A missense nucleotide substitution in codon -17 in the leader peptide results in the novel HLA-DRB1*04:354 allele.

Circularin A is a circular bacteriocin belonging to a subgroup of the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The post-translational biosynthesis of circular bacteriocins primarily consists of leader cleavage, core peptide circularization, and bacteriocin secretion. However, none of these processes have been fully elucidated due to the complex biosynthesis of such bacteriocins and the lack of homology to the functions of other known biosynthetic enzymes. In this study, we investigated the leader- and terminal residue requirements for the biosynthesis of circularin A by systematic mutational analyses, including the mutational effects of variable leader lengths, as well as site-directed substitutions of residues at positions near the leader cleavage site and the circularization site. Results show that the leader with only one Met residue, the shortest leader possible, is sufficient to produce mature circularin A; helix-forming short-sidechain hydrophobic residues are required at positions Val1 and Ala2 of the N-terminus to form active peptide derivatives, indicating the possible steric hindrance effect at these two positions; and an aromatic residue is required at the C-terminal Tyr69 position to produce a mature circular derivative. However, the requirements for residues at position Ala68 are much more relaxed relative to the positions of Val1 and Ala2, since even substitution with the largest possible residue, i.e., tryptophan, still allows the generation of an active Ala68Trp derivative. Our findings provide new perspectives for the biosynthesis of this short-leader circular bacteriocin, which enables the application of circular bacteriocin biosynthesis in rational modified peptide engineering.

Gene expression strategies ensuring bacterial survival and competitiveness rely on cis- and trans-acting RNA-regulators (riboregulators). Among the cis-acting riboregulators are transcriptional and translational attenuators, and antisense RNAs (asRNAs). The trans-acting riboregulators are small RNAs (sRNAs) that bind proteins or base pairs with other RNAs. This classification is artificial since some regulatory RNAs act both in cis and in trans, or function in addition as small mRNAs. A prominent example is the archetypical, ribosome-dependent attenuator of tryptophan (Trp) biosynthesis genes. It responds by transcription attenuation to two signals, Trp availability and inhibition of translation, and gives rise to two trans-acting products, the attenuator sRNA rnTrpL and the leader peptide peTrpL. In Escherichia coli, rnTrpL links Trp availability to initiation of chromosome replication and in Sinorhizobium meliloti, it coordinates regulation of split tryptophan biosynthesis operons. Furthermore, in S. meliloti, peTrpL is involved in mRNA destabilization in response to antibiotic exposure. It forms two types of asRNA-containing, antibiotic-dependent ribonucleoprotein complexes (ARNPs), one of them changing the target specificity of rnTrpL. The posttranscriptional role of peTrpL indicates two emerging paradigms: (1) sRNA reprograming by small molecules and (2) direct involvement of antibiotics in regulatory RNPs. They broaden our view on RNA-based mechanisms and may inspire new approaches for studying, detecting, and using antibacterial compounds. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Ribosome stalling on ermBL at the tenth codon (Asp) is believed to be a major mechanism of ermB induction by erythromycin (Ery). In this study, we demonstrated that the mechanism of ermB induction by Ery depends not only on ermBL expression but also on previously unreported ermBL2 expression. Introducing premature termination codons in ermBL, we proved that translation of the N-terminal region of ermBL is the key component for ermB induced by Ery, whereas translation of the C-terminal region of ermBL did not affect Ery-induced ermB. Mutation of the tenth codon (Asp10) of ermBL with other amino acids showed that the degree of induction in vivo was not completely consistent with the data from the in vitro toe printing assay. Alanine-scanning mutagenesis of ermBL demonstrated that both N-terminal residues (R7-K11) and the latter part of ermBL (K20-K27) are critical for Ery induction of ermB. The frameshifting reporter plasmid showed that a new leader peptide, ermBL2, exists in the ermB regulatory region. Further, introducing premature termination mutation and alanine-scanning mutagenesis of ermBL2 demonstrated that the N-terminus of ermBL2 is essential for induction by Ery. Therefore, the detailed function of ermBL2 requires further study.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides. Their precursor peptide comprises of an N-terminal leader peptide and a C-terminal core peptide. Here, the leader peptide is crucial for enzyme recognition especially for the modification enzymes and acts furthermore as a secretion signal for the lanthipeptide exporter. The core peptide is the target site for the posttranslational modifications and contains dehydrated amino acids and lanthionine rings. Nisin produced by the Gram-positive bacterium Lactococcus lactis is one of the best-studied lanthipeptides and used as a model system to study their modification and secretion processes. Nisin is secreted as a precursor peptide. Here, we present an in vivo secretion analysis of NisT in the absence of the modification machinery allowing the secretion of leader peptide mutants and their impact solely on the secretion activity of NisT. Additionally, we created leader peptide hybrids to provide new insights, how the secretion is effected by unnatural leader peptides. The focus on the secretion activity of the transporter alone enabled us to determine the recognition site of NisT within the leader peptide of nisin.

EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems.

Bacterial ribosome-dependent attenuators are widespread posttranscriptional regulators. They harbor small upstream open reading frames (uORFs) encoding leader peptides, for which no functions in trans are known yet. In the plant symbiont Sinorhizobium meliloti, the tryptophan biosynthesis gene trpE(G) is preceded by the uORF trpL and is regulated by transcription attenuation according to tryptophan availability. However, trpLE(G) transcription is initiated independently of the tryptophan level in S. meliloti, thereby ensuring a largely tryptophan-independent production of the leader peptide peTrpL. Here, we provide evidence for a tryptophan-independent role of peTrpL in trans We found that peTrpL increases the resistance toward tetracycline, erythromycin, chloramphenicol, and the flavonoid genistein, which are substrates of the major multidrug efflux pump SmeAB. Coimmunoprecipitation with a FLAG-peTrpL suggested smeR mRNA, which encodes the transcription repressor of smeABR, as a peptide target. Indeed, upon antibiotic exposure, smeR mRNA was destabilized and smeA stabilized in a peTrpL-dependent manner, showing that peTrpL acts in the differential regulation of smeABR Furthermore, smeR mRNA was coimmunoprecipitated with peTrpL in antibiotic-dependent ribonucleoprotein (ARNP) complexes, which, in addition, contained an antibiotic-induced antisense RNA complementary to smeRIn vitro ARNP reconstitution revealed that the above-mentioned antibiotics and genistein directly support complex formation. A specific region of the antisense RNA was identified as a seed region for ARNP assembly in vitro Altogether, our data show that peTrpL is involved in a mechanism for direct utilization of antimicrobial compounds in posttranscriptional regulation of multiresistance genes. Importantly, this role of peTrpL in resistance is conserved in other AlphaproteobacteriaIMPORTANCE Leader peptides encoded by transcription attenuators are widespread small proteins that are considered nonfunctional in trans We found that the leader peptide peTrpL of the soil-dwelling plant symbiont Sinorhizobium meliloti is required for differential, posttranscriptional regulation of a multidrug resistance operon upon antibiotic exposure. Multiresistance achieved by efflux of different antimicrobial compounds ensures survival and competitiveness in nature and is important from both evolutionary and medical points of view. We show that the leader peptide forms antibiotic- and flavonoid-dependent ribonucleoprotein complexes (ARNPs) for destabilization of smeR mRNA encoding the transcription repressor of the major multidrug resistance operon. The seed region for ARNP assembly was localized in an antisense RNA, whose transcription is induced by antimicrobial compounds. The discovery of ARNP complexes as new players in multiresistance regulation opens new perspectives in understanding bacterial physiology and evolution and potentially provides new targets for antibacterial control.

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5\'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.

Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. Not only are the corresponding genes intergenic but they are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.IMPORTANCE Proteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the functions of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification, and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.

The secretion of peptides and proteins is essential for survival and ecological adaptation of bacteria. Dual-functional ATP-binding cassette transporters export antimicrobial or quorum signaling peptides in Gram-positive bacteria. Their substrates contain a leader sequence that is excised by an N-terminal peptidase C39 domain at a double Gly motif. We characterized the protease domain (LahT150) of a transporter from a lanthipeptide biosynthetic operon in Lachnospiraceae and demonstrate that this protease can remove the leader peptide from a diverse set of peptides. The 2.0 Å resolution crystal structure of the protease domain in complex with a covalently bound leader peptide demonstrates the basis for substrate recognition across the entire class of such transporters. The structural data also provide a model for understanding the role of leader peptide recognition in the translocation cycle, and the function of degenerate, non-functional C39-like domains (CLD) in substrate recruitment in toxin exporters in Gram-negative bacteria.