The Bacillus subtilis tyrS gene is a member of a group of gram-positive aminoacyl-tRNA synthetase and amino acid biosynthesis genes which are regulated by transcription antitermination. Each gene in the group is specifically induced by limitation for the appropriate amino acid. This response is mediated by interaction of the cognate tRNA with the mRNA leader region to promote formation of an antiterminator structure. The tRNA interacts with the leader by codon-anticodon pairing at a position designated the specifier sequence which is upstream of the antiterminator. In this study, an additional site of possible contact between the tRNA and the leader was identified through covariation of leader mRNA and tRNA sequences. Mutations in the acceptor end of tRNAl',r could suppress mutations in the side bulge of the antiterminator, in a pattern consistent with base pairing. This base pairing may thereby directly affect the formation and/or function of the antiterminator. The discriminator position of the tRNA, an important identity determinant for a number of tRNAs, including tRNA"tF, was shown to act as a second specificity determinant for assuring response to the appropriate tRNA. Furthermore, overproduction of an unchargeable variant of tRNATYr resulted in antitermination in the absence of limitation for tyrosine, supporting the proposal that uncharged tRNA is the effector in this system.Most of the aminoacyl-tRNA synthetase genes identified to date in Bacillus subtilis are regulated by transcription antitermination (17,22,24,35). All of these genes exhibit very strong conservation of a set of primary-sequence and secondarystructure elements in their mRNA leader regions, upstream of the start of the coding region (17, 18). These elements, which include three stem-loop structures, a highly conserved 14-bp sequence designated the T box, and a factor-independent transcriptional terminator ( Fig. 1), are also found in certain amino acid biosynthesis genes and tRNA synthetase genes in Bacillus and other gram-positive species, including B. subtilis ilv-leu and cysE-cysS, Lactobacillus casei valS and trp, Lactococcus lactis trp and his, and Brevibacterium lactofermentum (and Corynebacterium glutamicum) argS-lysA, suggesting that this mechanism is widespread in gram-positive bacteria (18,22). For each gene that has been examined, readthrough of the leader region terminator is induced by limitation for the appropriate amino acid and not by general amino acid starvation (5,12,13,24,35). Analysis of the mechanism for the specific response of each gene to the cognate amino acid has been a major focus of attention in our laboratory.We noted that each leader region structure contains a triplet sequence corresponding to a codon specifying the appropriate amino acid, displayed at a specific position within a side bulge of the first stem-loop structure (17). Alteration of this triplet in the tyrS leader from a UAC tyrosine codon to a UUC phenylalanine codon was sufficient to switch the specificity of the response to amino acid limita...
SummaryThe Bacillus subtilis tyrS gene belongs to the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes, which are regulated by a common mechanism of transcriptional antitermination. Each gene is induced by specific amino acid limitation; the uncharged cognate tRNA is the effector inducing transcription of the full-length message. The leader regions of the genes in this family share a number of conserved primary sequence and secondary structural elements, the functions of which are unknown. In this study, we examine these regions and report the effects of mutations in several of these elements. In addition, two alternative basepairings in the F box region were found to be necessary for tyrS antitermination.
We applied an immunoscreening technique, in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed during human infection with Salmonella enterica serovar Typhi, the cause of typhoid fever. We were able to assign a functional classification to 25 of 35 proteins identified by IVIAT. Of these 25, the majority represent proteins with known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in fimbrial structure and biogenesis, antimicrobial resistance, heavy metal transport, bacterial adhesion, and extracytoplasmic substrate trafficking as well as secreted hydrolases. The 10 remaining antigens represent proteins with unknown functions. Of the 35 identified antigens, four had no immunoreactivity when probed with control sera from individuals never exposed to serovar Typhi organisms; these four included PagC, TcfB, and two antigens of unknown function encoded by STY0860 and STY3683. PagC is a virulence factor known to be upregulated in vivo in S. enterica serovar Typhimurium infection of mice. TcfB is the major structural subunit of a fimbrial operon found in serovar Typhi with no homolog in serovar Typhimurium organisms. By examining differential immunoreactivities in acute-versus convalescent-phase human serum samples, we found specific anti-PagC and anti-TcfB immunoglobulin G responses in patients with serovar Typhi bacteremia. Serovar Typhi antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and they may have diagnostic, therapeutic, or preventive uses.In humans, infection with Salmonella enterica serovar Typhi causes a spectrum of illness that includes diarrhea, a selflimited febrile illness, and most significantly, typhoid fever, a systemic infection characterized by persistent fever, lymphadenopathy, hepatosplenomegaly, encephalopathy, and intestinal hemorrhage and perforation (16). Worldwide, approximately 20 million cases of typhoid fever resulting in 200,000 deaths occur annually (2). The increasing incidence of disease caused by multidrug-resistant serovar Typhi organisms underscores the importance of developing novel approaches to the diagnosis, treatment, and prevention of typhoid fever (22).Although many S. enterica serovars infect a broad range of animal hosts and cause gastroenteritis in humans, serovar Typhi is among the few serovars for which natural infection appears to be limited to human hosts. In humans, serovar Typhi organisms penetrate the gastrointestinal epithelial barrier and infect phagocytes within the lamina propria. However, unlike organisms from other broad-host-range serovars, serovar Typhi organisms are adapted for prolonged intracellular survival in human macrophages, allowing the bacteria to spread to reticuloendothelial organs, including the liver, spleen, and bone marrow (16).Because serovar Typhi organisms are human specific, there is no optimal animal model of serovar Typhi infection, and presumed factors that contribute to the pathogenesis and immunology of...
SummaryIn vivo induced antigen technology (IVIAT) is a technique that identifies pathogen antigens that are immunogenic and expressed in vivo during human infection. IVIAT is complementary to other techniques that identify genes and their products expressed in vivo . Genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during human infection, and may be appropriate for inclusion in therapeutic, vaccine or diagnostic applications.
The Bacillus subtilis tyrS gene, encoding tyrosyl-tRNA synthetase, is a member of the T-box family of genes, which are regulated by control of readthrough of a leader region transcriptional terminator. Readthrough is induced by interaction of the cognate uncharged tRNA with the leader; the system responds to decreased tRNA charging, caused by amino acid limitation or insufficient levels of the aminoacyl-tRNA synthetase. Recognition of the cognate tRNA is mediated by pairing of the anticodon of the tRNA with the specifier sequence of the leader, a codon specifying the appropriate amino acid; a second interaction between the acceptor end of the tRNA and an antiterminator structure is also important. Certain switches of the specifier sequence to a new codon result in a switch in the specificity of the amino acid response, while other switches do not. These effects may reflect additional sequence or structural requirements for the mRNA-tRNA interaction. This study includes investigation of the effects of a large number of specifier sequence switches in tyrS and analysis of structural differences between tRNA Tyr and tRNA species which interact inefficiently with the tyrS leader to promote antitermination.Many aminoacyl-tRNA synthetase and amino acid biosynthesis operons in gram-positive bacteria are regulated by a common transcription antitermination system (11,12,16,28). The genes in this group share a number of features in the 5Ј region in their transcripts, including a set of conserved primary sequence and structural elements located upstream of the start of the coding sequence. The mRNA leader region structural elements include three large stem-loops, designated stems I, II, and III, preceding a factor-independent transcriptional terminator. The most prominent of the sequence elements, the 14-base T box, forms a portion of an antiterminator structure by pairing with conserved sequences on the 5Ј side of the terminator. Expression of the genes in this group is therefore controlled at the level of readthrough of the leader region transcriptional terminator, by a switch between the antiterminator and terminator forms of the transcript.Expression of several genes in this family has been shown to be induced in response to limitation for the cognate amino acid and not by general amino acid starvation (5,6,18,27,29). Examination of a number of the leader regions revealed a precisely located single codon, specifying the appropriate amino acid (11); the effector signalling amino acid limitation was therefore proposed to be tRNA, with the specificity of the amino acid response dictated by codon-anticodon pairing. The induction specificity of the Bacillus subtilis tyrS gene was switched from a response to tyrosine limitation to a response to phenylalanine limitation by a single base substitution in the UAC tyrosine codon of the tyrS leader to a UUC phenylalanine codon (11). These studies indicated that the leader region codon, termed the "specifier sequence," acts as a critical determinant of the specificity of the amino a...
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