Timothy J. Brickman

Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli

Transcriptional linkage of the enterobactin gene cluster entCEBA (P15) was confirmed by ent-lacZ gene fusion analysis. Control sequences directing iron-regulated expression of this polycistronic message were localized to the fepB-entC bidirectional promoter region. Transcriptional initiation sites defined by primer extension analysis were located 103 base-pairs apart for the divergent fepB and entC messages. Within this divergent regulatory region, strongly consensus -35 and -10 promoter determinants and potential Fur repressor-binding sequences were identified. A vector containing divergently oriented indicator gene fusions was constructed to monitor regulatory effects of mutations within this iron-responsive control region. The fepB-entC promoter-operator elements were confirmed by mutation, using the dual gene fusion system in multicopy and low copy number states. Mutations in the -35 and -10 regions of the fepB and entC promoters that decreased their similarity to consensus resulted in reduced promoter activity. Mutations in the Fur-controlled operators reduced induction ratios (iron-deficient levels/iron-rich levels) for the respective fusion gene activities by approximately sevenfold. Although operator mutants retained some degree of inducibility, complete relief of repression was observed for double operator mutants, suggesting that only minor regulatory influence is exerted by Fur occupation of the opposing operator site. DNase I footprinting experiments were performed to characterize the sequence-specific Fur interactions at the operator sequences. At the fepB operator, a 31 base-pair Fur-protected region was identified, corresponding to positions -19 to +12 with respect to the transcriptional start site. Similarly, Fur protected a 31 base-pair region in entC, corresponding to positions +1 to +31 in the message. A contiguous and sequentially occupied secondary Fur-binding site in entC was protected at higher Fur concentrations, extending the protected region to +49, and sequestering the putative Shine-Dalgarno sequence. Operator positional effects and co-operativity are discussed.

Hc1‐mediated effects on DNA structure: a potential regulator of chlamydial development

Chlamydiae are obligate intracellular bacteria which undergo a unique developmental cycle, alternating between non‐replicative elementary bodies (EBs) and replicatlve reticulate bodies (RBs). The transition from RB to EB is characterized by condensation of the chromosome into a dense nucleoid structure. The chlamydial histone homologue Hc1 is sufficient to induce formation of a similar structure in Escherichia coli. High‐level Hc1 expression in E. coli is self‐limiting and down‐regulates transcription, translation, and replication at concentrations similar to those observed in chlamydial elementary bodies. Expression of Hc1 at sub‐structural levels may have specific regulatory functions through its interaction with chromosomal DNA, In E. coli this is reflected in a dramatic shift in the pattern of gene expression. The differential expression of the outer membrane porin proteins OmpC and OmpF and analysis of lacZ fusions with promoter regions sensitive to supercoiling suggests that low‐level Hc1 expression results in a net relaxation of chromosomal DNA. Topological analysis of plasm id DNA from both E. coli and Chlamydia trachomatis supports a decrease in superhelicity preceding nucleoid formation. In vitro analysis of purified Hc1–DNA interactions supports preferential binding based upon DNA conformation. These results suggest a dual role in which Hc1‐mediated changes in gene expression may precede metabolic inactivity.

Heme Transport Contributes to In Vivo Fitness of Bordetella pertussis during Primary Infection in Mice

ABSTRACT Bordetella pertussis, the causative agent of whooping cough or pertussis, is an obligate human pathogen with multiple high-affinity iron transport systems. Maximal expression of the dedicated heme utilization functions encoded by the hurIR bhuRSTUV genes requires an iron starvation signal to relieve Fur repression at the hurIR promoter-operator and an inducing signal supplied by heme for HurI-mediated transcriptional activation at the bhuRSTUV promoter. The BhuR outer membrane receptor protein is required for heme uptake and for heme sensing for induction of bhuRSTUV transcription. It was hypothesized that heme utilization contributed to the success of B. pertussis as a pathogen. In this study, virulence attenuation resulting from inactivation of the B. pertussis heme system was assessed using mixed infection competition experiments in a mouse model. As a measure of in vivo fitness, the ability of a B. pertussis heme utilization mutant to colonize and persist was determined relative to that of an isogenic coinfecting wild-type strain. Relative fitness of the mutant strain declined significantly after 7 days postinfection and continued to decline throughout the remainder of the 28-day infection time course. In parallel infections using inocula supplemented with an inducing 2 μM concentration of hemin chloride, hemin coadministration augmented the competitive advantage of the wild-type strain over the mutant. The results confirm that heme utilization contributes to the pathogenesis of B. pertussis in the mouse infection model and indicate that heme utilization may be most important for adaptation to host conditions existing during the later stages of infection.

Transcriptional Activation of Bordetella Alcaligin Siderophore Genes Requires the AlcR Regulator with Alcaligin as Inducer

Genetic and biochemical studies have established that Fur and iron mediate repression of Bordetella alcaligin siderophore system (alc) genes under iron-replete nutritional growth conditions. In this study, transcriptional analyses using Bordetella chromosomal alc-lacZ operon fusions determined that maximal alc gene transcriptional activity under iron starvation stress conditions is dependent on the presence of alcaligin siderophore. Mutational analysis and genetic complementation confirmed that alcaligin-responsive transcriptional activation of Bordetella alcaligin system genes is dependent on AlcR, a Fur-regulated AraC-like positive transcriptional regulator encoded within the alcaligin gene cluster. AlcR-mediated transcriptional activation is remarkably sensitive to inducer, occurring at extremely low alcaligin concentrations. This positive autogenous control circuit involving alcaligin siderophore as the inducer for AlcR-mediated transcriptional activation of alcaligin siderophore biosynthesis and transport genes coordinates environmental and intracellular signals for maximal expression of these genes under conditions in which the presence of alcaligin in the environment is perceived.

Transcriptional profiling of the iron starvation response in Bordetella pertussis provides new insights into siderophore utilization and virulence gene expression

Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes.

Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica

Hydroxamate siderophores of virulent Bordetella pertussis and Bordetella bronchiseptica strains were purified using a simple large-scale isolation procedure, and identified by various spectroscopic techniques as the macrocyclic dihydroxamate siderophore trivially known as alcaligin, 1,8(S),11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, which was previously isolated from the taxonomically-related bacterial species Alcaligenes denitrificans subsp. xylosoxydans. Alcaligin purified from iron-depleted cultures of B. pertussis and B. bronchiseptica exhibited specific growth-promoting activity under iron-restricted conditions for Bordetella indicator strains, and were active in [55Fe]ferric alcaligin transport assays. Evidence suggests that several C2-symmetric conformations of alcaligin exist simultaneously in both methanolic and aqueous solution.

Bordetella AlcS Transporter Functions in Alcaligin Siderophore Export and Is Central to Inducer Sensing in Positive Regulation of Alcaligin System Gene Expression

Bordetella pertussis and Bordetella bronchiseptica, which are respiratory mucosal pathogens of mammals, produce and utilize the siderophore alcaligin to acquire iron in response to iron starvation. A predicted permease of the major facilitator superfamily class of membrane efflux pumps, AlcS (synonyms, OrfX and Bcr), was reported to be encoded within the alcaligin gene cluster. In this study, alcS null mutants were found to be defective in growth under iron starvation conditions, in iron source utilization, and in alcaligin export. trans complementation using cloned alcS genes of B. pertussis or B. bronchiseptica restored the wild-type phenotype to the alcS mutants. Although the levels of extracellular alcaligin measured in alcS strain culture fluids were severely reduced compared with the wild-type levels, alcS mutants had elevated levels of cell-associated alcaligin, implicating AlcS in alcaligin export. Interestingly, a deltaalcA mutation that eliminated alcaligin production suppressed the growth defects of alcS mutants. This suppression and the alcaligin production defect were reversed by trans complementation of the deltaalcA mutation in the double-mutant strain, confirming that the growth-defective phenotype of alcS mutants is associated with alcaligin production. In an alcA::mini-Tn5 lacZ1 operon fusion strain background, an alcS null mutation resulted in enhanced AlcR-dependent transcriptional responsiveness to alcaligin inducer; conversely, AlcS overproduction blunted the transcriptional response to alcaligin. These transcription studies indicate that the alcaligin exporter activity of AlcS is required to maintain appropriate intracellular alcaligin levels for normal inducer sensing and responsiveness necessary for positive regulation of alcaligin system gene expression.

Diversity in the Chlamydia trachomatis histone homologue Hc2

Chlamydia trachomatis elementary bodies contain two developmentally expressed histone H1 homologues. An 18-kDa histone homologue, Hc1, is conserved among C. trachomatis serovars and C. psittaci. The other histone homologue, Hc2 (encoded by hctB), varies in size between C. trachomatis serovars but is present in reduced amounts or absent from C. psittaci. The variation in Hc2 size among C. trachomatis serovars was found to be due to internal deletions from a region of the hctB gene encoding lysine- and alanine-rich pentameric repeats.

Bordetella iron transport and virulence

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are pathogens with a complex iron starvation stress response important for adaptation to nutrient limitation and flux in the mammalian host environment. The iron starvation stress response is globally regulated by the Fur repressor using ferrous iron as the co-repressor. Expression of iron transport system genes of Bordetella is coordinated by priority regulation mechanisms that involve iron source sensing. Iron source sensing is mediated by distinct transcriptional activators that are responsive to the cognate iron source acting as the inducer.

Impact of alcaligin siderophore utilization on in vivo growth of Bordetella pertussis.

ABSTRACT Bordetella pertussis, the causative agent of human whooping cough, or pertussis, is an obligate human pathogen with diverse high-affinity transport systems for the assimilation of iron, a biometal that is essential for growth. Under iron starvation stress conditions, B. pertussis produces the siderophore alcaligin. The alcaligin siderophore gene cluster, consisting of the alcABCDERS and fauA genes, encodes activities required for alcaligin biosynthesis, the export of the siderophore from the cell, the uptake of the ferric alcaligin complex across the outer membrane, and the transcriptional activation of alcaligin system genes by an autogenous mechanism involving alcaligin sensing. The fauA gene encodes a 79-kDa TonB-dependent outer membrane receptor protein required for the uptake and utilization of ferric alcaligin as an iron source. In this study, using mixed-infection competition experiments in a mouse respiratory model, inactivation of the B. pertussis ferric alcaligin receptor protein was found to have a profound impact on in vivo growth and survival of a fauA mutant compared with a coinfecting wild-type strain. The attenuating effect of fauA inactivation was evident early in the course of the infection, suggesting that the contribution of ferric alcaligin transport to the ecological fitness of B. pertussis may be important for adaptation to iron-restricted host conditions that exist at the initial stages of infection. Alcaligin-mediated iron acquisition by B. pertussis may be critical for successful host colonization and establishment of infection.