved by mutagenesis in vitro of a small fragment of DNA covering the promoter region. After insertion of the mutagenized gal promoter fragment into the gal-lac fusion plasmid, lac^?1 cells were transformed and screened for an altered Lac⁺ phenotype on indicator plates. Several mutants were isolated due to lesions mapping in the small fragment covering the galactose promoter. In these mutants, the level of β-galactosidase was between 15 and 50% of the wild-type level.The mutant promoters were subsequently reinserted into a plasmid containing the intact galactose operon. Cells harbouring such plasmids, reconstituted with mutant galactose promoters, contained decreased levels of galactokinase that paralleled the decreases in β-galactosidase. The biochemical properties of these mutants are reported in the accompanying paper (Busby et al., 1982).     

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.     

Influence of carbon monoxide on metabolite formation in Methanosarcina acetivorans

Methanogenic archaea conserve energy for growth by reducing some one- and two-carbon compounds to methane and concomitantly generating an ion motive force. Growth of Methanosarcina acetivorans on carbon monoxide (CO) is peculiar as it involves formation of, besides methane, formate, acetate and methylated thiols. It has been argued that methane formation is partially inhibited under carboxidotrophic conditions and that the other products result from either detoxification of CO or from bypassing methanogenesis with other pathways for energy conservation. To gain a deeper understanding of the CO-dependent physiology of M. acetivorans we analyzed metabolite formation in resting cells. The initial rates of methane, acetate, formate, and dimethylsulfide formation increased differentially with increasing CO concentrations but were maximal already at the same moderate CO partial pressure. Strikingly, further increase of the amount of CO was not inhibitory. The maximal rate of methane formation from CO was approximately fivefold lower than that from methanol, consistent with the previously observed significant downregulation of the energy converting sodium-dependent methyltransferase. The rate of dimethylsulfide formation from CO was only 1–2% of that of methane formation under any conditions tested. Implications of the data presented for previously proposed pathways of CO utilization are discussed.     

In vivo role of three fused corrinoid/methyl transfer proteins in Methanosarcina acetivorans

Methanosarcina acetivorans is able to use carbon monoxide (CO) as the sole source of energy for growth. Its carboxidotrophic growth is peculiar as it involves formation of acetate, formate and methylated thiols, besides methane. Under this condition three proteins homologous to both corrinoid proteins and methyltransferases (MA0859, MA4384 and MA4558) are highly abundant. To address their role in M. acetivorans , a set of single and double mutants, and the triple mutant, was constructed by deletion/disruption of the encoding genes. Phenotypic analysis of the mutants rules out an important role of the methyltransferase homologues in the CO₂ reduction pathway of methanogenesis. Instead, the single and double mutants were affected to various degrees in their capacity to generate dimethylsulphide (DMS) from CO and to form methane from DMS. The triple mutant was unable to produce or metabolize DMS, and could not grow with DMS as the sole energy source, which demonstrates that MA0859, MA4384 and MA4558 are involved in, and required for, methylsulphide metabolism of M. acetivorans . Based on these findings we propose to designate MA0859, MA4384 and MA4558 as m ethyl t ransferases specific for methyl s ulphides, MtsD, MtsF and MtsH respectively.     

Essential Components of the Ti Plasmid trb System, a Type IV Macromolecular Transporter

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.     

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.

The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.     

Lysine-2,3-aminomutase and beta-lysine acetyltransferase genes of methanogenic archaea are salt induced and are essential for the biosynthesis of Nepsilon-acetyl-beta-lysine and growth at high salinity

The compatible solute N(epsilon)-acetyl-beta-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of N(epsilon)-acetyl-beta-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and beta-lysine acetyltransferase (ablB), which are assumed to catalyze N(epsilon)-acetyl-beta-lysine formation from alpha-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei G?1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei G?1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Delta(abl) mutants of M. maripaludis no longer produced N(epsilon)-acetyl-beta-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for N(epsilon)-acetyl-beta-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.     

Identification of Functionally Related Genes That Stimulate Early Meiotic Gene Expression in Yeast

Meiosis and spore formation in the yeast Saccharomyces cerevisiae are associated with increased expression of sporulation-specific genes. One of these genes, IME2, encodes a putative protein kinase that is a positive regulator of other sporulation-specific genes. We have isolated mutations that cause reduced expression of an ime2-lacZ fusion gene. We found mutations in IME1, a known positive regulator of IME2, and MCK1, a known positive regulator of IME1. We also isolated recessive mutations in 12 other genes, which we designate RIM (Regulator of IME2) genes. Our analysis indicates that the defects in rim1, rim8, rim9 and rim13 mutants are a consequence of diminished IME1 expression and can be suppressed by expression of IME1 from the heterologous ACT1 promoter. These rim mutations also reduced expression of an ime1-HIS3 fusion, in which the HIS3 gene is expressed from the IME1 promoter, and caused reduced levels of IME1 RNA. Although the rim1, rim8, rim9 and rim13 mutant phenotypes are similar to those of mck1 mutants, we found that the defects in ime2-lacZ expression and sporulation of the mck1 rim double mutants were more severe than either single mutant. In contrast, the defects of the rim rim double mutants were similar to either single mutant. The rim1, rim8, rim9 and rim13 mutants also display slow growth at 17° and share a smooth colony morphology that is not evident in mck1 mutants or isogenic wild-type strains. We suggest that RIM1, RIM8, RIM9 and RIM13 encode functionally related products that act in parallel to MCK1 to stimulate IME1 expression.