Recent studies have indicated that nitrogen availability can be an important determinant of primary production in freshwater lakes and that herbivore growth can be limited by low dietary nitrogen ...availability. Furthermore, a lack of specific essential nitrogenous biochemicals (such as essential amino acids) might be another important constraint on the fitness of consumers. This might be of particular importance for cladoceran zooplankton, which can switch between two alternative reproductive strategies--the production of subitaneously developing and resting eggs. Here, we hypothesize that both the somatic growth and the type of reproduction of the aquatic keystone herbivore Daphnia is limited by the availability of specific essential amino acids in the diet. In laboratory experiments, we investigated this hypothesis by feeding a high quality phytoplankton organism (Cryptomonas) and a green alga of moderate nutritional quality (Chlamydomonas) to a clone of Daphnia pulex with and without the addition of essential amino acids. The somatic growth of D. pulex differed between the algae of different nutritional quality, but not dependent on the addition of dissolved amino acids. However, in reproduction experiments, where moderate crowding conditions at saturating food quantities were applied, addition of the essential amino acids arginine and histidine (but not lysine and threonine) increased the total number and the developmental stage of subitaneous eggs. While D. pulex did not produce resting eggs on Cryptomonas, relatively high numbers of resting eggs were released on Chlamydomonas. When arginine and histidine were added to the green algal diet, the production of resting eggs was effectively suppressed. This demonstrates the high, but previously overlooked importance of single essential amino acids for the reproductive strategy of the aquatic keystone herbivore Daphnia.
The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and ...proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis.
Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 +/- 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions.
Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense.
Corynebacterium glutamicum
grows with a variety of carbohydrates and carbohydrate derivatives as sole carbon sources; however, growth with glucosamine has not yet been reported. We isolated a ...spontaneous mutant (M4) which is able to grow as fast with glucosamine as with glucose as sole carbon source. Glucosamine also served as a combined source of carbon, energy and nitrogen for the mutant strain. Characterisation of the M4 mutant revealed a significantly increased expression of the
nagB
gene encoding the glucosamine-6P deaminase NagB involved in degradation of glucosamine, as a consequence of a single mutation in the promoter region of the
nagAB-scrB
operon. Ectopic
nagB
overexpression verified that the activity of the NagB enzyme is in fact the growth limiting factor under these conditions. In addition, glucosamine uptake was studied, which proved to be unchanged in the wild-type and M4 mutant strains. Using specific deletion strains, we identified the PTS
Glc
transport system to be responsible for glucosamine uptake in
C. glutamicum
. The affinity of this uptake system for glucosamine was about 40-fold lower than that for its major substrate glucose. Because of this difference in affinity, glucosamine is efficiently taken up only if external glucose is absent or present at low concentrations.
C. glutamicum
was also examined for its suitability to use glucosamine as substrate for biotechnological purposes. Upon overexpression of the
nagB
gene in suitable
C. glutamicum
producer strains, efficient production of both the amino acid
l
-lysine and the diamine putrescine from glucosamine was demonstrated.
For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top‐down approach starting from any existing organism. In this study, ...we initiated the top‐down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome‐reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild‐type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage‐free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application.
The construction of a chassis organism from Corynebacterium glutamicum ATCC 13032 was initiated in a top‐down approach, aiming for the relevant gene set to maintain its fast growth on defined medium. As a result, 26 gene clusters ranging from 3.7 kbp to 49.7 kbp were found to include exclusively irrelevant genes for growth on defined glucose medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage‐free strain, decrease the size of the native genome by about 722 kbp (22 %) to 2561 kbp. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application.
Succinic acid is excreted during anaerobiosis by many bacteria, and manifold applications are known making the biotechnological production of succinate attractive. Although the pathways for succinate ...formation are known, succinate export is not understood in most of the succinate producing bacteria. Here, we present a bioinformatic approach for identification of a putative succinate export system in Corynebacterium glutamicum. The subsequent screening revealed that a mutant in the gene cg2425 is impaired in succinate production or transport under anaerobic conditions. A function of the Cg2425 protein as import system was excluded. In contrast, a role of the Cg2425 protein as succinate export system was indicated by accumulation of increased amounts of internal succinate under anaerobic conditions in a Cg2425-dependent manner and a concomitant impairment of external succinate accumulation. In conclusion, we propose that Cg2425 participates in succinate export in C. glutamicum and suggest the name SucE for the protein.
Based on sequence similarity, the
mscCG gene product of
Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance ...of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an
E
scherichia
coli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant
E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from
E. coli. Deletion and complementation studies in
C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in
C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress.
α-D-Glucosylglycerol (αGG) has beneficial functions as a moisturizing agent in cosmetics and potential as a health food material, and therapeutic agent. αGG serves as compatible solute in various ...halotolerant cyanobacteria such as Synechocystis sp. PCC 6803, which synthesizes αGG in a two-step reaction: The enzymatic condensation of ADP-glucose and glycerol 3-phosphate by GG-phosphate synthase (GGPS) is followed by the dephosphorylation of the intermediate by the GG-phosphate phosphatase (GGPP). The Gram-positive Corynebacterium glutamicum, an industrial workhorse for amino acid production, does not utilize αGG as a substrate and was therefore chosen for the development of a heterologous microbial production platform for αGG.
Plasmid-bound expression of ggpS and ggpP from Synechocystis sp. PCC 6803 enabled αGG synthesis exclusively in osmotically stressed cells of C. glutamicum (pEKEx2-ggpSP), which is probably due to the unique intrinsic control mechanism of GGPS activity in response to intracellular ion concentrations. C. glutamicum was then engineered to optimize precursor supply for αGG production: The precursor for αGG synthesis ADP-glucose gets metabolized by both the glgA encoded glycogen synthase and the otsA encoded trehalose-6-phosphate synthase. Upon deletion of both genes the αGG concentration in culture supernatants was increased from 0.5 mM in C. glutamicum (pEKEx3-ggpSP) to 2.9 mM in C. glutamicum ΔotsA IMglgA (pEKEx3-ggpSP). Upon nitrogen limitation, which inhibits synthesis of amino acids as compatible solutes, C. glutamicum ΔotsA IMglgA (pEKEx3-ggpSP) produced more than 10 mM αGG (about 2 g L
).
Corynebacterium glutamicum can be engineered as efficient platform for the production of the compatible solute αGG. Redirection of carbon flux towards αGG synthesis by elimination of the competing pathways for glycogen and trehalose synthesis as well as optimization of nitrogen supply is an efficient strategy to further optimize production of αGG.
Metal ion uptake is crucial for all living cells and an essential part of cellular bioenergetic homeostasis. In this study the uptake and the impact of the most abundant internal cation, potassium, ...were investigated in
Actinobacteria, a group of high G
+
C Gram-positives with a number of prominent biotechnologically and medically important members. Genome analyses revealed a variety of different potassium uptake systems in this monophyletic group ranging from potassium channels common in virtually all
Actinobacteria to different active carriers that were present predominantly in pathogenic members able to cope with various stress conditions. By applying
Corynebacterium glutamicum as model system we provide experimental evidence that under optimal conditions a potassium channel is sufficient in bacteria for the maintenance of internal pH and membrane potential ensuring survival of cells under stress conditions. Under potassium limitation, however, viability of
C. glutamicum was increased under acidic stress or during desiccation when a functional KtrAB potassium transporter from the pathogen
Corynebacterium jeikeium was heterologously expressed. We provide experimental evidence that the KtrAB mediated enhanced potassium accumulation improved maintenance of internal pH and membrane potential. The results indicate that the occurrence of active potassium transport systems correlates with an improved potassium-dependent bioenergetic homeostasis and survival of bacterial cells under stress conditions.
► Active potassium carriers are present in pathogenic
Actinobacteria predominantly. ► Potassium uptake in the model system
C. glutamicum is facilitated by a channel. ► The heterologous transporter KtrAB improves potassium accumulation. ► Thus pH homeostasis and membrane potential adjustment were improved. ► Thus survival under stress conditions was improved.
PDF
