bLegionella pneumophila is a waterborne pathogen, and survival in the aquatic environment is central to its transmission to humans. Therefore, identifying genes required for its survival in water could help prevent Legionnaires' disease outbreaks. In the present study, we investigate the role of the sigma factor RpoS in promoting survival in water, where L. pneumophila experiences severe nutrient deprivation. The rpoS mutant showed a strong survival defect compared to the wild-type strain in defined water medium. The transcriptome of the rpoS mutant during exposure to water revealed that RpoS represses genes associated with replication, translation, and transcription, suggesting that the mutant fails to shut down major metabolic programs. In addition, the rpoS mutant is transcriptionally more active than the wild-type strain after water exposure. This could be explained by a misregulation of the stringent response in the rpoS mutant. Indeed, the rpoS mutant shows an increased expression of spoT and a corresponding decrease in the level of (p)ppGpp, which is due to the presence of a negative feedback loop between RpoS and SpoT. Therefore, the lack of RpoS causes an aberrant regulation of the stringent response, which prevents the induction of a successful response to starvation. L egionella pneumophila is the causative agent of Legionnaires' disease. It is widely distributed in natural freshwater systems (1) and readily colonizes man-made water systems such as cooling towers and water distribution systems (2). L. pneumophila persists in aquatic environments thanks to its ability to adapt to a variety of different ecological niches, either as an intracellular parasite of amoebae or ciliate protozoans, as a free-living member of complex biofilm communities, or as planktonic cells (3, 4). Amoebae support intracellular multiplication of L. pneumophila, and protect against suboptimal growth conditions and exposure to chlorine (5-7). Infection of HeLa cells and Tetrahymena tropicalis leads to the differentiation of L. pneumophila into mature infectious forms (MIFs), characterized by ultrastructural changes and accumulation of poly-␤-hydroxybutyrate (8-10). MIFs are highly infectious and are more resistant to antibiotics and detergent than other forms (9, 11). Increased resistance to antibiotics was also described after incubation of L. pneumophila in Acanthamoeba castellanii buffer for 16 h (12). MIFs are able to resist starvation better than stationary-phase forms in encystment buffer (11), but both forms show similar levels of resistance in distilled water (8).In the free-living state outside the amoebal host, L. pneumophila encounters stressful conditions, such as limited nutrient availability in aquatic systems (1, 13). Nevertheless, previous studies have shown that L. pneumophila is able to survive for long periods (up to 1.5 years) in sterilized tap, surface and estuarine waters (14-17). The genetic determinants underlying the ability of L. pneumophila to survive in its natural habitat of low-nutrient water for a...

The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp 0 ) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp 0 and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp 0 and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and ␣-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCEThe type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.E rwinia amylovora causes a devastating fire blight disease of apples and pears, which results in severe economic losses to growers around the world (1, 2). E. amylovora is closely related to members of the Enterobacteriaceae family, including many important human pathogens, such as Escherichia coli, Yersinia pestis, and Salmonella enterica (3). Studies have revealed that the hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a major pathogenicity factor in E. amylovora (4-7). The hrp T3SS genes are carried on a pathogenicity island (8), and the alternative sigma factor HrpL, a member of the ECF subfamily of sigma factors, serves as the master regulator to control the expression of the structural and effector genes by binding to a consensus sequence known as the hrp box (9-13)...

BODIPY-based ECL chemodosimeters for H2O2 were developed and applied for glucose detection in human serum in combination with GOx.

A new application of a fluorescent sensor (PyDPA) for the discrimination of redox-responsible molecules is reported. Nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD(+)/NADP(+)) and flavin mononucleotide/flavin adenine dinucleotide (FMN/FAD) were differentiated by means of ratiometric fluorescence change from excimer-monomer equilibrium and time-dependent fluorescence change, respectively.

Hydrogen sulfide (H2S) is a well-known toxic gas with the odor of rotten eggs. Several reaction-based electrochemiluminescence (ECL) chemosensors for H2S have been developed; however, no homogeneous ECL probe with high selectivity toward H2S in aqueous media has been reported. Herein, we report an iridium­(III) complex-based ECL chemodosimetric probe employing two 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) groups known as a photo-induced electron transfer quencher and a reaction site for the selective detection of H2S; the detection mechanism involves H2S being clearly distinguished from biothiols based on the different cleavage rates of the two NBD groups and extremely weak ECL interferences caused by reaction by-products. The probe was rationally designed to improve selectivity toward H2S within the ECL analysis platform by enabling the removal of nonspecific background signals observed via fluorescence analysis. This analytical system exhibited remarkable selectivity toward H2S, a rapid reaction rate, and high sensitivity (LOD = 57 nM) compared to conventional fluorescence methods. Furthermore, the probe could successfully quantify H2S in tap water samples and commercial ammonium sulfide solutions, which demonstrates the effectiveness of this probe in field monitoring.

A series of pyrophosphate (PPi) receptors were synthesized, and their binding affinities toward both PPi and adenosine triphosphate were evaluated in N-(2-hydroxyethyl)­piperazine-N′-ethanesulfonic acid-buffered solution. The presence of two hydrogen bond donors slightly retarded the reaction rate of anionic guest exchange, while four had a significant retardation effect. A macrocyclic receptor, 17, exhibited superior selectivity toward PPi compared with acyclic receptors, presumably due to effective steric interactions. Isothermal titration calorimetry experiments and potentiometric titration experiments revealed the effect of the substituent structure on the degree of anion guest stabilization and the deprotonation of water molecules bound to zinc­(II) ions, as well as the guest binding thermodynamics.

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