Moore Foundation MMI

CLOSELY INTERACTING MICROBES AS HOTSPOTS OF BIOGEOCHEMICAL ACTIVITY



The grant to the Scripps Institution of Oceanography supports investigating how closely coupled marine microorganisms interact physically and exchange nutrient molecules. By combining new microscopy tools with molecular and isotope techniques, this project aims to advance understanding of the mechanisms that drive biogeochemical cycles in the surface ocean.


 Atomic Force Microscope image of Synechoccocus cell associated with a heterotrophic marine bacterium  
 
Hypotheses:

Our overarching hypothesis is that the abundant cyanobacteria-bacteria associations display a range of fundamental adaptive strategies and underlying mechanisms and critically influence the oceanic biogeochemical dynamics and global climate


  1. *Bacteria-cyanobacteria associations are phylogenetically diverse


  1. * Bacteria-cyanobacteria associations involve nutrient exchange between partners


  1. *Bacteria-cyanobacteria associations involve structurally & biochemically specific interactions 



PROJECT DATA



Further information and data can be obtained by contacting Dr. Farooq Azam: fazam@ucsd.edu




  1. Bacteria-cyanobacteria associations are phylogenetically diverse



Determine the phylogenetic affiliation of naturally occurring conjoint cells via multiple displacement amplification and 16S rRNA sequencing.


Heterotrophic bacteria associated with Synechococcus:

γ Protobacteria, Methylococcales (91 %) 

α Protobacteria, candidatus Pelagibacter ubique (100 %) 

α Protobacteria, Rhizobilaes (99 %)

α Protobacteria, Rhizobilaes (99 %)


NBCI accession number of heterotrophic bacteria associated with SynechococcusJX489502-JX489503-JX489504-JX489505

Heterotrophic bacterium: KC758958.1




Discovery of antagonistic Synechococcus-heterotrophic bacteria interactions



We have successfully developed a protocol for growing co-cultures of heterotrophic bacteria andcyanobacteria. We tested phylogenetically diverse heterotrophic bacteria (Vibrio, Pseudoalteromonas, Alteromonas, Flavobacterium) with two cyanobacteria, one belonging to the coastal clade (CC9311) and the other to the oceanic clade (WH8102). Our data indicate that SWAT3 (Vibrio), ATW7 (Alteromonas),Pseudoalteromonas flavipulchra (Pesudoalteromonas) strongly inhibit the growth of CC9311 and WH8102 whereas BBFL7 (Flavobacterium) doesn’t. This suggests that some Synechococcus-bacteria associations are antagonistic in nature. Thus, some bacteria may regulate Synechococcus-based primary production as well as contribute to variations in clade-specific productivity. These are important findings and we are currently elucidating the mechanism by which bacteria inhibit/kill Synechococcus.



                                      Figure 1: Co-culture growth effect experiments among WH8102 and CC9311 Synechococcus strains and heterotrophic bacteria in SN medium.





Bacteria-cyanobacteria associations involve nutrients exchange between the partners



Measure individual cell carbon fixation of conjoint cyanobacteria and elemental fluxes between cyanobacteria and heterotrophic bacteria cells using NanoSIMS.


In our first stable isotope labeling experiments using natural microbial populations we did not find significant differences in 13C carbon and 15N nitrogen exchange in the free- versus cyanobacteria-associated heterotrophic bacteria. We are now optimizing culture conditions of the established co-culture system that we have developed with CC9311 (Synechococcus strain) and SWAT 3 and Pseudoalteromonas flavipulchra.


                                    Figure 2: NanoSIMS experiment: 15N and 13C individual cell uptake rate of Synechococcus cells and heterotrophic bacteria. In orange free Synechococcus,                                                                      in pink associated Synechococcus, in blue free heterotrophic bacteria, in green associated heterotrophic bacteria.




Individual cell growth rates using "click chemistry"



We have applied the highly sensitive fluorescent-based approach to quantify protein synthesis and individual cell growth rate in natural assemblage of associated and free heterotrophic bacteria. (This method was developed by Ty Samo, a graduate student in the laboratory who recently graduated and joined Dave Karl’s group). Bacteria are incubated with homopropargylglycine (HPG) that is a methionine analog and will be incorporated in the newly synthesized proteins. Click-IT chemistry was used to covalent bind Alexa Fluor® 488 hydrazide and HPG compounds through the reaction of alkyne and azide to form a stable triazole.


Using this method at the laser scanning confocal microscope A1R (Nikon), we have obtained a view of the activity continuum of bacteria off Scripps Pier. We found that 42% of the conjoint heterotrophic bacteria were growth-positive (positive for de novo protein synthesis) whereas 98% of the free heterotrophic bacteria were growth-positive. This result is consistent with the finding that some of the associations involve antagonistic interactions. This method also enables quantification of individual cell protein synthesis--whether the conjoint heterotrophic bacteria grew faster, on average, than the free bacteria. These data are currently being analyzed to determine growth advantage or disadvantage of bacteria associations with Synechococcus. Translating the protein synthesis rates into individual cell growth rates should enable us to determine the effect of individual microbes’ interactions on its growth rate.


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Figure 3: Epifluorescence image natural marine bacteria assemblage incorporating HPG, homopropargylglycine-ALEXAFluor 488.




Microbe-microbe interactions involving vitamin B1 exchange



Vitamin B1 (thiamine) Physiology of Marine Plankton


Expanding upon initial interests in bacterial picophytoplankton interactions, we are investigating vitamin B1 ecology of marine microbes, in particular the physiology of vitamin B1 auxotrophs (phytoplankton and bacterioplankton) that must obtain the thiamine or its forms/fragments from an external source.  



In collaboration with B. Palenik (SIO), we have established two model systems (two vitamin B1 auxotrophic axenic cultures) for learning more about the B1 physiology of marine microbes: one, the picoeukaryotic phytoplankton O. lucimarinus (CCE9901) (Fig.4) and two, a motile alphaproteobacterium named OUnk1.


Focusing on CCE9901, we confirmed it as a B1 auxotroph and then determined its ability to grow on varying concentrations of B1 (specifically, its half saturation growth constant, Ks) (Fig.5).  Interestingly, its Ks is in the upper range of B1 concentrations found in euphotic coastal waters where Ostreococcus spp. persist (e.g. Scripps Pier, Carlucci et al. 1970) (Fig.6) .  Based on this result, our working hypothesis is that CCE9901 experiences B1 growth limitation in coastal CA waters where Ostreococcus cells are endemic.  



Figure 5: Growth Kinetics Data for CCE9901.  The Michaelis Menten fit parameters are: R2 = 0.92, Df =19.  At the 95% CI, Ks= 24.94 to 59.10 pM.  


Figure 6: A comparison of published dissolved B1 concentrations from surface or upper euphotic zone marine waters alongside the determined Ks range (at a 95% C.I.) for CCE9901.  Presented symbols and abbreviations represent the following: ^ = B1 estimated from bioassay; # = B1 estimated via HPLC (Okbamichael and Sañudo-Wilhelmy, 2005); all other values were determined by LC/MS.  LIS = Long Island Sound, NY (Koch et al., 2012); La Jolla, CA (Carlucci, 1970); SCCS = Southern California Current System off Baja, Mexico (Sañudo-Wilhelmy et al., 2012); SBHC = Stony Brook Harbor (Okbamichael and Sañudo-Wilhelmy, 2005); WTNA = Western North Atlantic (Barada et al., 2013); NA = North Atlantic (Panzeca et al., 2008).


Microbial ‘producers’ of thiamine in the ocean


De novo synthesis by organisms is the source of vitamin B1 in the ocean.  It remains unclear if B1 becomes available largely through ‘active’ or ‘passive’ processes (e.g. continual exudation versus cell death).  We are investigating potential differences in vitamin B1 ‘production’ by B1 autotrophic bacteria.  Preliminary results indicate that B1 autotrophic bacteria vary in how much vitamin B1 they make available to CCE9901 (Fig. 7).  In particular, D. shibae supported the most CCE9901 biomass in co-culture.  Co-cultures with TW7 (Alteromonas representative) or SWAT3 (Vibrio representative) supported amount of CCE9901 biomass over the 13 day incubation.


We are further utilizing CCE9901 as a system to examine if certain conditions facilitate or dampen B1 production by the tested non-auxotrophic bacteria (D. shibae, TW7, SWAT3).




Figure 7: Fluorescence (FL) data for CCE9901 during co-culture experiments with B1 autotrophic bacteria.  Positive controls (1nM final B1 added at the start of the experiment) are noted with solid lines.  Cultures without a co-cultured bacterium are noted by Ost (O. lucimarinus, CCE9901): DSHI = Dinoroseobacter shibae; TW7 = Alteromonadales TW-7, SWAT3 = Vibrionales bacterium SWAT3. Equal amounts of CCE9901 and bacteria were combined at the start of the experiment.  


Genome data publicly available via NCBI:


SUBID 

 BioProject 

BioSample 

 Accession 

 Organism

---------------------------------------------------

 

SUB715623 

 PRJNA263854 

 SAMN03107778 

JSEN00000000 

Marinomonas sp. SBI22

SUB722628 

 PRJNA263856 

 SAMN03107782 

JSEO00000000 

Marinomonas sp. SBI8L

SUB722729 

 PRJNA263857 

 SAMN03107784 

JSEP00000000 

Labrenzia sp. OB1

SUB722726 

 PRJNA263859 

 SAMN03107786 

JSEE00000000 

Marinomonas sp. TW1




Atomic force microscopy of bacterial surface membranes


The morphology of several marine bacterial isolates (Alteromonas sp. AltSIO, Pseudoalteromonas TW7, Vibrio SWAT-3, Flavobacterium BBFL7) (Figs. A,B,&C) and marine bacterial natural assemblages were extensively studied with AFM at different growth stages under differing conditions. Treatments included the addition of antibacterial agents such as silver nanoparticles and antibiotics. AFM data of the bacteria was collected to observe the cell structure and investigate formation of blebs and morphological changes at the nanoscale. AFM probing was also used to investigate the bio-mechanical properties of marine bacteria. 

Raw AFM data is stored in Veeco Nanoscope format and available upon request to Dr. Azam 


Marine microbes rapidly turnover a high concentration of coral-spawn derived organic matter

Each year in the Caribbean, coral species of the genus Orbicella engage in well-predicted mass spawning reproductive events in which the organisms broadcast gametes into the water column. This study investigates the microbial response to the pulse of coral-spawn derived organic matter by taking advantage of this well-predicted influx of nascent carbon potentially available to heterotrophic bacteria. We collected seawater directly after an O. franksi mass-spawning event while the gametes in the water column were highly abundant. The experiments were conducted as 60-hour microcosms in order to evaluate the breakdown of particulate organic matter, with pre-spawn and post-spawn seawater microcosms used as controls. See below slides for more information.

A manuscript containing all results from this spawning study is currently in preparation for submission to a peer reviewed journal. 






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Recent presentations (presentations.pdf) and poster abstracts Pedler_ISME14.pdf and Samo_ISME14.pdf


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