Jeella Z. Acedo

Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II

Significance The increasing development of antimicrobial resistance is a major global concern, and there is an urgent need for the development of new antibiotics. We show that the antimicrobial lipopeptide tridecaptin A1 selectively binds to the Gram-negative analogue of peptidoglycan precursor lipid II, disrupting the proton motive force and killing Gram-negative bacteria. We present an example of the selective targeting of Gram-negative lipid II and a binding mode to this peptidoglycan precursor. No persistent resistance develops against tridecaptin A1 in Escherichia coli cells exposed to subinhibitory concentrations of this peptide during a 1-mo period. This study showcases the excellent antibiotic properties of the tridecaptins in an age where new antibiotics that target Gram-negative bacteria are desperately needed. Tridecaptin A1 (TriA1) is a nonribosomal lipopeptide with selective antimicrobial activity against Gram-negative bacteria. Here we show that TriA1 exerts its bactericidal effect by binding to the bacterial cell-wall precursor lipid II on the inner membrane, disrupting the proton motive force. Biochemical and biophysical assays show that binding to the Gram-negative variant of lipid II is required for membrane disruption and that only the proton gradient is dispersed. The NMR solution structure of TriA1 in dodecylphosphocholine micelles with lipid II has been determined, and molecular modeling was used to provide a structural model of the TriA1–lipid II complex. These results suggest that TriA1 kills Gram-negative bacteria by a mechanism of action using a lipid-II–binding motif.

Solution Structure of Acidocin B, a Circular Bacteriocin Produced by Lactobacillus acidophilus M46.

ABSTRACT Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ∼7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.

Nuclear Magnetic Resonance Solution Structures of Lacticin Q and Aureocin A53 Reveal a Structural Motif Conserved among Leaderless Bacteriocins with Broad-Spectrum Activity

Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless bacteriocins from Lactococcus lactis QU5 and Staphylococcus aureus A53, respectively. These bacteriocins are characterized by the absence of an N-terminal leader sequence and are active against a broad range of Gram-positive bacteria. LnqQ and AucA consist of 53 and 51 amino acids, respectively, and have 47% identical sequences. In this study, their three-dimensional structures were elucidated using solution nuclear magnetic resonance and were shown to consist of four α-helices that assume a very similar compact, globular overall fold (root-mean-square deviation of 1.7 Å) with a highly cationic surface and a hydrophobic core. The structures of LnqQ and AucA resemble the shorter two-component leaderless bacteriocins, enterocins 7A and 7B, despite having low levels of sequence identity. Homology modeling revealed that the observed structural motif may be shared among leaderless bacteriocins with broad-spectrum activity against Gram-positive organisms. The elucidated structures of LnqQ and AucA also exhibit some resemblance to circular bacteriocins. Despite their similar overall fold, inhibition studies showed that LnqQ and AucA have different antimicrobial potency against the Gram-positive strains tested, suggesting that sequence disparities play a crucial role in their mechanisms of action.

Diacylglycerol acyltransferase 1 is regulated by its N-terminal domain in response to allosteric effectors

The N-terminal domain of Brassica diacylglycerol acyltransferase1 binds CoA and acyl-CoA, contributing to feedback inhibition and activation, respectively, of triacylglycerol biosynthesis. Diacylglycerol acyltransferase 1 (DGAT1) is an integral membrane enzyme catalyzing the final and committed step in the acyl-coenzyme A (CoA)-dependent biosynthesis of triacylglycerol (TAG). The biochemical regulation of TAG assembly remains one of the least understood areas of primary metabolism to date. Here, we report that the hydrophilic N-terminal domain of Brassica napus DGAT1 (BnaDGAT11-113) regulates activity based on acyl-CoA/CoA levels. The N-terminal domain is not necessary for acyltransferase activity and is composed of an intrinsically disordered region and a folded segment. We show that the disordered region has an autoinhibitory function and a dimerization interface, which appears to mediate positive cooperativity, whereas the folded segment of the cytosolic region was found to have an allosteric site for acyl-CoA/CoA. Under increasing acyl-CoA levels, the binding of acyl-CoA with this noncatalytic site facilitates homotropic allosteric activation. Enzyme activation, on the other hand, is prevented under limiting acyl-CoA conditions (low acyl-CoA-to-CoA ratio), whereby CoA acts as a noncompetitive feedback inhibitor through interaction with the same folded segment. The three-dimensional NMR solution structure of the allosteric site revealed an α-helix with a loop connecting a coil fragment. The conserved amino acid residues in the loop interacting with CoA were identified, revealing details of this important regulatory element for allosteric regulation. Based on these results, a model is proposed illustrating the role of the N-terminal domain of BnaDGAT1 as a positive and negative modulator of TAG biosynthesis.

Bacillus amyloliquefaciens ssp. plantarum F11 isolated from Algerian salty lake as a source of biosurfactants and bioactive lipopeptides

In this study, we identified a new Bacillus strain isolated from an Algerian salty lake that produces metabolites that are active against Gram-positive and Gram-negative bacteria, as well as fungal pathogens. The draft genome sequence of the strain is presented herein. Genome sequence analysis identified the strain to be B. amyloliquefaciens subspecies plantarum F11, and showed that the strain carries the gene clusters for the production of a number of bioactive and surface-active compounds. These include the lipopeptides surfactin and fengycin, antibacterial polyketides macrolactin and bacillaene, and a putative novel lanthipeptide, among others. Through an activity-guided purification method using hydrophobic interaction chromatographic techniques, we confirmed the ability of the strain to produce fengycin lipopeptides. The identities of the isolated fengycin homologs were ascertained through tandem mass spectrometry.

Identification and three‐dimensional structure of carnobacteriocin XY, a class IIb bacteriocin produced by Carnobacteria

In this study, we report that CbnX (33 residues) and CbnY (29 residues) comprise a class IIb (two‐component) bacteriocin in Carnobacteria. Individually, CbnX and CbnY are inactive, but together act synergistically to exert a narrow spectrum of activity. The structures of CbnX and CbnY in structure‐inducing conditions were determined and strongly resemble other class IIb bacteriocins (i.e., LcnG, PlnEF, PlnJK). CbnX has an extended, amphipathic α‐helix and a flexible C terminus. CbnY has two α‐helices (one hydrophobic, one amphipathic) connected by a short loop and a cationic C terminus. CbnX and CbnY do not appear to interact directly and likely require a membrane‐bound receptor to facilitate formation of the bacteriocin complex. This is the first class IIb bacteriocin reported for Carnobacteria.

Insights into the draft genome sequence of bioactives-producing Bacillus thuringiensis DNG9 isolated from Algerian soil-oil slough

Bacillus thuringiensis is widely used as a bioinsecticide due to its ability to form parasporal crystals containing proteinaceous toxins. It is a member of the Bacillus cereus sensu lato, a group with low genetic diversity but produces several promising antimicrobial compounds. B. thuringiensis DNG9, isolated from an oil-contaminated slough in Algeria, has strong antibacterial, antifungal and biosurfactant properties. Here, we report the 6.06 Mbp draft genome sequence of B. thuringiensis DNG9. The genome encodes several gene inventories for the biosynthesis of bioactive compounds such as zwittermycin A, petrobactin, insecticidal toxins, polyhydroxyalkanoates and multiple bacteriocins. We expect the genome information of strain DNG9 will provide another model system to study pathogenicity against insect pests, plant diseases, and antimicrobial compound mining and comparative phylogenesis among the Bacillus cereus sensu lato group.

The expanding structural variety among bacteriocins from Gram-positive bacteria

Bacteria use various strategies to compete in an ecological niche, including the production of bacteriocins. Bacteriocins are ribosomally synthesized antibacterial peptides, and it has been postulated that the majority of Gram-positive bacteria produce one or more of these natural products. Bacteriocins can be used in food preservation and are also considered as potential alternatives to antibiotics. The majority of bacteriocins from Gram-positive bacteria had been traditionally divided into two major classes, namely lantibiotics, which are post-translationally modified bacteriocins, and unmodified bacteriocins. The last decade has seen an expanding number of ribosomally synthesized and post-translationally modified peptides (RiPPs) in Gram-positive bacteria that have antibacterial activity. These include linear azol(in)e-containing peptides, thiopeptides, bottromycins, glycocins, lasso peptides and lipolanthines. In addition, the three-dimensional (3D) structures of a number of modified and unmodified bacteriocins have been elucidated in recent years. This review gives an overview on the structural variety of bacteriocins from Gram-positive bacteria. It will focus on the chemical and 3D structures of these peptides, and their interactions with receptors and membranes, structure-function relationships and possible modes of action.