Identification of succinate exporter in Corynebacterium glutamicum and its physiological roles under anaerobic conditions

Corynebacterium glutamicum produces succinate from glucose via the reductive tricarboxylic acid cycle under microaerobic and anaerobic conditions. We identified a NCgl2130 gene of C. glutamicum as a novel succinate exporter that functions in succinate production, and designated sucE1. sucE1 expression levels were higher under microaerobic conditions than aerobic conditions, and overexpression or disruption of sucE1 respectively increased or decreased succinate productivity during fermentation. Under microaerobic conditions, the sucE1 disruptant sucE1Δ showed 30% less succinate productivity and a lower sugar-consumption rate than the parental strain. Under anaerobic conditions, succinate production by sucE1Δ ceased. The intracellular succinate and fructose-1,6-bisphosphate levels of sucE1Δ under microaerobic conditions were respectively 1.7-fold and 1.6-fold higher than those of the parental strain, suggesting that loss of SucE1 function caused a failure of succinate removal from the cells, leading to intracellular accumulation that inhibited upstream sugar metabolism. Homology and transmembrane helix searches identified SucE1 as a membrane protein belonging to the aspartate:alanine exchanger (AAE) family. Partially purified 6x-histidine-tagged SucE1 (SucE1-[His]₆) reconstituted in succinate-loaded liposomes clearly demonstrated counterflow and self-exchange activities for succinate. Together, these findings suggest that sucE1 encodes a novel succinate exporter that is induced under microaerobic conditions, and is important for succinate production under both microaerobic and anaerobic conditions.     

Ca requirement for aerobic nitrogen fixation by heterocystous blue-green algae

The requirement of Ca²⁺ for growth and nitrogen fixation has been investigated in two strains of heterocystous blue-green algae (Anabaena sp. and Anabaena ATCC 33047). With combined nitrogen (nitrate or ammonium) or with N₂ under microaerobic conditions, Ca²⁺ was not required for growth, at least in concentrations greater than traces. In contrast, Ca²⁺ was required as a macronutrient for growth and nitrogen fixation with air as the nitrogen source. Addition of Ca²⁺ to an aerobic culture without Ca²⁺ promoted, after a lag of several hours, development of nitrogenase activity and cell growth. Provision of air to a microaerobic culture in the absence of Ca²⁺ promoted a drastic drop in nitrogenase activity, which rapidly recovered its initial level upon restoration of microaerobic conditions. Development of nitrogenase activity in response to either Ca²⁺ or low oxygen tension was dependent on de novo protein synthesis. The role of Ca²⁺ seems to be related to protection of nitrogenase from inactivation, by conferring heterocysts resistance to oxygen.     

The Cgl1281-encoding putative transporter of the cation diffusion facilitator family is responsible for alkali-tolerance in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Mutants of Corynebacterium glutamicum that were unable to grow under mild alkaline pH conditions were isolated by mutagenesis. Strain AL-43 exhibiting the highest sensitivity to alkaline pH among the mutants was selected and used to clone a DNA fragment that could complement the phenotype. Sequencing and subcloning of the cloned 4.0-kb EcoRI DNA fragment showed that the Cgl1281 gene was responsible for the complementation. The deduced amino acid sequence of Cgl1281 was found to show significant sequence similarity with CzcD, a Me²⁺/H⁺(K⁺) antiporter, from Bacillus subtilis and also possess the features of the cation diffusion facilitator (CDF) family: the presence of 6 putative transmembrane segments and a signature sequence, indicating that the gene product is a member of the CDF family. Chromosomal disruption of the Cgl1281 rendered C. glutamicum cells sensitive to alkaline pH as well as cobalt, while expression of the gene from a plasmid restored alkali-tolerance to the wild-type level and also led to increased cobalt resistance. These results demonstrated that the putative transporter of the CDF family mediates resistance to cobalt and also plays a physiological role in alkaline pH tolerance in C. glutamicum.     

Structure and function of cytidine monophosphate kinase from Yersinia pseudotuberculosis,essential for virulence but not for survival

The need for new antibiotics has become pressing in light of the emergence of antibiotic-resistant strains of human pathogens. Yersinia pestis, the causative agent of plague, is a public health threat and also an agent of concern in biodefence. It is a recently emerged clonal derivative of the enteric pathogen Yersinia pseudotuberculosis. Previously, we developed a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. One such target was cytidine monophosphate (CMP) kinase, which is an essential gene in some organisms. Previously, we had thought CMP kinase was essential for Y. pseudotuberculosis, but by modification of the mutagenesis approach, we report here the production and characterization of a Δcmk mutant. The isogenic mutant had a growth defect relative to the parental strain, and was highly attenuated in mice. We have also elucidated the structure of the CMP kinase to 2.32 Å, and identified three key residues in the active site that are essential for activity of the enzyme. These findings will have implications for the development of novel CMP kinase inhibitors for therapeutic use.     

Utilization of the tricarboxylic acid cycle, a reactor design criterion for the microaerobic production of 2,3-butanediol

A new parameter, the relative utilization of tricarboxylic acid (TCA) cycle beta, is introduced to quantitatively account for the involvement of fermentation pathways and TCA cycle in the utilization of oxygen under oxygen-limiting (microaerobic) conditions. With the facultative anaerobe Enterobacter aerogenes, which produces 2,3-butanediol, a method is proposed to calculate beta from measurement of metabolites and exhaust gas. In continuous culture beta was found to be small under oxygen limitation, indicating that the fermentation pathways were preferred over the TCA cycle and oxygen was almost entirely consumed through oxidation of reduced nicotinamide adenine dinucleotide (NADH(2)) released by fermentation under these conditions. The increase of beta at high oxygen supply revealed a saturation of oxygen utilization through fermentation pathways. It could be concluded that, for the optimal performance of a microaerobic culture, oxygen uptake rate must be kept at such a level that as much NADH(2) as possible from fermentation pathways is oxidized by oxygen, and at the same time the utilization of TCA cycle is kept at a minimum. As the dynamics of the microaerobic culture can be fast, a significant effect of reactor hydrodynamics, i.e., mixing, on the overall performance can be expected. This was confirmed experimentally, and the parameter beta proved to be a useful reactor design criterium for the microaerobic cultivation. (c) 1992 John Wiley & Sons, Inc.     

Relationship between culture redox potential and culture fluorescence inCorynebacterium glutamicum

Summary Transient experiments were performed during a batch fermentation ofCorynebacterium glutamicum ATCC 14296 in which the flow of air to the reactor was cut off. The fluorescence of the culture was observed to vary linearly with the culture redox potential during the transient. This observation was made below the sensitivity limit of a dissolved oxygen probe. It appears that redox potential in conjunction with fluorescence measurements are of utility in studying extra- and intracellular reduction states, particularly in the microaerobic regime.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).     

Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum.     

Microbial production of L -glutamate and L -glutamine by recombinant Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb

Vitreoscilla hemoglobin (VHb) gene vgb equipped with a native promoter Pvgb or a tac promoter Ptac was introduced into Corynebacterium glutamicum ATCC14067, respectively. Ptac was proven to be more suitable for expressing VHb protein in higher concentration in both Escherichia coli and C. glutamicum strains compared with the native vgb promoter Pvgb. VHb-expressing C. glutamicum exhibited higher oxygen uptake rate and enhanced cell growth. Recombinant C. glutamicum harboring vgb gene equipped with Ptac promoter produced 23% more l-glutamate in shake-flask culture and grew to 30% more cell density and formed 22% more l-glutamate in fermentor studies compared with the wild-type strain. When a site-directed mutagenesis in which Tyr405 was replaced by a phenylalanine residue (Y405F) was performed on glutamine synthesis gene, recombinant C. glutamicum overexpressing the mutated gene glnA′ was able to produce l-glutamine effectively. Co-expression of vgb and glnA′ genes in C. glutamicum produced 17 g/l l-glutamine in shake flask culture, approximately 30% more than that produced by the recombinant harboring only glnA′ gene. In fermentor cultivation, the recombinant yielded 25% more cells and produced 40.5 g/l l-glutamine. In this study, it was clearly demonstrated that VHb significantly enhanced cell growth, l-glutamate, and l-glutamine production by recombinant C. glutamicum.     

Influence of SigB inactivation on Corynebacterium glutamicum protein secretion

The non-essential Corynebacterium glutamicum sigma factor, sigB, modulates global gene expression during the transition from exponential growth to the stationary phase. Utilizing a signal peptide derived from C. glutamicum R CgR_0949, a sigB disruption mutant able to secrete 3- to 5-fold more green fluorescence protein (GFP) and α-amylase than the wild type strain was isolated. The signal peptide selectively enabled the mutant to produce greater amounts of both proteins, which were in turn secreted in culture medium in greater quantities than previously acknowledged. A peak GFP productivity of 2.8 g/l was attained, representing the highest GFP productivity reported in C. glutamicum to date. CgR_0949 signal sequence length (30 residues), type (Tat) or the target protein identity (GFP or α-amylase) had no measurable effect on the magnitude of the protein accumulation and consequent secretion. It therefore follows that actual experimentation remains the fastest way to identify suitable signal sequences in C. glutamicum. More secretion studies may reveal even greater secretion productivity by C. glutamicum and consequently present an attractive avenue to further enhance the utility of C. glutamicum as an industrial workhorse.     

Monitoring enzyme expression of a branched respiratory chain of <Emphasis Type="Italic">corynebacterium glutamicum</Emphasis> using an EGFP reporter gene

To investigate the expressional control of branched respiratory chain complexes of the amino-acid producing bacterium Corynebacterium glutamicum according to growth conditions, the expression indexes of the ndh, sdh, qcrCAB, ctaCF, ctaD, ctaE, and cydAB genes were estimated under aerobic and microaerobic, and carbon-rich and -poor conditions. The promoter region of each target gene was cloned upstream of the EGFP gene on expression vector pVK6, and the nine reporter constructs were transformed into C. glutamicum ssp. lactofermentum. The cytochrome content of cellular membranes obtained from each growth phase closely corresponded to the expression indexes based on EGFP fluorescence and cell density, indicating that this rapid and convenient method is suitable for analyzing the expression levels of respiratory chain complexes. Using this method, we demonstrated that a reciprocal change in the expression levels of cytochrome bd-type and aa ₃-type oxidases occurs when C. glutamicum cells are held in stationary phase for extended periods.     

Oxygen-Dependent Growth of the Sulfate-Reducing Bacterium Desulfovibrio oxyclinae in Coculture with Marinobacter sp. Strain MB in an Aerated Sulfate-Depleted Chemostat

A chemostat coculture of the sulfate-reducing bacterium Desulfovibrio oxyclinae and the facultatively aerobic heterotroph Marinobacter sp. strain MB was grown for 1 week under anaerobic conditions at a dilution rate of 0.05 h⁻¹. It was then exposed to an oxygen flux of 223 μmol min⁻¹ by gassing the growth vessel with 5% O₂. Sulfate reduction persisted under these conditions, though the amount of sulfate reduced decreased by 45% compared to the amount reduced during the initial anaerobic mode. After 1 week of growth under these conditions, sulfate was excluded from the incoming medium. The sulfate concentration in the growth vessel decreased exponentially from 4.1 mM to 2.5 μM. The coculture consumed oxygen effectively, and no residual oxygen was detected during either growth mode in which oxygen was supplied. The proportion of D. oxyclinae cells in the coculture as determined by in situ hybridization decreased from 86% under anaerobic conditions to 70% in the microaerobic sulfate-reducing mode and 34% in the microaerobic sulfate-depleted mode. As determined by the most-probable-number (MPN) method, the numbers of viable D. oxyclinae cells during the two microaerobic growth modes decreased compared to the numbers during the anaerobic growth mode. However, there was no significant difference between the MPN values for the two modes when oxygen was supplied. The patterns of consumption of electron donors and acceptors suggested that when oxygen was supplied in the absence of sulfate and thiosulfate, D. oxyclinae performed incomplete aerobic oxidation of lactate to acetate. This is the first observation of oxygen-dependent growth of a sulfate-reducing bacterium in the absence of either sulfate or thiosulfate. Cells harvested during the microaerobic sulfate-depleted stage and exposed to sulfate and thiosulfate in a respiration chamber were capable of anaerobic sulfate and thiosulfate reduction.     

Cloning of the Malic Enzyme Gene from Corynebacterium glutamicum and Role of the Enzyme in Lactate Metabolism

Malic enzyme is one of at least five enzymes, known to be present in Corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. To date, no information is available concerning the physiological role of the malic enzyme in this bacterium. The malE gene from C. glutamicum has been cloned and sequenced. The protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. Biochemical characteristics indicate a decarboxylation role linked to NADPH generation. Strains of C. glutamicum in which the malE gene had been disrupted or overexpressed showed no detectable phenotype during growth on either acetate or glucose, but showed a significant modification of growth behavior during lactate metabolism. The wild type showed a characteristic brief period of exponential growth on lactate followed by a linear growth period. This growth pattern was further accentuated in a malE-disrupted strain (ΔmalE). However, the strain overexpressing malE maintained exponential growth until all lactate had been consumed. This strain accumulated significantly larger amounts of pyruvate in the medium than the other strains.     

Stepwise reduction of the culture redox potential allows the analysis of microaerobic metabolism and photosynthetic membrane synthesis in Rhodospirillum rubrum

Bacterial growth under oxygen‐limited (microaerobic) conditions is often accompanied by phenomena of great interest for fundamental research and industrial application. The microaerobic lifestyle of anoxygenic photosynthetic bacteria like Rhodospirillum rubrum harbors such a phenomenon, as it allows the formation of photosynthetic membranes and related interesting products without light. However, due to the technical difficulties in process control of microaerobic cultivations and the limited sensitivity of available oxygen sensors, the analysis of microaerobic growth and physiology is still underrepresented in current research. The main focus of the present study was to establish an experimental set‐up for the systematic study of physiological processes, associated with the growth of R. rubrum under microaerobic conditions in the dark. For this purpose, we introduce a robust and reliable microaerobic process control strategy, which applies the culture redox potential (CRP) for assessing different degrees of oxygen limitation in bioreactor cultivations. To describe the microaerobic growth behavior of R. rubrum cultures for each of these defined CRP reduction steps, basic growth parameters were experimentally determined. Flux variability analysis provided an insight into the metabolic activity of the TCA cycle and implied its connection to the respiratory capacity of the cells. In this context, our results suggest that microaerobic growth of R. rubrum can be described as an oxygen‐activated cooperative mechanism. The present study thus contributes to the investigation of metabolic and regulatory events responsible for the redox‐sensitive formation of photosynthetic membranes in facultative photosynthetic bacteria. Furthermore, the introduced microaerobic cultivation setup should be generally applicable for any microbial system of interest which can be cultivated in common stirred‐tank bioreactors. Biotechnol. Bioeng. 2013; 110: 573–585. © 2012 Wiley Periodicals, Inc.     

The glycosylated cell surface protein Rpf2, containing a resuscitation-promoting factor motif,is involved in intercellular communication of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The genome of Corynebacterium glutamicum ATCC 13032 contains two genes, rpf1 and rpf2, encoding proteins with similarities to the essential resuscitation-promoting factor (Rpf) of Micrococcus luteus. Both the Rpf1 (20.4 kDa) and Rpf2 (40.3 kDa) proteins share the so-called Rpf motif, a highly conserved protein domain of approximately 70 amino acids, which is also present in Rpf-like proteins of other gram-positive bacteria with a high G+C content of the chromosomal DNA. Purification of the C. glutamicum Rpf2 protein from concentrated supernatants, SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified modified Rpf2 variants with increased or reduced mobility when compared with the calculated size of Rpf2. A Western blot-based enzyme immunoassay demonstrated glycosylation of the Rpf2 variants with higher molecular masses. Galactose and mannose were identified as two components of the oligosaccharide portion of the Rpf2 glycoprotein by capillary gas chromatography coupled to mass spectrometry. The Rpf2 protein was localized on the surface of C. glutamicum with the use of immuno-fluorescence microscopy. C. glutamicum strains with defined deletions in the rpf1 or rpf2 gene or simultaneous deletions in both rpf genes were constructed, indicating that the rpf genes are neither individually nor collectively essential for C. glutamicum. The C. glutamicum rpf double mutant displayed slower growth and a prolonged lag phase after transfer of long-stored cells into fresh medium. The addition of supernatant from exponentially growing cultures of the rpf double mutant, the wild type or C. glutamicum strains with increased expression of the rpf1 or rpf2 gene significantly reduced the lag phase of long-stored wild-type and rpf single mutant strains, but addition of purified His-tagged Rpf1 or Rpf2 did not. In contrast, the lag phase of the C. glutamicum rpf double mutant was not affected upon addition of these culture supernatants.     

Effect of NADH dehydrogenase-disruption and over-expression on respiration-related metabolism in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> KY9714

The function of type II NADH dehydrogenase (NDH-2) in Gram-positive Corynebacterium glutamicum was investigated by preparing strains with ndh, the NDH-2 gene, disrupted and over-expressed. Although disruption showed no growth defects on glucose minimum medium, the growth rate of the over-expressed strain was lower compared with its parent, C. glutamicum KY9714. Ndh-disruption and over-expression did not lead to a large change in the respiratory chain and energetics, including the cytochrome components and the H⁺/O ratio. However, in the strain that lacked NDH-2, membrane l-lactate oxidase activity increased, while NDH-2 over-expression led to decreased l-lactate and malate oxidase activities. In addition, relatively high cytoplasmic lactate dehydrogenase (LDH) activity was always present as was malate dehydrogenase, irrespective of NDH-2 level. Furthermore, l-lactate or malate-dependent NADH oxidase activity could be reproduced by reconstitution with the membranes and the cytoplasmic fraction isolated from the disruptant. These results suggest that coupling of LDH and the membrane l-lactate oxidase system, together with the malate-dependent NADH oxidase system, operates to oxidize NADH when the NDH-2 function is defective in C. glutamicum.