Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibrio spp. Isolated on Phytopathogenic Bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ PFU g⁻¹. A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

Three-Membered Parasitic System: a Bacteriophage, Bdellovibrio bacteriovorus, and Escherichia coli

Two bacteriophages for Bdellovibrio bacteriovorus were isolated. One of the phages (VL-1) was isolated on a host-independent Bdellovibrio strain, and the other (VL-2) was isolated on a host-dependent strain. Both phages grew on host-dependent as well as on host-independent Bdellovibrio strains. The development of the phages in host-dependent bdellovibrios occurred only when the phage-infected bdellovibrios parasitized cells of other bacteria. In the absence of other bacteria, the phages adsorbed to the bdellovibrios and killed them and in the process lost their own plaque-forming ability.     

Design and uses of Bdellovibrio 16S rRNA-targeted oligonucleotides

An 18-mer oligonucleotide almost exclusively targeting Bdellovibrio spp. was designed based on available 16S rRNA sequence data. The specificity of this oligonucleotide used as a PCR primer in combination with a Bacteria domain-targeted primer as well as used as a probe in rRNA dot blot hybridizations was experimentally confirmed using a variety of alpha-, beta-, gamma-, delta-Proteobacteria and Gram-positive bacteria. Similarly, combinations of the Bacteria primer with oligonucleotides targeting the 16S rRNA gene of Bdellovibrio bacteriovorus and Bdellovibrio stolpii were shown to be species specific by PCR. Positive amplification products were obtained from an irrigation water sample in which a low level of bdellovibrios was detected by plating as well as from soil detached from potato tubers, using the Bdellovibrio spp.-Bacteria and the B. bacteriovorus-Bacteria primer pairs.     

Verification of the protein in the outer membrane of Bdellovibrio bacteriovorus as the OmpF protein of its Escherichia coli prey.

Two research groups showed that several Bdellovibrio strains incorporated into their outer membranes intact OmpF porin proteins derived from their Escherichia coli prey. These results could not be reproduced by another group using Bdellovibrio bacteriovorus 109J. They showed that a major protein appearing in the Bdellovibrio Triton X-100-insoluble outer membrane was coded for by the bdellovibrios. We reconciled these results by examining the strain used by this group and by reviving a freeze-dried culture of strain 109J which had been stored for almost 9 years. B. bacteriovorus 109J failed to acquire substantial amounts of the OmpF protein from E. coli ML35, and a protein coded for by the bdellovibrios was expressed in its place. However, B. bacteriovorus 109J incorporated the OmpF protein from rough K-12 strains of E. coli, and the revived 9-year-old culture of B. bacteriovorus 109J incorporated more of the OmpF protein from the smooth E. coli ML35 than did its contemporary counterpart. The protein isolated from the outer membrane of the bdellovibrios was identified as the OmpF protein of E. coli by its protease peptide profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot analysis. This confirmed that bdellovibrios relocalize outer membrane proteins from their prey, but relocalization may be an unstable trait which can be influenced by the prey.     

Chemotaxis by Bdellovibrio bacteriovorus toward prey.

A chemotaxis assay system that uses a modified Boyden chamber was characterized and used for measurements of chemotaxis by Bdellovibrio bacteriovorus strain UKi2 toward several bacterial species. Bacteria tested included both susceptible and nonsusceptible cells (Escherichia coli, Pseudomonas fluorescens, Bacillus megaterium, and B. bacteriovorus strains UKi2 and D). None was attractive to bdellovibrios when present at densities below 10(7) cells per ml. Chemotaxis toward E. coli was studied most extensively; under conditions that minimized effects of osmotic shock to the cells, E. coli and exudates from E. coli at densities as high as 10(8) cells per ml failed to elicit a chemotactic response. Cell-free filtrates from mixed cultures of bdellovibrios and E. coli neither attracted nor repelled bdellovibrios. The data indicate that bdellovibrios do not use chemotaxis to locate prey cells.     

Acquisition of apparently intact and unmodified lipopolysaccharides from Escherichia coli by Bdellovibrio bacteriovorus.

The ability of Bdellovibrio bacteriovorus to relocalize the OmpF major outer membrane porins from its Escherichia coli prey to its own outer membranes is diminished in prey expressing smooth lipopolysaccharide (S-LPS). Since porins exist in the membrane complexed with LPS, we examined the LPS associated with relocalized porin to determine whether it had been acquired intact, mixed or replaced with Bdellovibrio LPS, or derivatized by the bdellovibrios. The relocalized trimers were found associated with the same LPS originally bound to them in the E. coli. The bulk-phase LPS from bdellovibrios grown on various chemotypes of rough prey was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine whether more than the trimer-bound LPS was acquired by the bdellovibrios. This analysis revealed bands of Bdellovibrio LPS matching the LPS chemotype of the prey. One or two other bands were identical in migration to the LPS of prey-independent mutants of B. bacteriovorus and represented bdellovibrio-synthesized LPS. The LPS of bdellovibrios grown on prey with radiolabeled lipid A showed radioactivity only in gel band positions identical with those of the prey's LPS. The amount of this prey-derived LPS was shown by enzyme-linked immunosorbent assay to reach a constant value during the purification of the bdellovibrios, and it represented approximately 25% of the total Bdellovibrio LPS. Immunoelectron microscopy confirmed the presence of prey-derived LPS on the cell surface of bdellovibrios, and no evidence could be found for bdellovibrio-induced modifications of the relocalized prey LPS.     

Molecular Heterogeneity of the Bdellovibrios: Evidence of Two New Species

A systematic examination of a variety of isolates of the bacterial endoparasite Bdellovibrio has revealed extensive molecular diversity. The quantity of deoxyribonucleic acid (DNA) polynucleotide homology ranges from more than 90% among the isolates with DNA containing 50 to 51% guanine plus cytosine (GC) to undetectable levels between the 43% GC and 51% GC isolates. The two isolates with low GC-containing DNA (H-I Bdellovibrio A3.12 and UKi2) have only 16% DNA homology. H-I Bdellovibrio A3.12 and 109 have barely detectable ribosomal ribonucleic acid (rRNA) homology, whereas the homology approaches 100% among all the high GC isolates tested. Cases of high DNA/DNA and DNA/rRNA homologies are reflected in low dissimilarities of enzyme migration patterns in starch gel electrophoresis. The dissimilarities exhibited among the high GC Bdellovibrio isolates are as low as those previously reported for different Escherichia coli strains. The zymograms of H-I Bdellovibrio A3.12 and UKi2 are completely different from each other as well as from all other bdellovibrios (100% dissimilarity). Genome sizes determined for the representative isolates demonstrate three size ranges which coincide with group differences based on the above measurements. Enzyme assays reveal that all isolates possess a tricarboxylic acid cycle and most contain an alanine and glutamic dehydrogenase. We conclude that the use of bacterial endoparasitism as a defining trait has resulted in a molecularly diverse collection of isolates. It is recommended that the specific epitaph bacteriovorus be used only for the type specimen (Bdellovibrio 100 of Stolp and Starr, 1963) and for other related 50 to 51% GC isolates. The heterogeneity of the group warrants two new species. We designate Bdellovibrio A3.12 as the nomenclatural type of B. starrii sp. n. and Bdellovibrio UKi2 as the nomenclatural type of B. stolpii sp. n.     

Taxonomic studies of predatory bdellovibrios based on 16S rRNA analysis, ribotyping and the hit locus and characterization of isolates from the gut of animals

The aim of our study was to obtain data for the molecular characterization of bdellovibrio bacteria, which were recently split into the genus Bdellovibrio and the newly designated genus Bacteriovorax. We determined the 16S rDNA sequences of five reference strains and performed a phylogenetic analysis including published 16S rRNA sequences of bdellovibrios. A comparison of the secondary structure showed significant differences in two regions of the 16S rRNAs of the species Bdellovibrio bacteriovorus, Bacteriovorax starrii, and Bacteriovorax stolpii. In addition, ribotyping techniques gave specific hybridization patterns and revealed that two rRNA operons are present in the investigated strains. A hybridization probe derived from the genetic locus hit, associated with the host independent (HI) phenotype of B. bacteriovorus, was found to be specific for this species. Sequence comparison of the hit locus revealed few base pair changes between host independent (HI) and host dependent (HD) strains. Ribotyping and hybridization experiments using the hit probe were applied to characterize bdellovibrio strains isolated from the gut of animals and humans and one isolate from sewage.     

Serogrouping of Halophilic Bdellovibrios from Chesapeake Bay and Environs by Immunodiffusion and Immunoelectrophoresis

Little has been reported on the serological relationship of halophilic bdellovibrios (Bd). Immunodiffusion analysis performed with rabbit or mouse Bd antisera developed against eight halophilic Bd isolates and one terrestrial Bd isolate, when reacted with soluble antigen preparations of 45 isolates of halophilic Bd, allowed separation into seven serogroups, which were distinct from the terrestrial isolate. Soluble antigen preparations of prey bacteria, Vibrio parahaemolyticus P-5 (P-5) and Escherichia coli ML 35 (ML 35), exhibited no reactivity with the antisera by immunodiffusion. Immunoelectrophoresis revealed the presence of three distinct antigens in homologous reactions and one shared antigen in heterologous Bd reactions. Shared antigens were noted between halophilic and terrestrial Bd, in addition to between halophilic Bd strains, indicating the possible existence of an antigen(s) which may be shared among all Bd. Again, no shared antigen was noted when P-5 or ML 35 was allowed by immunoelectrophoresis to react with the antisera. Prey susceptibility testing of the seven distinct groups of halophilic Bd, using 20 test prey, produced essentially identical spectra for each group, indicating that this was not a useful technique in delineating the Bd. While immunoelectrophoresis was able to demonstrate an antigen common to all Bd tested, immunodiffusion was able to delineate strains on the basis of a “serogroup specific” antigen. This suggests that immunological tools may serve as important means to study the taxonomy of halophilic Bd, as well as in the formation of a clearer taxonomic picture of the genus Bdellovibrio.     

A new model for the penetration of prey cells by bdellovibrios.

Bdellovibrio bacteriovorus 109J and most other bdellovibrios cause prey cells to round following penetration. Bdellovibrio sp. strain W does not cause rounding of the prey. Analysis of enzyme activities during the early stages of bdellovibrio attack indicated that strain W differs from most other bdellovibrios in that there is no glycanase activity produced during penetration. Likewise, heat-killed prey were penetrated normally by strain 109J, but the resulting bdelloplast did not become round and no glycanase was detected, indicating that glycanase is not essential for penetration. Peptidoglycan from prey cells penetrated by strain W was sensitive to lysozyme, but these cells were not susceptible to attack and penetration by strain 109J, indicating that peptidoglycan deacetylation is not the primary exclusion mechanism. We propose a model in which it is the peptidase activity of the bdellovibrios which allows them to breach the peptidoglycan of their prey and in which the glycanase activity exhibited by strain 109J and other bdellovibrios is responsible for the rounding of the bdelloplast.     

Acquisition of Escherichia coli outer membrane proteins by Bdellovibrio sp. strain 109D

The ability of Bdellovibrio sp. to acquire the OmpF major outer membrane protein from its Escherichia coli prey was examined to determine if there were other outer membrane proteins which could or could not be acquired. Growth of bdellovibrios on mutant prey which were defective in the expression of outer membrane proteins revealed that Bdellovibrio sp. could acquire the OmpC protein in the absence of the OmpF protein. However, the OmpA, LamB, and protein 2 proteins could not be found in the Bdellovibrio Triton-insoluble outer membrane. The disappearance of the OmpF and OmpC proteins from the bdelloplast surface was measured, and it was determined that Bdellovibrio sp. exhibited a kinetic and temporal preference for the OmpF protein. Bdellovibrios could be grown on porin-deficient prey, and the progeny bdellovibrios possessed outer membranes with a protein mass deficiency.     

Characterization of Rhizobium etli and other Rhizobium spp. that nodulate Phaseolus vulgaris L. in an Austrian soil

Phaseolus vulgaris L. (common bean) is nodulated by rhizobia present in the fields around the Seibersdorf laboratory despite the fact that common bean has not been grown for a long time. Using PCR analysis with repetitive primers, plasmid profiles, nifH profiles, PCR-RFLP analysis of the 16S rRNA gene and of the 16S rRNA-23S rRNA intergenic spacer and the nodulation phenotype, two well-differentiating groups could be distinguished. One group showed high similarity to Rhizobium sp. R602sp, isolated from common bean in France, while the other showed the same characteristics as R. etli . We detected little variation in the symbiotic regions but found higher diversity when using approaches targeting the whole genome. Many isolates obtained in this study might have diverged from a limited number of strains, therefore the Austrian isolates showed high saprophytic and nodulation competence in that particular soil.     

Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains

Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and labeled transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions.     

Role of dissimilatory fermentative iron-reducing bacteria in Fe uptake by common bean (Phaseolus vulgaris L.) plants grown in alkaline soil

Iron (Fe) is an essential element for plant growth and development. Some plant growth-promoting rhizobacteria can increase Fe uptake by plants through reduction of Fe(III) to Fe(II) at the root surface. The aim of this work was to identify novel bacterial strains with high Fe(III) reduction ability and to evaluate their role in plant Fe uptake. Four bacterial strains (UMCV1 to UMCV4) showing dissimilatory Fe-reducing activity were isolated from the rhizosphere of bean and maize plants and further identified by 16S rDNA amplification and sequence analysis. From these analyses, UMCV1 and UMCV2 isolates were identified as Bacillus megaterium and Arthrobacter spp., respectively, whereas UMCV3 and UMCV4 were identified as Stenotrophomonas maltophilia. All four isolates showed Fe reduction in a nonflooded soil and when associated with roots of bean plants grown in alkaline soil or in mineral medium. In addition, the bacterial isolates were able to stimulate plant growth in vitro and on a broad level, plants grown in inoculated soil were generally bigger and with higher Fe content than those grown in sterilized soil. These results indicate that bacterial species isolated from the rhizosphere of bean and maize plants contribute significantly to Fe uptake by plants likely through increased Fe(III) reduction in the rhizosphere.     

Peculiarities of the fatty acid composition ofBdellovibrio

The fatty acid composition of twelve Bdellovibrio strains isolated upon the growth on bacteria of various taxonomic groups was studied. A dependence of the lipid composition of bdellovibrios on that of bacteria they were parasitizing on was shown. Data pointing to the selective incorporation of fatty acids of host bacteria by bdellovibrios were obtained. Bdellovibrio membranes were shown to contain monounsatured fatty acids with different positions of double bonds indicating that there are at least two alternative mechanisms of synthesis of these acids in the parasites.     

Isolation and Characterization of Host-Independent Bdellovibrios

A reliable method has been developed for the isolation of host-independent (H-I; i.e., "saprophytic") strains of Bdellovibrio from host-dependent (H-D; i.e., "parasitic") cultures. The technique involves growing streptomycin-resistant (Sm(r)) H-D cultures on streptomycin-susceptible (Sm(8)) host cells. A lysate containing large numbers of the Sm(r) H-D cells and some remaining Sm(8) host cells is transferred to a selection medium which contains the antibiotic. The Sm(8) host cells in the lysate are killed, and the Sm(r) H-I strains develop in broth within 3 to 6 days. By use of this method, it has been possible to isolate H-I strains from 16 different H-D Bdellovibrio strains studied. The frequency of occurrence of host independence is in the range of one H-I colony per 10(6) to 10(7) plaque-forming units of H-D bdellovibrios. The H-I cultures are nonfermentative, do not reduce nitrate, are strongly proteolytic, are oxidase-positive, and do not utilize 14 different carbon compounds as sources of energy for growth. Most H-I cultures are catalase-positive upon initial isolation from H-D lysates, but some cultures lose this enzyme upon subsequent transfers through host-free media. Most H-I bdellovibrios are pleomorphic, consisting of vibrio- to spiral-shaped cells typically measuring 0.3 to 0.4 mum in width and 1 to 10 mum in length. All H-I bdellovibrios have a cytochrome a and c component (H-I A3.12 differs from the other strains in the location of the peaks of the cytochrome spectrum). All are sensitive to oxytetracycline and (except for strain H-I A3.12) to the vibriostatic pteridine 0/129; most bdellovibrios, except for H-I A3.12, are generally uniformly resistant or susceptible to a given antibiotic. Bdellovibrio and Vibrio spp. have common cytochrome difference spectra and susceptibilities to oxytetracycline and to the vibriostatic pteridine 0/129. All H-I bdellovibrios examined produce an exocellular protease which digests heat-killed host cells. Bdellovibrios possessing predatory and bacteriolytic properties could be reselected from H-I bdellovibrio cultures growing in the presence of living host cells. Attempts to select for bacteriolytic isolates from Vibrio and Spirillum spp. were unsuccessul.     

热带药用植物根际放线菌的分离、鉴定及生物活性分析

从海南热带植物园采集12种药用植物的根际土样,采用选择性分离方法,分离得到400株根际放线菌。使用5种活性筛选模型对分离菌株进行生物活性评价,154株放线菌在一个或多个活性筛选模型中显示为阳性,菌株初筛阳性率达38.5%;根据菌株形态特征并结合代谢产物的生物活性,从中挑选出28株菌进行16S rRNA基因序列分析,发现其分属于链霉菌属、诺卡氏菌属、小单孢菌属和野野村菌属。     

Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum.

The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.     

Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum.

The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.     

Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms.

Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 x 10(8) cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4', 6-diamidino-2-phenylindole staining, was 4.8 x 10(8) cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050-5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.