1;  <JL2>OLDTEXTS.SET;7804   Fri 7-Apr-78 2:31PM                             PAGE 39:1

        Application for Renewal, Dec. 1, 1977-Nov. 30, l978

        to National Aeronautics and Space Administration

        Cytochemical Studies of Planetary Microorganisms.
        -------------------------------------------------

        GENERAL INTRODUCTION.
.fi
        As has been the case for some time, our research effort is
divided into a) microbiological, and b) analytical-chemical studies.
These are discussed in two main partitions of this renewal application
and progress report. On this occasion, all of the methodologically
oriented efforts of past years have been 'spun off' into programs
supported from other sources; our current proposals -- truncated by
progressive inflation at level-dollar support -- converge on the theme
of the origin of life and fundamental issues in the evolution of
primitive microorganisms.

        The overall program of work summarized here far exceeds what
could be accomplished within the actual budget requested from NASA.
We have the advantage, however, of considerable momentum from previous
experience and equipment. We also anticipate an advantageous overlap
with support that we are planning to request from ERDA and from the
NIEHS with respect to health- and energy-related aspects of the biology
and chemistry of polymeric carbon. {Only the most preliminary contacts
have been made with those agencies at this time.} The funds requested
will enable us to sustain the key professional staff during this transition,
and to continue to focus on those issues that are of specific interest
to the NASA program office. If other support cannot be obtained, we will
be constrained to an opportunistic pursuit of only the most promising
research gambles that present themselves during our explorations.

                --------------------------------


          Research plans 1977-78.


          A.     Microbiological and genetic studies
     For several years, the main focus of microbiological
research in this laboratory has been on the construction of
simple model systems for the genetic diversification of
simple genetic codes. This work entailed the use of 'recombinant DNA'
technology to construct bacteria that had
simple monotonous synthetic sequences, whose further
evolution could then be studied in detail. The technical
task has proven to be far more difficult than was
originally envisaged; but on the way we have made a number
of important advances in a) methods of DNA segmentation and
implantation, b) understanding of the conditions for
expression of genes exchanged among widely diverse
bacterial species, and c) the prevalence of mechanisms for
promiscuous mixing of genes among bacteria believed to be
quite separate in an evolutionary sense -- e.g. Bacillus
and Staphyloccus. In a sense this last finding may
unexpectedly prove to be the most important of our
contributions to the theme of the origin and evolution of
life.


     In the light of the unexpected ferocity and
hostility of contention about the public health hazards
of work with recombinant DNA,
-- although we have endeavored to stay as far as possible
from situations with credible pathogenic potential -- we
have decided to phase out our work under NASA support that
directly involves recombinant DNA; and this will no longer
be part of our continuation proposal. However, we will
comtinue efforts along the lines of c) above, namely to
seek still broader understanding of the extent to which
genetic information is shared with a common gene pool among
microbes. Julian Davies, for example, has long speculated
that antibiotic-producing streptomycetes are the primary
reservoir of plasmids that recur in a wide range of
pathogenic bacteria, and by conferring resistance to the
antibiotics result in serious clinical problems. However,
direct support for this degree of panmixis has yet to be
achieved experimentally.  In the course of our observations
of the Staphylococcus x Bacillus exchanges mentioned above,
we believe we may have some hints how to overcome these
experimental barriers, and will apply these ideas to
building general "plasmid-traps" to capture the movement of
plasmids among a variety of species in their natural soil
habitats. The insight sought here would of course be of the
greatest importance for a) the theory of evolution of
microbes (if not eukaryotes as well); b) practical problems
of the sources of antibiotic-resistance; and c)
common-sense tempering of regulatory controls that have
become a serious hindrance to research in this field.
     This shift of emphasis to natural habitats is coupled
with an effort to get a better understanding of the role of
solid surfaces in microbial physiology, ecology and
genetics. Some years ago, in connection with studies that
helped to prove that penicillin acted as an inhibitor of
cell-wall synthesis, I was impressed by the role that
solid-agar had in controlling the morphogenesis of
spheroplasts or L-forms in E. coli.  Knowing that many,
perhaps most, of the microbes resident in soils have never
been isolated in pure culture, I wondered whether much of
laboratory microbiology -- using homogeneous liquid media
-- is not an artefact, far removed from conditions of the
natural habitat. In the interval, many soil microbiologists
have made similar observations (summarized in Marshall's
book), at least of the influence of interfaces on the
growth and physiology of particular species. The most
extreme speculation that I propose to investigate is
whether there are not many species that have an absolute
requirement for a solid substrate -- from which it would
follow that conventional methods have been actively
selecting against the recognition of an important part of
our biosphere. An even more interesting proposition is that
these organisms may also include representatives of the
simplest, most primitive categories that could give further
clues about the baffling missing links in the 'Scala
Naturae'. Some (feeble) support for such a speculation can
be gathered from the fact that Mycoplasmas, which have the
smallest genome-sizes of any presently known freeliving
organisms, lack a rigid cell wall. Some of these have, to
be sure, been cultivated in suspension, but often only with
difficulty; and analogous L-phases of larger bacteria
generally require an agar substrate. The implication is
that a rigid wall, and the ability to proliferate readily
in suspension, are later evolved attributes of
the 'typical' bacteria compared to more primitive forms. So
far, the most primitive mycoplasmas still have the complete
repertoire of the genetic code, and therefore can give us
little insight into its primary evolution (unless one takes
this as evidence of special creation or of panspermia]).
     We must concede that we have made little progress
since Oparin in bridging the hiatus between the
chemosynthesis of nucleotides and their assembly into
meaningful, autocatalytic sequences -- in part for lack of
observable representatives of the intermediate stages.
     While this would be the most rewarding outcome, one
does not have to stake one's faith in that speculation to
justify the research plan. The strategy will be to
construct media and devices that offer large surface areas
to organisms in soil habitats, but which can be extracted
for closer study-- e.g. glass films either bare or with
various mineral coatings. These will be manipulated so as
to encourage organisms that have a preference for sessile
growth on the surface, and then sampled further for
organisms that may have a near- or total- dependence on
such surfaces. {One may not have to look far: witness algal
and cyanophytic films in aquaria; but I do not know of
critical comparisons of their growth potential in adherent
vs. suspended mode. It is known that the sheathed habit, associated with
attachment, of Sphaerotilus natans, is suppressed by high
nutrient concentrations -- there may well be many organisms
that correspond to the attached phase of Sphaerotilus, being
unable to grow except as epibionts The association of bacteria with
surfaces has also attracted renewed interest in recent years
in connection with dental microbiology, and with other aspects
of infectious disease..}
     The associations of blue-green algae with fissures and
voids in the immediate sub-surface of dry Antarctic soils
has been pointed out, by E.I. Friedmann, to refute the
asserted sterility of this terrestrial habitat, and to
point to some caveats in the design of life-detection
strategies for planetary exploration. Saprophytic bacteria
that would exploit the primary production of the algae
would be very difficult to ascertain by conventional
methods if they were interface-dependent. This gap in our
appreciation of the soil habitat is thus equally important
for laying the groundwork for future planetary biological
science and engineering. Furthermore, there are many
anecdotal observations of the enhanced resistance to
thermal sterilization of microbes in soil matrices; but the
phenomenon has not been systematically investigated with
regard to mechanism. Thus, our projected studies of
interface biology bear on problems of survival in hostile
environments (terrestrial and extra-terrestrial) as well as
upon practical problems of disinfection, e.g. for planetary
quarantine. The methods of microscopic examination of soils
that will be developed for these studies should also have
some bearing on planning for instruments appropriate to
future planetary and return-sample missions from an exobiological
perspective.

     Finally, few students of the origin of life have
FAILED to comment on the probable role of clay and other
heterogeneous surfaces for the local concentration of
chemosynthetic precursors of life. We thus note that, while
the central ideas of this research plan are widely
entertained, they have only rarely been the subject of
concerted investigation: doubtless because of the
overwhelming convenience of working with transparent,
homogenous media.

        Besides the minerals commonly associated with soil,
we will also give particular attention to polymeric carbon
as an interphase -- the prevalence, and adsorptive and
catalytic properties of this material which justify this
choice are elaborated in part B. of this proposal.
  ----------

     Bibliography: Part I.

(This is not a systematic reference list, but the following
sources will help to illustrate and enlarge upon the ideas
just summarized.)

Courvalin,P., Weisblum,B., and Davies,J.  (1977). Aminoglycoside-
modifying enzyme of an antibiotic-producing bacterium acts as a determinant
of antibiotic resistance in Escherichia coli. Proc. Nat. Acad. Sci. , US.,
74:999-1003.

Ehrlich,S.D. (1977) Replication and expression of plasmids from Staphylococcus
aureus in Bacillus subtilis. Proc. Nat. Acad. Sci. , US., 74:1680-1682.
                        {Reprint enclosed, from this laboratory}

Lederberg,J. and St. Clair,J. (1958) Protoplasts and L-type growth of E. coli.
J. Bact. 75:143-160.

Lederberg,J. (1952) Preservation of bacterial cultures on silica gel.
(unpublished note). Cf. Hunt,G.A.,Gourevitch,A., and Lein,J. (1958)
Preservation of cultures by drying on porcelain beads. J. Bact.,
76:453-454.

Wallace,D.C. and Morowitz,H.J. (1973) Genome size and evolution.
Chromosoma, 40:121-126.  {This article points out that Mycoplasma probably
represents a primitive genome size of about 1/2 megadalton, with other
bacterial species tending to be integral multiples.  Prof. Wallace is now
a member of the faculty of this department, and will consult imformally
with us.}

Gaudy,E. and Wolfe,R.S. (1961) Factors affecting filamentous growth of
Sphaerotilus natans. Applied Microbiol., 9:580-584.

Landman,O.E.,Ryter,A.,and Frehel,C. (1968) Gelatin-induced reversion
of protoplasts of Bacillus subtilis to the bacillary form. J. Bact.
96:2154-2170.

deCastro-Costa and Landman,O.E. (1977) Inhibitory protein controls the
reversion of protoplasts and L-forms of Bacillus subtilis to the walled
state.  J. Bact., 129:678-689.

Ou,Li-Tse and Alexander,M. (1974) Effect of glass microbeads on growth,
activity and morphological changes of Bacillus megaterium. Arch. Microbiol.
101:35-44.

Friedmann,E.I. and Ocampo,R., (1976) Endolithic blue-green algae in the
dry valleys: primary producers in the Antarctic desert ecosystem.
Science 193:1247-1249.

Marshall,K.C. Interfaces in microbial ecology. Harvard,1976.

Hattori,T.  Microbial life in the soil. Dekker, 1973.

 [added later:  Zobell, CE  J Bact 46:39-56 (1943)]