CRISPR/Cas12a-mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus.
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ABSTRACT:
SUBMITTER: Zhang Y
PROVIDER: S-EPMC8601187 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rhodobacter capsulatus SB 1003 belongs to the group of purple nonsulfur bacteria. Its genome consists of a 3.7-Mb chromosome and a 133-kb plasmid. The genome encodes genes for photosynthesis, nitrogen fixation, utilization of xenobiotic organic substrates, and synthesis of polyhydroxyalkanoates. These features made it a favorite research tool for studying these processes. Here we report its complete genome sequence.
| S-EPMC2897665 | biostudies-literature
Project description:The long-range movement of Rhodobacter capsulatus cells in the glass-agar interstitial region of borosilicate Petri plates was found to be due to a subset of the cells inoculated into plates. The macroscopic appearance of plates indicated that a small group of cells moved in a coordinated manner to form a visible satellite cluster of cells. Satellite clusters were initially separated from the point of inoculation by the absence of visible cell density, but after 20 to 24 hours this space was colonized by cells apparently shed from a group of cells moving away from the point of inoculation. Cell movements consisted of flagellum-independent and flagellum-dependent motility contributions. Flagellum-independent movement occurred at an early stage, such that satellite clusters formed after 12 to 24 hours. Subsequently, after 24 to 32 hours, a flagellum-dependent dispersal of cells became visible, extending laterally outward from a line of flagellum-independent motility. These modes of taxis were found in several environmental isolates and in a variety of mutants, including a strain deficient in the production of the R. capsulatus acyl-homoserine lactone quorum-sensing signal. Although there was great variability in the direction of movement in illuminated plates, cells were predisposed to move toward broad spectrum white light. This predisposition was increased by the use of square plates, and a statistical analysis indicated that R. capsulatus is capable of genuine phototaxis. Therefore, the variability in the direction of cell movement was attributed to optical effects on light waves passing through the plate material and agar medium.
| S-EPMC3087807 | biostudies-literature
Project description:A flavodoxin was isolated from iron-sufficient, nitrogen-limited cultures of the photosynthetic bacterium Rhodobacter capsulatus. Its molecular properties, molecular weight, UV-visible absorption spectrum, and amino acid composition suggest that it is similar to the nif-specific flavodoxin, NifF, of Klebsiella pneumoniae. The results of immunoblotting showed that R. capsulatus flavodoxin is nif specific, since it is absent from ammonia-replete cultures and is not synthesized by the mutant strain J61, which lacks a nif-specific regulator (NifR1). Growth of cultures under iron-deficient conditions causes a small amount of flavodoxin to be synthesized under ammonia-replete conditions and increases its synthesis under N2-fixing conditions, suggesting that its synthesis is under a dual system of control with respect to iron and fixed nitrogen availability. Here we show that flavodoxin, when supplemented with catalytic amounts of methyl viologen, is capable of efficiently reducing nitrogenase in an illuminated chloroplast system. Thus, this nif-specific flavodoxin is a potential in vivo electron carrier to nitrogenase; however, its role in the nitrogen fixation process remains to be established.
| S-EPMC206800 | biostudies-other
Project description:A first glimpse into the proteome of Rhodobacter capsulatus revealed more than 450 (with over 210 cytoplasmic and 185 extracytoplasmic known as well as 55 unknown) proteins that are identified with high degree of confidence using nLC-MS/MS analyses. The accumulated data provide a solid platform for ongoing efforts to establish the proteome of this species and the cellular locations of its constituents. They also indicate that at least 40 of the identified proteins, which were annotated in genome databases as unknown hypothetical proteins, correspond to predicted translation products that are indeed present in cells under the growth conditions used in this work. In addition, matching the identification labels of the proteins reported between the two available R. capsulatus genome databases (ERGO-light with RRCxxxxx and NT05 with NT05RCxxxx numbers) indicated that 11 such proteins are listed only in the latter database.
| S-EPMC3075558 | biostudies-literature
Project description:The physiological function of Rhodobacter capsulatus FdVI, a [2Fe-2S] ferredoxin, was investigated by the cloning, sequence analysis, and mutagenesis of its structural gene, called fdxE. The DNA region surrounding fdxE was mapped, and the nucleotide sequence of a 4.2-kb fragment was determined. fdxE is preceded by a sequence that is very similar to a sigma54 recognition site and is followed by a putative transcription stop signal, suggesting that fdxE forms a separate cistron. Two open reading frames were identified upstream and downstream of fdxE and were named ORFE0 and ORFE1, respectively. The former may encode a polypeptide having 34% similarity with HtrA, a serine protease found in enteric bacteria. ORFE1 is homologous to purU, a gene involved in purine biosynthesis. Interposon mutagenesis of fdxE was unsuccessful when attempted on the wild-type strain B10. Disruption of fdxE could be achieved only in strains harboring an additional copy of fdxE on a plasmid. Mutants obtained in this way and carrying a plasmid-borne copy of fdxE under the control of the nifH promoter grew only in N-free medium, thus demonstrating that fdxE expression is required for growth. Nevertheless, such mutants were found to spontaneously revert at a frequency of 5 x 10(-6) to an apparent wild-type phenotype, although they contained no detectable amount of FdVI. Taken together, the results indicate that FdVI is required for an essential metabolic function in R. capsulatus and that this FdVI dependence could be relieved by a single-mutation event. In accordance, FdVI biosynthesis was found to be constitutive in R. capsulatus.
| S-EPMC179111 | biostudies-other
Project description:Photocaged inducer molecules, especially photocaged isopropyl-β-d-1-thiogalactopyranoside (cIPTG), are well-established optochemical tools for light-regulated gene expression and have been intensively applied in Escherichia coli and other bacteria including Corynebacterium glutamicum, Pseudomonas putida or Bacillus subtilis. In this study, we aimed to implement a light-mediated on-switch for target gene expression in the facultative anoxygenic phototroph Rhodobacter capsulatus by using different cIPTG variants under both phototrophic and non-phototrophic cultivation conditions. We could demonstrate that especially 6-nitropiperonyl-(NP)-cIPTG can be applied for light-mediated induction of target gene expression in this facultative phototrophic bacterium. Furthermore, we successfully applied the optochemical approach to induce the intrinsic carotenoid biosynthesis to showcase engineering of a cellular function. Photocaged IPTG thus represents a light-responsive tool, which offers various promising properties suitable for future applications in biology and biotechnology including automated multi-factorial control of cellular functions as well as optimization of production processes.
| S-EPMC9561348 | biostudies-literature
Project description:A Rhodobacter capsulatus hemC mutant has been isolated and used to show that oxygen regulates the intracellular levels of porphobilinogen. Experiments using a hemB-cat gene fusion demonstrated that oxygen does not transcriptionally regulate hemB transcription. Porphobilinogen synthase activity is not regulated by oxygen nor is the enzyme feedback inhibited by hemin or protoporphyrin IX. It was demonstrated that less than 20% of [(14)C]aminolevulinate was incorporated into bacteriochlorophyll, suggesting that the majority of the aminolevulinate is diverted from the common tetrapyrrole pathway. Porphobilinogen oxygenase activity was not observed in this organism; however, an NADPH-linked aminolevulinate dehydrogenase activity was demonstrated. The specific activity of this enzyme increased with increasing oxygen tension. The results presented here suggest that carbon flow over the common tetrapyrrole pathway is regulated by a combination of feedback inhibition of aminolevulinate synthase and diversion of aminolevulinate from the pathway by aminolevulinate dehydrogenase.
| S-EPMC134899 | biostudies-literature
Project description:Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt c(y). In species like R. capsulatus, cyt c(y) functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c(y) (S-c(y)), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt c(y). This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c(y) regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c(y) variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c(y) was about 340 mV, similar to that of cyts c2 or c(y). Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-c(y) was distinct from those seen with the cyt c2 or cyt c(y). The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c(y) interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-c(y) revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt c(y), whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.
| S-EPMC2376234 | biostudies-literature
Project description:Clostridium species have gained attention in industrial and medical applications, and the development of genetic tools has enabled the advancement of the CRISPR-Cas systems for these bacteria. Compared to the primarily used Cas9 from Streptococcus pyogenes, the utilization of Cas12a (previously known as Cpf1) proteins remains incomplete in clostridia, although they exhibit potential advantages, including T-rich recognition for Clostridium genomes and lower toxicity. In this study, we expanded the CRISPR-Cas tools in clostridia by establishing a CRISPR-Cas12a system with two different cas12a genes (Ascas12a from Acidaminococcus and Fncas12a from Francisella novicida). The optimized tetracycline-inducible systems were initially determined by the glucuronidase reporter and were used to drive expression of the cas12a genes and crRNAs. Our results demonstrate that the CRISPR-FnCas12a system offers flexible target selection in clostridia, and a specific folding pattern of the precursor crRNA is important to enable high mutation generation efficiency. By using sacB (encoding levansucrase) as a negative marker for plasmid curing and determining the optimal size of the donor DNA template for gene integration in the CRISPR-FnCas12a system, we achieved highly efficient and rapid genome modification, exemplified by the successful engineering of clostridia (Clostridium butyricum and Clostridium sporogenes) to produce near-infrared fluorescence from biliverdin and hemin.IMPORTANCEContinued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes, which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.
| S-EPMC10715149 | biostudies-literature
Project description:CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)-RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site-is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype-phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.
| S-EPMC4877446 | biostudies-other