A Small RNA, UdsC, Interacts with the RpoHII mRNA and Affects the Motility and Stress Resistance of Rhodobacter sphaeroides

Spanka, Daniel-Timon; Grützner, Julian; Jäger, Andreas; Klug, Gabriele

doi:10.3390/ijms232415486

Cited by 4 publications

(2 citation statements)

References 49 publications

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“…E.g. sRNAs were shown to make important contributions to the regulated formation of the photosynthetic apparatus and to regulation of stress responses in R. sphaeroides ( Mank et al, 2012 ; Adnan et al, 2015 ; Billenkamp et al, 2015 ; Müller et al, 2016 ; Peng et al, 2016 ; Eisenhardt et al, 2018 , 2021 ; Reuscher and Klug, 2021 ; Spanka et al, 2022 ). Many sRNAs were detected in the R. capsulatus transcriptome ( Licht et al, 2020 ), which are still awaiting functional characterization.…”

Section: Resultsmentioning

confidence: 99%

Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus

Kretz,

Börner,

Friedrich

et al. 2024

Front. Microbiol.

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Bacteria use CRISPR Cas systems to defend against invading foreign nucleic acids, e.g., phage genomes, plasmids or mobile genetic elements. Some CRISPR Cas systems were reported to have physiological importance under a variety of abiotic stress conditions. We used physiological tests under different stress conditions and RNA-seq analyses to address the possible function of the RNA-targeting class 2 type VI CRISPR Cas system of the facultative phototrophic α-proteobacterium Rhodobacter capsulatus. Expression of the system was low under exponential non-stress conditions and high during oxidative stress, membrane stress and in stationary phase. Induction of the CRISPR Cas system in presence of a target protospacer RNA resulted in a growth arrest of R. capsulatus. RNA-seq revealed a strong alteration of the R. capsulatus transcriptome when cas13a was induced in presence of a target protospacer. RNA 5′ end mapping indicated that the CRISPR Cas-dependent transcriptome remodeling is accompanied by fragmentation of cellular RNAs, e.g., for mRNAs originating from a genomic locus which encodes multiple ribosomal proteins and the RNA polymerase subunits RpoA, RpoB and RpoC. The data suggest a function of this CRISPR Cas system in regulated growth arrest, which may prevent the spread of phages within the population.

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Section: Resultsmentioning

confidence: 99%

Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus

Kretz,

Börner,

Friedrich

et al. 2024

Front. Microbiol.

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“…For instance, the wild-type lac promoter from E. coli is reported to function poorly in R. sphaeroides (26). While no inducible promoters have been examined in N. aromaticivorans to our knowledge, promoters inducible by light, oxygen, crystal violet, and IPTG have been tested in R. sphaeroides (26)(27)(28)(29). Although these foreign promoters can respond to an array of possible inducers, some inducers inhibit R. sphaeroides growth (crystal violet) (26) or substantially alter physiological processes of interest (light, oxygen).…”

Section: Introductionmentioning

confidence: 99%

Tools for Genetic Engineering and Gene Expression Control inNovosphingobium aromaticivoransandRhodobacter sphaeroides

Hall,

Kinney

et al. 2023

Preprint

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Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria,Novosphingobium aromaticivorans,Rhodobacter sphaeroides, andZymomonas mobilis, show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit forZ. mobilis,N. aromaticivoransandR. sphaeroidesstill need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools forN. aromaticivoransandR. sphaeroidesto address these issues. We demonstrate that Tn7transposition is an effective approach for introducing engineered DNA into the chromosome ofN. aromaticivoransandR. sphaeroides. We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn7integration with promoters from our library, we establish CRISPR interference systems forN. aromaticivoransandR. sphaeroidesthat can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria.IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise excellent properties for conversion are challenging to genetically engineer. Here, we report systems to overcome that barrier in the Alphaproteobacteria,Novosphingobium aromaticivoransandRhodobacter sphaeroides, by producing genetic tools that allow easy insertion of engineered pathways into their genomes and to precisely control gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to optimize yield of biofuels and bioproducts.

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Tools for genetic engineering and gene expression control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides

Hall,

Kinney

et al. 2024

Appl Environ Microbiol

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Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans , Rhodobacter sphaeroides , and Zymomonas mobilis show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis , N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn 7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides . We screen a synthetic promoter library to identify isopropyl β-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in N. aromaticivorans and ~5-fold induction in R. sphaeroides ). Combining Tn 7 integration with promoters from our library, we establish CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference systems for N. aromaticivorans and R. sphaeroides (up to ~10-fold knockdown in N. aromaticivorans and R. sphaeroides ) that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these species and other Alphaproteobacteria. IMPORTANCE It is important to increase our understanding of the microbial world to improve health, agriculture, the environment, and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise promising properties for conversion are challenging to genetically engineer. Here we report genetic tools for Novosphingobium aromaticivorans and Rhodobacter sphaeroides that add to the burgeoning set of tools available for genome engineering and gene expression in Alphaproteobacteria. Our approaches allow straightforward insertion of engineered pathways into the N. aromaticivorans or R. sphaeroides genome and control of gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to aid in optimizing yield of biofuels and bioproducts.

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A Small RNA, UdsC, Interacts with the RpoHII mRNA and Affects the Motility and Stress Resistance of Rhodobacter sphaeroides

Cited by 4 publications

References 49 publications

Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus

Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus

Tools for Genetic Engineering and Gene Expression Control inNovosphingobium aromaticivoransandRhodobacter sphaeroides

Tools for genetic engineering and gene expression control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides

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