Characterization of the mannitol catabolic operon of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum encodes a mannitol catabolic operon, which comprises three genes: the DeoR-type repressor coding gene mtlR (sucR), an MFS transporter gene (mtlT), and a mannitol 2-dehydrogenase gene (mtlD). The mtlR gene is located upstream of the mtlTD genes in the opposite orientation. In spite of this, wild-type C. glutamicum lacks the ability to utilize mannitol. This wild-type phenotype results from the genetic regulation of the genes coding for mannitol transport and catalytic proteins mediated by the autoregulated MtlR protein since mtlR mutants grow on mannitol as the sole carbon source. MtlR binds to sites near the mtlR (two sites) and mtlTD promoters (one site downstream of the promoter), with the consensus sequence 5′-TCTAACA-3′ being required for its binding. The newly discovered operon comprises the three basic functional elements required for mannitol utilization: regulation, transport, and metabolism to fructose, further processed to the common intermediate of glycolysis fructose-6-phosphate. When relieved from MtlR repression, C. glutamicum, which lacks a functional fructokinase, excretes the fructose derived from mannitol and imports it by the fructose-specific PTS. In order to use mannitol from seaweed biomass hydrolysates as a carbon source for the production of useful commodity chemicals and materials, an overexpression system using the tac promoter was developed. For congruence with the operon, we propose to rename sucR as the mtlR gene.     

Cloning and molecular analysis of a mannitol operon of phosphoenolpyruvate-dependent phosphotransferase (PTS) type from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EII^Mtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a mannitol operon repressor (MtlR). The transport of [³H]mannitol by the cloned mannitol operon in E. coli was 13.8 ± 1.4 nmol/min/mg protein. The insertional inactivation of EII^Mtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram-positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon.     

Engineering Lactococcus lactis for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS^Mtl). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (ΔldhΔmtlA and ΔldhΔmtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo ¹³C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS^Mtl. Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.     

Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter,EIImtl, of Escherichia coli

The mannitol transporter from Escherichia coli, EII^mtl, belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII^mtl is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC^mtl). To localize this binding site, 19 single Trp-containing mutants of EII^mtl were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IIC^mtl. Combined with the available two-dimensional projection maps of IIC^mtl, it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EII^mtl becomes phosphorylated at Cys³⁸⁴ in the cytoplasmic B domain. Stably phosphorylated EII^mtl mutants were constructed, and FRET experiments showed that the position of mannitol in IIC^mtl remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.     

Analysis of the regulatory mechanisms controlling the synthesis of the hexitol transport systems in Escherichia coli K12.

Summary The synthesis of the transport systems (enzymeII-complexes) coded for in the mtl and in the gut (srl) operon was found to be induced by unphosphorylated D-mannitol and D-glucitol respectively. Induction from the outside however is only possible if these polyols are taken up into the cells. Induction of the D-mannitol system is immediate, resistant against catabolite repression, relatively insensitive towards transient repression and starts from a high uninduced level (5–30%). By contrast, the induction of the D-glucitol system starts at a low basal level (0.5–2.5%), does show a pronounced lag from 25 to 90 min, and is hypersensitive towards catabolite and transient repression. These differences apparently reflect primarely differences in the corresponding operator-promotor genes mtl(P,O) and gut(P,O) as well as differences in the uptake of the first, inducing hexitol molecules. For each operon additional regulatory genes exist, called mtlR and gutR respectively, in which transrecessive, temperature sensitive mutations leading to a constitutive expression of the corresponding operon can be found. The influence of these regulatory mechanisms in diauxie experiments and their importance for the differentiation of the three operons during evolution from apparently one common ancestor operon will be discussed.     

Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12

Summary The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cm^r structural gene coding for chloramphenicol acetyl transferase, CAT). The promoters (P₁, P₂, P₃, P_a, P_b, P_hs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene. Promoter occlusion by P₁ can be demonstrated within this operon. Regions 5kb upstream have a profound effect on operon gene expression. There is a thermoinducible promoter located within the dnaG structural gene. One of the macromolecular synthesis operon promoters is under lexA control. Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions.Abbreviations Ap^r ampicillin resistance - CAT chloramphenicol acetyl transferase - Cm^r chloramphenicol resistance - kb kilobase pair - orf open reading frame - P promoter - T terminator - Tc^r tetracycline resistance     

Domain complementation studies reveal residues critical for the activity of the mannitol permease from Escherichia coli

This paper presents domain complementation studies in the mannitol transporter, EII^mtl, from Escherichia coli. EII^mtl is responsible for the transport and concomitant phosphorylation of mannitol over the cytoplasmic membrane. By using tryptophan-less EII^mtl as a basis, each of the four phenylalanines located in the cytoplasmic loop between putative transmembrane helices II and III in the membrane-embedded C domain were replaced by tryptophan, yielding the mutants W97, W114, W126, and W133. Except for W97, these single-tryptophan mutants exhibited a high, wild-type-like, binding affinity for mannitol. Of the four mutants, only W114 showed a high mannitol phosphorylation activity. EII^mtl is functional as a dimer and the effect of these mutations on the oligomeric activity was investigated via heterodimer formation (C/C domain complementation studies). The low phosphorylation activities of W126 and W133 could be increased 7-28 fold by forming heterodimers with either the C domain of W97 (IIC^mtlW97) or the inactive EII^mtl mutant G196D. W126 and W133, on the other hand, did not complement each other. This study points towards a role of positions 97, 126 and 133 in the oligomeric activation of EII^mtl. The involvement of specific residue positions in the oligomeric functioning of a sugar-translocating EII protein has not been presented before.     

Isolation and functional characterization of a novel rice constitutive promoter

Key message

A novel rice constitutive promoter (P _OsCon1 ) was isolated. The molecular mechanism of the promoter activity was investigated. P _OsCon1 could be used as an alternative constitutive promoter for crop transgenic engineering.

Abstract

Monocot constitutive promoter is an important resource for crop transgenic engineering. In this report, we isolated a novel promoter, Oscon1 promoter (P _OsCon1 ), from the 5′ upstream region of a constitutively expressed rice gene OsDHAR1. In P _OsCon1 ::GUS transgenic rice, we showed that P _OsCon1 had a broad expression spectrum in all tested tissues. The expression of the promoter was further analyzed in comparison with the previously characterized strong constitutive promoters. P _OsCon1 exhibited comparable activity to OsCc1, OsAct1 or ZmUbi promoters in most tissues, and more active than 35S promoter in roots, seeds, and calli. Further quantitative assays indicated that P _OsCon1 activity was not affected by developmental stages or by environmental factors. Further, 5′-deletions analysis indicated that the distinct regions might contribute to the strong expression of P _OsCon1 in different tissues. Overall, our results suggest that P _OsCon1 is a novel constitutive promoter, which could potentially use in transgenic crop development.     

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).     

trans-Acting factors and cis elements involved in glucose repression of arabinan degradation in Bacillus subtilis

In Bacillus subtilis, the synthesis of enzymes involved in the degradation of arabinose-containing polysaccharides is subject to carbon catabolite repression (CCR). Here we show that CcpA is the major regulator of repression of the arabinases genes in the presence of glucose. CcpA acts via binding to one cre each in the promoter regions of the abnA and xsa genes and to two cres in the araABDLMNPQ-abfA operon. The contributions of the coeffectors HPr and Crh to CCR differ according to growth phase. HPr dependency occurs during both exponential growth and the transitional phase, while Crh dependency is detected mainly at the transitional phase. Our results suggest that Crh synthesis may increase at the end of exponential growth and consequently contribute to this effect, together with other factors.     

Tuning the Expression Level of a Gene Located on a Bacterial Chromosome

A new method of constructing a set of bacterial cell clones varying in the strength of a promoter upstream of the gene of interest was developed with the use of Escherichia coli MG1655 and lacZ as a reporter. The gist of it lies in constructing a set of DNA fragments with tac-like promoters by means of PCR with the consensus promoter P _tac and primers ensuring randomization of the four central nucleotides in the ?35 region. DNA fragments containing the tac-like promoters and a selective marker (Cm ^R) were used to replace lacI and the regulatory region of the lactose operon in E. coli MG1655. Direct LacZ activity assays with independent integrant clones revealed 14 new promoters (out of 4⁴ = 256 possible variants), whose strength varied by two orders of magnitude: LacZ activity in the corresponding strains gradually varied from 10² Miller units with the weakest promoter to 10⁴ Miller units with consensus P _tac Sequencing of the modified promoters showed that randomization of three positions in the ?35 region is sufficient for generating a representative promoter library, which reduces the number of possible variants from 256 to 64. The method of constructing a set of clones varying in expression of the gene or operon of interest is promising for modern metabolic engineering.