Seminar Minutes of 9-19-74

I. Hela: Dinitrosoguanidine treated B. subtilis on minimal media having
Galactose instead of Glucose, shows a very slow utilization of the Galactose.

l.

2.

This may be due to contamination by trace amounts of Glucose in

the Galactose. Therefore, to the minimal media having galactose

add trace amounts of glucose and observe the difference in growth.

Does this represent a lack of metabolic steps or inhibition?

Gal” may be self inhibiting and also for SB19 on Gelactose medium.
Therefore, cross streak the two; plate mixtures of Gal” and SB19

in different proportions. +
Various percentages of lactose were tested in selecting for the Lac
marker. The wild type grows only on 5% , and not with other concentrations
of lactose. It may have the enzyme B galactosidase (which can be
determined by colorimetric assay), but be missing the enzyme permease
(the B. subtilis membrane may not be able to accomodate permease).

Grow cells on glucose and a low level of lactose.

Try to get maltose mutants and cellobiose mutants.

Get a collection of other Bacillus strains in order to look for

crossing with the whole group of Bacillus. Transformation with high

M.W. DNA may accomplish the same purpose in studying cross transformability
(to get in + alleles by transformations). Two sets of reliable mutants
are needed (there are problems if either one reverts). Build up
multiple marker strains for recombination. Streptomycin Resistance

is a good reliable auxotroph to cross with Streptomycin Sensitive
prototroph; then look for Streptomycin Resistant prototroph. Order a
dozen type species strains that are different from one another from ATTC.

II. Hela: Salmonella Rough cells + P22 DNA result in Transfection; compiete
phage are released providing plaques. The cells must first be made
competent. The rough Salmonella have lost the surface polysaccharide
and therefore cannot absorb free phage but can be transfected with the DNA.

Ll.

Observation of the cells under the microscope after UV radiation

following Transfection, showed clumps. Clumping occurs in Catlos
resuspension after one hour of shaking (which separates the clumped cells)
could explain the later increase in the number of cell infectious centers
(but the sharp increase of plaques after after one hour argues against
this). When put in broth with growth for 21/2 hours a greater number

of elongated cells were seen. It is desired that the radiation should
have no effect other than to prevent cell division; the problem with UV

is that it has a number of other effects. Therefore, look for other
inhibitors to prevent cell division (e.g., crystal violet) and that
operate at the level of the cell wall and not the DNA replication.

There is delayed bursting in about an hour; eventually the phage goes
through the vegetative cycle. After Transfection perhaps there are
lysogenic complexes that are not fully stabilized. There may be more
lysogenic ceils than cells with lytic phage. After lysogeny has

occurred, irradiate the culture to induce lysis. Try induction wigh
Mitomycin or other efficient inducers. Prepare lawns with 16°- 10
bacterial colonies on each plate; these should be the bacterial survivers
of the competency treatment and the exposure to DNA. Grow the cells for
8 hours and wash them well to eldminate excess free phage. Then test for
lysogenics in the bacterial population. Look for clones that can still
produce phage and release them upon induction. Use pour plates to see
plaques in the vicinity of rough clones that produce phage; it is necessary
to look for a later stage of phage release. Partially stabilized
lysogenic complexes may not produce the normal amount of repressor
that stable lysogenics do.

nent
Seminar Minutes of 9-19-74 continued, p.2

3. In assaying for infectious centers, observe the cells under the
minroscope for clumping at the various steps in the competency
treatment. See if there is any correlation between the efficiency

of
III. Hela:
1,
2,
3.

Transfection and the qualitative level of clumping.

There are indications that the DNA alone may be toxic to the cells.
Results of the experiments to test this were not reproducible.

The sensitivity of the method is the crucial point.

Always do viable counts before and after adding the DNA.

IV. There was a discussion of terminal splicing of DNA,

1.

The argument for end to end joining, that P22 is subject to

random permutattbns of the duplicated ends, is not a strong argument
since it is not a random ending.
2. There is an inability to get T7-P22 dimers. Also, Dush¢o reports

3.

that T7 is not joinable to itself.

J.L. questions whether fhe difference in behavior of E.coli and
T4 ligases (as reportec“by Sgaramella:~ T4& ligase with P22 DNA
provides terminal joining of fully base paired terminii, whereas
T4 ligase or E. Goli ligase with SV40:DNA provides cohesive
joining of complementary single-stranded sticky ends) is truly
the Phenomenon that war: reported. Even a minimal amount of
erosion should destroy the flush endedness, and then there should
be no more end to end Linking. The behavior of the two ligases
should then be alike, with sticky end joining predominating.

It is questionable whether end to end joining does occur, the
evidence being very weak.

V. Betty: Superbug Search

°

4.

Samptes were obtained from a vatiety of sources; creeks along

. Campus Drive, Stanford Ave., and Galvez and Arboretum; Baylands

swamp and duck pond; and soils from garden, Medical School
courtyard and Baylands swamp area.

Different type colonies were isolated and described both on

pour plates (with 4mm soft agar depth) and streaked plates.

From a number of the samples, large abundantly growing colonies

were obtained with overnight incubation.

Growth ofi various media was compared: that on SOminimal media +
0.5% glucose gave the best growth, Glycerol + Vitamin Mix gave

good growth with smaller colonies than on SO, Ethanol + Vitamin Mix
gave good growth with colonies smaller than in Glycerol, and
Ammonium Acetate + Vitamin Mix gave either very slight growth

of tiny colonies or no growth at all.

Cell morphology of the isolated colonies are being described

by gram stains.

Generation Time studies in Spizizen Minimal Media +0.5% glucose

and in TB Complete Media are in progress for all the isolated
colonies. Ohe of the colonies isolated from the creek along Galvez
gave a doubling time of 23 minutes in minimal media and a

doubling time of 21 minutes in complete media (as compared to 28-30
minutes for E. coli, which was tested simultaneously). Its colonies
are larger and grow more amply than those of E. coli on the same
media incubated for the same period of time.

Suggestions made include: identify the above bacteria taxonomically,
do viable counts fmom the minimal flask, try growth in a mineral

mix recommended by J.L. , try Spizizen Minimal without the glucose
(the citrate in the Spizizen may be serving as the carbon source),
try growth in minimal media of different pHs, test the colonies that
are difficult to separate and purify for symbiosis, follow daily
viability counts of newly gathered samples.

wane