Waveform analysis and structure of flagella and basal complexes from Bdellovibrio bacteriovorus 109J.

The structure of sheathed flagella from Bdellovibrio bacteriovorus was investigated. The first three periods of these flagella were characterized by progressively smaller wavelengths and amplitudes in periods more distal to the cell. The damped appearance was due to a single nonrandom transition between two helical structures within each filament. The intersection of the two helices, one of which was a threefold-reduced miniature of the other, occurred at a fixed distance along the filament and resulted in a shift in the flagellar axis. Flagella increased in length as the cells aged and assumed a constant miniature waveform at their distal ends. The core filament was the principal determinant of flagellar morphology. It was composed of 28,000- and 29,500-dalton polypeptides. The 28,000-dalton subunits were located in the cell-proximal segment of the filament, and the 29,500-dalton subunits were located in the more distal region. The heteromorphous appearance of bdellovibrio flagella arose from the sequential assembly of these subunits. The basal complex associated with core filaments was examined because of its potential involvement in sheath formation. Bdellovibrio basal organelles were generally similar to those of other gram-negative species, but appeared to lack a disk analogous to the outer membrane-associated L ring which is a normal component of gram-negative basal complexes.

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.

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.

Attachment of diaminopimelic acid to bdelloplast peptidoglycan during intraperiplasmic growth of Bdellovibrio bacteriovorus 109J.

An early event in the predatory lifestyle of Bdellovibrio bacteriovorus 109J is the attachment of diaminopimelic acid (DAP) to the peptidoglycan of its prey. Attachment occurs over the first 60 min of the growth cycle and is mediated by an extracellular activity(s) produced by the bdellovibrio. Some 40,000 DAP residues are incorporated into the Escherichia coli bdelloplast wall, amounting to ca. 2 to 3% of the total initial DAP content of its prey cells. Incorporation of DAP occurs when E. coli, Pseudomonas putida, or Spirillum serpens are the prey organisms. The structurally similar compounds lysine, ornithine, citrulline, and 2,4-diaminobutyric acid are not attached. The attachment process is not affected by heat-killing the prey nor by the addition of inhibitors of either energy generation (cyanide, azide, or arsenate), protein or RNA synthesis (chloramphenicol and rifamycin), or de novo synthesis of cell wall (penicillin or vancomycin). Approximately one-third of the incorporated DAP is exchangeable with exogenously added unlabeled DAP, whereas the remaining incorporated DPA is solubilized only during the lysis of the bdelloplast wall. Examination of DAP incorporation at low prey cell densities suggests that bdellovibrios closely couple the incorporation to an independent, enzymatic solubilization of DAP by a peptidase. The data indicate that DAP incorporation is a novel process, representing the second example of the ability of the bdellovibrio to biosynthetically modify the wall of its prey.

Permeability of the boundary layers of Bdellovibrio bacteriovorus 109J and its bdelloplasts to small hydrophilic molecules.

Measurements of the sucrose-permeable and -impermeable volumes during Bdellovibrio bacteriovorus attack on Escherichia coli or Pseudomonas putida showed that the volume of the bdelloplast increased over that of the substrate cell. Although the pattern of the increase differed with the two organisms, the volumes reached maximum at about 60 min into the bdellovibrio growth cycle. By this time, the cytoplasmic membranes of the attacked cells were completely permeable to sucrose. The kinetics of increase in sucrosepermeable volumes were similar to the kinetics of attachment and penetration (Varon and Shilo, J. Bacteriol. 95:744-753, 1968). These data show that the original cytoplasmic and periplasmic compartmentalization of the substrate cell ceases to exist with respect to small hydrophilic molecules during bdellovibrio attack. In contrast, the effective pore size of the outer membrane of the substrate cell to small oligosaccharides remains unaltered during bdelloplast formation as was shown by direct measurements of its exclusion limits. The major porin protein of E. coli, OmpF, was recoverable from the bdelloplast outer membrane fraction until the onset of lysis. The Braun lipoprotein was removed from the bdelloplast wall early, and OmpA was lost in the terminal part of the bdellovibrio growth cycle.

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.

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.

Partial characterization of lipid A of intraperiplasmically grown Bdellovibrio bacteriovorus.

The lipid A components of substrate cell origin incorporated by Bdellovibrio bacteriovorus during intraperiplasmic growth (D. R. Nelson and S. C. Rittenberg, J. Bacteriol. 147:860-868, 1981) were shown to be integrated into its lipopolysaccharide structure. Lipid A isolated from bdellovibrios grown on Escherichia coli was resolved into two fractions by thin-layer chromatography. Fraction 2 had the same Rf as the single lipid A fraction of axenicaly grown bdellovibrios, and both stained identically with aniline-diphenylamine reagent. Fraction 1 resembled, in Rf and staining reaction, the slower migrating of two lipid A fractions obtained from the E.coli used as the substrate cell. Both fractions 1 and 2 contained glucosamine, a substrate cell-derived compound. Greater than 65% of the fatty acids in fraction 1 were derived from the substrate cell, whereas more than 60% of the fatty acids of fraction 2 were synthesized by the bdellovibrio. Nevertheless, each fraction contained significant amounts of fatty acid of both origins. The substrate cell-derived fatty acids had the same distribution of N-acyl and O-acyl linkages as in E. coli lipid A. The data indicate that the two lipid A moieties in lipopolysaccharide of intraperiplasmically grown bdellovibrios are hybrids of substrate cell-derived and bdellovibrio-synthesized components. The data also suggest that disaccharide units and N- and O-acyl linkages preexisting in the substrate cell lipid A may be conserved. A possible explanation for the unequal distribution of substrate cell-derived material in the two lipid A fractions of the bdellovibrio is suggested.

Incorporation of substrate cell lipid A components into the lipopolysaccharide of intraperiplasmically grown Bdellovibrio bacteriovorus.

The composition of Bdellovibrio bacteriovorus lipopolysaccharide (LPS) was determined for cells grown axenically and intraperiplasmically on Escherichia coli or Pseudomonas putida. The LPS of axenically grown bdellovibrios contained glucose and fucosamine as the only detectable neutral sugar and amino sugar, and nonadecenoic acid (19:1) as the predominant fatty acid. Additional fatty acids, heptose, ketodeoxyoctoic acid, and phosphate were also detected. LPS from bdellovibrios grown intraperiplasmically contained components characteristic of both axenically grown bdellovibrios and the substrate cells. Substrate cell-derived LPS fatty acids made up the majority of the bdellovibrio LPS fatty acids and were present in about the same proportions as in the substrate cell LPS. Glucosamine derived from E. coli LPS amounted to about one-third of the hexosamine residues in intraperiplasmically grown bdellovibrio LPS. However, galactose, characteristic of the E. coli outer core and O antigen, was not detected in the bdellovibrio LPS, suggesting that only lipid A components of the substrate cell were incorporated. Substrate cell-derived and bdellovibrio-synthesized LPS materials were conserved in the B. bacteriovorus outer membrane for at least two cycles of intraperiplasmic growth. When bdellovibrios were grown on two different substrate cells successively, lipid A components were taken up from the second while the components incorporated from the lipid A of the first were conserved in the bdellovibrio LPS. The data show that substrate cell lipid A components were incorporated into B. bacteriovorus lipid A during intraperiplasmic growth with little or no change, and that these components, fatty acids and hexosamines, comprised a substantial portion of bdellovibrio lipid A.

Pyrimidine metabolism of Bdellovibrio bacteriovorus grown intraperiplasmically and axenically.

Bdellovibrio bacteriovorus grown axenically or intraperiplasmically on Escherichia coli has pathways for the interconversion of pyrimidines and the synthesis of pyrimidine nucleoside 5'-triphosphates similar to those found in the enteric bacteria. Minimal differences in enzyme activities were observed for axenically and intraperiplasmically grown cells. As might be expected for an organism which takes up deoxyribonucleoside 5'-monophosphates per se, high levels of enzymes which catalyze the generation of deoxyribonucleoside triphosphates from monophosphates were found. In addition, all enzymes of the thymine salvage pathway, except for thymidine kinase, were directly demonstrated in wild-type strains. It was possible to demonstrate this activity only indirectly owing to an inhibitor in wild-type extracts. Investigations with inhibitors of pyrimidine interconversion reactions showed that essentially all B. bacteriovorus deoxyribonucleic acid not synthesized from units derived from E. coli deoxyribonucleic acid is made from components of the substrate organism's ribonucleic acid. Evidence for de novo pyrimidine synthesis from the amino acid level was not found for B. bacteriovorus grown on E. coli that had a high protein/deoxyribonucleic acid ratio or on normal E. coli. The potential for de novo pyrimidine synthesis by intraperiplasmically grown B. bacteriovorus, however, cannot be totally ruled out on the basis of these investigations.

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.

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.

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.

Penicillin-induced formation of osmotically stable spheroplasts in nongrowing Bdellovibrio bacteriovorus.

Bdellovibrio peptidoglycan is of typical gram-negative composition. The molar ratios of alanine:glutamic acid:diaminopimelic acid:muramic acid:glucosamine were about 2:1:1:1:1. Nascent, nongrowing Bdellovibrio bacteriovorus 109J were converted from highly motile vibrios to highly motile spheres when shaken in dilute buffer plus penicillin, cephalothin, bacitracin, or D-cycloserine. The spherical forms contained essentially no sedimentable peptidoglycan; i.e., they were spheroplasts. Spheroplasts induced by penicillin, D-cycloserine, and lysozyme were stable in dilute buffer and did not lyse when subjected to osmotic shock. Normal Bdellovibrio suspended in buffer turned over their peptidoglycan at a rate of approximately 30% h during the initial 120 min of starvation. Chloramphenicol and sodium azide strongly inhibited Bdellovibrio peptidoglycan turnover and the induction of spheroplasts by penicillin. The data indicate that nongrowing B. bacteriovorus are sensitive to penicillin and other antibiotics affecting cell walls because of their high rate of peptidoglycan turnover. It is also concluded that an intact peptidoglycan layer is required for maintaining cell shape, but is not required for osmotic stability of B. bacteriovorus.

Phenotypic changes in the resistance of Neisseria gonorrhoeae to killing by normal human serum.

Gonococci adapted to growth in chambers implanted subcutaneously into guinea pigs are resistant to killing by human serum. This resistance is lost after a few generations in vitro both in culture medium and in fluid taken from guinea-pig chambers. The rate of loss is too rapid to occur by mutation and selection. Furthermore, the resistance is regained after a few generations when bacteria from the first in vitro culture are inoculated back into guinea-pig chambers in vivo. Hence the loss of serum resistance in vitro and the gain in vivo are probably due to phenotypically controlled events. Such events could be important in the pathogenicity of Neisseria gonorrhoeae.

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.

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.

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.