The cssR gene of Corynebacterium glutamicum plays a negative regulatory role in stress responses.

BACKGROUND: CssR, the product of the Corynebacterium glutamicum ncgl1578 gene cotranscribed with ncgl1579, is a TetR (tetracycline regulator) family repressor. Although many TetR-type regulators in C. glutamicum have been extensively described, members of the TetR family involved in the stress response remain unidentified. RESULTS: In this study, we found that CssR regulated the transcription of its own gene and the ncgl1576-ncgl1577 operon. The ncgl1576-ncgl1577 operon, which is located upstream of cssR in the orientation opposite that of the cssR operon, encodes an ATP-binding cassette (ABC), some of which are involved in the export of a wide range of antimicrobial compounds. The cssR-deletion (ΔcssR) mutant displayed increased resistance to various stresses. An imperfect palindromic motif (5'-TAA(G)TGN13CA(G)TTA-3'; 25 bp) located at the intergenic region between cssR and ncgl1577 was identified as the sole binding site for CssR. Expression of cssR and ncgl1577 was induced by antibiotics and heavy metals but not H2O2 or diamide, and the DNA-binding activity of CssR was impaired by antibiotics and heavy metals but not H2O2. Antibiotics and heavy metals caused CssR dissociation from target gene promoters, thus derepressing their transcription. Oxidant treatment neither altered the conformation of CssR nor modified its cysteine residues, indicating that the cysteine residues in CssR have no redox activity. In the ΔcssR mutant strain, genes involved in redox homeostasis also showed increased transcription levels, and the NADPH/NADP+ ratio was higher than that of the parental strain. CONCLUSION: The stress response mechanism of CssR in C. glutamicum is realized via ligand-induced conformational changes of the protein, not via cysteine oxidation-based thiol modification. Moreover, the crucial role of CssR in the stress response was demonstrated by negatively controlling the expression of the ncgl1576-ncgl1577 operon, its structural gene, and/or redox homeostasis-related genes.

Diosmetin ameliorate type 2 diabetic mellitus by up-regulating Corynebacterium glutamicum to regulate IRS/PI3K/AKT-mediated glucose metabolism disorder in KK-Ay mice.

BACKGROUND AND PURPOSE: Diosmetin (Dios), a flavonoid compound with multiple pharmacological activities. However, fewer studies have reported its effects on type 2 diabetic mellitus (T2DM). Here, we address the effect of Dios on glucose metabolism and gut microbiota in KK-Ay diabetic mice. METHOD: Wild type C57BL/6 J mice or diabetic KK-Ay mice were treated with vehicle or Dios for one month. The ELISA kit and fluorescence microscope system were respectively employed to the evaluation of serum biochemical indicators and histopathological changes. Liver RNA-Seq and western blot were used to reveal the key signaling pathway. The effects of Dios on gut microbiota was investigated by the 16S rRNA gene sequencing, as well as the relationship between Dios and C. glu on glucose metabolism was explored with the C. glu transplantation. RESULTS: Dios treatment significantly decreased blood glucose and increased serum insulin concentrations. RNA-Seq analysis found that the underlying action mechanism of Dios on T2DM was via modulating glucose metabolism, which was proved by up-regulating IRS/PI3K/AKT signaling pathway to promote glycogen synthesis and GLUT4 translocation. Besides, Dios treatment reshaped the unbalanced gut microbiota by suppressing the ratio of Firmicutes/Bacteroidetes and markedly increasing the richness of C. glu. Moreover, treatment with C. glu and Dios together could markedly ameliorate glucose metabolism by up-regulating IRS/PI3K/AKT signaling pathway to promote glycogen synthesis and GLUT4 translocation. CONCLUSIONS: Dios treatment remarkably ameliorated glucose metabolism in KK-Ay diabetic mice by the regulation of C. glu via IRS/PI3K/AKT signaling pathway and reshaped the unbalanced gut microbiota. Our study provided evidence for the application of Dios to the treatment of T2DM.

Multistep optimization of a cell-penetrating peptide towards its antimicrobial activity.

Multidrug resistant (MDR) bacteria have adapted to most clinical antibiotics and are a growing threat to human health. One promising type of candidates for the everlasting demand of new antibiotic compounds constitute antimicrobial peptides (AMPs). These peptides act against different types of microbes by permeabilizing pathogen cell membranes, whereas being harmless to mammalian cells. Contrarily, another class of membrane-active peptides, namely cell-penetrating peptides (CPPs), is known to translocate in eukaryotic cells without substantially affecting the cell membrane. Since CPPs and AMPs share several physicochemical characteristics, we hypothesized if we can rationally direct the activity of a CPP towards antimicrobial activity. Herein, we describe the screening of a synthetic library, based on the CPP sC18, including structure-based design to identify the active residues within a CPP sequence and to discover novel AMPs with high activity. Peptides with increased hydrophobicity were tested against various bacterial strains, and hits were further optimized leading to four generations of peptides, with the last also comprising fluorinated amino acid building blocks. Interestingly, beside strong antibacterial activities, we also detected activity in cancer cells, while non-cancerous cells remained unharmed. The results highlight our new candidates, particularly those from generation 4, as a valuable and promising source for the development of future therapeutics with antibacterial activity and beyond.

Effect of sodium dodecyl sulfate on the production of L-isoleucine by the fermentation of Corynebacterium glutamicum.

Corynebacterium glutamicum is a safe and popular industrial microorganism that it is gram-positive bacteria with thick cell walls, which hinder the extracellular secretion of products. Surfactant has good surface or interface activity and can destroy the cell membrane of microorganisms. In this study, the surfactant SDS was used to artificially destroy the cell membrane of Corynebacterium glutamicum, increase the permeability of the cell membrane, and increase the ability of the strain to secrete L-isoleucine. This is the first time that surfactants have been applied to the fermentation of Corynebacterium glutamicum. Results indicated that after optimization, the output of L-isoleucine reached 43.67 g/L, which was 13.01% higher than that without sodium dodecyl sulfate. The yield of the by-products, such as valine, leucine, and alanine, was reduced by 72.30%, 64.30%, 71.70%, respectively. This method can promote the production of L-isoleucine while minimizing the damage of SDS to the strain.

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.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance.

MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to ß-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Identification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae.

Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603-1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division.

An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial microorganism, but the availability of tools for its genetic modification has lagged compared to other model microorganisms such as Escherichia coli. Despite great progress in CRISPR-based technologies, the most feasible genome editing method in C. glutamicum is suicide plasmid-mediated, the editing efficiency of which is low due to high false-positive rates of sacB counter selection, and the requirement for tedious two-round selection and verification of rare double-cross-over events. In this study, an rpsL mutant conferring streptomycin resistance was harnessed for counter selection, significantly increasing the positive selection rate. More importantly, with the aid of high selection efficiencies through the use of antibiotics, namely kanamycin and streptomycin, the two-step verification strategy can be simplified to just one-step verification of the final edited strain. As proof of concept, a 2.5-kb DNA fragment comprising aroGfbr pheAfbr expressing cassettes was integrated into the genome of C. glutamicum, with an efficiency of 20% out of the theoretical 50%. The resulting strain produced 110 mg l-1  l-tyrosine in shake-flask fermentation. This updated suicide plasmid-mediated genome editing system will greatly facilitate genetic manipulations including single nucleotide mutation, gene deletion and gene insertion in C. glutamicum and can be easily applied to other microbes.

Study of the effect of culture mediums on the amino acid metabolites for Corynebacterium glutamicum using high-speed micellar electrokinetic chromatography.

Corynebacterium glutamicum (C. glutamicum) is a well-known workhorse for the industrial production of amino acids. Different carbon, nitrogen, and sulfur source may force the bacterium to produce specific metabolites. In this work, a method of high-speed MEKC with LIF detection was developed to rapidly analyze the amino acid metabolites released by C. glutamicum, which is fed with different culture mediums. Corynebacterium glutamicum was cultured in microbial fuel cells to monitor its metabolism process and collect its metabolites. In the CE system, a microliter-scale sample reservoir was designed and applied to perform tiny volume sample injection. With the assistance of microwave, the derivatization time for amino acids with FITC was greatly shortened to 6 min. Under the optimized condition, the eight candidate amino acids of metabolites could be rapidly separated within 2 min. The whole analysis process for real samples, from sampling to determination, could be shortened to less than 10 min. The results showed that C. glutamicum could produce additional l-lysine and l-valine as the metabolites when fed with glucose and l-methionine, respectively. The method proved that culture mediums used to feed C. glutamicum had great effect on the bacterium's metabolites.

Bacterial Cell Wall Modification with a Glycolipid Substrate.

Despite the ubiquity and importance of glycans in biology, methods to probe their structures in cells are limited. Mammalian glycans can be modulated using metabolic incorporation, a process in which non-natural sugars are taken up by cells, converted to nucleotide-sugar intermediates, and incorporated into glycans via biosynthetic pathways. These studies have revealed that glycan intermediates can be shunted through multiple pathways, and this complexity can be heightened in bacteria, as they can catabolize diverse glycans. We sought to develop a strategy that probes structures recalcitrant to metabolic incorporation and that complements approaches focused on nucleotide sugars. We reasoned that lipid-linked glycans, which are intermediates directly used in glycan biosynthesis, would offer an alternative. We generated synthetic arabinofuranosyl phospholipids to test this strategy in Corynebacterium glutamicum and Mycobacterium smegmatis, organisms that serve as models of Mycobacterium tuberculosis. Using a C. glutamicum mutant that lacks arabinan, we identified synthetic glycosyl donors whose addition restores cell wall arabinan, demonstrating that non-natural glycolipids can serve as biosynthetic intermediates and function in chemical complementation. The addition of an isotopically labeled glycan substrate facilitated cell wall characterization by NMR. Structural analysis revealed that all five known arabinofuranosyl transferases could process the exogenous lipid-linked sugar donor, allowing for the full recovery of the cell envelope. The lipid-based probe could also rescue wild-type cells treated with an inhibitor of cell wall biosynthesis. Our data indicate that surrogates of natural lipid-linked glycans can intervene in the cell's traditional workflow, indicating that biosynthetic incorporation is a powerful strategy for probing glycan structure and function.

A simple dual-inducible CRISPR interference system for multiple gene targeting in Corynebacterium glutamicum.

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.

Impaired oxidative stress and sulfur assimilation contribute to acid tolerance of Corynebacterium glutamicum.

The industrial organism Corynebacterium glutamicum is often subjected to acid stress during large-scale fermentation for the production of bio-based chemicals. The capacity of the cells to thrive in acidic environments is a prerequisite for achieving high product yields. In this study, we obtained an acid-adapted strain using an adaptive laboratory evolution strategy. Physiological characterizations revealed that the adapted strain achieved improved cell viability after acid-stress challenge, with a higher cytoplasmic pHin level, a lower intracellular reactive oxygen species (ROS), and an enhanced morphological integrity of the cells, when compared to those of the original control strain. Transcriptome analysis indicated that several important cellular processes were altered in the adapted strain, including sulfur metabolism, iron transport, and central metabolic pathways. Further research displayed that KatA and Dps cooperatively mediated intracellular ROS scavenging, which was required for resistance to low-pH stress in C. glutamicum. Furthermore, the repression of sulfur assimilation by the McbR regulator also contributed to the improvement of acid-stress tolerance. Moreover, two copper chaperone genes cg1328 and cg3292 were found to be involved in promoting cell survival under acid-stress conditions. Finally, a new recombinant C. glutamicum strain with enhanced acid tolerance was generated by the combined overexpression of katA, dps, mcbR, and cg1328, showing 18.4 ± 2.5% higher biomass yields than the wild-type strain under acid-stress conditions. These findings will provide new insights into the understanding and genetic improvement of acid tolerance in C. glutamicum.

The accD3 gene for mycolic acid biosynthesis as a target for improving fatty acid production by fatty acid-producing Corynebacterium glutamicum strains.

We have recently developed Corynebacterium glutamicum strains that produce free fatty acids in culture supernatant due to enhanced fatty acid biosynthesis. Of these producing strains, the basic producer PAS-15 has a defect in the gene for a fatty acid biosynthesis repressor protein, and the advanced producer PCC-6 has two additional mutations to augment the production by strain PAS-15. The aim of the present study was to obtain novel genetic traits for improving fatty acid production by these producers. A new mutant with increased production derived from strain PAS-15 had a missense mutation in the accD3 gene (mutation accD3A433T), which is involved in the biosynthesis of mycolic acids that are cell envelope lipids of C. glutamicum, as the causal mutation. Mutation accD3A433T was verified to reduce the AccD3 enzymatic activity and increase fatty acid production in strain PAS-15 by 1.8-fold. Deletion of the accD3 gene in strain PAS-15, which was motivated by the characteristic of mutation accD3A433T, increased fatty acid production by 3.2-fold. Susceptibility of strain PAS-15 to vancomycin was significantly increased by accD3 gene deletion and by mutation accD3A433T to the intermediate level, suggesting that the cell envelope permeability barrier by mycolic acids is weakened by this engineering. Furthermore, mutation accD3A433T also increased fatty acid production in strain PCC-6 by 1.3-fold. These increased production levels were suggested to be involved not only in the redirection of carbon flux from mycolic acid biosynthesis to fatty acid production but also in the permeability of the cell envelope.

Natural biocide cocktails: Combinatorial antibiotic effects of prodigiosin and biosurfactants.

Bacterial secondary metabolites are naturally produced to prevail amongst competitors in a shared habitat and thus represent a valuable source for antibiotic discovery. The transformation of newly discovered antibiotic compounds into effective drugs often requires additional surfactant components for drug formulation. Nature may also provide blueprints in this respect: A cocktail of two compounds consisting of the antibacterial red pigment prodigiosin and the biosurfactant serrawettin W1 is naturally produced by the bacterium Serratia marcescens, which occurs in highly competitive habitats including soil. We show here a combinatorial antibacterial effect of these compounds, but also of prodigiosin mixed with other (bio)surfactants, against the soil-dwelling bacterium Corynebacterium glutamicum taken as a model target bacterium. Prodigiosin exerted a combinatorial inhibitory effect with all tested surfactants in a disk diffusion assay which was especially pronounced in combination with N-myristoyltyrosine. Minimal inhibitory and bactericidal concentrations (MIC and MBC) of the individual compounds were 2.56 µg/mL prodigiosin and 32 µg/mL N-myristoyltyrosine, and the MIC of prodigiosin was decreased by 3 orders of magnitude to 0.005 µg/mL in the presence of 16 µg/mL N-myristoyltyrosine, indicative of synergistic interaction. Investigation of bacterial survival revealed similar combinatorial effects; moreover, antagonistic effects were observed at higher compound concentrations. Finally, the investigation of microcolony formation under combined application of concentrations just below the MBC revealed heterogeneity of responses with cell death or delayed growth. In summary, this study describes the combinatorial antibacterial effects of microbial biomolecules, which may have ecological relevance by inhibiting cohabiting species, but shall furthermore inspire drug development in the combat of infectious disease.

Glutamine-rich toxic proteins GrtA, GrtB and GrtC together with the antisense RNA AsgR constitute a toxin-antitoxin-like system in Corynebacterium glutamicum.

The Corynebacterium glutamicum R grtA (cgR_2936), grtB (cgR_2934) and grtC (cgR_2933) genes were identified as paralogs encoding glutamine-rich toxic proteins. We also identified a new antisense small RNA AsgR (antisense sRNA for grtA) that overlaps the 3' end of the grtA gene. Single over-expressions of grtA, grtB and grtC resulted in complete inhibition of Escherichia coli cell growth. This growth was rescued by co-expression of AsgR. Similar effects were observed in C. glutamicum, although the toxicities of these proteins were moderate. Inhibition of AsgR transcription resulted in increased levels and prolonged half-lives of grtA, grtB and grtC mRNAs. We also found that the expression levels of grtA, grtB and grtC were increased in an RNase III deletion mutant. Primer extension analysis revealed the RNase III cleavage site to be in the 3' untranslated region (3'-UTR) of the grtA mRNA. The expression levels of grtA, grtB and grtC were increased after exposure to several stresses, including heat shock, treatment with penicillin G, lysozyme or H2 O2 . The deletions of grtABC and asgR genes resulted in decreased survival rate under several stresses. These results indicate that GrtABC and AsgR constitute a type I toxin-antitoxin-like system in C. glutamicum.

Construction of pOGOduet - An inducible, bicistronic vector for synthesis of recombinant proteins in Corynebacterium glutamicum.

The Gram-positive Corynebacterium glutamicum is widely known for its application in the industrial production of amino acids and as a non-pathogenic model organism for cell wall biosynthesis in the group of CMN bacteria. For biotechnological and physiological studies often co-expression of recombinant genes is required, however for C. glutamicum no vector for the independent co-expression of two genes was described. We here created the novel expression vector pOGOduet for C. glutamicum, which carries the ColE1 replicon of E. coli and the pBL1 replicon of C. glutamicum and two independently inducible promoters Ptac and Ptet each followed by unique multiple cloning sites. Functionality of pOGOduet is tested by coexpression of genes for the fluorescent proteins eCFP and mVenus; fluorescence of the reporters varies in dependence of the inducer concentrations present in the culture broth. These experiments demonstrate that the vector pOGOduet fulfills the task for individually inducible expression of two genes of interest in C. glutamicum.

Integrated Analysis of the Transcriptome and Metabolome of Corynebacterium glutamicum during Penicillin-Induced Glutamic Acid Production.

Corynebacterium glutamicum is known for its ability to produce glutamic acid and has been utilized for the fermentative production of various amino acids. Glutamic acid production in C. glutamicum is induced by penicillin. In this study, the transcriptome and metabolome of C. glutamicum is analyzed to understand the mechanism of penicillin-induced glutamic acid production. Transcriptomic analysis with DNA microarray revealed that expression of some glycolysis- and TCA cycle-related genes, which include those encoding the enzymes involved in conversion of glucose to 2-oxoglutaric acid, is upregulated after penicillin addition. Meanwhile, expression of some TCA cycle-related genes, encoding the enzymes for conversion of 2-oxoglutaric acid to oxaloacetic acid, and the anaplerotic reactions decreased. In addition, expression of NCgl1221 and odhI, encoding proteins involved in glutamic acid excretion and inhibition of the 2-oxoglutarate dehydrogenase, respectively, is upregulated. Functional category enrichment analysis of genes upregulated and downregulated after penicillin addition revealed that genes for signal transduction systems are enriched among upregulated genes, whereas those for energy production and carbohydrate and amino acid metabolisms are enriched among the downregulated genes. As for the metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry, the intracellular content of most metabolites of the glycolysis and the TCA cycle decreased dramatically after penicillin addition. Overall, these results indicate that the cellular metabolism and glutamic acid excretion are mainly optimized at the transcription level during penicillin-induced glutamic acid production by C. glutamicum.

Tolerance improvement of Corynebacterium glutamicum on lignocellulose derived inhibitors by adaptive evolution.

Robustness of fermenting strains to lignocellulose derived inhibitors is critical for efficient biofuel and biochemical productions. In this study, the industrial fermenting strain Corynebacterium glutamicum S9114 was evolved for improved inhibitor tolerance using long-term adaptive evolution by continuously transferring into the inhibitors containing corn stover hydrolysate every 24 h, and finally a stably evolved C. glutamicum was obtained after 128 days of serial transfers. The evolved strain exhibited the highly increased conversion rate to the typical lignocellulose derived inhibitors including furfural, 5-hydroxymethylfurfural, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, and acetic acid. Glucose consumption was obviously accelerated, and 22.4 g/L of glutamic acid was achieved in the corn stover hydrolysate, approximately 68.4% greater than that by the original strain. Whole genome re-sequencing revealed various mutations with the potential connection to the improved performance of the evolved strain. Transcriptional analysis further demonstrated that the glucose-PTS transport and the pentose phosphate pathway were significantly upregulated in the evolved strain, which very likely contributed to the accelerated glucose consumption, as well as sufficient NAD(P)H supply for aldehyde inhibitors reduction conversion and thus enhanced the inhibitor tolerance. This study provided important experimental evidences and valuable genetic information for robust strain construction and modification in lignocellulose biorefining processes.

Biological consequences of improving the structural stability of hairpins that have antimicrobial activity.

Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non-naturally occurring AMPs. We have used a backbone-cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity. We focused on beta-hairpin-like structuring features. Improvements to the structure of this peptide reduced the activity of the peptide against gram-negative, Escherichia coli but improved the activity against gram-positive, Corynebacterium glutamicum. Distinctions in structuring effects on gram-negative versus gram-positive activity were also seen in a second peptide system. Structural improvements resulted in a peptide that was more active than the native against gram-positive bacterium but less active against gram-negative bacterium. Our results show that there is not always a correlation between improved hairpin-structuring and activity. Other factors such as the type of bacteria being targeted as well as net positive charge can play a role in the potency of AMPs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria.

Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked; this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome). IMPORTANCE: Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation.