Molecular approaches to marine microbial ecology and the mar

Molecular approaches to Marine Microbial Ecology and Biogeochemical cycling of Nitrogen and CarbonAmal Jayakumar and Bess B. Ward

Department of Geosciences Princeton University

Traditional methods of bacterial enumeration, culturing and microscopy are often insufficient to understand some biogeochemical processes– by g y complex mixed microbial population- the black box Insights on the complexity of the microbial population and its interaction with the environment can be probed with molecular tools Molecular tools have been developed and used to understand various groups that catalyze environmental function its activity and abundance function, Largely, these techniques rely on PCR amplification of low abundance gene in a mixed soup of abundant other genes Functions and activity of microbial populations can also be understood by using reverse transcriptase coupled with PCR and other techniques y g p p q

Annual Global Primary Production Sept 1998-August 1999

Source: http://marine.rutgers.edu/opp/swf/Production/results/all2_swf.html

Annual Mean DO at 150m

Intermediate Nephloid Layer and Cell AbundanceThe occurrence of pronounced subsurface particle maxima embedded within the core of suboxic layers of Eastern Tropical Pacific (Pak t l 1980, Garfield t l 1983, T i l P ifi (P k et al 1980 G fi ld et al 1983 Spinrad et al 1989)

Arabian Sea, Naqvi et al 1993

(Ward et al unpublished)

Key Questions What is the composition of the Nepheloid layer Are they all denitrifiers or are there others g p groups as well Is there persity within various functional groups so If so, who are the key players that contribute to carbon and N cycling in the OMZs

Nitrate Respiring Microbes t ate esp g c obes

Organisms that can remove Oxidized forms of N from the environment include,– heterotrophic denitrifying bacteria– anammox bacteria– archaea and autotrophic nitrifiers– foraminifera– thioploca(?)

Diversity Studies Techniques: q– Traditional- Isolation, Microscopy– Molecular: TRFLP, RFLP, Sequencing, Pyrosequencing. Several genes may be targeted depending on the question: e.g.16S rRNA or a function (e.g.. nir, rbcl) Sequence and Types of Suboxic Respiration (Codispoti et al 2005) O2> IO3-> NO3-> Mn (IV)> Fe (III)> SO42 We have targeted the nirS, nirK and AMX 16S rRNA

nirS gene d e s ty in Arabian Sea O S ge e persity ab a OMZ

Sample-DNA extraction- 3 stations with varying suboxia/anoxia PCR amplification- nirS 1F 6R lifi ti i S 1F-6R (Braker et al 1998) -clone library-Sequencing Based on 890 bp Can compare sequences within as well as from database 250 seqs grouped into 7 clusters Low nitrite sample- high Diversity High nitrite sample- low persityDominance of one group

A gene found not only in denitrifiers, but also nitrifiers, (wide range of physiological groups)

Phylogeny SummaryThe overall persity of nitrite reductase genes are limited in the open Ar

abian Sea - WRT sedimentary and terrestrial environments and CAS Most nirK or nirS genes from the Arabian Sea do not show any close identities to other published sequences One clone from Coastal AS- 97% identical to waste water organism (Enterococcus ) Some clades are unique to the open ocean and some are unique to the coastal regions, only two groups appears to be cosmopolitan Some groups of nirS common to both Open Ocean AS and ETSP Cannot usually detect nirS i oxic environments, nirK can b C t ll d t t i S in i i t iK be Both nirS and nirK show bloom dynamics