Heat shock proteome analysis of wild-type Corynebacterium glutamicum ATCC 13032 and a spontaneous mutant lacking GroEL1, a dispensable chaperone.

Proteome analysis of Corynebacterium glutamicum ATCC 13032 showed that levels of several proteins increased drastically in response to heat shock. These proteins were identified as DnaK, GroEL1, GroEL2, ClpB, GrpE, and PoxB, and their heat response was in agreement with previous transcriptomic results. A major heat-induced protein was absent in the proteome of strain 13032B of C. glutamicum, used for genome sequencing in Germany, compared with the wild-type ATCC 13032 strain. The missing protein was identified as GroEL1 by matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting, and the mutation was found to be due to an insertion sequence, IsCg1, that was integrated at position 327 downstream of the translation start codon of the groEL1 gene, resulting in a truncated transcript of this gene, as shown by Northern analysis. The GroEL1 chaperone is, therefore, dispensable in C. glutamicum. On the other hand, GroEL2 appears to be essential for growth. Based on these results, the role of the duplicate groEL1 and groEL2 genes is analyzed.

Transcriptional analysis of the groES-groEL1, groEL2, and dnaK genes in Corynebacterium glutamicum: characterization of heat shock-induced promoters.

The appropriate conditions to switch on the heat shock promoters in Corynebacterium glutamicum were defined by Northern blot analysis. Transcriptional patterns were characterized for the groEL2 gene and the groES-groEL1 and dnaK operons. Transcriptional start points of these genes were determined by primer extension analysis, allowing the identification of CIRCE and HAIR boxes close to the -10 and -35 regions of the promoters. The presence of both CIRCE and HAIR sequences within a single promoter (P-groEL2) in bacteria is described for the first time. In addition, the dnaK promoter showed -10 and -35 sequences similar to those recognized by SigH of Mycobacterium and SigR of Streptomyces close to a second transcription start region with -10 and -35 boxes typical of promoters for housekeeping genes.

The neurotrophic effect of oleic acid includes dendritic differentiation and the expression of the neuronal basic helix-loop-helix transcription factor NeuroD2.

We have shown recently that the presence of albumin in astrocytes triggers the synthesis and release of oleic acid, which behaves as a neurotrophic factor for neurons. Thus, oleic acid promotes axonal growth together with the expression of the axonal growth-associated protein, GAP-43. Here we attempted to elucidate whether the neurotrophic effect of oleic acid includes dendritic differentiation. Our results indicate that oleic acid induces the expression of microtubule associated protein-2 (MAP-2), a marker of dendritic differentiation. In addition, the presence of oleic acid promotes the translocation of MAP-2 from the soma to the dendrites. The time course of MAP-2 expression during brain development coincides with that of stearoyl-CoA desaturase, the limiting enzyme of oleic acid synthesis, indicating that both phenomena coincide during development. The effect of oleic acid on MAP-2 expression is most probably independent of autocrine factors synthesized by neurons because this effect was also observed at low cellular densities. As oleic acid is an activator of protein kinase C, the possible participation of this transduction pathway was studied. Our results indicate that added oleic acid or oleic acid endogenously synthesized by astrocytes exerts its neurotrophic effect through a protein kinase C-dependent mechanism as the effect was inhibited by sphingosine or two myristoylated peptide inhibitors of protein kinase C. The transduction pathway by which oleic acid induces the expression of genes responsible for neuronal differentiation appears to be mediated by the transcription factor NeuroD2, a regulator of terminal neuronal differentiation.

Ribosomal RNA and ribosomal proteins in corynebacteria.

Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation. Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies. All six copies of the C. glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA. The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3'. Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C. glutamicum pre-rRNA. A secondary structure of the C. glutamicum 16S rRNA is proposed. The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species. In C. glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins. The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different. Some specific ribosomal protein genes are located in a different cluster in C. glutamicum when compared with E. coli, indicating that the control of expression of these genes is different in E. coli and C. glutamicum.

Albumin promotes neuronal survival by increasing the synthesis and release of glutamate.

It is well known that the presence of albumin within the brain and the CSF is developmentally regulated. However, the physiological relevance of this phenomenon is not well established. We have previously shown that albumin specifically increases the flux of glucose and lactate through the pyruvate dehydrogenase reaction in astrocytes. Here we show that, in neurones, albumin also increases the oxidation of glucose and lactate through the pyruvate dehydrogenase-catalysed reaction, the final purpose of this being the synthesis of glutamate. Thus, in neurones, the presence of albumin strongly increased the synthesis and release of glutamate to the extracellular medium. Our results also suggest that glutamate release caused by albumin is designed to promote neuronal survival. Thus, under culture conditions in which neurones die by apoptosis, the presence of albumin promoted neuronal survival and maintained the differentiation programme of these cells, as judged by the expression of the axonal protein, GAP-43. The effect of albumin on neuronal survival was counteracted by the presence of DNQX, an antagonist of non-NMDA-glutamate receptors, suggesting that the glutamate synthesized and released due to the presence of albumin is responsible for neuronal survival. In addition, the effect of albumin seemed to depend on the activity of the NGF receptor, TrkA, suggesting that the glutamate synthesized and released due to the presence of albumin promotes neuronal survival through the activity of TrkA.

Transcytosis of albumin in astrocytes activates the sterol regulatory element-binding protein-1, which promotes the synthesis of the neurotrophic factor oleic acid.

We have recently reported that albumin, a serum protein present in the developing brain, stimulates the synthesis of oleic acid by astrocytes, which promotes neuronal differentiation. In this work, we gain insight into the mechanism by which albumin induces the synthesis of this neurotrophic factor. Our results show that astrocytes internalize albumin in vesicle-like structures by receptor-mediated endocytosis. Albumin uptake was followed by transcytosis, including passage through the endoplasmic reticulum, which was required to induce the synthesis of oleic acid. Oleic acid synthesis is feedback-regulated by the sterol regulatory element-binding protein-1, which induces the transcription of stearoyl-CoA 9-desaturase, the key rate-limiting enzyme for oleic acid synthesis. In our research, the presence of albumin activated the sterol regulatory element-binding protein-1 and increased stearoyl-CoA 9-desaturase mRNA. Moreover, when the activity of sterol regulatory element-binding protein-1 was inhibited by overexpression of a truncated form of this protein, albumin did not affect stearoyl-CoA 9-desaturase mRNA, indicating that the effect of albumin is mediated by this transcription factor. The effect of albumin was abolished when traffic to the endoplasmic reticulum was prevented or when albumin was accompanied with oleic acid. In conclusion, our results suggest that the transcytosis of albumin includes passage through the endoplasmic reticulum, where oleic acid is sequestrated, initiating the signal cascade leading to an increase in its own synthesis.