Exploration of the role of the penicillin binding protein 2c (Pbp2c) in inducible ß-lactam resistance in Corynebacteriaceae.

Six genes encoding putative high molecular weight penicillin-binding proteins (Pbp) are present in the genome of the ß-lactam-resistant strain Corynebacterium jeikeium K411. In this study, we show that pbp2c, one of these six genes, is present in resistant strains of Corynebacteriaceae but absent from sensitive strains. The molecular study of the pbp2c locus from C. jeikeium and its heterologous expression in Corynebacterium glutamicum allowed us to show that Pbp2c confers high levels of ß-lactam resistance to the host and is under the control of a ß-lactam-induced regulatory system encoded by two adjacent genes, jk0410 and jk0411. The detection of this inducible resistance may require up to 48 h of incubation, particularly in Corynebacterium amycolatum. Finally, the Pbp2c-expressing strains studied were resistant to all the ß-lactam antibiotics tested, including carbapenems, ceftaroline, and ceftobiprole.

Carbon-flux distribution within Streptomyces coelicolor metabolism: a comparison between the actinorhodin-producing strain M145 and its non-producing derivative M1146.

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and (13)C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined.

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD+-dependent L-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-ß-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator D-fructose-1,6-bisphosphate (Fru-1,6-P2). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with Vmax for the reduction of pyruvate being approximately tenfold that of the value for L-lactate oxidation at pH 7.5. No deviation from Michaelis-Menten kinetics was observed in the presence of Fru-1,6-P2, while a sigmoidal response (indicative of positive cooperativity) was seen towards L-lactate without Fru-1,6-P2. Strikingly, when introduced into an lldD- strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on L-lactate as the sole carbon source.

Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum.

Corynebacterium glutamicum is a biotin-auxotrophic bacterium and some strains efficiently produce glutamic acid under biotin-limiting conditions. In an effort to understand C. glutamicum metabolism under biotin limitation, growth of the type strain ATCC 13032 was investigated in batch cultures and a time-course analysis was performed. A transient excretion of organic acids was observed and we focused our attention on lactate synthesis. Lactate synthesis was due to the ldh-encoded l-lactate dehydrogenase (Ldh). Features of Ldh activity and ldh transcription were analysed. The ldh gene was shown to be regulated at the transcriptional level by SugR, a pleiotropic transcriptional repressor also acting on most phosphotransferase system (PTS) genes. Electrophoretic mobility shift assays (EMSAs) and site-directed mutagenesis allowed the identification of the SugR-binding site. Effector studies using EMSAs and analysis of ldh expression in a ptsF mutant revealed fructose 1-phosphate as a highly efficient negative effector of SugR. Fructose 1,6-bisphosphate also affected SugR binding.

Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling.

A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

Ketopantoate reductase activity is only encoded by ilvC in Corynebacterium glutamicum.

Ketopantoate reductase catalyzes the second step of the pantothenate pathway after ketoisovalerate, common intermediate in valine, leucine and pantothenate biosynthesis. We show here that the Corynebacterium glutamicum ilvC gene is able to complement a ketopantoate reductase deficient Escherichia coli mutant. Thus ilvC, encoding acetohydroxyacid isomeroreductase, involved in the common pathway for branched-chained amino acids, also exhibits ketopantoate reductase activity. Enzymatic activity was confirmed by biochemical analysis in C. glutamicum. Furthermore, inactivation of ilvC in C. glutamicum leads to auxotrophy for pantothenate, indicating that ilvC is the only ketopantoate reductase- encoding gene in C. glutamicum.

Promoters of Corynebacterium glutamicum.

Regulation of gene expression in Corynebacterium glutamicum represents an important issue since this Gram-positive bacterium is a notable industrial amino acid producer. Transcription initiation, beginning by binding of RNA polymerase to the promoter DNA sequence, is one of the main points at which bacterial gene expression is regulated. More than 50 transcriptional promoters have so far been experimentally localized in C. glutamicum. Most of them are assumed to be promoters of vegetative genes recognized by the main sigma factor. Although transcription initiation rate defined by many of these promoters may be affected by transcription factors, which activate or repress their function, the promoter regions share common sequence features, which may be generalized in a consensus sequence. In the consensus C. glutamicum promoter, the prominent feature is a conserved extended -10 region tgngnTA(c/t)aaTgg, while the -35 region is much less conserved. Some commonly utilized heterologous promoters were shown to drive strong gene expression in C. glutamicum. Conversely, some C. glutamicum promoters were found to function in Escherichia coli and in other bacteria. These observations suggest that C. glutamicum promoters functionally conform with the common bacterial promoter scheme, although they differ in some sequence structures.

Lipoamide dehydrogenase from Corynebacterium glutamicum: molecular and physiological analysis of the lpd gene and characterization of the enzyme.

Lipoamide dehydrogenase (LPD) is an essential component of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, both playing a crucial role within the central metabolism of aerobic organisms. Using oligonucleotides designed according to conserved regions of LPD amino acid sequences from several organisms, the lpd gene from Corynebacterium glutamicum was identified and subsequently subcloned. The cloned lpd gene expressed in C. glutamicum cells harbouring the gene on a plasmid showed a 12-fold higher specific LPD activity when compared to the wild-type strain. DNA sequence analysis of a 4524 bp segment containing the lpd gene and adjacent regions revealed that the lpd gene is not flanked by genes encoding other subunits of the pyruvate or 2-oxoglutarate dehydrogenase complexes and predicted an LPD polypeptide of 469 amino acids with an M(r) of 50619. The amino acid sequence of this polypeptide shows between 26 and 58% identity when compared to LPD enzymes from other organisms. Transcriptional analyses revealed that the lpd gene from C. glutamicum is monocistronic (1.45 kb mRNA) and that its transcription is initiated exactly at the nucleotide defined as the translational start. LPD was purified and biochemically characterized. This analysis revealed that the enzyme catalyses the reversible reoxidation of dihydrolipoic acid and NADH:NAD(+) transhydrogenation, and is able to transfer electrons from NADH to various redox-active compounds and quinones. An in vivo participation of C. glutamicum LPD in facilitation of quinone redox cycling is proposed.

S-layer protein production by Corynebacterium strains is dependent on the carbon source.

Three strains of Corynebacterium producing various amounts of PS2 S-layer protein were studied. For all strains, more PS2 was produced if the bacteria were grown in minimal medium supplemented with lactate than if they were grown in minimal medium supplemented with glucose. The consumption of substrate and PS2 production was studied in cultures with mixed carbon sources. It was found that the inhibitory effect of glucose consumption was stronger than the stimulatory effect of lactate in one strain, but not in the other two strains. The regulation of gene expression involved in S-layer formation may involve metabolic pathways, which probably differ between strains. S-layer organization was also studied by freeze-fracture electron microscopy. It was found that low levels of PS2 production correlated with the partial covering of the cell surface by a crystalline array. Finally, it was found that PS2 production was mainly regulated by changes in gene expression and that secretion was probably not a limiting step in PS2 accumulation.