Escherichia coli exhibits a membrane-related response to a small arginine- and tryptophan-rich antimicrobial peptide

Since multiresistant bacterial strains are more widespread and the victim numbers steadily increase, it is very important to possess a broad bandwidth of antimicrobial substances. Antibiotics often feature membrane-associated effect mechanisms. So, we present a membrane proteomic approach to shed light on the cellular response of Escherichia coli as model organism to the hexapeptide MP196, which is arginine and tryptophan rich. Analyzing integral membrane proteins are still challenging, although various detection strategies have been developed in the past. In particular, membrane proteomics in bacteria have been conducted very little due to the special physical properties of these membrane proteins. To obtain more information on the cellular response of the new compound group of small peptides, the tryptophan- and arginine-rich hexapeptide MP196 was subject to a comprehensive quantitative membrane proteomic study on E. coli by means of metabolic labeling in combination with membrane lipid analyses. This study provides in total 767 protein identifications including 185 integral membrane proteins, from which 624 could be quantified. Among these proteins, 134 were differentially expressed. Thereby, functional groups such as amino acid and membrane biosynthesis were affected, stress response could be observed, and the lipid composition of the membrane was significantly altered. Especially, the strong upregulation of the envelope stress induced protein. Spy indicates membrane damage, as well as the downregulation of the mechano-sensitive channel MscL beside others. Finally, the exceptional downregulation of transport systems strengthens these findings.     

Investigation of ptsG gene in response to xylose utilization in Corynebacterium glutamicum

Corynebacterium glutamicum strains NC-2 were able to grow on xylose as sole carbon sources in our previous work. Nevertheless, it exhibited the major shortcoming that the xylose consumption was repressed in the presence of glucose. So far, regarding C. glutamicum, there are a number of reports on ptsG gene, the glucose-specific transporter, involved in glucose metabolism. Recently, we found ptsG had influence on xylose utilization and investigated the ptsG gene in response to xylose utilization in C. glutamicum with the aim to improve xylose consumption and simultaneously utilized glucose and xylose. The ptsG-deficient mutant could grow on xylose, while exhibiting noticeably reduced growth on xylose as sole carbon source. A mutant deficient in ptsH, a general PTS gene, exhibited a similar phenomenon. When complementing ptsG gene, the mutant ΔptsG-ptsG restored the ability to grow on xylose similarly to NC-2. These indicate that ptsG gene is not only essential for metabolism on glucose but also important in xylose utilization. A ptsG-overexpressing recombinant strain could not accelerate glucose or xylose metabolism. When strains were aerobically cultured in a sugar mixture of glucose and xylose, glucose and xylose could not be utilized simultaneously. Interestingly, the ΔptsG strain could co-utilize glucose and xylose under oxygen-deprived conditions, though the consumption rate of glucose and xylose dramatically declined. It was the first report of ptsG gene in response to xylose utilization in C. glutamicum.     

Metabolic engineering of Corynebacterium glutamicum by synthetic small regulatory RNAs

Journal of Industrial Microbiology & Biotechnology - Corynebacterium glutamicum is an important platform strain that is wildly used in industrial production of amino acids and various other...     

Sodium and proline accumulation in Corynebacterium glutamicum as a response to an osmotic saline upshock

In order to understand the role of the medium osmolality on the metabolism of glumate-producing Corynebacterium glutamicum, effects of saline osmotic upshocks from 0.4 osnol. kg^–1 to 2 osmol. kg^–1 have been investigated on the growth kinetics and the intracellular content of the bacteria. Addition of a high concentration of NaCl after a few hours of batch culture results in a temporary interruption of the cellular growth. Cell growth resumes after about 1 h but at a specific rate that decreases with increasing medium osmolality. Investigation of the intracellular content showed, during the first 30 min following the shock, a rapid but transient influx of sodium ions. This was followed by a strong accumulation of proline, which rose from 5 to 110 mg/g dry weight at the end of the growth phase. A slight accumulation of intracellular glutamate from 60 to 75 mg/g dry weight was also observed. Accordingly, for Corynebacterium glutamicum an increased osmolality in the glutamate and proline synthesis during the growth phase.     

Gene expression of Corynebacterium glutamicum in response to the conditions inducing glutamate overproduction

AIM: The ultimate aim is to elucidate the molecular mechanisms for glutamate overproduction by Corynebacterium glutamicum. METHODS AND RESULTS: Gene expression in response to the conditions inducing glutamate overproduction was investigated by using a DNA microarray technique. Most genes involved in the EMP pathway, the PPP, and the TCA cycle were downregulated, while five genes that were highly upregulated (NCgl0917, NCgl2944, NCgl2945, NCgl2946, and NCgl2975) were identified under all the three conditions for overproduction that are studied here. Gene products of NCgl2944, NCgl2945, and NCgl2946 were highly homologous to each other, did not resemble any other protein, and have remained uncharacterized thus far. The product of NCgl0917 showed a similarity to a few hypothetical and uncharacterized proteins. NCgl2975 was homologous to metal-binding proteins. CONCLUSIONS: The decrease in the activity of 2-oxoglutarate dehydrogenase complex, a key enzyme that is downregulated during glutamate overproduction, can be mainly attributed to the downregulation of odhA and sucB. Five highly upregulated genes were also identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Although fermentative production of glutamate has been carried out for more than 45 years, information on the molecular mechanisms of glutamate overproduction is still limited. This study further elucidates these mechanisms.     

Impact of transport processes in the osmotic response of Corynebacterium glutamicum

Osmoregulation, the adaptation of cells to changes in the external osmolarity, is an important aspect of the bacterial stress response, in particular for a soil bacterium like Corynebacterium glutamicum. Consequently, this organism is equipped with several redundant systems for coping with both hyper- and hypoosmotic stress. For the adaptation to hypoosmotic stress C. glutamicum possesses at least three different mechanosensitive (MS) channels. To overcome hyperosmotic stress C. glutamicum accumulates so-called compatible solutes either by means of biosynthesis or by uptake. Uptake of compatible solutes is in general preferred to de novo synthesis because of lower energy costs. Noticeable, only secondary transporters belonging to the MHS (ProP) or the BCCT-family (BetP, EctP and LcoP) are involved in the uptake of proline, betaine and ectoine. In contrast to Escherichia coli or Bacillus subtilis no ABC-transporters were found catalyzing uptake of compatible solutes. BetP was one of the first examples of the growing group of osmosensory proteins to be analyzed in detail. This transporter is characterized, besides the catalytic activity of betaine uptake, by the ability to sense osmotic changes (osmosensing) and to respond to the extent of osmotic stress by adaptation of transport activity (osmoregulation). BetP detects hyperosmotic stress via an increase in the internal K(+) concentration following a hyperosmotic shift, and thus acts as a chemosensor.     

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 Δ whcA 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 Δ whcA 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 Δ sigH mutant cells. Collectively, these data suggest that the whcA gene of C. glutamicum plays a negative role in the sigH -mediated stress response pathway.     

Corynebacterium glutamicum: a dissection of the PTS

The high-GC Gram-positive actinomycete Corynebacterium glutamicum is commercially exploited as a producer of amino acids that are used as animal feed additives and flavor enhancers. Despite its beneficial role, carbon metabolism and its possible influence on amino acid metabolism is poorly understood. We have addressed this issue by analyzing the phosphotransferase system (PTS), which in many bacteria controls the flux of nutrients and therefore regulates carbon metabolism. The general PTS phosphotransferases enzyme I (EI) and HPr were characterized by demonstration of PEP-dependent phosphotransferase activity. An EI mutant exhibited a pleiotropic negative phenotype in carbon utilization. The role of the PTS as a major sugar uptake system was further demonstrated by the finding that glucose and fructose negative mutants were deficient in the respective enzyme II PTS permease activities. These carbon sources also caused repression of glutamate uptake, which suggests an involvement of the PTS in carbon regulation. The observation that no HPr kinase/phosphatase could be detected suggests that the mechanism of carbon regulation in C. glutamicum is different to the one found in low-GC Gram-positive bacteria.     

Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation

As a response to hyperosmotic stress bacterial cells accumulate compatible solutes by synthesis or by uptake. Beside the instant activation of uptake systems after an osmotic upshift, transport systems show also a second, equally important type of regulation. In order to adapt the pool size of compatible solutes in the cytoplasm to the actual extent of osmotic stress, cells down-regulate solute uptake when the initial osmotic stress is compensated. Here we describe the role of the betaine transporter BetP, the major uptake carrier for compatible solutes in Corynebacterium glutamicum, in this adaptation process. For this purpose, betP was expressed in cells (C. glutamicum and Escherichia coli), which lack all known uptake systems for compatible solutes. Betaine uptake mediated by BetP as well as by a truncated form of BetP, which is deregulated in its response to hyperosmotic stress, was dissected into the individual substrate fluxes of unidirectional uptake, unidirectional efflux and net uptake. We determined a strong decrease of unidirectional betaine uptake by BetP in the adaptation phase. The observed decrease in net uptake was thus mainly due to a decrease of Vmax of BetP and not a consequence of the presence of separate efflux system(s). These results indicate that adaptation of BetP to osmotic compensation is different from activation by osmotic stress and also different from previously described adaptation mechanisms in other organisms. Cytoplasmic K+, which was shown to be responsible for activation of BetP upon osmotic stress, as well as a number of other factors was ruled out as triggers for the adaptation process. Our results thus indicate the presence of a second type of signal input in the adaptive regulation of osmoregulated carrier proteins.     

Triple deletion of clpC,porB, and mepA enhances production of small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide in Corynebacterium glutamicum

Journal of Industrial Microbiology & Biotechnology - In our previous work, a two-plasmid CRISPR/Cas9 system was constructed for genome editing in Corynebacterium glutamicum. To increase the...     

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.     

Corynebacterium glutamicum whcB, a stationary phase-specific regulatory gene

The function of whcB, one of the four whiB homologues of Corynebacterium glutamicum, was assessed. Cells carrying the P(180)-whcB clone, and thus overexpressing the whcB gene, showed retarded growth, probably due to increased sensitivity to oxidants, whereas cells lacking whcB (ΔwhcB) did not. However, growth retardation was not observed in cells with additionally whcE deleted. Furthermore, the ΔwhcE phenotype, characterized by slow growth and sensitivity to oxidants, was reversed in cells carrying P(180)-whcB. Like the whcE gene, which is also known as a whiB homologue, the whcB gene was preferentially expressed in stationary phase. Determination of the genes under regulation of whcB using two-dimensional polyacrylamide gel electrophoresis identified several genes involved in electron transfer reactions that were regulated in cells carrying P(180)-whcB. Collectively, these findings indicate that whcB function requires whcE. Furthermore, whcB and whcE are paralogues but perform distinct regulatory roles during growth under oxidative stress.     

Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.     

Structure of a GTP-dependent bacterial PEP-carboxykinase from Corynebacterium glutamicum

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2₁ with four molecules per asymmetric unit. The 2.3 Å resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guanine binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.