Salmonella pathogenicity island 2 (SPI-2) encodes a putative, two-component regulatory system, SsrA-SsrB, which regulates a type III secretion system needed for replication inside macrophages and systemic infection in mice. The sensor and regulator homologs, ssrAB (spiR), and genes within the secretion system, including the structural gene ssaH, are transcribed after Salmonella enters host cells. We have studied the transcriptional regulation of ssrAB and the secretion system by using gfp fusions to the ssrA and ssaH promoters. We found that early transcription of ssrA, after entry into macrophages, is most efficient in the presence of OmpR. An ompR mutant strain does not exhibit replication within cultured macrophages. Furthermore, footprint analysis shows that purified OmpR protein binds directly to the ssrA promoter region. We also show that minimal medium, pH 4.5, induces SPI-2 gene expression in wild-type but not ompR mutant strains. We conclude that the type III secretion system of SPI-2 is regulated by OmpR, which activates expression of ssrA soon after Salmonella enters the macrophage.

Enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were compared for analyzing microcystins in water. ELISA results of microcystin‐LR spiked into raw water samples were close to the spike concentrations, but method variability was ±25%. However, ELISA‐derived microcystin‐LA concentrations were two to three times higher than the spike concentrations obtained using the kit‐provided microcystin‐LR standards, indicating the need for variant‐appropriate ELISA standards. LC/MS/MS results agreed with spike concentrations for all variants in reagent water, but matrix suppression was observed in some raw waters. In bench‐scale studies, ozonated microcystins generated low‐level positive responses by ELISA and a protein phosphatase inhibition assay, even though microcystins were not detected by LC/MS/MS. These findings indicate that ELISA results—particularly in treated water—should be interpreted with caution because of the possibility of false‐positives, relatively high variability, and differential detection of some variants.

We have characterized a host-induced virulence gene, mig-14, that is required for fatal infection in the mouse model of enteric fever. mig-14 is present in all Salmonella enterica subspecies I serovars and maps to a region of the chromosome that appears to have been acquired by horizontal transmission. A mig-14 mutant replicated in host tissues early after infection but was later cleared from the spleens and livers of infected animals. Bacterial clearance by the host occurred concomitantly with an increase in gamma interferon levels and recruitment of macrophages, but few neutrophils, to the infection foci. We hypothesize that the mig-14 gene product may repress immune system functions by interfering with normal cytokine expression in response to bacterial infections.There are six subspecies of Salmonella enterica that are capable of colonizing both warm-and cold-blooded animals (1, 9). S. enterica subspecies I serovars are strictly associated with infection of warm-blooded animals and can cause a wide a range of diseases, including gastroenteritis, bacteremia, and typhoid fever (11). S. enterica serovar Typhimurium (from here on referred to as serovar Typhimurium) is the causative agent of gastroenteritis in humans and a typhoid-like disease in mice (11). Serovar Typhimurium survives and replicates within phagocytic cells of the reticuloendothelial system, resulting in the release of proinflammatory cytokines in response to bacterial compounds such as lipopolysaccharide (LPS) and peptidoglycan (11,16,17,20). Two of these inflammatory cytokines, tumor necrosis factor alpha (TNF-␣) and gamma interferon (IFN-␥), are required for host clearance of Salmonella infections (11,12,16,18). Treatment of infected animals with IFN-␥ decreases the numbers of bacteria found in the spleen early in infection, and injection of anti-TNF-␣ or anti-IFN-␥ abolishes the ability of mice to clear sublethal doses of serovar Typhimurium (12,26,27). Recently, it has been reported that modified Salmonella lipid A (an LPS component) can reduce the LPS-mediated expression of TNF-␣ by human monocytes and E-selectin by endothelial cells (14). These lipid A modifications are regulated by the PhoP/PhoQ virulence regulon (7, 13), suggesting a potential role for PhoP/PhoQ-activated genes not only in intracellular survival but also in lowering cytokine and chemokine production.In the present study we characterized the virulence properties and evolutionary history of a host-induced serovar Typhimurium factor with potential immunomodulatory functions. A previous study described a PhoP/PhoQ-dependent, macrophage-inducible promoter, fmi-14, which exhibited a 22-fold induction within murine macrophages (36). To identify the open reading frame (ORF) associated with fmi-14, we isolated an adjacent 2.2-kb serovar Typhimurium DNA fragment by recombinational cloning (6). Sequence analysis of this fragment revealed a single ORF (mig-14) encoding a putative 298-amino-acid (aa) soluble polypeptide with limited homology to Bacillus subtilis RecG, an ATP-dependen...

The green fluorescent protein (GFP) gene was expressed on a plasmid in B. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter. Promoter fusions of B. henselaechromosomal DNA to gfp were examined by flow cytometry, and a B. henselae groEL promoter fusion which induced expression at 37°C was isolated.

Environmental iron concentrations coordinately regulate transcription of genes involved in iron acquisition and virulence via the ferric uptake regulation (fur) system. We identified and sequenced the fur gene and flanking regions of three Bartonella species. The most notable difference between Bartonella Fur and other Fur proteins was a substantially higher predicted isoelectric point. No promoter activity or Fur autoregulation was detected using a gfp reporter gene fused to the 204 nucleotides immediately upstream of the Bartonella fur gene. Bartonella henselae fur gene expression complemented a Vibrio cholerae fur mutant.Bartonella, an extremely fastidious gram-negative bacillus, causes cat scratch disease, bacillary angiomatosis, and other syndromes (2,13,15,29). Few data exist regarding the pathogenic mechanisms of this hemophilic bacterium (5), which can occupy two alternate niches: the iron-rich gut of obligately hematophagous arthropods and the iron-restricted bloodstream of mammals (11,32). Acquisition of iron and expression of many virulence factors are under transcriptional regulation by the fur gene product, the ferric uptake regulation (Fur) protein, and its homodimeric complex (7). At sufficient intracellular iron levels, the corepressors Fur and Fe 2ϩ form an active Fur-Fe 2ϩ complex that binds a consensus sequence ("iron box," a 19-bp hyphenated dyad repeat [22] or three repeats of 6 bp of the sequence NAT[A,T]AT [7]) in the promoter region of genes regulated by Fur, down-regulating genes encoding iron-scavenging proteins (7,22). We hypothesized that Bartonella species possess a fur gene homolog with a gene regulatory system influenced by iron levels.Bacterial strains. Strains and plasmids used in this study are listed in Table 1. All Bartonella strains were used at low passage numbers (passes 1 through 3). B. henselae and B. quintana strains were grown on fresh chocolate agar (14), which provided a replete iron source. B. bacilliformis was grown on fresh heart infusion agar supplemented with 5% defibrinated rabbit blood (Hemostat Labs, Dixon, Calif.). Plates were incubated at 34°C (B. henselae and B. quintana) or 29°C (B. bacilliformis) in an enriched CO 2 environment for 5 to 7 days. Iron availability to B. henselae and B. quintana was restricted by adding the ferric-specific chelating agent EDDHA (ethylene diamine dihydroxy-o-phenylacetic acid) (The Complete Green Company, El Segundo, Calif.) (25) or by decreasing the hemoglobin (Hb) concentration in agar.Vibrio strains were grown overnight in Luria-Bertani medium with the appropriate selective antibiotic(s), with or without the iron chelator 2,2-dipyridyl (Sigma-Aldrich, Inc., St. Louis, Mo.), as previously described (8). Because of limited growth at 37°C by CML13(pSYP4), all strains were grown at 30°C and then incubated at 37°C for 60 to 90 min before assays. Selective antibiotics were added to growth media as required, at the following concentrations: ampicillin, 100 g/ml; chloramphenicol, 25 g/ml; kanamycin, 50 g/ml; streptomycin, 100 g/ml...