The Impact of Heterologous Regulatory Genes from Lipodepsipeptide Biosynthetic Gene Clusters on the Production of Teicoplanin and A40926.

StrR-like pathway-specific transcriptional regulators (PSRs) function as activators in the biosynthesis of various antibiotics, including glycopeptides (GPAs), aminoglycosides, aminocoumarins, and ramoplanin-like lipodepsipeptides (LDPs). In particular, the roles of StrR-like PSRs have been previously investigated in the biosynthesis of streptomycin, novobiocin, GPAs like balhimycin, teicoplanin, and A40926, as well as LDP enduracidin. In the current study, we focused on StrR-like PSRs from the ramoplanin biosynthetic gene cluster (BGC) in Actinoplanes ramoplaninifer ATCC 33076 (Ramo5) and the chersinamycin BGC in Micromonospora chersina DSM 44151 (Chers28). Through the analysis of the amino acid sequences of Ramo5 and Chers28, we discovered that these proteins are phylogenetically distant from other experimentally investigated StrR PSRs, although all StrR-like PSRs found in BGCs for different antibiotics share a conserved secondary structure. To investigate whether Ramo5 and Chers28, given their phylogenetic positions, might influence the biosynthesis of other antibiotic pathways governed by StrR-like PSRs, the corresponding genes (ramo5 and chers28) were heterologously expressed in Actinoplanes teichomyceticus NRRL B-16726 and Nonomuraea gerenzanensis ATCC 39727, which produce the clinically-relevant GPAs teicoplanin and A40926, respectively. Recombinant strains of NRRL B-16726 and ATCC 39727 expressing chers28 exhibited improved antibiotic production, although the expression of ramo5 did not yield the same effect. These results demonstrate that some StrR-like PSRs can "cross-talk" between distant biosynthetic pathways and might be utilized as tools for the activation of silent BGCs regulated by StrR-like PSRs.

Actinoplanes oblitus sp. nov., producing the glycopeptide antibiotic A477.

In 1973, Eli Lilly and Company described the filamentous actinomycete producing the glycopeptide antibiotic A477 as an Actinoplanes species on the basis of its morphological and physiological features and deposited it as NRRL 3884T. In this paper, we report that the phylogenetic analysis based on the 16S rRNA gene sequence and the whole genome phylogenomic study indicate that NRRL 3884T forms a distinct monophyletic line within the genus Actinoplanes, being most closely related to Actinoplanes octamycinicus NBRC 14524T [99.6 % 16S rRNA gene similarity, 89.4 % average nucleotide identity (ANI), 46.0 % digital DNA-DNA hybridization (dDDH)] and Actinoplanes ianthinogenes NBRC 13996T (98.8 % 16S rRNA gene similarity, 89.0 % ANI, 47.0 % dDDH). NRRL 3884T forms an extensively branched, non-fragmented vegetative mycelium; either sterile aerial hyphae or regular subglobose sporangia are formed depending on cultivation conditions. The cell wall contains meso-2,6-diaminopimelic acid and 2,6-diamino-3-hydroxypimelic acid and the diagnostic sugars are glucose, mannose and ribose with a minor amount of rhamnose. The predominant menaquinone (MK) is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H8). Mycolic acids are absent. The diagnostic phospholipids are diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids are anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0, with moderate amounts of anteiso-C15 : 0 and iso-C17 : 0. The genomic G+C content is 71.5 mol%. Significant differences in the genomic, morphological, chemotaxonomic and biochemical data between NRRL 3884T and the two most closely related Actinoplanes type strains clearly demonstrate that NRRL 3884T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes oblitus sp. nov. is proposed. The type strain is NRRL 3884T (=DSM 116196T).

Cross-Talking of Pathway-Specific Regulators in Glycopeptide Antibiotics (Teicoplanin and A40926) Production.

Teicoplanin and A40926 (natural precursor of dalbavancin) are clinically relevant glycopeptide antibiotics (GPAs) produced by Actinoplanes teichomyceticus NRRL B-16726 and Nonomuraea gerenzanensis ATCC 39727. Their biosynthetic enzymes are coded within large biosynthetic gene clusters (BGCs), named tei for teicoplanin and dbv for A40926, whose expression is strictly regulated by pathway-specific transcriptional regulators (PSRs), coded by cluster-situated regulatory genes (CSRGs). Herein, we investigated the "cross-talk" between the CSRGs from tei and dbv, through the analysis of GPA production levels in A. teichomyceticus and N. gerenzanensis strains, with knockouts of CSRGs cross-complemented by the expression of heterologous CSRGs. We demonstrated that Tei15* and Dbv4 StrR-like PSRs, although orthologous, were not completely interchangeable: tei15* and dbv4 were only partially able or unable to cross-complement N. gerenzanensis knocked out in dbv4 and A. teichomyceticus knocked out in tei15*, implying that the DNA-binding properties of these PSRs are more different in vivo than it was believed before. At the same time, the unrelated LuxR-like PSRs Tei16* and Dbv3 were able to cross-complement corresponding N. gerenzanensis knocked out in dbv3 and A. teichomyceticus knocked out in tei16*. Moreover, the heterologous expression of dbv3 in A. teichomyceticus led to a significant increase in teicoplanin production. Although the molecular background of these events merits further investigations, our results contribute to a deeper understanding of GPA biosynthesis regulation and offer novel biotechnological tools to improve their production.

A novel promising laccase from the psychrotolerant and halotolerant Antarctic marine Halomonas sp. M68 strain.

Microbial communities inhabiting the Antarctic Ocean show psychrophilic and halophilic adaptations conferring interesting properties to the enzymes they produce, which could be exploited in biotechnology and bioremediation processes. Use of cold- and salt-tolerant enzymes allows to limit costs, reduce contaminations, and minimize pretreatment steps. Here, we report on the screening of 186 morphologically diverse microorganisms isolated from marine biofilms and water samples collected in Terra Nova Bay (Ross Sea, Antarctica) for the identification of new laccase activities. After primary screening, 13.4 and 10.8% of the isolates were identified for the ability to oxidize 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and the dye azure B, respectively. Amongst them, the marine Halomonas sp. strain M68 showed the highest activity. Production of its laccase-like activity increased six-fold when copper was added to culture medium. Enzymatic activity-guided separation coupled with mass spectrometry identified this intracellular laccase-like protein (named Ant laccase) as belonging to the copper resistance system multicopper oxidase family. Ant laccase oxidized ABTS and 2,6-dimethoxy phenol, working better at acidic pHs The enzyme showed a good thermostability, with optimal temperature in the 40-50°C range and maintaining more than 40% of its maximal activity even at 10°C. Furthermore, Ant laccase was salt- and organic solvent-tolerant, paving the way for its use in harsh conditions. To our knowledge, this is the first report concerning the characterization of a thermo- and halo-tolerant laccase isolated from a marine Antarctic bacterium.

Occurrence of vanHAX and Related Genes beyond the Actinobacteria Phylum.

Clinically relevant glycopeptide antibiotics remain among the most successful classes of natural antibacterials. This success, however, is endangered by the spread of glycopeptide resistance genes, also known as van genes. Thus, it is important to trace and comprehend possible routes of van gene dissemination. In the current work, we present a comprehensive bioinformatic analysis aimed at mapping the occurrence of van genes beyond the Actinobacteria phylum-the most likely natural reservoir of van genes. We show that two additional classes of Gram-positive bacteria, Erysipelotrichia and Ktedonobacteria, as well as one class of Gram-negative bacteria, Anaerolineae, carry van genes. Additionally, we demonstrate that various new genera belonging to the classes Clostridia and Bacilli also carry van genes. The majority of discovered van loci are co-localized with MGE-related genes of various types. Finally, we propose a phylogeny-based scenario for the spread of van genes, unraveling a network of consequential horizontal gene transfer events linking the phylum Actinobacteria with the five other bacterial classes carrying van genes.

Genomic Insights into the Distribution and Phylogeny of Glycopeptide Resistance Determinants within the Actinobacteria Phylum.

The spread of antimicrobial resistance (AMR) creates a challenge for global health security, rendering many previously successful classes of antibiotics useless. Unfortunately, this also includes glycopeptide antibiotics (GPAs), such as vancomycin and teicoplanin, which are currently being considered last-resort drugs. Emerging resistance towards GPAs risks limiting the clinical use of this class of antibiotics-our ultimate line of defense against multidrug-resistant (MDR) Gram-positive pathogens. But where does this resistance come from? It is widely recognized that the GPA resistance determinants-van genes-might have originated from GPA producers, such as soil-dwelling Gram-positive actinobacteria, that use them for self-protection. In the current work, we present a comprehensive bioinformatics study on the distribution and phylogeny of GPA resistance determinants within the Actinobacteria phylum. Interestingly, van-like genes (vlgs) were found distributed in different arrangements not only among GPA-producing actinobacteria but also in the non-producers: more than 10% of the screened actinobacterial genomes contained one or multiple vlgs, while less than 1% encoded for a biosynthetic gene cluster (BGC). By phylogenetic reconstructions, our results highlight the co-evolution of the different vlgs, indicating that the most diffused are the ones coding for putative VanY carboxypeptidases, which can be found alone in the genomes or associated with a vanS/R regulatory pair.

Laboratory scale cultivation of Salinispora tropica in shake flasks and mechanically stirred bioreactors.

OBJECTIVE: Marine actinomycetes from the genus Salinispora have an unexploited biotechnological potential. To accurately estimate their application potential however, data on their cultivation, including biomass growth kinetics, are needed but only incomplete information is currently available. RESULTS: This work provides some insight into the effect of temperature, salinity, nitrogen source, glucose concentration and oxygen supply on growth rate, biomass productivity and yield of Salinispora tropica CBN-440T. The experiments were carried out in unbaffled shake flasks and agitated laboratory-scale bioreactors. The results show that the optimum growth temperature lies within the range 28-30 °C, salinity is close to sea water and the initial glucose concentration is around 10 g/L. Among tested nitrogen sources, yeast extract and soy peptone proved to be the most suitable. The change from unbaffled to baffled flasks increased the volumetric oxygen transfer coefficient (kLa) as did the use of agitated bioreactors. The highest specific growth rate (0.0986 h-1) and biomass productivity (1.11 g/L/day) were obtained at kLa = 28.3 h-1. A further increase in kLa was achieved by increasing stirrer speed, but this led to a deterioration in kinetic parameters. CONCLUSIONS: Improvement of S. tropica biomass growth kinetics of was achieved mainly by identifying the most suitable nitrogen sources and optimizing kLa in baffled flasks and agitated bioreactors.

Genomic-Led Discovery of a Novel Glycopeptide Antibiotic by Nonomuraea coxensis DSM 45129.

Glycopeptide antibiotics (GPAs) are last defense line drugs against multidrug-resistant Gram-positive pathogens. Natural GPAs teicoplanin and vancomycin, as well as semisynthetic oritavancin, telavancin, and dalbavancin, are currently approved for clinical use. Although these antibiotics remain efficient, emergence of novel GPA-resistant pathogens is a question of time. Therefore, it is important to investigate the natural variety of GPAs coming from so-called "rare" actinobacteria. Herein we describe a novel GPA producer-Nonomuraea coxensis DSM 45129. Its de novo sequenced and completely assembled genome harbors a biosynthetic gene cluster (BGC) similar to the dbv BGC of A40926, the natural precursor to dalbavancin. The strain produces a novel GPA, which we propose is an A40926 analogue lacking the carboxyl group on the N-acylglucosamine moiety. This structural difference correlates with the absence of dbv29-coding for an enzyme responsible for the oxidation of the N-acylglucosamine moiety. Introduction of dbv29 into N. coxensis led to A40926 production in this strain. Finally, we successfully applied dbv3 and dbv4 heterologous transcriptional regulators to trigger and improve A50926 production in N. coxensis, making them prospective tools for screening other Nonomuraea spp. for GPA production. Our work highlights genus Nonomuraea as a still untapped source of novel GPAs.

Streptomycetes: Attractive Hosts for Recombinant Protein Production.

Enzymes are increasingly applied as biocatalysts for fulfilling industrial needs in a variety of applications and there is a bursting of interest for novel therapeutic proteins. Consequently, developing appropriate expression platforms for efficiently producing such recombinant proteins represents a crucial challenge. It is nowadays widely accepted that an ideal 'universal microbial host' for heterologous protein expression does not exist. Indeed, the first-choice microbes, as Escherichia coli or yeasts, possess known intrinsic limitations that inevitably restrict their applications. In this scenario, bacteria belonging to the Streptomyces genus need to be considered with more attention as promising, alternative, and versatile platforms for recombinant protein production. This is due to their peculiar features, first-of-all their natural attitude to secrete proteins in the extracellular milieu. Additionally, streptomycetes are considered robust and scalable industrial strains and a wide range of tools for their genetic manipulation is nowadays available. This mini-review includes an overview of recombinant protein production in streptomycetes, covering nearly 100 cases of heterologous proteins expressed in these Gram-positives from the 1980s to December 2019. We investigated homologous sources, heterologous hosts, and molecular tools (promoters/vectors/signal peptides) used for the expression of these recombinant proteins. We reported on their final cellular localization and yield. Thus, this analysis might represent a useful source of information, showing pros and cons of using streptomycetes as platform for recombinant protein production and paving the way for their more extensive use in future as alternative heterologous hosts.

Glycopeptide Antibiotic Resistance Genes: Distribution and Function in the Producer Actinomycetes.

Glycopeptide antibiotics (GPAs) are considered drugs of "last resort" for the treatment of life-threatening infections caused by relevant Gram-positive pathogens (enterococci, staphylococci, and clostridia). Driven by the issue of the never-stopping evolution of bacterial antibiotic resistance, research on GPA biosynthesis and resistance is developing fast in modern "post-genomic" era. It is today widely accepted that resistance mechanisms emerging in pathogens have been acquired from the soil-dwelling antibiotic-producing actinomycetes, which use them to avoid suicide during production, rather than being orchestrated de novo by pathogen bacteria upon continued treatment. Actually, more and more genomes of GPA producers are being unraveled, carrying a broad collection of differently arranged GPA resistance (named van) genes. In the producer actinomycetes, van genes are generally associated with the antibiotic biosynthetic gene clusters (BGCs) deputed to GPA biosynthesis, being probably transferred/arranged together, favoring a possible co-regulation between antibiotic production and self-resistance. GPA BGC-associated van genes have been also found mining public databases of bacterial genomic and metagenomic sequences. Interestingly, some BGCs for antibiotics, seemingly unrelated to GPAs (e.g., feglymycin), carry van gene homologues. Herein, we would like to cover the recent advances on the distribution of GPA resistance genes in genomic and metagenomics datasets related to GPA potential/proved producer microorganisms. A thorough understanding of GPA resistance in the producing microorganisms may prove useful in the future surveillance of emerging mechanisms of resistance to this clinically relevant antibiotic class.

New Molecular Tools for Regulation and Improvement of A40926 Glycopeptide Antibiotic Production in Nonomuraea gerenzanensis ATCC 39727.

Genome sequencing has revealed that Nonomuraea spp. represent a still largely unexplored source of specialized metabolites. Nonomuraea gerenzanensis ATCC 39727 is the most studied representative species since it produces the glycopeptide antibiotic (GPA) A40926 - the precursor of the clinically relevant antibiotic dalbavancin, approved by the FDA in 2014 for the treatment of acute skin infections caused by multi-drug resistant Gram-positive pathogens. The clinical relevance of dalbavancin has prompted increased attention on A40926 biosynthesis and its regulation. In this paper, we investigated how to enhance the genetic toolkit for members of the Nonomuraea genus, which have proved quite recalcitrant to genetic manipulation. By constructing promoter-probe vectors, we tested the activity of 11 promoters (heterologous and native) using the GusA reporter system in N. gerenzanensis and in Nonomuraea coxensis; this latter species is phylogenetically distant from N. gerenzanesis and also possesses the genetic potential to produce A40926 or a very similar GPA. Finally, the strongest constitutive promoter analyzed in this study, aac(3)IVp, was used to overexpress the cluster-situated regulatory genes controlling A40926 biosynthesis (dbv3 and dbv4 from N. gerenzanensis and nocRI from N. coxensis) in N. gerenzanensis, and the growth and productivity of the best performing strains were assessed at bioreactor scale using an industrial production medium. Overexpression of positive pathway-specific regulatory genes resulted in a significant increase in the level of A40926 production in N. gerenzanensis, providing a new knowledge-based approach to strain improvement for this valuable glycopeptide antibiotic.

Specificity of Induction of Glycopeptide Antibiotic Resistance in the Producing Actinomycetes.

Glycopeptide antibiotics are drugs of last resort for treating severe infections caused by Gram-positive pathogens. It is widely believed that glycopeptide-resistance determinants (van genes) are ultimately derived from the producing actinomycetes. We hereby investigated the relationship between the antimicrobial activity of vancomycin and teicoplanins and their differential ability to induce van gene expression in Actinoplanes teichomyceticus—the producer of teicoplanin—and Nonomuraea gerenzanensis—the producer of the teicoplanin-like A40926. As a control, we used the well-characterized resistance model Streptomyces coelicolor. The enzyme activities of a cytoplasmic-soluble d,d-dipeptidase and of a membrane-associated d,d-carboxypeptidase (corresponding to VanX and VanY respectively) involved in resistant cell wall remodeling were measured in the actinomycetes grown in the presence or absence of subinhibitory concentrations of vancomycin, teicoplanin, and A40926. Results indicated that actinomycetes possess diverse self-resistance mechanisms, and that each of them responds differently to glycopeptide induction. Gene swapping among teicoplanins-producing actinomycetes indicated that cross-talking is possible and provides useful information for predicting the evolution of future resistance gene combinations emerging in pathogens.

Old and new glycopeptide antibiotics: From product to gene and back in the post-genomic era.

Glycopeptide antibiotics are drugs of last resort for treating severe infections caused by multi-drug resistant Gram-positive pathogens. First-generation glycopeptides (vancomycin and teicoplanin) are produced by soil-dwelling actinomycetes. Second-generation glycopeptides (dalbavancin, oritavancin, and telavancin) are semi-synthetic derivatives of the progenitor natural products. Herein, we cover past and present biotechnological approaches for searching for and producing old and new glycopeptide antibiotics. We review the strategies adopted to increase microbial production (from classical strain improvement to rational genetic engineering), and the recent progress in genome mining, chemoenzymatic derivatization, and combinatorial biosynthesis for expanding glycopeptide chemical diversity and tackling the never-ceasing evolution of antibiotic resistance.

Classification of Actinoplanes sp. ATCC 33076, an actinomycete that produces the glycolipodepsipeptide antibiotic ramoplanin, as Actinoplanes ramoplaninifer sp. nov.

Strain ATCC 33076, which produces the antibiotic ramoplanin, was isolated from a soil sample collected in India, and it was classified as a member of the genus Actinoplanes on the basis of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain forms a distinct clade within the genus Actinoplanes, and it is most closely related to Actinoplanes deccanensis IFO 13994T (98.71 % similarity) and Actinoplanes atraurantiacus Y16T (98.33 %). The strain forms an extensively branched substrate mycelium; the sporangia are formed very scantily and are globose with irregular surface. Spores are oval and motile. The cell wall contains meso-diaminopimelic acid and the diagnostic sugars are xylose and arabinose. The predominant menaquinone is MK-9(H6), with minor amounts of MK-9(H4) and MK-9(H2). Mycolic acids are absent. The diagnostic phospholipids are phosphatidylethanolamine, hydroxyphosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids are anteiso-C17 : 0 and iso-C16 : 0, followed by iso-C15 : 0 and moderate amounts of anteiso-C15 : 0, iso-C17 : 0 and C18 : 1ω9c. The genomic DNA G+C content is 71.4 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA-DNA relatedness between strain ATCC 33076 and closely related type strains, clearly demonstrated that strain ATCC 33076 represents a novel species of the genus Actinoplanes, for which the name Actinoplanes ramoplaninifer sp. nov. is proposed. The type strain is ATCC 33076T (=DSM 105064T=NRRL B-65484T).

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.

Extraction and Analysis of Peptidoglycan Cell Wall Precursors.

Extraction and analysis by LC-MS of peptidoglycan precursors represent a valuable method to study antibiotic mode of action and resistance in bacteria. Here, we describe how to apply this method for: (1) testing the action of different classes of antibiotics inhibiting cell wall biosynthesis in Bacillus megaterium; (2) studying the mechanism of self-resistance in mycelial actinomycetes producing glycopeptide antibiotics.

Classification of Nonomuraea sp. ATCC 39727, an actinomycete that produces the glycopeptide antibiotic A40926, as Nonomuraea gerenzanensis sp. nov.

Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic dalbavancin), was isolated from a soil sample collected in India, and it was originally classified as a member of the genus Actinomadura on the base of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicates that the strain forms a distinct clade within the genus Nonomuraea, and it is most closely related to Nonomuraea angiospora DSM 43173T (98.72 % similarity) and Nonomuraea jabiensis A4036T (98.69 %). The strain forms an extensively branched substrate mycelium and aerial hyphae that form spiral chains of spores with ridged surfaces. The cell wall contains meso-diaminopimelic acid and the whole-cell sugars are glucose, ribose, galactose, mannose and madurose (madurose as the diagnostic sugar). The N-acyl type of muramic acid is acetyl. The predominant menaquinone is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H0). The polar-lipid profile includes diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylmethylethanolamine, phosphatidylinositol and a series of uncharacterized phospholipids, glycolipids and phosphoglycolipids. The major cellular fatty acids are iso-C16 : 0 and 10-methyl C17 : 0. The genomic DNA G+C content is 71.2 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA-DNA relatedness between strain ATCC 39727 and closely related type strains, clearly demonstrated that strain ATCC 39727 represents a novel species of the genus Nonomuraea, for which the name Nonomuraea gerenzanensis sp. nov. is proposed. The type strain is ATCC 39727T ( = DSM 100948T).

Characterization of SCO4439, a D-alanyl-D-alanine carboxypeptidase involved in spore cell wall maturation, resistance, and germination in Streptomyces coelicolor.

This work contributes to the understanding of cell wall modifications during sporulation and germination in Streptomyces by assessing the biological function and biochemical properties of SCO4439, a D-alanyl-D-alanine carboxypeptidase (DD-CPase) constitutively expressed during development. SCO4439 harbors a DD-CPase domain and a putative transcriptional regulator domain, separated by a putative transmembrane region. The recombinant protein shows that DD-CPase activity is inhibited by penicillin G. The spores of the SCO4439::Tn5062 mutant are affected in their resistance to heat and acid and showed a dramatic increase in swelling during germination. The mycelium of the SCO4439::Tn5062 mutant is more sensitive to glycopeptide antibiotics (vancomycin and teicoplanin). The DD-CPase domain and the hydrophobic transmembrane region are highly conserved in Streptomyces, and both are essential for complementing the wild type phenotypes in the mutant. A model for the biological mechanism behind the observed phenotypes is proposed, in which SCO4439 DD-CPase releases D-Ala from peptidoglycan (PG) precursors, thereby reducing the substrate pool for PG crosslinking (transpeptidation). PG crosslinking regulates spore physical resistance and germination, and modulates mycelium resistance to glycopeptides. This study is the first demonstration of the role of a DD-CPase in the maturation of the spore cell wall.