A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro.

Incorporation of selenocysteine (Sec) in bacteria requires a UGA codon that is reassigned to Sec by the Sec-specific elongation factor SelB and a conserved mRNA motif (SECIS element). These requirements severely restrict the engineering of selenoproteins. Earlier, a synthetic tRNASec was reported that allowed canonical Sec incorporation by EF-Tu; however, serine misincorporation limited its scope.

Reducing the genetic code induces massive rearrangement of the proteome.

Expanding the genetic code is an important aim of synthetic biology, but some organisms developed naturally expanded genetic codes long ago over the course of evolution. Less than 1% of all sequenced genomes encode an operon that reassigns the stop codon UAG to pyrrolysine (Pyl), a genetic code variant that results from the biosynthesis of Pyl-tRNA(Pyl). To understand the selective advantage of genetically encoding more than 20 amino acids, we constructed a markerless tRNA(Pyl) deletion strain of Methanosarcina acetivorans (ΔpylT) that cannot decode UAG as Pyl or grow on trimethylamine.

Transfer RNA misidentification scrambles sense codon recoding.

Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused (or rarely used) codon, and an orthogonal tRNA synthetase:tRNA pair with the complementary anticodon. The Mycoplasma capricolum genome contains just six CGG arginine codons, without a dedicated tRNA(Arg).

Carbon source-dependent expansion of the genetic code in bacteria.

Despite the fact that the genetic code is known to vary between organisms in rare cases, it is believed that in the lifetime of a single cell the code is stable. We found Acetohalobium arabaticum cells grown on pyruvate genetically encode 20 amino acids, but in the presence of trimethylamine (TMA), A. arabaticum dynamically expands its genetic code to 21 amino acids including pyrrolysine (Pyl).

Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion.

Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent.

An unusual tandem-domain rhodanese harbouring two active sites identified in Desulfitobacterium hafniense.

The rhodanese protein domain is common throughout all kingdoms of life and is characterized by an active site cysteine residue that is able to bind sulfane sulfur and catalyse sulfur transfer. No unique function has been attributed to rhodanese-domain-containing proteins, most probably because of their diversity at both the level of sequence and protein domain architecture. In this study, we investigated the biochemical properties of an unusual rhodanese protein, PhsE, from Desulfitobacterium hafniense strain TCE1 which we have previously shown to be massively expressed under anaerobic respiration with tetrachloroethene.

Physiological adaptation of Desulfitobacterium hafniense strain TCE1 to tetrachloroethene respiration.

Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed.

Proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm formation at the alkane-water interface reveals novel proteins and cellular processes involved in hexadecane assimilation.

Many hydrocarbon-degrading bacteria form biofilms at the hydrocarbon-water interface to overcome the weak accessibility of these poorly water-soluble substrates. In order to gain insight into the cellular functions involved, we undertook a proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm developing at the hexadecane-water interface. Biofilm formation on hexadecane led to a global change in cell physiology involving modulation of the expression of 576 out of 1144 detected proteins when compared with planktonic cells growing on acetate.