Salmonella typhimurium is a facultative intracellular pathogen capable of surviving within phagocytic cells of the reticuloendothelial system. To identify the genes important for intracellular survival, 9516 independent Tn1O insertional mutations were isolated in a virulent strain of S.typhimurium. By using an in vitro assay for survival within macrophages, 83 TnlO mutants have been identified that have a diminished capacity for intracellular survival (designated MS or macrophage survival mutants). All of the MS mutants are less virulent than the parent strain in vivo, demonstrating that, for Salmonella, survival within the macrophage is essential for virulence. Thirty-seven of the MS mutants have been characterized as to their phenotype, including several mutations that confer sensitivity to specific microbiocidal mechanisms of the macrophage.Phagocytic cells are one of the first lines of defense in the body against invading organisms (1); they engulf and kill nonpathogenic and some pathogenic microbes by oxygendependent and -independent mechanisms (2, 3). Intracellular pathogens have evolved means to evade killing by professional phagocytes, thereby allowing them to survive within phagocytic cells (4). Although intracellular pathogens are a clinically important group of microorganisms that cause lingering diseases, such as tuberculosis and typhoid fever, little is known about the molcular mechanisms they employ to interfere with normal phagocyte function.Salmonella typhimurium is a facultative intracellular pathogen of mice (5) 5189The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Facultative intracellular pathogens pose an important health problem because they circumvent a primary defense mechanism of the host: killing and degradation by professional phagocytic cells. A gene of the intracellular pathogen Salmonella typhimurium that is required for virulence and intracellular survival was identified and shown to have a role in resistance to defensins and possibly to other microbicidal mechanisms of the phagocyte. This gene may prove to be a regulatory element in the expression of virulence functions.

Proteins of the LuxR family detect the presence of N-acylhomoserine lactones (AHLs) and regulate transcription accordingly. When AHLs are synthesized by the same species that detects them, the system allows a bacterium to measure the population density of its own species, a phenomenon known as quorum sensing. The sdiA genes of Escherichia coli and Salmonella enterica serovar Typhimurium are predicted to encode LuxR homologs. However, these species do not appear to synthesize AHLs or any other molecule detected by SdiA. It has previously been demonstrated that overexpression of sdiA results in the activation of the ftsQAZ locus in E. coli and four other loci in Salmonella serovar Typhimurium. Here we report that transcriptional fusions to these five loci fall into two classes. The first class requires overexpression of sdiA for activation. The second class responds to sdiA expressed from its natural position in the chromosome if the appropriate AHLs are added to the culture. The only member of the second class is a series of Prck-luxCDABE fusions in Salmonella serovar Typhimurium. SdiA responds with highest sensitivity to AHLs that have a keto modification at the third carbon and an acyl chain length of 6 or 8 (half-maximal response between 1 and 5 nM). Growth of Salmonella in proximity to species known to synthesize these AHLs results in sdiA-dependent activation of the Prck-luxCDABE fusions. SdiA appears to be the first AHL receptor discovered that detects signals emanating exclusively from other species.

A fragment of the Salmonella typhimurium ethanolamine utilization operon was cloned and characterized. The 6.3-kb nucleotide sequence encoded six complete open reading frames, termed cchA, cchB, eutE, eutJ, eutG, and eutH. In addition, the nucleotide sequences of two incomplete open reading frames, termed eutX and eutI, were also determined. Comparison of the deduced amino acid sequences and entries in the GenBank database indicated that eutI encodes a phosphate acetyltransferase-like enzyme. The deduced amino acid sequences of the EutE and EutG proteins revealed a significant degree of homology with the Escherichia coli alcohol dehydrogenase AdhE sequence. Mutations in eutE or eutG completely abolished the ability of mutants to utilize ethanolamine as a carbon source and reduced the ability to utilize ethanolamine as a nitrogen source. The product of eutE is most probably an acetaldehyde dehydrogenase catalyzing the conversion of acetaldehyde into acetyl coenzyme A. The product of the eutG gene, an uncommon iron-containing alcohol dehydrogenase, may protect the cell from unconverted acetaldehyde by converting it into an alcohol. The deduced amino acid sequence of cchA resembles that of carboxysome shell proteins from Thiobacillus neapolitanus and Synechococcus sp. as well as that of the PduA product from S. typhimurium. CchA and CchB proteins may be involved in the formation of an intracellular microcompartment responsible for the metabolism of ethanolamine. The hydrophobic protein encoded by the eutH gene possesses some characteristics of bacterial permeases and might therefore be involved in the transport of ethanolamine. Ethanolamine-utilization mutants were slightly attenuated in a mouse model of S. typhimurium infection, indicating that ethanolamine may be an important source of nitrogen and carbon for S. typhimurium in vivo.Ethanolamine is, in the form of phosphatidylethanolamine, an essential component of both prokaryotic and eukaryotic membranes (32). The efficient recycling of components such as membrane lipids and proteins is of great importance for survival of microorganisms living in natural habitats (3,23,25,39). A metabolic pathway for ethanolamine utilization might therefore be an important survival strategy against the constant famine that microorganisms face in nature, although so far there is no experimental evidence to support this hypothesis (13,24). In addition, ethanolamine found in the mammalian gastrointestinal tract may present an important alternative source of nitrogen and carbon for bacteria living in the gut. Therefore, it should not be surprising that many enterobacteria, including Escherichia coli and Salmonella typhimurium, can use ethanolamine as a source of both carbon and nitrogen (5, 43). Roof and Roth isolated a large number of S. typhimurium mutants which were deficient in different stages of ethanolamine utilization (36). An elegant genetic analysis of these mutants revealed six complementation groups in the ethanolamine utilization operon located at 50 min on the S. typhi...