ABSTRACT
Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.
ABSTRACT
BACKGROUND: Whole-cell biosensors are a powerful and easy-to-use screening tool for the fast and sensitive detection of chemical compounds, such as antibiotics. ß-Lactams still represent one of the most important antibiotic groups in therapeutic use. They interfere with late stages of the bacterial cell wall biosynthesis and result in irreversible perturbations of cell division and growth, ultimately leading to cell lysis. In order to simplify the detection of these antibiotics from solutions, solid media or directly from producing organisms, we aimed at developing a novel heterologous whole-cell biosensor in Bacillus subtilis, based on the ß-lactam-induced regulatory system BlaR1/BlaI from Staphylococcus aureus. RESULTS: The BlaR1/BlaI system was heterologously expressed in B. subtilis and combined with the luxABCDE operon of Photorhabdus luminescens under control of the BlaR1/BlaI target promoter to measure the output of the biosensor. A combination of codon adaptation, constitutive expression of blaR1 and blaI and the allelic replacement of penP increased the inducer spectrum and dynamic range of the biosensor. ß-Lactams from all four classes induced the target promoter P blaZ in a concentration-dependent manner, with a dynamic range of 7- to 53-fold. We applied our biosensor to a set of Streptomycetes soil isolates and demonstrated its potential to screen for the production of ß-lactams. In addition to the successful implementation of a highly sensitive ß-lactam biosensor, our results also provide the first experimental evidence to support previous suggestions that PenP functions as a ß-lactamase in B. subtilis. CONCLUSION: We have successfully established a novel heterologous whole-cell biosensor in B. subtilis that is highly sensitive for a broad spectrum of ß-lactams from all four chemical classes. Therefore, it increases the detectable spectrum of compounds with respect to previous biosensor designs. Our biosensor can readily be applied for identifying ß-lactams in liquid or on solid media, as well as for identifying potential ß-lactam producers.
ABSTRACT
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
ABSTRACT
A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.
ABSTRACT
The bacterial cell wall separates the cell from its surrounding and protects it from environmental stressors. Its integrity is maintained by a highly regulated process of cell wall biosynthesis. The membrane-located lipid II cycle provides cell wall building blocks that are assembled inside the cytoplasm to the outside for incorporation. Its carrier molecule, undecaprenyl phosphate (UP), is then recycled by dephosphorylation from undecaprenyl pyrophosphate (UPP). In Bacillus subtilis, this indispensable reaction is catalyzed by the UPP phosphatases BcrC and UppP. Here, we study the physiological function of both phosphatases with respect to morphology, cell wall homeostasis and the resulting cell envelope stress response (CESR). We demonstrate that uppP and bcrC represent a synthetic lethal gene pair, which encodes an essential physiological function. Accordingly, cell growth and morphology were severely impaired during exponential growth if the overall UPP phosphatase level was limiting. UppP, but not BcrC, was crucial for normal sporulation. Expression of bcrC, but not uppP, was upregulated in the presence of cell envelope stress conditions caused by bacitracin if UPP phosphatase levels were limited. This homeostatic feedback renders BcrC more important during growth than UppP, particularly in defense against cell envelope stress.
ABSTRACT
Bacillus subtilis combines natural competence for genetic transformation with highly efficient homologous recombination. These features allow using vectors that integrate into the genome via double homologous recombination. So far, their utilization is restricted by the fixed combination of resistance markers and integration loci, as well as species- or strain-specific regions of homology. To overcome these limitations, we developed a toolbox for the creation of personalized Bacillus vectors in a standardized manner with a focus on fast and easy adaptation of the sequences specifying the integration loci. We based our vector toolkit on the Standard European Vector Architecture (SEVA) to allow the usage of their vector parts. The Bacillus SEVA siblings are assembled via efficient one-pot Golden Gate reactions from four entry parts with the choice of four different enzymes. The toolbox contains seven Bacillus resistance markers, two Escherichia coli origins of replication, and a free choice of integration loci. Vectors can be customized with a cargo, before or after vector assembly, and could be used in different B. subtilis strains and potentially beyond. Our adaptation of the SEVA-standard provides a powerful and standardized toolkit for the convenient creation of personalized Bacillus vectors.
