The Search for Hunters: Culture-Dependent and -Independent Methods for Analysis of Bdellovibrio and Like Organisms

Koval, Susan F.

doi:10.1007/7171_057

Cited by 6 publications

(14 citation statements)

References 37 publications

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“…In September 2015, we collected 50 g of soil from a bioswale on the Providence College campus (lat 41.84277, lon −71.43944). To isolate predatory bacteria from the soil sample, we adapted standard methods [33,34]. We combined the soil sample with 500 ml of sterile HM buffer (25 mM HEPES adjusted to pH 7.4 and supplemented with 3 mM calcium chloride dihydrate and 2 mM magnesium chloride hexahydrate).…”

Section: Isolation and Identification Of Bdellovibrio Sp Nc01 From Bmentioning

confidence: 99%

“…We obtained Bdellovibrio bacteriovorus HD100 from Mark Martin (University of Puget Sound, Puget Sound, WA, USA) for predatory phenotype comparisons. To conduct phenotype assays, we adapted standard methods [33,34,53]. To culture Bdellovibrio NC01 and HD100, we added a small amount of −80 °C stocks to 20 ml HM buffer combined with 1.5 ml of an overnight culture of E. coli ML35.…”

Section: Phenotype Assays To Test Prey Range Differences and Predatiomentioning

confidence: 99%

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Variation in genome content and predatory phenotypes between Bdellovibrio sp. NC01 isolated from soil and B. bacteriovorus type strain HD100

et al. 2019

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Defining phenotypic and associated genotypic variation among Bdellovibrio may further our understanding of how this genus attacks and kills different Gram-negative bacteria. We isolated Bdellovibrio sp. NC01 from soil. Analysis of 16S rRNA gene sequences and average amino acid identity showed that NC01 belongs to a different species than the type species bacteriovorus. By clustering amino acid sequences from completely sequenced Bdellovibrio and comparing the resulting orthologue groups to a previously published analysis, we defined a 'core genome' of 778 protein-coding genes and identified four proteincoding genes that appeared to be missing only in NC01. To determine how horizontal gene transfer (HGT) may have impacted NC01 genome evolution, we performed genome-wide comparisons of Bdellovibrio nucleotide sequences, which indicated that eight NC01 genomic regions were likely acquired by HGT. To investigate how genome variation may impact predation, we compared protein-coding gene content between NC01 and the B. bacteriovorus type strain HD100, focusing on genes implicated as important in successful killing of prey. Of these, NC01 is missing ten genes that may play roles in lytic activity during predation. Compared to HD100, NC01 kills fewer tested prey strains and kills Escherichia coli ML35 less efficiently. NC01 causes a smaller log reduction in ML35, after which the prey population recovers and the NC01 population decreases. In addition, NC01 forms turbid plaques on lawns of E. coli ML35, in contrast to clear plaques formed by HD100. Linking phenotypic variation in interactions between Bdellovibrio and Gram-negative bacteria with underlying Bdellovibrio genome variation is valuable for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in clinical applications.

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Section: Isolation and Identification Of Bdellovibrio Sp Nc01 From Bmentioning

confidence: 99%

Section: Phenotype Assays To Test Prey Range Differences and Predatiomentioning

confidence: 99%

Variation in genome content and predatory phenotypes between Bdellovibrio sp. NC01 isolated from soil and B. bacteriovorus type strain HD100

et al. 2019

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“…Previous work in our laboratory had elucidated an appropriate method for the enrichment of both raw sewage and activated sludge with BALOs (19,20). Therefore, these two environmental samples were selected for further analysis and were enriched using both A. serpens and E. coli as prey cells and dilute nutrient broth (0.08% nutrient broth [Difco] with 1 mM CaCl 2 and 0.1 mM MgSO 4 ) (7).…”

mentioning

confidence: 99%

“…These flow cytometry techniques may allow the detection of Bdellovibrio in environmental samples without the expense associated with peptide nucleic acid probes or multilaser flow cytometry or the technical demands of catalyzed reporter deposition FISH (24). If successful, these techniques would allow the identification and enumeration of Bdellovibrio cells without the prey bias associated with enrichment cultures (19). Despite the difficulties, the identification of Bdellovibrio in environmental samples, although not directly, has been made possible through the use of FISH.…”

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confidence: 99%

Design and Performance of a 16S rRNA-Targeted Oligonucleotide Probe for Detection of Members of the Genus Bdellovibrio by Fluorescence In Situ Hybridization

Mahmoud

McNeely

Elwood

et al. 2007

Appl Environ Microbiol

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A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment.Bdellovibrio and like organisms (BALOs) are predatory gram-negative bacteria that are ubiquitous in terrestrial and aquatic environments. Prey include free-living bacteria as well as plant, animal, and human pathogens (12,13,17). The life cycle of Bdellovibrio has two major stages: a motile, nonreplicative stage spent searching for prey (the attack phase) and a stage spent inside the periplasm of the gram-negative prey cell after the formation of an osmotically stable body termed the bdelloplast (the growth phase) (15,27). Until recently, all bacteria that exhibited these life cycle traits were included in the genus Bdellovibrio. Phylogenetic studies based on 16S rRNA sequences have demonstrated extensive diversity among the organisms in this genus, and two new genera have been established: Bacteriovorax (4) and Peredibacter (8). To date, the genus Bdellovibrio comprises only one species, Bdellovibrio bacteriovorus. However, a Bdellovibrio-like organism (BLO) with a different life cycle was isolated from sewage on lawns of Caulobacter crescentus cells. This epibiotic strain, JSS, does not enter the periplasmic space of the prey cell (20,26). Phylogenetic studies show that it belongs to the genus Bdellovibrio (8) and is a new species (S. F. Koval, unpublished data). Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has become a commonly used technique for the direct identification of prokaryotes and is widely used to determine bacterial community composition (2, 10). Previously designed BALO-targeted oligonucleotides for use in FISH were not specific enough for the identification of the genus Bdellovibrio, as the targets detected included non-BALOs (8, 16). However, Bdellovibrio-specific primers were used to detect Bdellovibrio sequences in soil (9). In this study, an oligonucleotide probe designated BDE525, targeting a region of the 16S rRNA gene sequence of Bdellovibrio, was designed. The probe was used in a FISH procedure with B. bacteriovorus strains, other BLO isolates, and environmental samples.Cultures. B. bacteriovorus strain 109J was cultured on Escherichia coli ML35 according to the method of Flannagan et al.(11). B. bacteriovorus 6-5-S was cultured on Aquaspirillum serpens VHL and BLO strains JSS and KL1 were cultured on C. crescentus CB2A as described previously by Koval and Hynes (20). BLO strains DM7C, DM8A, and DM11A were cultured on Burkholderia cenocepacia strains grown in Lennox LuriaBertani medium (Difco) for 24 h at 30°C. For the maintenance of predators, 3 ml of prey cells and 1 ml of predators were mixed in 20 ml of HM buffer (3 mM HEPES [Sigma], pH 7.6, with 1 mM CaCl 2 and 0.1 mM MgSO 4 ...

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“…So far, the double‐layer agar technique remains the most popular method used to isolate and enumerate BALOs. The selection of appropriate prey bacteria is a critical step for isolation of BALOs from environmental samples as recovery efficiency may be influenced (Koval 2007; Williams and Piñeiro 2007). In the case of the saltwater BALOs, V. parahaemolyticus or other Vibrio spp.…”

Section: Discussionmentioning

confidence: 99%

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

Wen

Lai

Xue

et al. 2009

Journal of Applied Microbiology

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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.

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The Search for Hunters: Culture-Dependent and -Independent Methods for Analysis of Bdellovibrio and Like Organisms

Cited by 6 publications

References 37 publications

Variation in genome content and predatory phenotypes between Bdellovibrio sp. NC01 isolated from soil and B. bacteriovorus type strain HD100

Variation in genome content and predatory phenotypes between Bdellovibrio sp. NC01 isolated from soil and B. bacteriovorus type strain HD100

Design and Performance of a 16S rRNA-Targeted Oligonucleotide Probe for Detection of Members of the Genus Bdellovibrio by Fluorescence In Situ Hybridization

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

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