Norspermidine is not a self-produced trigger for biofilm disassembly.

Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 µM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50-80 µM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 µM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.

Lateral interactions govern self-assembly of the bacterial biofilm matrix protein BslA.

The soil bacterium Bacillus subtilis is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give the B. subtilis biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in bslA deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.

Nucleotide signaling pathway convergence in a cAMP-sensing bacterial c-di-GMP phosphodiesterase.

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.

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent.

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.

Genomic Differences between Listeria monocytogenes EGDe Isolates Reveal Crucial Roles for SigB and Wall Rhamnosylation in Biofilm Formation.

Listeria monocytogenes is a Gram-positive firmicute that causes foodborne infections, in part due to its ability to use multiple strategies, including biofilm formation, to survive adverse growth conditions. As a potential way to screen for genes required for biofilm formation, we harnessed the ability of bacteria to accumulate mutations in the genome over time, diverging the properties of seemingly identical strains. By sequencing the genomes of four laboratory reference strains of the commonly used L. monocytogenes EGDe, we showed that each isolate contains single nucleotide polymorphisms (SNPs) compared with the reference genome. We discovered that two SNPs, contained in two independent genes within one of the isolates, impacted biofilm formation. Using bacterial genetics and phenotypic assays, we confirmed that rsbU and rmlA influence biofilm formation. RsbU is the upstream regulator of the alternative sigma factor SigB, and mutation of either rsbU or sigB increased biofilm formation. In contrast, deletion of rmlA, which encodes the first enzyme for TDP-l-rhamnose biosynthesis, resulted in a reduction in the amount of biofilm formed. Further analysis of biofilm formation in a strain that still produces TDP-l-rhamnose but which cannot decorate the wall teichoic acid with rhamnose (rmlT mutant) showed that it is the decorated wall teichoic acid that is required for adhesion of the cells to surfaces. Together, these data uncover novel routes by which biofilm formation by L. monocytogenes can be impacted.IMPORTANCE Biofilms are an important mode of growth in many settings. Here, we looked at small differences in the genomes of the bacterium Listeria monocytogenes isolate EGDe and used them to find out how biofilms form. This important fundamental information may help new treatments to be developed and also highlights the fact that isolates of the same identity often diverge.

Dual Predation by Bacteriophage and Bdellovibrio bacteriovorus Can Eradicate Escherichia coli Prey in Situations where Single Predation Cannot.

Bacteria are preyed upon by diverse microbial predators, including bacteriophage and predatory bacteria, such as Bdellovibrio bacteriovorus While bacteriophage are used as antimicrobial therapies in Eastern Europe and are being applied for compassionate use in the United States, predatory bacteria are only just beginning to reveal their potential therapeutic uses. However, predation by either predator type can falter due to different adaptations arising in the prey bacteria. When testing poultry farm wastewater for novel Bdellovibrio isolates on Escherichia coli prey lawns, individual composite plaques were isolated containing both an RTP (rosette-tailed-phage)-like-phage and a B. bacteriovorus strain and showing central prey lysis and halos of extra lysis. Combining the purified phage with a lab strain of B. bacteriovorus HD100 recapitulated haloed plaques and increased killing of the E. coli prey in liquid culture, showing an effective side-by-side action of these predators compared to their actions alone. Using approximate Bayesian computation to select the best fitting from a variety of different mathematical models demonstrated that the experimental data could be explained only by assuming the existence of three prey phenotypes: (i) sensitive to both predators, (ii) genetically resistant to phage only, and (iii) plastic resistant to B. bacteriovorus only. Although each predator reduces prey availability for the other, high phage numbers did not abolish B. bacteriovorus predation, so both predators are competent to coexist and are causing different selective pressures on the bacterial surface while, in tandem, controlling prey bacterial numbers efficiently. This suggests that combinatorial predator therapy could overcome problems of phage resistance.IMPORTANCE With increasing levels of antibiotic resistance, the development of alternative antibacterial therapies is urgently needed. Two potential alternatives are bacteriophage and predatory bacteria. Bacteriophage therapy has been used, but prey/host specificity and the rapid acquisition of bacterial resistance to bacteriophage are practical considerations. Predatory bacteria are of interest due to their broad Gram-negative bacterial prey range and the lack of simple resistance mechanisms. Here, a bacteriophage and a strain of Bdellovibrio bacteriovorus, preyed side by side on a population of E. coli, causing a significantly greater decrease in prey numbers than either alone. Such combinatorial predator therapy may have greater potential than individual predators since prey surface changes selected for by each predator do not protect prey against the other predator.

Social behaviour and making attachments: a report from the fifth 'Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis'.

The fifth Young Microbiologists Symposium was held in Queen's University Belfast, Northern Ireland, in late August 2018. The symposium, focused on 'Microbe signalling, organization and pathogenesis', attracted 121 microbiologists from 15 countries. The meeting allowed junior scientists to present their work to a broad audience, and was supported by the European Molecular Biology Organization, the Federation of European Microbiological Societies, the Society of Applied Microbiology, the Biochemical Society and the Microbiology Society. Sessions covered recent advances in areas of microbiology including gene regulation and signalling, secretion and transport across membranes, infection and immunity, and antibiotics and resistance mechanisms. In this Meeting Report, we highlight some of the most significant advances and exciting developments communicated during talks and poster presentations.

Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR.

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.

Interfacial self-assembly of a bacterial hydrophobin.

The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded ß-sheet. This form then self-assembles into a well-ordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.

Identification and Characterization of Two Klebsiella pneumoniae lpxL Lipid A Late Acyltransferases and Their Role in Virulence.

Klebsiella pneumoniae causes a wide range of infections, from urinary tract infections to pneumonia. The lipopolysaccharide is a virulence factor of this pathogen, although there are gaps in our understanding of its biosynthesis. Here we report on the characterization of K. pneumoniaelpxL, which encodes one of the enzymes responsible for the late secondary acylation of immature lipid A molecules. Analysis of the available K. pneumoniae genomes revealed that this pathogen's genome encodes two orthologues of Escherichia coli LpxL. Using genetic methods and mass spectrometry, we demonstrate that LpxL1 catalyzes the addition of laureate and LpxL2 catalyzes the addition of myristate. Both enzymes acylated E. coli lipid A, whereas only LpxL2 mediated K. pneumoniae lipid A acylation. We show that LpxL1 is negatively regulated by the two-component system PhoPQ. The lipid A produced by the lpxL2 mutant lacked the 2-hydroxymyristate, palmitate, and 4-aminoarabinose decorations found in the lipid A synthesized by the wild type. The lack of 2-hydroxymyristate was expected since LpxO modifies the myristate transferred by LpxL2 to the lipid A. The absence of the other two decorations is most likely caused by the downregulation of phoPQ and pmrAB expression. LpxL2-dependent lipid A acylation protects Klebsiella from polymyxins, mediates resistance to phagocytosis, limits the activation of inflammatory responses by macrophages, and is required for pathogen survival in the wax moth (Galleria mellonella). Our findings indicate that the LpxL2 contribution to virulence is dependent on LpxO-mediated hydroxylation of the LpxL2-transferred myristate. Our studies suggest that LpxL2 might be a candidate target in the development of anti-K. pneumoniae drugs.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms.

Biofilm formation is a social behaviour that generates favourable conditions for sustained survival in the natural environment. For the Gram-positive bacterium Bacillus subtilis the process involves the differentiation of cell fate within an isogenic population and the production of communal goods that form the biofilm matrix. Here we review recent progress in understanding the regulatory pathways that control biofilm formation and highlight developments in understanding the composition, function and structure of the biofilm matrix.

Phosphorylated DegU manipulates cell fate differentiation in the Bacillus subtilis biofilm.

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation.

Discrete cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.

Genome analysis of a simultaneously predatory and prey-independent, novel Bdellovibrio bacteriovorus from the River Tiber, supports in silico predictions of both ancient and recent lateral gene transfer from diverse bacteria.

BACKGROUND: Evolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as "obligate predators" because only by mutations, often in gene bd0108, are 1 in ~1x10(7) of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome. RESULTS: To test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired. CONCLUSIONS: The natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria.

Mutagenesis of RpoE-like sigma factor genes in Bdellovibrio reveals differential control of groEL and two groES genes.

BACKGROUND: Bdellovibrio bacteriovorus HD100 must regulate genes in response to a variety of environmental conditions as it enters, preys upon and leaves other bacteria, or grows axenically without prey. In addition to "housekeeping" sigma factors, its genome encodes several alternate sigma factors, including 2 Group IV-RpoE-like proteins, which may be involved in the complex regulation of its predatory lifestyle. RESULTS: We find that one sigma factor gene, bd3314, cannot be deleted from Bdellovibrio in either predatory or prey-independent growth states, and is therefore possibly essential, likely being an alternate sigma 70. Deletion of one of two Group IV-like sigma factor genes, bd0881, affects flagellar gene regulation and results in less efficient predation, although not due to motility changes; deletion of the second, bd0743, showed that it normally represses chaperone gene expression and intriguingly we find an alternative groES gene is expressed at timepoints in the predatory cycle where intensive protein synthesis at Bdellovibrio septation, prior to prey lysis, will be occurring. CONCLUSIONS: We have taken the first step in understanding how alternate sigma factors regulate different processes in the predatory lifecycle of Bdellovibrio and discovered that alternate chaperones regulated by one of them are expressed at different stages of the lifecycle.

Three motAB stator gene products in Bdellovibrio bacteriovorus contribute to motility of a single flagellum during predatory and prey-independent growth.

The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.

Predatory Bdellovibrio bacteria use gliding motility to scout for prey on surfaces.

Bdellovibrio bacteriovorus is a famously fast, flagellate predatory bacterium, preying upon Gram-negative bacteria in liquids; how it interacts with prey on surfaces such as in medical biofilms is unknown. Here we report that Bdellovibrio bacteria "scout" for prey bacteria on solid surfaces, using slow gliding motility that is present in flagellum-negative and pilus-negative strains.

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth.

Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron-sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks.

Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.