The genetics of lantibiotic biosynthesis

The lantibiotics are a rapidly expanding group of biologically active peptides produced by a variety of Gram-positive bacteria, and are so-called because of their content of the thioether amino acids lanthionine and β-methyllanthionine. These amino acids, and indeed a number of other unusual amino acids found in the lantibiotics, arise following post-translational modification of a ribosomally synthesised precursor peptide. A number of genes involved in the biosynthesis of these highly modified peptides have been identified, including genes encoding the precursor peptide, enzymes responsible for specific amino acid modifications, proteases able to remove the leader peptide, ABC-superfamily transport proteins involved in lantibiotic translocation, regulatory proteins controlling lantibiotic biosynthesis and proteins that protect the producing strain from the action of its own lantibiotic. Analysis of these genes and their products is allowing greater understanding of the complex mechanism(s) of the biosynthesis of these unique peptides.     

N-terminal acetylation in paenibacillin, a novel lantibiotic

N-terminal acetylation was uncovered in paenibacillin, a novel lantibiotic recently reported as a product of Paenibacillus polymyxa OSY-DF. This N-terminal modification is unprecedented among bacteria-derived antimicrobial peptides and further illustrates the broad range of modifications that can occur in lantibiotics. Additionally, the primary structure of paenibacillin has been finally determined unequivocally by the extensive NMR analysis taken together with previous MS/MS results. These analyses revealed the structure of paenibacillin as one of the most post-translationally modified lantibiotics.     

The solution structure of the lantibiotic gallidermin

The 21-peptide amide antibiotic gallidermin is a potential therapeutic against acne disease. It belongs to the class of polycyclic lanthionine and alpha,beta-didehydroamino acids containing polypeptides, which were named "lantibiotics." The structural gene of the recently elucidated lantibiotic gallidermin encodes a precursor peptide containing Ser, Thr, and Cys residues in the C-terminal prolantibiotic part, and an unusually hydrophilic leader peptide. The ribosomally synthesized pregallidermin is posttranslationally modified and processed to a complex peptide antibiotic with four sulfide rings and two unsaturated residues. The complete solution structure of gallidermin was determined in trifluoroethanol: water (95:5) and dimethylsulfoxide by two-dimensional 1H-nmr at 500 MHz, using a combination of double quantum filtered correlated spectroscopy, homonuclear Hartman-Hahn, and nuclear Overhauser enhancement spectroscopy experiments. Using a total number of 152 distance constraints from NOEs and 14 torsional constraints, derived from coupling constants, we obtained a screwlike solution structure of gallidermin. Restrained molecular dynamics simulations yielded a set of five converging structures with an atomic rms difference of 1.7 A for the backbone atoms, not dependent on the starting structure. The spatial structure model is in excellent agreement with the amphiphilic and channel-forming properties of gallidermin on membranes and its tryptic cleavage at the exposed site between residues 13 and 14.     

The lantibiotic mersacidin is an autoinducing peptide

The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.     

C-di-GMP: the dawning of a novel bacterial signalling system

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has come to the limelight as a result of the recent advances in microbial genomics and increased interest in multicellular microbial behaviour. Known for more than 15 years as an activator of cellulose synthase in Gluconacetobacter xylinus, c-di-GMP is emerging as a novel global second messenger in bacteria. The GGDEF and EAL domain proteins involved in c-di-GMP synthesis and degradation, respectively, are (almost) ubiquitous in bacterial genomes. These proteins affect cell differentiation and multicellular behaviour as well as interactions between the microorganisms and their eukaryotic hosts and other phenotypes. While the role of GGDEF and EAL domain proteins in bacterial physiology and behaviour has gained appreciation, and significant progress has been achieved in understanding the enzymology of c-di-GMP turnover, many questions regarding c-di-GMP-dependent signalling remain unanswered. Among these, the key questions are the identity of targets of c-di-GMP action and mechanisms of c-di-GMP-dependent regulation. This review discusses phylogenetic distribution of the c-di-GMP signalling pathway in bacteria, recent developments in biochemical and structural characterization of proteins involved in its metabolism, and biological processes affected by c-di-GMP. The accumulated data clearly indicate that a novel ubiquitous signalling system in bacteria has been discovered.     

Bacterial protein acetylation: the dawning of a new age

Protein acetylation has historically been considered a predominantly eukaryotic phenomenon. Recent evidence, however, supports the hypothesis that acetylation broadly impacts bacterial physiology. To explore more rapidly the impact of protein acetylation in bacteria, microbiologists can benefit from the strong foundation established by investigators of protein acetylation in eukaryotes. To help advance this learning process, we will summarize the current understanding of protein acetylation in eukaryotes, discuss the emerging link between acetylation and metabolism and highlight the best‐studied examples of protein acetylation in bacteria.     

The role of coccolithophore calcification in bioengineering their environment

Coccolithophorids are enigmatic plankton that produce calcium carbonate coccoliths, which over geological time have buried atmospheric CO₂ into limestone, changing both the atmosphere and geology of the Earth. However, the role of coccoliths for the proliferation of these organisms remains unclear; suggestions include roles in anti-predation, enhanced photosynthesis and sun-screening. Here we test the hypothesis that calcification stabilizes the pH of the seawater proximate to the organisms, providing a level of acidification countering the detrimental basification that occurs during net photosynthesis. Such bioengineering provides a more stable pH environment for growth and fits the empirical evidence for changes in rates of calcification under different environmental conditions. Under this scenario, simulations suggest that the optimal production ratio of inorganic to organic particulate C (PIC : POC_prod) will be lower (by approx. 20%) with ocean acidification and that overproduction of coccoliths in a future acidified ocean, where pH buffering is weaker, presents a risk to calcifying cells.     

Chitinases in bioengineering research

Techniques of introduction of foreign genes into the plant genome have been intensely developed in order to directionally improve properties of crops. One of the key directions in plant bioengineering is searching for and analyzing promising genes, in particular, to construct genotypes with high resistance to pathogens and pests. In this review, the use for this purpose of transgenes coding for chitinase family enzymes is considered. Many of these transgenes have proved to be efficient factors for elevating plant resistance to pathogenic fungi.     

Photosynthetic characteristics of the leaves of 'Golden Delicious' appletrees

Abstract Using an open-system leaf chamber, gas exchange measurements on attached leaves of 3-4-year-old Golden Delicious apple trees, made through two seasons, provided data from which the parameters of a leaf photosynthesis model could be derived. The equation is: where C₁ is internal CO₂ concentration and Q_p is the incident quantum flux. There was considerable leaf to leaf variation in the values of the parameters but no clear seasonal trends were established. The initial slope (a) had an average value of about 2.5 × 10^?3 mg μmol^?1? (i.e. quantum yield ～ 0.057); the mesophyll conductance (g_m) was about 3.5 mm s^?1 in extension leaves of trees carrying fruit and 2.5 mm s^?1 in extension leaves of defruited trees. Differences between the values of g_m for spur leaves with and without subtending fruits were not significant; 2.5 mm s^?1 may be used. Dark respiration (R_d, mg m^?2 s^?1) increased exponentially with temperature (T°C); R_d～ 0.006 exp (0.09 T). At saturating photon flux density P_n was linearly related to C_i, up to C_i～ 250 mg m^?3. Optimum temperatures for P_n were slightly different in the two years and were in the range 16-26°C.     

Sublancin is not a lantibiotic but an S-linked glycopeptide

Sublancin is shown to be an S-linked glycopeptide containing a glucose attached to a cysteine residue, establishing a new post-translational modification. The activity of the S-glycosyl transferase was reconstituted in vitro, and the enzyme is shown to have relaxed substrate specificity, allowing the preparation of analogs of sublancin. Glycosylation is essential for its antimicrobial activity.     

In vitro reconstitution and substrate specificity of a lantibiotic protease

Lacticin 481 is a lanthionine-containing bacteriocin (lantibiotic) produced by Lactococcus lactis subsp. lactis. The final steps of lacticin 481 biosynthesis are proteolytic removal of an N-terminal leader sequence from the prepeptide LctA and export of the mature lantibiotic. Both proteolysis and secretion are performed by the dedicated ATP-binding cassette (ABC) transporter LctT. LctT belongs to the family of AMS (ABC transporter maturation and secretion) proteins whose prepeptide substrates share a conserved double-glycine type cleavage site. The in vitro activity of a lantibiotic protease has not yet been characterized. This study reports the purification and in vitro activity of the N-terminal protease domain of LctT (LctT150), and its use for the in vitro production of lacticin 481. The G(-2)A(-1) cleavage site and several other conserved amino acid residues in the leader peptide were targeted by site-directed mutagenesis to probe the substrate specificity of LctT as well as shed light upon the role of these conserved residues in lantibiotic biosynthesis. His 10-LctT150 did not process most variants of the double glycine motif and processed mutants of Glu-8 only very slowly. Furthermore, incorporation of helix-breaking residues in the leader peptide resulted in greatly decreased proteolytic activity by His 10-LctT150. On the other hand, His 10-LctT150 accepted all peptides containing mutations in the propeptide or at nonconserved positions of LctA. In addition, the protease domain of LctT was investigated by site-directed mutagenesis of the conserved residues Cys12, His90, and Asp106. The proteolytic activities of the resulting mutant proteins are consistent with a cysteine protease.     

Solution structure of plantaricin C, a novel lantibiotic.

Plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin, is a lantibiotic. One dehydroalanine, one lanthionine and three beta-methyl-lanthionine residues were found in its 27 amino acid sequence. The plantaricin C structure has two parts: the first comprises the six NH2-terminal residues, four of which are lysines, which confer a strong positive charge to this stretch. The amino acids in positions 7 and 27 form the lanthionine bridge, giving a globular conformation to the rest of the molecule. The beta-methyl-lanthionine bridges are established between residues 12-15, 13-18 and 23-26. This central region has a charge distribution compatible with an amphipathic alpha-helix, through which plantaricin C would become inserted into the membrane matrix of sensitive organisms, provoking the opening of pores and leakage of the cytoplasmic content.     

Immunomodulatory efficacy of nisin—a bacterial lantibiotic peptide

Nisin is a peptide bacteriocin, grouped under the category of lantibiotics. It is naturally produced by Lactococcus lactis to eliminate other competing gram‐positive bacteria from its vicinity. Moreover under certain conditions it is reported to be effective against a broad range of gram‐negative bacteria as well. Thus, it has been widely used as a safe food preservative especially in the dairy industry. Because of its wide‐scale consumption, its effect on eukaryotic cells should be of great concern. Here we examine the immunomodulatory efficacy of nisin in vitro. MTT‐based cytotoxicity assay demonstrated nisin's cytotoxicity on human T‐cell lymphoma Jurkat cells, Molt‐4 cells and freshly cultured human lymphocytes at over 200 µM concentration (IC₅₀225 µM ). The cell death mechanism induced by nisin in all these lymphocyte types was independent of oligonucleosomal DNA fragmentation, as analyzed by agarose gel electrophoresis and comet assay. Additionally, scanning electron microscope and fluorescence microscopy demonstrated the ability of nisin to activate human PMNs in vitro. Nisin‐activated neutrophils extruded intact nuclear chromatin to form NETs, well known for neutralization of virulence factors and extermination of bacterial pathogens. Nisin's presence also elevated neutrophil intracellular superoxide levels, normally produced by activated NADPH oxidase and prerequisite to NET formation. These nisin‐induced responses in cellular representatives of two separate branches of human immune system—adaptive and innate—although leading to cell death, did not include DNA fragmentation. From these findings, we propose that nisin might trigger similar AICD mechanisms in lymphocytes and neutrophils, different from conventional apoptosis which involves DNA fragmentation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.