Regulated breakdown of Escherichia coli deoxyribonucleic acid during intraperiplasmic growth of Bdellovibrio bacteriovorus 109J.

During growth of Bdellovibrio bacteriovorus on [2-14C]deoxythymidine-labeled Escherichia coli, approximately 30% of the radioactivity was released to the culture fluid as nucleoside monophosphates and free bases; the remainder was incorporated by the bdellovibrio. By 60 min after bdellovibrio attack, when only 10% of the E. coli deoxyribonucleic acid (DNA) had been solubilized, the substrate cell DNA was degraded to 5 X 10(5)-dalton fragments retained within the bdelloplast. Kinetic studies showed these fragments were formed as the result of sequential accumulation of single- and then double-strand cuts. DNA fragments between 2 X 10(3) and 5 X 10(5) daltons were never observed. Chloramphenicol, added at various times after initiation of bdellovibrio intraperiplasmic growth on normal or on heated E. coli, which have inactivated deoxyribonucleases, inhibited further breakdown and solubilization of substrate cell DNA. Analysis of these intraperiplasmic culture deoxyribonuclease activities showed that bdellovibrio deoxyribonucleases are synthesized while E. coli nucleases are inactivated. It is concluded that continuous and sequential synthesis of bdellovibrio deoxyribonucleases of apparently differing specificities is necessary for complete breakdown and solubilization of substrate cell DNA, and that substrate cell deoxyribonucleases are not involved in any significant way in the degradation process.     

Ribonucleic acid destruction and synthesis during intraperiplasmic growth of Bdellovibrio bacteriovorus.

During growth of Bdellovibrio bacteriovorus on (2-14C)uracil-labeled Escherichia coli approximately 50% of the radioactivity is incorporated by the bdellovibrio and most of the remainder is released as free nucleic acid bases. Kinetic studies showed that 50 and 30S ribosomal particles and 23 and 16S ribosomal ribonucleic acid (RNA) of E. coli are almost completely degraded by the first 90 min in a 210- to 240-min bdellovibrio developmental cycle. Synthesis of bdellovibrio ribosomal RNA was first detected after 90 min. The specific activity and the ratio of radioactivity in the bases of the synthesized bdellovibrio RNA was essentially the same as those of the substrate E. coli. The total radioactivity of the bdellovibrio deoxyribonucleic acid (DNA) exceeded that in the DNA of the substrate E. coli cell, and the ratio of radioactivity of cytosine to thymine residues differed. Intraperiplasmic growth of B. bacteriovorus in the presence of added nucleoside monophosphates (singly or in combination) significantly decreased the uptake of radioactivity from (2-14C)uracil-labeled E. coli; nucleosides or nucleic acid bases did not. It is concluded that the RNA of the substrate cell, in the form of nucleoside monophosphates, is the major or exclusive precursor of the bdellovirbrio RNA and also serves as a precursor for some of the bdellovibrio DNA.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: solubilization of Escherichia coli peptidoglycan.

During penetration of Bdellovibrio bacteriovorus into Escherchia coli, two enzymatic activities, a glycanase and a peptidase, rapidly solubilized some 10 to 15% of the E. coli peptidoglycan. The glycanase activity, which solubilizes peptidoglycan amino sugars, came to a sharp halt with completion of the penetration process. Peptidase activity, which cleaves diaminopimelic acid residues from the peptidoglycan, continued, but at a decreasing rate. By 90 min after bdellovibrio attack, some 30% of the initial E. coli diaminopimelic acid residues were solubilized and present in the culture fluid as free diaminopimelic acid. During bdellovibrio penetration some 25% of the lipopolysaccharide glucosamine was also solubilized by an as yet undefined enzymatic activity that yielded products having molecular weights below 2,000. The solubilization of E. coli lipopolysaccharide glucosamine also terminated at completion of bdellovibrio penetration. At the end of bdellovibrio growth, a second period of rapid solubilization of bdelloplast peptidoglycan began which resulted in lysis of the bdelloplast and complete solubilization of the peptidoglycan amino sugars and diaminopimelic acid. The final lytic enzyme(s) was synthesized just before the time of lysis.     

Change in the surface hydrophobicity of substrate cells during bdelloplast formation by Bdellovibrio bacteriovorus 109J.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J, the substrate cell surface becomes more hydrophobic. This was shown (i) by comparing the sensitivity to hydrophobic antibiotics of wild-type and lipopolysaccharide mutant strains of Salmonella typhimurium to that of the bdellovibrio growing on these strains and (ii) by measuring the binding efficiency of these strains, Escherichia coli, and their derived bdelloplasts to octyl Sepharose. The kinetics of increase in surface hydrophobicity was similar to the kinetics of the conversion of the substrate cell peptidoglycan to a lysozyme-resistant form (M. Thomashow and S. Rittenberg, J. Bacteriol. 135:1008-1014, 1978), and hydrophobicity reached a maximum at about 60 min in a synchronous culture. The change in hydrophobicity was inhibited by chloramphenicol, suggesting that bdellovibrio protein synthesis was required. Control experiments revealed that the free-swimming bdellovibrio had a more hydrophobic surface than the deep rough mutants of S. typhimurium.     

Energy efficiency of intraperiplasmic growth of Bdellovibrio bacteriovorus.

The Y-ATP (energy efficiency) of intraperiplasmic growth of Bdellovibrio bacteriovorus was determined from the distribution of radioactivity of the substrate organism ([U-14C]Escherichia coli) btween CO2 and bdellovibrio cells at the end of growth. A "best" Y-ATP value of 18.5 was obtained from single growth cycle experiments and an average value of 25.9 from multicycle experiments. Both values are much higher than the usual value of 10.5 for bacteria growing in rich media. The bases for the unusual energy efficiency for growth of B. bacteriovorus are discussed.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: N-deacetylation of Escherichia coli peptidoglycan amino sugars.

During intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli, the substrate cell peptidoglycan is extensively modified as it is converted to bdelloplast peptidoglycan. The initially lysozyme-sensitive peptidoglycan of E. coli was rapidly converted to a lysozyme-resistant form. The conversion was due to the N-deacetylation of a large portion of the peptidoglycan amino sugars. Chemically acetylating the isolated peptidoglycan restored its sensitivity to lysozyme digestion. However, approximately half of the products of lysozyme digestion exhibited hydrophobic interactions that were shown not to be due to the presence of protein. This suggests that a molecule capable of hydrophobic interactions, other than protein, becomes linked to the bdelloplast peptidoglycan. The data also suggest that much of the Braun lipoprotein is removed from the E. coli peptidoglycan early during bdellovibrio development.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: attachment of long-chain fatty acids to escherichia coli peptidoglycan.

During the initial stages of intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli, the peptidoglycan of the E. coli becomes acylated with long-chain fatty acids, primarily palmitic acid (60%) and oleic acid (20%). The attachment of the fatty acids to the peptidoglycan involves a carboxylic-ester bond, i.e., they were removed by treatment with alkaline hydroxylamine. Their linkage to the peptidoglycan does not involve a protein molecule. When the bdelloplast peptidoglycan was digested with lysozyme, the fatty acid-containing split products behaved as lipopeptidoglycan, i.e., they were extracted into the organic phase of 1-butanol:acetic acid:water (4:15) two-phase system; all of the lysozyme split products generated from normal E. coli peptidoglycan were extracted into the water phase. It is suggested that the function of the acylation reaction is to help stabilize the bdelloplast outer membrane against osmotic forces. In addition, a model is presented to explain how a bdellovibrio penetrates, stabilizes, and lyses a substrate cell.     

Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent or very low in cell extracts prepared from heat-treated E. coli cells. It is concluded that during intraperiplasmic growth B. bacteriovorus degrades some 20 to 40% of the ribonucleotides derived from the initial E. coli RNA into the base and ribose-1-phosphate moieties. The ribose-1-phosphate is further metabolized by B. bacteriovorus both for energy production and for biosynthesis, of non-nucleic acid cell material. In addition, the data indicate that during intraperiplasmic growth B. bacteriovorus can metabolize ribose only if this compound is available to it as the ribonucleoside monophosphate.     

Glycolytic and tricarboxylic acid cycle enzyme activities during intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli.

Selected enzyme activities were measured in extracts of the total cell pellets obtained at various times during aerobic intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on anaerobically grown Escherichia coli substrate cells. Initially, the glycolytic enzyme activities were associated with the input of E. coli and the tricarboxylic acid cycle enzyme activities with the input of bdellovibrios. During the first 90 min of Bdellovibrio development, the glycolytic activities declined about 25 to 60%, whereas the tricarboxylic acid cycle activities increased about 10%. Between 110 and 180 min, the glycolytic activities decreased to trace levels and tricarboxylic acid cycle activities increased about 50 to 90%. Both bdellovibrio cell extracts and the cell-free growth menstruum (obtained after bdellovibrio growth on E. coli) caused the inactivation of glycolytic enzymes in E. coli extracts.     

Differential Predation by Bdellovibrio bacteriovorus 109J

Bdellovibrio bacteriovorus is a predatory bacterium that can replicate only inside Gram-negative bacteria. We incubated B. bacteriovorus 109J in a mixture of two prey cells present in equal numbers and enumerated prey cells after 3 h of predation. In multiple prey pairings, B. bacteriovorus preferentially lysed on one prey over the other. When prey were individually incubated with B. bacteriovorus, they were preyed on with different efficiencies. Three prey had only 5–8% of cells remaining after Bdellovibrio predation and the other three prey had 37–43% of cells remaining. Timing of attachment of B. bacteriovorus to prey cells also varied with Bdellovibrio attachment to more preferred prey occurring the fastest. These results suggest that B. bacteriovorus 109J does not randomly infect prey cells but infects and kills some prey more readily than others.     

Effects of methotrexate on intraperiplasmic and axenic growth of Bdellovibrio bacteriovorus.

The intraperiplasmic growth rate and cell yield of wild-type Bdellovibrio bacteriovorus 109J, growing on Escherichia coli of normal composition as the substrate, were not markedly inhibited by 10-3 M methotrexate (4-amino-N10-methylpteroylglutamic acid). In contrast, the growth rate and cell yield of the mutant 109Ja, growing axenically in 0.5% yeast extract +0.15% peptone, were strongly inhibited by 10-4 and 10-3 M methotrexate. Thymine, thymidine, and thymidine-5'-monophosphate, in increasing order of effectiveness, partially or completely reversed the inhibition. E. coli depleted of tetrahydrofolate and having an abnormally high protein/deoxyribonucleic acid (DNA) ratio was obtained by growing it in the presence of methotrexate. B. bacteriovourus grew at a normal rate on these depleted E. coli cells but with somewhat reduced cell yield. Mexthotrexate (10-3 M) inhibited intraperiplasmic growth of bdellovibrio on the depleted E. coli somewhat more than it inhibited growth on normal E. coli, but the effects were small compared with inhibition of axenic growth of the mutant. Total bdellovibrio DNA after growth on the depleted E. coli in the presence or absence of methotrexate exceeded the initial quanity of E. coli DNA present. Thymidine-5'-monophosphate (10-3 M) largely reversed the inhibition and increased the amount of net synthesis of DNA. The data are consistent with the prediction that intraperiplasmic growth of B. bacteriovorus should be insensitive to all metabolic inhibitors that act by specifically preventing synthesis of essential monomers. The data also indicate that B. bacteriovorus possesses thymidylate synthetase, thymidine phosphorylase, and thymidine kinase, and has the potential to carry out de novo DNA synthesis from non-DNA precursors during intraperiplasmic growth. The results also suggest that methionyl tRNAfMet is not required for initiation of protein synthesis by B. bacteriovorus.     

Nonidentity of Bdellovibrio bacteriovorus Strains 109D and 109J

Two strains of Bdellovibrio bacteriovorus, both designated as 109 in the literature, differ. They should be referred to as 109D and 109J to avoid further confusion.     

Incorporation of long-chain fatty acids of the substrate organism by Bdellovibrio bacteriovorus during intraperiplasmic growth.

Data are presented showing that a large proportion of the fatty acids of Bdellovibrio bacteriovorus grown intraperiplasmically are derived unaltered from the fatty acids of its substrate organism. Those fatty acids of the bdellovibrio not homologous with those of the substrate organism are derived mainly by metabolic alteration of preexisting fatty acids in the latter. De novo synthesis from acetate occurs only to a small extent. These characteristics of bdellovibrio physiology are in part responsible for its minimal energy expenditure for intraperiplasmic growth. The data presented also indicate that B. bacteriovorus is capable of hydrogenating unsaturated fatty acids, of beta-oxidation of fatty acids, and of regulating the proportion of saturated and unsaturated fatty acids in the lipids.     

A soluble enzyme activity that attaches free diaminopimelic acid to bdelloplast peptidoglycan

An enzyme activity, responsible for the attachment of diaminopimelic acid (DAP) to bdelloplast wall peptidoglycan, was studied in an in vitro, cell-free system. Most of the activity was found in the high-speed (20000g) supernatant fraction of homogenates of bdelloplasts prepared from a culture of the intracellular bacterium Bdellovibrio bacteriovorus 109J, growing synchronously within cells of Escherichia coli. Peptidoglycan preparations obtained either from E. coli ML35 or from the walls of bdelloplasts synchronously cultured for 40 or 90 min served as the acceptors in this reaction, whereas cell wall or peptidoglycan preparations obtained from Gram-positive bacteria could not function as acceptors of DAP. The attachment activity had an apparent Km value for DAP of 10 microM; for bdelloplast peptidoglycan, it was approximately 0.43 mg/mL, which is 13 microM with respect to peptidoglycan disaccharide peptide units. DAP attachment was partially inhibited by the structural analogues lanthionine, L-ornithine, beta-aminobutyric acid, and D-serine, as well as the cell wall synthesis inhibitors penicillin G, ampicillin, and cephalexin. This enzyme activity is present only during the intracellular stage of the bdellovibrio's developmental growth cycle and may serve a stage-specific function of biochemically modifying the cell in which it grows.     

Bdellovibrio bacteriovorus synthesizes an OmpF-like outer membrane protein during both axenic and intraperiplasmic growth.

Outer membrane preparations of Bdellovibrio bacteriovorus grown intraperiplasmically on Escherichia coli containing OmpF were prepared by the Triton X-100 procedure of Schnaitman (J. Bacteriol. 108:545-552, 1971). They contained a protein that migrated to almost the same position as E. coli OmpF in sodium dodecyl sulfate-acrylamide gradient gel electrophoresis and to the same position as E. coli OmpF when urea was incorporated into the gel. The mobility of this protein increased relative to that of OmpC in urea-containing gels as does E. coli OmpF. However, the same protein was also produced during axenic growth and during intraperiplasmic growth on prey lacking OmpF. The peptide profile generated by partial proteolysis of this protein showed no homology to that produced from E. coli OmpF. We conclude that B. bacteriovorus synthesizes an OmpF-like protein. Previous claims that the bdellovibrio incorporates an intact E. coli OmpF are not consistent with these observations.     

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).     

Utilization of nucleoside monophosphates per Se for intraperiplasmic growth of Bdellovibrio bacteriovorus.

During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E. coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing. This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus. Exogenous thymidine-5'-monophosphate competed effectively with [3H]thymine residues of E. coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all. A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E. coli phosphorus as a phosphorus source for B. bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids. A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E. coli phosphorus by B. bacteriovorus, as did orthophosphate alone. A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E. coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid. These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B. bacteriovorus growing intraperiplasmically. As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth. The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved.     

Uptake of intact nucleoside monophosphates by Bdellovibrio bacteriovorus 109J

The degraded nucleic acids and ribosomes of its prey cell provide Bdellovibrio bacteriovorus 109J with a source of ribonucleoside monophosphates and deoxyribonucleoside monophosphates for biosynthesis and respiration. We demonstrate that bdellovibrios, in contrast to almost all other bacteria, take up these nucleoside monophosphates into the cell in an intact, phosphorylated form. In this way they are able to assimilate more effectively the cellular contents of their prey. Studies with UMP and dTMP demonstrate that they are transported and accumulated against a concentration gradient, achieving internal levels at least 10 times the external levels. Treatment of the bdellovibrios with azide or carbonyl cyanide m-chlorophenylhydrazone eliminates their ability to either transport or maintain accumulated UMP and suggests the presence of a freely reversible exchange mechanism. There are at least two separate classes of transport systems for nucleoside monophosphates, each exhibiting partial specificity for either ribonucleoside monophosphates or deoxyribonucleoside monophosphates. Kinetic analyses of UMP transport in different developmental stages of strain 109J indicate that each stage expresses a single, saturable uptake system with a distinct apparent substrate affinity constant (Kt) of 104 microM in attack phase cells and 35 microM in prematurely released growth phase filaments. The capacity for transport of UMP by the growth phase filaments was 2.4 times that of the attack phase cells. These data, in addition to the apparent lack of environmental control of UMP transport capacity in attack phase cells, suggest that there are two transport systems for UMP in bdellovibrios and that the high-affinity, high-capacity growth phase system is developmentally regulated.     

Isolation and composition of sheathed flagella from Bdellovibrio bacteriovorus 109J.

A procedure was developed for the purification of sheathed flagella from Bdellovibrio bacteriovorus 109J. Preparations of isolated flagella appeared as filaments 28 nm in diameter, did not vary in sheath content by more than 10% from the mean, and contained 50% protein, 38% phospholipid, and 12% lipopolysaccharide (LPS) by weight. The sheath was readily solubilized by Triton X-100, whether or not EDTA was present, and contained all of the LPS and phospholipid and 30 to 40% of the protein of the intact flagella; sedimentable core filament polypeptides accounted for the remainder. Flagellar LPS was significantly enriched in nonadecenoic acid (19:1) and depleted in beta-hydroxymyristic acid relative to outer membrane LPS from intraperiplasmically grown bdellovibrios. These observations suggest that the sheath is a stable domain distinct from the bulk of the outer membrane. The sheath also contained substantially more phospholipid (57%) and less protein (26%) of a more heterogeneous composition than that of previously described outer membranes. This unusual balance of constituents was predicted to result in a fluid membrane compatible with a model for the generation of motility by rotation of the core filament within a highly flexible sheath.     

Analysis of phenotypic diversity among host-independent mutants of Bdellovibrio bacteriovorus 109J

Host-independent (H-I) mutants of the obligate bacterial parasite Bdellovibrio bacteriovorus were isolated from wild-type strain 109J. Seven H-I mutants differed in morphological features such as cell length (2-30 microm) and shape (short or long spirals or rod-like), plaque size, and pigmentation (from almost colorless to bright orange). The mutants exhibited widely different growth capabilities in rich medium, with biomass doubling times and final biomass varying by a factor of two or more. Growth was always enhanced by the addition of host cell extract or divalent cations to the growth medium, but the effect varied widely between the mutants. Analysis of the hit region, mutations in which were previously proposed to be associated with the H-I phenotype, revealed that changes in the nucleotide sequence in this region occurred only in three of the seven mutants.