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.

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted vs. wild-type M. alcaliphilum strains (DASS vs. DSM19304). Overall, a ∼ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression.

The GhNAC2 transcription factor identified from G. herbaceum improves root growth and drought tolerance through transcriptional reprogramming of phytohormone signaling. The promoter of such a versatile gene could serve as an important genetic engineering tool for biotechnological application. In this study, we identified and characterized the promoter of GhNAC2 to understand its regulatory mechanism. GhNAC2 transcription factor increased in root tissues in response to GA, ethylene, auxin, ABA, mannitol, and NaCl. In silico analysis revealed an overrepresentation of cis-regulatory elements associated with hormone signaling, stress responses and root-, pollen-, and seed-specific promoter activity. To validate their role in GhNAC2 function/regulation, an 870-bp upstream regulatory sequence was fused with the GUS reporter gene (uidA) and expressed in Arabidopsis and cotton hairy roots for in planta characterization. Histochemical GUS staining indicated localized expression in root tips, root elongation zone, root primordia, and reproductive tissues under optimal growth conditions. Mannitol, NaCl, auxin, GA, and ABA, induced the promoter-driven GUS expression in all tissues while ethylene suppressed the promoter activity. The results show that the 870 nt fragment of the GhNAC2 promoter drives root-preferential expression and responds to phytohormonal and stress signals. In corroboration with promoter regulation, GA and ethylene pathways differentially regulated root growth in GhNAC2-expressing Arabidopsis. The findings suggest that differential promoter activity governs the expression of GhNAC2 in root growth and stress-related functions independently through specific promoter elements. This multifarious promoter can be utilized to develop yield and climate resilience in cotton by expanding the options to control gene regulation.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s12298-024-01411-2.

Plant cells accumulate osmotic substances (e.g., sugar) to protect cell components and maintain osmotic balance under drought stress conditions. Previous studies found that pOsHAK1:OsFLN2 promotes sugar metabolism and improves the drought tolerance of rice plants under drought stress. This study further evaluated the effect of the ectopic expression of the OsSUT1 gene driven by the OsHAK1 promoter on the sugar transport and drought tolerance of rice. The results showed that the net photosynthetic rate and sucrose phosphate synthase activity of plants expressing the OsSUT1 gene were not significantly different from those of wild-type (WT) rice plants under drought conditions. However, the sucrose transport rate in the phloem increased in the transgenic plants, and the sucrose contents were significantly lower in the leaves but significantly higher in the roots of transgenic plants than those in WT plants. The pOsHAK1:OsSUT1 and pOsHAK1:OsFLN2 transgenic lines had similar rates of long-distance sucrose transport and drought tolerance, which were higher than those of the WT plants. The relative water content of the transgenic plants was higher, while their water loss rate, hydrogen peroxide (H2O2), and malondialdehyde (MDA) contents were lower than those of the WT plants. The stress-responsive gene OsbZIP23 and the antioxidant-related gene OsCATB were significantly upregulated in the drought-treated transgenic lines, while the senescence indicator gene SGR and the stress-responsive gene OsNAC2 were down-regulated compared to WT plants. These results showed that promoting the long-distance sugar transport through the expression of pOsHAK1:OsSUT1 could produce an improved drought tolerance effect similar to that of pOsHAK1:OsFLN2, providing an effective way to improve the drought tolerance of cereal crops at the seedling stage.

Lactic acid bacteria (LAB) are important for many biotechnological applications such as bioproduction and engineered probiotics for therapy. Inducible promoters are key gene expression control elements, yet those available in LAB are mainly based on bacteriocin systems and have many drawbacks, including large gene clusters, costly inducer peptides, and little portability to in vivo settings. Using Lactobacillus gasseri, a model commensal bacteria from the human gut, we report the engineering of synthetic LactoSpanks promoters (Pls), a collection of variable strength inducible promoters controlled by the LacI repressor from E. coli and induced by isopropyl β-d-1-thiogalactopyranoside (IPTG). We first show that the Phyper-spank promoter from Bacillus subtilis is functional in L. gasseri, albeit with substantial leakage. We then construct and screen a semirational library of Phyper-spank variants to select a set of four IPTG-inducible promoters that span a range of expression levels and exhibit reduced leakages and operational dynamic ranges (from ca. 9 to 28 fold-change). With their low genetic footprint and simplicity of use, LactoSpanks will support many applications in L. gasseri, and potentially other lactic acid and Gram-positive bacteria.

Actinomyces are gram-positive bacteria known for their valuable secondary metabolites. Redirecting metabolic flux towards desired products in actinomycetes requires precise and dynamic regulation of gene expression. In this study, we integrated the CRISPR interference (CRISPRi) system with a cumate-inducible promoter to develop an inducible gene downregulation method in Saccharopolyspora erythraea, a prominent erythromycin-producing actinobacterium. The functionality of the cumate-inducible promoter was validated using the gusA gene as a reporter, and the successful inducible expression of the dCas9 gene was confirmed. The developed inducible CRISPRi strategy was then employed to downregulate the expression of target genes rppA in the wild-type strain NRRL2338 and sucC in the high erythromycin-producing strain E3. Through dynamic control of sucC expression, a significant enhancement in erythromycin production was achieved in strain E3. This study demonstrated the effectiveness of an inducible gene downregulation approach using CRISPRi and a cumate-inducible promoter, providing valuable insights for optimizing natural product production in actinomyces.

The mechanical damage of plant tissues leads to the activation of methanol production and its release into the atmosphere. The gaseous methanol or vapors emitted by the damaged plant induce resistance in neighboring intact plants to bacterial pathogens but create favorable conditions for viral infection spread. Among the Nicotiana benthamiana methanol-inducible genes (MIGs), most are associated with plant defense and intercellular transport. Here, we characterize NbMIG21, which encodes a 209 aa protein (NbMIG21p) that does not share any homology with annotated proteins. NbMIG21p was demonstrated to contain a nucleolus localization signal (NoLS). Colocalization studies with fibrillarin and coilin, nucleolus and Cajal body marker proteins, revealed that NbMIG21p is distributed among these subnuclear structures. Our results show that recombinant NbMIG21 possesses DNA-binding properties. Similar to a gaseous methanol effect, an increased NbMIG21 expression leads to downregulation of the nuclear import of proteins with nuclear localization signals (NLSs), as was demonstrated with the GFP-NLS model protein. Moreover, upregulated NbMIG21 expression facilitates tobacco mosaic virus (TMV) intercellular transport and reproduction. We identified an NbMIG21 promoter (PrMIG21) and showed that it is methanol sensitive; thus, the induction of NbMIG21 mRNA accumulation occurs at the level of transcription. Our findings suggest that methanol-activated NbMIG21 might participate in creating favorable conditions for viral reproduction and spread.

Small-molecule control of gene expression underlies the function of numerous engineered gene circuits that are capable of environmental sensing, computation, and memory. While many recently developed inducible promoters have been tailor-made for bacteria or mammalian cells, relatively few new systems have been built for Saccharomyces cerevisiae, limiting the scale of synthetic biology work that can be done in yeast. To address this, we created the yeast Tunable Expression Systems Toolkit (yTEST), which contains a set of five extensively characterized inducible promoter systems regulated by the small-molecules doxycycline (Dox), abscisic acid (ABA), danoprevir (DNV), 1-naphthaleneacetic acid (NAA), and 5-phenyl-indole-3-acetic acid (5-Ph-IAA). Assembly was made to be compatible with the modular cloning yeast toolkit (MoClo-YTK) to enhance the ease of use and provide a framework to benchmark and standardize each system. Using this approach, we built multiple systems with maximal expression levels greater than those of the strong constitutive TDH3 promoter. Furthermore, each of the five classes of systems could be induced at least 60-fold after a 6 h induction and the highest fold change observed was approximately 300. Thus, yTEST provides a reliable, diverse, and customizable set of inducible promoters to modulate gene expression in yeast for applications in synthetic biology, metabolic engineering, and basic research.

Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter PQ2148 used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens.

Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys-), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).