Mark Martin

A. Introduction and overview
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 symbioses.

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 students.

  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.
  3. 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.

B. 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 predators
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 planktonic cells.

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 bacterial predators
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 role.

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 extant biofilm?

Useful references

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, New York.

Martin, M.O. (2002). “Predatory prokaryotes: an emerging research opportunity.” J. Mol. Microbiol. Biotechnol. 4: 467 - 477.

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 - 3388.

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.