Complementation between mutants of Phycomyces deficient with respect to carotenogenesis

Summary White and red mutants of Phycomyces, derived from two independent wild types (yellow) by mutagenesis using nitrosoguanidine, either in a single step (26 white, 5 red mutants), or in two steps (10 white mutants, from one of the red mutants) were studied with respect to complementation in heterokaryons. The tests clearly establish the involvement of three and only three genes, here named carA, carB, and carB. The carA and the carR mutants are white, the carA mutants do not accumulate phytoene, the carB mutants do. The carR mutants are red and accumulate lycopene. The two step mutants are either carA and carR, or carB and carR double mutants. A few of the white mutants obtained in a single mutagenization step are affected in carA and carR. They may be polar mutants in an operon or accidental double mutants.     

Accumulation of carotenoids in structural and regulatory mutants of the bacterium Myxococcus xanthus

Summary Accumulation of carotenoids in Myxococcus xanthus is absolutely dependent on illumination with blue light. We report the analysis of the carotenoids of dark- and light-grown cultures of the wild type and several previously characterized mutants. A carR mutant produces the same carotenoids in the dark as the wild type grown in the light. This agrees with previous evidence indicating that the carR gene codes for a general negative regulator of the system. A cis-dominant mutation in the gene carA causes constitutive expression of the light-inducible gene carB, which is linked to carA. In the dark, the carA mutant produces high levels of phytoene, the first C₄₀ colourless carotenoid precursor; in the light, it produces the same carotenoids as the wild type. Since a mutation in carB blocks accumulation of phytoene, we propose that carB, and probably other linked genes also controlled by carA, code for enzymes involved in the synthesis of phytoene. This is virtually the only carotene accumulated by strains mutated in the gene carC, which is unlinked to the others. Thus carC codes for phytoene dehydrogenase, the enzyme that converts phytoene into coloured carotenoids. The results presented here also provide evidence for control of carotenogenesis by an endproduct that is independent of the blue light effect.     

Light-induced carotenogenesis in Myxococcus xanthus: light-dependent membrane sequestration of ECF sigma factor CarQ by anti-sigma factor CarR

Light-induced carotenogenesis in Myxococcus xanthus is under the control of the carQRS operon. CarQ, a proposed extracytoplasmic (ECF) RNA polymerase sigma factor, is required for expression of the operon and the carC gene that encodes phytoene dehydrogenase. CarR, an inner membrane protein in Escherichia coli, is essential for carQRS promoter inactivation in the dark. CarS is required for the light-dependent expression of the promoter of the carB gene cluster that encodes the rest of the structural genes for carotenogenesis. Regulation of carQRS is dependent on the stoichiometry of CarQ and CarR. Increasing the copy number of carQ over carR led to constitutive carotenogenesis, as did loss of translational coupling between carQ and carR. The severity of the constitutive phenotype depended on the distance between the uncoupled genes. When expressed in M. xanthus, a CarR:β-galactosidase fusion protein disappeared in the light. We propose that anti-sigma factor CarR sequesters CarQ to the membrane in the dark, but, in the light, loss of CarR leads to release of the sigma factor.     

Light-induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region

The carR region encodes a light-inducible promoter, a negative regulator of the promoter and a trans-acting activator that controls the light-inducible Myxococcus xanthus carotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS. Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed that carS was the trans-acting activator and that carQ was a positive regulator of the promoter. Neither gene encodes proteins with known sequence-specific DNA-binding motifs. The sequence of the light-inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response (‘heat-shock’) promoters.     

Genetic analysis of carbamoylphosphate synthesis in Rhizobium meliloti 104A14

We have previously isolated ineffective (Fix-) mutants of Rhizobium meliloti 104A14 requiring both arginine and uracil, and thus probably defective in carbamoylphosphate synthetase. We describe here the molecular and genetic analysis of the R. meliloti genes coding for carbamoylphosphate synthetase. Plasmids that complement the mutations were isolated from a R. meliloti gene bank. Restriction analysis of these plasmids indicated that complementation involved two unlinked regions of the R. meliloti chromosome, carA and carB. Genetic complementation between the plasmids and mutants demonstrated a single complementation group for carA, but two overlapping complementation groups for carB. The cloned R. meliloti genes hybridize to the corresponding E. coli carA and carB genes which encode the two subunits of carbamoylphosphate synthetase. Transposon Tn5 mutagenesis was used to localize the carA and carB genes on the cloned R. meliloti DNA. The cloned R. meliloti carA and carB genes were unable to complement E. coli carA or carB mutants alone or in combination. We speculate on the mechanism of the unusual pattern of genetic complementation at the R. meliloti carB locus.     

A carotenoid synthesis gene cluster from Algoriphagus sp. KK10202C with a novel fusion-type lycopene β-cyclase gene

A carotenoid synthesis gene cluster was isolated from a marine bacterium Algoriphagus sp. strain KK10202C that synthesized flexixanthin. Seven genes were transcribed in the same direction, among which five of them were involved in carotenoid synthesis. This cluster had a unique gene organization, with an isoprenoid gene, ispH (previously named lytB), being present among the carotenoid synthesis genes. The lycopene β-cyclase encoded by the crtY _cd gene appeared to be a fusion of bacterial heterodimeric lycopene cyclase CrtY_c and CrtY_d. This was the first time that a fusion-type of lycopene β-cyclase was reported in eubacteria. Heterologous expression of the Algoriphagus crtY _cd gene in lycopene-accumulating Escherichia coli produced bicyclic β-carotene. A biosynthesis pathway for monocyclic flexixanthin was proposed in Algoriphagus sp. strain KK10202C, though several of the carotenoid synthesis genes not linked with the cluster have not yet been cloned.     

Cloning and organization of seven arginine biosynthesis genes from Neisseria gonorrhoeae.

A genomic library for Neisseria gonorrhoeae, constructed in the lambda cloning vector EMBL4, was screened for clones carrying arginine biosynthesis genes by complementation of Escherichia coli mutants. Clones complementing defects in argA, argB, argE, argG, argIF, carA, and carB were isolated. An E. coli defective in the acetylornithine deacetylase gene (argE) was complemented by the ornithine acetyltransferase gene (argJ) from N. gonorrhoeae. This heterologous complementation is reported for the first time. The carAB operon from E. coli hybridized with the gonococcal clones that carried carA or carB genes under conditions of high stringency, detecting 80% or greater similarity and showing that the nucleotide sequence of the carbamoylphosphate synthetase genes is very similar in these two organisms. Under these conditions for hybridization, the gonococcal clones carrying argB or argF genes did not hybridize with plasmids containing the corresponding E. coli gene. Cocomplementation experiments established gene linkage between carA and carB. Clones complementing a gene defect in argE were also able to complement an argA mutation. This suggests that the enzyme ornithine acetyltransferase from N. gonorrhoeae (encoded by argJ) may be able to complement both argA and argE mutations in E. coli. The arginine biosynthesis genes in N. gonorrhoeae appear to be scattered as in members of the family Pseudomonadaceae.     

Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa.

The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated.     

A carotenogenic gene cluster from Brevibacterium linens with novel lycopene cyclase genes involved in the synthesis of aromatic carotenoids

The carotenogenic (crt) gene cluster from Brevibacterium linens, a member of the commercially important group of coryneform bacteria, was cloned and identified. An expression library of B. linens genes was constructed and a fragment of the crt cluster was obtained by functional complementation of a colourless B. flavum mutant, screening transformed cells for production of a yellow pigment. Subsequent screening of a cosmid library resulted in the cloning of the wholecrt cluster from B. linens. All genes necessary for the synthesis of the aromatic carotenoid isorenieratene were identified on the basis of sequence homologies. In addition a novel type of lycopene cyclase was identified by complementation of a lycopene-accumulating B. flavum mutant. Two genes, named crtYc and crtYd, which code for polypeptides of 125 and 107 amino acids, respectively, are necessary to convert lycopene to β-carotene. The amino acid sequences of these polypeptides show no similarity to any of the known lycopene cyclases. This is the first example of a carotenoid biosynthetic conversion in which two different gene products are involved, probably forming a heterodimer. Received: 17 July 1999 / Accepted: 7 December 1999     

Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.

Cumulative repression of Escherichia coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.     

Alfalfa (Medicago sativa) carbamoylphosphate synthetase gene structure records the deep lineage of plants

Given the central role of carbamoylphosphate synthetases in pyrimidine and arginine metabolism in all living organisms, the absence of fundamental information regarding plant CPSase genes is a striking omission [Lawson et al., Mol. Biol. Evol. 13 (1996) 970-977; van den Hoff et al., J. Mol. Evol. 41 (1995) 813-832]. Whereas CPSase gene architecture and aa sequence have proven to be useful characters in establishing ancient and modern genetic affinities, phylogenetic analysis cannot be completed without the inclusion of plant CPSases. We describe the first isolation by molecular cloning of a plant CPSase gene (CPAII) derived from alfalfa (Medicago sativa). DNA sequence analysis reveals a proteobacterial architecture, namely closely linked carA and carB coding domains separated by a short intergenic region, and transcribed as a polycistronic mRNA. CPAII encodes the amino acid residues that typify a CPSase type II enzyme. In addition, an ancient internal duplication has been retained in the plant carB sequence. Partial nucleotide sequencing of additional clones reveals that the alfalfa genome contains multiple CPSase II gene copies which may be tissue-specific in their expression. It appears that with respect to CPSase genes, CPAII resembles the carAB gene of bacteria, and may have preserved much of this ancient gene structure in the alfalfa genome.     

A caffeyl-coenzyme A synthetase initiates caffeate activation prior to caffeate reduction in the acetogenic bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii couples the reduction of caffeate with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism using sodium ions as coupling ions, but the enzymes involved remain to be established. Previously, the electron transfer flavoproteins EtfA and EtfB were found to be involved in caffeate respiration. By inverse PCR, we identified three genes upstream of etfA and etfB: carA, carB, and carC. carA encodes a potential coenzyme A (CoA) transferase, carB an acyl-CoA synthetase, and carC an acyl-CoA dehydrogenase. carA, -B, and -C are located together with etfA/carE and etfB/carD on one polycistronic message, indicating that CarA, CarB, and CarC are also part of the caffeate respiration pathway. The genetic data suggest an initial ATP-dependent activation of caffeate by CarB. To prove the proposed function of CarB, the protein was overproduced in Escherichia coli, and the recombinant protein was purified. Purified CarB activates caffeate to caffeyl-CoA in an ATP- and CoA-dependent reaction. The enzyme has broad pH and temperature optima and requires K(+) for activity. In addition to caffeate, it can use ρ-coumarate, ferulate, and cinnamate as substrates, with 50, 15, and 9%, respectively, of the activity obtained with caffeate. Expression of the car operon is induced not only by caffeate, ρ-coumarate, ferulate, and cinnamate but also by sinapate. There is no induction by ρ-hydroxybenzoate or syringate.     

Use of Transposon Promoter-Probe Vectors in the Metabolic Engineering of the Obligate Methanotroph Methylomonas sp. Strain 16a for Enhanced C40 Carotenoid Synthesis

The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C₄₀ carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations—the fliCS, hsdM, ccp-3, cysH, and nirS regions—were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.