An Interplay of Multiple Positive and Negative Factors Governs Methicillin Resistance in Staphylococcus aureus.

The development of resistance to ß-lactam antibiotics has made Staphylococcus aureus a clinical burden on a global scale. MRSA (methicillin-resistant S. aureus) is commonly known as a superbug. The ability of MRSA to proliferate in the presence of ß-lactams is attributed to the acquisition of mecA, which encodes the alternative penicillin binding protein, PBP2A, which is insensitive to the antibiotics. Most MRSA isolates exhibit low-level ß-lactam resistance, whereby additional genetic adjustments are required to develop high-level resistance. Although several genetic factors that potentiate or are required for high-level resistance have been identified, how these interact at the mechanistic level has remained elusive. Here, we discuss the development of resistance and assess the role of the associated components in tailoring physiology to accommodate incoming mecA.

Evolving MRSA: High-level ß-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine tuning of gene expression.

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level ß-lactam resistance (oxacillin MIC 2-4 µg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 µg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit ß) or rpoC (RNA polymerase subunit ß') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.

Regulation of Antimycin Biosynthesis Is Controlled by the ClpXP Protease.

The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntAin vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation.IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the ∼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins.

Fluorescent proteins are a major cell biology tool to analyze protein sub-cellular topology. Here we have applied this technology to study protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, a widely used host for heterologous protein secretion biotechnology. Green and monomeric red fluorescent proteins were fused behind Sec (SPSec) or Tat (SPTat) signal peptides to direct them through the respective export pathway. Significant secretion of fluorescent eGFP and mRFP was observed exclusively through the Tat and Sec pathways, respectively. Plasmid over-expression was compared to a chromosomally integrated spSec-mRFP gene to allow monitoring secretion under high and low level synthesis in various media. Fluorimetric detection of SPSec-mRFP recorded folded states, while immuno-staining detected even non-folded topological intermediates. Secretion of SPSec-mRFP is unexpectedly complex, is regulated independently of cell growth phase and is influenced by the growth regime. At low level synthesis, highly efficient secretion occurs until it is turned off and secretory preforms accumulate. At high level synthesis, the secretory pathway overflows and proteins are driven to folding and subsequent degradation. High-level synthesis of heterologous secretory proteins, whether secretion competent or not, has a drastic effect on the endogenous secretome, depending on their secretion efficiency. These findings lay the foundations of dissecting how protein targeting and secretion are regulated by the interplay between the metabolome, secretion factors and stress responses in the S. lividans model.

Regulation of antibiotic production in Actinobacteria: new perspectives from the post-genomic era.

Covering: 2000 to 2018 The antimicrobial activity of many of their natural products has brought prominence to the Streptomycetaceae, a family of Gram-positive bacteria that inhabit both soil and aquatic sediments. In the natural environment, antimicrobial compounds are likely to limit the growth of competitors, thereby offering a selective advantage to the producer, in particular when nutrients become limited and the developmental programme leading to spores commences. The study of the control of this secondary metabolism continues to offer insights into its integration with a complex lifecycle that takes multiple cues from the environment and primary metabolism. Such information can then be harnessed to devise laboratory screening conditions to discover compounds with new or improved clinical value. Here we provide an update of the review we published in NPR in 2011. Besides providing the essential background, we focus on recent developments in our understanding of the underlying regulatory networks, ecological triggers of natural product biosynthesis, contributions from comparative genomics and approaches to awaken the biosynthesis of otherwise silent or cryptic natural products. In addition, we highlight recent discoveries on the control of antibiotic production in other Actinobacteria, which have gained considerable attention since the start of the genomics revolution. New technologies that have the potential to produce a step change in our understanding of the regulation of secondary metabolism are also described.

Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions.

Alternative sigma factors control numerous aspects of bacterial life, including adaptation to physiological stresses, morphological development, persistence states and virulence. This is especially true for the physiologically complex actinobacteria. Here we report the development of a robust gene deletions system for Streptomyces lividans TK24 based on a BAC library combined with the λ-Red recombination technique. The developed system was validated by systematically deleting the most highly expressed genes encoding alternative sigma factors and several other regulatory genes within the chromosome of S. lividans TK24. To demonstrate the possibility of large scale genomic manipulations, the major part of the undecylprodigiosin gene cluster was deleted as well. The resulting mutant strains were characterized in terms of morphology, growth parameters, secondary metabolites production and response to thiol-oxidation and cell-wall stresses. Deletion of SLIV_12645 gene encoding S. coelicolor SigR1 ortholog has the most prominent phenotypic effect, resulted in overproduction of actinorhodin and coelichelin P1 and increased sensitivity to diamide. The secreted proteome analysis of SLIV_12645 mutant revealed SigR1 influence on trafficking of proteins involved in cell wall biogenesis and refactoring. The reported here gene deletion system will further facilitate work on S. lividans strain improvement as a host for either secondary metabolites or protein production and will contribute to basic research in streptomycetes physiology, morphological development, secondary metabolism. On the other hand, the systematic deletion of sigma factors encoding genes demonstrates the complexity and conservation of regulatory processes conducted by sigma factors in streptomycetes.

Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074.

BACKGROUND: Efforts to construct the Streptomyces host strain with enhanced yields of heterologous product have focussed mostly on engineering of primary metabolism and/or the deletion of endogenous biosynthetic gene clusters. However, other factors, such as chromosome compactization, have been shown to have a significant influence on gene expression levels in bacteria and fungi. The expression of genes and biosynthetic gene clusters may vary significantly depending on their location within the chromosome. Little is known about the position effect in actinomycetes, which are important producers of various industrially relevant bioactive molecules. RESULTS: To demonstrate an impact of the chromosomal position effect on the heterologous expression of genes and gene clusters in Streptomyces albus J1074, a transposon mutant library with randomly distributed transposon that includes a ß-glucuronidase reporter gene was generated. Reporter gene expression levels have been shown to depend on the position on the chromosome. Using a combination of the transposon system and a φC31-based vector, the aranciamycin biosynthetic cluster was introduced randomly into the S. albus genome. The production levels of aranciamycin varied up to eightfold depending on the location of the gene cluster within the chromosome of S. albus J1074. One of the isolated mutant strains with an artificially introduced attachment site produced approximately 50% more aranciamycin than strains with endogenous attBs. CONCLUSIONS: In this study, we demonstrate that expression of the reporter gene and aranciamycin biosynthetic cluster in Streptomyces albus J1074 varies up to eightfold depending on its position on the chromosome. The integration of the heterologous cluster into different locations on the chromosome may significantly influence the titre of the produced substance. This knowledge can be used for the more efficient engineering of Actinobacteria via the relocation of the biosynthetic gene clusters and insertion of additional copies of heterologous constructs in a suitable chromosomal position.

An influence of the copy number of biosynthetic gene clusters on the production level of antibiotics in a heterologous host.

Streptomyces albus J1074 is a well-known host for heterologous expression of secondary metabolites. To further increase its potential and to study the influence of cluster multiplication, additional φC31-attachment site was integrated into its genome using a system for transposon mutagenesis. Four secondary metabolite clusters were expressed in strains with different numbers of attachment sites, ranging from one to three copies of the site. Secondary metabolite production was examined and a new compound could be detected, purified and its structure was elucidated.

Unusual site-specific DNA integration into the highly active pseudo-attB of the Streptomyces albus J1074 genome.

The φC31-encoded recombination system has become a widely used tool for genetic analysis of streptomycetes, gene therapy and generation of transgenic animals. However, the application of this system, even in the context of its natural host genus, Streptomyces, may require a specific approach for each species. In this study, we have identified a novel pseudo-attB site, called pseB4, for integration of vectors using the φC31 system. More than 90 % of clones contained two copies of pSET152- or pOJ436-based cosmids, after their introduction into S. albus. The efficiency of the integration of φC31-based vectors into pseB4 is therefore comparable to that of the integration into attB. Moreover, in contrast with integration into the native attB, integration into pseB4 is not polar and does not require a complementary sequence in the TT-core region. Furthermore, an analysis of conjugation frequency revealed mutual inhibition of plasmid integration into either site when both the attB and pseB4 sites were present in the genome.

In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1.

We report here the in vivo expression of the synthetic transposase gene himar1(a) in Streptomyces coelicolor M145 and Streptomyces albus. Using the synthetic himar1(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the streptomycetes genome. The insertion frequency for the Himar1-derived minitransposons is nearly 100 % of transformed Streptomyces cells, and insertions are stably inherited in the absence of an antibiotic selection. The minitransposons contain different antibiotic resistance selection markers (apramycin, hygromycin, and spectinomycin), site-specific recombinase target sites (rox and/or loxP), I-SceI meganuclease target sites, and an R6Kγ origin of replication for transposon rescue. We identified transposon insertion loci by random sequencing of more than 100 rescue plasmids. The majority of insertions were mapped to putative open-reading frames on the S. coelicolor M145 and S. albus chromosomes. These insertions included several new regulatory genes affecting S. coelicolor M145 growth and actinorhodin biosynthesis.