Overall strategy used in genetic complementation analysis of the iron reduction-deficient mutants of Alteromonas putrefaciens 200. Arnold and co-workers (1986) investigated the iron reduction system of the non-fermenting chemotroph Alteromonas putrefaciens 200.
INTRODUCTION
The goal of this part of the project was to clarify uncertainties regarding the physiological role of iron reduction in relation to No3 and Oz utilization. Second, the effect of Oz on iron reduction activity was investigated using cyanide as an inhibitor of electron transport to O2.
MATERIALS AND METHODS
In the third (control) experiment, No3- was added up to 1.86 mM while Fe(III) was omitted. Ferrozine (up to 7.2 mM) was added to the batch prior to simultaneous addition of FeCl3·6H2O and KNO3.
Fe(III) Reduction in the Presence of Molecular Oxygen and Cyanide
RESULTS AND DISCUSSION
Simultaneous Reduction of Fe(III) and No3- by Alteromonas putrefaciens 200
SUMMARY
The combined effects of nitrate, molecular oxygen and cyanide on iron reduction activity were used to study mechanistic aspects of this. Molecular oxygen completely inhibited the Fe(III) reduction activity; however, when electron transport to Oz was blocked by the addition of cyanide, Fe(III) reduction activity was restored, although not at maximal rates.
The availability of iron reduction-deficient mutants will facilitate both genetic and biochemical characterization (see Chapter 5) of the iron reduction system of Alteromonas putrefaciens 200. The detection of respiratory-defective mutants is often complicated by the existence of secondary metabolic systems (e.g., multi-branched respiratory chains, fermentation pathways) that can support normal growth even in the absence of the respiratory component of interest. After transfer of the donor plasmid to an appropriate host strain, transposons are thought to autoreplicate (via self-encoded replicases) and insert a second copy into the host genome.
Thus, class IB-type transposable elements (Table 3.1) appear to be the best candidates for use as mutagens in Alteromonru putrefaciens 200.
Simon
Nutrient agar was supplemented with filter sterilized FeCl3·6H2O (to 0.15 mM as Fe) for use in screening experiments.
Lidstrom D. Bartlett
HQNO was added to several wells at a level (lo-4 M) previously shown to be inhibitory to constitutive iron reduction activity. A fine mist of filter-sterilized ferrozine (37.2 mM) was immediately sprayed onto the filter paper to wet the colonies. Each oxide (except ferric hydroxide, added as FeCl3·6H2O) was prepared synthetically (Pfizer Corp.) and added to sterile molten nutrient agar cooled to 50°C to avoid problems associated with high-temperature conversion of the iron oxides to hematite (a -Fe2O3), the thermodynamically favored oxide form.
When the colonies were of sufficient size (i.e., 0.5 mm), each was checked for the presence of clearing zones (indicating iron reduction activity).
Mutagenesis Procedures
Chemical Mutagenesis (Ethyl Methane Sulfonate)
Specific auxotrophs were identified by replica plating master plates onto MSSG plates supplemented with various combinations of amino acids, vitamins and purine/pyrimidine bases. Specific auxotrophs were picked from the master plates and stored in LB:glycerol (85:15 vol%) at -8o0c for future use. EMS-treated cells were washed and resuspended in MSS before plating serial dilutions on nutrient agar supplemented with FeCl 3 6H 2 O (to 0.15 mM as Fe).
Those colonies showing aberrant magenta color formation were picked onto nutrient agar plates and rescreened for subsequent verification as mutants lacking Fe(III) reducing ability.
Transposon Mutagenesis
The screening technique used to detect the abnormal iron reduction activity of the Tn.5 mutants was identical to that technique (#3) previously used in the EMS mutagenesis experiments. A fine mist of ferrozine (30 mM) was then sprayed onto the resulting individual colonies (i.e., Tn.5 recipients) and all bright magenta colored colonies were picked onto NFRK plates. Stable Tn.5 mutants showing bright crimson staining were screened for iron-reducing activity in batch liquid culture as described above for iron-reduction-deficient strains generated by EMS.
To ensure that Km did not affect iron reduction activity in liquid culture, control experiments were performed with Tn.5 mutants that had previously shown a positive phenotype on NFRK plates.
Iron Reduction Activity of Potential Iron-Reductase-Deficient Mutants
RESULTS AND DISCUSSION A. Screening Technique
Each of the three methods developed and tested for use in this study was capable of detecting iron reduction activity by this method. The intensity was highest for the middle cells and decreased radially until the cells of the budding outer ring appeared almost colorless (see Figure 3.2). In addition, the degree of magenta color intensity depended on the identity of the iron oxide added to the nutrient agar (results not shown).
2, the intensity was highest for the middle cells and decreased radially until the nascent outer ring cells appeared almost colorless.
Mutagenesis Procedures
Four of the isolates (false negatives) were capable of iron reduction and oxygen utilization at rates almost identical to those of the wild-type strain; however, the four other isolates (F18, F37, Wanda, Flamingo) showed extremely low iron reduction activity by the constitutive system. The genetic "fingerprints" of the four Tn.5 iron reduction-deficient mutants (Figure 3.9) were identical to the wild-type A. Of the five classes of broad host range plasmids isolated from gram-negative bacteria (IncC, N, P , Q and W), three (IncP, Q and W) were used in the construction of broad host range cloning vehicles.
The trfB operon consists of the A, B, C, and D kor (kill on horse) genes whose gene products control the expression of their respective kil gene complements, (A, B, C, and D).
Gene Transfer Techniques 1. Generalized Transduction
Direct Transformation
The procedures followed for large-scale preparations of pSP329 (inE. coli HB101) and pVKlO0 (inE. coli HB101) were essentially those of Maniatis et al. After the heat pulse, the mixtures were allowed to cool to room temperature for 10 min before adding 3 ml of lactate medium to each. The mutagenesis procedures used in generating DNase-deficient mutants were essentially identical to those used in previous experiments to generate iron-deficient mutants of A .
However, instead of plating EMS-treated liquid cultures on nutrient agar (supplemented with ferric iron), mutagenized cells were plated on DNase test agar (Difeo) supplemented with methyl green (0.01.
Conjugative Gene Transfer Systems
DNA fragments in the size range of 15-20 kb were obtained by size fractionation of partial HindIII digestion of the wild-type DNA preparation. After each processing step (linearization and dephosphorylation), an aliquot (2 µg) of the cosmid preparation was loaded on an agarose gel to check plasmid size and yield. An aliquot (1 µL) of the ligation mixture was loaded onto an agarose gel to check concatamer size and yield.
The positive lambda control (i.e. the packaging of wild-type lambda DNA) was also used by the.
Development of a Gene Transfer System
A random sample of 25 members of each clone bank was checked for the presence of p VK.100 and the size of the cloned insert. Results of the liquid culture technique used to determine the immunity range of Pseudomonas aeruginosa phage B3. IncPl-based cloning vectors (i.e., those vectors containing the mob site of the broad host range plasmid RK2) can be mobilized in a wide range of gram-negative bacteria (Barth, 1979) suggesting that such cloning systems may be applicable to A.
It is interesting to note that the frequency of conjugal transmission of pVK100 is independent of the type of mating system used in vector mobilization (see Table 4.3).
Construction of an Alteromonas putrefaciens 200 Gene Clone Bank
A random sample of 25 members from each clone bank was checked for the presence of pVK1O0 and the size of the cloned insert (see Figure 4.10). With the ability to transfer cloned DNA into selected (mutant) strains, complementation analysis of the iron reduction system A. Analysis of the origin of vegetative replication of the RK2 plasmid of a broad host range by transposon mutagenesis.
Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria.
MUTANT
MATERIALS AND METHODS A. Complementation Experiments
After the 18 h incubation period, a previously selected iron reduction-deficient (Rfr) mutant [was grown to log phase in LB. The iron reduction activities of the complemented transconjugate were 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. Each iron-reduction-deficient mutant was individually mated to any member of the clone bank that had previously restored the positive iron-reduction phenotype to a mutant strain.
To test the effect of the cosmid vector on iron reduction activity, pVKlO0 was mobilized into A.
Biochemical Analyses
3 (see Chapter 3) was used to detect any T121 transconjugates in which the positive iron reduction phenotype was restored. Of the 192 clones mated to T121 and screened for iron reduction activity via Screening Technique No. 3, two [designated S4-E-2 and SlS-F-4 (plate-row-column)] have a positive iron reduction recovery phenotype to mutant T121.
To test the effect of the cosmid vector on the positive iron reduction phenotype, pVK1O0 was mobilized into A.
I UNITS 0.10
- PROJECT SUMMARY
- EXPERIMENTAL DESIGN
- Defined Lactate Medium as Substrate
- Primary Effluent Wastewater as Substrate
Reduced-minus-oxidized difference spectra of cell-free extracts of iron reduction-deficient (transposon) mutant Tl21 grown at low oxygen tension (data not shown for cells grown at high oxygen tension). These results suggest that b- and c-type cytochromes may be involved in the low-rate iron reduction system of. Complementary biochemical and genetic analyzes were used to study the molecular basis of the (dissimilative) iron reduction system of A.
A suite of iron-reduction-deficient mutants were generated via chemical (EMS) and transposon (Tnj) mutagenesis procedures.
Time (hours)
The initial reductive dissolution rates (1.0 x 10 -5 M.hr -1 ) were approximately 33% of the measured rate when batch cultures were grown on defined lactate media. viii) The oxygen utilization rates (3 × 10-4 M hr-1) of batch cultures grown on wastewater media were also 33% of the measured rate when batch cultures were grown on defined lactate media. Results from the batch experiments described above indicate that the rate of reductive dissolution of iron from low grade ore is influenced by several factors. A decrease in iron ore particle size increases reducing dissolution rates, probably by increasing the surface area available for microbial attachment.
Substitution of primary effluent wastewater with lactate as substrate during growth and reductive dissolution experiments resulted in a 67% reduction in reductive digestion rates.
