Structure analysis of a soil community of predatory bacteria using culture-dependent and culture-independent methods reveals a hitherto undetected diversity of Bdellovibrio-and-like organisms

Bdellovibrio-and-like organisms (BALOs) are widespread obligatory predators of other Gram-negative bacteria. Their detection by culture-dependent methods is complicated as their replication is totally dependent upon the availability of an appropriate prey. Because BALOs do not form numerically dominant groups within microbial communities, non-specific 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 electrophoresis (DGGE) were combined, yielding partial 16S rDNA sequences. This simple method that allows specific 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 different potential preys exhibited eight different spectra of prey utilization and formed nine operational taxonomic units (OTUs). These OTUs were affiliated with the Bdellovibrionaceae, Bacteriovorax, Peredibacter or Micavibrio, i.e. the known BALO groups. In comparison, 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 suggests that predation occurs on a much wider range of prey than can be detected by the classical culture-dependent technique.     

Interactions between Bdellovibrio and like organisms and bacteria in biofilms: beyond predator–prey dynamics

Bdellovibrio and like organisms (BALOs) prey on Gram-negative bacteria in the planktonic phase as well as in biofilms, with the ability to reduce prey populations by orders of magnitude. During the last few years, evidence has mounted for a significant ecological role for BALOs, with important implications for our understanding of microbial community dynamics as well as for applications against pathogens, including drug-resistant pathogens, in medicine, agriculture and aquaculture, and in industrial settings for various uses. However, our understanding of biofilm predation by BALOs is still very fragmentary, including gaps in their effect on biofilm structure, on prey resistance, and on evolutionary outcomes of both predators and prey. Furthermore, their impact on biofilms has been shown to reach beyond predation, as they are reported to reduce biofilm structures of non-prey cells (including Gram-positive bacteria). Here, we review the available literature on BALOs in biofilms, extending known aspects to potential mechanisms employed by the predators to grow in biofilms. Within that context, we discuss the potential ecological significance and potential future utilization of the predatory and enzymatic possibilities offered by BALOs in medical, agricultural and environmental applications.     

In and out: an analysis of epibiotic vs periplasmic bacterial predators

Bdellovibrio and like organisms (BALO) are obligate predators of Gram-negative bacteria, belonging to the α- and δ-proteobacteria. BALO prey using either a periplasmic or an epibiotic predatory strategy, but the genetic background underlying these phenotypes is not known. Here we compare the epibiotic Bdellovibrio exovorus and Micavibrio aeruginosavorus to the periplasmic B. bacteriovorus and Bacteriovorax marinus. Electron microscopy showed that M. aeruginosavorus, but not B. exovorus, can attach to prey cells in a non-polar manner through its longitudinal side. Both these predators were resistant to a surprisingly high number of antibiotic compounds, possibly via 26 and 19 antibiotic-resistance genes, respectively, most of them encoding efflux pumps. Comparative genomic analysis of all the BALOs revealed that epibiotic predators have a much smaller genome (ca. 2.5 Mbp) than the periplasmic predators (ca. 3.5 Mbp). Additionally, periplasmic predators have, on average, 888 more proteins, at least 60% more peptidases, and one more rRNA operon. Fifteen and 219 protein families were specific to the epibiotic and the periplasmic predators, respectively, the latter clearly forming the core of the periplasmic ‘predatome'', which is upregulated during the growth phase. Metabolic deficiencies of epibiotic genomes include the synthesis of inosine, riboflavin, vitamin B6 and the siderophore aerobactin. The phylogeny of the epibiotic predators suggests that they evolved by convergent evolution, with M. aeruginosavorus originating from a non-predatory ancestor while B. exovorus evolved from periplasmic predators by gene loss.     

Hydrothermal vent gastropods from the same family (Provannidae) harbour epsilon- and gamma-proteobacterial endosymbionts

The discovery of new hydrothermal vent systems in the back-arc basins of the Western Pacific revealed chemosynthesis-based faunal communities distinct from those of other vents. These vents are dominated by two related gastropods (Alviniconcha spp. and Ifremeria nautilei) that harbour symbiotic bacteria in their gills. We used comparative 16S ribosomal RNA (rRNA) gene sequencing and in situ hybridization with rRNA-targeted probes to characterize the bacterial symbionts of Alviniconcha sp. and I. nautilei from the Manus Basin in the Western Pacific. The analyses revealed that these two gastropod species, although affiliated with the same family, harbour phylogenetically distant chemosymbionts, suggesting independent origins of these endosymbioses. The I. nautilei endosymbiont clusters with sulfur-oxidizing bacteria within the gamma-Proteobacteria, as is the case for all previously characterized endosymbionts from a wide diversity of host taxa harbouring thioautotrophic prokaryotes. In contrast, the Alviniconcha endosymbiont is affiliated with sulfur-oxidizing bacteria within the epsilon-Proteobacteria. These results show that bacteria from the epsilon-Proteobacteria are also capable of forming endosymbiotic associations with marine invertebrates from chemosynthetic environments. More generally, the endosymbiotic lifestyle is now shown to be distributed throughout all recognized classes of the Proteobacteria.     

Biostimulation of Estuarine Microbiota on Substrate Coated Agar Slides: A Novel Approach to Study Diversity of Autochthonous Bdellovibrio- and Like Organisms

Characterization of Bdellovibrio- and like organisms (BALOs) from environmental samples involves growing them in the presence of Gram-negative prey bacteria and isolation of BALO plaques. This labor-intensive enrichment and isolation procedure may impede the detection and phylogenetic characterization of uncultivable BALOs. In this article, we describe a simple slide biofilm assay to improve detection and characterization of BALO microbiota. Agar spiked with biostimulants such as yeast extract (YE), casamino acids (CA), or concentrated cells of Vibrio parahaemolyticus P5 (most widely used prey bacteria for isolation of halophilic BALOs) was plated onto buffed glass slides and exposed to water samples collected from Apalachicola Bay, Florida. After incubating for a week, diversity of the biofilm bacterial community was studied by culture-dependent and culture-independent molecular methods. The results revealed that most probable numbers (MPNs) of BALOs and total culturable bacteria recovered from YE agar slide were significantly higher than the numbers on CA- or P5-spiked agar slides. Polymerase chain reaction–restriction fragment length polymorphism followed by 16S rDNA sequencing of clones from different biostimulants resulted in identification of a plethora of Gram-negative bacteria predominantly from the alpha, gamma, delta-proteobacteria, and the Cytophaga–Flavobacterium–Bacteroides group. Corresponding to the higher biomass on the YE agar slide, the BALO clone library from YE was most diverse, consisting of Bacteriovorax spp. and a novel clade representing Peredibacter spp. Microbiota from all three biostimulated biofilms were exclusively Gram-negative, and each bacterial guild represented potential prey for BALOs. We propose the use of this simple yet novel slide biofilm assay to study oligotrophic aquatic bacterial diversity which could also potentially be utilized to isolate marine bacteria with novel traits.     

Microbial diversity in biofilms from corroding heating systems

Culture-independent investigations of the bacterial diversity and activity in district heating systems with and without corrosion did not make it possible to relate one group of microorganisms with the observed corrosion. Fluorescence in situ hybridization by oligonucleotide probes revealed the dominance of beta-proteobacteria, sulphate reducing prokaryotes and alpha-proteobacteria. Analysis of a clone library from one Danish heating (DH) system showed that the most sequences formed two clusters within the alpha-proteobacteria affiliated to the families Rhizobiaceae and Acetobacteraceae and two clusters within the beta-proteobacteria belonging to the family Comamonadaceae. Functional groups were determined by microautoradiography showing aerobic and anaerobic bacteria (sulphate reducing and methanogenic bacteria). The corrosion study showed that pitting corrosion rates were five to ten times higher than the general corrosion rates, suggesting the presence of biocorrosion. The results indicate that several bacterial groups could be involved in corrosion of DH system piping including sulphate reducing prokaryotes, Acidovorax (within the beta-proteobacteria), methanogenic bacteria and others.     

Sequencing and analysis of a 63 kb bacterial artificial chromosome insert from the Wolbachia endosymbiont of the human filarial parasite Brugia malayi.

Wolbachia endosymbiotic bacteria are widespread in filarial nematodes and are directly involved in the immune response of the host. In addition, antibiotics which disrupt Wolbachia interfere with filarial nematode development thus, Wolbachia provide an excellent target for control of filariasis. A 63.1 kb bacterial artificial chromosome insert, from the Wolbachia endosymbiont of the human filarial parasite Brugia malayi, has been sequenced using the New England Biolabs Inc. Genome Priming System() transposition kit in conjunction with primer walking methods. The bacterial artificial chromosome insert contains approximately 57 potential ORFs which have been compared by individual protein BLAST analysis with the 35 published complete microbial genomes in the Comprehensive Microbial Resource database at The Institute for Genomic Research and in the NCBI GenBank database, as well as to data from 22 incomplete genomes from the DOE Joint Genome Institute. Twenty five of the putative ORFs have significant similarity to genes from the alpha-proteobacteria Rickettsia prowazekii, the most closely related completed genome, as well as to the newly sequenced alpha-proteobacteria endosymbiont Sinorhizobium meliloti. The bacterial artificial chromosome insert sequence however has little conserved synteny with the R. prowazekii and S. meliloti genomes. Significant sequence similarity was also found in comparisons with the currently available sequence data from the Wolbachia endosymbiont of Drosophila melanogaster. Analysis of this bacterial artificial chromosome insert provides useful gene density and comparative genomic data that will contribute to whole genome sequencing of Wolbachia from the B. malayi host. This will also lead to a better understanding of the interactions between the endosymbiont and its host and will offer novel approaches and drug targets for elimination of filarial disease.     

Phylogenetic relationships of organellar Hsp90 homologs reveal fundamental differences to organellar Hsp70 and Hsp60 evolution

In agreement with endosymbiont theory for the origin of organelles, mitochondria and chloroplasts (plastids) are universally accepted to have monophyletically arisen from within alpha-proteobacteria and cyanobacteria, respectively. Convincing particular evidence in support of this theory emerged from phylogenetic analysis of highly conserved, ubiquitous heat shock proteins (Hsps) chaperonin 60 and Hsp70. These apparently indispensable general chaperones have proven to be highly useful molecular tracers of organellar origin. Phylogenetic relationships of Hsp90--a less conserved and less widely distributed general chaperone--are reported here that are strikingly incongruent with canonical patterns of endosymbiotic ancestry. It appears that Hsp90 of chloroplasts derives from the endoplasmic reticulum-specific isoform while mitochondrial Hsp90 homologs affiliate with a eubacterial lineage other than alpha subdivision of proteobacteria. These data suggest that endosymbiont htpG genes, encoding Hsp90, have been either functionally displaced by pre-existing nuclear genes or completely lost during establishment of organelles and subsequently added to initial organellar complement.     

Single eubacterial origin of eukaryotic sulfide:quinone oxidoreductase,a mitochondrial enzyme conserved from the early evolution of eukaryotes during anoxic and sulfidic times

Mitochondria occur as aerobic, facultatively anaerobic, and, in the case of hydrogenosomes, strictly anaerobic forms. This physiological diversity of mitochondrial oxygen requirement is paralleled by that of free-living alpha-proteobacteria, the group of eubacteria from which mitochondria arose, many of which are facultative anaerobes. Although ATP synthesis in mitochondria usually involves the oxidation of reduced carbon compounds, many alpha-proteobacteria and some mitochondria are known to use sulfide (H2S) as an electron donor for the respiratory chain and its associated ATP synthesis. In many eubacteria, the oxidation of sulfide involves the enzyme sulfide:quinone oxidoreductase (SQR). Nuclear-encoded homologs of SQR are found in several eukaryotic genomes. Here we show that eukaryotic SQR genes characterized to date can be traced to a single acquisition from a eubacterial donor in the common ancestor of animals and fungi. Yet, SQR is not a well-conserved protein, and our analyses suggest that the SQR gene has furthermore undergone some lateral transfer among prokaryotes during evolution, leaving the precise eubacterial lineage from which eukaryotes obtained their SQR difficult to discern with phylogenetic methods. Newer geochemical data and microfossil evidence indicate that major phases of early eukaryotic diversification occurred during a period of the Earth's history from 1 to 2 billion years before present in which the subsurface ocean waters contained almost no oxygen but contained high concentrations of sulfide, suggesting that the ability to deal with sulfide was essential for prokaryotes and eukaryotes during that time. Notwithstanding poor resolution in deep SQR phylogeny and lack of a specifically alpha-protebacterial branch for the eukaryotic enzyme on the basis of current lineage sampling, a single eubacterial origin of eukaryotic SQR and the evident need of ancient eukaryotes to deal with sulfide, a process today germane to mitochondrial quinone reduction, are compatible with the view that eukaryotic SQR was an acquisition from the mitochondrial endosymbiont.     

Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters

Aims:  To apply and compare two PCR-based methods for typing saltwater Bdellovibrio- and-like organisms (BALOs) and to understand ecological and phylogenetic aspects of the BALOs isolated from shrimp mariculture systems.
Methods and Results:  Using double-layer agar technique, the numbers of culturable BALOs that lyse Vibrio alginolyticus were found to be 10–10³ PFU ml⁻¹ in the surface water samples. A total of 130 BALOs isolates were differentiated into five phylotypes by denaturing gradient gel electrophoresis targeting the 16S rDNA V3 region and four phylotypes by amplified rDNA restriction analysis of the Bacteriovoracaceae -specific 16S rDNA fragment respectively. Phylogenetic analysis of representative isolates showed that all of them were identified as Bacteriovorax spp., but affiliated with four different clusters in the family Bacteriovoracaceae.
Conclusions:  The two PCR-based methods both can be chosen to rapidly type the saltwater BALOs at cluster level. And the relatively large numbers of BALOs with various phylotypes recovered from the same habitats suggested that these predators might play important ecological role in shrimp mariculture environments.
Significance and Impact of the Study:  We proposed two effective methods to distinguish rapidly large numbers of BALOs isolates and our results would be helpful to understand the diversity and function of BALOs in mariculture environments.     

Evolution of mitochondria

Until recently, the origin and evolution of mitochondria was explained by the serial endosymbiosis hypothesis. This hypothesis posits that contemporary mitochondria are the direct descendants of a bacterial endosymbiont, which was settled in a nucleus-containing amitochondriate host cell. Results of the mitochondrial gene sequences support a monophyletic origin of the mitochondria from a single eubacterial ancestor shared with a subdivision of the alpha-proteobacteria. In recent years, the complete sequences of the vast variety of mitochondrial and eubacterial genomes were determined. These data indicate that the mitochondrial genome evolved from a common ancestor of all extant eukaryotes and assume a possibility that the mitochondrial and nuclear constituents of the eukaryotic cell originated simultaneously.     

Members of the Cytophaga–Flavobacterium–Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host ( Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga – Flavobacterium – Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as ' Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of ' Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.     

两种培养基对对虾苗池海洋蛭弧菌的分离及其多样性分析

【目的】明确海水(Sw)和聚蛋白胨20(Pp20)两种双层琼脂培养基对海洋蛭弧菌的分离计数效果,了解对虾苗池可培养海洋蛭弧菌多样性。【方法】采用双层平板法,比较Sw和Pp20培养基对2株海洋蛭弧菌和对虾苗池未知海洋蛭弧菌的计数效果。通过宿主范围测试和16S rRNA基因序列分析评估两种培养基分离苗池海洋蛭弧菌的多样性。【结果】宿主菌含量高时,Sw培养基对两株已知海洋蛭弧菌的计数值均显著高于(P0.05)Pp20。Sw和Pp20培养基从同一苗池水样分别分离得到21和22株蛭弧菌。根据宿主裂解范围差异,43株分离物可分为15种裂解模式,其中Sw和Pp20培养基各分离到12和8种。16S rRNA基因序列分析表明,所有分离物都被鉴定为噬菌弧菌属(Bacteriovorax)菌株,并可分为6个类群,Sw和Pp20培养基分别分离到6和4个类群。【结论】Sw培养基在分离计数海洋蛭弧菌及其多样性检测上效果均优于Pp20;对虾苗池可培养海洋蛭弧菌具有较高多样性,并以类群XIII、X及一个潜在新类群为优势种群。     

COMMON EVOLUTIONARY ORIGIN OF STARCH BIOSYNTHETIC ENZYMES IN GREEN AND RED ALGAE1

Plastidic starch synthesis in green algae and plants occurs via ADP‐glucose in likeness to prokaryotes from which plastids have evolved. In contrast, floridean starch synthesis in red algae proceeds via uridine diphosphate‐glucose in semblance to eukaryotic glycogen synthesis and occurs in the cytosol rather than the plastid. Given the monophyletic origin of all plastids, we investigated the origin of the enzymes of the plastid and cytosolic starch synthetic pathways to determine whether their location reflects their origin—either from the cyanobacterial endosymbiont or from the eukaryotic host. We report that, despite the compartmentalization of starch synthesis differing in green and red lineages, all but one of the enzymes of the synthetic pathways shares a common origin. Overall, the pathway of starch synthesis in both lineages represents a chimera of the host and endosymbiont glycogen synthesis pathways. Moreover, host‐derived proteins function in the plastid in green algae, whereas endosymbiont‐derived proteins function in the cytosol in red algae. This complexity demonstrates the impacts of integrating pathways of host with those of both primary and secondary endosymbionts during plastid evolution.     

Predation between prokaryotes and the origin of eukaryotes

Accumulating data suggest that the eukaryotic cell originated from a merger of two prokaryotes, an archaeal host and a bacterial endosymbiont. However, since prokaryotes are unable to perform phagocytosis, the means by which the endosymbiont entered its host is an enigma. We suggest that a predatory or parasitic interaction between prokaryotes provides a reasonable explanation for this conundrum. According to the model presented here, the host in this interaction was an anaerobic archaeon with a periplasm‐like space. The predator was a small (facultative) aerobic α‐proteobacterium, which penetrated and replicated within the host periplasm, and later became the mitochondria. Plausible conditions under which this interaction took place and circumstances that may have led to the contemporary complex eukaryotic cell are discussed.     

Endosymbiotic Bacteroidales bacteria of the flagellated protist Pseudotrichonympha grassii in the gut of the termite Coptotermes formosanus

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.     

Origins of hydrogenosomes and mitochondria

Complete genome sequences for many oxygen-respiring mitochondria, as well as for some bacteria, leave no doubt that mitochondria are descendants of alpha-proteobacteria, a finding for which the endosymbiont hypothesis can easily account. Yet a wealth of data indicate that mitochondria and hydrogenosomes - the ATP-producing organelles of many anaerobic protists - share a common ancestry, a finding that traditional formulations of the endosymbiont hypothesis less readily accommodates. Available evidence suggests that a more in-depth understanding of the origins of eukaryotes and their organelles will hinge upon data from the genomes of protists that synthesize ATP without the need for oxygen.     

Functional annotation of class I lysyl-tRNA synthetase phylogeny indicates a limited role for gene transfer

Functional and comparative genomic studies have previously shown that the essential protein lysyl-tRNA synthetase (LysRS) exists in two unrelated forms. Most prokaryotes and all eukaryotes contain a class II LysRS, whereas most archaea and a few bacteria contain a less common class I LysRS. In bacteria the class I LysRS is only found in the alpha-proteobacteria and a scattering of other groups, including the spirochetes, while the class I protein is by far the most common form of LysRS in archaea. To investigate this unusual distribution we functionally annotated a representative phylogenetic sampling of LysRS proteins. Class I LysRS proteins from a variety of bacteria and archaea were characterized in vitro by their ability to recognize Escherichia coli tRNA(Lys) anticodon mutants. Class I LysRS proteins were found to fall into two distinct groups, those that preferentially recognize the third anticodon nucleotide of tRNA(Lys) (U36) and those that recognize both the second and third positions (U35 and U36). Strong recognition of U35 and U36 was confined to the pyrococcus-spirochete grouping within the archaeal branch of the class I LysRS phylogenetic tree, while U36 recognition was seen in other archaea and an example from the alpha-proteobacteria. Together with the corresponding phylogenetic relationships, these results suggest that despite its comparative rarity the distribution of class I LysRS conforms to the canonical archaeal-bacterial division. The only exception, suggested from both functional and phylogenetic data, appears to be the horizontal transfer of class I LysRS from a pyrococcal progenitor to a limited number of bacteria.     

Bacterial predation under changing viscosities

Bdellovibrio and like organisms (BALOs) are largely distributed in soils and in water bodies obligate predators of gram-negative bacteria that can affect bacterial communities. Potential applications of BALOs include biomass reduction, their use against pathogenic bacteria in agriculture, and in medicine as an alternative against antibiotic-resistant pathogens. Such different environments and uses mean that BALOs should be active under a range of viscosities. In this study, the predatory behaviour of two strains of the periplasmic predator B. bacteriovorus and of the epibiotic predator Micavibrio aeruginosavorus was examined in viscous polyvinylpyrrolidone (PVP) solutions at 28 and at 37°C, using fluorescent markers and plate counts to track predator growth and prey decay. We found that at high viscosities, although swimming speed was largely decreased, the three predators reduced prey to levels similar to those of non-viscous suspensions, albeit with short delays. Prey motility and clumping did not affect the outcome. Strikingly, under low initial predator concentrations, predation dynamics were faster with increasing viscosity, an effect that dissipated with increasing predator concentrations. Changes in swimming patterns and in futile predator–predator encounters with viscosity, as revealed by path analysis under changing viscosities, along with possible PVP-mediated crowding effects, may explain the observed phenomena.