2004 Chaston, John by wfq74180

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									                                  John Chaston
  The Secondary Phase of Photorhabdus luminescens TT01 Is
Closely Tied to the Sexual Form of Heterorhabditis bacteriophora
                       Byron Adams, Microbiology and Molecular Biology

Heterorhabditis bacteriophora and Photorhabdus luminescens are a symbiotically associated
nematode/bacterium pair that act together as potent insect killers and have been cultured en
masse for distribution as an insect control agent in agricultural settings. Heterorhabditis is a
nematode species that acts as a vector to penetrate the insect exoskeleton through existing
openings where it subsequently releases Photorhabdus, the bacterial symbiont that it carries in its
gut, into the haemocoel of the invaded insect. The bacteria produce a variety of proteases and
toxins that digest the insides of the insect into nutrients they use as they rapidly replicate. The
nematode then feeds on the proliferating bacteria until all available nutrients are used up. In an
unknown process the nematode eventually “saves” some of the bacteria, upon which it has been
feeding, in its gut and leaves the nutrient depleted cadaver in search of a new insect host.

My original intent was to generate mutant bacteria by using a special protein/DNA complex
called a transposase that would randomly integrate itself into the bacterial genome and prevent
proper expression of any gene it interrupted in the process. These mutants would be
subsequently screened for symbiotic ability by exposing sterile nematode eggs to a monoculture
of the mutant bacteria; however, the protocol that I used did not work with this taxa of bacteria,
and I was unable to generate any mutants. My proof-of-concept work performing symbiosis
assays was quite instructive, however, and by carefully noting interactions between the nematode
and bacterium under normal conditions I designed several assays that allowed me to propose a
mechanism for the interaction between the two organisms.

There are several unique features of the Heterorhabditis/Photorhabdus system. First, the
nematode Heterorhabditis has a life cycle that involves both a sexual and an asexual life form.
The sexual form is only found inside of a parasitized insect host during the process of infection
while several generations of reproduction occur inside the insect host. However, it is the asexual
form that is released from the host and that subsequently penetrates into a new insect host when
found. The life cycle follows a very specific timeline, where the asexual nematode enters and
produces asexual progeny. These asexual progeny produce sexual progeny for one generation,
and then asexual progeny are produced during several more rounds of reproduction until all
available nutrients in the insect cadaver have been exploited. The process by which the
nematode “decides” to go through a sexual life cycle for one generation as yet is unknown, but
there is a clear evolutionary benefit to the process in that it prevents genetic bottlenecking that
would normally occur in an entirely asexual species by allowing for sexual recombination of
alleles. Interestingly, this sexual period of reproduction does not occur when the nematode is
cultured in vitro.

The bacteria also have an interesting dual-life cycle and exist in two specific phases, creatively
termed the primary and secondary phases. The primary phase has been classically characterized
as the “useful” phase because the asexual form of the nematode (which is the free living form
and as such has been subject to more scientific investigation) requires the primary phase of the
bacterium for growth and reproduction – in the absence of the bacteria in primary phase the
nematode survives for a brief period of time on food stores inside its body and subsequently dies.
In the laboratory the primary phase often switches to the secondary phase, thus far observed to
be an irreversible process, a phase for which no specific function has been described. My
observations regarded the interactions between the two organisms and their different life cycles
(asexual vs. sexual and primary vs. secondary).

I designed several assays that allowed me to observe the effects of the nematode on bacterial
nematode phase switching and the effects of the bacterial phases on nematode growth and
maturation. The assays with their results are shown in the table below:
The effect of the primary phase of the bacterium on the growth and   Primary phase kills and solubilizes sexual form within 48
maturation of both the sexual and asexual forms of the nematode      hours while asexual form survives. No maturation of
                                                                     either form observed
The effect of the secondary phase of the bacterium on the growth     Secondary phase kills and solubilizes asexual form within
and maturation of both the sexual and asexual forms of the           48 hours while sexual form survives. No maturation of
nematode                                                             either form observed
The effect of soluble proteins produced by the primary phase of      Primary phase proteins kill and solubilize sexual form
the bacterium on the growth and maturation of both the sexual and    within 48 hours while asexual form survives. No
asexual forms of the nematode                                        maturation of either form observed
The effect of soluble proteins produced by the secondary phase of    Secondary phase proteins kill and solubilize asexual form
the bacterium on the growth and maturation of both the sexual and    within 48 hours while sexual form survives. No
asexual forms of the nematode                                        maturation of either form observed
The effect of the sexual from of the nematode on phase switching     Sexual form stimulates rapid conversion of primary to
of the primary to the secondary phase                                secondary phase.
The effect of the asexual from of the nematode on phase switching    Asexual form prolongs the persistence of bacteria in
of the primary to the secondary phase                                primary phase.
The effect of gut proteins from the sexual form of the nematode on   Sexual form proteins stimulate rapid conversion of primary
phase switching of the primary to the secondary phase                to secondary phase.
The effect of gut proteins from the asexual form of the nematode     Asexual form proteins prolong the persistence of bacteria
on phase switching of the primary to the secondary phase             in primary phase.


From these observations I concluded that the secondary phase of Photorhabdus luminescens
TT01 was closely tied to the sexual form of the bacteria. In order to confirm this expectation, I
infected a number of insects with the nematode/bacterium complex at the same time and
extracted bacteria from these cadavers at 12 hour intervals following infection. If the secondary
phase were tied to the sexual form of the nematode, it would be expected to be found inside the
insect host only during the period during which the sexual form predominates inside the host. As
expected, the primary phase was found at all times in the cadaver except during a brief period 4
½ to 5 days following infection, during which the sexual form of the nematode was present.

These observations suggest a reason for the evolutionary preservation of a phase of the bacterium
that has classically been described as useless to the nematode. However, as my results above
indicate, I was not able to observe the development of the nematode from its juvenile form to a
more advanced stage before all nematodes were killed in broth culture. New assays need to be
developed that simulate an in vivo system more closely and would therefore more appropriately
represent what occurs inside the insect cadaver. My results however, clearly suggest an
alternative to the null hypothesis that has prevailed during the last 25 years of work on this
system that only the primary phase is required for nematode growth and development. Further
investigation of the interaction between the nematode and bacteria will lead to a greater
understanding of the mechanism of symbiosis in the field of “pure” science and are important to
the applied science of creating a more perfect bioinsecticide.

								
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