A new scientific study has revealed unique life strategies of two major groups of microbes that live below Earth’s surface.
A publication in Frontiers in Microbiology reports that these groups, originally thought to rely on symbiotic relationships with other organisms, may also live independently and use an ancient mode of energy production.
“These microbes, which belong to the groups Patescibacteria and DPANN, are really special, really exciting examples of the early evolution of life,” said Ramunas Stepanauskas, a senior research scientist at Bigelow Laboratory for Ocean Sciences and an author of the paper.
“They may be remnants of ancient forms of life that had been hiding and thriving in the Earth’s subsurface for billions of years.”
Stepanauskas led a research team that used advanced molecular techniques and bioinformatics to analyze thousands of microbial genomes and learn about their evolutionary history.
Reading their genetic code revealed that these two groups of abundant microbes lack the capability to breathe in order to synthesize ATP, the common energy currency of life.
The team found that these microbes, which live in a variety of environments in Earth’s interior, appear to gain energy only through the process of fermentation.
Many organisms are capable of fermentation, including humans when their muscles run out of oxygen during intense exercise – but they use it only as a supplementary source of energy.
“Our findings indicate that Patescibacteria and DPANN are ancient forms of life that may have never learned how to breathe,” Stepanauskas said.
“These two major branches of the evolutionary tree of life constitute a large portion of the total microbial diversity on the planet – and yet they lack some capabilities that are typically expected in every form of life.”
The researchers found that the most recent common ancestors of these two lineages lacked the ability to breathe, just as their modern descendants do. For the first two billion years of Earth’s existence, there was no oxygen in the atmosphere.
Today, oxygen is a key component of Earth’s atmosphere and essential to the life it can support – but just a few hundred feet underground, conditions have not changed, and this recent discovery suggests that some subsurface life hasn’t, either.
Scientists had previously speculated that because Patescibacteria and DPANN have very simple genetic features and metabolism, they must live symbiotically and depend upon host organisms to survive.
In the new study, the research team found no evidence that Patescibacteria and DPANN are dominated by symbionts – most of them seem to live as free cells and rely on the primitive pathway of fermentation to supply themselves with energy.
“Dependence on other organisms is a feature of life,” said Jacob Beam, a former postdoctoral researcher at Bigelow Laboratory and the lead author of this study.
“There are no absolutes in biology, and our research shows that microbes can vary along the spectrum of interdependencies.”
Scientists analyzed microbes from diverse environments around the globe, including a mud volcano at the bottom of the Mediterranean Sea, hydrothermal vents in the Pacific, and the world’s deepest gold mines in South Africa.
Bigelow Laboratory Bioinformatics Scientist Julie Brown, Research Scientist Nicole Poulton, former Postdoctoral Research Scientists Eric Becraft and Oliver Bezuidt, and Research Experience for Undergraduates intern Kayla Clark worked on this project, alongside with an international team of scientists who contributed to fieldwork, laboratory, and computational analyses.
In addition to revealing the inner workings of Earth’s subsurface and the evolution of life, these findings can provide a model system of what life on other planets may look like.
Environments on Mars and other bodies in the solar system likely resemble Earth’s subsurface, and Patescibacteria and DPANN represent examples of life that appear to require very little energy to survive, which scientists expect would be a requirement for life on other planets.
“This project would not have been possible without the collaboration of this diverse group of scientists collecting samples around the world and uniting their expertise,” Beam said. “Through the collaboration of a global group of scientists working together, we know more about the inner workings of these microbes that form a major fraction of the total biodiversity on our planet.”
In our study, we acquired 158 good-quality MAGs from groundwater samples and analyzed them with MAGs of previous studies. We found that the Patescibacteria superphylum has highly reduced redundant functions of metabolisms, cellular activities, and stress response, while retaining the essential functions such as genetic information processing.
The Patescibacteria has ultra-small cell sizes and simplified membrane structures including diminished flagellar assembly, transporters, and two-component systems. Although the bacteria lack CRISPR, which is important for phage defense, they could have alternative strategies to resist phage infection.
Previously, Brown et al. proposed 26 candidate phyla from groundwater samples belonging to the super phyla Parcubacteria and Microgenomates .
They discovered certain usual features including ultra-small cell sizes, insertions inside 16S rRNA genes, missing ribosomal proteins L30, small genome sizes, and metabolic limitations.
The Parcubacteria and Microgenomates belong to the super phyla Patescibacteria and account for about a half of the super phyla in our study (Fig. 1). We here conducted a more extensive genomic comparison by including the additional sequences recovered from Oak Ridge.
In general, members of the Patescibacteria superphylum have retained basic metabolic functions centered on glucose and pyruvate, and lost numerous functions related to motility, chemotaxis, outer membrane function, polysaccharide metabolism, biosynthesis, and nutrient transport.
They have retained basic systems for gene expression and replication, especially the surprisingly conserved ribosomal proteins despite highly reduced genome size.
Anantharaman et al. proposed 47 candidate phyla from groundwater and sediment samples through metagenomic binning  and they found the interactions of microorganisms in terms of biogeochemical processes such as nitrogen cycle.
Through phylogenetic analysis and genomic comparison, we found that ten of the 47 candidate phyla belong to the Patescibacteria because they form an independent lineage with the super phyla Parcubacteria and Microgenomates as well as MAGs of this study consistently based on both nearly full-length 16S rRNA gene and concatenated RP16 genes.
In addition, similar to the other Patescibacteria phyla, they also share the features of reduced non-essential functions and metabolisms and lack of CRISPR among others.
In addition to the bacterial cell size study of Brown et al. , Luef et al. also proved the ultra-small cell size of Microgenomates (OP11), Parcubacteria (OD1), and Katanobacteria (WWE3) from groundwater using cryogenic transmission election microscope .
Their cell size was about 0.009 ± 0.002 μm3. These small cells do not have outer membrane and are inferred to be gram-positive bacteria.
They also found pili-like structures of these ultra-small microorganisms and inferred inter-organism substance exchange through it. The electron microscope images showed no flagellum and outer membrane of Microgenomates and Parcubacteria, which is consistent to our genomic results.
In our study, we found that the Patescibacteria superphylum has reduced functions of cell motility and flagellum, outer membrane, polysaccharide metabolism, biosynthesis process, transporter for nutrient uptake, and retained metabolisms of simple metabolites such as glucose and pyruvate.
The bacteria may use the pili-like structure for nutrient transport from hosts (could be either bacteria or protists), because they have reduced functions of biosynthesis and transporter for nutrient uptake according to our genomic comparison.
Based on environmental condition of groundwater and the metabolic and functional features of Patescibacteria, we proposed that the adaptation of Patescibacteria to groundwater environments facilitates the features of small genome size, lack of CRISPR viral defense, and ultra-small cell size (Fig. 5) as below.
The groundwater is an environment with nutrients (including C, N, S, and P) in low concentration and low diversity (e.g., in comparison to soil).
The low and less nutrients may have reduced the metabolic capacity of Patescibacteria and thus the carbohydrate utilization genes for polysaccharides, disaccharides, and amino sugar among others were streamlined in the genomes. Chemoautotrophic metabolisms (such as sulfur oxidation, ammonia oxidation, and nitrite oxidation) were not detected either.
Some of the Patescibacteria may rely on hosts such as larger bacteria or protist (Parcubacteria  for nutrients supply (they have pili-like structures  for nutrient uptake). According to the Black Queen Hypothesis , bacteria with reduced genomes may rely on bacterial community with full metabolism capacity for “public goods.”
The Patescibacteria with highly reduced genomes may rely on simple intermediate metabolites from “host” community for energy because the metabolic pathways for simple metabolites were retained in the genomes, which facilitates the genomic simplicity.
The environment of low nutrient concentration requires bacteria to increase absorption rate. The Patescibacteria seem to have adopted the strategy of shrinking cell size (~ 0.3 μm). Small cell size has been proved to increase metabolic rate  because smaller cells have a higher ratio of surface area to volume, which speeds up the substance exchange across cell membrane.
Cell membrane of Patescibacteria was simplified because of cell size reduction.
As a result, the reductions of some membrane structures, such as flagellum, capsule, and outer membrane protein which could be taken advantage by phage as receptors [22, 23], in turn make the bacteria escape from phage attachment, thus the bacteria are less invaded by phage (indicated by the less phage-associated proteins detected in genomes).
Because phage receptors such as flagellin and capsule proteins have only been identified in model phages, the role of these proteins as phage receptors in Patescibacteria still needs to be verified.
Small cell size could serve as a merit to escape from phage adsorption. There are studies in the effects of bacterial cell size on the phage adsorption and burst. Hilla Hadas et al.  found that adsorption rate of T4 phage was positively correlated to Escherichia coli cell size, suggesting that smaller cell size prevents phages from attaching.
Charles Choi et al.  investigated the effect of cell size of E. coli B23 on the T4 phage burst size and found that larger cell has increased phage burst.
Moreover, physically, ultra-small cell size makes it hard for phages to target, and also there is no sufficient space for the phages (~ 0.2 μm) to attach. These alternative strategies might have complemented the lack of CRISPR which plays important roles of viral defense in other phyla but has been deleted in Patescibacteria.
The lack of CRISPR and reduced phage receptors are not considered evidences of the resistance to phages, but they could be taken as the effects of any possible phage resistance, because resistance to phage would cause reduced CRISPR to save genetic materials and energy, reduced phage receptors on membrane to save space for other membrane proteins due to reduced surface area, and less prophage proteins.
Groundwater has a low oxygen concentration  because it is underground and there is no light for plankton to generate oxygen through photosynthesis.
The 93 wells of this study had a dissolved oxygen (DO) concentration of 1.1 ± 1.7 mg/L (Table S1) and light intensity is considered zero in the natural groundwater. Because of the low oxygen concentration, Patescibacteria only rely on anaerobic respiration (lacking oxidative phosphorylation pathway) which provides less energy than aerobic respiration.
Functions requiring much energy such as flagellar motility and secondary metabolisms were thus reduced in Patescibacteria. Dark environment also reduces functions involving light energy.
There were no photosynthetic pathways and corresponding CO2 fixation pathways detected in Patescibacteria genomes. Dark environment also reduces functions of light repair of DNA damage.
Without ultraviolet (UV) radiation, functions involved in UV stress were also reduced in Patescibacteria (data not shown). The anaerobic and dark environment and all these effects contributed to the reduced genome size of Patescibacteria.
Stability of environmental conditions may also have contributed to reduced genome size of Patescibacteria. The uncontaminated wells of this study had very stable conditions such as temperature (16.6 ± 3.9 °C, Table S1) and pH (6.5 ± 0.7, Table S1).
Less variability of environment was demonstrated to select bacteria with smaller genomes . The stable physical conditions such as temperature and pH allow Patescibacteria to survive without investing in an adaptive response to environmental perturbation.
This is verified by the fact that Patescibacteria was sensitive to contamination probably because of the reduced metabolic potential and stress response due to genome reduction.
reference link : https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-00825-w
More information: Jacob P. Beam et al, Ancestral Absence of Electron Transport Chains in Patescibacteria and DPANN, Frontiers in Microbiology (2020). DOI: 10.3389/fmicb.2020.01848