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.     

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.     

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.     

Symbiosis-independent and symbiosis-incompetent mutants of Bdellovibrio bacteriovorus 109J.

Symbiosis-independent (Sin) mutants were isolated from the symbiosis-dependent and symbiosis-competent (Sdcomp+) Bdellovibrio bacteriovorus 109J. Independently isolated Sin mutants were examined for their symbiosis competence and most were found to be comp+. Bdellovibrios comp- were selected from the Sincomp+ mutants. The Sincomp+ bdellovibrios are always at a selective disadvantage, either against Sincomp- bdellovibrios (in organic medium) or against Sdcomp+ bdellovibrios (in buffer with Escherichia coli cells).     

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.     

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.     

A Comparative Study of the Effect of Certain Pollutants on Free-living and Immobilized Bdellovibrio

The paper deals with a comparative study of the growth of free-living and immobilized predatory bacteria of the genus Bdellovibrio in the presence of toxic concentrations of urea and phenol. It was found that the cell wall of bdelloplasts plays a protective role in the adaptation of bdellovibrios to xenobiotics. The attachment of bdellovibrios to solid surfaces allows them to survive under unfavorable environmental conditions.     

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.     

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.     

Bdellovibrio-like parasite of cyanobacteria symbiotic in marine sponges

A bdellovibrio-like bacterium was observed infecting unicellular symbiotic cyanobacteria in two coral reef sponges, Neofibularia irata and Jaspis stellifera. The infecting bacterium, which was located between the cell wall and the cytoplasmic membrane of the cyanobacteria, was similar in size and appearance to previously described bdellovibrios. This observation is believed to extend the host range of the bdellovibrios.     

Bdellovibrios in Callinectus sapidus, the Blue Crab

Bdellovibrios were recovered from the gill tissue of all of 31 crabs sampled and from all samples of epibiota obtained from the ventral shell surface of 15 crabs. The results suggest that the blue crab is a reservoir for bdellovibrios. The association with crabs may be an important factor in the ecology of the bdellovibrios.     

Recovery of bdellovibrios from submerged surfaces and other aquatic habitats

The distribution of bdellovibrios was investigated over a wide geographical area of the Chesapeake Bay including some tributaries and subestuaries. Bdellovibrios were recovered from five aquatic habitats; water, sediment, oyster shell surface biofilm, zooplankton, and plants over a wide range of temperature and salinity measurements. Consistently, the greatest number of the predators was recovered from samples of biofilm irrespective of temperature and salinity. A decrease in the numbers and frequency of predators recovered from all habitats was observed at temperatures below 10°C. Only the shell surface biofilm samples yielded bdellovibrios 100% of the time. The organisms were recovered from 79% of water samples and 44% of sediment samples. The results reveal that bdellovibrios are surface-associated organisms and that this association appears to provide some protection for the predators at low temperatures.     

A comparison of the survival of intraperiplasmic and attack phase bdellovibrios with reduced oxygen

The ability of intraperiplasmic and attack phase bdellovibrios to survive and/or grow under anoxic and microaerobic conditions was examined. Both halotolerant and nonhalotolerant bdellovibrio strains were examined. In all instances, the bdellovibrio strains were unable to grow under anoxic conditions, but were able to survive for periods of time in both the extracellular and intraperiplasmic forms. However, the intraperiplasmic organisms were observed to survive longer. Increased temperature hastened the loss of viability of both forms of the predatory bacteria in oxic and anoxic environments. Under microaerobic conditions, halotolerant bdellovibrios were observed to grow, although at a slightly reduced rate than in atmospheric oxygen, while two nonhalotolerant isolates survived but did not grow. The ability of attack phase bdellovibrios to survive in an anoxic environment for up to nine days and their growth or survival under microaerobic conditions greatly expands the possible ecological niches in which the predators may be active members of the microbial community. Correspondence to: A.J. Schoeffield.     

Differentiation after premature release of intraperiplasmically growing Bdellovibrio bacteriovorous.

Bdellovibrio bacteriovorous attacks and penetrates other gram-negative bacteria, creating a growth chamber termed a bdelloplast. We have found that exposing the bdelloplasts to EDTA, followed by treatment with a lytic enzyme concentrate derived from bdellovirio cultures, prematurely released the intraperiplasmically growing bdellovibrios at any time during their growth cycle. Upon release, the growth-form bdellovibrios terminated any initiated rounds of DNA synthesis and differentiated into motile attack-form cells. The ability of growth-form cells to synthesize DNA appears to depend upon an initiation signal that is not received until about 60 min after attack. Each subsequent round of DNA synthesis by the growing bdellovibrio filaments seems to require an additional initiation signal that is provided by their intraperiplasmic environment. Differentiation included fragmentation into multiple progeny cells to a degree proportional to the extent of intraperiplasmic growth. This differentiation could be performed totally at the expense of cellular reserves. The significance of these data to an understanding of the regulation of differentiation in bdellovibrios is discussed.     

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.     

Evaluation of the effect of ecologically hazardous pollutants on the bacteriolytic activity of the predatory bacterium Bdellovibrio

We studied the effect of various concentrations of ecologically hazardous pollutants, urea, phenol, diuron, and cadmium ions, on the physiological activity and survival of the parasitic bacterium Bdellovibrio. Experiments showed that the survival of bdellovibrios in the presence of the pollutants was two times higher when they were cultivated on agar than when they were cultivated in liquid medium. The data obtained are in agreement with the recent concept of the surface-associated state as a survival strategy of bdellovibrios in various ecosystems.     

Inhibition of the predatory activity ofBdellovibrio by various environmental pollutants

The predatory activity of bdellovibrios is affected by various environmental pollutants such as detergents, heavy metals, and pesticides. This was shown in a two-membered system ofBdellovibrio andPhotobacterium, in which the effect of the predator on the bioluminescence of the prey indicated the activity of the former. The high sensitivity of the bdellovibrios toward certain chemicals (e.g., CdCl₂) indicates the possibility of using the system for biological monitoring of those chemicals.     

Attempts to grow bdellovibrios micurgically-injected into animal cells

Incubation in buffer of Bdellovibrio bacteriovorus 109J, B. stolpii UKi2, or B. starrii A3.12 with washed eucaryotic animal cells (mouse liver, hamster kidney, or bovine mammary gland) resulted in neither attachment nor growth of the bdellovibrios. When cells of these bdellovibrio strains were incubated with erythrocyte suspensions (bovine or rabbit) a very low level of bdellovibrio attachment and penetration occurred, but no growth could be detected. Using micurgical procedures, bdellovibrios were injected into the perivetelline space or the cytoplasm of rabbit ova. After 18–24h incubation, neither a significant loss nor increase of injected, intracellular bdellovibrios was observed. Limited axenic growth of bdellovibrios (109J or UKi2) occurred in media containing rabbit ova extracts and dilute nutrient broth. It is concluded that eucaryotic rabbit ova do not provide a suitable environment for intracellular bdellovibrio growth.     

Evaluation of the effect of ecologically hazardous pollutants on the bacteriolytic activity of the predatory bacteriumBdellovibrio

We studied the effect of various concentrations of ecologically hazardous pollutants, urea, phenol, diuron, and cadmium ions, on the physiological activity and survival of the parasitic bacteriumBdellovibrio. Experiments showed that the survival of bdellovibrios in the presence of the pollutants was two times higher when they were cultivated on agar than when they were cultivated in liquid medium. The data obtained are in agreement with the recent concept of the surface-associated state as a survival strategy of bdellovibrios in various ecosystems.     

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.