It has been estimated that there may be tenfold more viruses than microorganisms in certain environments. The microbial mats which we study in Yellowstone National Park may be no exception but we know little about host – cyanophage interactions in these environments. Several phage resistance mechanisms are well-characterized; but the recent discovery that bacteria and archaea may have what has been called “adaptive immunity” against phages has created a new excitement in the world of phage–host dynamics. We have recently begun the study of this “CRISPR mediated resistance” in cyanobacteria, within the broader context of phages in natural microbial mats. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to phage. The mechanism requires acquisition of phage nucleic acid by the host. This acquired genetic information is used by the host to recognize and destroy incoming DNA from an attacking phage. There are some intriguing parallels to the RNAi system in eukaryotes, although the details differ.
We analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B’) as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B’ genomes. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences, (between the repeats) are unique. There were no matches of these spacers against the GENBANK database, but good matches to some viral metagenome sequences derived from the hotsprings. Translation of some CRISPR spacer sequences led to the identification of a lysozyme like protein which may play a role in infection. Further analysis of the viral metagenome identified several mutations in the lysozyme sequences which translate into silent or conservative mutations. These are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a evolving phage population, and support the notion that the host CRISPR system must be able to adapt quickly to provide immunity. The ability of metagenomics to track population-level variation in sequences allows for a culture-independent method for evaluating the fast co-evolution of host and phage genomes and its consequence on the structuring of complex microbial communities.
These preliminary analyses suggest that there is a great deal of genetic diversity in both host and phage populations and that we will need to adopt some new technologies to examine this in greater detail. One of these is single cell capture via microfluidic devices: In a collaboration with Dick Zare’s group in Chemistry we used microfluidic cell chambers and single cell capture, followed by capillary electrophoresis to count single molecules (in this case the highly fluorescent phycobiliproteins in cyanobacteria. We intend to modify this strategy to query both genomic and proteomic diversity.
RELEVANT PUBLICATIONS (PDF version available under Publications)
1. Bhaya D., Grossman, A.R., Anne-Soisig Steunou, N.Khuri, F.M. Cohan, N. Hamamura, M. C. Melendrez, M. M. Bateson, D. M. Ward, J.F. Heidelberg (2007) Genomic, metagenomic and functional analyses of cyanobacteria from hot-spring microbial mats reveal an unexpected diversity in nutrient utilization strategies ISME J: 1(8):703-13
2. Huang, B., Wu H., Bhaya D., Grossman A. R., Granier, S., Kobilka B.K. and Zare R. N.(2007) Counting low-copy number proteins in a single cell. Science 315: 81-84
3. Heidelberg J.F., W.C. Nelson, T. Schoenfeld and D. Bhaya Germ warfare in a microbial mat community: CRISPRs provide insights into the co-evolution of host and viral genomes. PloS ONE 2009; 4(1):e4169.