The Predatory Organism: Bdellovibrio

Identifying the genes and the proteins to be expressed by this organism during pore formation in the host cell will be of significance to bacteriology. The possible use of this information can be seen in the study done by Kadouri and O’toole. It was established in this report that B. bacteriovorus can be used as a control agent against certain biofilm communities. It was observed that biofilm populations of Escherichia coli and Pseudomonas fluorescens were significantly reduced when exposed to B. bacteriovorus.

Since biofilm formation is unnecessary and damaging in some instances, inhibition of its formation can be done through natural elimination of the biofilm organisms, and that is through the inoculation of B. bacteriovorus. Another possible application of this predatory organism is through its use as an anti-microbial therapeutic agent. It had been suggested by Hobley et al. that the introduction of this organism to a microbial-infected wound can lead to the reduction of pathogenic organisms in the site of inoculation.

In this way, the use of antibiotics can be avoided and possible resistance of disease-causing organisms to the applied drug can be avoided. If the genes responsible for pore formation in the host cell will be identified, the organism can be manipulated to increase the genes’ expression. These can then be enhanced and controlled in accordance to the desired amount and time of expression. II. Aims This paper aims to propose a method on how to determine the necessary and significant enzymes involved in pore formation of the host cell by Bdellovibrio bacteriovorus H100.

The specific aims are as follows: 1. Identify the genes of B. bacteriovorus H100 specifically expressed during pore formation in the host cell during the predatory life cycle phase of the organism using time course Microarray analysis. 2. Identify specific hydrolytic enzymes expressed by the identified genes of B. bacteriovorus H100 involved in pore formation on host cell membrane. III. Background The discovery of Bdellovibrio by Stolp and Starr in 1962 opened the scientific world to the dawn of a new organism- a bacterium which preys on its own kind (12).

This bacterium is a curved rod, Gram negative organism classified under the group of delta-proteobacteria. It is a motile bacterium; monotrichously flagellated; obligately aerobic; small in size (0. 3 ? m in width and 1-2 ? m in length); and is obligately predatory. Its genome consists of 3. 85 mega base pairs. Most of these genes encode for enzymes involved in hydrolysis and flagella involved in host sensing (3). The best known species of Bdellovibrio is B. bacteriovorus, observed to prey on other Gram negative organisms such as Salmonella, Escherichia coli, Sphaerotilus natans and Pseudomonas fluorescens (1).

The distribution of Bdellovibrio bacteriovorus is observed in diverse environments; therefore, it is considered to be an ubiquitous bacterium. It was associated in dry environment such as the soil; in wet locations such as brackish water, sewage, fresh water, pooled reservoirs and sea water; and in unique microbial niches such as biofilms (3). The life cycle of this bacterium consists of two stages. The first stage is the so-called free-swimming attack phase and the second stage is known as the intraperiplasmic replication phase. Figure 2 shows a graphical representation of these two stages.

The life cycle of B. bacteriovorus takes about three hours to complete. For the initiation of the attack, the monotrichous flagellum is an important facet to consider. Movement is essential in finding the suitable host in the environment. The predatory organism moves towards a region with a high prey concentration. This process is meditated by chemotaxis. It can be seen from the illustration that a critical stage in the predation of B. bacteriovorus is the ability of the organism to penetrate its host bacterium. Upon contact with another Gram negative, B.

bacteriovorus then forms a pore in the cell membrane of its host. Initial entrance of B. bacteriovorus is followed by the organism’s penetration of the periplasm. Bdellovibrio bacteriovorus then occupies the periplasmic space of the host cell (3). Without this phase, the other steps in the whole life cycle of the organism cannot be ensured to occur. Figure 3 and 4 shows an electronmicrograph of B. bacteriovorus attaching to a host cell. A specific strain of the bacterium, B. bacteriovorus HD100, was studied by Rendulic, et al. This strain of Bdellovibrio was found to have an unusually large genome.

Though this bacterium preys on other Gram negative organisms, its genetic make-up did not comprise of any gene from its host. Furthermore, it was elucidated that the genes present in B. bacteriovorus HD100 are made up of gene families coding for enzymes such as hydrolases and transporters, important in the penetration and killing of the host. These genes also code for enzymes needed for uptake of complex molecules (6). One hurdle in studying the molecular characteristics of this organism is its host dependent nature. Without a suitable host, growth cannot be ensured, thus, elucidation of its genetic make-up may be difficult to achieve.

Further studies using this bacterium revealed that Bdellovibrio can generate mutant cells that do not require host cells for growth and are therefore known as host independent (HI) strains. Despite this, they were able to retain the ability to grow on prey and hence are termed as facultative predators. For gene manipulation techniques, HI strains are usually used (8). Despite the fact that the complete genome of the organism was already sequenced, the specific genes coding for the needed enzymes to form pores in the host cell were still unidentified.

With this lack of information, this study is formulated and designed. IV. Research Design and Methodology Culturing of B. bacteriovorus HD100 on prey dependent and prey independent set-ups: Predatory (HD) cultures of B. bacteriovorus HD100 will be grown on E. coli in Ca2_-HEPES buffer at 30°C, with shaking at 200 rpm (8). Escherichia coli ML35 and E. coli W7-M5 (10) will be used as the prey throughout the experiments. Escherichia coli ML35 will be cultured in nutrient broth (Difco Laboratories), and E. coli W7-M5, a lysine and DAP auxotroph, will be cultured in nutrient broth supplemented with 0.

2 mM lysine and 0. 1 mM DAP at 37°C with shaking at 200 rpm. Prey-independent HI strains will be plated on rich peptone-yeast extract (PY) medium (8). Synchronous cultures: Synchronous cultures will be used for performing various experiments as described below. Briefly, fresh bdellovibrios will be added to prey cells in HM buffer (3 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-1 mM CaCl. LQ. One mM of MgCl2 will be adjusted to pH 7. 6 using NaOH (10). The organisms will be grown until a final concentration of 1010 bdellovibrios per ml and 5 x 109 E.

coli per ml is reached. For proper aeration, volumes will be kept to ? 20% of the flask’s volume and incubated at 30°C with shaking at 400 rpm. Synchronous cultures will be examined at intervals for attachment and penetration with a Nikon model L-Ke microscope (Nippon Kogaku Inc. ) equipped with phase-contrast optics and a Nikon model AF camera. Time course Microarray analysis. Time course Microarray analysis will be performed to identify the genes to be expressed during the entry phase, specifically during pore formation on the host cell membrane of B.

bacterovorus H100. Microarray slides of B. bacteriovorus H100 will be ordered from Advanced Throughput, Inc Services. Total cellular RNA will be extracted from B. bacteriovorus H100 cells at entry phase using the RNeasy mid kit (Qiagen). The RNA of the organism will also be extracted during the other stages of infection. This will serve as a reference for comparison of the genes expressed and not expressed at the desired stage. Complementary DNA synthesis, fragmentation, labeling, hybridization, staining and washing will be performed according to the Affymetrix B.

bacteriovorus H100 GeneChip array expression analysis protocol (Affymetrix). Briefly, cDNA will be synthesized from RNA using Superscript II (Invitrogen) according to the manufacturer’s instructions. RNA will be removed by alkaline treatment and subsequent neutralization. Complementary DNA will be purified with QIAquick PCR purification columns (Qiagen). Purified cDNA will be fragmented by DNase I (Amersham) at 37°C for 10 min followed by end labeling with biotinddUTP, using an Enzo BioArray terminal labeling kit (Affymetrix), at 37°C for 60 min.

Hybridization will be performed in an Affymetrix GeneChip hybridization Oven 640. Washing and staining will be performed using an Affymetrix Fluidics Station 400. Arrays will be scanned with an Agilent GeneArray Scanner G2500A. GeneChip scans will be initially analyzed using the Affymetrix Microarray Suite 5. 1 software, from which PivotData tables will be exported. Raw data from the PivotData Tables will be analyzed in GeneSpring software version 6 (Silicon Genetics), using the parameters suggested by Silicon Genetics for analysis of Affymetrix Microarrays. Real-time PCR:

Real-time PCR using the Applied Biosystems 7500 Real-time PCR system will be performed to confirm microarray results. RNA will be extracted from B. bacteriovorus H100 at initial phases of predatory life cycle up to entry phase as described above. RNA will be reverse transcribed into cDNA and simultaneously labelled using the iScript One-step RT-PCR kit with SYBR Green (Biorad). RT-PCR reactions will also be performed to amplify cDNA of housekeeping genes (identified from micro array studies) for normalization of fluorescence values. Identifying the specific hydrolytic enzymes of B.

bacteriovorus which are involved in pore formation on host cell membrane. Many experiments showed that B. bacteriovorus H100 releases hydrolytic enzymes during predatory life cycle. According to Thomashow and Ritterberg, glycanases and lipopolysaccharideases are required for pore formation in the prey’s peptidoglycan and LPS layers respectively. The glycanase and/or peptidase could be responsible for weakening the peptidoglycan layer of the prey and thereby responsible for permitting conversion of the substrate cell to a spherical shape (10).

Tudor et al. proposed another model for penetration. According to them peptidase is responsible for pore formation but not glycanase (11). Specific enzymes involved in pore formation are not known. The genes identified from the time course micro array technique will be mutated as described previously using suicide vector pSSK10. Resulting mutants will be complemented by using vector pMMB206 (8). Mutants will be analysed for the specific enzymes (using 2D-gel electrophoresis) and their actions on host cell i.

e, as a glycanase, LPSase or peptidase will be observed by radio labelling experiments (10). Wild-type B. bacteriovorus H100 and complemented strains will be used as controls. Radio labeling experiments: Escherichia. coli W7-M5, auxotroph for lysine and DAP and cannot metabolize glucosamine, will be radiolabelled as described previously (9,10). Peptide portion of E. coli W7-M5 peptidoglycan will be labelled with [3H] DAP and the lipopolysaccharides and glycan portions of the peptidoglycan will be labeled with [3H]glucosamine.

Various mutants and wild-type strains will be tested for predation using this radiolabelled strain. Solubilisation of glucosamine and DAP from labelled prey peptidoglycan will be measured as described previously (11). Briefly, samples taken at intervals will be precipitated with an equal volume of cold 10% trichloroacetic acid for 30 min followed by centrifugation. Resulting supernatants will be assayed for soluble radioactivity in a scintillation counter (Rackbeta II). Two-dimensional gel electrophoresis: The hydrolytic enzymes released by B.

bacteriovorus H100 during its predatory life cycle will be analyzed by performing two-dimensional gel electrophoresis. Sample preparation for 2D-gel electrophoresis: Escherichia coli ML35 cells will be challenged with B. bacteriovorus H100 wild-type as well as the mutant strain. Culture fluid will be drawn from synchronous cultures during attachment and entry phases of B. bacteriovorus H100. Culture fluid will be centrifuged to discard any cell debris. Proteins in the supernatant will be precipitated using cold acetone. The precipitated proteins will be separated by centrifugation.

The precipitated pellet will be air dried and will be dissolved in rehydration solution (8M urea, 2% CHAPS {3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 18 mM DTT, 0. 5% IPG buffer pH range 4-7; Amersham Biosciences), plus a trace of bromophenol blue. Sample protein concentrations will be determined using the BCA protein assay (Pierce). Resulting protein pellet will be subjected to 2D-gel electrophoresis. 2D-gel electrophoresis: Two-dimensional (2-D) gel electrophoresis will be performed according to the method of O’Farrell.

Proteins present in the pellet will be resolved on two-dimensional gels using the products and protocols of Amersham Pharmacia Biotech. In the first-dimension, proteins will be resolved by isoelectric focusing on a precast Immobiline DryStrip with a linear pH gradient. This will be followed by electrophoresis using sodium dodecyl sulfate polyacrylamide gel on 12. 5% acrylamide gel. For analytical 2-D gel electrophoresis, 100 ? g of sample protein will be applied to the gels and the proteins will be stained with Pharmacia Biotech silver stain kit.

For preparative two-dimensional protein gel electrophoresis, 500 ? g of the sample protein will be loaded on the gels and proteins will be visualized using Coomassie blue R-350 (Phast Gel BlueR; Amersham Pharmacia Biotech). Spot analysis will be carried out using PDQuest Image Analysis software (BioRad). Spots absent from the mutant sample gel will be manually excised from the wild-type B. bacteriovorus H100 sample gel for identification. Mass spectrometry and protein identification: Excised protein spots of interest will be destained, reduced, carboxymethylated, and digested with trypsin in situ.

This will be done overnight with a temperature of 37°C as described (8). Gel digests will be centrifuged, and an aliquot of the supernatant will be taken for analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Lists of peptide masses will be generated and searched against the NCBInr database using the Mascot protein identification system (Matrix Science; http://www. matrixscience. com). Sequences of proteins identified by Mascot will be analyzed for a predicted signal sequence using SignalP 3. 0 (http://www. cbs. dtu.

dk/services/SignalP/). V. Conclusion The identification of genes responsible for the expression of hydrolytic enzymes of Bdellovibrio bacteriovorus is of significant importance. Since the organism preys on other bacteria, its special attributes must be thoroughly studied and appreciated for possible useful applications. As mentioned earlier, this predatory organism can be used as a control agent against biofilm, as well as pathogenic organisms. Through the use of natural agents to control destructive and disease-causing bacteria, the use of chemicals and antibiotics can be lessened or avoided.

Since the rapid evolution of organisms towards resistance and tolerance to anti-microbial substances is becoming more widespread, an alternative approach to these problems can be useful in the world of bacteriology. Thus, the identification of these genes and hydrolytic enzymes is of utmost importance. The elucidation of this information can be used as a primary defence against harmful microbes if this knowledge will be taken advantage. A more effective predatory role can be performed by Bdellovibrio bacteriovorus if the genes and the hydrolytic enzymes of the organism will be expressed efficiently and constitutively.