Thiol-specific oxidant diamide downregulates whiA gene of Corynebacterium glutamicum, thereby suppressing cell division and metabolism.

The whiA (NCgl1527) gene from Corynebacterium glutamicum plays a crucial role during cell growth, and WhiA is recognized as the transcription factor for genes involved in cell division. In this study, we assessed the regulatory role of the gene in cell physiology. Transcription of the gene was specifically downregulated by the thiol-specific oxidant, diamide, and by heat stress. Cells exposed to diamide showed decreased transcription of genes involved in cell division and these effects were more profound in ΔwhiA cells. In addition, the ΔwhiA cells showed sensitivity to thiol-specific oxidants, DNA-damaging agents, and high temperature. Further, downregulation of sigH (NCgl0733), the central regulator in stress responses, along with master regulatory genes in cell metabolism, was observed in the ΔwhiA strain. Moreover, the amount of cAMP in the ΔwhiA cells in the early stationary phase was only at 30% level of that for the wild-type strain. Collectively, our data indicate that the role of whiA is to downregulate genes associated with cell division in response to heat or thiol-specific oxidative stress, and may suggest a role for the gene in downshifting cell metabolism by downregulating global regulatory genes when growth condition is not optimal for cells.

The Iron Deficiency Response of Corynebacterium glutamicum and a Link to Thiamine Biosynthesis.

The response to iron limitation of the Gram-positive soil bacterium Corynebacterium glutamicum was analyzed with respect to secreted metabolites, the transcriptome, and the proteome. During growth in glucose minimal medium, iron limitation caused a shift from lactate to pyruvate as the major secreted organic acid complemented by l-alanine and 2-oxoglutarate. Transcriptome and proteome analyses revealed that a pronounced iron starvation response governed by the transcriptional regulators DtxR and RipA was detectable in the late, but not in the early, exponential-growth phase. A link between iron starvation and thiamine pyrophosphate (TPP) biosynthesis was uncovered by the strong upregulation of thiC As phosphomethylpyrimidine synthase (ThiC) contains an iron-sulfur cluster, limiting activities of the TPP-dependent pyruvate-2-oxoglutarate dehydrogenase supercomplex probably cause the excretion of pyruvate and 2-oxoglutarate. In line with this explanation, thiamine supplementation could strongly diminish the secretion of these acids. The upregulation of thiC and other genes involved in thiamine biosynthesis and transport is presumably due to TPP riboswitches present at the 5' end of the corresponding operons. The results obtained in this study provide new insights into iron homeostasis in C. glutamicum and demonstrate that the metabolic consequences of iron limitation can be due to the iron dependency of coenzyme biosynthesis.IMPORTANCE Iron is an essential element for most organisms but causes problems due to poor solubility under oxic conditions and due to toxicity by catalyzing the formation of reactive oxygen species (ROS). Therefore, bacteria have evolved complex regulatory networks for iron homeostasis aiming at a sufficient iron supply while minimizing ROS formation. In our study, the responses of the actinobacterium Corynebacterium glutamicum to iron limitation were analyzed, resulting in a detailed view on the processes involved in iron homeostasis in this model organism. In particular, we provide evidence that iron limitation causes TPP deficiency, presumably due to insufficient activity of the iron-dependent phosphomethylpyrimidine synthase (ThiC). TPP deficiency was deduced from the upregulation of genes controlled by a TPP riboswitch and secretion of metabolites caused by insufficient activity of the TPP-dependent enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. To our knowledge, the link between iron starvation and thiamine synthesis has not been elaborated previously.

Live cell imaging of macrophage/bacterium interaction demonstrates cell lysis induced by Corynebacterium diphtheriae and Corynebacterium ulcerans.

OBJECTIVES: In frame of a study to characterize the interaction of human macrophage-like cells with pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, live cell imaging experiments were carried out and time lapse fluorescence microscopy videos were generated, which are presented here. DATA DESCRIPTION: The time lapse fluorescence microscopy data revealed new insights in the interaction of corynebacteria with human macrophage-like THP-1 cells. In contrast to uninfected cells and infections with non-pathogenic C. glutamicum used as a control, pathogenic C. diphtheriae and C. ulcerans showed highly detrimental effects towards human cells and induction of cell death of macrophages.

Myo-inositol-1-phosphate synthase (Ino-1) functions as a protection mechanism in Corynebacterium glutamicum under oxidative stress.

Reactive oxygen species (ROS) generated in aerobic metabolism and oxidative stress lead to macromolecules damage, such as to proteins, lipids, and DNA, which can be eliminated by the redox buffer mycothiol (AcCys-GlcN-Ins, MSH). Myo-inositol-phosphate synthase (Ino-1) catalyzes the first committed step in the synthesis of MSH, thus playing a critical role in the growth of the organism. Although Ino-1s have been systematically studied in eukaryotes, their physiological and biochemical functions remain largely unknown in bacteria. In this study, we report that Ino-1 plays an important role in oxidative stress resistance in the gram-positive Actinobacteria Corynebacterium glutamicum. Deletion of the ino-1 gene resulted in a decrease in cell viability, an increase in ROS production, and the aggravation of protein carbonylation levels under various stress conditions. The physiological roles of Ino-1 in the resistance to oxidative stresses were corroborated by the absence of MSH in the Δino-1 mutant. In addition, we found that the homologous expression of Ino-1 in C. glutamicum yielded a functionally active protein, while when expressed in Escherichia coliBL21(DE3), it lacked measurable activity. An examination of the molecular mass (Mr) suggested that Ino-1 expressed in E. coliBL21(DE3) was not folded in a catalytically competent conformation. Together, the results unequivocally showed that Ino-1 was important for the mediation of oxidative resistance by C. glutamicum.

Transcriptomic analysis for elucidating the physiological effects of 5-aminolevulinic acid accumulation on Corynebacterium glutamicum.

5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthetic pathway, attracts close attention among researchers because of its potential applications to cancer treatment and agriculture. Overexpression of heterologous hemA and hemL, which encode glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase, respectively, in Corynebacterium glutamicum produces ALA, although whether ALA accumulation causes unintended effects on the host is unknown. Here we used an integrated systems approach to compare global transcriptional changes induced by the expression of hemA and hemL. Metabolic pathway such as glycolysis was inhibited, but tricarboxylic acid cycle, pentose phosphate pathway, and respiratory metabolism were stimulated. Moreover, the transcriptional levels of certain genes involved in heme biosynthesis were up-regulated, and the data implicate the two-component system (TCS) HrrSA was involved in the regulation of heme synthesis. With these understandings, it is proposed that ALA accumulation stimulates heme synthesis pathway and respiratory metabolism. Our study illuminates the physiological effects of overexpressing hemA and hemL on the phenotype of C. glutamicum and contributes important insights into the regulatory mechanisms of the heme biosynthetic pathways.

Improvement of the intracellular environment for enhancing l-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply.

l-arginine, a semi essential amino acid, is an important amino acid in food flavoring and pharmaceutical industries. Its production by microbial fermentation is gaining more and more attention. In previous work, we obtained a new l-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through mutation breeding. In this work, we enhanced l-arginine production through improvement of the intracellular environment. First, two NAD(P)H-dependent H2O2-forming flavin reductases Frd181 (encoded by frd1 gene) and Frd188 (encoded by frd2) in C. glutamicum were identified for the first time. Next, the roles of Frd181 and Frd188 in C. glutamicum were studied by overexpression and deletion of the encoding genes, and the results showed that the inactivation of Frd181 and Frd188 was beneficial for cell growth and l-arginine production, owing to the decreased H2O2 synthesis and intracellular reactive oxygen species (ROS) level, and increased intracellular NADH and ATP levels. Then, the ATP level was further increased by deletion of noxA (encoding NADH oxidase) and amn (encoding AMP nucleosidase), and overexpression of pgk (encoding 3-phosphoglycerate kinase) and pyk (encoding pyruvate kinase), and the l-arginine production and yield from glucose were significantly increased. In fed-batch fermentation, the l-arginine production and yield from glucose of the final strain reached 57.3g/L and 0.326g/g, respectively, which were 49.2% and 34.2% higher than those of the parent strain, respectively. ROS and ATP are important elements of the intracellular environment, and l-arginine biosynthesis requires a large amount of ATP. For the first time, we enhanced l-arginine production and yield from glucose through reducing the H2O2 synthesis and increasing the ATP supply.

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones.

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35mg/L naringenin and 37mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.

Corynebacterium glutamicum methionine sulfoxide reductase A uses both mycoredoxin and thioredoxin for regeneration and oxidative stress resistance.

Oxidation of methionine leads to the formation of the S and R diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in Actinomycetes. Here, we report that a Corynebacterium glutamicum methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the msrA gene in C. glutamicum resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an S-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditions in vivo, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed.

Enhancing Corynebacterium glutamicum robustness by over-expressing a gene, mshA, for mycothiol glycosyltransferase.

Over-expression of the gene, mshA, coding for mycothiol glycosyl transferase improved the robustness of Corynebacterium glutamicum to various stresses. Intracellular mycothiol (MSH) content was increased by 114 % in WT(pXMJ19-mshA) compared to WT(pXMJ19). Survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to WT(pXMJ19) under stress by H2O2 (40 mM), methylglyoxal (5.8 mM), erythromycin (0.08 mg ml(-1)), streptomycin (0.005 mg ml(-1)), Cd(2+) (0.01 mM), Mn(2+) (2 mM), formic acid (0.05 %), acetic acid (0.15 %), levulinic acid (0.25 %), furfural (7.2 mM), and ethanol (10 % v/v), respectively. Increased MSH content also decreased the concentration of reactive oxygen species in the presence of the above stresses. Our results may open a new avenue for enhancing robustness of industrial bacteria for production of commodity chemicals.

Transcriptional control of the F0F1-ATP synthase operon of Corynebacterium glutamicum: SigmaH factor binds to its promoter and regulates its expression at different pH values.

Corynebacterium glutamicum used in the amino acid fermentation industries is an alkaliphilic microorganism. Its F(0)F(1)-ATPase operon (atpBEFHAGDC) is expressed optimally at pH 9.0 forming a polycistronic (7.5 kb) and a monocistronic (1.2 kb) transcripts both starting upstream of the atpB gene. Expression of this operon is controlled by the SigmaH factor. The sigmaH gene (sigH) was cloned and shown to be co-transcribed with a small gene, cg0877, encoding a putative anti-sigma factor. A mutant deleted in the sigH gene expressed the atpBEFHAGDC operon optimally at pH 7.0 at difference of the wild-type strain (optimal expression at pH 9.0). These results suggested that the SigmaH factor is involved in pH control of expression of the F(0) F(1) ATPase operon. The SigmaH protein was expressed in Escherichia coli fused to the GST (glutathione-S-transferase) and purified to homogeneity by affinity chromatography on a GSTrap HP column. The fused protein was identified by immunodetection with anti-GST antibodies. DNA-binding studies by electrophoretic mobility shift assays showed that the SigH protein binds to a region of the atpB promoter containing the sigmaH recognition sequence (-35)TTGGAT…18nt…GTTA(-10). SigmaH plays an important role in the cascade of control of pH stress in Corynebacterium.

Physiology and global gene expression of a Corynebacterium glutamicum ΔF(1)F(O)-ATP synthase mutant devoid of oxidative phosphorylation.

A mutant of Corynebacterium glutamicum ATCC 13032 with a deletion of the atpBEFHAGDC genes encoding F(1)F(O)-ATP synthase was characterized. Whereas no growth was observed with acetate as sole carbon source, the ΔF(1)F(O) mutant reached 47% of the growth rate and 65% of the biomass of the wild type during shake-flask cultivation in glucose minimal medium. Initially, the mutant strain showed a strongly increased glucose uptake rate accompanied by a high oxygen consumption rate and pyruvate secretion into the medium. When oxygen became limiting, the glucose consumption rate was reduced below that of the wild type and pyruvate was consumed again. The ΔF(1)F(O) mutant had increased levels of b- and d-type cytochromes and a significantly increased proton motive force. Transcription of genes involved in central carbon metabolism was essentially unchanged, whereas genes for cytochrome bd oxidase, pyruvate:quinone oxidoreductase, oxidative stress response, and others showed increased mRNA levels. On the other hand, genes for amino acid biosynthesis and ribosomal proteins as well as many genes involved in transport displayed decreased mRNA levels. Several of the transcriptional changes were reflected at the protein level, but there were also discrepancies between the mRNA and protein levels suggesting some kind of posttranscriptional regulation. The results prove for the first time that F(1)F(O)-ATP synthase and oxidative phosphorylation are in general not essential for growth of C. glutamicum.

Regulation of the nitrate reductase operon narKGHJI by the cAMP-dependent regulator GlxR in Corynebacterium glutamicum.

The Corynebacterium glutamicum anaerobic nitrate reductase operon narKGHJI is repressed by a transcriptional regulator, ArnR, under aerobic conditions. A consensus binding site of the cAMP receptor protein (CRP)-type regulator, GlxR, was recently found upstream of the ArnR binding site in the narK promoter region. Here we investigated the involvement of GlxR and cAMP in expression of the narKGHJI operon in vivo. Electrophoretic mobility shift assays showed that the putative GlxR binding motif in the narK promoter region is essential for the cAMP-dependent binding of GlxR. Promoter-reporter assays showed that mutation in the GlxR binding site resulted in significant reduction of narK promoter activity. Furthermore, a deletion mutant of the adenylate cyclase gene cyaB, which is involved in cAMP synthesis, exhibited a decrease in both narK promoter activity and nitrate reductase activity. These results demonstrated that C. glutamicum GlxR positively regulates narKGHJI expression in a cAMP-dependent manner.

Regulation of the expression of genes involved in NAD de novo biosynthesis in Corynebacterium glutamicum.

Three genes, nadA, nadB, and nadC, involved in NAD de novo biosynthesis are broadly conserved in the genomes of numerous bacterial species. In the genome of Corynebacterium glutamicum, nadA and nadC but not nadB are annotated. The nadA and nadC genes are located in a gene cluster containing two other genes, designated ndnR and nadS herein. ndnR encodes a member of the Nudix-related transcriptional regulator (NrtR) family. nadS encodes a homologue of cysteine desulfurase involved in Fe-S cluster assembly. The gene cluster ndnR-nadA-nadC-nadS is genetically characterized herein. Mutant strains deficient in nadA, nadC, or nadS required exogenous nicotinate for growth, and the nicotinate auxotrophy was complemented by introduction of the corresponding gene in trans, indicating that each of these genes is essential for growth in the absence of an exogenous source of NAD biosynthesis. The results of reverse transcriptase PCR analyses and ndnR promoter-lacZ expression analyses revealed that the expression of ndnR, nadA, nadC, and nadS genes was markedly and coordinately repressed by nicotinate. The expression of these genes was enhanced by the disruption of ndnR, resulting in the loss of the nicotinate-responsive regulation of gene expression. These results suggest that NdnR acts as a transcriptional repressor of NAD de novo biosynthesis genes and plays an essential role in the regulation of the response to nicotinate.

The whcA gene plays a negative role in oxidative stress response of Corynebacterium glutamicum.

In this study, we analyzed the whcA gene from Corynebacterium glutamicum, which codes for a homologue of the WhiB-family of proteins. Deletion of the gene did not affect the growth of the mutant cells, indicating that the whcA gene was not essential under ordinary growth conditions. However, cells overexpressing the protein not only showed retarded growth as compared with the wild-type or the DeltawhcA mutant cells but also showed increased sensitivity to a variety of oxidants, such as diamide, menadione, and hydrogen peroxide. Thioredoxin reductase activity was repressed in the whcA-overexpressing cells, whereas its activity in the DeltawhcA mutant strain was derepressed regardless of the presence of oxidative stress. The whcA gene was constitutively expressed throughout the growth phase and its expression level was not affected by oxidative stress. A set of proteins under the control of whcA were identified by two-dimensional polyacrylamide gel electrophoresis and they were annotated as NADH oxidase, alcohol dehydrogenase, quinone reductase, and cysteine desulfurase. The corresponding genes encoding the identified proteins were not transcribed in DeltasigH mutant cells. Collectively, these data suggest that the whcA gene of C. glutamicum plays a negative role in the sigH-mediated stress response pathway.

Physiological response of Corynebacterium glutamicum to oxidative stress induced by deletion of the transcriptional repressor McbR.

In the present work the metabolic response of Corynebacterium glutamicum to deletion of the global transcriptional regulator McbR, which controls, e.g. the expression of enzymes of L-methionine and L-cysteine biosynthesis and sulfur assimilation, was studied. Several oxidative stress proteins were significantly upregulated among about 40 proteins in response to deletion of McbR. Linked to this oxidative stress, the mutant exhibited a 50 % reduced growth rate, a 30 % reduced glucose uptake rate and a 30 % reduced biomass yield. It also showed metabolic flux rerouting in response to the deletion. NADPH metabolism was strongly altered. In contrast to the wild-type, the deletion strain supplied significantly more NADPH than required for anabolism, indicating the activity of additional NADPH-consuming reactions. These involved enzymes of oxidative stress protection. Through redirection of metabolic carbon flux in the central catabolism, including a 40 % increased tricarboxylic acid (TCA) cycle flux, the mutant revealed an enhanced NADPH supply to provide redox power for the antioxidant systems. This, however, was not sufficient to compensate for the oxidative stress, as indicated by the drastically disturbed redox equilibrium. The NADPH/NADP+ ratio in C. glutamicum DeltamcbR was only 0.29, and thus much lower than that of the wild-type (2.35). Similarly, the NADH/NAD+ ratio was substantially reduced from 0.18 in the wild-type to 0.08 in the mutant. Deletion of McbR is regarded as a key step towards biotechnological L-methionine overproduction in C. glutamicum. C. glutamicum DeltamcbR, however, did not overproduce L-methionine; this was very likely linked to the low availability of NADPH. Since oxidative stress is often observed in industrial production processes, engineering of NADPH metabolism could be a general strategy for improvement of production strains. Unlike the wild-type, C. glutamicum DeltamcbR contained large granules with high phosphorus content. The storage of these energy-rich polyphosphates is probably the result of a large excess of formation of ATP, as revealed by estimation of the underlying fluxes linked to energy metabolism.

Corynebacterium glutamicum sigmaE is involved in responses to cell surface stresses and its activity is controlled by the anti-sigma factor CseE.

In this study, we demonstrate that sigma(E), an alternative sigma factor of Corynebacterium glutamicum, is involved in cell surface stresses. Cells in which the sigE gene was deleted evidenced increased sensitivity to magnesium deficiency, as well as to SDS, lysozymes, EDTA and heat. We utilized physiological analyses to show that the downstream gene, designated cseE, encodes an anti-sigma factor. The retarded growth of the cseE mutant cells under ordinary growth conditions could be recovered by an additional deletion of sigE encoding sigma(E). Under stress conditions, the phenotype of the cseE-overexpressing cells mimicked that of the sigE mutant. The sigE and cseE genes were transcribed into a single transcript, and gene transcription was stimulated by heat. The SigE and CseE proteins interacted physically in vitro, in the form of glutathione S-transferase (GST) and maltose binding protein (MBP) fusion proteins, respectively. 2D-PAGE analysis of the wild-type and mutant crude extracts showed that the sigE mutant failed to synthesize a 34 kDa polypeptide that was normally induced in wild-type cells grown under heat (or SDS)-stressed conditions. The protein turned out to be expressed from ORF NCgl1070 and showed similarity to methyltransferases which may confer resistance to antibiotics. Accordingly, the sigE mutant evidenced extreme sensitivity to antibiotics, including nalidixic acid, penicillin and vancomycin. Finally, we present a discussion of the possible role of the sigE and cseE genes in the acclimation of C. glutamicum to cell surface stress conditions.

Osmolality, temperature, and membrane lipid composition modulate the activity of betaine transporter BetP in Corynebacterium glutamicum.

The gram-positive soil bacterium Corynebacterium glutamicum, a major amino acid-producing microorganism in biotechnology, is equipped with several osmoregulated uptake systems for compatible solutes, which is relevant for the physiological response to osmotic stress. The most significant carrier, BetP, is instantly activated in response to an increasing cytoplasmic K(+) concentration. Importantly, it is also activated by chill stress independent of osmotic stress. We show that the activation of BetP by both osmotic stress and chill stress is altered in C. glutamicum cells grown at and adapted to low temperatures. BetP from cold-adapted cells is less sensitive to osmotic stress. In order to become susceptible for chill activation, cold-adapted cells in addition needed a certain amount of osmotic stimulation, indicating that there is cross talk of these two types of stimuli at the level of BetP activity. We further correlated the change in BetP regulation properties in cells grown at different temperatures to changes in the lipid composition of the plasma membrane. For this purpose, the glycerophospholipidome of C. glutamicum grown at different temperatures was analyzed by mass spectrometry using quantitative multiple precursor ion scanning. The molecular composition of glycerophospholipids was strongly affected by the growth temperature. The modulating influence of membrane lipid composition on BetP function was further corroborated by studying the influence of artificial modulation of membrane dynamics by local anesthetics and the lack of a possible influence of internally accumulated betaine on BetP activity.

An experimental comparison of respiration measuring techniques in fermenters and shake flasks: exhaust gas analyzer vs. RAMOS device vs. respirometer.

Respiration measurement is applied as a universal tool to determine the activity of biological systems. The measurement techniques are difficult to compare, due to the vast variety of devices and analytical procedures commonly in use. They are used in fields as different as microbiology, gene engineering, toxicology, and industrial process monitoring to observe the physiological activity of living systems in environments as diverse as fermenters, shake flasks, lakes and sewage plants. A method is introduced to determine accuracy, quantitation limit, range and precision of different respiration measurement devices. Corynebacterium glutamicum cultures were used to compare an exhaust gas analyzer (EGA), a RAMOS device (respiration measurement in shake flasks) and a respirometer. With all measuring devices it was possible to determine the general culture characteristics. The EGA and the RAMOS device produced almost identical results. The scatter of the respirometer was noticeably higher. The EGA is the technique of choice, if the reaction volume is high or a short reaction time is required. The possibility to monitor cultures simultaneously makes the RAMOS device an indispensable tool for media and strain development. If online monitoring is not compulsive, the respiration of the investigated microbial system extremely low, or the sample size small, a respirometer is recommended.

Osmosensing by bacteria.

Osmosensors are proteins that sense environmental osmotic pressure. They mediate or direct osmoregulatory responses that allow cells to survive osmotic changes and extremes. Bacterial osmosensing transporters sense high external osmotic pressure and respond by mediating organic osmolyte uptake, hence cellular rehydration. Detailed studies of osmosensing transporters OpuA, BetP, and ProP suggest that they sense and respond to different osmotic pressure-dependent cellular properties. These studies also suggest that each protein has a cytoplasmic osmosensory or osmoregulatory domain, but that these domains differ in structure and function. It is not yet clear whether each transporter represents a distinct osmosensory mechanism or whether different research groups are approaching the same mechanism by way of different paths. Principles emerging from this research will apply to other osmosensors, including those that initiate signal transduction cascades in prokaryotes and eukaryotes.

Functional analysis of L-serine O-acetyltransferase from Corynebacterium glutamicum.

We report here the function of L-serine O-acetyltransferase (SAT) from the glutamic acid-producing bacterium Corynebacterium glutamicum. Based on the genome sequence of C. glutamicum and the NH(2)-terminal amino-acid sequence, the gene encoding SAT (cysE) was cloned and expressed in C. glutamicum. Deletion analysis of the 5'-noncoding region showed a putative -10 region ((-27)TTAAGT(-22) or (-26)TAAGTC(-21)) and a possible ribosome-binding site ((-12)AGA(-10)) just upstream from the start codon. We found that the SAT activity was sensitive to feedback inhibition by L-cysteine, and that SAT synthesis was repressed by L-methionine. Further, cysE-disrupted cells showed L-cysteine auxotrophy, indicating that C. glutamicum synthesizes L-cysteine from L-serine via O-acetyl-L-serine through the pathway involving SAT and O-acetyl-L-serine sulfhydrylase in the same manner as Escherichia coli.