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considered to be the highest quality, ash-free paper then available. The first 50 ml
of the filtered medium was discarded as this has come into contact with measur-
ing cylinder, the filter funnel and the filter paper itself. The remaining medium was
then dispensed in 100 ml lots into cleaned 250 ml conical flasks. Cleaning the flasks
necessitated devising another strategy. Initially flasks were cleaned with chromic
acid although this procedure was soon replaced by filling the flasks with alcoholic
KOH and leaving overnight. This was followed by washing them in distilled water
then standing for another night in 2 M HNO 3 . The flasks were finally thoroughly
rinsed in distilled water and allowed to drain upside down before being filled with
medium the next day. The procedure was extremely tedious and time-consuming and
services of a dedicated technician were then needed to do all the preparatory work.
Winder and O'Hara [ 11 ] also carried out some significant analytical work on the
cellular content of iron in the mycobacterial cells. Under the most stringent iron
deficient growth conditions, M. smegmatis contained 64 μ g Fe/g cell dry weight
suggesting that this was the lowest possible concentration needed for the cells to
function. Obviously, as the mycobacteria have an essential requirement for iron, the
cells would not be able to grow if there was, literally, no iron in the medium. They
could not synthesize the various cytochromes and iron-containing enzymes that are
vital for cell metabolism and growth. If iron was not limiting, then the iron content
of the cells rose to 224 μ g/g cell dry wt. Values for the zinc content of the cells
were simultaneously calculated as 11 and 43 μ g/g cell dry weight, respectively. It
was evident, however, that iron-deficiently growing cells were adapting to allow
some growth to occur but clearly major changes were occurring within the meta-
bolic pathways to minimize the detrimental effects of iron deficiency.
One of the main effects of iron deficiency on metabolism appeared to be a decrease
in the DNA to protein ratio [ 8 ]. This was subsequently attributed to there being a
considerable increase in the activity of an ATP-dependent DNAase [ 12 , 13 ] and a
DNA polymerase [ 14 ]. Further work on the DNAase [ 15 , 16 ] considered that it was
involved in recombination repair and possibly in excision repair. Interestingly, how the
increased activities of both these enzymes then correlated with the original discovery,
that there were low concentrations of DNA in iron-deficient cells, was resolved by
Winder and Barber [ 17 ] who reported that hydroxyurea could induce the same effects
as iron deficiency, including cell elongation. However, one cause of DNA degrada-
tion might be in the lowered activity of ribonucleotide reductase which, in E. coli , is
known to contain iron as an essential co-factor [ 18 ] and would therefore lead to an
alteration in the pool of nucleotides. This possibility, though, does not appear to have
been followed up in M. smegmatis and the general consensus was that the decrease in
DNA during iron deficient growth was a secondary but not a primary effect.
The paper by Winder and Barber [ 17 ] and a subsequent one by MacNaughton
and Winder [ 19 ] were the last papers that Frank would write on aspects of his
work connected to iron deficiency in the mycobacteria. He then, with respect to
his continuing interest in the mycobacteria, concentrated on trying to unravel the
mechanism of action of isoniazid (INH) as one of the more potent anti-TB com-
pounds then available. This work had also begun in the 1960s and was carried out
in parallel with the iron deficiency studies.
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