Bacterial second messengers are important for regulating diverse bacterial lifestyles. Cyclic di-GMP (c-di-GMP) is produced by diguanylate cyclase enzymes, named GGDEF proteins, which are widespread ...across bacteria. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been described in some bacteria, which produce both c-di-GMP and a more recently identified bacterial second messenger, 3',3'-cyclic-GMP-AMP (cGAMP). One of these proteins was found in the predatory Bdellovibrio bacteriovorus, Bd0367. The bd0367 GGDEF gene deletion strain was found to enter prey cells, but was incapable of leaving exhausted prey remnants via gliding motility on a solid surface once predator cell division was complete. However, it was unclear which signal regulated this process. We show that cGAMP signalling is active within B. bacteriovorus and that, in addition to producing c-di-GMP and some c-di-AMP, Bd0367 is a primary producer of cGAMP in vivo. Site-directed mutagenesis of serine 214 to an aspartate rendered Bd0367 into primarily a c-di-GMP synthase. B. bacteriovorus strain bd0367S214D phenocopies the bd0367 deletion strain by being unable to glide on a solid surface, leading to an inability of new progeny to exit from prey cells post-replication. Thus, this process is regulated by cGAMP. Deletion of bd0367 was also found to be incompatible with wild-type flagellar biogenesis, as a result of an acquired mutation in flagellin chaperone gene homologue fliS, implicating c-di-GMP in regulation of swimming motility. Thus the single Bd0367 enzyme produces two secondary messengers by action of the same GGDEF domain, the first reported example of a synthase that regulates multiple second messengers in vivo. Unlike roles of these signalling molecules in other bacteria, these signal to two separate motility systems, gliding and flagellar, which are essential for completion of the bacterial predation cycle and prey exit by B. bacteriovorus.
Bacterial usage of the cyclic dinucleotide c‐di‐GMP is widespread, governing the transition between motile/sessile and unicellular/multicellular behaviors. There is limited information on c‐di‐GMP ...metabolism, particularly on regulatory mechanisms governing control of EAL c‐di‐GMP phosphodiesterases. Herein, we provide high‐resolution structures for an EAL enzyme Bd1971, from the predatory bacterium Bdellovibrio bacteriovorus, which is controlled by a second signaling nucleotide, cAMP. The full‐length cAMP‐bound form reveals the sensory N‐terminus to be a domain‐swapped variant of the cNMP/CRP family, which in the cAMP‐activated state holds the C‐terminal EAL enzyme in a phosphodiesterase‐active conformation. Using a truncation mutant, we trap both a half‐occupied and inactive apo‐form of the protein, demonstrating a series of conformational changes that alter juxtaposition of the sensory domains. We show that Bd1971 interacts with several GGDEF proteins (c‐di‐GMP producers), but mutants of Bd1971 do not share the discrete phenotypes of GGDEF mutants, instead having an elevated level of c‐di‐GMP, suggesting that the role of Bd1971 is to moderate these levels, allowing “action potentials” to be generated by each GGDEF protein to effect their specific functions.
Synopsis
The cyclic‐di‐GMP hydrolase Bd1971 is a sensor‐enzyme fusion protein controlling turnover and predatory behavior of the bacterium Bdellovibrio bacteriovorus. Here, structural work combined with enzyme assays and genetics reveal regulation of Bd1971 by cAMP, with important consequences for signalling during the Bdellovibrio lifecycle.
The Bd1971 structure identifies a sensor adapted from the dimeric CRP superfamily.
Bd1971 bound to stimulus cAMP renders the protein active to hydrolyse cyclic‐di‐GMP.
Empty and part‐occupied sensor structures reveal a “swing‐out” mechanism of regulation.
Bd1971 can bind GGDEF synthases and putatively allow “action potential” spikes of cyclic‐di‐GMP to be generated.
Structural and biochemical work shows how usage of the second messenger cyclic dinucleotide c‐di‐GMP is controlled by upstream cAMP.
Cyclic-di-GMP is a ubiquitous second messenger regulating lifestyles across a wide range of bacteria, most notably motility and virulence. The components of c-di-GMP signalling networks are made up ...of: diguanylate cyclases (DGCs) that contain a GGDEF domain and make c-di-GMP, phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain and break-down c-di-GMP and PilZ domains (or degenerate DGCs/PDEs) which are receptors for c-di-GMP. Bdellovibrio bacteriovorus HD100 is a predatory Gram-negative bacterium that preys upon other Gram-negative bacteria. B. bacteriovorus HD100 has a complex c-di-GMP signalling network, including four DGCs; one EAL domain protein, six HD-GYP proteins, 19 PilZ domain-proteins, a degenerate DGC and a proposed riboswitch all of which can act as cdi-GMP receptors. Bd0367, a DGC, regulates gliding motility and prey cell exit in B. bacteriovorus. Bd0367 has a GGDEF domain but also a response regulator domain, which suggests that c-di-GMP production by Bd0367 may be regulated by a phosphorylation event by a cognate sensor kinase. This thesis investigated the Bd0367 signalling pathway through analysis of Bd0367 protein interaction networks, Bd0367 DGC c-di-GMP production and searched for the cognate histidine kinase for Bd0367. This study confirmed that a conserved aspartate residue is required for Bd0367 to be an active DGC via mutational studies and cellular cdi-GMP extractions. It also demonstrated that Bd0367 enzymatic activity is essential for prey cell exit, but is less important for gliding motility, this was tested by a Bd0367D63A mutation. Which suggests that localised protein interactions formed by Bd0367 (independent of its enzymatic role) may be more important for gliding motility than the Bd0367 specific c-di-GMP generation. This concept was supported by other data as deletion of a gene encoding a PilZ domain protein that interacted with Bd0367 by bacterial two hybrid analysis, caused hyperreversals during gliding motility. Future work is needed to dissect the exact mechanism by which the PilZ domain protein influences the gliding machinery. A PDE protein Bd1971 interacts by bacterial two hybrid assay with Bd0367. This study found that ∆bd1971 resulted in impaired gliding motility, suggesting that a high global level of c-di-GMP in B. bacteriovorus cells can negatively affect motility as seen in many other bacteria. The molecular reason for impaired gliding at high c-di-GMP levels in B. bacteriovorus is yet to be determined and requires further study.
Instructive feedback (IF) is a modification to discrete trial instruction that may increase instructional efficiency for individuals with autism spectrum disorder. Several variations of IF have ...recently been evaluated in the literature; however, few studies have assessed the effectiveness and efficiency of presenting secondary targets on continuous versus intermittent presentation schedules. The current study evaluated the effectiveness and efficiency of various presentation schedules of secondary targets during discrete trial instruction. Specifically, we replicated and extended Griffen et al. (1998) by comparing a condition in which secondary targets were presented during each trial of a session, a condition in which secondary targets were presented every other trial, and a condition in which secondary targets were presented about every 4 trials. Within-subject replications were included for both participants. One of the intermittent presentation schedules was associated with the most optimal outcomes in all 4 comparisons.
The level of Salmonella contamination in retail chicken products was investigated bimonthly in two counties in the UK over 12 consecutive months. In total 325 chicken and 35 giblet samples were ...tested. Supermarket chicken, fresh chilled and frozen, and chicken from local butcher shops were compared for their rates of Salmonella contamination which were, respectively, 18.6, 25.5, and 24.5%. These differences were not found to be significantly different. The overall level of contamination for the three groups combined was 22.8%. Giblets (supermarket frozen and butcher shop combined) were contaminated at a rate of 37.1%, which was not significantly different from the combined rate for the chicken samples. Amongst all salmonellae isolated, Salmonella enteritidis was present in 51.4% and S. typhimurium in 12.2% of the chicken samples and 23.1% and 0% respectively of the giblet samples
Cardiovascular risk factors identified in major studies of children include a family history of heart disease, smoking, blood pressure, dietary factors, exercise or fitness level, and stress. The ...purpose of this descriptive study was to identify differences in children's physical measurements with regard to six cardiovascular risk factors. A descriptive design using three questionnaires and physical measurements was used. The questionnaires included the Bloomsday Cardiovascular Fitness Questionnaire, the Coronary Risk Profile, and the Diet Habit Survey. Physical measurements included blood pressure readings, weight, total serum cholesterol, and high-density lipoprotein levels. A convenience sample of 78, predominantly Caucasian children between the ages of 7 and 18 years (M = 13.13, SD = 2.79) participated. Results indicated that differences in physical measurements were significantly for children with respect to the cardiovascular risk factors, including dietary factors, blood pressure, exercise, and stress. Nursing implications for thorough assessment and intervention through education are discussed.
Provider: - Institution: - Data provided by Europeana Collections- Magura Buduiasca site is located on the Teleorman lower terrace, 10 km north-east from Alexandria. Researches began in 2001 ...following surveys that evidenced Starcevo-Cris and Dudesti cultures materials. Since 2003, the site has become the main objective of SRAP Romanian-British project. The exceptional importance of the site is due to the existence of occupation levels from the early to the late Neolithic representing Starcevo-Cris, Dudesti and Vadastra cultures. Objectives: researching the specific occupation levels from the early to the late Neolithic; finding out the limits of the site and the elements of the internal organization; conducting interdisciplinary studies and researches. In the north part of the survey S10/2003, a 12x12 m area was opened, divided in 4 equal 6x6 m squares (S19-S22), delimited by 0.5 m baulks. We uncovered 18 archaeological complexes, 15 from the Neolithic period and three from the post-Neolithic period. The researches conducted during this campaign confirm the Magura-Buduiasca Neolithic occupation stratigraphy: the early Neolithic occupation belongs to the Starcevo-Cris culture, 3rd phase, and the late Neolithic one to the Dudesti (with an early and a late phase) and Vadastra (early phase, Vadastra 1) cultures. The 14C test dates the Starcevo-Cris occupation level to 5700 B.C. Regarding the researched complexes, some preliminary observations can be made so far: - few identified complexes can be considered to be dwellings; - most complexes seem to be waste pits. That is proved by the artifacts found on the site: animal bones, a lot of sherds, different unusable fragmentary tools, building material and kiln fragments. The lack of occupation and the grouping of waste pits can suggest, at least for the late Neolithic, the existence of waste areas, located either on the outskirts of the settlement or at a certain distance from the occupation area.- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
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