The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens.

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Aims-To assess the potential antibacterial eVect of omeprazole, a benzimidazole proton pump inhibitor, on the growth of Helicobacter pylori in vitro and to evaluate the eVect of this compound on bacterial urease activity. Methods-The growth of H pylori was observed in liquid culture in the presence and absence of omeprazole (0.8 mg/ml). Urease activity was evaluated in aliquots removed from two hour cultures by monitoring the initial change in absorbency at 560 nm in the presence of 0.02% phenol red. Results-The minimum inhibitory concentration of omeprazole against H pylori was 0.8 mg/ml. The concentration of omeprazole required to inhibit growth was dependent on inoculum density: omeprazole (0.8 mg/ml) prevented growth from a 1 × 10 6 cfu/ml inoculum, but not from the higher inocula of 10 7 or 10 8 cfu/ml. This is the first study to demonstrate that omeprazole exerts a bacteriocidal eVect against low bacterial densities and a bacteriostatic eVect when bacterial density is high. When used at the onset of growth, this concentration of omeprazole has a bacteriocidal eVect after four hours, although it exerts a bacteriostatic eVect when added to cultures after the exponential phase. Bacterial urease activity is competitively inhibited by omeprazole in a dose dependent manner. Conclusion-The results suggest that omeprazole exerts both a bacteriocidal and a bacteriostatic eVect against H pylori and competitively inhibits bacterial extracellular urease activity (J Clin Pathol 1998;51:220-224)

Poly[(R)-3-hydroxybutyrate] (PHB) is a microbial biopolymer that has been commercialized as biodegradable plastics. The key enzyme for the degradation is PHB depolymerase (PhaZ). A new intracellular PhaZ from Bacillus thuringiensis (BtPhaZ) has been screened for potential applications in polymer biodegradation. Recombinant BtPhaZ was crystallized using 25% polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M bis-tris pH 6.5 at 288 K. The crystals belonged to space group P1, with unit-cell parameters a = 42.97, b = 83.23, c = 85.50 Å , = 73.45, = 82.83, = 83.49 . An X-ray diffraction data set was collected to 1.42 Å resolution with an R merge of 6.4%. Unexpectedly, a molecular-replacement solution was obtained using the crystal structure of Streptomyces lividans chloroperoxidase as a template, which shares 24% sequence identity to BtPhaZ. This is the first crystal structure of an intracellular poly(3-hydroxybutyrate) depolymerase.

Genomic DNAs containing the gene encoding apolipoprotein A-I (apoA-1) from four strains of mice and three strains of rats have been sequenced. Some peculiar repetitive sequences were found in the third intron of apoA-1 of the murine species. The striking features include regular tandem repeats of C(A)4 and C(A)6 in mice and long A-tracts in rats. Not completely identical but very similar motifs were found in mice or rats belonging to the same species while repetitive elements from different species show some variation from their speciesspecific consensus sequences. These repetitive motifs are very similar to the sequences flanking human Alu and rodent B 1 repetitive elements, although no evidence for the existence of Alu or B 1 was found near the peculiar repetitive DNA sequences in apoA-1 gene. BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONALMice and rats are useful experimental models for studying the genetic factors that may be

The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50 % of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.

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