A. Introduction and
Research in my undergraduate-based laboratory group here at the
University of Puget Sound focuses on a very unusual and
relatively understudied area of microbial molecular biology and
genetics which will I believe will shortly "bloom" due to recent
advances in genomics: the genetics and other characteristics of
predatory bacteria, such as Bdellovibrio bacteriovorus
and Ensifer adhaerens. I also have interests in
microbial evolution, unusual microbes, microbial ecology, and
Below you will find a general statement of my research
interests, my philosophy regarding student research, and finally
a description of the directions I believe my research program
will take in the next few years. Students will find that work in
my lab can teach important skills in microbiology, genetics,
molecular biology, genomics, and general laboratory practices.
The goal in the laboratory will be to work toward publishable
material, carried out by undergraduate students.
In a nutshell, I have three research interests over the long
term, all of which could be carried out by undergraduate
1. Investigating the genetic and biochemical mechanisms
controlling predation by prokaryotic organisms (both
Bdellovibrio and Ensifer) and developing genetic
systems for the analysis of these microbes.
2. Using newly available genomic data from various species of
Bdellovibrio to search for genes involved in predation and
intraperiplasmic metabolism, and to investigate the evolution of
the predation phenotype.
3. Searching for more examples of predatory prokaryotes from the
environment and investigating how such organisms---as well as
Bdellovibrio and Ensifer--- impact bacterial
populations in nature and the laboratory.
It is important to note that there are many interconnections
between these three general areas, and that I am involved in
several related research collaborations with scientists at other
institutions. Overall, my research program has an effective
mixture of observational microbiology, genetics, biochemistry,
and genome analysis. Students working in my research group will
thus be exposed to many up to date techniques and ideas that
will help them better prepare for careers as scientists, as well
as allow them to do valuable and interesting research.
Role of students in my research program:
I consider research to be an excellent form of teaching, whether
it is part of a long-term research project, a senior thesis, or
even a laboratory class assignment. I know as a certainty that
undergraduate students can carry out research on predatory
prokaryotes, present posters at national meetings, give
seminars, and co-author journal articles---because they have
done those things while working in my laboratory. My research on
Bdellovibrio was initiated in a small liberal arts
college environment, and was not “carried over” from a previous
project at a large institution; I developed the program de
novo at Occidental College before coming to the University
of Puget Sound.
Thus, I am confident that I can continue to mentor students into
doing research of genuine scientific value, teaching both the
method and culture of science effectively and with humor.
There are a wide variety of projects available for students,
depending on their energy levels and background, beginning with
first year students having little exposure to molecular biology
to very advanced students. For beginning students, it is
important to begin by working with a more advanced student (or
me) in tandem, learning the basic “tools of the trade” (sterile
technique, media preparation, plasmid, DNA, and RNA isolation,
gel electrophoresis, using BLAST to search for genes of
interest, reading of the critical literature, etc). As the
student develops more autonomy and understanding, the projects
can become more long term and individualized. Weekly lab
meetings keep everyone (including me) “in the loop,” promoting
interaction and discussion. Essential to the overall process
with undergraduates is summer research, when students can devote
40+ hours a week to research in what I consider to be a
“graduate school boot camp” atmosphere.
C. Research Area #1 (Molecular and genetic studies of bacterial
This is the area in which most of my previous work with
Bdellovibrio and Ensifer has taken place.
My laboratory group has demonstrated that, in Bdellovibrio,
transposon mutagenesis and allelic exchange are possible. The
use of reporter genes such as beta-galactosidase, GFP, RFP, and
luciferase has been demonstrated. My research group has
successfully cloned a variety of Bdellovibrio genes. We
have recently shown that we can move mutations from
host-independent to wild type Bdellovibrio using
generalized transduction. These results make a number of
fascinating experiments both possible and straightforward.
Certainly, I remain interested in learning the role that global
regulation has in predation (following up on our cloning of the
adenylate cyclase gene from Bdellovibrio). Are other
global regulators, such as rpoS, part of this
transition from attack phase to intraperiplasmic growth in
Bdellovibrio? These are certainly projects well within the
abilities of engaged and hard working undergraduates.
Another question relates to the nature of the prey cell
periplasm itself; what is the periplasmic environment "sensed"
by Bdellovibrio during invasion of prey cells? The
temporal expression of the Bdellovibrio proU,
uspA, groEL, and grpE genes could be
analyzed in synchronized attack cultures using gene fusions or
RT-PCR. This is of particular comparative interest, as many
intracellular symbionts and pathogens of eukaryotic cells
overexpress or otherwise regulate stress or osmoticum-related
genes. Thus, is the periplasm of prey cells at all similar to
the cytoplasm of eukaryotic cells? Perhaps Bdellovibrio
can “tell” us.
Given what has been learned about the genome of Bdellovibrio,
questions of basic metabolism during predation remain of
interest (for example, are amino acid biosynthetic or catabolic
genes constitutive or expressed in a stage specific fashion?).
We have already cloned the citrate synthase gene of
Bdellovibrio; is it under tight regulation or does it play
more of a "housekeeping" role?
Of course, we are continuing our efforts to learn more about the
amylase-like gene we have discovered in Bdellovibrio,
and what role it plays, if any, in predatory activities. Since
Bdellovibrio's metabolism is clearly non-sacchrolytic,
this has led us to questions about differences between the
enzymatic and transport activities of intraperiplasmic and free
living Bdellovibrio cells, gluconeogenic enzyme
activities, and the presence and function of glycogen-like
compounds in Bdellovibrio.
Using a combination of our previous work, and data mined from
the genome sequence of the closely related strain 100 of
Bdellovibrio, many student accessible questions can be
investigated, ranging from the relatively simple (are flagella
necessary for predation?) to the more complex (is a specific two
component histidine kinase gene expressed in a stage specific
fashion, and does it play any role in predation?).
D. Research Area #2 (Genomic studies of bacterial predators):
I consider this to be an essential part of the long-term future
of my research program.
The published genome of Bdellovibrio bacteriovorus type
strain 100 has answered some questions, but raised many more.
Several amino acid biosynthetic and catabolic pathways are
missing (there are only complete biosynthetic pathways for
eleven amino acids, and pathways for degrading ten). Yet the
genome size is large (3.8 MB), and there appears to be no
evidence of horizontal gene transfer (based on local deviations
in G+C composition). There is much complexity in flagellar
structure (six copies of flagellin genes at four loci), and for
pilin genes (four clusters plus many dispersed pilin genes).
There exist a wealth of hydrolytic enzyme genes, as expected
from Bdellovibrio's predatory lifestyle.
As Bdellovibrio is supposedly obligately associated
with prey cells for its growth and reproduction, it would be
particularly interesting to compare the genome of
Bdellovibrio species with other obligate bacterial
pathogens and symbionts. Certainly obligate symbionts such as
Nanoarchaeum have lost most biosynthetic capacities
(and in fact has the smallest genome thus known of roughly 500
KB). Buchnera, Wolbachia, and Mycobacterium leprae
(as well as several examples of Chlamydia and
Rickettsia) have undergone massive gene loss, duplications,
and rearrangement as either symbiont or pathogen. Why is
Bdellovibrio so different?
This is where comparisons of multiple genomes from
Bdellovibrio like organisms become vital. One genome is
complete (Bdellovibrio bacteriovorus strain 100), while
three more are in progress (Bdellovibrio strain "W,"
Bacteriovorax marinus, and Bacteriovorax stolpii).
In addition, I have great concerns about heterogeneity between
strains, species, and isolates, since historically,
Bdellovibrio strains were "passaged" from culture to
culture once a week over decades; there may well be significant
genomic differences between "cultivated" and "fresh" strains of
the same species!
In any event, genomic comparisons will be helpful. Are the
patterns of gene loss/presence for amino acid metabolism common
to all species, or a pattern specific to strain 100? Are
specific sets of genes organized in synteny between different
predators? Is there truly no evidence of horizontal gene
transfer? Are there similarities or differences in the
"catalogue" of hydrolytic enzymes encoded among the genomes? Can
any of these data shed light on how the predation phenotype
evolved: is intraperiplasmic growth an ancient or recent
phenotype, are there common mechanisms for attack and metabolic
access of prey cells, and are there control mechanisms shared by
all predators? Comparisons between genomes, genomic mining for
genes of interest, and use of genetic tools to create mutants
could help provide an answer.
Finally, the genome sequences and their careful analysis will be
key to understanding the basic genetics and biochemistry of this
species. For example, the genome sequence of Bdellovibrio
strain 100 has revealed several genes similar to the
gliding motility loci used by Myxococcus xanthus (a
fellow alpha-proteobacter) for locomotion over surfaces (gldA,
gldF, gldG, and ftsX). My hypothesis
is that Bdellovibrio uses a very different predatory
strategy for attacking biofilm-associated prey cells than for
planktonic cells. I plan to create "knockout" mutations in one
or several of these genes and determine if the mutants are
impaired in their ability to attack biofilm associated versus
In the near future, I would like to become involved in
microarray analysis as a strategy to identify periplasmically
regulated genes in Bdellovibrio. It would be
interesting to learn if any such "predation" gene was
co-regulated by oligotrophic (i.e., low) nutrient conditions. I
also believe that a nice "first step" in this area would be to
use a commercial E. coli microarray to determine if any
E. coli genes are up or down regulated in prompt response
to attack by bacterial predators such as Bdellovibrio.
Considering that 11% of the 3,584 genes identified in
Bdellovibrio strain 100 are homologous to unknown genes
found in other microbes, and 34% of the genes are completely
unknown in GENBANK thus far, I can predict that genomic analysis
will provide researchers with a great deal of new information
worth pursuing in the laboratory for quite some time.
E. Research Area #3 (Ecological studies of known and unknown
This is an area that suggests possible practical applications to
the study of predatory prokaryotes.
First and foremost, I intend to have my research team continue
to look for new predatory microbes. Unusual relatives of
Bdellovibrio (that is, organisms first found because of a
periplasmic life cycle) may shed light on any number of
evolutionary questions about organisms in the genus
Bdellovibrio or Bacteriovorax. Searching for new
predators is straightforward and remains an excellent “starter
project” for students. Currently, I am interested in learning
more about BLOs (Bdellovibrio like organisms) capable
of attacking Gram positive, archaean, or anaerobic microbes.
The role that Bdellovibrio or Ensifer have in
modulating microbial community structure remains unknown.
Discovering BLOs or 16s rRNA evidence of such organisms in the
mammalian gut and deep ocean sediments suggests that these
predators are common in many microbial communities. I intend to
investigate how introduction of these kinds of organisms can
change the prevalence and diversity of microbial populations in
soil, gut, or aquatic environments. Just as bacteriophages have
been shown to modulate microbial community structure in the
ocean, we may find that prokaryotic predators play a similar
A good deal of work has been done in recent years using nematode
or insect models of bacterial pathogenesis. It would be
fascinating and perhaps even medically relevant to learn if BLOs
could be “used” to protect such model organisms against
“challenge” by a pathogenic microbe, or even therapeutically.
Thus far, BLOs are only known to attack Gram-negative organisms,
and certainly there are a large number of such microbes that are
currently being used as pathogens in nematode or insect systems.
Again, the fact that BLOs are found the intestinal tracts of
animals (including humans) argues that predatory prokaryotes are
often present, and may play a role in maintaining a specific
microbial community structure within an animal.
Finally, we and others have uncovered evidence that
Bdellovibrio can effectively attack and consume biofilm
associated prey cells. Are the predatory mechanisms the same as
when Bdellovibrio attacks planktonic cells? Do biofilm
associated Bdellovibrio cells use "gliding motility" to
move within the biofilm and consume prey? We have also shown
that Bdellovibrio itself generates a biofilm. Are the
genes involved in this phenotype similar to the biofilm related
genes of other bacteria, or is it simply a strategy to allow
Bdellovibrio to become "recruited" into a developing or
1. General references:
Ruby, E.G. (1992). The genus Bdellovibrio, pages 3400 -
3415. In: A. Balows, H.G. Truper, M. Dworkin, W. Harder, and K.H.
Schliefer (editors), The Prokaryotes, 2nd Edition. Springer-Verlag,
Martin, M.O. (2002). “Predatory prokaryotes: an emerging
research opportunity.” J. Mol. Microbiol. Biotechnol. 4: 467 -
2. Genomics issues:
Campoy, S., Salvador, N., Cortes, P. Erill, I., and J. Barbe.
(2005). “Expression of canonical SOS genes is not under LexA
repression in Bdellovibrio bacteriovorus.” J. Bacteriol.
187: 5367 – 5375.
Rendulic S, Jagtap P, Rosinus A, Eppinger M, Baar C, Lanz C,
Keller H, Lambert C, Evans KJ, Goesmann A, Meyer F, Sockett RE,
Schuster SC. (2004). “A predator unmasked: life cycle of
Bdellovibrio bacteriovorus from a genomic perspective.”
Science. 303: 689 -692.
3. Molecular genetics and basic biology of Bdellovibrio:
Nunez, M.E., Martin, M.O., Chan, P.H., and E.M. Spain (2005).
“Predation, death, and survival in a biofilm: Bdellovibrio
investigated by atomic force microscopy.” Colloids Surf. B.
Biointerfaces. 42: 263 - 271.
Kadouri, D., and G.A. O’Toole. (2005). “Susceptibility of
biofilms to Bdellovibrio bacteriovorus attack.” Appl.
Environ. Microbiol. 71: 4044 – 4051.
Flannagan, R.S., Valvano, M.A., and S.F. Koval. (2004).
“Downregulation of the motA gene delays the escape of
the obligate predator Bdellovibrio bacteriovorus 109J
from bdelloplasts of bacterial prey cells.” Microbiology. 150:
649 - 656.
Nunez, M.E., Martin, M.O., Duong, L.K., Ly, E., and E.M. Spain.
(2003). “Investigations into the life cycle of the bacterial
predator Bdellovibrio bacteriovorus 109J at an
interface by atomic force microscopy.” Biophys. J. 84: 3379 -
4. Phylogeny issues:
Davidov Y., and E. Jurkevitch. (2004). “Diversity and evolution
of Bdellovibrio-and-like organisms (BALOs),
reclassification of Bacteriovorax starrii as
Peredibacter starrii gen. nov., comb. nov., and description
of the Bacteriovorax-Peredibacter clade as
Bacteriovoracaceae fam. nov.” Int J Syst Evol Microbiol.
54:1439 - 1452.
Snyder, A.R., Williams, H.N., Baer, M.L., Walker, K.E., and O.C.
Stine. (2002). “16S rDNA sequence analysis of environmental
Bdellovibrio-and-like organisms (BALO) reveals extensive
diversity.” Int J Syst Evol Microbiol. 52: 2089 - 2094.