Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the “FilmArray”, which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

The signal recognition particle (SRP) plays a central role in directing the export of nascent proteins from the cytoplasm of mammalian cells. An SRP-dependent translocation machinery in bacteria has not been demonstrated in previous genetic and biochemical studies. Sequence comparisons, however, have identified (i) a gene in Escherichia coli (ffh) whose product is homologous to the 54-kilodalton subunit (SRP54) of SRP, and (ii) an RNA encoded by the ffs gene (4.5S RNA) that shares a conserved domain with the 7SL RNA of SRP. An antiserum to Ffh precipitated 4.5S RNA from E. coli extracts, implying that the two molecules reside in a complex. The 4.5S RNA can also bind to SRP54 and can replace 7SL RNA in an enzymatic assay. The product of a dominant mutation in the ffs gene (4.5S RNAdl1) is also coprecipitated by the antiserum to Ffh protein and is lethal when expressed from an inducible promoter. After induction of 4.5S RNAdl1, the earliest observed phenotype was a permanent induction of the heat shock response, suggesting that there was an accumulation of aberrant proteins in the cytoplasm. Late after induction, translocation of beta-lactamase was impaired; this may be an indirect effect of heat shock, however, because translocation of ribose binding protein or of the porin, OmpA, was unaffected. An unusual separation of the inner and outer membranes, suggestive of a defect in cell envelope, was also observed. Protein synthesis did not cease until very late, an indication that 4.5S RNA probably does not have a direct role in this process.

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Inc., Salt Lake City, UT) Blood Culture (BC) panel can identify > 25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 hour. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 of 92 pathogens (91%) covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven MRSA and VRE. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.

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