No abstract

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. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids.

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). A. arabaticum is the only known organism that modulates the size of its genetic code in response to its environment and energy source. The gene cassette pylTSBCD , required to biosynthesize and genetically encode UAG codons as Pyl, is present in the genomes of 24 anaerobic archaea and bacteria. Unlike archaeal Pyl-decoding organisms that constitutively encode Pyl, we observed that A. arabaticum controls Pyl encoding by down-regulating transcription of the entire Pyl operon under growth conditions lacking TMA, to the point where no detectable Pyl-tRNA Pyl is made in vivo. Pyl-decoding archaea adapted to an expanded genetic code by minimizing TAG codon frequency to typically ∼5% of ORFs, whereas Pyl-decoding bacteria (∼20% of ORFs contain in-frame TAGs) regulate Pyl-tRNA Pyl formation and translation of UAG by transcriptional deactivation of genes in the Pyl operon. We further demonstrate that Pyl encoding occurs in a bacterium that naturally encodes the Pyl operon, and identified Pyl residues by mass spectrometry in A. arabaticum proteins including two methylamine methyltransferases.

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. We report a superior tRNASec variant (tRNAUTuX) that facilitates EF-Tu dependent stoichiometric Sec insertion in response to UAG both in vivo in Escherichia coli and in vitro in a cellfree protein synthesis system. We also demonstrate recoding of several sense codons in a SelB supplemented cell-free system. These advances in Sec incorporation will aid rational design and directed evolution of selenoproteins.

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. Mycoplasma capricolum contains only 6 CGG arginine codons without a dedicated tRNAArg. We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (tRNAPylCCG), and the genes for pyrrolysine biosynthesis. Here we show that tRNAPylCCG is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry reveals that in the presence of tRNAPylCCG, CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.

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. Soluble and membrane-associated proteins obtained from cells of Desulfitobacterium hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the WoodLjungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.The first members of the genus Desulfitobacterium have been isolated as organohalide-respiring bacteria able to use chlorinated aliphatic (chloroethenes and -ethanes) and/or aromatic compounds as terminal electron acceptor (6,19,39). The variety of environments from which Desulfitobacterium strains have been isolated suggests that they are ubiquitous organisms (for a review, see reference 34). Their ability to use such toxic chlorinated compounds as electron acceptors has probably allowed them to colonize a number of particular environmental niches. However, they survive probably thanks to their metabolic versatility using various nonchlorinated electron acceptors, such as fumarate, nitrate, sulfite, thiosulfate, humic acids, and metals (reviewed in reference 39).The recently sequenced genomes of Desulfitobacterium hafniense strains Y51 (NCBI reference strain NC_007907) (23) and DCB-2 (NCBI reference strain NC_011830) (DOE Joint Genome Institute) have unraveled additional aspects of the metabolic versatility of this genus. A total of 59 members of the conserved iron-sulfur molybdoenzyme (CISM) family (28), previously known as the dimethyl sulfoxide (DMSO) reductase family, have been identified in the genome of strain Y51, among which are enzymes with a possible role in anaerobic respiration of DMSO, trimethylamine N-oxide, polysulfide, selenate, and arsenate. This genome also contains 18 paralogues of the fumarate reductase, indicating a gene reservoir far beyond the recognized physiological capabilities of Desulfitobacterium spp., especially with respect to their already considerable respiratory flexibility.Anaerobic respiration implies the establishment of a structured electron transport chain within the cytoplasmic membrane enabling energy conservation via the proton motive force, and the electron transport chains can sometimes ...

Many hydrocarbon degrading bacteria form biofilms at the hydrocarbon-water interface to overcome the low 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 of the cell physiology involving modulation of the expression of 573 out of 1144 detected proteins when compared with planktonic cells growing on acetate. Biofilm cells overproduced a protein, encoded by MARHY0478 that contains a conserved domain belonging to the family of the outer membrane transporters of hydrophobic compounds. Homologs of MARHY0478 were exclusively found in marine bacteria degrading alkanes or possessing alkane degradation genes and hence presumably constitute a family of alkane transporter specific to marine bacteria. Interestingly, we also found that sessile cells growing on hexadecane overexpressed type VI secretion system components. This secretion system has been identified as a key factor in virulence and in symbiotic interaction with host organisms. This observation is the first experimental evidence of the contribution of a type VI secretion system to environmental adaptation and raises the intriguing question about the role of this secretion machine in alkane assimilation.

Selenium is an essential micronutrient for animals. [1] Humans contain 25 presumably essential selenoproteins [2] in which selenium is found in the form of Sec. [3] In this 21 st genetically encoded amino acid [4] the thiol moiety of Cys is replaced by a selenol group. In all Sec-decoding organisms, Sec biosynthesis (Scheme 1B) starts with the acylation of tRNA Sec by seryl-tRNA synthetase (SerRS) to form Ser-tRNA Sec (reviewed in [5]). In bacteria, conversion of Ser-tRNA Sec to Sec-tRNA Sec is achieved by SelA (reviewed in [4]). In contrast, archaea and eukaryotes employ an additional phosphorylation step. Ophosphoseryl-tRNA Sec kinase (PSTK) phosphorylates the tRNA-bound Ser moiety of Ser-tRNA Sec to form O-phosphoseryl-tRNA Sec (Sep-tRNA Sec), [6] the substrate for SepSecS that catalyzes the tRNA-dependent Sep to Sec conversion. [7] The selenium donor for both SelA and SepSecS is selenophosphate (reviewed in [4, 7b]). During selenoprotein synthesis Sec is co-translationally incorporated by a reprogrammed UGA stop codon. A specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element) located within the bacterial ORF sequence are required for unambiguous Stop to Sec recoding. [4] EF-Tu does not recognize Sec-tRNA Sec and also discriminates against Ser-tRNA Sec.[4] Selenium and sulfur are in the same group of elements in the periodic table and share certain properties (e.g., size, electronegativity, major oxidation states); yet, Cys and Sec are distinguished by different electrode potentials, [8] nucleophilicity (Cys < Sec), [9] and sidechain pK a (8.3 for Cys vs 5.2 for Sec). [10] Thus, selenoproteins have unique properties [11]. Sec is frequently found as an enzyme active site residue endowing these proteins (e.g., redox enzymes) with superior catalytic activities. Sec to Cys replacements in selenoenzymes may lead to 10 to 1,000-fold activity loss (reviewed in [11b]). While disulfides occur frequently in proteins to increase stability or provide redox functions, diselenides are much less frequent. [12] The occurrence of diselenides in proteins has exciting biological and biomedical significance, as they are more stable than disulfides [13] and sometimes even resistant to reduction by DTT. [12]