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,...