Involvement of ERK-1/2 in IL-21-induced cytokine production in leukemia cells and human monocytes.

Cytokines play an important role in the immune system, and abnormalities in their production have been found in many human diseases. Interleukin-21 (IL-21), a type I cytokine produced by activated T cells, has diverse effects on the immune system, but its ability to induce production of other cytokines is not well delineated. Furthermore, the signaling pathway underlying its action is poorly understood. Here, we have evaluated IL-21-induced cytokine production in human monocytes and U937 leukemia cells. We found that IL-21 induces upregulation of a variety of cytokines from multiple cytokine families. We also found that IL-21 triggers rapid activation of ERK1/2. Neutralizing antibody to the IL-21R prevented both IL-21-induced cytokine production and IL-21-induced activation of ERK1/2. Inhibition of ERK1/2 activity by the ERK-selective inhibitor U0126 reverses the ability of IL-21 to upregulate cytokine production, suggesting that IL-21-induced cytokine production is dependent on ERK1/2 activation.

Microbial communities and interactions in the lone star tick, Amblyomma americanum.

To quantify microbial composition and interactions, we identified prokaryotic communities in the lone star tick (Amblyomma americanum) based on 16S rRNA gene sequences and direct probing. The lone star tick is the vector of emerging diseases and host to additional symbionts of unknown activity, and is representative of other blood-sucking arthropods. We evaluated the potential for vertical (transovarial) transmission by molecular analysis of microbial symbionts from egg and larval clutches. Direct probing of adults (N = 8 populations from the southeastern and midwestern USA, 900 ticks total) revealed three vertically transmitted symbionts: a Coxiella symbiont occurred at 100% frequency, Rickettsia species occurred in 45-61% of all ticks in every population and an Arsenophonus symbiont occurred in 0-90% of ticks per population. Arsenophonus and Rickettsia exhibited significant heterogeneity in frequency among populations. The human pathogens Ehrlichia chafeensis and Borrelia lonestari were rare in most populations. Additional microbes were detected sporadically. Most ticks (78%) were co-infected by two or three microbes but statistical analysis indicated no significant deviation from random co-occurrence. Our findings indicate that microbial communities within lone star ticks are diverse, and suggest that direct probing for a wider range of prokaryotes and application of quantitative polymerase chain reaction (PCR) may provide further insights into microbial interactions within disease vectors. Our results also emphasize the close phylogenetic relationship between tick symbionts and human pathogens, and consistent differences in their prevalence.

Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing.

Quorum sensing is an example of community behavior prevalent among diverse bacterial species. The term "quorum sensing" describes the ability of a microorganism to perceive and respond to microbial population density, usually relying on the production and subsequent response to diffusible signal molecules. A significant number of gram-negative bacteria produce acylated homoserine lactones (acyl-HSLs) as signal molecules that function in quorum sensing. Bacteria that produce acyl-HSLs can respond to the local concentration of the signaling molecules, and high population densities foster the accumulation of inducing levels of acyl-HSLs. Depending upon the bacterial species, the physiological processes regulated by quorum sensing are extremely diverse, ranging from bioluminescence to swarming motility. Acyl-HSL quorum sensing has become a paradigm for intercellular signaling mechanisms. A flurry of research over the past decade has led to significant understanding of many aspects of quorum sensing including the synthesis of acyl-HSLs, the receptors that recognize the acyl-HSL signal and transduce this information to the level of gene expression, and the interaction of these receptors with the transcriptional machinery. Recent studies have begun to integrate acyl-HSL quorum sensing into global regulatory networks and establish its role in developing and maintaining the structure of bacterial communities.

Inhibition of the Agrobacterium tumefaciens TraR quorum-sensing regulator. Interactions with the TraM anti-activator.

The Agrobacterium tumefaciens quorum-sensing transcriptional regulator TraR and its inducing ligand 3-oxo-octanoyl-l-homoserine lactone control conjugal transfer of the tumor-inducing plasmid, the primary virulence factor responsible for crown gall disease of plants. This regulatory system enables A. tumefaciens to express its conjugal transfer regulon preferentially at high population densities. TraR activity is antagonized by a second tumor-inducing plasmid-encoded protein designated TraM. TraM and TraR are thought to form an anti-activation complex that prevents TraR from recognizing its target DNA-binding sites. The formation and inhibitory function of the TraM-TraR anti-activation complex was analyzed using several different assays for protein-protein interaction, including surface plasmon resonance. The TraR-TraM complex forms readily in solution and is extremely stable (K(D) of 1-4 x 10(-9) m). Directed mutational analysis of TraM identified a number of amino acids that play important roles in the inhibition of TraR, clustering in two regions of the protein. Interestingly, several mutants were identified that proficiently bound TraR but were unable to inhibit its activity. This observation suggests a mechanistic separation between the initial assembly of the complex and conversion of TraR to an inactive form.

Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator.

We describe the development and analysis of broad-host-range (BHR) cloning vectors that carry the araC-PBAD controlled expression cassette from Escherichia coli. These plasmids are designed to facilitate l-arabinose-responsive control of target genes in a variety of Gram-negative bacterial hosts. BHR PBAD::lacZ fusions were used to analyze the utility of this controlled expression system in the plant pathogen Agrobacterium tumefaciens. In A. tumefaciens, the level of control afforded is significant, although less stringent than that observed in E. coli. The BHR PBAD vectors offer a useful alternative to currently used controlled expression systems, and can be employed in conjunction with other regulated promoters to simultaneously regulate expression of multiple genes. Addition of a variety of carbon sources, namely C4 acids and the anti-inducer d-fucose, allows modulation of l-arabinose induction. Activation of PBAD expression in A. tumefaciens requires a plasmid-borne copy of araC, and is not affected by endogenous regulators.

Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens.

The TraR and TraI proteins of Agrobacterium tumefaciens mediate cell-density-dependent expression of the Ti plasmid tra regulon. TraI synthesizes the autoinducer pheromone N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), while TraR is an 3-oxo-C8-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C8-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only 3-oxo-C8-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive. Furthermore, many of these compounds were potent antagonists. In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist. We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action. Wild-type A. tumefaciens released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. However, under all conditions tested, 3-oxo-C8-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with tra gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C8-HSL significantly stimulates tra gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers. When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.

Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro.

Acylated homoserine lactones (AHLs) are chemical signals that mediate population density-dependent (quorum-sensing) gene expression in numerous gram-negative bacteria. In this study, gram-negative bacilli isolated from catheters were screened for AHL production by a cross-feeding assay utilizing an AHL-responsive Agrobacterium tumefaciens reporter strain. Positive reactions were obtained from 14 isolates of Pseudomonas aeruginosa; negative or weakly positive reactions were recorded for isolates of five other species. P. aeruginosa biofilms were then produced on catheters in a physical model of the bladder. Sections of colonized all-silicone catheters gave positive reactions for the quorum-sensing signal molecules as did sections that had been cleaned of biofilm and autoclaved. Control sections of unused catheters were negative in the tests. Sections from four of nine catheters that had been freshly removed from patients gave positive reactions for AHLs. Cleaned autoclaved sections of three of these catheters also gave strongly positive reactions for AHLs. These results demonstrate that AHLs are produced by biofilms as they develop on the catheters both in vitro in the model and in vivo in the patient's bladder. They represent the first demonstration of AHL production by biofilms in a clinical setting.

Self perception in bacteria: quorum sensing with acylated homoserine lactones.

A variety of Gram-negative bacteria produce membrane permeant, acylated homoserine lactone (HL) pheromones that act as cell density cues. Synthesis and response to these factors requires proteins homologous to the Luxl acylhomoserine lactone synthase and the LuxR transcription factor from Vibrio fischeri. Recent genetic and biochemical studies have begun to provide a mechanistic understanding of acyl HL dependent gene regulation. Examination of the role of acyl HLs in diverse bacteria positions LuxR-Luxl type systems within an increasingly broad regulatory context and suggests that, in some bacteria, they comprise a global regulatory circuit.

Transcriptional regulation and locations of Agrobacterium tumefaciens genes required for complete catabolism of octopine.

By screening for octopine-inducible gene expression, we previously identified all the genes required for utilization of octopine as a source of carbon, nitrogen, and energy. They are (i) octopine oxidase, which converts octopine to arginine and pyruvate and is encoded by the ooxAB operon, (ii) arginase, which converts arginine to ornithine and urea and is encoded by arcA, (iii) ornithine cyclodeaminase, which converts ornithine to proline and ammonia and is encoded by the homologous arcB and ocd genes, and (iv) proline dehydrogenase, which converts proline to glutamate and is encoded by putA. Here we describe the regulation and localization of each of these genes. The ooxA-ooxB-ocd operon was previously shown to reside on the Ti plasmid and to be directly inducible by octopine. The arcAB operon is directly inducible by arginine, while it is induced by octopine only in strains that can convert octopine to arginine. Ornithine may also be a direct inducer of arcAB. putA is directly inducible by proline, while induction by octopine and by arginine (and probably by ornithine) requires their conversion to proline. Genetic studies indicate that arcAB and putA are localized on a conjugal genetic element. This element can be transferred to other Agrobacterium tumefaciens strains by a mechanism that does not require recA-dependent homologous recombination. Transfer of this genetic element from A. tumefaciens R10 requires at least one tra gene found on its Ti plasmid, indicating that this element is not self-transmissible but is mobilizable by the Ti plasmid. The DNA containing the arcAB and putA genes comigrates with a 243-kb linear molecular weight standard on field inversion electrophoretic gels.

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.

Localization of OccR-activated and TraR-activated promoters that express two ABC-type permeases and the traR gene of Ti plasmid pTiR10.

Conjugation of Agrobacterium tumefaciens wide-host-range octopine-type Ti plasmids is regulated by the LuxR-type transcriptional activator TraR in conjunction with an acylated homoserine lactone designated AAI. Expression of traR in octopine-type Ti plasmids is stimulated by OccR in response to octopine, an opine released from crown gall tumours, and is also positively autoregulated by TraR and AAI. Genetic and physical mapping of these promoters indicates that the OccR-activated promoter lies 14.5 kb upstream of traR, while the TraR-activated promoter lies 6 kb upstream. The upstream portion of the 14.5 kb operon contains seven previously characterized genes that direct the uptake and catabolism of octopine. The TraR-activated promoter lies just downstream from the octopine catabolic genes, and transcribes six genes in addition to traR, including five genes (ophABCDE) that show strong homology to oligo-peptide permeases of Salmonella typhimurium and Bacillus subtilis. Several TraR-regulated promoters overlap with 18 bp inverted repeats called tra boxes. In contrast, the traR autoregulatory promoter is not associated with a consensus tra box.

Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates.

Many bacteria, including several pathogens of plants and humans, use a pheromone called an autoinducer to regulate gene expression in a cell density-dependent manner. Agrobacterium autoinducer [AAI, N-(3-oxo-octanoyl)-L-homoserine lactone] of A. tumefaciens is synthesized by the Tral protein, which is encoded by the tumor-inducing plasmid. Purified hexahistidinyl-Tral (H6-Tral) used S-adenosylmethionine to make the homoserine lactone moiety of AAI, but did not use related compounds. H6-Tral used 3-oxo-octanoyl-acyl carrier protein to make the 3-oxo-octanoyl moiety of AAI, but did not use 3-oxo-octanoyl-coenzyme A. These results demonstrate the enzymatic synthesis of an autoinducer through the use of purified substrates.

Identification of Agrobacterium tumefaciens genes that direct the complete catabolism of octopine.

Agrobacterium tumefaciens R10 was mutagenized by using the promoter probe transposon Tn5-gusA7, and a library of approximately 5,000 transcriptional fusions was screened for octopine-inducible patterns of gene expression. Twenty-one mutants carrying strongly inducible gusA fusions, 20 of which showed defects in the catabolism of octopine or its metabolites, were obtained. One group of mutants could not use octopine as a carbon source, while a second group of mutants could not utilize arginine or ornithine and a third group could not utilize octopine, arginine, ornithine, or proline as a carbon source. Utilization of these compounds as nitrogen sources showed similar but not identical patterns. Fifteen fusions were subcloned together with adjacent DNA. Sequence analysis and further genetic analysis indicated that insertions of the first group are localized in the occ region of the Ti plasmid. Insertions of the second group were localized to a gene encoding ornithine cyclodeaminase. This gene is very similar to, but distinct from, a homolog located on the Ti plasmid. This gene is located immediately downstream from a gene encoding an arginase. Genetic experiments indicated that this arginase gene is essential for octopine and arginine catabolism. Insertions of the third group was localized to a gene whose product is required for degradation of proline. We therefore have identified all steps required for the catabolism of octopine to glutamate.

Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators.

The importance of accurate demographic information is reflected in the United States Constitution, Article 1, which provides for a decennial census of this country's human population. Bacteria also conduct a census of their population and do so more frequently, more efficiently, and as far we know, with little if any of the political contentiousness caused by human demographers. Many examples have been found of particular bacterial genes, operons, or regulons that are expressed preferentially at high cell densities. Many of these are regulated by proteins related to the LuxR and LuxI proteins of Vibrio fischeri, and by a diffusible pheromone called an autoinducer. LuxR and LuxI and their cognate autoinducer (3-oxohexanoyl homoserine lactone, designated VAI-1) provide an important model to describe the functions of this family of proteins. LuxR is a VAI-1 receptor and a VAI-1-dependent transcriptional activator, and LuxI directs the synthesis of VAI-1. VAI-1 diffuses across the bacterial envelope, and intracellular concentrations of it are therefore strongly increased by nearby VAI-1-producing bacteria. Similar systems regulate pathogenesis factors in Pseudomonas aeruginosa and Erwinia spp., as well as T1 plasmid conjugal transfer in Agrobacterium tumefaciens, and many other genes in numerous genera of gram-negative bacteria. Genetic analyses of these systems have revealed a high degree of functional conservation, while also uncovering features that are unique to each.

Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes.

Ti plasmids of Agrobacterium tumefaciens, in addition to transferring oncogenic DNA to the nuclei of infected plant cells, can conjugally transfer between agrobacteria. Conjugation of wide-host-range octopine-type Ti plasmids requires a tumor-released arginine derivative called octopine. Octopine stimulates expression of the traR gene, whose product directly activates other tra genes in the presence of an acylated homoserine lactone called Agrobacterium autoinducer (AAI). We have localized the transcription starts of three tra promoters and find conserved elements (tra boxes) at virtually identical positions upstream of each promoter. Disruption of these tra boxes abolished induction of each promoter. Deletion analysis of the traI promoter indicates that tra boxes are the only upstream elements required for transcriptional activation. Since Ti plasmid donor cells both produce and respond to AAI, we tested whether expression of tra promoters was enhanced by high concentrations of bacteria. Both tra gene expression and conjugation itself were strongly stimulated either by high donor densities or by exogenous AAI.

Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene.

The Agrobacterium Ti plasmid tra regulon was previously found to be positively regulated by the TraR protein in the presence of a diffusible N-acyl homoserine lactone designated Agrobacterium autoinducer (AAI). TraR and AAI are similar to LuxR from Vibrio fischeri and the Vibrio autoinducer (VAI), which regulate target bioluminescence (lux) genes in a cell density-dependent manner. We now show that tra genes are also regulated by a second protein, designated TraM, which acts to antagonize TraR-dependent activation. The traM gene is closely linked to traR, and the two genes are transcribed convergently. The predicted TraM proteins of two different Ti plasmids are 77% identical but are not significantly similar to other protein sequences in the database, and thus TraM may represent a novel regulatory protein. Null mutations in traM cause strongly increased conjugation, tra gene transcription, and AAI production. A functional copy of traM introduced into traM mutants decreased conjugation, tra gene transcription, and AAI synthesis. TraM inhibits transcription of traA, traI, and traM. Although traM was first identified by its octopine-inducible promoter, we now show that induction by octopine requires traR, strongly suggesting that TraR is the direct traM activator.

Effects of light conditioning on reproduction in partridge.

Reproductive response was measured in two lines of chukar partridge given conditioning light treatments of either 8 hr light:16 hr dark (8L:16D) at 50 lx or 16L:8D at .1 lx for 4, 6, or 8 weeks, respectively. Birds were recycled to lay using the same conditioning light treatments. During both lay cycles, all birds received 16L:8D at 100 lx. The results indicate that both conditioning light regimens were effective in terminating postjuvenile and postlay refractoriness. Hens given a conditioning light treatment of 8L:16D at 50 lx reached sexual maturity earlier, and produced more eggs and more viable chicks, and males maintained fertility longer, compared to birds conditioned under 16L:8D at .1 lx. Light conditioning periods of 6 and 8 weeks were superior to those of 4 weeks. For maximum reproductive performance in partridges, a short day photoperiod of 8L:16D at 50 lx is recommended for successful interruption of either postjuvenile or postlay refractoriness.