Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA.

Abstract

Fingerprinting of RNA by arbitrarily primed PCR (RAP) can be used to identify conditionaily expressed genes in prokaryotes. Differential gene expression in SalmoneUa typhimurium LT2 in response to peroxide treatment was examined as a system in which to demonstrate this strategy. This treatment models the induction of bacterial protective proteins that may occur when mammalian phagocytes use peroxide to fight S. typhimurium infection. To identify genes inducible by hydrogen peroxide stress, total RNA from peroxide-treated and untreated bacterial cultures were RAP rmgerprinted with six different arbitrarily selected primers. A 435-base RAP product that was differentialy amplified by RAP using the reverse sequencing primer was cloned and sequenced. Northern blot analysis confirmed that the RNA corresponding to this clone, RSP435, was induced when bacteria were treated with hydrogen peroxide. The RNA was not induced in an oxyRI mutant that constitutively expresses a subset of hydrogen peroxide-inducible genes. Using pulsedfield gel electrophoresis and dot blot hybridization to an array of induced Mud-P22 integrations, the gene corresponding to RSP435 was mapped to two places, one between 19 and 21.5 min and one between 56 and 57 min. Thus, two similar or identical stress-inducible genes were found in different parts of the genome. Identification, cloning, and mapping of the conditionaHly expressed RSP435 cDNA were performed entirely by physical means, demonstrating that the strategy should complement genetic methods for many prokaryotic or archaebacterial systems and should be applicable to organisms in which genetic methods are difficult to perform or have not yet been developed.Two similar methods for fingerprinting of eukaryotic RNA have been developed (1, 2) based on arbitrarily primed PCR (RAP) (3, 4). In principle, fingerprinting of RNA by RAP (1, 5) can be used on prokaryotic RNAs because it does not rely on poly(A)-tailed RNA that is rare in mRNAs isolated from bacteria (6). To demonstrate that the method can be used to identify differentially expressed genes of bacteria we searched for stress-induced RNAs in Salmonella typhimurium.Enterobacteria respond to various stimuli such as oxidative stress, extreme pH, anaerobiosis, heat shock, osmotic shock, and starvation, by changing the expression of groups of genes termed "stimulons" (7,29). Assorted genetic tools have been used to study these regulatory networks, including operon fusions (8), promoter probe plasmids (9, 10), and positively selected promoter probes (11). An example of the differential expression of sets of genes due to external stimuli is the response of S. typhimurium to oxidative stress. When bacteria invade the mammalian host, activated granulocytes may employ millimolar concentrations of peroxide and superoxide anion during phagocytosis in an attempt to kill the bacteria (12, 13). In response, the bacteria can induce stimu-The publication costs of this article were defrayed in part by page charge payment. This article must therefore...

A novel tRNA-associated locus (trl) from Helicobacter pylori is co-transcribed with tRNAGly and reveals genetic diversity

“…In contrast to other reports on the use of arbitrarily primed PCR of RNA to identify differentially expressed genes in prokaryotes (Wong & McClelland, 1994;Kwaik & Pederson, 1996;Peek et al, 1998;Thies et al, 1998;Yuk et al, 1998), few arbitrarily primed PCR products were seen in the fingerprints produced in the present work. This is most likely due to the fact that total prokaryotic RNA contains large amounts of rRNA species which reduces the efficiency of obtaining arbitrarily primed PCR products (Wong & McClelland, 1994).…”

“…There are therefore probably several other genes in addition to cagA, iceA and those identified by Akopyants et al (1998) Arbitrarily primed PCR of RNA has been used successfully in eukaryotes to identify genes that are differentially expressed under varying environmental conditions (Welsh et al, 1992). The technique has been further developed for the identification of differentially expressed genes in prokaryotes (Wong & McClelland, 1994;Kwaik & Pederson, 1996;Thies et al, 1998;Yuk et al, 1998), including H . pylori (Peek et al, 1998).…”

Molecular analysis of the adaptive response in Salmonella Typhimurium after starvation in salty conditions

“…Typhimurium responds to various stimuli such as oxidative stress, extreme pH, heat shock, osmotic conditions, and starvation by changing the expression of groups of genes termed "stimulons" [9]. Genes that contribute to the fitness of the pathogen within the host have been classified as virulence factors [10,11].…”

Transcriptome profiling of lactococcal mixed culture activity in milk by fluorescent RNA arbitrarily primed-PCR

“…Microarrays [26], which analyze whole transcriptomes, can be applied when the genome is known. However, if the genome has not been sequenced, the techniques of differential display [30,31] can be applied to evaluate modifications in transcriptomes according to various conditions either in eucaryotes [10,29] or in procaryotes [32]. The two molecular techniques of cDNA amplified fragment length polymorphism (cDNA-AFLP) [1] and RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) [32] are the most frequently used for evaluating modifications in transcriptomes [15].…”

“…However, if the genome has not been sequenced, the techniques of differential display [30,31] can be applied to evaluate modifications in transcriptomes according to various conditions either in eucaryotes [10,29] or in procaryotes [32]. The two molecular techniques of cDNA amplified fragment length polymorphism (cDNA-AFLP) [1] and RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) [32] are the most frequently used for evaluating modifications in transcriptomes [15]. However, cDNA-AFLP requires restriction sites so transcripts without correct sites will not be revealed at all, while RAP-PCR uses acrylamide electrophoresis that lacks the resolution necessary to efficiently separate amplimers [2].…”