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International audience; When considering microbial biotic interactions, viruses as well as eukaryotic grazers are known to be important components of aquatic microbial food webs. It might be the same for bacterivorous bacteria but these groups have been comparatively less studied. This is typically the case of the Bdellovibrio and like organisms (BALOs), which are obligate bacterial predators of other bacteria. Recently, the abundance and distribution of three families of this functional group were investigated in perialpine lakes, revealing their presence and quantitative importance. Here, a more in-depth analysis is provided for Lake Geneva regarding the diversity of these bacterial predators at different seasons, sites and depths. We reveal a seasonal and spatial (vertical) pattern for BALOs. They were also found to be relatively diverse (especially Bdellovibrionaceae) and assigned to both known and unknown phylogenetic clusters. At last we found that most BALOs were positively correlated to other bacterial groups, mainly Gram-negative, in particular Myxococcales (among which many are predators of other microbes). This study is the first shedding light on this potentially important bacterial killing group in a large and deep lake.

International audience; Microbes drive a variety of ecosystem processes and services, but many of them remain largely unexplored because of a lack of knowledge on both the diversity and functionality of some potentially crucial microbiological compartments. This is the case with and within the group of bacterial predators collectively known as Bdellovibrio and like organisms (BALOs). Here, we report the abundance, distribution, and diversity of three families of these obligate predatory Gram-negative bacteria in three perialpine lakes (Lakes Annecy, Bourget, and Geneva). The study was conducted at different depths (near-surface versus 45 or 50 m) from August 2015 to January 2016. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and cloning-sequencing approaches, we show that the diversity of BALOs is relatively low and very specific to freshwaters or even the lakes themselves. While the Peredibacteraceae family was represented mainly by a single species (Peredibacter starrii), it could represent up to 7% of the total bacterial cell abundances. Comparatively, the abundances of the two other families (Bdellovibrionaceae and Bacteriovoracaceae) were significantly lower. In addition, the distributions in the water column were very different between the three groups, suggesting various life strategies/niches, as follows: Peredibacteraceae dominated near the surface, while Bdellovibrionaceae and Bacteriovoracaceae were more abundant at greater depths. Statistical analyses revealed that BALOs seem mainly to be driven by depth and temperature. Finally, this original study was also the opportunity to design new quantitative PCR (qPCR) primers for Peredibacteraceae quantification. IMPORTANCE This study highlights the abundance, distribution, and diversity of a poorly known microbial compartment in natural aquatic ecosystems, the Bdellovibrio and like organisms (BALOs). These obligate bacterial predators of other bacteria may have an important functional role. This study shows the relative quantitative importance of the three main families of this group, with the design of a new primer pair, and their diversity. While both the diversity and the abundances of these BALOs were globally low, it is noteworthy that the abundance of the Peredibacteraceae could reach important values.

Microbes drive a variety of ecosystem processes and services but still many of them remain largely unexplored because of our lack of knowledge on both diversity and functionality of some potentially key microbiological compartments. This is typically the case with and within the group of bacterial predators collectively known as Bdellovibrio and like organisms (BALOs). Here we report for the first time the abundance, distribution and diversity of the three main families of these natural and obligatory predators of gram negative bacteria in three peri-alpine lakes (e.g. lakes Annecy, Bourget and Geneva) at different depths (surface vs. 45 or 50 m) and along a few months (from August 2015 to January 2016). We show that, using PCR-DGGE and cloning-sequencing approaches, the diversity appeared relatively low and very specific to fresh waters or even of the lakes themselves. While the Peredibacteraceae family was represented mainly by a single species (i.e. Peredibacter starii), it could constitute up to 7% of the total bacterial cell abundances. Comparati vel y, the abundances of the two other families (referred to as Bdellovibrionaceae and Bacteriovaracaceae) were significantly lower. More interestingly, the distribution in the water column was very different between the three groups suggesting various life strategies/niches for each of them: Peredibactereacea dominated near surface while the Bdellovibrionaceae and the Bacteriovaracaceae were more abundant at depth. All in all, our results suggest that these bacterial predators are likely to play a significant role in mortality, carbon fluxes and prokaryotic community structure in lakes.

Standard aquaculture generates large-scale pollution and strains water resources. In aquaculture using zero discharge systems (ZDS), highly efficient fish growth and water recycling are combined. The wastewater stream is directed through compartments in which beneficial microbial activities induced by creating suitable environmental conditions remove biological and chemical pollutants, alleviating both problems. Bacterial predators, preying on bacterial populations in the ZDS, may affect their diversity, composition and functional redundancy, yet in-depth understanding of this phenomenon is lacking. The dynamics of populations belonging to the obligate predators Bdellovibrio and like organisms (BALOs) were analyzed in freshwater and saline ZDS over a 7-month period using QPCR targeting the Bdellovibrionaceae, and the Bacteriovorax and Bacteriolyticum genera in the Bacteriovoracaeae. Both families co-existed in ZDS compartments, constituting 0.13-1.4% of total Bacteria. Relative predator abundance varied according to the environmental conditions prevailing in different compartments, most notably salinity. Strikingly, the Bdellovibrionaceae, hitherto only retrieved from freshwater and soil, also populated the saline system. In addition to the detected BALOs, other potential predators were highly abundant, especially from the Myxococcales. Among the general bacterial population, Flavobacteria, Bacteroidetes, Fusobacteriaceae and unclassified Bacteria dominated a well mixed but seasonally fluctuating diverse community of up to 238 operational taxonomic units, as revealed by 16S rRNA gene sequencing.

Quantification of Bdellovibrio-and-like organisms (BALOs) by microbial culturing has a number of substantial drawbacks. Therefore a quantitative PCR (qPCR) assay was designed for the culture-independent enumeration of the Bdellovibrionaceae. After optimization, the dynamic range of the qPCR assay was assessed, the specificity was evaluated and a comparison with quantitative microbial culturing was made. To evaluate the suitability of the qPCR assay for analysing environmental samples, fresh water samples were investigated by microbial culturing and by the qPCR assay. The results revealed a substantial difference between the two techniques and indicate that most Bdellovibrionaceae cells are left undetected in environmental samples when only current microbial culturing techniques are used. The application of this new technique is therefore likely to confirm the hitherto underestimated sizes and roles of predatory bacterial populations in nature. ispartof: Environmental microbiology reports vol:1 issue:4 pages:228-233 ispartof: location:United States status: published

Summary Bdellovibrio -and-like organisms (BALOs) are wide- spread obligatory predators of other Gram-negative bacteria. Their detection by culture-dependent meth- ods is complicated as their replication is totally dependent upon the availability of an appropriate prey. Because BALOs do not form numerically domi- nant groups within microbial communities, non-spe- cific culture-independent tools also generally fail to detect them. We designed sets of 16S rRNA primers that specifically target BALOs. Polymerase chain reaction (PCR) and denaturing gradient gel electro- phoresis (DGGE) were combined, yielding partial 16S rDNA sequences. This simple method that allows spe- cific in situ culture-independent detection of BALOs was applied to the soil environment. Bdellovibrio -and- like organisms were also isolated from the same soil and the phylogeny and prey range of the isolates analysed. Seventeen isolates retrieved using five dif- ferent potential preys exhibited eight different spectra of prey utilization and formed nine operational taxo- nomic units (OTUs). These OTUs were affiliated with the Bdellovibrionaceae , Bacteriovorax , Peredibacter or Micavibrio , i.e. the known BALO groups. In com- parison, 15 OTUs including 10 that were not detected by the culture-dependent approach were obtained using the specific primers in a PCR-DGGE approach. The occurrence of a complex BALO community sug- gests that predation occurs on a much wider range of prey than can be detected by the classical culture- dependent technique.

The predation of Bdellovibrio bacteriovorus on different periodontal pathogens has already been described. However, it is necessary to consider the polymicrobial nature of periodontal disease. The current study explores the predation of Bdellovibrio on oral pathogens organized in multispecies communities. The effect of the predator was evaluated on in vitro six species communities with microbial culturing. Additionally, the effect on ex vivo subgingival plaque and saliva samples from periodontitis patients was assessed. In the latter experiment results were examined with microbial culturing, quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). The latter technique was used to get an overview of the whole mixed microbial population. Results showed that even in more complex models, B. bacteriovorus was still able to predate on F. nucleatum and A. actinomycetemcomitans. However predation on P. intermedia and P. gingivalis could not be validated in multispecies models. The effect of Bdellovibrio was not restricted to the target bacteria. Changes in the overall ecology of the different models were evident. It could be concluded that the efficiency of predation decreased when complexity of the models increased. However, B. bacteriovorus was able to attack two important oral pathogens, F. nucleatum, and A. actinomycetemcomitans, even when present in ex vivo clinical samples. These effects still have to be validated in in vivo models to see the impact of Bdellovibrio on the whole bacterial ecology. publisher: Elsevier articletitle: Effect of Bdellovibrio bacteriovorus HD100 on multispecies oral communities journaltitle: Anaerobe articlelink: http://dx.doi.org/10.1016/j.anaerobe.2014.09.011 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved. ispartof: Anaerobe vol:35 issue:Pt A pages:45-53 ispartof: location:England status: published

Bdellovibrio and like organisms (BALOs) are gram-negative, obligate predators of other gram-negative bacteria. These small bacteria interact with their prey as highly motile attack phase cells, attaching to the outer membrane and consuming the prey extracellularly (epibiotic predation) or penetrating their periplasm (periplasmic predation). The former divides in a binary fashion, while the latter grows as a polynucleotide filament to finally split as progeny attack cells. High-resolution microscopy, molecular genetics, genomics, and functional genomics have been applied to study the cell cycle of BALOs, revealing functions required for predation and for cellular organization. Until recently, Bdellovibrio bacteriovorus was the only recognized species of BALOs. Culture-dependent and culture-independent approaches have shown that these predators form diverse monophyletic groups, including the three families Bdellovibrionaceae, Bacteriovoraceae, and Peridibacteraceae in the d-proteobacteria, and the genus Micavibrio in the a-proteobacteria. Based on this detailed taxonomical knowledge, it has become possible to track predator and prey interactions in natural systems, providing first evaluations of the impact of bacterial predation on community structure.

Bdellovibrio-and-like organisms (BALO) are a phylogenetically diverse group of predatory prokaryotes that consists of the two families Bdellovibrionaceae and Bacteriovoracaceae. We investigated the phospholipid composition of the three important BALO strains Bacteriovorax stolpii (DSM 12778), Bdellovibrio bacteriovorus HD100 (DSM 50701) and Peredibacter starrii (DSM 17039). We confirmed the presence of sphingophosphonolipids in B. stolpii, while we characterized sphingophosphonolipids with a 2-amino-3-phosphonopropanate head group for the first time. In B. bacteriovorus HD100 phosphatidylthreonines were found and, thus, B. bacteriovorus is the second prokaryote investigated so far possessing this rare lipid class. In the third analyzed organism, P. starrii, we observed phosphatidylethanolamine structures with an additional N-glutamyl residue, which form the first reported class of amino acid-containing phosphatidylethanolamines.

A phylogenetic analysis of Bdellovibrio-and-like organisms (BALOs) was performed. It was based on the characterization of 71 strains and on all consequent 16S rRNA gene sequences available in databases, including clones identified by data-mining, totalling 120 strains from very varied biotopes. Amplified rDNA restriction analysis (ARDRA) accurately reflected the diversity and phylogenetic affiliation of BALOs, thereby providing an efficient screening tool. Extensive phylogenetic analysis of the 16S rRNA gene sequences revealed great diversity within the Bdellovibrio (>14 % divergence) and Bacteriovorax (>16 %) clades, which comprised nine and eight clusters, respectively, exhibiting more than 3 % intra-cluster divergence. The clades diverged by more than 20 %. The analysis of conserved 16S rRNA secondary structures showed that Bdellovibrio contained motifs atypical of the δ-Proteobacteria, suggesting that it is ancestral to Bacteriovorax. While none of the Bdellovibrio strains were of marine origin, Bacteriovorax included separate soil/freshwater and marine-specific groups. On the basis of their extensive diversity and the large distance separating the groups, it is proposed that Bacteriovorax starrii be placed into a new genus, Peredibacter gen. nov., with Peredibacter starrii A3.12T (=ATCC 15145T=NCCB 72004T) as its type strain. Also proposed is a redefinition of the Bdellovibrio and the Bacteriovorax–Peredibacter lineages as two different families, i.e. ‘Bdellovibrionaceae’ and a new family, Bacteriovoracaceae. Also, a re-evaluation of oligonucleotides targeting BALOs is presented, and the implications of the large diversity of these organisms and of their distribution in very different environments are discussed.

The Bdellovibrionaceae are predatory, intraperiplasmic bacteria that prey upon a variety of Gram-negative bacteria. The prey susceptibility pattern is frequently used to characterize new isolates. The objective in this study was to isolate and characterize predators from the Great Salt Lake (GSL) by prey susceptibility testing. To recover the predators, water samples were inoculated into an enrichment medium with Vibrio parahaemolyticus as prey. After several days of incubation, the predators were isolated, pure DNA was extracted, and partial 16S rDNA gene was sequenced. Water samples were also plated for isolation of heterotrophic bacteria. The susceptibility of bacterial isolates from the lake and other sources to each predator isolate was determined. The results revealed that there are predators in the GSL, and they preferentially prey on bacteria from the lake. This is the first report of the isolation of Bdellovibrionaceae from GSL and the predators showing preferences for bacteria from the same habitat.

Bdellovibrionaceae sind außergewöhnliche Bakterien, da sie als die kleinsten bekannten räuberischen Organismen gelten. Seit ihrer Entdeckung in Bodenproben im Jahr 1962 durch Stolp und Starr konnte eine weite Verbreitung in der Natur nachgewiesen werden. Besonderes Interesse erlangten Bdellovibrionaceae durch ihre Fähigkeit bakterielle Erreger wie Escherichia coli, Salmonella und Yersinia zu attackieren. Der bestuntersuchte Vertreter der Familie Bdellovibrionaceae ist Bdellovibrio bacteriovorus. Er durchläuft einen komplexen Lebenszyklus, der nur partiell verstanden ist. In der Attack-Phase werden durch einen noch nicht aufgeklärten Mechanismus potentielle Beutebakterien erkannt und der interzelluläre Kontakt hergestellt. Nachdem eine Pore in der Zellwand der ausschließlich Gram-negativen Beutebakterien erzeugt wurde, dringt B. bacteriovorus in den periplasmatischen Raum ein. Nach etwa 30 Minuten ist der Invasionsprozess abgeschlossen und man kann die Ausbildung sphärischer Bdelloblasten beobachten. Im Beutebakterium verdaut B. bacteriovorus makromolekulare Bestandteile des Wirtes und wächst zu einem langen spiralförmigen Stäbchen aus. Es setzt schließlich die Zellteilung ein bei der 5 bis 30 Tochterzellen in Abhängigkeit zur Größe des Beutebakteriums freiwerden. Mit der Reproduktion ist der parasitäre Lebenszyklus nach etwa 3 Stunden beendet. Die komplexe Regulation der Expression von Proteinen konnte für die einzelnen Wachstumsphasen durch ein- sowie zweidimensionale Gelelektrophorese nachgewiesen werden. In der Vergangenheit haben verschiedene Studien die Komplexität der Wechselwirkung mit den Beutebakterien belegt. Hierbei wurde, neben dem offensichtlichen Abbau makromolekularer Bestandteile der Beutebakterien, ein Recycling und Einbau von Membranbestandteilen wie Lipopolysacchariden (LPS) und Outer Membrane Proteine (OmP) in das Membransystem von B. bacteriovorus diskutiert. Die biologische Interpretation dieses Phänomens ist eine erhöhte Effizienz in der Reproduktion von B. bacteriovorus wenn es Bestandteile des Wirtsbakteriums wiederverwertet im Gegensatz zur Eigensynthese. Trotz der morphologischen Ähnlichkeit und des ungewöhnlichen Lebensstils wurde festgestellt, dass die Familie der Bdellovibrionaceae phylogenetisch sehr heterogen ist. Es werden zur Zeit zwei Gattungen unterschieden Bdellovibrio und Bacteriovorax. Aufgrund von 16S rRNA Untersuchungen konnten auch innerhalb der Gattungen eine Vielzahl von phylogenetisch distinkten Arten nachgewiesen werden. In der Literatur wird eine regulierende Wirkung auf pathogene Gram-negative Bakterien für aquatische Systeme durch Bdellovibrionaceae beschrieben. Weiterhin konnten sie bei verschiedenen Nutztieren im Verdauungstrakt mikrobiologisch nachgewiesen werden. Ein positiver Einfluss auf die Gesundheit der Tiere wurde bei Vorkommen von B. bacteriovorus festgestellt. Für eine Charakterisierung von Umweltisolaten werden in der vorliegenden Arbeit genetische Methoden vorgestellt. Hierfür wurden Hybridisierungsmethoden und PCR-methoden entwickelt, die es ermöglichen aus der Umwelt isolierte Bdellovibrionaceae phylogenetisch einzuordnen. Es konnte gezeigt werden, das sowohl B. bacteriovorus als auch Bacteriovorax stolpii im Verdauungstrakt von Nutztieren vorkommen. Es ist weiterhin gelungen eine PCR-methode für Kotproben zu entwickeln, die einen direkten Nachweis ermöglicht ohne mikrobiologische Anzucht. B. bacteriovorus ist ein Gram-negatives Bakterium, allein dieser Sachverhalt spiegelt die Schwierigkeit wider, wenn der enzymattische Abbau von Zellwandbestandteilen der Beutebakterien Ziel der Untersuchung ist, da auch die Beutebakterien Gram-negativ sind. Es ergibt sich aufgrund eines ähnlichen Zellwandaufbaus ein immenses analytisches Problem die makromolekularen Bestandteile von Wirtsbakterium und Jäger zu trennen. Um dem Lebensstil angepasst Modifikationen von B. bacteriovorus zu untersuchen, wurde in dieser Arbeit LPS, charakteristischer Bestandteil der Äußeren Membran, von Wildtypstamm B. bacteriovorus HD100 und seiner wirtsunabhängigen Mutante HI100 isoliert und das Lipid A strukturell aufgeklärt. Die Isolation gelang durch die Ausnutzung unterschiedlicher Fällungseigenschaften des LPS von B. bacteriovorus HD100 gegenüber des E. coli K-12 LPS aus dem Extraktionsmittel. Weiterhin konnte nachgewiesen werden, das B. bacteriovorus S-Form LPS besitzt. Die außergewöhnliche Struktur des Lipid A von B. bacteriovorus wurde im Detail mit massenspektrometrischen Methoden, ein- und zweidimensionalen NMR Methoden sowie mikrochemischer Methoden in Kombination mit der GC/MS charakterisiert. Der Fettsäureanker besteht aus a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. Dies stellt eine neuartige Struktur dar, da an allen bisher bekannten Lipid A´s sich Brönstedtsäuren am hydrophilen Fettsäureanker befinden, die in physiologischer Lösung durch Protonenabgabe negative Ladungen tragen. Im Lipid A von B. bacteriovorus sind diese Substituenten durch den Neutralzucker Mannose ersetzt. Weiterhin wurden als Fettsäuren nur 3-Hydroxyfettsäuren nachgewiesen, wobei sich auf die 6 gebunden Fettsäuren etwa 1,5 Doppelbindungen verteilen. Dieses Lipid A zeigt eine wesentlich reduzierte endotoxische Aktivität im Vergleich zu E. coli Lipid A und weist als biophysikalische Besonderheit eine erhöhte Fluidität über einen weiten Temperaturbereich auf. Die vorgestellte 16S rRNA Analyse und die Strukturanalyse des Lipid A von B. bacteriovorus belegen seine besondere Stellung in der Welt der Bakterien., Bdellovibrionaceae are extraordinary bacteria known as the smallest predatory organism so far. Since their discovery in soil samples by Stolp and Starr 1963 they have been detected in a wide range of other natural habitats. Bdellovibrionaceae became the focus of attention concerning their ability to attack pathogens like Escherichia coli, Salmonella and Yersinia. For Bdellovibrio bacteriovorus the most detailed studies are available. The up to now only partially understood lifecycle consists of several complex phases. In the attack phase B. bacteriovorus is motile possessing flagella and the preys are recognized by an unknown mechanism. After attachment on the cell wall within 15 to 30 minutes a pore is formed which is used as entrance to the periplasmatic space of the Gram-negative prey bacteria. The completion of the invasion process can be observed by the change of the prey s shape to spherical bdelloblasts. Inside of the prey B. bacteriovorus degrades macromolecular compounds and transforms into a long spirally shaped rod. At the end of the lifecycle the rod divides yielding 5 up to 30 daughter cells depending on the size of the prey bacteria. This reproduction phase is completed within 3 hours. The complex regulation of expression of a certain number of proteins was observed by one and two dimensional gelelectrophoresis. The complexity of the interaction between predator and prey were examined in several studies. Besides the obvious degradation of macromolecular compounds of the prey, reutilising of lipopolysaccharides (LPS) and Outer Membrane Proteins (OmP) into the membrane system of B. bacteriovorus was discussed. The biological interpretation of such behaviour was that it is more efficient for reproduction to recycle components of the prey than to perform de novo synthesis. Unexpectedly, despite the unique predatory lifecycle and common morphological features, Bdellovibrionaceae show a great phylogenetical diversity based on 16S rRNA analyses. Bdellovibrionaceae are divided into the three species Bdellovibrio bacteriovorus, Bacteriovorax stolpii, Bacteriovorax starrii and some strains yet to be assigned. In two studies Bdellovibrionaceae were found as part of regulation processes for decreasing the number of pathogenic Gram-negative bacteria in aquatic systems. Furthermore, B. bacteriovorus were found in the intestinal tract of several domestic animals showing a positive influence on the state of health. For the phylogenetic characterization of environmental isolates techniques were developed based on hybridisation methods and the PCR. In this work we detected B. bacteriovorus and B. stolpii strains in the gut of animals. Furthermore, a PCR method for direct detection of Bdellovibrionaceae in fecal samples was developed. B. bacteriovorus are Gram-negative bacteria. This fact complicates the study of the degradation of the prey s cell wall as it possesses the architecture of Gram-negative bacteria also. Furthermore, the search for important modifications of the cell wall of B. bacteriovorus concerning the predatory lifestyle becomes an analytical problem. In this study we isolated LPS of the wild type strain B. bacteriovorus HD100 and its host independent mutant strain HI100. For the isolation of pure B. bacteriovorus HD100 S-form LPS we took advantage of different precipitation properties in the extraction solvent of E. coli K-12 LPS and the predator s LPS. The structure of the lipid A was examined in detail by mass spectrometric methods, one- and two-dimensional NMR and chemical analytical techniques. The novel structure consists of backbone built of a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. This is the first known natural lipid A without negatively charged substituents in physiological solution. The lipid A of B. bacteriovorus carries the neutral sugar mannose instead of Brönstedt acids. Furthermore, the lipid A exclusively consists of 3-hydroxy fatty acids with approximately 1.5 double bounds distributed on six bounded fatty acids. This lipid A shows a significant decreased endotoxic activity in comparison to E. coli lipid A and revealed increased fluidity over broad temperature range as further remarkable biophysical property. The 16S rRNA analysis and the structural analysis of the lipid A of B. bacteriovorus document the unique position in the world of bacteria.