Isolation of Host-Dependent and Nonparasitic Mutants of the Facultative Parasitic Bdellovibrio UKi2

Obligate host-dependent and nonparasitic mutants were isolated from a facultative parasitic Bdellovibrio strain. Thus it is possible to separate host-dependency from the ability to parasitize in bdellovibrios.     

Identification of a Bdellovibrio bacteriovorus genetic locus, hit, associated with the host-independent phenotype.

Bdellovibrios invade and grow within the periplasmic space of suitable gram-negative bacteria. Wild-type bdellovibrios are obligately dependent on host cells for growth, but spontaneous host-independent (H-I) mutants that grow axenically on standard rich culture media can be isolated. Such mutants generally retain the ability to grow intraperiplasmically, although the plaques that they produce on lawns of host cells are smaller and more turbid than those produced by wild-type bdellovibrios. Here, we identify the first genetic locus associated with the H-I phenotype: hit (host interaction). We show that three individual H-I mutants suffered mutations at the hit locus and that recombination of wild-type hit sequences into the genomes of the H-I mutants greatly enhanced their plaquing ability. DNA sequence analysis localized the hit mutation in each of the H-I mutants to a 135-bp region of the genome. Mutations at hit may not fully account for the H-I phenotype, however, as recombination of wild-type hit sequences into the genomes of the H-I mutants had little effect on the axenic-growth phenotype of the mutants. Possible explanations for this result and potential roles for the hit locus are discussed.     

Distribution of bdellovibrios in the water column of an estuary

The distribution of bdellovibrios in the water column of the Miles River has been studied. Water samples were collected every 4 h over a 24-h period from five depths in the water column. The samples were cultured for the recovery of bdellovibrios lytic against Vibrio parahaemolyticus. Environmental parameters, i.e., salinity, temperature, turbidity, and dissolved oxygen (DO) were measured for each sample. Bdellovibrios were observed to be uniformly distributed at all depths measured in the water columns. There were no significant differences between the number of bdellovibrios recovered at the various depths. There were significant differences between the number of bdellovibrios recovered at various sampling times. However, no basis for these significant differences could be established. No association was found between the number of bdellovibrios recovered and the environmental parameters measured. Of interest was the observation that the distribution of the aerobic bdellovibrios did not correlate with DO measurements. The results suggest that neither depth nor DO content influenced the recovery of bdellovibrios from the Miles River.     

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.     

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.     

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 x 10(2) to 6 x 10(3) and 2.8 x 10(2) to 2.3 x 10(4) 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.     

Antigenicity of bdellovibrios.

Antigenic relationships between 12 locally isolated bdellovibrios and 3 established reference strains (109D, 6-5-S, and UKi2) were investigated. Antigenicity of the strains was examined by use of the micro-complement fixation test, the serum and complement bactericidal test, and the immunodiffusion test. Antisera were prepared against one of the local strains (MS7) and against one of the established reference strains (UKi2). The complement fixation titers suggest a close relationship among all strains. Immunodiffusion tests produced lines of identity between the homologous strain MS7 and all other strains. It is suggested on the basis of these results that bdellovibrio may possess a common antigen.     

Dependence of Marine Bdellovibrios on Potassium, Calcium, and Magnesium Ions

Marine bdellovibrios show a specific requirement for K⁺, Ca²⁺, and Mg²⁺. Potassium is essential for high velocity and seems to be necessary for attachment of the free bdellovibrios. Calcium and magnesium are necessary for attachment and penetration. Magnesium also plays a role in maintaining the integrity of the bdelloplast. The adaptation of these bdellovibrios to the marine environment is manifested by their stringent cation requirements.     

Antigenicity of bdellovibrios.

Antigenic relationships between 12 locally isolated bdellovibrios and 3 established reference strains (109D, 6-5-S, and UKi2) were investigated. Antigenicity of the strains was examined by use of the micro-complement fixation test, the serum and complement bactericidal test, and the immunodiffusion test. Antisera were prepared against one of the local strains (MS7) and against one of the established reference strains (UKi2). The complement fixation titers suggest a close relationship among all strains. Immunodiffusion tests produced lines of identity between the homologous strain MS7 and all other strains. It is suggested on the basis of these results that bdellovibrio may possess a common antigen.     

Effects of Temperature, Salinity, and Substrate on the Colonization of Surfaces In Situ by Aquatic Bdellovibrios

Recent studies suggest that surfaces are a more conducive habitat than the water column for the proliferation of bdellovibrios in the aquatic environment. The effect of temperature and salinity on the colonization of bdellovibrios on oyster shell, glass, and polystyrene surfaces in situ was investigated over an annual cycle. Sterile surfaces were suspended in various bodies of water for intervals ranging from 24 to 120 h. The results revealed that bdellovibrios associated with different types of surfaces over a broad temperature and salinity range. After 24 h of submersion in waters with temperatures from 9.0 to 26.7(deg)C, the ranges in log(inf10) values per square centimeter for the three surfaces were as follows: oyster shell, 2.2 to 2.5; glass, 0.3 to 2.2; and polystyrene, 0.7 to 1.6. Bdellovibrios were not recovered from surfaces submerged in water at temperatures below 8(deg)C during the 120-h experimental cycle. The number of bdellovibrios and culturable bacteria on oyster shells was significantly higher than the numbers on glass and polystyrene at all time intervals. The number of bdellovibrios was positively correlated with temperature and salinity on all surfaces. A positive correlation between the number of recoverable bacteria and temperature was observed, but the results with respect to salinity were diverse. The numbers of bdellovibrios recovered from oyster shells (up to 48 h) and water samples were significantly increased at salinities greater than 11(permil) compared to those in lower-salinity environments. The results of this study reveal that like many other bacteria in the aquatic environment, bdellovibrios prefer to associate with surfaces. This association provides the predators a rich source of prey bacteria in surface biofilms and perhaps protection in the gel-like matrix of the biofilm.     

Facultatively Parasitic Strain of Bdellovibrio bacteriovorus

A strain of Bdellovibrio bacteriovorus (designated strain UKi2) was isolated which was capable of growing either saprophytically in host-free medium or endoparasitically in Escherichia coli B/r. It was quantitatively determined that each bdellovibrio could develop in solid medium to produce a colony, and 65% of the cells in a late exponential-phase culture were capable of inducing E. coli B/r spheroplasts. A photomicrographic sequence of single E. coli spheroplasts containing bdellovibrios demonstrated that parasitically derived B. bacteriovorus UKi2 could develop saprophytically after release from the host cells. Strain UKi2 appears to be morphologically quite similar to previously described obligately parasitic bdellovibrios; biochemical data on this strain suggests its close relationship to some of the previously described host-independent strains of Bdellovibrio.     

A study of the occurrence and distribution of bdellovibrios in estuarine sediment over an annual cycle

The recovery of bdellovibrios from estuarine sediments over an annual cycle was studied. Greater numbers of the predators were recovered in sediment than in the water column. Increases in the number of bdellovibrios recovered from sediment over various periods of time suggest that multiplication of the predators occurred. Sediment was observed to be an important ecosystem for the survival of bdellovibrios in the winter months. As has been observed in water, the number of bdellovibrios in sediment fluctuated, with seasonal and temperature changes declining to very low numbers during the winter months. In the colder months, low numbers of the predators appeared to winter-over in sediment, with greater numbers of the organisms being recovered from deeper sediment. As the water temperature warmed in the spring, increases in the number of bdellovibrios occurred first in sediment and subsequently in water. This increase of bdellovibrios in sediment may have resulted in the shedding of the organisms into the water column where their numbers subsequently increased. Population fluctuations of bdellovibrios were similar in both water and sediment. Although the temperature may account for much of the observed fluctuation in the number of bdellovibrios, other factors, including salinity and the number of host bacteria, may also play a major role. The number of bdellovibrios recovered from sediment correlated positively with the water temperature, and negatively with the water salinity and the number of bacterial colony-forming units from sediment. The results of this study revealed the significance of sediment to the seasonal cycle, survival, and growth of the bdellovibrios in an estuarine environment.     

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.     

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.     

Incidence of marine bdellovibrios lytic against Vibrio parahaemolyticus in Chesapeake Bay.

The incidence of marine bdellovibrios at selected sampling sites in the Chesapeake Bay during the months of June 1978 and 1979 was studied. Bdellovibrios were isolated from eight of nine sampling stations in the bay. Higher numbers than previously reported with sea or ocean water were recovered in the midregion of the bay.     

mTORC2 targets AGC kinases through Sin1-dependent recruitment

The protein kinase TOR (target of rapamycin) is a key regulator of cell growth and metabolism with significant clinical relevance. In mammals, TOR signals through two distinct multi-protein complexes, mTORC1 and mTORC2 (mammalian TOR complex 1 and 2 respectively), the subunits of which appear to define the operational pathways. Rapamycin selectively targets mTORC1 function, and the emergence of specific ATP-competitive kinase inhibitors has enabled assessment of dual mTORC1 and mTORC2 blockade. Little is known, however, of the molecular action of mTORC2 components or the relative importance of targeting this pathway. In the present study, we have identified the mTORC2 subunit Sin1 as a direct binding partner of the PKC (protein kinase C) ε kinase domain and map the interaction to the central highly conserved region of Sin1. Exploiting the conformational dependence for PKC phosphorylation, we demonstrate that mTORC2 is essential for acute priming of PKC. Inducible expression of Sin1 mutants, lacking the PKC-interaction domain, displaces endogenous Sin1 from mTORC2 and disrupts PKC phosphorylation. PKB (protein kinase B)/Akt phosphorylation is also suppressed by these Sin1 mutants, but not the mTORC1 substrate p70(S6K) (S6 kinase), providing evidence that Sin1 serves as a selectivity adaptor for the recruitment of mTORC2 targets. This inducible selective mTORC2 intervention is used to demonstrate a key role for mTORC2 in cell proliferation in three-dimensional culture.     

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.     

构巢曲霉胞质分裂SIN信号途径中sepH的反向调节子研究

在构巢曲霉Aspergillusnidulans的细胞隔膜开始网(septation initiation network,SIN)的信号途径中,sepH基因对胞质分裂起着关键的正调节作用,但对该途径中同样起着十分重要作用的有关反向调节子(suppressors)的研究目前还不清楚。本课题采用UV诱导点突变法成功筛选到sepH突变株的反向调节子116株,这些突变株共分为三类。通过对突变株进行杂交和回交等遗传学分析,排除了sepH回复突变菌株,获得了能使sepH胞质分裂恢复正常的温度敏感菌株Sin110,证实了SIN途径具有对应反向的旁路途径(bypass)的存在。