Common thiolation mechanism in the biosynthesis of tRNA thiouridine and sulphur-containing cofactors

2-Thioribothymidine (s2T), a modified uridine, is found at position 54 in transfer RNAs (tRNAs) from several thermophiles; s2T stabilizes the L-shaped structure of tRNA and is essential for growth at higher temperatures. Here, we identified an ATPase (tRNA-two-thiouridine C, TtuC) required for the 2-thiolation of s2T in Thermus thermophilus and examined in vitro s2T formation by TtuC and previously identified s2T-biosynthetic proteins (TtuA, TtuB, and cysteine desulphurases). The C-terminal glycine of TtuB is first activated as an acyl-adenylate by TtuC and then thiocarboxylated by cysteine desulphurases. The sulphur atom of thiocarboxylated TtuB is transferred to tRNA by TtuA. In a ttuC mutant of T. thermophilus, not only s2T, but also molybdenum cofactor and thiamin were not synthesized, suggesting that TtuC is shared among these biosynthetic pathways. Furthermore, we found that a TtuB–TtuC thioester was formed in vitro, which was similar to the ubiquitin-E1 thioester, a key intermediate in the ubiquitin system. The results are discussed in relation to the mechanism and evolution of the eukaryotic ubiquitin system

Mechanism of Therapeutic Effectiveness of Cefixime against Typhoid Fever

β-Lactams have been considered ineffective against organisms growing inside mammalian cells because of their poor penetration into cells. However, cefixime has been shown to be clinically effective against typhoid fever. The probable mechanism of therapeutic effectiveness of cefixime against typhoid fever was investigated using Salmonella enterica serovar Typhimurium instead of S. enterica serovar Typhi both in a cellular and in a mouse infection model. Cefixime was able to inhibit the growth of serovar Typhimurium inhabiting monocyte-derived THP-1 cells. Elongation of serovar Typhimurium in THP-1 cells was observed microscopically. Apparent morphological changes of serovar Typhimurium in THP-1 cells were also observed by electron microscopy. The concentration of cefixime inside THP-1 cells was almost half (46 to 48%) of the concentration outside the cells when serovar Typhimurium coexisted in the solution. The length of time after oral dosing (8 mg/kg) that cefixime was present—calculated from levels in serum—at a concentration above the MIC at which 90% of the serovar Typhi organisms inside human cells were inhibited was presumed to be more than 12 h. Cefixime also showed excellent activity in the mouse systemic and oral infection models based on infections caused by serovar Typhimurium. It is concluded that a fair amount of cefixime can enter mammalian cells and inhibit the growth of bacteria inside cells when the bacteria are sensitive enough to cefixime, as are serovars Typhimurium and Typhi

Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA

Ubiquitin-like proteins (UBLs) can change protein function, localization or turnover by covalent attachment to lysine residues. Although UBLs achieve this conjugation through an intricate enzymatic cascade, their bacterial counterparts MoaD and ThiS function as sulphur carrier proteins. Here we show that Urm1p, the most ancient UBL, acts as a sulphur carrier in the process of eukaryotic transfer RNA (tRNA) modification, providing a possible evolutionary link between UBL and sulphur transfer. Moreover, we identify Uba4p, Ncs2p, Ncs6p and Yor251cp as components of this conserved pathway. Using in vitro assays, we show that Ncs6p binds to tRNA, whereas Uba4p first adenylates and then directly transfers sulphur onto Urm1p. Finally, functional analysis reveals that the thiolation function of Urm1p is critical to regulate cellular responses to nutrient starvation and oxidative stress conditions, most likely by increasing translation fidelity

Quantative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g to 4.70, 5.85 and 3.36 log cfu g. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage