Exploiting unassigned codons in Micrococcus luteus for tRNA-based amino acid mutagenesis

Kowal, Anne K.; Oliver, John S.

doi:10.1093/nar/25.22.4685

Cited by 22 publications

(22 citation statements)

References 26 publications

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“…Possibly, the presence of both functional rrs genes was detrimental to the functionality of the ribosome and the transposon insertion in Mlut_14290 rescued fitness. Forty-eight tRNA genes were identified encoding tRNAs theoretically capable of translating all codons including AGA, which had previously been found to be untranslatable using an in vitro system derived from a M. luteus strain (61). Eighteen additional RNA genes were predicted by using models from the RNA families database (Rfam) (40).…”

Section: Resultsmentioning

confidence: 99%

Genome Sequence of the Fleming Strain of Micrococcus luteus , a Simple Free-Living Actinobacterium

et al. 2010

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Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G؉C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to ␤-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.Micrococcus luteus, the type species of the genus Micrococcus (family Micrococcaceae, order Actinomycetales) (117), is an obligate aerobe. Three biovars have been distinguished (138). Its simple, coccoid morphology delayed the recognition of its relationship to actinomycetes, which are typically morphologically more complex. In the currently accepted phylogenetic tree of the actinobacteria, Micrococcus clusters with Arthrobacter and Renibacterium. Some other coccoid actinobacteria originally also called Micrococcus, but reclassified into four new genera (Kocuria, Nesterenkonia, Kytococcus, and Dermacoccus), are more distant relatives (121). The genus Micrococcus now includes only five species: M. luteus, M. lylae, M. antarcticus, M. endophyticus, and M. flavus (20, 69, 70, 121).We report here the genome sequence of Micrococcus luteus NCTC2665 (DSM 20030 T ), a strain of historical interest, since Fleming used it to demonstrate bacteriolytic activity (due to lysozyme) in a variety of body tissues and secretions (29,3...

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Section: Resultsmentioning

confidence: 99%

Genome Sequence of the Fleming Strain of Micrococcus luteus , a Simple Free-Living Actinobacterium

et al. 2010

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“…Beispielsweise greifen Freisetzungsfaktoren an einigen Positionen derart effektiv ein, dass die natürliche Funktion des Amber-Stoppcodons, die Freisetzung des Proteins, nicht supprimiert wird, -selbst dann nicht, wenn die Suppressor-tRNA mit derjenigen Aminosäure beladen ist, die normalerweise im Wildtyp-Protein an dieser Stelle eingebaut wird. [173] Weitere ungelöste Probleme sind die mangelnde Stabilität von mRNA mit Stoppcodons sowie die Stabilität von Proteinen mit nichtkanonischen Aminosäuren.…”

Section: Die Grenzen Suppressionsbasierter Methodenunclassified

“…Die Verwendung nichtkanonischer Nucleotide zur Bildung eines neuen Watson-Crick-Basenpaars wird bereits ausführlich diskutiert. [205][206][207] Um die Probleme zu umgehen, die mit der Konkurrenz durch Freisetzungsfaktoren in suppressionsbasierten Methoden einhergehen, erforschten Kowal und Oliver [173] …”

Section: Weitere Ansätzeunclassified

Prolegomena zum experimentellen Engineering des genetischen Codes durch Erweiterung seines Aminosäurerepertoires

Budiša

2004

Angewandte Chemie

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Die Proteinsynthese und ihre Verbindung mit dem genetischen Code spielten lange Zeit eine zentrale Rolle in der Biologie. Der schnelle experimentelle Fortschritt führte im vergangenen Jahrzehnt zu einer nahezu kompletten Beschreibung dieser Prozesse. Weitere wichtige Experimente ergaben Hinweise dafür, dass die natürliche Protein‐Translationsmaschinerie neu programmiert werden kann, um viele nichtkanonische Aminosäuren genetisch zu codieren. Tatsächlich lassen die 20 kanonischen Aminosäuren, die im universellen genetischen Code verwendet werden, viele wünschenswerte Funktionalitäten vermissen, etwa Halogen‐, Keto‐, Cyan‐, Azido‐, Nitroso‐, Nitro‐ oder Silylgruppen sowie CC‐ oder CC‐Bindungen. Die Fähigkeit, genetisch eine derartige chemische Vielfalt zu codieren, wird uns ermöglichen, lebende Zellen wie Bakterien neu zu programmieren, sodass sie maßgeschneiderte Proteine mit vielseitigen Funktionen exprimieren. An der Schnittstelle von Biologie, Chemie und Physik entwickelt sich mit dem Engineering des genetischen Codes ein aufstrebendes Forschungsgebiet.

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“…The observation that some microbial genomes lack the cognate tRNAs required to read some sense codons lead to the idea that these sense codons are open or blank, and perhaps could be prime targets for reassignment. Micrococcus luteus is one such example that was reported to have several rare codons and missing cognate tRNAs, and was used to demonstrate insertion of a standard amino acid into these unassigned sense codons in vitro ( [26] and references within).…”

Section: Evolving (Reduced) Genomes and Sense Codon Recodingmentioning

confidence: 99%

Experimental challenges of sense codon reassignment: An innovative approach to genetic code expansion

Krishnakumar

Ling

2013

FEBS Letters

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a b s t r a c tThe addition of new and versatile chemical and biological properties to proteins pursued through incorporation of non-canonical amino acids is at present primarily achieved by stop codon suppression. However, it is critical to find new ''blank'' codons to increase the variety and efficiency of such insertions, thereby taking 'sense codon recoding' to center stage in the field of genetic code expansion. Current thought optimistically suggests the use of the pyrrolysine system coupled with re-synthesis of genomic information towards achieving sense codon reassignment. Upon review of the serious experimental challenges reported in recent studies, we propose that success in this area will depend on the re-synthesis of genomes, but also on 'rewiring' the mechanism of protein synthesis and of its quality control.

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Exploiting unassigned codons in Micrococcus luteus for tRNA-based amino acid mutagenesis

Cited by 22 publications

References 26 publications

Genome Sequence of the Fleming Strain of Micrococcus luteus , a Simple Free-Living Actinobacterium

Genome Sequence of the Fleming Strain of Micrococcus luteus , a Simple Free-Living Actinobacterium

Prolegomena zum experimentellen Engineering des genetischen Codes durch Erweiterung seines Aminosäurerepertoires

Experimental challenges of sense codon reassignment: An innovative approach to genetic code expansion

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