Ronald M. Weiner
Office Phone: 301.405.5446
Graduate Program Affiliations
There is an ongoing and active research program centered about two areas, marine biofilms and complex carbohydrate degradation in the mare environment.
We are learning how prokaryotes adhere to, and colonize, marine surfaces. We are focusing on capsular exopolysaccharide (EPS) which may be a primary adhesion and which is a dominant component of marine biofilms. We most commonly study a paradigm, marine genus which we named Hyphomonas and which has species isolated from deep-sea hydrothermal vents. It's species have biphasic life cycles with a pelagic and adherent stage. This complex prokaryotic life cycle and its evolution and taxonomy have been elucidated by my laboratory. These bacteria tolerate a strikingly wide range of environmental conditions and have both autotrophic and heterotrophic capability. We are examining the molecular mechanisms and developmental cell biology that confer a potent survival advantage upon this taxon. We are using these mechanisms and biofilm probes that we have developed to screen for biofilm forming pathogens in the environment and on contaminated food products, in collaboration with Dr. S Joseph.
As part of complex biofilms, we have investigated the way that periphytic bacteria signal invertebrates to colonize surfaces. We have shown that some Shewanella and Hyphomonas species synthesize enzymes involved in tyrosine degradation and oxidation and that a primary product, L-dihydroxyphenyl alanine (L-Dopa), can cue invertebrate set and enhance the integrity of biofilms. We have cloned the parahydroxy-phenyl-pyruvate oxidase (PHPPO) operon and isolated its product, homogentisic acid. The function and expression of PHPPO among diverse marine species has been determined.
We are also studying enzymes that degrade relatively insoluble complex polysaccharides (CP) that are important in marine biofilms and marine plant and animal symbioses. These include cellulases, chitinases, agarases and alginases. We are learning about their expression, mode of action and role in inanimate surface or algal colonization. The laboratory is focusing on cell biological adaptations that optimize enzyme function. Briefly we have discovered that the marine bacterium, strain 2-40 (we proposed to name it to the genus, Microbulbifer), synthesizes complex structures that house, organize, and protect consortia of hydrolytic enzymes that bring to bear a concerted attack on the CP.
This is very important because in terrestrial habitats, a specialized group of microorganisms degrade a significant portion of the more than 52 billion tons of cellulose that accumulate yearly by producing structures termed cellulosomes. These structures bind and organize the cellulolytic enzymes for a concerted attack on the CP, making the degradative process much more efficient. In the oceans, on the other hand, much less is known about how the annual production of the more than 25 billion tons of ICP, found in biofilms, associated with algae, and in invertebrate shells, is recycled to usable carbon.
It turns out that 2-40 belongs to a recently discovered group of bacteria that includes degradative species such as shipworm symbionts, that recycle complex carbohydrates. 2-40, was isolated from decaying marine grass, Spartina, and appeared capable of single-handedly decomposing the plant. It was found to synthesize a battery of proteases, lipases, and at least 10 different enzyme systems that attack complex carbohydrates, including agarases, chitinases, alginases, pullulanases and laminarinases. The enzymes were best induced by the homologous substrate and their synthesis was repressed by glucose.
Marine bacterium 2-40 was found, by scanning and transmission electron microscopy, to produce surface protuberances (termed hydrolysomes), when growing on complex carbohydrates but not when growing on simple sugars. Structurally the hydrolysomes resembled cellulosomes.The isolated hydrolysomes have potential applications in the bioremediation of biofouled surfaces, aquaculture wastes (e.g. shrimp and crab-shell) and plants efficiently recycling these to usable feedstock. They have medical applications as well.
In collaboration with DOE, we sequenced the genome of 2-40. In collaboration with Dr. S. Hutcheson we have initiated a genome wide functional genomics and proteomics study. At least 150 open reading frames coding for CP depolymerases have been identified thus far. Of these, 22 have been identified as likely cellulases, cellobiases, or cellodextrinases and at least 7 identified as xylanases. Additionally, there are as many as 15 likely pectin lyases in the draft sequence.
The completed draft sequence of the 2-40 genome enabled the first genome-wide analysis of a microbial chitinolytic system. The chitinase system of 2-40 was found to include secreted chitin depolymerases (ChiA, ChiB, ChiC), a secreted chitin binding protein (CbpA), periplasmic chitooligosaccharide modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in GlcNAc metabolism. Each chitin depolymerase was detectable in culture supernatants of chitin grown 2-40 and was active against chitin and glycol chitin.
The agarase system was also examined to elucidate the constituent enzymes and functional domains. Several strategies, including genome analysis, revealed five enzymes apparently involved in the degradation of agar: AgaA, AgaB, AgaC, AgaD, AgaE. These all shared at least 40% similarity to a known agarase. The genes for these agarases were found dispersed within the 2-40 genome. Zymograms and thin layer chromatography of cloned AgaB and AgaF revealed that they are beta-agarases. The major product of AgaB activity is neoagarotretraose, indicating that it is a beta-agarase I.
To our knowledge, M. degradans 2-40 is the first marine bacterium shown to have a complete enzymatic system capable of completely degrading all three of the major components of plant cell walls. Based upon the number of its degradative enzyme systems, 2-40 appears to be a versatile saprophyte and a significant participant in the marine carbon cycle.
Weiner, R. 2004. The Hyphomonas. in: Bergey's Manual of Determinative Bacteriology. 10th ed., Vol 3, Williams and Wilkins
Howard M, N Ekborg, L Taylor, S Hutcheson and R Weiner. 2004. Genome-wide identification and analysis polyserine linker domains in prokaryotes with emphasis on the marine bacterium, Microbulbifer degradans. J Protein Sci 13: 1422 -1425.
Howard M, N Ekborg, L Taylor, R Weiner and S Hutcheson. 2004. Chitinase B of Microbulbifer degradans strain 2-40 contains two catalytic domains with different chitinolytic activities. J Bacteriol 186: 1297-1303.
de Renzende, C, L Carr, S Joseph and R Weiner. 2005. A capsular polysaccharide surrounds smooth and rugose types of Salmonella. enterica serovar Typhimurium DT104 Appl Env Microbiol. 71:7345-7351.
Ekborg, N, JM Gonzalez M Howard, L Taylor, S Hutcheson and RM Weiner. 2005. Saccharophagus degradans gen. nov., sp. nov., A versatile marine bacterial degrader of complex polysaccharides. Int J Syst Evolut Microbiol 55: 1545-1549.
Ekborg, N, Larry E. Taylor, A Longmire, B Henrissat, R Weiner and S Hutchison. 2006. Genomic and proteomic analysis of the agarolytic system encoded by Saccharophagus degradans 2-40. Appl Env Microbiol 72:3396-33405.
Badger, JH, T. R. Hoover, YV Brun, RM Weiner, MT Laub, G Alexandre, J Mrázek, Q Ren, IT Paulsen, KE Nelson, HM Khouri, D Radune, J Sosa, RJ Dodson, SA Sullivan, MJ Rosovitz, R Madupu, LM Brinkac, AS Durkin, SC Daugherty, SP Kothari, MG Giglio, L Zhou, D H. Haft, JD Selengut, TM Davidsen, Q Yang, N Zafar, and NL Ward. 2006. Comparative genomic evidence for a close relationship between the dimorphic prosthecate bacteria Hyphomonas neptunium and Caulobacter crescentus. J. Bacteriol. 188: 6841- 6850.
J Henshaw, A Horne-Bitschy, Al LAmmerts van Bueren, V Money, D Bolam, M Czjzek, N. Ekborg, RWeiner, S Hutcheson, G Davies, A Boraston and HGilbert. 2006. Family 6 Carbohydrate Binding Modules in β-Agarases Display Exquisite Selectivity for the Non-Reducing Termini of Agarose chains. JBC. 281:17099-17107
Taylor l, B Henrissat, P Coutinho, N. Ekborg, S.Hutcheson, and R Weiner. 2006. A complete cellulase system in the marine bacterium Saccharophagus degradans strain 2-40T. J Bacteriol. 188:3849-3861.
Weiner R , L Taylor II, B Henrissat, L Hauser, M Land, P Coutinho, CRancurel, E Saunders, A Longmire, H Zhang, E Bayer, H Gilbert, F Larimer, I Zhulin, N. Ekborg, R Lamed, P Richardson, I Borovok, and S Hutcheson, 2008 Complete Genome Sequence of the Complex Carbohydrate-Degrading Marine Bacterium, Saccharophagus degradans strain 2-40T PLoS Genetics. http://www.plosgenetics.org/doi/pgen.1000087.
Ph.D., Iowa State University, 1970