Periplasmic enzymes in Bdellovibrio bacteriovorus and Bdellovibrio stolpii.

When cells of either Bdellovibrio bacteriovorus 109J or Bdellovibrio stolpii UKi2 were subjected to osmotic shock by treatment with sucrose-EDTA and MgCl2 solutions, only trace amounts of proteins or enzyme activities were released into the shock fluid. In contrast, when nongrowing cells were converted to motile, osmotically stable, peptidoglycan-free spheroplasts by penicillin treatment, numerous proteins were released into the suspending fluid. For both species, this suspending fluid contained substantial levels of 5'-nucleotidase, purine phosphorylase, and deoxyribose-phosphate aldolase. Penicillin treatment also released aminoendopeptidase N from B. bacteriovorus, but not from B. stolpii. Penicillin treatment did not cause release of cytoplasmic enzymes such as malate dehydrogenase. The data indicated that bdellovibrios possess periplasmic enzymes or peripheral enzymes associated with the cell wall complex. During intraperiplasmic bdellovibrio growth, periplasmic and cytoplasmic enzymes of the Escherichia coli substrate cell were not released upon formation of the spherical bdelloplast during bdellovibrio penetration. Most of the E. coli enzymes were retained within the bdelloplast until later in the growth cycle, when they became inactivated or released into the suspending buffer or both.     

Interaction of Bdellovibrio bacteriovorus and Host Bacteria II. Intracellular Growth and Development of Bdellovibrio bacteriovorus in Liquid Cultures

The intracellular life cycle of Bdellovibrio bacteriovorus 109 growing on Escherichia coli in a dilute nutrient medium exhibits a period of constant infective titer while the parasite grows and elongates inside the host cell. This period is terminated after 2 to 4 hr, and the number of the plaque-forming units in the culture rises rapidly to as much as six times the initial titer. The growth pattern of Bdellovibrio is similar with actively growing or resting host cells, or with host cells killed by ultraviolet irradiation or by heating at 70 C. The yield of B. bacteriovorus strain 109 in two-membered cultures with E. coli B depends on the host concentration and may reach 7.5 x 10(10) cells per ml. Penicillin, which has no effect on the attachment and penetration of Bdellovibrio, inhibits its multiplication.     

Interaction of Bdellovibrio bacteriovorus and Host Bacteria I. Kinetic Studies of Attachment and Invasion of Escherichia coli B by Bdellovibrio bacteriovorus

Quantitative methods were developed for the study of the early stages in the interaction of Bdellovibrio bacteriovorus and host bacteria. Attachment measurements were based on the differential filtration of host and parasite. Invasion was measured by estimation of radioactively labeled Bdellovibrio cells remaining attached to the host cells after mechanical agitation. The kinetics of attachment and the final number of Bdellovibrio cells attached were dependent on the multiplicity of the parasite, the composition and pH of the medium, and the incubation temperature. Inhibitors of Bdellovibrio motility, including chelating agents, NaN(3), and low pH, all inhibited attachment, as did anaerobiosis. Ultraviolet-killed host cells retained their competence for attachment of Bdellovibrio cells, whereas heat-killed cells lost it. Invasion was selectively inhibited by inhibitors of protein synthesis, such as streptomycin, puromycin, and chloramphenicol. These antibiotics had no effect on attachment.     

Modifications of hydrophobicity, in vitro adherence and cellular aggregation of Streptococcus mutans by Helichrysum italicum extract

AIMS: The purpose of the present study was to examine whether sublethal concentrations of Helichrysum italicum extract could affect some of the cariogenic properties of Streptococcus mutans. METHODS AND RESULTS: We studied the antibacterial activity of H. italicum (ethanolic extract) against oral streptococci (Strep. mutans ATCC 35668, Strep. salivarius ATCC 13419 and Strep. sanguis ATCC 10556) and its influence on cell-surface hydrophobicity, in vitro sucrose-dependent adherence to glass surface and cellular aggregation of Strep. mutans. The results indicate that all streptococci were susceptible to ethanolic extract with minimum inhibitory concentration (MIC) values of 31.25-62.50 microg x ml(-1). Sub-MIC concentrations of H. italicum (7.81-31.25 microg x ml(-1)) reduced the hydrophobicity and the adherence (almost 90%) to glass surface of Strep. mutans. The aggregation in the presence of dextran T2000 was also affected. CONCLUSION: The inhibitory activity of H. italicum extract on Strep. mutans is worthy of further study. SIGNIFICANCE AND IMPACT OF THE STUDY: There is considerable interest in the use of natural compounds as alternative methods to control undesirable micro-organisms.     

Energy metabolism of Bdellovibrio bacteriovorus

The P/O ratio of Bdellovibrio bacteriovorus, strain Bd 109 Sa, was evaluated by two different methods based on the determination of energy-rich phosphate bonds and either NADH oxidation or oxygen-uptake. P/O values calculated on the basis of NADH oxidation were up to 6, which has to be regarded as being overestimated. P/O values calculated from energy-rich phosphate bonds and oxygen uptake were around 2. The P/O values determined for Escherichia coli B were similar. The loss of phosphorylation efficiency at one site is discussed.The ATP pool turnover rate of Bdellovibrio was 8/min during endogenous respiration and 24/min during substrate respiration. The corresponding values in Escherichia coli B were 3/min and 38/min.This study was performed at the University of Hamburg (Institut für Allgemeine Botanik, Abteilung Mikrobiologic).     

Elongation and cell division in Bdellovibrio bacteriovorus

Elongation and division of Bdellovibrio bacteriovorus were studied in axenic synchronous cultures. The cells elongate unidirectionally from one end attaining a length of several unit cells, and then divide into the corresponding number of cells. The length of the filament and, consequently, the progeny number, vary within the range of two to several dozen cells, according to the conditions used. A protein and a low molecular weight component are required for normal division.     

Spiral architecture of the nucleoid in Bdellovibrio bacteriovorus

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.     

Rapid isolation of host-independent Bdellovibrio bacteriovorus

A new method of isolating host-independent Bdellovibrio bacteriovorus has been developed. Filtered suspensions of host-dependent cells are dropped in small volumes onto 0.2 μm membranes laid on rich media agar. Significant growth is observed within 1–2 days; these cells were confirmed to be B. bacteriovorus using microscopic observations and PCR.     

The influence of saliva on the hydrophobic surface properties of bacteria isolated from oral sites of macaque monkeys

Abstract The surface hydrophobicity of 64 bacterial strains isolated from discrete, intra-oral sites of monkeys ( Macaca fascicularis ) was determined by measuring their affinity for hexadecane. Bacteria were also exposed to monkey saliva which either increased or reduced the surface hydrophobicity of the cells. After exposure to saliva those bacteria isolated solely from the mucosal surfaces were significantly more hydrophobic than bacteria ( Streptococcus mutans and Actinomyces spp.) whose major habitat was the dentition. Streptococcus sanguis strains isolated from all intra-oral sites and among the early plaque formers were as hydrophobic as the organisms isolated only from the mucosal surfaces.     

噬菌蛭弧菌的分离及初步鉴定

采用双层平板法,以滤膜过滤的方法来收集噬菌蛭弧菌,以嗜水气单胞菌(Aeromonas hudrophila)、荧光假单胞菌(Pseudomonas fluorescent)和绿脓杆菌(Pseudomonas aeruginosa)为宿主菌,进行噬菌蛭弧菌的分离研究;并在此基础上,通过接触酶检测和寄生性确认对噬菌蛭弧菌(Bdellovibrio bacteriovorus)进行了初步的鉴定.结果表明未使用滤膜过滤,采用自来水琼脂双层平板法分离噬菌蛭弧菌的效果较好;并经过接触酶和寄生性检测初步鉴定此BD-SPOI菌株为噬菌蛭弧菌.     

Ancient lateral gene transfer in the evolution of Bdellovibrio bacteriovorus

The recently sequenced genome of the predatory delta-proteobacterium Bdellovibrio bacteriovorus provides many insights into its metabolism and evolution. Because its genes are reasonably uniform in G+C content, it was suggested that B. bacteriovorus actively resists recombination with foreign DNA and horizontal transfer of DNA from other bacteria. To investigate this further, we carried out a variety of phylogenetic and comparative genomics analyses using data from >200 microbial genomes, including several published delta-proteobacteria. Although there might be little evidence for the extensive recent transfer of genes, we demonstrate that ancient lateral gene acquisition has shaped the B. bacteriovorus genome to a great extent.     

Early host damage in the infection cycle of Bdellovibrio bacteriovorus

The effects of bdellovibrio infection on host permeability and respiration were investigated by measuring respiration rates and the rate of o-nitrophenyl-beta-d-galactopyranoside hydrolysis during the course of single infection cycles of Bdellovibrio bacteriovorus strain 109 growing on Escherichia coli ML 35 (lac i(-)z(+)y(-)). The data show that among the very early consequences of parasite attack on the host are an increase in permeability and a general disruption of respiratory activity of the host, and it is suggested that both phenomena stem from early damage to host membrane. The rapid onset of damage after inception of the cycle and the failure of streptomycin to prevent the damage indicate that complete penetration of the parasite into the host is not a requirement for the observed effects. The data also show that bdellovibrio does not use host energy-generating mechanisms for its growth and suggest that the parasites may have a search mechanism that permits them, to some degree, to distinguish between infected and uninfected hosts.     

Occurrence of Phosphonosphingolipids in Bdellovibrio bacteriovorus Strain UKi2

The major phospholipids of two strains of Bdellovibrio bacteriovorus were characterized. Both strain UKi1, which is obligately saprophytic, and strain UKi2, which is facultatively parasitic, contained phosphatidylethanolamine and phosphatidylglycerol as their major glycerophosphatides. A branched, 15-carbon fatty acid is the major component of these alkali-labile lipids. Absent from UKi1 but present in UKi2 were three alkali-stable lipids (compounds 8, 9, and 11) which appear to be phosphosphingolipids. After acid hydrolysis, both compound 8 and 9 yield the identical phosphorus-containing substance that is water soluble, dipolar ionic, and ninhydrin positive. This substance appears to contain a C-P bond since P(i) could not be released from this substance by treatment with alkaline phosphatase or by very harsh mineral acid treatment. Based on chromatographic comparisons, this phosphonate appears to be a novel lipid constituent. Upon degradation, compound 8 yields 1 mol of dihydroxy long-chain base and compound 9 yields 1 mol of a trihydroxy long-chain base. These bases appear to have a 17-carbon, possibly branched, structure based on gas-liquid chromatography retention times. Degradation of both sphingolipids yields a mixture of hydroxy fatty acids, the major component being a branched, 15-carbon hydroxy acid.     

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.     

Changes in cell composition and viability of Bdellovibrio bacteriovorus during starvation

1. Washed cell suspensions of Bdellovibrio bacteriovorus harvested shortly after lysis of their substrate organisms and shaken in buffer have a constant and high endogenous respiration rate for a bout 6 h which then declines sharply to a rate approximately 10% of the original. Viability of cell suspensions shows little change over the first 4–6 h and then decreases by some 50% in 10 h.
2. Over the first 5–6 h of starvation there is a loss of about 50% of total cell carbon. This loss is distributed about equally between CO₂ and small molecules released into the suspending buffer. The protein and nucleic acid contents of the cells decrease concomitantly from time zero during starvation while DNA content remains constant. Ribosomal profiles show a rapid degradation of ribosomes.
3. In the presence of glutamate or glutamate plus a balanced amino acid mixture, loss of cell material and loss of viability is partially or completely prevented. There is extensive protein turnover when glutamate and an amino acid mixture are available to the bdellovibrio.
4. The pattern of changes observed in B. bacteriovorus during starvation is compared to reported changes in other species of bacteria, and the significances of its high endogenous respiration and sensitivity to starvation are discussed.

     

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.