Expression in Escherichia coli and Pseudomonas aeruginosa of Methylophilus methylotrophus AS1 genes carried on prime plasmids

Prime plasmids carrying chromosomal fragments of Methylophilus methylotrophus AS1 were transferred to Pseudomonas aeruginosa and Escherichia coli auxotrophs. It was shown that with argF (ornithine transcarbamylase), pyrB (aspartate transcarbamylase) and trpB (tryptophan synthetase, β-subunit) mutants of E. coli and P. aeruginosa complementation was accompanied by the synthesis of the corresponding M. methylotrophus gene product. These results support the basis of complementation mapping by prime plasmids previously used for M. methylotrophus. The level of expression of the M. methylotrophus genes varied in the two hosts but the regulation of the M. methylotrophus genes under these conditions was less responsive to cultural conditions than their expression in M. methylotrophus.     

Regulation of methanol and methylamine dehydrogenases in Methylophilus methylotrophus

Abstract Methylophilus methylotrophus can use methylamine as sole source of carbon and nitrogen. Measurements of the specific activity of methylamine dehydrogenase (MNDH) in bacteria grown in batch or chemostat culture showed that MNDH was induced by methylamine and repressed when methanol or NH₄⁺ were provided as alternative carbon or nitrogen sources. The degree of repression varied with the growth conditions. Methanol dehydrogenase (MDH) was present in bacteria growtn on methylamine as sole carbon source, but the specific activity was low compared with that in bacteria grown on medium containing methanol, indicating that this enzyme is induced by methanol.     

Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP-Encoded Transporter of Escherichia coli

The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, which encodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Km^r marker, and electroporated into an aroP carrier recipient strain. This approach led to the construction of a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.The nonpathogenic Gram-negative bacterium Methylophilus methylotrophus is able to grow efficiently using C₁ substrates (methanol, methylamine, or trimethylamine) as the sole source of carbon and energy, and it uses the ribulose monophosphate pathway for fixation of formaldehyde produced by the oxidation of methanol (36). Methanol has received considerable attention by the fermentation industry as an alternative substrate to the more generally used sugars from agricultural crops. It can be synthesized either from petrochemicals or renewable resources, such as biogas (48), and therefore the production of methanol does not compete directly with human food supplies. Methylotrophs can therefore be considered potentially useful strains for industrial biotechnology. M. methylotrophus AS1 is an obligate methylotroph originally isolated from activated sludge, and it has been deposited in the National Collections of Industrial, Marine and Food Bacteria (NCIMB; no. 10515). This organism was extensively studied in the 1970s and has been industrialized on a large scale for the manufacturing of single-cell proteins (SCP) from methanol (56, 63). During that period, a significant amount of research was conducted on the direct production of amino acids by fermentation from methanol (3, 58). Although initially promising, these efforts ultimately proved relatively unsatisfactory and impractical, due primarily to the rather poor set of genetic tools that had been developed for methylotrophs.Over the last 5 years, several genomes of methylotrophs have been sequenced (8, 20, 29, 37, 65, 67), and significant progress in elucidating their metabolism has been achieved (14). The number of tools available for the genetic and metabolic engineering of methylotrophic bacteria has been expanded greatly (1, 15, 21, 43), and strategies to produce fine and bulk chemicals by methylotrophs have been described (5, 42, 57, 61). All of these factors led to renewed interest in the construction of methylotrophic strain producers, and the larger knowledge base has enabled more targeted engineering of these bacteria (55).Although M. methylotrophus AS1 has been extensively studied with regard to the industrial scale production of SCP (56, 63) and the oxidation of methanol at the initial stages of carbon and energy metabolism (13, 28), there has been little metabolic analysis of amino acid biosynthesis in this organism. Moreover, selection of auxotrophic mutants of obligate methylotrophs for broadening convenient genetic tools remains a particularly complicated task (19). Although the isolation of several auxotrophs for M. methylotrophus AS1 has been described (6, 23, 40), their numbers are limited. Development of different methods for the isolation of the mutants did not lead to construction of a collection of auxotrophic mutants that could assist in the investigation of amino acid biosynthetic pathways in M. methylotrophus AS1.As for the l-lysine (Lys) synthesis, systematic research was carried out by specialists at Ajinomoto Co., Inc., Japan, beginning with the investigation of the Lys biosynthetic pathway in M. methylotrophus AS1 (23, 61) and continuing with the construction and improvement of a Lys producer (22, 24, 33, 34). This was followed by optimization of fed-batch fermentation for overproduction of Lys from methanol (35).The aim of our investigation was to generate strains based on M. methylotrophus AS1 with the potential for industrial production of aromatic amino acids (AroAAs). It is known that mutants with relaxed feedback inhibition of key biosynthetic enzymes should be isolated at the initial steps of the construction of the amino acid producers and that the relevant degradation pathways should be blocked due to selection of the corresponding auxotrophic strains (7, 31, 49).In this study, a novel method for the construction of AroAA auxotrophic mutants of M. methylotrophus AS1 is described. This method is based on the introduction of a foreign gene encoding a specific amino acid transporter into the genome of M. methylotrophus AS1. The resulting recombinant methylotrophic strain, which possesses increased permeability to the AroAAs and their analogues, was mutated by recombination-mediated substitution of the target chromosomal genes of aromatic pathways by a flippase recombinase (FLP)-excisable marker from artificial linear DNA. This approach led to the construction of a set of M. methylotrophus AS1 marker-less mutants with destroyed genes of AroAA biosynthesis. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.     

The autoreducible cytochromes c of the methylotrophs Methylophilus methylotrophus and Pseudomonas AM1.

The two types of soluble cytochrome c (cytochrome cH and cytochrome cL) found in methylotrophs are completely distinct proteins; one type is not a dimer or degradation product of the other. Free thiol groups are probably not involved in the unusually rapid autoreduction of the cytochromes at high pH. The axial ligands to the haem iron, histidine and methionine, are the same as in other low-spin cytochromes c. The methionine ligand is displaced at high pH by an alternative strong-field ligand. This displacement does not occur on reduction of cytochrome cL by methanol dehydrogenase, but this does not rule out the possibility that the autoreduction mechanism is involved in the interaction of the dehydrogenase and cytochrome c.     

Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis

Summary Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the -galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.     

Enhancement of L-lysine production in methylotroph Methylophilus methylotrophus by introducing a mutant LysE exporter

The obligate methylotroph Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine could secrete L-lysine into the medium, but also maintained a high concentration of intracellular L-lysine. To improve the yield from excretion, we attempted to introduce an L-lysine/L-arginine exporter (LysE) from Corynebacterium glutamicum 2256 into M. methylotrophus. We were unable to stably transform M. methylotrophus with a plasmid expressing the wild type lysE gene, but happened to obtain a transformant carrying a spontaneously mutated lysE gene (designated lysE24) which could induce L-lysine production even in the wild type strain. The transformant also possessed increased tolerance to S-(2-aminoethyl)-L-cysteine (an L-lysine analog). lysE24 has a single-base insertion mutation in the middle of the lysE gene, and its product is presumably quite different in structure from wild-type LysE. When lysE24 was introduced into an L-lysine producer of M. methylotrophus carrying dapA24, the level of intracellular L-lysine fell. During fermentation, M. methylotrophus carrying both lysE24 and dapA24 produced 10-fold more L-lysine (11.3 gl(-1) in jar-fermentation) than the parent producer carrying only dapA24 or lysE24. These results show the importance of the factor (lysE24) involved in the excretion of L-lysine on its overproduction in M. methylotrophus.     

Organization of the methylamine utilization (mau) genes in Methylophilus methylotrophus W3A1-NS.

The organization of genes involved in utilization of methylamine (mau genes) was studied in Methylophilus methylotrophus W3A1. The strain used was a nonmucoid variant termed NS (nonslimy). The original mucoid strain was shown to be identical to the NS strains on the basis of chromosomal digest and hybridization patterns. An 8-kb PstI fragment of the chromosome from M. methylotrophus W3A1-NS encoding the mau genes was cloned and a 6,533-bp region was sequenced. Eight open reading frames were found inside the sequenced area. On the basis of a high level of sequence identity with the Mau polypeptides from Methylobacterium extorquens AM1, the eight open reading frames were identified as mauFBEDAGLM. The mau gene cluster from M. methylotrophus W3A1 is missing two genes, mauC (amicyanin) and mauJ (whose function is unknown), which have been found between mauA and mauG in all studied mau gene clusters. Mau polypeptides sequenced so far from five different bacteria show considerable identity. A mauA mutant of M. methylotrophus W3A1-NS that was constructed lost the ability to grow on all amines as sources of nitrogen but still retained the ability to grow on trimethylamine as a source of carbon. Thus, unlike M. extorquens AM1 and Methylobacillus flagellatum KT, M. methylotrophus W3A1-NS does not have an additional methylamine dehydrogenase system for amine oxidation. Using a promoter-probe vector, we identified a promoter upstream of mauF and used it to construct a potential expression vector, pAYC229.     

Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis

We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis. A "suicide plasmid" mutagenesis procedure was used to generate TN-5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random TN5 insertions. Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-). We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9. In approximately 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R. meliloti genome contiguous with Tn5. These later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion. In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.     

UV and chemical mutagenesis in rev7 mutants of yeast

Summary We have examined induced mutagenesis in rev7-1 mutants of Baker's yeast' Saccharomyces cerevisiae, using a variety of contrasting test systems and several different mutagens. UV-induced reversion frequencies of the ochre allele arg4-17, the putative missense allele ilv1-92 and the frameshift allele his4-38 were 10 to 200 fold lower in haploid and diploid rev7-1 mutants compared with wild type strains, but UV-induced reversion frequencies of the frameshift allele leu2-3 and the proline missense allele cyc1-115 were reduced only a few fold. Ilv1-92 reversion frequencies induced by methyl methane sulfonate or by N-methyl-N-nitro-N-nitrosoguanidine were 10 to 20 times lower in rev7-1 mutants, but normal frequencies of these revertants were induced with ethyl methane sulfonate, even though rev7-1 strains are slightly sensitive to this mutagen as well as to the others tested. We conclude that the rev7 mutants, like the rev3 mutants they closely resemble, have a substantial but not total deficiency concerning induced mutagenesis.     

Improvement of L-lysine production by Methylophilus methylotrophus from methanol via the Entner-Doudoroff pathway, originating in Escherichia coli

To improve the amino acid production by metabolic engineering, eliminating the pathway bottleneck is known to be very effective. The metabolic response of Methylophilus methylotrophus upon the addition of glucose and of pyruvate was investigated in batch cultivation. We found that the supply of pyruvate is a bottleneck in L-lysine production in M. methylotrophus from methanol as carbon source. M. methylotrophus has a ribulose monophosphate (RuMP) pathway for methanol assimilation, and consequently synthesized fructose-6-phosphate is metabolized to pyruvate via the Entner-Doudoroff (ED) pathway, and the ED pathway is thought to be the main pathway for pyruvate supply. An L-lysine producer of M. methylotrophus with an enhanced ED pathway was constructed by the introduction of the E. coli edd-eda operon encoding the enzyme involving the ED pathway. In this strain, the overall enzymatic activity of ED pathway, which is estimated by measuring the activities of 6-phosphogluconate dehydrogenase plus 2-keto-3-deoxy-6-phosphogluconate aldolase, was about 20 times higher than in the parent. This strain produced 1.2 times more L-lysine than the parent producer. Perhaps, then, the supply of pyruvate was a bottleneck in L-lysine production in the L-lysine producer of M. methylotrophus.     

Purification, properties and physiological regulation of a putative outer-membrane porin for methanol in Methylophilus methylotrophus

A major outer-membrane protein was purified and partially characterised from the methylotrophic bacterium Methylophilus methylotrophus. The protein had a subunit M_r of 38 000 and was similar in terms of its biochemical properties to the recently characterised amide-urea porin (FmdC) from the same organism. Expression of the protein, as determined by SDS-PAGE and Western blotting of cells grown in continuous culture under various nutrient limitations, varied in a similar manner to that of methanol dehydrogenase and was maximal under methanol limitation. It was concluded that the protein is probably an outer-membrane porin for methanol.     

Ethyl methanesulfonate mutagenesis in extremely halophilic archaebacteria: Isolation of auxotrophic mutants ofHaloferax mediterranei andHaloferax gibbonsii

The lethality and mutagenicity in ethyl methanesulfonate (EMS)-treated cells of five archaebacterial strains belonging to each of the three described genera of non-alkaliphilic halobacteria were investigated. In order to test the efficiency of the mutagenesis under a variety of experimental conditions, we chose the fast-growing halobacteriumHaloferax mediterranei as a model strain. A strong induced mutagenicity was found, since the spontaneous mutation rate (expressed as the rate of resistance to the antibiotic josamycin) increased up to 500-fold after mutagen exposure. The mutagenesis was also successfully used in obtaining auxotrophic mutants. Although a heterogeneous response to the induced effects caused after EMS exposure was detected for the other halophilic archaebacteria tested, a clear, efficient mutagenicity ofHalobacterium halobium andHaloferax gibbonsii was observed; auxotrophic mutants of this halobacterium were also produced. Optimal experimental conditions for EMS mutagenesis of some halobacteria were determined.     

The periplasmic modifier protein for methanol dehydrogenase in the methylotrophs Methylophilus methylotrophus and Paracoccus denitrificans.

A modifier protein (M-protein), which increases the affinity of methanol dehydrogenase (MDH) for alcohols but decreases its affinity for formaldehyde, has been partially purified from Methylophilus methylotrophus and Paracoccus denitrificans. Analysis was complicated by non-protein factors in bacterial extracts that are able to mimic M-protein in one of its functions-that of increasing the activity of MDH with butane-1,3-diol in the dye-linked assay system. The 67 kDa polypeptide, previously identified as a subunit of the M-protein, is an unrelated cytoplasmic protein. The M-protein is exclusively periplasmic and is a multimeric protein with subunits of 45 kDa. The M-protein is active in the 'physiological' assay system with the specific cytochrome c electron acceptor for MDH, lowering its affinity for formaldehyde. It has its maximum effect when the ratio of M-protein:MDH is 1:5 but its concentration in the periplasm is much lower than 20% of that of MDH.     

Isolation of Physarum polycephalum plasmodial mutants altered in sporulation by chemical mutagenesis of flagellates

Plasmodia are giant, multinucleate single cells which develop from mononucleate amoebae during the developmental cycle of Physarum polycephalum. In visible light, starving plasmodia lose their unlimited replicative potential and terminally differentiate into fruiting bodies (sporulation). Aiming at genetic dissection of the circuits controlling commitment and differentiation, we worked out a standardized procedure for the generation and screening of plasmodial mutants altered in sporulation by mutagenesis with ethylnitrosourea. To obtain a homogeneous population of cells of those strains which cannot grow axenically, we describe a protocol for preparing a suspension of flagellates to be used as starting material for mutagenesis. Flagellates can transform into plasmodia via the amoebal stage. Pilot phenotypic screening yielded plasmodial mutants altered in the photocontrol of sporulation or with disturbed developmental program. The existence of mutants with a disturbed developmental program indicates that the sequence and synchrony of morphogenetic steps of fruiting body formation can be uncoupled through mutation. Complementation testing by plasmodial fusion identified three complementation groups of non-sporulating mutants. The work described provides an experimental basis for performing mass screens for Physarum mutants altered in sporulation.     

Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c'.

Methylophilus methylotrophus cytochrome c' is an unusual monohaem protein (15 kDa) undergoing a redox-linked spin-state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Acta 954, 277-286]. The midpoint redox potential of cytochrome c" was measured over the pH range 4-10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the protein. This analysis led to the following pKa values within the pH range studied: pKa10 = 6.4, pKa1r = 5.4 and pKa2r = 8.1. Proton-NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox-Bohr effect: the group ionizing with a lower pKar was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pKa0 too low to be measured. It is shown that M. methylotrophus cytochrome c" is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haem-iron coordination. Possible implications for the physiological role of the protein are discussed.     

The effect of EDTA and related chelating agents on the oxidation of methanol by the methylotrophic bacterium, Methylophilus methylotrophus

The effects of EDTA and related metal-chelating agents on the respiratory system of the methylotrophic bacterium Methylophilus methylotrophus have been investigated. EDTA completely inhibited whole cell methanol oxidase activity and concomitantly decreased the aerobic steady-state reduction level of cytochrome c, but only partially inhibited methanol dehydrogenase activity. Analysis of the inhibition kinetics of EDTA and other chelating agents on methanol oxidase activity indicated that they were effective in the order CDTA greater than EDTA greater than HEDTA greater than EGTA greater than EDDA, and that they inhibited in an uncompetitive manner. Inhibition by EDTA varied as a function of the ambient cell mass in the assay, and could be partly reversed by the addition of divalent cations. EDTA had no effect on the activity of solubilised methanol dehydrogenase. These and other results indicate that EDTA binds a divalent metal ion, probably Mg2+, which is involved in the functional association of methanol dehydrogenase with the respiratory membrane. This concept is discussed in terms of the electron transfer properties and spatial organisation of the terminal respiratory chain of M. methylotrophus.