Bdellovibrio Proteobacteria In Gram Positive Bacteria Biology Essay

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Bdellovibrio are delta-proteobacteria that penetrate other gram-negative bacteria and use the cytoplasmic contents as substrates for growth and reproduction. As Figure 1 shows, Bdellovibrio are vibroid in shape and measure approximately 0.3 µm by 2 µm. Despite their small size, the Bdellovibrio genome consists of 3.78 kilobases that code for over 3000 proteins They also possess an unusually thick polar flagellum, of around 24-27 nm, with distinctive motility. These characteristics, along with the distinctive chemical composition of the Bdellovibrio outer membrane, aid the bacteria in their survival. The distinct biphasic life cycle of Bdellovibrio is summarised in Figures 2 and Table 1. Another distinguishing characteristic of genus Bdellovibrio is the host lethal growth cycle, whereas other parasitic bacteria continue to sequester nutrients from host cells for an extended time, Bdellovibrio uses the limited host resources before repeating the process in a new host. For this reason, Bdellovibrio's relationship with other bacteria may be described as both predatory and parasitic.

Bdellovibrio are ubiquitous in soil, the rhizosphere and aquatic environments and have been isolated from sewage and the guts of some vertebrates, suggesting that they are part of the commensal flora of these species. These bacteria possess lytic activity against a variety of Gram-negative plant, animal and human pathogens, although they cannot infect Gram-positive species since they lack the periplasm in which the growth phase occurs. Table 2 demonstrates the number of species Bdellovibrio is able to infect as well as the differences in prey range between certain strains. It is not completely understood what mechanisms are responsible for prey specificity, although they may involve prey surface receptors.

Heinz Stolp was the first to describe Bdellovibrio in 1962 during his investigation into the isolation of soil bacteriophage. The developmental stages were then studied extensively by microscopic observation and the limited biochemical methods available. It was not until the genetic studies by Thomashow and Cotter and the publication of the Bdellovibrio HD100 genome by Rendulic et al. (2004) that interest in these unique predatory organisms was re-ignited. This genome sequence revealed a larger number of biosynthetic and hydrolytic genes than had been previously estimated and a genome size similar to that of some prey organisms, which had implications on theories of acquisition of nutrients from the host. Advances in genomic sequencing have also revealed large differences in 16S rRNA and GC content in two species, Bdellovibrio starrii and Bdellovibrio stolpii, and has resulted in their reclassification to the new genus Bacteriovorax.

Since the genome of Bdellovibrio HD100 was sequenced, a large amount of information has now come to light about the molecular genetics of this genus, which can be linked to experimental observations of structure, prey range, and the life cycle of this unique organism, including possible chemotaxis mechanisms and the energetics of predation, In light of ever increasing resistance to traditional antibiotics, it is hoped that knowledge of the genes involved in predation will allow researchers to develop Bdellovibrio for use as a novel antimicrobial based on these characteristics.

The life-cycle of Bdellovibrio consists of two distinct stages, the extraperiplasmic attack phase and the intraperiplasmic growth phase. During the first phase, the predator locates and collides with bacterial prey, attaches and penetrates the membrane. Subsequently, the Bdelloplast is formed, in which the predator assimilates host macromolecules for development of the filament inside the host cells, followed by the lysis of the host and release of progeny cells.

Bdellovibrio bacteriovorus exhibits a single, polar flagellum (Figure 1) with an unusual thickness and dampened waveform morphology, which allows them motility at speeds of up to 160 mm sec-1 and is essential for predation in liquid environments The flagellar filament of Bdellovibrio bacteriovorus HD100 and 109J is composed of six flagellins, encoded by a cluster of six genes, fliC 1-6. Lambert et al. (2006) determined which of these genes were most important for motility by systematically inactivating each of the flagellin genes, the results of which are summarised in Figure 3. Inactivation of fliC3 produced non-motile cells with severely truncated flagella that did not survive, indicating that motility is essential for the location of, and attachment to, prey. Additionally, Q-PCR experiments revealed that fliC3 and filC5 were expressed more than the other flagellar proteins. These observations demonstrate that only the flagellins encoded by fliC3 and filC5 are required for correct flagellar function and that the remaining flagellins are expressed in small quantities in the filament and are possibly redundant for motility.

Due to their high rate of respiration, described later in this essay, attack-phase Bdellovibrio must locate and infect prey within a few hours or risk starvation It has been noted that the probability of encountering a prey cell by random collision alone is approximately 3.0% and that a very high concentration of prey bacteria, of approximately 3.0 x 106 cells/ml, would be required for Bdellovibrio to encounter cells by random collision aloneSince Bdellovibrio lacks genes for known quorum-sensing compounds, it seems unlikely that it responds to high densities of prey cells It still remains unclear whether Bdellovibrio employ a chemotactic mechanism to locate and respond to high concentrations of potential prey bacteria, although it can be argued that this would be of great selective advantage and would greatly increase survival rate.

Studies have demonstrated that Bdellovbrio is attracted to specific amino acids and other compounds such as NH4+, Mg2+ and K+. Furthermore, B. Bacteriovorus 109J with a defect in the mcp2 gene that encodes a methyl-accepting chemotactic protein show a reduction in predatory efficiency. A further seventeen uncharacterised MCP genes have been found in the B. Bacteriovorus HD100 genome. Together, this evidence appears to indicate that chemotaxis does play a role in the predation by attack-phase Bdellovibrio It may be that attraction to these compounds allows the predatory cells to maintain themselves while in starvation conditions, rather than acting directly as chemo-attractants, or rather that prey bacteria themselves are attracted to these compounds, and that chemoattraction simply serves to increase the probability of random collision.

Upon location of potential prey, Bdellovibrio attaches at the non-flagellated pole by colliding violently with the cell. Approximately 5-10 minutes after attachment to a suitable host, the Bdellovibrio enters the cell. This stage in the life cycle comprises two stages of attachment, both of which require continued motility a primary, reversible attachment, where the Bdellovibrio either remains attached to or detaches from the cell. The second stage is an effective and specific attachment, where the Bdellovibrio uses various hydrolytic enzymes to disrupt the host membrane, including glycanase which solubilises peptidoglycan amino sugars and peptidase that solubilises DAPA residues. The Bdellovibrio uses its powerful flagellum to rotate the two cells as one, while the it pushes through the host membrane, possibly by spreading the non-cross linked polymers through its rotation, and enters the periplasm.

It is not fully understood what causes Bdellovibrio prey upon some species, but not others. The first stage of attachment involves no specific interactions, so it is an event occurring during second, irreversible stage which determines whether Bdellovibrio will initiate infection. Due to the large variation in specificity between different strains isolated from different environments, it has been speculated that a specific receptor on the cell surface of Bdellovibrio is involved. This hypothesis is evidenced by Dunn et al.'s (1974) finding that the irreversible, but not the reversible attachment stage was prevented in temperature-sensitive strains of Bdellovibrio, suggesting that a mutation in one or more genes could affect its predatory ability. Recent investigations suggest that the interaction involves prey LPS core oligosaccharides. Thus, it can be concluded that it is the irreversible attachment of a specific molecule on the surface of Bdellovibrio to LPS antigens that is the signal of attachment to a suitable prey cell that leads to establishment of infection.) A variation in this receptor would account for the different prey ranges among strains, although the receptor molecule itself has not yet been identified.

The role of pilus-like structures present on the non-flagellate pole of Bdellovibrio, shown in Figure 4, in the attachment process is also debated. The work of Renduluc et al. (2004) revealed that B. bacteriovorus HD100 has numerous pil genes encoding type IV pili. Similar structures are involved in cell-cell contact in other bacteria, and have been proposed to contribute to formation of the pore in the membrane via shearing forces. Lack of these genes, especially of pilA in HD100 strains has been shown to disrupt predation, demonstrating that type IV pili are critical in the attachment and penetration of prey by Bdellovibrio.

Approximately 30-45 minutes after penetration, Bdellovibrio anchors to the outer membrane and forms the bdelloplast, an invagination of the prey membrane that is secure from outside interference and osmotically stable (shown at an early stage in figure 5.B. ) The periplasm is as rich in solutes, enzymes, and oligosaccharides as the cytoplasm, and thus the bdelloplast presents an ideal environment for growth. The prey cell protoplast contracts as host respiration, DNA and RNA replication and translation cease. The Bdellovibrio receives an elongation signal from the host, which initiates the intraperiplasmic growth phase

Once the parasite has formed the bdelloplast, it enters the non-motile replicative growth phase. Generally, this process begins at around 45 minutes into the infectious cycle of Bdellovibrio. (Figure 2) Inside the bdelloplast, Bdellovibrio begins to hydrolyse cytoplasmic macromolecules and use the resulting monomers for energy generation or formation of new filament molecules. Certain substrate cell macromolecules, such as fatty acids, proteins and peptides can be used by Bdellovibrio with little or no modification, while others must be degraded. The filament itself is coiled, coencytic and many cell units in length. A chromosome is formed for each progeny cell inside the filament, and prey material is distributed evenly between them throughout the growth periodThe size of the prey cell determines the number of progeny cells released upon lysis. An average of 4-5 progeny cells may be released from a single E. coli cell with a genome size of 4Mb, while larger cells, such as larger species of Aquaspirillum can support the growth of up to 30 progeny cells.

One of the earliest events after establishment within the bdelloplast is the rapid disruption of prey nucleic acids by both prey DNases freed by destruction of the cell and Bdellovibrio-encoded DNases. Figure 4 shows that host DNA degradation is complete by 150 minutes post-infection and Bdellovibrio DNA synthesis increases rapidly after this point. Some prey nucleotide monophosphates are hydrolysed from host DNA and are incorporated directly, as shown by labelling experiments. However, Table 3 shows that the total amount of DNA found in progeny is greater than that in original E. coli. Additionally, the work of Rendulic et al. (2004) revealed that the Bdellovibrio genome is approximately 3.78 Mb, similar in size to many of its prey. Clearly the pool of available nucleotides is not sufficient for the formation of multiple progeny, and therefore Bdellovibrio must synthesize a proportion of these nucleotides itself from substrate cell monomers. Similar mechanisms are thought to exist for the synthesis of RNA and protein, although the amount of ribonucleotides appears to be sufficient for the needs of Bdellovibrio. (Table 3) Some remaining nucleotides and ribonuclotides are metabolized for energy or used for the synthesis of other predator macromolecules.

Bdellovibrio is an obligate anaerobe, and generates ATP by the oxidation of substrate cell amino acids, glutamate and protein, rather than scavenging prey ATP directly; this is clear from the fact that no ATP uptake mechanisms have been found. Table 4 shows that Bdellovibrio requires much less ATP for the synthesis of many macromolecules, due to its utilization of intact nucleoside monophosphates for synthesis of DNA and RNA and selected fatty acids for synthesis of lipids, although this is not fully understood. It is important to note that the ATP required for Protein synthesis is the same, although this does not take into account proteolytic degradation of host proteins to yield amino acids. Consequently, it exhibits an unusually high YATP compared to other bacteria, as shown in the last column of table 4. Additionally, it has been shown that that ATP expenditure during the growth phase increases slightly. This, together with the large number of metabolic and hydrolytic enzymes encoded by the HD100 genome, and the presence of some lipids unique to Bdellovibrio found in its membrane appear to further the concept that Bdellovibrio transports some of the necessary host molecules from the prey for intraperiplasmic growth and uses some to re-synthesise the remainder of the molecules required.

Polymer Synthesized

ATP required

E. coli

B. bacteriovorus

























Growth of the Filament continues until the supply of nutrients is exhausted, at 180-240 minutes post-infection. The coencytic filament septates synchronously into motile progeny and flagellar synthesis begins. A recent study has revealed that the progeny cells lyse the bdelloplast and enter the environment via discrete pores, rather than lysing the entire cell, as previously thought. From fluorescence studies, it has been found that the time interval between septation and lysis correlates with the number of progeny per prey cell, the average being 26 minutes, and that lysis occurs more rapidly the greater the number of progeny produced. The progeny that are released are mature attack-phase cells, ready to locate and infect new prey and begin the cycle again.

The rapid emergence of bacterial resistance to traditional antibiotics in recent years has prompted research into the possibility of using bacterial predators, especially Bdellovibrio strains, as live antimicrobial agents. It is thought that Bdellovibrio could also be applied in industry or in agriculture, and these possibilities are being explored. The recent genome sequencing of strain HD100 by Rendulic et al (2004) revealed its large repertoire of hydrolytic and proteolytic enzymes, and revived the interest in this genus and will allow researchers to evaluate its interaction with pathogenic bacteria and its potential for controlling bacterial populations.

The ecological implications of the uses of pesticides have prompted interest in the use of Bdellovibrio strains as agricultural biocontrol agents. The abundance of Bdellovibrio in the soil and rhizosphere indicates the importance of these organisms in protecting plants against disease. Indeed, successful attempts have been made at controlling bacterial blight of soy bean plants by Pseudomonas glycinea, and Xanthomonas oryzae infection of rice plants, which suggests the possibility of Bdellovibrio as a means of controlling plant pathogens Sockett et al. note the surprising lack of further work in this area, but mention that, as with human pathogens, it is unclear whether Bdellovibrio will affect beneficial microorganisms, such as Sinorhizobium present in the same environment

Numerous studies have demonstrated the potential of Bdellovibrio as a biocontrol agent in animal populations. Chu and Zhu (2010) have demonstrated the use of Bdellovibrio to reduce Aeromonas hydrophila infections of fish in aquaculture. A further example is the treatment of keratoconjunctivitis in rabbits caused by Shigella flexineri.

Many studies have demonstrated that Bdellovibrio do not infect mammalian cells since they lack type III and IV secretion systems necessary for invasion. This is vital if it is to be used as an antibiotic.Since the prey range of Bdellovibrio species includes a variety of human pathogens, including Shigella species and E. coli, many studies have focused on its development as a novel 'antibiotic.' Sockett has suggested that the organism might have applications in the treatment of nosocomial Pseudomonas infection in HIV and cystic fibrosis patients, especially in light of emergence of Vancomycin resistant strains. The most promising application appears to be the topical application to burn wounds to prevent Pseudomonas infection.

Although Westergaard and Kramer's (1977) early studies suggested that obligately anaerobic Bdellovibrio was unable to colonize mammals due to the uneven distribution of oxygen in the gastrointestinal tract, in more recent investigations, certain strains of Bdellovibrio have been isolated from the faeces of humans and animals which indicates that some strains are able to colonise animals and are believed to be integral components of the microbial flora. It remains to be established whether there is any interaction between Bdellovibrio and commensal intestinal organism, and whether it could be applied as a probiotic in future.

Another possibility is the use of Bdellovibrio in the sanitation of equipment in hospitals and in the food industry, since they can attack pathogens growing in biofilms on medical equipment that are also a significant cause of infection. Fratamico and Whiting (1995) demonstrated the possibility of utilising Bdellovibrio to control food-bourne pathgens that cause food poisoning, including E. coli, by adding Bdellovibrio to food itself or to stainless steel equipment used in the production of foods.

Thus far, prey resistance to Bdellovibrio strains has been found to be very rare (which, in light of the issue of pathogen development of resistance to 'traditional' antibiotics, gives it great potential for antibiotic use. This property also gives Bdellovibrio an advantage over bacteriophages that have also been considered as antimicrobial agents, since bacterial resistance to bacteriophage is relatively common and bacteriophages are much more species-specific. It will be necessary to monitor bacterial genomes of prey species in order to detect resistance mutations as they arise year-term experiments of the like are currently being carried out, in which Bdellovibrio and certain hosts are routinely combined and changes in the host genome, indicating prey adaption, is identified.

The ability of Bdellovibrio to prey upon a large variety of bacteria and use their constituents with such energetic efficiency has ensured their success, and hence their distribution in many diverse environments. However, the predatory nature of these bacteria hindered early studies, but due to the advances in biochemical techniques, including gene-manipulation and molecular phylogenetic techniques, our understanding of the unique association that Bdellovibrio develops with its prey has greatly increased. The complete sequencing of the B. bacteriovorus HD100 genome has brought about a revival in interest in these unique bacteria and allowed researchers to evaluate their potential as antimicrobial agents with a variety of applications. Further studies of gene expression and the fates of their products will help to reveal the processes involved in the development inside the bdelloplast. Despite the extensive research that is still required if Bdellovibrio are to be used as therapeutic agents, the possibility remains that it could be the answer the ever growing number of antibiotic-resistant pathogens.