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.
Summary
Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. ...Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non‐glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form.
Bacterial swimming is mediated by the rotation of a flagellar filament. Many bacteria are now known to be able to O‐glycosylate their flagellins, the proteins that make up the flagellar filament. For ...bacteria that use nonulosonic acid sugars such as pseudaminic acid, this glycosylation process is essential for the formation of a functional flagellum. However, the specific role of glycosylation remains elusive. Aeromonas caviae is a model for this process as it has a genetically simple glycosylation system. Here, we investigated the localization of the glycans on the A. caviae flagellum filament. Using mass spectrometry it was revealed that pseudaminic acid O‐glycosylation was heterogeneous with no serine or threonine sites that were constantly glycosylated. Site‐directed mutagenesis of particular glycosylation sites in most cases resulted in strains that had reduced motility and produced less detectable flagellin on Western blots. For flagellin O‐linked glycosylation, there is no known consensus sequence, although hydrophobic amino acids have been suggested to play a role. We, therefore, performed site‐directed mutagenesis of isoleucine or leucine residues flanking the sites of glycosylation and demonstrated a reduction in motility and the amount of flagellin present in the cells, indicating a role for these hydrophobic amino acids in the flagellin glycosylation process.
Flagellin glycosylation is required for swimming motility in Aeromonas. Here, we show that O‐glycosylation was heterogeneous with no serine or threonine sites that were constantly glycosylated, and mutation of several sites was found to affect motility. Data herein suggest that hydrophobic amino acids play a role in the glycosylation process.
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.
Bdellovibrio bacteriovorus is a predatory deltaproteobacterium that encounters individual Gram-negative prey bacteria with gliding or swimming motility, and then is able to invade such prey cells via ...type IVa pilus-dependent mechanisms. Movement control (pili or gliding) in other deltaproteobacteria, such as the pack hunting Myxococcus xanthus, uses a response regulator protein, RomR
(which dynamically relocalises between the cell poles) and a GTPase, MglA
, previously postulated as an interface between the Frz
chemosensory system and gliding or pilus-motility apparatus, to produce regulated bidirectional motility. In contrast, B. bacteriovorus predation is a more singular encounter between a lone predator and prey; contact is always via the piliated, non-flagellar pole of the predator, involving MglA
, but no Frz system. In this new study, tracking fluorescent RomR
microscopically during predatory growth shows that it does not dynamically relocalise, in contrast to the M. xanthus protein; instead having possible roles in growth events. Furthermore, transcriptional start analysis, site-directed mutagenesis and bacterial two-hybrid interaction studies, indicate an evolutionary loss of RomR
activation (via receiver domain phosphorylation) in this lone hunting bacterium, demonstrating divergence from its bipolar role in motility in pack-hunting M. xanthus and further evolution that may differentiate lone from pack predators.
Complex 3′-5′-cyclic diguanylic acid (c-di-GMP) responsive regulatory networks that are modulated by the action of multiple diguanylate cyclases (DGC; GGDEF domain proteins) and phosphodiesterases ...(PDE; EAL domain proteins) have evolved in many bacteria. YfgF proteins possess a membrane-anchoring domain (MASE1), a catalytically inactive GGDEF domain and a catalytically active EAL domain. Here, sustained expression of the Salmonella enterica spp. Enterica ser. Enteritidis YfgF protein is shown to mediate inhibition of the formation of the aspartate chemotactic ring on motility agar under aerobic conditions. This phenomenon was c-di-GMP-independent because it occurred in a Salmonella strain that lacked the ability to synthesize c-di-GMP and also when PDE activity was abolished by site-directed mutagenesis of the EAL domain. YfgF-mediated inhibition of aspartate chemotactic ring formation was impaired in the altered redox environment generated by exogenous p-benzoquinone. This ability of YfgF to inhibit the response to aspartate required a motif, 213Lys-Lys-Glu215, in the predicted cytoplasmic loop between trans-membrane regions 5 and 6 of the MASE1 domain. Thus, for the first time the function of a MASE1 domain as a redox-responsive regulator of bacterial responses to aspartate has been shown.
Aeromonas caviae is motile via a polar flagellum in liquid culture, with a lateral flagella system used for swarming on solid surfaces. The polar flagellum also has a role in cellular adherence and ...biofilm formation. The two subunits of the polar flagellum, FlaA and FlaB, are posttranslationally modified by O‐linked glycosylation with pseudaminic acid on 6–8 serine and threonine residues within the central region of these proteins. This modification is essential for the formation of the flagellum. Aeromonas caviae possesses the simplest set of genes required for bacterial glycosylation currently known, with the putative glycosyltransferase, Maf1, being described recently. Here, we investigated the role of the AHA0618 gene, which shares homology (37% at the amino acid level) with the central region of a putative deglycosylation enzyme (HP0518) from the human pathogen Helicobacter pylori, which also glycosylates its flagellin and is proposed to be part of a flagellin deglycosylation pathway. Phenotypic analysis of an AHA0618 A. caviae mutant revealed increased swimming and swarming motility compared to the wild‐type strain but without any detectable effects on the glycosylation status of the polar flagellins when analyzed by western blot analysis or mass spectroscopy. Bioinformatic analysis of the protein AHA0618, demonstrated homology to a family of l,d‐transpeptidases involved in cell wall biology and peptidoglycan cross‐linking (YkuD‐like). Scanning electron microscopy (SEM) and fluorescence microscopy analysis of the wild‐type and AHA0618‐mutant A. caviae strains revealed the mutant to be subtly but significantly shorter than wild‐type cells; a phenomenon that could be recovered when either AHA0618 or H. pylori HP0518 were introduced. We can therefore conclude that AHA0618 does not affect A. caviae behavior by altering polar flagellin glycosylation levels but is likely to have a role in peptidoglycan processing at the bacterial cell wall, consequently altering cell length and hence influencing motility.
Mutation of the AHA0618 gene in Aeromonas caviae caused an increase in motility. Similar to the mutation of a putative deglycosylation gene from Helicobacter pylori. However, in A. caviae there appeared to be an effect on flagellin glycosylation, but a small change in cell size was observed.
Abstract
A
eromonas caviae
is motile via a polar flagellum in liquid culture, with a lateral flagella system used for swarming on solid surfaces. The polar flagellum also has a role in cellular ...adherence and biofilm formation. The two subunits of the polar flagellum, FlaA and FlaB, are posttranslationally modified by O‐linked glycosylation with pseudaminic acid on 6–8 serine and threonine residues within the central region of these proteins. This modification is essential for the formation of the flagellum.
A
eromonas caviae
possesses the simplest set of genes required for bacterial glycosylation currently known, with the putative glycosyltransferase, Maf1, being described recently. Here, we investigated the role of the
AHA0618
gene, which shares homology (37% at the amino acid level) with the central region of a putative deglycosylation enzyme (HP0518) from the human pathogen
H
elicobacter pylori
, which also glycosylates its flagellin and is proposed to be part of a flagellin deglycosylation pathway. Phenotypic analysis of an
AHA0618 A. caviae
mutant revealed increased swimming and swarming motility compared to the wild‐type strain but without any detectable effects on the glycosylation status of the polar flagellins when analyzed by western blot analysis or mass spectroscopy. Bioinformatic analysis of the protein AHA0618, demonstrated homology to a family of
l
,
d
‐transpeptidases involved in cell wall biology and peptidoglycan cross‐linking (YkuD‐like). Scanning electron microscopy (
SEM
) and fluorescence microscopy analysis of the wild‐type and
AHA0618‐
mutant
A. caviae
strains revealed the mutant to be subtly but significantly shorter than wild‐type cells; a phenomenon that could be recovered when either
AHA0618
or
H. pylori HP0518
were introduced. We can therefore conclude that AHA0618 does not affect
A. caviae
behavior by altering polar flagellin glycosylation levels but is likely to have a role in peptidoglycan processing at the bacterial cell wall, consequently altering cell length and hence influencing motility.
The Court determined that states must provide juvenile non-homicide offenders with a "meaningful opportunity to obtain release," but left the method for providing this opportunity to the states, ...which has led to a lack of consensus in court decisions and divergent holdings. This Note suggests that a constitutional boundary needs to be set in order to give states guidance in complying with Terrance Jamar Graham and proposes that the boundary be set at thirty years. Part I discusses the development of the Court's "kids are different" decisions. Part II argues that the rationale behind Graham applies not only to life-without-parole sentences but also to lengthy term-of-years sentences for juvenile non-homicide offenders. Part III suggests a constitutional mandate as to when states must provide a meaningful opportunity for release and explores other legislative action states can employ to comply with Graham.
is a small Gram-negative, obligate predatory bacterium that is largely found in wet, aerobic environments (e.g., soil). This bacterium attacks and invades other Gram-negative bacteria, including ...animal and plant pathogens. The intriguing life cycle of
consists of two phases: a free-living nonreplicative attack phase, in which the predatory bacterium searches for its prey, and a reproductive phase, in which
degrades a host's macromolecules and reuses them for its own growth and chromosome replication. Although the cell biology of this predatory bacterium has gained considerable interest in recent years, we know almost nothing about the dynamics of its chromosome replication. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells of
Our results show that in
, chromosome replication takes place only during the reproductive phase and exhibits a novel spatiotemporal arrangement of replisomes. The replication process starts at the invasive pole of the predatory bacterium inside the prey cell and proceeds until several copies of the chromosome have been completely synthesized. Chromosome replication is not coincident with the predator cell division, and it terminates shortly before synchronous predator filament septation occurs. In addition, we demonstrate that if this
life cycle fails in some cells of
, they can instead use second prey cells to complete their life cycle.
New strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacterium
, which kills other bacteria, including pathogens, is considered promising for combating bacterial infections. The
life cycle consists of two phases, a free-living, invasive attack phase and an intracellular reproductive phase, in which this predatory bacterium degrades the host's macromolecules and reuses them for its own growth. To understand the use of
as a "living antibiotic," it is first necessary to dissect its life cycle, including chromosome replication. Here, we present a real-time investigation into subcellular localization of chromosome replication in a single cell of
This process initiates at the invasion pole of
and proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells of
require two prey cells sequentially to complete their life cycle.
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