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a wild-type gene; Beckwith et al., 1985). In theory, the recircularized recombinant plasmid carrying a DNA fragment containing the wild-type gene will complement the previously inactivated loci.

Both chemical ( ethyl methane sulfonate) and transposon (Tn_;i) mutagenesis procedures have been used to generate a suite of A. putrefaciens 200 mutants deficient in iron reduction activity. Mutants in both high-rate (induced) and

low-rate (constitutive) iron reduction activity were identified via a newly developed technique for screening mutagenized A. putrefaciens 200 cultures. The broad host range cosmid cloning vector p VKlO0 (IncPl) was mobilized into A. putrefaciens at relatively high frequencies ( one transconjugate per every 100 potential recipients) by either two-way or three-way mating ( conjugation) procedures. An A. putrefaciens 200 gene clone bank was constructed in p VKlO0 and housed in E. coli mobilizing strain S17-1, thus facilitating complementation studies via two-way crosses between the gene clone bank and the suite of iron-reduction mutants. The present study details the results of such complementation analysis, and in addition, outlines preliminary biochemical characterization of the iron-reduction-deficient mutants.

II. MATERIALS AND METHODS A. Complementation Experiments

Figure 5.2 outlines the overall strategy followed in complementation analysis of the previously generated suite of iron reduction-deficient mutants of Alteromonas putrefaciens 200. The A. putrefaciens 200 gene clone bank housed in E. coli

mobilizing strain S17-1 (and stored permanently in the individual wells of the Micro Test plates) was used in two-way matings to transfer the clone bank to the suite of iron-reduction-deficient mutants. A 96-pronged "Clonemaster" stamp (Immusine Laboratories, Inc.) was used to transfer the 96 clone bank members contained in a single Micro Test plate to an identical plate containing 50 µLLB in each well. It was estimated that each prong transferred approximately 10 µL of cells

COMP~EMENTATION STRATEGY NC!onemoster" Transfer

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Figure 5.2. Overall strategy used in complementation of the iron-reduction-deficient mutants of Alteromonas putrefaciens 200.

during inoculation. The 96 clone bank members were subsequently incubated 18 hours at 37°C without shaking. After the 18 hour incubation period, a previously selected iron-reduction-deficient (Rfr) mutant [grown to log-phase in LB

supplemented with rifamycin (100 mg/L) and kanamycin (SO mg/L if required), washed and resuspended in equal volume LB] was added to each well of the MicroTest plate with an automated 8-pronged pipettor (EDP-MS; Rainin

Instrument Co.). The mixture ofrecipient and donor cells ( 4: 1 ratio; 0.1 ml total volume) was then incubated at 30°C for 4 hours. After the 4 hour mating period, the "Clonemaster" stamp was used to transfer the mating mixture from each of the 96 wells to selective media. The selective media consisted of nutrient agar

supplemented with rifamycin SV (100 mg/L), kanamycin (SO mg/L if required), tetracycline (10 mg/L) and ferric chloride (to 0.15 mM as Fe). The selection plates were incubated 50 hours at 30°C and the resulting colonies sprayed with a fine mist of filter-sterilized ferrozine (0.15 mM; Sigma). The formation of magenta-colored colonies was monitored for 15 minutes and any positive phenotypes picked to identical selection media for single-colony isolation. After two single-colony

isolations, each transconjugate was tested in liquid culture for iron reduction activity after growth at either high ( > 200 µM) or low ( < 2 µM) oxygen tension. A 5 ml liquid culture grown overnight in lactate medium supplemented with rifamycin SV (100 mg/L), kanamycin (SO mg/L if required) and tetracycline (5 mg/L) was used to inoculate a 1.5 L batch reactor (Biostat M, B. Braun Co.) containing 1 L of identical medium. At an optical density of A600

=

0.25 both oxygen utilization and iron reduction rates were measured as previously described (see Chapter 3). The iron reduction activities of the complemented transconjugate was compared to the activity of the original iron-reduction-deficient mutant, and to the activity of a randomly selected transconjugate that produced a negative iron reduction phenotype during complementation experiments.

In order to classify the mutants into complementation groups, an array of two-way matings were carried out. Each iron-reduction-deficient mutant was mated individually with each clone bank member that had previously restored the positive iron reduction phenotype to a mutant strain. Experimental procedures were

identical to those used to mobilize pVKlO0 from£. coli mobili⁷;ng strain S17-1 into A. putrefaciens 200 (see Chapter 4 ). Negative controls consisted of individual

two-way matings between each mutant and either E.coli S17-1 (pVKlO0) or a randomly selected clone bank member that had previously failed to restore a

positive iron reduction phenotype to that particular mutant. To test the effect of the cosmid vector on iron reduction activity, p VKlO0 was mobilized into A. putrefaciens 200 (via the two-way mating procedures described above ),and a resulting

transconjugate screened for iron reduction activity on solid media via Screening Technique No. 3 (see Chapter 3) and tested for iron reduction activity in liquid culture.

The recombinant cosmids harbored in each clone were isolated via the mini-prep procedures described previously (see Chapter 4). DNA from each mini-prep was digested with Hindlll and the cloned DNA fragments analyzed via agarose gel (0.8%) electrophoresis.