Kay Marin

Identification of histidine kinases that act as sensors in the perception of salt stress in Synechocystis sp. PCC 6803

In plants and microorganisms, salt stress regulates the expression of large numbers of genes. However, the machinery that senses salt stress remains to be characterized. In this study we identified sensory histidine kinases that are involved in the perception of salt stress in the cyanobacterium Synechocystis sp. strain PCC 6803. A library of strains with mutations in all 43 histidine kinases was screened by DNA microarray analysis of genomewide gene expression under salt stress. The results suggested that four histidine kinases, namely, Hik16, Hik33, Hik34, and Hik41, perceived and transduced salt signals. However, Hik33, Hik34, and Hik16 acting with Hik41 regulated the expression of different sets of genes. These histidine kinases regulated the expression of ≈20% of the salt-inducible genes, whereas the induction of the remaining salt-inducible genes was unaffected by mutations in any of the histidine kinases, suggesting that additional sensory mechanisms might operate in the perception of salt stress. We also used DNA microarrays to investigate the effect of various salts on gene expression. Our results indicate that Hik33 responds to sodium salts and not to KCl, whereas the Hik16/Hik41 system responds only to NaCl.

Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803.

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.

Glucosylglycerol, a Compatible Solute, Sustains Cell Division under Salt Stress

The cyanobacterium Synechocystis sp. PCC 6803 accumulates the compatible solute glucosylglycerol (GG) and sucrose under salt stress. Although the molecular mechanisms for GG synthesis including regulation of the GG-phosphate synthase (ggpS) gene, which encodes GgpS, has been intensively investigated, the role of GG in protection against salt stress remains poorly understood. In our study of the role of GG in the tolerance to salt stress, we found that salt stress due to 450 mm NaCl inhibited cell division and significantly increased cell size in ΔggpS mutant cells, whereas the inhibition of cell division and increase in cell size were observed in wild-type cells at high concentrations of NaCl, such as 800 mm. Electron microscopy revealed that, in ΔggpS cells, separation of daughter cells was incomplete, and aborted division could be recognized by the presence of a structure that resembled a division ring. The addition of GG to the culture medium protected ΔggpS cells against salt stress and reversed the adverse effects of NaCl on cell division and cell size. These observations suggest that GG is important for salt tolerance and thus for the proper division of cells under salt stress conditions.

Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum

Many bacteria can utilize C(4)-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na(+) symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, L-malate, and likely also oxaloacetate in a sodium-dependent manner.

Glucosamine as carbon source for amino acid-producing Corynebacterium glutamicum

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 PTSGlc 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.

The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation

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 Escherichiacoli 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.

Chassis organism from Corynebacterium glutamicum--a top-down approach to identify and delete irrelevant gene clusters.

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.

Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis.

BackgroundThe 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.ResultsHere 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.ConclusionsNovel 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.

Ammonia Triggers Photodamage of Photosystem II in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Ammonia has long been known to be toxic for many photosynthetic organisms; however, the target for its toxicity remains elusive. Here, we show that in the cyanobacterium Synechocystis sp. strain PCC 6803, ammonia triggers a rapid photodamage of photosystem II (PSII). Whereas wild-type cells can cope with this damage by turning on the FtsH2-dependent PSII repair cycle, the FtsH2-deficient mutant is highly sensitive and loses PSII activity at millimolar concentration of ammonia. Ammonia-triggered PSII destruction is light dependent and occurs already at low photon fluence rates. Experiments with monochromatic light showed that ammonia-promoted PSII photoinhibition is executed by wavebands known to directly destroy the manganese cluster in the PSII oxygen-evolving complex, suggesting that the oxygen-evolving complex may be a direct target for ammonia toxicity.

Characterization of the Dicarboxylate Transporter DctA in Corynebacterium glutamicum

Transporters of the dicarboxylate amino acid-cation symporter family often mediate uptake of C(4)-dicarboxylates, such as succinate or l-malate, in bacteria. A member of this family, dicarboxylate transporter A (DctA) from Corynebacterium glutamicum, was characterized to catalyze uptake of the C(4)-dicarboxylates succinate, fumarate, and l-malate, which was inhibited by oxaloacetate, 2-oxoglutarate, and glyoxylate. DctA activity was not affected by sodium availability but was dependent on the electrochemical proton potential. Efficient growth of C. glutamicum in minimal medium with succinate, fumarate, or l-malate as the sole carbon source required high dctA expression levels due either to a promoter-up mutation identified in a spontaneous mutant or to ectopic overexpression. Mutant analysis indicated that DctA and DccT, a C(4)-dicarboxylate divalent anion/sodium symporter-type transporter, are the only transporters for succinate, fumarate, and l-malate in C. glutamicum.