nai

Project Progress

Aim 1 of this CAN-7 project is development of the model- co-operative growth between Escherichia coli and its predator Bdellovibrio bacteriovorus. As a prelude to establishment of the model, a fluorescent marker system to assist in cell tracking and selection is highly desirable. Tn7-based chromosomal tagging of E. coli with green fluorescent protein (GFP) has been completed and analogous marking of B. bacteriovorus with mCherry is close to being comlete. Although chromosomal gene insertion has not previously been reported for Bdellovibrio, the Tn7 system has repeatedly been shown to be useful for introducing genes into the chromosomes of a wide variety of gram-negative and gram-positive bacteria [1]. To date we have successfully delivered an antibiotic resistance cassette (KmR) coupled to the mCherry gene into the correct location the B. bacteriovorus chromosome but have not detected fluorescence. Alternate promoter-mCherry combinations are currently being constructed to see if better transcription enhances fluorescent gene production and detection. We are also in the midst of developing a reliable protocol for labeling and detection of 16s DNA sequences in fixed cells (fluorescence in situ hybridization or FISH) that can be coupled with fluorescence activated cell sorting (FACS). To assist in detection of B. bacteriovorus with reduced virulence toward its E. coli host (and thus B. bacteriovorus en route to symbiosis with E. coli), a luminescence-based assay for high-throughput measurement of virulence developed by the Sockett lab (University of Nottingham, [2]) has also been adapted for use with our E. coli strain, CV103.

Two essential characteristics of mutualistic endosymbioses are (1) a commitment of the attenuated parasite to existence inside of the host cell and (2) positive reinforcement of co-existence via benefit conferred to the host cell. In order to experimentally “push” B. bacteriovorus toward an intracellular lifestyle, selective regimes that either penalize parasites that exit their hosts, reward hosts for harboring parasites and/or reward parasites for not harming their hosts have been pursued. Experiments in which a set of 20 E. coli auxotrophs unable to synthesize necessary amino acids that B. bacteriovorus is able to produce were individually co-cultured with B. bacteriovorus initially yielded encouraging results (i.e. growth of the E. coli auxotrophs on minimal media in the presence of the parasite) but no molecular evidence that growth of the E. coli auxotrophs was due to presence of the parasite rather than reversion of the auxotrophy via mutation could be established. A mutual positive-reinforcement scheme in which E. coli auxotrophs engineered to excrete an ampicillin detoxification enzyme (β-lactamase) are cultured on amino acid-depleted ampicillin-laced media alongside B. bacteriovorus has also been developed. To aid in detection of symbioses and de-couple resource sharing from the harmful effects of parasitism, a host-independent derivative of B. bacteriovorus in being used. Formation of colonies in close proximity to one another under the above experimental conditions would be an indication of co-operative growth, at which point the host-independent B. bacteriovorus could be genetically converted back into an attenuated parasitic form to test the viability of such a symbiosis under a single membrane.

With the view that understanding how the parasite-host relationship is maintained will aid in attenuating it, we have undertaken a number of experiments aimed at identifying genes necessary for predation/virulence and host resistance to predation. To identify genes necessary for virulence, Tn5 insertion libraries were constructed using host-independent B. bacteriovorus. These libraries were screened for mutants that, through the deletion of a gene, produced lower levels of two main virulence factor- proteases and nucleases. No isolates with statistically significant reduced protease production were found among approximately 1100 mutants screened. The reasons for this could be many, but it may be that growth of B. bacteriovorus on the standard media for growth, which includes polypeptides from yeast extract and tryptone, may require protease production. To circumvent this issue, we have been developing an alternative media formulation that consists only of free amino acids. Screening for isolates with reduced nuclease production has been attempted, but only minor production of nuclease from control isolates has hampered identification of mutants on standard indicator plates.

To better understand the evolutionary trajectory of long-term association between E. coli and B. bacteriovorus, evolution experiments in which the two are co-cultured in their un-altered state (ie. not attenuated) are underway. Serial passaging of the two potential partners over only a few cycles has yielded isolates of E. coli that appear to be less receptive to B. bacteriovorus infection that their ancestor. Testing of these isolates to conform their phenotype and analogous experiments under more controlled culture conditions (chemostats) are underway. Confirmation on typing of these E. coli variants will assist in disentangling the complex relationship between host and parasite and provide clues to how the host must change in order to live in “harmony” with a lethal parasite.

1. Choi K, Gaynor J, White K, Lopez C, Bosio C, et al. (2005) A Tn7 -based broad-range bacterial cloning and expression system. Nat Methods 2: 443 – 448.
2. Lambert C, Smith MC, Sockett RE (2003) A novel assay to monitor predator-prey interactions for Bdellovibrio bacteriovorus 109 J reveals a role for methyl-accepting chemotaxis proteins in predation. Environ Microbiol 5: 127-132.