QUARTERLY REPORT

November 14, 1969

Jane P . Foster

i . Terpene Metabolism

Extensive work was done by Dr . Benko, Dr . Ayres, and myself to

establish a t},in layer chromatographic method of detecting and ident-
ifying 2,4-DNP (di-nitro-phenol) positive compounds . Then fungal
cultures were fed 0 .2% and 0 .02% alpha ionone as carbon source and
the chloroform extracts of these cultural filtrates were examined by
thin layer chromatography for 2,4 -D :vP positive compounds indicating
the conversion of alpha ionone to other, more polar compounds .

An enrichment technique was set up to select soil microorganisms

which would utilize alpha ionone as their carbon source . Ten samples
of soil from a pine forest were tested and 9 out of 10 samples showed
bacterial growth after being shaken at 25°C in medium with 0 .2% alpha
ionone as sole carbon source and NH4N03 as nitrogen source ; and also
in medium with tryptone and yeast extract (0 .01%) plus 0 .2% alpha ionone
and NH4C1 as nitrogen source . Growth was much better in the latter .
When chloroform extracts of the cultural filtrates were examined by
thin layer chromatography there was no evidence of the appearance of
new 2,4-DNP positive or H2SO4 positive compounds . Enrichment cultures
are continuing and the supernates will be examined for other compounds .

II . Lysis of Arthrobacter Cells to Release Glucose Isomerase

A simple and fast screening method for detecting microorganisms

perhaps capable of lysing Arthrobacter cells has been devised . A
culture plate consisting of 1 .5% agar + 0 .5% peptone + heat killed
Arthrobacter cells (10 ml . washed, resuspended & boiled cells per 100 ml .
agar) is spread with bacteria . After as little as 24 hours at 30°C,
some bacterial colonies show a zone of clearing, presumably indicating
lysis of the Arthrobacter cells . Suspected colonies can then be isolated
and purified on the same agar and then grown up in shake culture (nutrient
broth, 1% yeast extract, or nutrient broth + 2% Arthrobacter cell suspension) .

Water extracts of soil samples and rabbit pellets have been examined
in this way . Also, rabbit pellets have been pre-enriched in nutrient
broth for varying times at 30°C, which doesn't increase the number of
lytic colonies . The best method to obtain lytic colonies from the rabbit
pellets seems to be placing the pellet directly on the agar plates described
above and incubating approximately 48 hours at 30°C .

To test the eolonies for lysis of Arthrobacter cells, the supernate

of the 48 hour old organism in shake culture was used . The substrate is
a 72 hour culture of Arthrobacter cells grown in a shake medium devised by
Larry Hayes . The Arthrobacter cal.lz; are washed and resuspended irn di ;- ;iilled
water . The cells + supernate are incubated together 1 ho,ir at 37°i : -nd the
supernatea tested on the autotechnicon for fructose, indicating thc F .resence
of glucose isomerase .

So far , na colonies have beer, found which release glucose isomerase

from the Arthrobncter celis f ~-~o thc su r orn .ate . ~ ;I
' c ~ -L % ~ t
Jane i' . Foster