The biotechnology industry has become a key element in modern societies. Within this industry, the production of recombinant enzymes and biopharmaceutical proteins is of major importance. The global markets for such recombinant proteins are growing rapidly and, accordingly, there is a continuous need for new production platforms that can deliver protein products in greater yields, with higher quality and at lower costs. This calls for the development of next-generation super-secreting cell factories. One of the microbial cell factories that can meet these challenges is the Gram-positive bacterium Bacillus subtilis, an inhabitant of the upper layers of the soil that has the capacity to secrete proteins in the gram per litre range. The engineering of B. subtilis into a next-generation super-secreting cell factory requires combined Systems and Synthetic Biology approaches. In this way, the bacterial protein secretion machinery can be optimized from the single molecule to the network level while, at the same time, taking into account the balanced use of cellular resources. Although highly ambitious, this is an achievable objective due to recent advances in functional genomics and Systems- and Synthetic Biological analyses of B. subtilis cells.

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria. The major human pathogen Staphylococcus aureus can survive under a wide range of conditions, both inside and outside the human body. The goal of this study was to determine how S. aureus adapts to such different conditions and, additionally, we wanted to identify general factors governing the staphylococcal transcriptome architecture. Therefore, we performed a precise analysis of all RNA transcripts of S. aureus across experimental conditions ranging from in vitro growth in different media to internalization by eukaryotic host cells. We systematically mapped all transcription units, annotated non-coding RNAs, and assigned promoters controlled by particular RNA polymerase sigma factors and transcription factors. By a comparison with data available for the related Gram-positive bacterium Bacillus subtilis, we made key observations concerning the abundance and origin of antisense RNAs. Intriguingly, these findings support the view that many antisense RNAs in a bacterium like B. subtilis could be byproducts of spurious promoter recognition by condition-specific alternative sigma factors. We also report that the transcription termination factor Rho prevents widespread antisense transcription, presumably caused by pervasive transcription initiation in the A+T-rich genome of S. aureus. Altogether our study presents new perspectives on the biological significance of antisense and pervasive transcription in bacteria.

SUMMARY: The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus . The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus , but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis , are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative “secretomics” approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the “gadgets” that S. aureus needs to conquer these well-protected niches.

Staphylococcus aureus is an important human and livestock pathogen that is well-protected against environmental insults by a thick cell wall. Accordingly, the wall is a major target of present-day antimicrobial therapy. Unfortunately, S. aureus has mastered the art of antimicrobial resistance, as underscored by the global spread of methicillin-resistant S. aureus (MRSA). The major cell wall component is peptidoglycan. Importantly, the peptidoglycan network is not only vital for cell wall function, but it also represents a bacterial Achilles’ heel. In particular, this network is continuously opened by no less than 18 different peptidoglycan hydrolases (PGHs) encoded by the S. aureus core genome, which facilitate bacterial growth and division. This focuses attention on the specific functions executed by these enzymes, their subcellular localization, their control at the transcriptional and post-transcriptional levels, their contributions to staphylococcal virulence and their overall importance in bacterial homeostasis. As highlighted in the present review, our understanding of the different aspects of PGH function in S. aureus has been substantially increased over recent years. This is important because it opens up new possibilities to exploit PGHs as innovative targets for next-generation antimicrobials, passive or active immunization strategies, or even to engineer them into effective antimicrobial agents.

Bacterial infections represent an increasing problem in modern health care, in particular due to ageing populations and accumulating bacterial resistance to antibiotics. Diagnosis is rarely straightforward and consequently treatment is often delayed or indefinite. Therefore, novel tools that can be clinically implemented are urgently needed to accurately and swiftly diagnose infections. Especially, the direct imaging of infections is an attractive option. The challenge of specifically imaging bacterial infections in vivo can be met by targeting bacteria with an imaging agent. Here we review the current status of targeted imaging of bacterial infections, and we discuss advantages and disadvantages of the different approaches. Indeed, significant progress has been made in this field and the clinical implementation of targeted imaging of bacterial infections seems highly feasible. This was recently highlighted by the use of so-called smart activatable probes and a fluorescently labelled derivative of the antibiotic vancomycin. A major challenge remains the selection of the best imaging probes, and we therefore present a set of target selection criteria for clinical implementation of targeted bacterial imaging. Altogether, we conclude that the spectrum of potential applications for targeted bacterial imaging is enormous, ranging from fundamental research on infectious diseases to diagnostic and therapeutic applications.