We determined that LVS and Schu S4 strains of the human pathogen Francisella tularensis express a siderophore when grown under iron-limiting conditions. We purified this siderophore by conventional column chromatography and high-pressure liquid chromatography and used mass spectrometric analysis to demonstrate that it is structurally similar to the polycarboxylate siderophore rhizoferrin. The siderophore promoted the growth of LVS and Schu S4 strains in iron-limiting media. We identified a potential siderophore biosynthetic gene cluster encoded by fslABCD in the F. tularensis genome. The first gene in the cluster, fslA, encodes a member of the superfamily of nonribosomal peptide synthetase-independent siderophore synthetases (NIS synthetases) characterized by the aerobactin synthetases IucA and IucC. We determined that fslA is transcribed as part of an operon with downstream gene fslB and that the expression of the locus is induced by iron starvation. A targeted in-frame nonpolar deletion of fslA in LVS resulted in the loss of siderophore expression and in a reduced ability of F. tularensis to grow under conditions of iron limitation. Siderophore activity and the ability to grow under iron limitation could be regained by introducing the fslA ؉ gene on a complementing plasmid. Our results suggest that the fslA-dependent siderophore is important for survival of F. tularensis in an iron-deficient environment.

Amebiasis is the third leading parasitic cause of death worldwide, and it is not known whether immunity is acquired from a previous infection. An investigation was done to determine whether protection from intestinal infection correlated with mucosal or systemic antibody responses to the Entamoeba histolytica GalNAc adherence lectin. E. histolytica colonization was present in 0% (0/64) of children with and 13.4% (33/246) of children without stool IgA anti-GalNAc lectin antibodies (P= .001). Children with stool IgA lectin-specific antibodies at the beginning of the study had 64% fewer new E. histolytica infections by 5 months (3/42 IgA(+) vs. 47/227 IgA(-); P= .03). A stool antilectin IgA response was detected near the time of resolution of infection in 67% (12/18) of closely monitored new infections. It was concluded that a mucosal IgA antilectin antibody response is associated with immune protection against E. histolytica colonization. The demonstration of naturally acquired immunity offers hope for a vaccine to prevent amebiasis.

Background: FslE and FupA are Francisella-specific paralogous proteins involved in iron acquisition. Results: fslE mutation disrupts siderophore-mediated ferric iron uptake, fupA mutation impairs high affinity ferrous iron uptake, and both mutations impact virulence. Conclusion: Optimal iron acquisition and virulence require both paralogs. Significance: Iron acquisition mechanisms are potential targets for preventive or therapeutic intervention in F. tularensis infections.

Caulobacter crescentus flagellar (fla, fib, or flg) genes are periodically expressed in the cell cycle and they are organized in a regulatory hierarchy. We have analyzed the genetic interactions required forfla gene expression by determining the effect of mutations in 30 known fla genes on transcription from four operons in the hook gene cluster. These results show that the flaO (transcription unit Im) and flbF (transcription unit IV) operons are located at or near the top of the hierarchy. They also reveal an extensive network of negative transcriptional controls that are superimposed on the positive regulatory cascade described previously. The strong negative autoregulation observed for the flaN (transcription unit I),flbG (transcription unit II), andfiaO (transcription unit III) promoters provides one possible mechanism for turning off fla gene expression at the end of the respective synthetic periods. We suggest that these positive and negative transcriptional interactions are components of genetic switches that determine the sequence in whichfIa genes are turned on and off in the C. crescentus cell cycle.Cell differentiation in Caulobacter crescentus results from the repeated asymmetric division of a stalked cell to produce the parent stalked cell plus a new, motile swarmer cell. The flagellum is the most prominent and best-studied of several polar structures that characterize the newly differentiated swarmer cell. Understanding the biosynthesis of this complex organelle in C. crescentus is a challenging problem in morphogenesis and gene regulation, requiring the products of at least 30 flagellar (fla, flb, or flg) genes and spatial information for targeting the subunits to one of the cell poles. In addition, there is a temporal component of regulation, since flagellum biosynthesis, like other developmental events in C. crescentus, is stage specific in the cell cycle (see refs. 1 and 2 for reviews).The C. crescentus genes encoding flagellar subunits that have been examined are periodically expressed in the cell cycle, generally at times of gene product assembly. Thus, the synthesis of the 70-kDa hook protein precedes that of the 27-and 25-kDa flagellins (3-5) and the 29-, 27-, and 25-kDa flagellin gene transcripts appear in the same order that the protein products are assembled into the flagellar filament (6); theflaD transcript, which may encode one of the basal body ring subunits, appears earlier in the cell cycle (7).A major question in C. crescentus development is how the complex temporal pattern of periodic fla gene expression is programmed in the cell cycle and coordinated with flagellum morphogenesis. A number of results indicate that the fla genes are organized in a regulatory hierarchy similar to that described in Escherichia coli (8) and that the expression of these genes in C. crescentus is controlled by a cascade of positive transcriptional activators (1, 9-12). Recent studies have shown that flbG (hook operon; see refs. 5 and 13) and flaN (14), two transcription units in the hook gene c...

The periodic transcription of flagellar genes in the Caulobacter crescentus cell cycle is controlled, in part, by their organization in a regulatory hierarchy. The flbG (hook operon), flaN, and flagellin gene operons, which are at the lowest levels of the hierarchy and expressed late in the cell cycle, contain Ntr-like promoters. We report thatflbD, one of the early genes required in trans for expression of these operons, codes for a 52-kDa protein homologous to the transcriptional activators NtrC (NRI), NifA, DctD, HydG, and XylR. Our results show that in Escherchia co~flbD partially complements ginG (ntrC) mutations and stimulates transcription of the C. crescentus am RNA polymerase-dependentflbG gene. Additionally, the sequence predicts that FIbD protein, along with NtrC, DctD, and HydG proteins, is structurally related at the amino-terminal domain to a larger family of response regulators that mediate cellular responses to environmental stimuli. FIbD may be a singular member of this large protein family in that its function is tied to an internal cell-cycle signal. FIbD is also unusual in that its amino-terminal domain contains only one of the three residues conserved in previously described members of this family of response regulators.Differentiation in the dimorphic bacterium Caulobacter crescentus results from a sequence of discontinuous, stagespecific events that lead to the production of a new swarmer cell after each asymmetric cell division of the stalked cell. The best understood of these events is formation of the polar flagellum, which occurs late in the cell cycle and requires the products of >30 flagellar (fla, flb, flg) genes. Thesefla genes are periodically transcribed in the cell cycle, generally at the times of gene-product assembly. A question central to morphogenesis is how the sequential, stage-specific expression of this large fla gene family is controlled in the cell cycle (for reviews, see refs. 1 and 2).The temporal pattern of fla gene expression in C. crescentus depends, in part, on the organization of these genes in a regulatory hierarchy, in which genes at each level are required for expression of genes at lower levels (for summary, see refs. 1 and 3). Analysis of the 5' regulatory sequences has suggested that the resulting transcriptional cascade is mediated by the sequential synthesis of transcription activators and the use of alternative ao factors (4,5). fla genes at the two lowest levels of the hierarchy, including flbG and flaN of the hook gene cluster, contain nucleotide sequence elements at -12 and -24 (4, 6) that are strikingly similar to the ntr/nifpromoters in enteric bacteria recognized by the specialized 54 (NtrA) RNA polymerase (7,8). Results of site-specific mutagenesis (9) Severalfla genes, including those in the adjacentflaO and flbF operons, are required in trans for expression from the flaN andflbG promoters (1, 3). We report thatflbD,t the last gene in the flaO operon, codes for a protein homologous to the o.54specific transcriptional activators NtrC,...

Strains of Francisella tularensis secrete a siderophore in response to iron limitation. Siderophore production is dependent on fslA, the first gene in an operon that appears to encode biosynthetic and export functions for the siderophore. Transcription of the operon is induced under conditions of iron limitation. The fsl genes lie adjacent to the fur homolog on the chromosome, and there is a canonical Fur box sequence in the promoter region of fslA. We generated a ⌬fur mutant of the Schu S4 strain of F. tularensis tularensis and determined that siderophore production was now constitutive and no longer regulated by iron levels. Quantitative reverse transcriptase PCR analysis with RNA from Schu S4 and the mutant strain showed that Fur represses transcription of fslA under iron-replete conditions. We determined that fslE (locus FTT0025 in the Schu S4 genome), located downstream of the siderophore biosynthetic genes, is also under Fur regulation and is transcribed as part of the fslABCDEF operon. We generated a defined in-frame deletion of fslE and found that the mutant was defective for growth under iron limitation. Using a plate-based growth assay, we found that the mutant was able to secrete a siderophore but was defective in utilization of the siderophore. FslE belongs to a family of proteins that has no known homologs outside of the Francisella species, and the fslE gene product has been previously localized to the outer membrane of F. tularensis strains. Our data suggest that FslE may function as the siderophore receptor in F. tularensis.

Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the ␤2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin's role in virulence.

The periodic and sequential expression of flagellar (fla) genes in the Caulobacter crescentus cell cycle depends on their organization into levels I to IV of a regulatory hierarchy in which genes at the top of the hierarchy are expressed early in the cell cycle and are required for the later expression of genes below them. In these studies, we have examined the regulatory role of level IIfliF operon, which is located near the top of the hierarchy. The last gene in the fliF operon, flbD, encodes a transcriptional factor required for activation of j54-dependent promoters at levels III and IV and negative autoregulation of the level II fliF promoter. We have physically mapped the fliF operon, identified four new genes in the transcription unit, and determined that the organization of these genes is 5'-fliF-fliG-flbE-fliN-flbD-3'. Three of the genes encode homologs of the MS ring protein (FliF) and two switch proteins (FliG and FliN) of enteric bacteria, and the fourth encodes a predicted protein (FlbE) without obvious similarities to known bacterial proteins. We have introduced nonpolar mutations in each of the open reading frames and shown that all of the newly identified genes (fliFfliG,flbE, andfliN) are required in addition toflbD for activation of the u54-dependentflgK andflbG promoters at level III. In contrast,fliFfliG, andflbE, but notfliN, are required in addition toflbD for negative autoregulation of the level II fliF promoter. The simplest interpretation of these results is that the requirements of FlbD in transcriptional activation and repression are not identical, and we speculate that FlbD function is subject to dual or overlapping controls. We also discuss the requirement of multiple structural genes for regulation of levels II and III genes and suggest thatfla gene expression in C. crescentus may be coupled to two checkpoints in flagellum assembly.The procaryotic flagellum is a complex organelle composed of three structural elements: the basal body or motor, which is embedded in the membrane and peptidoglycan layers of the cell envelope; the external hook, which attaches the basal body to the flagellar filament; and the filament itself, which rotates to move the cell (35, 62). In the dimorphic bacterium Caulobacter crescentus, the flagellum is assembled at one pole of the dividing cell late in the cell cycle and then segregates with the motile swarmer cell at division. Its biosynthesis requires the activity of 40 to 50 flagellar (fla) genes (15), and, as in the enteric bacteria, expression of these genes is regulated by their organization in a regulatory hierarchy in which expression of genes at the top of the hierarchy is required for expression of genes lower in the hierarchy (reviewed in references 7 and 44). In the C. crescentus hierarchy, there is also evidence that fla gene interactions mediate the negative, as well as the positive, transcriptional control of gene expression (45,49,70).A unique feature of flagellum biosynthesis in C. crescentus is the cell cycle regulation of fla gene expre...