Screening for novel bacteriophage infecting various eubacteria

Steven Slater

Bacteriophage can be exceedingly useful organisms forstudying both the physiology and genetics of bacteria. Much of ourcurrent knowledge regarding DNA replication, genetic regulation,mutagenesis and other cellular processes in Gram-negative entericshas been gleaned from extensive analysis of phage such as lambda,phiXl74 and fi. In addition, phage can be of enormous practicalimportance, allowing genetic transduction (e.g.that performed by P1and P22) or providing powerful tools for molecular biology ( e.g. thelambda- and M13-based cloning vectors). Given the extensiveutility of phage in the study of enterics, phage can also be expectedto contribute significantly to characterization of numerous otherorganisms. Thus, I have attempted to isolate bacteriophage capableof growth on several organisms for which genetic systems arerudimentary or nonexistent.

Methods

Host Organisms: Attempts were made to isolate bacteriophagecapable of growth on Xenorhabdis luminescens, Rhodospirillumrubrum, Clostridium mayombii , two newly isolated Clostridiumspecies, and three newly isolated luminescentVibrio species. Thegeneral procedure described below was essentially identical for allattempts at phage isolation, except that all Clostridiummanipulations were performed anaerobically, and Rhodospirillumplates were incubated in gas-pack jars.

Inoculum: The inocula were homogenized, centrifuged in amicrofuge to remove particulate matter, and filtered through a 0.45micron filter prior to dilution and plating. In general, inocula wereinitially added to log-phase cultures of each test organism andgrown for 1-2 days in an attempt to amplify phage originally presentat low titer. The resulting cultures were cleared of cells bycentrifugation, filtered and used to inoculate top-agar cultures ofthe test organism. Serial dilutions were performed in NutrientBroth (Difco) and 0.1 ml of each dilution was added to 3 ml of theappropriate top agar (plus or minus 10 mM calcium chloride)containing 0.2 ml of an overnight culture of the test organism. Thesolutions were mixed, plated on the appropriate medium, and

S..-) incubated at room temperature for several days. The following

inocula were screened for phage capable of infecting all testorganisms

Sea water from garbage beachFresh water from numerous small ponds in the Woods Hole areaSoil samples extracted with nutrient broth

The Clostridium species were additionally inoculated with gutextracts of the North American termites Cryptotermes cavitansand Reticulotermes flavipes., and with filter-sterilized supernatantsolution of each of the other Clostridium species. Both the termiteguts and the Clostridium species serving as inoculum were derivedfrom cultures grown in media with or without 0.1 mg/mI mitomycinC in an attempt to induce any lysogenic phage present in the hosts.

Results

Initial attempts to detect phage in termite-gut extractswere hampered by extremely poor lawn formation, but this problemwas circumvented by carefully maintaining anaerobic conditions inthe top agar and cooling the top agar to about 45 C before addingcells. Despite these precautions, no plaques were ever detected inthe Clostridium cultures. Similarly, none of the proceduresdescribed above yielded any identifiable phage as monitored byplaque formation on any of the other tested strains.

C

Transposon M utagenesis of Xenorhabdis lurninescens.

Xenorhabdus luminescens is a Gram negative rod that has asymbiotic relationship with certain parasitic nematodes. Theorganism has two different physiological states, known as theprimary and secondary forms. These forms are distinguished by anumber of physiological parameters, including pigment production,lipase production, protease production and antibiotic production.Organisms of the primary form spontaneously convert to secondaryform, but those in the secondary form are incapable of conversion tothe primary form. The mechanism by which this switch occurs isunknown, and very little work has been done regarding any aspect ofX. luminescens physiology or genetics.

Methods

As a first step toward isolation mutant of Xenorhabdis withaltered developmental regulation, an attempt was made to introducetransposons into X. luminescens using three separate vectorsharbored in E. coiL These systems included phage P1 carrying Tn5,F+ carrying Tn5, and a plasmid encoded defective, chloramphenicolresistant TnlO with transposase expressed from the same plasmid(pJD12). Attempts to introduce these transposons were made usingstandard protocols for P1 transduction, E. coil mating, andtransformation, respectively. After 1 hour of outgrowth, theresulting cells were plated on media containing the appropriateantibiotics (kan for Tn5, cam for TnlO) in an attempt to selectorganisms that had received the transposon.

Results

Xenorhabdis was susceptible to both kanamycin andchloramphenicol, although spontaneous Kan resistant colonies aroseat a rate of approximately one in io7. No spontaneously cam-resistant strains were detected on the control plates. In no casewere the presence of Tn hops ever demonstrated. There are a numberof possible problems that could have negatively effected the results.In all cases, a strong restriction barrier may have destroyed allincoming DNA, thus making the possibility of a Tn hop remote. Inaddition, bacteriophage P1 may not adsorb to Xenorhabdis, or E. coilmay not be capable of mating with Xenorhabdis. Sincetransformation ofXenorhabdis has been demonstrated, it is unlikelythat the cells were not competent for uptake of DNA. Rather, a

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restriction barrier remains the most likely cause for the failure ofX. luminescens to be transformed by pJD12.

It may be possible to overcome a strong restriction barrier bydramatically increasing the efficiency of transformation. Thus,electroporation is a likely method for getting around theseproblems, and will be attempted in the next few weeks.