Regulation of \u3cem\u3eVibrio anguillarum empA\u3c/em\u3e Metalloprotease Expression and Its Role in Virulence

Atlantic salmon (Salmo salar) were challenged with Vibrio anguillarum strains M93Sm and NB10 and empA null mutants M99 and NB12. Both wild types were virulent when administered by intraperitoneal (i.p.) injection or anal intubation. NB12 was avirulent via either route of infection. M99 virulence was attenuated when delivered by intubation, but fully virulent by i.p. injection. Northern blot analysis revealed empA expression in M93Sm and NB10 cells incubated in mucus, while incubation in Luria-Bertani broth plus 2% NaCl (LB20) induced empA expression only in NB10. Nucleotide differences between M93Sm and NB10 empA sequences were found in regions located 207 and 229 bp upstream of the empA translational start. Reverse transcription-PCR and 5′ rapid amplification of cDNA ends revealed the empA transcriptional start site 85 bp upstream of the translational start for both strains. A putative σS-dependent promoter was identified upstream of the transcriptional start in both strains. Site-directed mutagenesis was used to create rpoS mutants of M93Sm and NB10. Neither rpoS mutant exhibited protease activity. Since empA is expressed during stationary phase, the effects of conditioned medium on protease activity were examined. M99 conditioned LB20 supernatants stimulated protease activity in NB10 while allowing M93Sm to produce protease in LB20. Neither acyl homoserine lactones nor AI-2 induced protease activity. Conditioned LB20 supernatant from a V. anguillarum luxS mutant caused a more rapid induction of protease activity in wild-type cells. Our data show that expression of empA is differentially regulated in V. anguillarum strains NB10 and M93Sm and requires σS, quorum-sensing molecules, and gastrointestinal mucus

Regulation of the \u3cem\u3eVibrio anguillarum\u3c/em\u3e Metalloprotease EmpA by Posttranslational Modification

The zinc metalloprotease EmpA is a virulence factor in the fish pathogen Vibrio anguillarum. Previous studies have shown that two strains of V. anguillarum regulate empA differently. Strain M93Sm exhibits protease activity only in the presence of fish gastrointestinal mucus, while protease activity is detected in NB10 culture supernatant under all stationary-phase conditions. In this study, we use real-time reverse transcription-PCR to show that even in conditions where no protease activity is detected, empA transcription occurs. Western blot analysis revealed that EmpA is secreted as a ∼48-kDa proenzyme and that activation occurs extracellularly by the removal of a ∼10-kDa peptide. The presence of stable extracellular pro-EmpA in M93Sm culture supernatants suggests that activation of EmpA is not autolytic

Gel Shift Analysis of the empA Promoter Region in \u3cem\u3eVibrio Anguillarum\u3c/em\u3e

Background: The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements. Results: Two strains of V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 μg/ml mucus (NSSM), 3M (marine minimal medium), or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSMgrown M93Sm cells were mixed with the DIG-labeled empA oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the luxR homologue in V. anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create vanT mutants in V. anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer. Conclusions: The data demonstrate that protein extracts of V. anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region. NB10 cells express empA during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm

Gel shift analysis of the empA promoter region in Vibrio anguillarum

BACKGROUND: The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements. RESULTS: Two strains of V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 μg/ml mucus (NSSM), 3M (marine minimal medium), or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled empA oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the luxR homologue in V. anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create vanT mutants in V. anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer. CONCLUSIONS: The data demonstrate that protein extracts of V. anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region. NB10 cells express empA during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm

Haplotypes at the Tas2r locus on distal chromosome 6 vary with quinine taste sensitivity in inbred mice

BACKGROUND: The detection of bitter-tasting compounds by the gustatory system is thought to alert animals to the presence of potentially toxic food. Some, if not all, bitter stimuli activate specific taste receptors, the T2Rs, which are expressed in subsets of taste receptor cells on the tongue and palate. However, there is evidence for both receptor-dependent and -independent transduction mechanisms for a number of bitter stimuli, including quinine hydrochloride (QHCl) and denatonium benzoate (DB). RESULTS: We used brief-access behavioral taste testing of BXD/Ty recombinant inbred (RI) mouse strains to map the major quantitative trait locus (QTL) for taste sensitivity to QHCl. This QTL is restricted to a ~5 Mb interval on chromosome 6 that includes 24 genes encoding T2Rs (Tas2rs). Tas2rs at this locus display in total 307 coding region single nucleotide polymorphisms (SNPs) between the two BXD/Ty RI parental strains, C57BL/6J (quinine-sensitive) and DBA/2J (quinine insensitive); approximately 50% of these mutations are silent. Individual RI lines contain exclusively either C57BL/6J or DBA/2J Tas2r alleles at this locus, and RI lines containing C57BL/6J Tas2r alleles are more sensitive to QHCl than are lines containing DBA/2J alleles. Thus, the entire Tas2r cluster comprises a large haplotype that correlates with quinine taster status. CONCLUSION: These studies, the first using a taste-salient assay to map the major QTL for quinine taste, indicate that a T2R-dependent transduction cascade is responsible for the majority of strain variance in quinine taste sensitivity. Furthermore, the large number of polymorphisms within coding exons of the Tas2r cluster, coupled with evidence that inbred strains exhibit largely similar bitter taste phenotypes, suggest that T2R receptors are quite tolerant to variation

Fisheries Assessment and Management Synthesis: Lessons for Chesapeake Bay

Describes the basic approaches that are used for stock assessment of the fish and shellfish stocks of the Chesapeake Bay system. The authors summarize the principal methods of stock assessment and fisheries management that have been and are being-applied to Bay fisheries, with particular emphasis on data collection and the use of models. Three case studies of critical species are presented - the striped bass, the blue crab and the Eastern oyster.https://scholarworks.wm.edu/vimsbooks/1168/thumbnail.jp

Identification and Characterization of Epp, the Secreted Processing Protease for the \u3cem\u3eVibrio anguillarum\u3c/em\u3e EmpA Metalloprotease

The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ∼10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease

Inbred mouse strains C57BL/6J and DBA/2J vary in sensitivity to a subset of bitter stimuli

BACKGROUND: Common inbred mouse strains are genotypically diverse, but it is still poorly understood how this diversity relates to specific differences in behavior. To identify quantitative trait genes that influence taste behavior differences, it is critical to utilize assays that exclusively measure the contribution of orosensory cues. With a few exceptions, previous characterizations of behavioral taste sensitivity in inbred mouse strains have generally measured consumption, which can be confounded by post-ingestive effects. Here, we used a taste-salient brief-access procedure to measure taste sensitivity to eight stimuli characterized as bitter or aversive in C57BL/6J (B6) and DBA/2J (D2) mice. RESULTS: B6 mice were more sensitive than D2 mice to a subset of bitter stimuli, including quinine hydrochloride (QHCl), 6-n-propylthiouracil (PROP), and MgCl(2). D2 mice were more sensitive than B6 mice to the bitter stimulus raffinose undecaacetate (RUA). These strains did not differ in sensitivity to cycloheximide (CYX), denatonium benzoate (DB), KCl or HCl. CONCLUSION: B6-D2 taste sensitivity differences indicate that differences in consumption of QHCl, PROP, MgCl(2 )and RUA are based on immediate orosensory cues, not post-ingestive effects. The absence of a strain difference for CYX suggests that polymorphisms in a T2R-type taste receptor shown to be differentially sensitive to CYX in vitro are unlikely to differentially contribute to the CYX behavioral response in vivo. The results of these studies point to the utility of these common mouse strains and their associated resources for investigation into the genetic mechanisms of taste

Problems With the Vortex-Boson Mapping in 1+1 Dimensions

Using the well known boson mapping, we relate the transverse magnetic susceptibility of a system of flux vortices in 1+1 dimensions to an appropriately defined conductivity of a one-dimensional boson system. The tilt response for a system free of disorder is calculated directly, and it is found that a subtle order of limits is required to avoid deceptive results.Comment: 4 Pages (REVTeX 3.0). Postscript file for this paper is available on the World Wide Web at http://cmtw.harvard.edu/~simon/

Spatial and temporal characteristics of error-related activity in the human brain

A number of studies have focused on the role of specific brain regions, such as the dorsal anterior cingulate cortex during trials on which participants make errors, whereas others have implicated a host of more widely distributed regions in the human brain. Previous work has proposed that there are multiple cognitive control networks, raising the question of whether error-related activity can be found in each of these networks. Thus, to examine error-related activity broadly, we conducted a meta-analysis consisting of 12 tasks that included both error and correct trials. These tasks varied by stimulus input (visual, auditory), response output (button press, speech), stimulus category (words, pictures), and task type (e.g., recognition memory, mental rotation). We identified 41 brain regions that showed a differential fMRI BOLD response to error and correct trials across a majority of tasks. These regions displayed three unique response profiles: (1) fast, (2) prolonged, and (3) a delayed response to errors, as well as a more canonical response to correct trials. These regions were found mostly in several control networks, each network predominantly displaying one response profile. The one exception to this “one network, one response profile” observation is the frontoparietal network, which showed prolonged response profiles (all in the right hemisphere), and fast profiles (all but one in the left hemisphere). We suggest that, in the place of a single localized error mechanism, these findings point to a large-scale set of error-related regions across multiple systems that likely subserve different function