May I Borrow a Cup of Catalase? Prochlorococcus and Its Helpful Neighbors

Dec. 7, 2018

What examples spring to mind when you think about microbe-microbe interactions? Likely we first think of the microbial chemical warfare that gives us our antibiotics when bacteria such as Streptomyces or fungi such as Penicillium battle for resources with their neighbors. But it’s not all antagonism in these tiny communities: there are also cooperative microbial neighborhoods. One example with global implications is the marine cyanobacteria, Prochlorococcus, and its associated heterotrophic bacteria.
 
Prochlorococcus, an ironically large number of syllables for the world’s smallest oxygen-producing (as opposed to non-oxygen producing) photosynthetic organism (0.5-0.7 mm), is highly abundant in the world’s ocean with estimates of up to 2.9 x 1027 cells. Moreover, its ecotypes (distinct lineages not represented by 16S rRNA) have given researchers an important new lens through which to examine microbial genetics and ecology. By virtue of its autotrophic lifestyle and widespread abundance, Prochlorococcus plays an important role in maintaining the health of our planet. Not only do marine algae serve as the base for many marine food webs, they produce 50% of the oxygen we breathe and cycle multiple tons of elements such as carbon, iron, nitrogen, phosphorus and sulfur (which all living things need). Additionally, by releasing compounds that stimulate cloud formation, marine algae affect Earth’s global climate through rain and the reflection of solar radiation. Despite their far-reaching influence across our planet, even these powerful organisms need some help now and then, and the relationship of Prochlorococcus with its heterotrophic bacterial neighbors reveals an intriguing story about microbial community dynamics in the ocean.
 
Figure 1. TEM images of Prochlorococcus MED4. Source: Luke Thompson, Chisholm lab and Nikki Watson, Whitehead Institute for Biomedical Research at MIT, 2006.

While Prochlorococcus may be self-sufficient in terms of making its own food, it’s highly vulnerable to reactive oxygen species (ROS), because it lacks ROS-protective enzymes such as catalase. ROS cause life-threatening damage in cells by damaging DNA, proteins, etc. In the ocean’s surface waters, ROS (most commonly in the form of hydrogen peroxide) are created via sunlight, from microbial activity, and introduced through rainfall. In our daily lives, we encounter ROS from normal cellular metabolism, from pollution such as tobacco smoke, pesticides, and heavy metals, and from certain drugs, which is why it’s a good thing we have proteins and enzymes that protect us and repair damage. Without a mechanism to defend against ROS, Prochlorococcus is vulnerable to ROS-mediated damage. However, associated heterotrophic bacteria relieve that burden by lending their own ROS-countering enzymes. This relationship has been both utilized by researchers as a culturing tool and as the focus of microbial physiology studies.

“Helper” Heterotrophic Bacteria as a Culturing Tool

For decades, studies of Prochlorococcus were hampered by the inability to obtain pure cultures for most of the 30+ isolates (exceptions being strains PCC 9511 and MIT 9313). Even for pure (axenic) cultures, large initial inocula were needed in transferring cells to fresh medium. While Prochlorococcus can be grown on agar plates, attempts at single colony formation were largely unsuccessful. For example, some strains could be grown but only with ~0.1-10% recovery efficiency or the yield expected based on initial numbers plated. Clearly both Prochlorococcus and researchers needed some help. Enter friendly heterotrophic neighbors.
Figure 2. Liquid cultures of Prochlorococcus. Source: Allison Coe, Chisholm lab at MIT, 2017.

It’s not uncommon to find heterotrophic bacteria associated with cyanobacteria and eukaryotic microalgae. After all, hanging out next to a food source is a great survival strategy. Such associated heterotrophs cover different genera and vary based on the original environment of isolation, such as the Atlantic or Pacific Oceans, or the Mediterranean or Arabian Seas. Researchers noted that Prochlorococcus strains preferred to grow on plates near the largest populations of heterotrophic contaminants. Given the difficulty in generating axenic cultures of Prochlorococcus, researchers surmised that these heterotrophs might be providing a necessary service to the algae, such as delivering a growth factor or reducing a growth barrier.

To test this, Morris et al. deliberately covered agar plates (that lacked a carbon source) with an isolated heterotrophic neighbor, Alteromonas strain EZ55, before plating Prochlorococcus strain MIT 9215 at different dilutions on the same plates. While Prochlorococcus didn’t grow on the heterotroph-minus control plates, researchers observed nearly full recovery efficiency (up to 100% in some cases) on the heterotroph-treated plates. Subsequent tests with other strains of Prochlorococcus, other Prochlorococcus-associated heterotrophs, and marine bacterial heterotrophs not found naturally associated with Prochlorococcus revealed a complex web of relationships.  Some pairings stimulated Prochlorococcus growth faster than others and some pairings did not support Prochlorococcus growth at all. While one heterotroph, Silicibacter lacuscaerulensis ITI 1157, did aid all Prochlorococcus strains tested, it was not always the best partner for supporting Prochlorococcus growth.  For example, it took 14 weeks before visible growth of Prochlorococcus strain MIT 9211 was observed when co-cultured with S. lacuscaerulensis strain ITI 1157, but only 9 weeks with Vibrio harveyi B392. No single heterotroph stood out as providing the best growth support overall.
Figure 3. A non-axenic culture of Prochlorococcus MIT9215 showing both the cyanobacteria and associated heterotrophic bacteria. Source: Anne Thompson, Chisholm lab at MIT, 2008.

Even though there were no clear trends and no ‘best’ heterotrophic helper, these results still provide valuable insights. It is notable that heterotrophs not originally isolated from Prochlorococcus were supportive of Prochlorococcus growth and that natural heterotrophic neighbors of Prochlorococcus did not always stimulate cyanobacterial growth. This illustrates that such a mutually beneficial relationship is not necessarily a given outcome from natural associations and that there is more at play than possible coevolution factors. That coevolution is at least one contributing factor is supported by additional research showing how strains of Prochlorococcus are affected differently by different bacterial heterotrophs. Also, work by Sison-Mangus et al. examining diatoms and their heterotrophic bacterial neighbors suggests the two have co-evolved a certain level of specificity for one another. Therefore it is reasonable to expect similar specificity in other microbial phototroph-heterotroph relationships especially given the opportunity provided by the distinct niches that algae, including different strains of Prochlorococcus, occupy in the ocean.

Morris et al. went on to observe that only living heterotrophs could help Prochlorococcus grow, indicating that Prochlorococcus needs ongoing support, not a one-off addition. While investigating the possibility of stress reduction as a potential MO for the heterotrophs, the researchers found that the Prochlorococcus genome did not contain catalase homologues or heme-containing peroxidases necessary to prevent ROS-mediated damage. Because catalase is light sensitive (and Prochlorococcus needs light for photosynthesis), it is not feasible to merely add catalase to Prochlorococcus media. Thus, the addition of heterotrophic isolates has become a standard in Prochlorococcus culturing, with researchers using phage and/or antibiotics to remove the heterotrophs once the cyanobacteria have reached a concentration where they can survive without heterotrophic help. This allows for physiological studies that need axenic cultures. Because of the clear importance of helper heterotrophs in the lab and in nature, they too have been a part of studies targeting Prochlorococcus physiology.

Neighboring Microbes Lend Insight into Prochlorococcus Physiology

To further uncover the nature of the symbiotic Prochlorococcus-heterotroph relationship, Biller et al. used transcriptome data to determine the impact of Alteromonas macleodii MIT1002 (a naturally occurring associate) on Prochlorococcus NATL2A. Researchers compared the transcriptome of axenic NATL2A to NATL2A reunited with MIT1002 at 2, 4, 6, 12, 24, and 48 hours post reunification. While growth curves didn’t show any changes in Prochlorococcus growth after its heterotroph was returned, the transcriptome data revealed dramatic changes over time. In total, researchers identified 375 Prochlorococcus genes that were differentially expressed at some point during the studied timeframe. Of these, 192 have homologs across the 41 Prochlorococcus genomes sequenced at the time of the study, marking them as core genes. They also found that nearly half of all the differentially expressed genes (~43%) displayed a consistent abundance after the initial shift in expression, suggesting that the associated physiological changes are continuous rather than temporary. But what changes do these data suggest?

Of the first set of transcripts to change after reunification (at the 6 hour timepoint), all decreased in abundance as compared to the control. The majority belong to a family of proteins called high light inducible (hli) proteins that respond to a variety stresses, including the shock of high amounts of light for which they were originally named. The other transcript to change at this time has a currently unknown function. Additionally at later timepoints (12 and 24 hours), more hli transcripts decreased indicating that Alteromonas helps to alleviate certain stresses, not just those associated with oxidative stress as uncovered by Morris et al. Providing further support to the conclusions of Morris et al. a decrease in transcripts related to oxidative stress, such as DNA repair enzymes, was also observed. Other stress-related transcripts were also decreased, such as those for proteins involved in the refolding of damaged proteins and in purine nucleotide salvage. However, it is curious to note that at the 24 hour timepoint one hli-related transcript increased in abundance, suggesting that although Alteromonas aids in the relief of a wide range of Prochlorococcus stress, it may also cause some of its own.

But stress relief wasn’t the only thing Biller, et al. observed. Co-cultured Prochlorococcus cells experienced changes in the expression of photosynthesis-related genes, suggesting a restructuring of their photosystems. The most likely explanation for this is that Alteromonas is consuming the exudates of Prochlorococcus, triggering Prochlorococcus to increase production. Based on the increases of transcripts involved in vitamin B12 production, it is also possible that Alteromonas receives B12 from its algal associate as well. Even though Alteromonas does not require B12 for growth, it is capable of utilizing it. While cyanobacterial growth rates between the 2 conditions did not differ during the experiment, co-cultured Prochlorococcus cells had increases in transcripts related to a number of biosynthesis pathways, such as those for ribosomal proteins and for amino acids. The most likely explanation is that these increases were supporting the protein synthesis needs of the other transcripts whose abundance increased (e.g., photosynthesis genes).

Overall we can tell that Prochlorococcus NTAL2A has an intricate and beneficial relationship with its Alteromonas MIT1002 neighbor. Prochlorococcus may be borrowing more than just a cup of catalase from Alteromonas, but the situation is win-win. Zooming out from this microbial neighborhood and remembering the global impact of Prochlorococcus underscores how valuable this cooperation is to the world at large.  Furthermore, considering that microbiologists are only able to culture ~1% of the microorganisms out there, this relationship and the underlying science behind it may help a broad range of microbial research finally bring more microbes into the lab. So while many aspects of nature can seem harsh and competitive, you can breathe easy thanks to these friendly neighbors supporting life on our planet and lending a helping hand to researchers.

Further Reading:
Morris, J., et al. 2011. Dependence of the Cyanobacterium Prochlorococcus on Hydrogen Peroxide Scavenging Microbes for Growth at the Ocean’s Surface. PLoS ONE. 6: e16805 
 

Author: Janet Goins

Janet Goins
Dr. Janet Goins is Assistant Director of UCLA's Undergraduate Research Center.