Engineering and modifying synthetic microbial chassis is one of the best ways not only to unravel the fundamental principles of life but also to enhance applications in the health, medicine, agricultural, veterinary, and food industries. The two primary strategies for constructing a microbial chassis are the top-down approach (genome reduction) and the bottom-up approach (genome synthesis). Research programs on this topic have been funded in several countries. The ‘Minimum genome factory’ (MGF) project was launched in 2001 in Japan with the goal of constructing microorganisms with smaller genomes for industrial use. One of the best examples of the results of this project is E. coli MGF-01, which has a reduced-genome size and exhibits better growth and higher threonine production characteristics than the parental strain [1]. The ‘cell factory’ project was carried out from 1998 to 2002 in the Fifth Framework Program of the EU (European Union), which tried to comprehensively understand microorganisms used in the application field. One of the outstanding results of this project was the elucidation of proteins secreted by Bacillus subtilis, which was summarized as the ‘secretome’ [2]. The GTL (Genomes to Life) program began in 2002 in the United States. In this program, researchers aimed to create artificial cells both in silico and in vitro, such as the successful design and synthesis of a minimal bacterial genome by John Craig Venter's group [3]. This review provides an update on recent advances in engineering, modification and application of synthetic microbial chassis, with particular emphasis on the value of learning about chassis as a way to better understand life and improve applications.
Apramycin is aclinically promising aminoglycoside antibiotic (AGA). To date,m echanisms underlying the biosynthesis and self-resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps,i nw hich an ovel aminoglycoside phosphotransferase (AprU), ap utative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5-OH of ap seudotrisaccharide intermediate,w hose N-7' acetyl group is subsequently hydrolyzed by AprP.A prZ is located extracellularly where it removes the phosphate group from ap seudotetrasaccharide intermediate,leading to the maturation of apramycin. Intriguingly,7'-N-acetylated and 5-O-phosphorylated apramycin that were accumulated in DaprU and DaprZ respectively exhibited significantly reduced antibacterial activities,i mplying Streptomyces tenebrarius employs C-5 phosphorylation and N-7' acetylation as two strategies to avoid auto-toxicity.S ignificantly,t his study provides insight into the design of new generation AGAs to circumvent the emergence of drugresistant pathogens.
CRISPR defense systems such as the well-known DNA-targeting Cas9 and the RNAtargeting type III systems are widespread in prokaryotes 1,2 . The latter can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition 3-5 . Among a large set of proteins that were linked to type III systems and predicted to bind cOAs 6,7 , a CRISPR associated Lon protease (CalpL) stood out to us. The protein contains a sensor domain of the SAVED (SMODSassociated and fused to various effector domains) family 7 , fused to a Lon protease effector domain. However, the mode of action of this effector was unknown. Here, we report the structure and function of CalpL and show that the soluble protein forms a stable tripartite complex with two further proteins, CalpT and CalpS, that are encoded in the same operon. Upon activation by cA4, CalpL oligomerizes and specifically cleaves the MazFhomolog CalpT, releasing the extracytoplasmic function (ECF) sigma factor CalpS from the complex. This provides a direct connection between CRISPR-based foreign nucleic acid detection and transcriptional regulation. Furthermore, the presence of a cA4-binding SAVED domain in a CRISPR effector reveals an unexpected link to the cyclic oligonucleotide-based antiphage signaling system (CBASS).3 Main CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptative immune system that enables microorganisms to fend off attacks from mobile genetic elements such as phages, viruses, or plasmids 8 . The protein complex Cas1-Cas2 captures short DNAs from invaders and integrates them as "memories" into a CRISPR locus 9 . Transcripts of these "memories" are processed into small CRISPR RNAs (crRNAs) and integrated into large ribonucleoprotein (RNP) complexes, which can sense the presence of a matching foreign nucleic acid in the cell 10 . Once an invading sequence is detected, an antiviral response is triggered. Depending on the type of CRISPR system 1 , this response can be markedly different, ranging from cleavage of the invading nucleic acid by the RNP as in the case of Cas9 11 , to a complex multipronged defense strategy as found in type III CRISPR systems 12 . For the latter, the Cas10 subunit of the RNP has a cyclase activity that converts ATP into a recently discovered class of cyclic oligoadenylates (cOAs) upon viral RNA recognition [3][4][5] . The cOAs are constructed from 3 to 6, 3'-5' linked AMP units 13 and act as second messengers, typically by binding to proteins harboring a CARF (CRISPR-associated Rossmann-fold) domain 14 . There is a wide variety of CARF proteins linked to effector domains with functions ranging from RNA cleavage, supercoiled DNA nicking, dsDNA cleavage to transcription modulation 12,[15][16][17][18][19][20] . The downstream effects of those cOA-activated proteins can lead to viral clearance, an abortive infection or a dormant state of the cell, enabling it to weather the phage attack 18,21 .Recently, two bioinformatic teams cataloged C...
Apramycin is aclinically promising aminoglycoside antibiotic (AGA). To date,m echanisms underlying the biosynthesis and self-resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps,i nw hich an ovel aminoglycoside phosphotransferase (AprU), ap utative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5-OH of ap seudotrisaccharide intermediate,w hose N-7' acetyl group is subsequently hydrolyzed by AprP.A prZ is located extracellularly where it removes the phosphate group from ap seudotetrasaccharide intermediate,leading to the maturation of apramycin. Intriguingly,7'-N-acetylated and 5-O-phosphorylated apramycin that were accumulated in DaprU and DaprZ respectively exhibited significantly reduced antibacterial activities,i mplying Streptomyces tenebrarius employs C-5 phosphorylation and N-7' acetylation as two strategies to avoid auto-toxicity.S ignificantly,t his study provides insight into the design of new generation AGAs to circumvent the emergence of drugresistant pathogens.
CRISPR antiviral defense systems such as the well-known DNA-targeting Cas9- and the more complex RNA-targeting type III systems are widespread in bacteria and archea. The type III systems can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition. These second messenger molecules bind to the CARF (CRISPR associated Rossmann-fold) domains of dedicated effector proteins that are often DNAses, RNAses, or putative transcription factors. The activated effectors interfere with cellular pathways of the host, inducing cell death or a dormant state of the cell that is better suited to avoid propagation of the viral attack. Among a large set of proteins that were predicted to be linked to the type III systems, the CRISPR-Lon protein caught our attention. The protein was predicted to be an integral membrane protein containing a SAVED- instead of a CARF-domain as well as a Lon protease effector domain. Here, we report the crystal structure of CRISPR-Lon. The protein is a soluble monomer and indeed contains a SAVED domain that accommodates cA4. Further, we show that CRISPR-Lon forms a stable complex with the 34 kDa CRISPR-T protein. Upon activation by cA4, CRISPR-Lon specifically cleaves CRISRP-T, releasing CRISPR-T23, a 23 kDa fragment that is structurally very similar to MazF toxins and is likely a sequence specific nuclease. Our results describe the first cOA activated proteolytic enzyme and provide the first example of a SAVED domain connected to a type III CRISPR defense system. The use of a protease as a means to unleash a fast response against a threat has intriguing parallels to eukaryotic innate immunity.
CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements (MGE) 1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA (crRNA) to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against MGE either directly, via the integral HD nuclease domain 3-5 or indirectly, via synthesis of cyclic oligonucleotide (cOA) second messengers to activate diverse ancillary effectors 6-9. A subset of type III CRISPR systems encode an uncharacterised CorA-family membrane protein and an associated NrN family phosphodiesterase predicted to function in antiviral defence. Here, we demonstrate that the CorA associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against MGE when expressed in E. coli. However, B. fragilis Cmr does not synthesise cOA species on activation, instead generating a previously undescribed signalling molecule, SAM-AMP (adenylyl-AdoMet) by conjugating ATP to S-adenosyl methionine via a phosphodiester bond. Once synthesised, SAM-AMP binds to the CorA effector, presumably leading to cell death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR associated phosphodiesterases or a SAM-AMP lyase, providing an off switch analogous to cOA specific ring nucleases 10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.
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