AI-2 is an autoinducer made by many bacteria. LsrB binds AI-2 in the periplasm, and Tsr is the L-serine chemoreceptor. We show that AI-2 strongly attracts Escherichia coli. Both LsrB and Tsr are necessary for sensing AI-2, but AI-2 uptake is not, suggesting that LsrB and Tsr interact directly in the periplasm.Many functions in bacteria are regulated by population density, including formation of biofilms and production of virulence factors (5). Assessment of population density, known as quorum sensing, relies on the ability of cells to determine the concentrations of compounds known as autoinducers (AIs). As the cell density increases, an AI accumulates to a concentration that triggers a quorum-sensing response. Autoinducers activate some genes and repress others. Induced genes typically include those responsible for production of the autoinducer, resulting in a positive feedback loop. Cell densities required to accumulate enough AI for good induction are 10 8 per ml or higher.AIs are of two basic types: species specific and general (22). Species-specific AI-1s are acyl homoserine lactones in Gramnegative bacteria and modified peptides in Gram-positive bacteria. Full induction of bioluminescence in the marine bacterium Vibrio harveyi, which colonizes dead organic matter, requires both a specific AI-1 inducer and a general autoinducer, called AI-2 (6). AI-2 is derived from spontaneous cyclization of 4,5-dihydroxy-2,3-pentanedione (DPD). DPD is made from S-ribosylhomocysteine by the enzyme LuxS (25). S-Ribosylhomocysteine is an intermediate in the breakdown of S-adenosylhomocysteine, the product remaining after methyl group donation by S-adenosylmethionine.AI-2 is produced by a wide range of Gram-negative and Gram-positive bacteria and exists in multiple forms that are in equilibrium with each other (5). The form that is active in V. harveyi is (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate (S-THMF borate) (8), which binds to the periplasmic protein LuxP. In S. enterica serovar Typhimurium, a boron-free isomer of AI-2 [(2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetra-hydrofuran (R-THMF)] binds to the periplasmic LsrB protein (21). LsrB is the recognition component of an ABC transporter for AI-2. LsrACD are the membrane-bound components of the ABC transporter for AI-2. AI-2 is generated by the enzyme LuxS, and the YdgG (TqsA) protein has been implicated in AI-2 export from the cytoplasm (14).AI-2 is a known chemoattractant for Escherichia coli (4, 10), but the receptor(s) involved in AI-2 sensing has not been identified. This work was initiated to characterize the proteins involved in AI-2 recognition by E. coli strain CV1, which is equivalent to the standard wild-type chemotaxis strain RP437. The strains used in this study are shown in Table 1.The microplug (Plug) assay (9), a modified plug-in-pond assay, provides a qualitative but highly visual representation of chemotaxis. Cells containing the green fluorescent protein (GFP)-encoding plasmid pCM18 were grown overnight at 32°C in tryptone broth (TB) (20) co...

Norepinephrine (NE), the primary neurotransmitter of the sympathetic nervous system, has been reported to be a chemoattractant for enterohemorrhagic Escherichia coli (EHEC). Here we show that nonpathogenic E. coli K-12 grown in the presence of 2 M NE is also attracted to NE. Growth with NE induces transcription of genes encoding the tyramine oxidase, TynA, and the aromatic aldehyde dehydrogenase, FeaB, whose respective activities can, in principle, convert NE to 3,4-dihydroxymandelic acid (DHMA). Our results indicate that the apparent attractant response to NE is in fact chemotaxis to DHMA, which was found to be a strong attractant for E. coli. Only strains of E. coli K-12 that produce TynA and FeaB exhibited an attractant response to NE. We demonstrate that DHMA is sensed by the serine chemoreceptor Tsr and that the chemotaxis response requires an intact serine-binding site. The threshold concentration for detection is <5 nM DHMA, and the response is inhibited at DHMA concentrations above 50 M. Cells producing a heterodimeric Tsr receptor containing only one functional serine-binding site still respond like the wild type to low concentrations of DHMA, but their response persists at higher concentrations. We propose that chemotaxis to DHMA generated from NE by bacteria that have already colonized the intestinal epithelium may recruit E. coli and other enteric bacteria that possess a Tsr-like receptor to preferred sites of infection.T he human gastrointestinal (GI) tract harbors an assortment of bacteria, most of which are harmless or helpful commensals. However, infection of the GI tract by pathogenic bacteria can have devastating consequences. It has been suggested that norepinephrine (NE), the predominant neurotransmitter of the enteric sympathetic nervous system, promotes growth and virulence of enteric bacteria (1) through signaling via adrenergic receptors located either on intestinal epithelial cells (2) or in the bacteria themselves (3, 4). In particular, the bacterial quorum sensor kinase QseC has been implicated in the NE-induced expression of genes whose products are involved in adherence, motility, and pathogenesis (4, 5). However, the concentrations of NE required for effective induction of virulence genes, 50 M in one recent study (6), are higher than those that are expected to occur in the intestinal lumen (7,8). Thus, for NE to activate expression of virulence factors, bacteria would have to navigate to regions of the intestinal epithelium that have locally high concentrations of NE. An obvious candidate for directing such migration is chemotaxis.Chemotaxis in Escherichia coli is well understood at the molecular level. However, the compounds that have been reported as chemoattractants (9) are primarily nutrients: serine and related amino acids, sensed by the chemoreceptor Tsr; aspartate and maltose, sensed by Tar; ribose and galactose, sensed by Trg; and dipeptides and pyrimidines, sensed by Tap. NE has been reported to be an interdomain signaling molecule (5, 10, 11). NE serves as an inducer of v...

Tumor-promoting phorbol esters and insulin produce similar effects in Reuber H35 rat hepatoma cell proliferation, including increased ornithine decarboxylase (ODC) enzyme activity, DNA synthesis, and mitogenesis. We investigated ODC mRNA accumulation in cells treated with either insulin or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Both agents caused rapid accumulation of ODC mRNA: for TPA, it was maximal 3 hr after treatment (4-6-fold greater than control cells) and returned quickly to control levels; for insulin, it was significantly longer, continuing to increase for at least 6 hr. Simultaneous treatment with TPA and insulin led to additive effects on ODC mRNA. Induction of ODC by TPA was blocked by down-regulation or inhibition of protein kinase C (PKC), consistent with a PKC-mediated mechanism. In contrast, PKC down-regulation had little effect on ODC induction by insulin. Furthermore, although both agents stimulated ribosomal S6 protein phosphorylation in cells containing normal amounts of PKC, the response to TPA was abolished in PKC-depleted cells; the effect of insulin was only slightly inhibited. TPA caused a rapid redistribution of essentially all of the PKC activity from the cytosolic to the membrane fraction of the cells, whereas insulin had no effect on PKC distribution. These results suggest that although insulin and TPA share some common cytoplasmic signalling pathways, their effects on phosphorylation of nuclear proteins and transcription of ODC may be mediated by distinct factors.

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Full-length cDNAs for thyrotropin β (TSHβ) and glycoprotein hormone α (GSUα) subunits were cloned and sequenced from the red drum (Sciaenops ocellatus). The cDNAs for TSHβ (877 bp) and GSUα (661 bp) yielded predicted coding regions of 126 and 94 amino acid proteins, respectively. Both sequences contain all invariant cysteine and putative glycosylated asparagines characteristic of each as deduced by comparison with other GSUα and TSHβ sequences from representative vertebrate species. Multiple protein sequence alignments show that each subunit shares highest identity (79% for the TSHβ and 86% for the GSUα) with perciform fish. Furthermore, in a single joint phylogenetic analysis, each subunit segregates most closely with corresponding GSUα and TSHβ subunit sequences from closely related fish. Tissue-specific expression assays using RT-PCR showed expression of the TSHβ subunit limited to the pituitary. GSUα mRNA was predominantly expressed in the pituitary but was also detected in the testis and ovary of adult animals. Northern hybridization revealed the presence of a single transcript for both TSHβ and GSUα, each close in size to mRNA transcripts from other species. Dot blot assays from total RNA isolated from S. ocellatus pituitaries showed that in vivo T3 administration significantly diminished mRNA expression of both the TSHβ and GSUα subunits and that goitrogen treatment caused a significant induction of TSHβ mRNA only. Both TSHβ and GSUα mRNA expression in the pituitary varied significantly in vivo over a 24-h period. Maximal expression for both TSHβ and GSUα occurred during the early scotophase in relation to a peak in T4 blood levels previously documented. These results suggest the production of TSH in this species which may serve to drive daily cycles of thyroid activity. Readily quantifiable, variable, and thyroid hormone-responsive pituitary TSH expression, coupled with previously described dynamic daily cycles of circulating T4 and extensive background on the growth, nutrition, and laboratory culture of red drum, suggests that this species will serve as a useful model for experimental studies of the physiological regulation of TSH production.

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