Genome-based analyses to learn from and about Paenibacillus sonchi genomovar Riograndensis SBR5T.

Paenibacillus sonchi genomovar Riograndensis SBR5T is a plant growth-promoting rhizobacterium (PGPR) isolated in the Brazilian state of Rio Grande do Sul from the rhizosphere of Triticum aestivum. It fixes nitrogen, produces siderophores as well as the phytohormone indole-3-acetic acid, solubilizes phosphate and displays antagonist activity against Listeria monocytogenes and Pectobacterium carotovorum. Comprehensive omics analysis and the development of genetic tools are key to characterizing and engineering such non-model microorganisms. Therefore, the complete genome of SBR5T was sequenced, and shown to encode 6,705 proteins, 87 tRNAs, and 27 rRNAs and it enabled a landscape transcriptome analysis that unveiled conserved transcriptional and translational patterns and characterized operon structures and riboswitches. The pangenome of P. sonchi species is open with a stable core pangenome. At the same time, the analysis of genes coding for nitrogenases revealed that the trait of nitrogen fixation is sparse within the Paenibacillaceae family and the presence of Fe-only nitrogenase in the P. sonchi group was exclusive to SBR5T. The development of genetic tools for SBR5T enabled genetic transformation, plasmid construction for constitutive and inducible gene expression, and gene repression using the CRISPRi system. Altogether, the work with P. sonchi can guide the study of non-model bacteria with economic potential.

Genomic and Transcriptomic Investigation of the Physiological Response of the Methylotroph Bacillus methanolicus to 5-Aminovalerate.

The methylotrophic thermophile Bacillus methanolicus can utilize the non-food substrate methanol as its sole carbon and energy source. Metabolism of L-lysine, in particular its biosynthesis, has been studied to some detail, and methanol-based L-lysine production has been achieved. However, little is known about L-lysine degradation, which may proceed via 5-aminovalerate (5AVA), a non-proteinogenic ω-amino acid with applications in bioplastics. The physiological role of 5AVA and related compounds in the native methylotroph was unknown. Here, we showed that B. methanolicus exhibits low tolerance to 5AVA, but not to related short-chain (C4-C6) amino acids, diamines, and dicarboxylic acids. In order to gain insight into the physiological response of B. methanolicus to 5AVA, transcriptomic analyses by differential RNA-Seq in the presence and absence of 5AVA were performed. Besides genes of the general stress response, RNA levels of genes of histidine biosynthesis, and iron acquisition were increased in the presence of 5AVA, while an Rrf2 family transcriptional regulator gene showed reduced RNA levels. In order to test if mutations can overcome growth inhibition by 5AVA, adaptive laboratory evolution (ALE) was performed and two mutants-AVA6 and AVA10-with higher tolerance to 5AVA were selected. Genome sequencing revealed mutations in genes related to iron homeostasis, including the gene for an iron siderophore-binding protein. Overexpression of this mutant gene in the wild-type (WT) strain MGA3 improved 5AVA tolerance significantly at high Fe2+ supplementation. The combined ALE, omics, and genetics approach helped elucidate the physiological response of thermophilic B. methanolicus to 5AVA and will guide future strain development for 5AVA production from methanol.

Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway.

Methanol is a sustainable substrate for biotechnology. In addition to natural methylotrophs, metabolic engineering has gained attention for transfer of methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes.

CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5.

Gene repression using the endonucleolytically deactivated dCas9 protein and sgRNAs (CRISPR interference or CRISPRi) is a useful approach to study gene functions. Here, we established CRISPRi in Paenibacillus sonchi genomovar Riograndensis SBR5, a plant growth promoting bacterium. CRISPRi system with sgRNAs targeting SBR5 endogenous genes spo0A, yaaT and ydjJ and plasmid-borne gfpUV was constructed and analyzed. Flow cytometry analysis revealed a significant decrease of reporter protein GFPUV signal in P. sonchi strains expressing gfpUV sgRNA in comparison with non-targeting controls. CRISPRi-based repression of chromosomal genes for regulation of sporulation spo0A and yaaT decreased sporulation and increased biofilm formation in SBR5. Repression of the sorbitol catabolic gene ydjJ revealed decreased specific activity of YdjJ in crude cell extracts and reduced biomass formation from sorbitol in growth experiments. Our work on CRISPRi-based gene repression serves as basis for gene function studies of the plant growth promoter P. sonchi SBR5. To our knowledge, the present study presents the first tool for gene repression established in Paenibacillus species.Key points• CRISPRi toward gene repression was applied for the first time in Paenibacillus.• CRISPRi of spo0A and yaaT depleted spores and increased biofilms in SBR5.• CRISPRi-based ydjJ repression decreased specific activity of sorbitol dehydrogenase.

Impact of CRISPR interference on strain development in biotechnology.

Genetic perturbation systems are of great interest to redirect metabolic fluxes for value-added production, as well as genetic screening for the development of new drugs, or to identify new targets for biotechnological applications. Here, we review CRISPR interference (CRISPRi), a method for gene expression using a catalytically inactive version of the CRISPR-associated protein 9 (dCas9) of the widely applied CRISPR-Cas9 genome editing system. In combination with the appropriate sgRNA, dCas9 binds to specific DNA sequences without causing double-stranded DNA breakage but interfering with transcription initiation or elongation. Besides manifold uses to interrogate the physiology of a bacterial cell, CRISPRi is used in applications for metabolic engineering and strain development in industrial biotechnology. Albeit in its infancy, CRISPRi has already delivered the first success stories; however, we also analyze limitations of the CRISPRi system and give future perspectives.

Inorganic Phosphate Solubilization by Rhizosphere Bacterium Paenibacillus sonchi: Gene Expression and Physiological Functions.

Due to the importance of phosphorus (P) in agriculture, crop inoculation with phosphate-solubilizing bacteria is a relevant subject of study. Paenibacillus sonchi genomovar Riograndensis SBR5 is a promising candidate for crop inoculation, as it can fix nitrogen and excrete ammonium at a remarkably high rate. However, its trait of phosphate solubilization (PS) has not yet been studied in detail. Here, differential gene expression and functional analyses were performed to characterize PS in this bacterium. SBR5 was cultivated with two distinct P sources: NaH2PO4 as soluble phosphate source (SPi) and hydroxyapatite as insoluble phosphate source (IPi). Total RNA of SBR5 cultivated in those two conditions was isolated and sequenced, and bacterial growth and product formation were monitored. In the IPi medium, the expression of 68 genes was upregulated, whereas 100 genes were downregulated. Among those, genes involved in carbon metabolism, including those coding for subunits of 2-oxoglutarate dehydrogenase, were identified. Quantitation of organic acids showed that the production of tricarboxylic acid cycle-derived organic acids was reduced in IPi condition, whereas acetate and gluconate were overproduced. Increased concentrations of proline, trehalose, and glycine betaine revealed active osmoprotection during growth in IPi. The cultivation with hydroxyapatite also caused the reduction in the motility of SBR5 cells as a response to Pi depletion at the beginning of its growth. SBR5 was able to solubilize hydroxyapatite, which suggests that this organism is a promising phosphate-solubilizing bacterium. Our findings are the initial step in the elucidation of the PS process in P. sonchi SBR5 and will be a valuable groundwork for further studies of this organism as a plant growth-promoting rhizobacterium.

Characterization of D-Arabitol as Newly Discovered Carbon Source of Bacillus methanolicus.

Bacillus methanolicus is a Gram-positive, thermophilic, methanol-utilizing bacterium. As a facultative methylotroph, B. methanolicus is also known to utilize D-mannitol, D-glucose and, as recently discovered, sugar alcohol D-arabitol. While metabolic pathways for utilization of methanol, mannitol and glucose are known, catabolism of arabitol has not yet been characterized in B. methanolicus. In this work we present the elucidation of this hitherto uncharted pathway. In order to confirm our predictions regarding genes coding for arabitol utilization, we performed differential gene expression analysis of B. methanolicus MGA3 cells grown on arabitol as compared to mannitol via transcriptome sequencing (RNA-seq). We identified a gene cluster comprising eight genes that was up-regulated during growth with arabitol as a sole carbon source. The RNA-seq results were subsequently confirmed via qRT-PCR experiments. The transcriptional organization of the gene cluster identified via RNA-seq was analyzed and it was shown that the arabitol utilization genes are co-transcribed in an operon that spans from BMMGA3_RS07325 to BMMGA3_RS07365. Since gene deletion studies are currently not possible in B. methanolicus, two complementation experiments were performed in an arabitol negative Corynebacterium glutamicum strain using the four genes discovered via RNA-seq analysis as coding for a putative PTS for arabitol uptake (BMMGA3_RS07330, BMMGA3_RS07335, and BMMGA3_RS07340 renamed to atlABC) and a putative arabitol phosphate dehydrogenase (BMMGA3_RS07345 renamed to atlD). C. glutamicum is a natural D-arabitol utilizer that requires arabitol dehydrogenase MtlD for arabitol catabolism. The C. glutamicum mtlD deletion mutant was chosen for complementation experiments. Heterologous expression of atlABCD as well as the arabitol phosphate dehydrogenase gene atlD from B. methanolicus alone restored growth of the C. glutamicum ΔmtlD mutant with arabitol. Furthermore, D-arabitol phosphate dehydrogenase activities could be detected in crude extracts of B. methanolicus and these were higher in arabitol-grown cells than in methanol- or mannitol-grown cells. Thus, B. methanolicus possesses an arabitol inducible operon encoding, amongst others, a putative PTS system and an arabitol phosphate dehydrogenase for uptake and activation of arabitol as growth substrate.

Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus.

Bacillus methanolicus is a thermophilic, Gram-positive, rod-shaped bacterium. It is a facultative methylotroph which can use carbon and energy sources including mannitol and the one-carbon (C1) and non-food substrate methanol for growth and overproduction of amino acids, which makes it a promising candidate for biotechnological applications. Despite a growing tool box for gene cloning and expression, tools for targeted chromosomal gene knockouts and gene repression are still missing for this organism. Here, the CRISPRi-dCas9 technique for gene repression was established in B. methanolicus MGA3. Significantly reduced spore formation on the one hand and increased biofilm formation on the other hand could be demonstrated when the stage zero sporulation protein A gene spo0A was targeted. Furthermore, when the mannitol-1-phosphate 5-dehydrogenase gene mtlD was targeted by CRISPRi, mtlD RNA levels, and MtlD specific activities in crude extracts were decreased to about 50 % which resulted in reduced biomass formation from mannitol. As a third target, the catalase gene katA was chosen. Upon targeting katA by CRISPRi, catalase activity was decreased to about 25 % as shown in H2O2 drop assays and by determination of specific catalase activity in crude extracts. Our results support the predicted functions of Spo0A in sporulation and biofilm formation, of MtlD for mannitol catabolism, and of catalase in hydrogen peroxide dismutation. Thus, CRISPR interference as developed here serves as basis for the functional characterization of B. methanolicus physiology as well as for its application in biotechnology.

Function of L-Pipecolic Acid as Compatible Solute in Corynebacterium glutamicum as Basis for Its Production Under Hyperosmolar Conditions.

Pipecolic acid or L-PA is a cyclic amino acid derived from L-lysine which has gained interest in the recent years within the pharmaceutical and chemical industries. L-PA can be produced efficiently using recombinant Corynebacterium glutamicum strains by expanding the natural L-lysine biosynthetic pathway. L-PA is a six-membered ring homolog of the five-membered ring amino acid L-proline, which serves as compatible solute in C. glutamicum. Here, we show that de novo synthesized or externally added L-PA partially is beneficial for growth under hyper-osmotic stress conditions. C. glutamicum cells accumulated L-PA under elevated osmotic pressure and released it after an osmotic down shock. In the absence of the mechanosensitive channel YggB intracellular L-PA concentrations increased and its release after osmotic down shock was slower. The proline permease ProP was identified as a candidate L-PA uptake system since RNAseq analysis revealed increased proP RNA levels upon L-PA production. Under hyper-osmotic conditions, a ΔproP strain showed similar growth behavior than the parent strain when L-proline was added externally. By contrast, the growth impairment of the ΔproP strain under hyper-osmotic conditions could not be alleviated by addition of L-PA unless proP was expressed from a plasmid. This is commensurate with the view that L-proline can be imported into the C. glutamicum cell by ProP and other transporters such as EctP and PutP, while ProP appears of major importance for L-PA uptake under hyper-osmotic stress conditions.

Reclassification of Paenibacillus riograndensis as a Genomovar of Paenibacillus sonchi: Genome-Based Metrics Improve Bacterial Taxonomic Classification.

Species from the genus Paenibacillus are widely studied due to their biotechnological relevance. Dozens of novel species descriptions of this genus were published in the last couple of years, but few utilized genomic data as classification criteria. Here, we demonstrate the importance of using genome-based metrics and phylogenetic analyses to identify and classify Paenibacillus strains. For this purpose, Paenibacillus riograndensis SBR5T, Paenibacillus sonchi X19-5T, and their close relatives were compared through phenotypic, genotypic, and genomic approaches. With respect to P. sonchi X19-5T, P. riograndensis SBR5T, Paenibacillus sp. CAR114, and Paenibacillus sp. CAS34 presented ANI (average nucleotide identity) values ranging from 95.61 to 96.32%, gANI (whole-genome average nucleotide identity) values ranging from 96.78 to 97.31%, and dDDH (digital DNA-DNA hybridization) values ranging from 68.2 to 73.2%. Phylogenetic analyses of 16S rRNA, gyrB, recA, recN, and rpoB genes and concatenated proteins supported the monophyletic origin of these Paenibacillus strains. Therefore, we propose to assign Paenibacillus sp. CAR114 and Paenibacillus sp. CAS34 to P. sonchi species, and reclassify P. riograndensis SBR5T as a later heterotypic synonym of P. sonchi (type strain X19-5T), with the creation of three novel genomovars, P. sonchi genomovar Sonchi (type strain X19-5T), P. sonchi genomovar Riograndensis (type strain SBR5T), P. sonchi genomovar Oryzarum (type strain CAS34T = DSM 102041T; = BR10511T).