When I say chromosome, what image pops into your head? Is it a chunky x-marks-the-spot, reminiscent of a wiggly car dealership tube man?
Do your chromosomes look like this? Source
Many of us have been exposed to eukaryotic (multicellular) mitosis and meiosis again and again, each time learning a little more about the ways our own cells copy their genetic material and pass along information to the next generation. In the process of DNA replication, which occurs in both single celled and multicellular organisms, DNA makes a copy of itself to pass along to the newly formed daughter cell. The microbial world, though it follows the same template, offers a whole new system of replication, one we are just beginning to understand fully.
Remember this chromosome? Source
Bacteria have a multitude of DNA organization techniques, though most use a single, circular chromosome to package DNA. Today, we dive into the bacterial species Sinorhizobium meliloti, which has one circular chromosome along with two megaplasmids. Megaplasmids, circular extrachromosomal packets of DNA, replicate separately from the main chromosome by remaining spatially distant and managing their DNA replication independent of the chromosome. How does a bacterial cell organize replication amidst the chaos of multiple DNA storage systems?
Generalized DNA organization of bacteria, with a single circular chromosome along with separate, smaller plasmids. Source
Before delving into the replication of Sinorhizobium meliloti, I would like to share a little more about what makes this bacterium unique, useful, and worthy of study. S. meliloti is part of the Rhizobia community of bacteria, known for forming symbiotic relationships with legumes.This particular relationship represents a mutualistic symbiosis, in which both the bacteria and the plant benefit from their interaction. Here, the symbiotic relationship of S. meliloti and its host plant involves the efficiency of nitrogen fixation, a process integral for plant growth and development. A balance of environmental nitrogen has been essential to the agricultural industry for thousands of years, and the nitrogen cycle helps to control levels of nitrogen across the planet. Plants frequently fix environmental nitrogen through nitrates and ammonia in the soil, but nitrogen gas is also readily available if the plant can convert it to a usable form. S. meliloti has the ability to metabolize nitrogen by converting nitrogen gas to ammonia, creating a new source of nitrogen for the legume.
Sinorhizobium meliloti, part of the rhizobium class of bacteria that exist within soil and form symbiotic relationships with legumes. Source
Rhizobia are soil bacteria that come in contact with the root systems of legumes. When signal molecules pass between the bacteria and plant cells, the legume forms root nodules where S. meliloti resides and fixes nitrogen for the plant. The roots themselves release chemo-attractant signal molecules that turn on nodulation genes within the bacteria. These genes, or Nod factors, trigger formation of the nodule in the host plant. Bacteria/host interactions are highly specialized to the species engaging in symbiosis. For example, when S. meliloti joins with M. truncatula, one of its plant hosts that is related to alfalfa, the bacteria terminally differentiate into bacteroids specialized for nitrogen fixation. Bacterial cells within the nodules elongate and can even become polyploid by copying their genomes many times over without physically dividing, which amplifies their genetic material for maximum symbiotic advantage.
Root nodules caused by symbiosis between the plant and rhizobia bacteria. Source
How does the S. meliloti bacteria pass along genes that control for Nod factors and other indispensable components of its symbiosis? As I referenced earlier, the genome of S. meliloti is split between a single, circular chromosome, made up of 3.65 million base pairs, and two megaplasmids, pSymA (1.35 million base pairs) and pSymB (1.68 million base pairs). These megaplasmids are integral to the symbiotic function of S. meliloti. Studies have shown that genes on the megaplasmid pSymA code for nitrogen and carbon metabolism. The presence of the megaplasmid thus ensures that S. meliloti will successfully establish its symbiotic relationship by supplying the plant with ammonia. For the symbiosis between plant species and S. meliloti to be successful, the chromosome and megaplasmids must be seamlessly replicated each cycle, perfectly coordinating the separate processes of chromosome and megaplasmid replication.
A 2016 study by Frage et al, “Spatiotemporal choreography of chromosome and megaplasmids in the Sinorhizobium meliloti cell cycle,” examines the replication patterns of S. meliloti by tracing the chromosomal and megaplasmid origins of replication in cells as they move through time (temporal-) and space (spatio-). The origin of replication marks where a set of molecular machinery attaches to the DNA to begin the process of DNA replication. The megaplasmids and chromosome of S. meliloti replicate once per cell cycle, and it is integral that all components of the bacterial genome are passed down to daughter cells. Frage et al. begin by tagging a subunit of the enzyme DNA polymerase III, known as DnaN, with a fluorescent molecular tag known as mCherry. Using fluorescence microscopy to view the location of mCherry allowed Frage et al. to follow the movement of the attached DnaN. Thus, the authors studied the movement and timing of different origins of replication within the chromosome and megaplasmids.
In the first figure of their study, Frage et al. observe up to five unique locations of DnaN-mCherry in replicating S. meliloti, indicating that multiple origins of replication act within replicating S. meliloti chromosome and megaplasmids. In a separate strain of S. meliloti without the pSymA megaplasmid, Frage et al. find less DnaN (and therefore fewer origins of replication), indicating that DnaN is associated not just with the replication of the chromosome, but also that of the megaplasmids. Additionally, the authors observe that the megaplasmids and chromosome have origins of replication that move at separate times, which indicates that there may be a pattern of when each piece replicates. Below, a part of the first figure shows several places where DnaN is present, as we can tell by the glowing mCherry tags.
Replication of S. meliloti, with DnaN-mCherry visible under fluorescent microscopy. Replication is initiated at different times and different locations within the dividing cells. Source
The second figure of the study draws crucial distinctions between the origins of the chromosome, pSymA, and pSymB during replication. Frage et al. label and trace the origins of the chromosome and megaplasmids throughout replication, proving that replication is orchestrated in time and space - a pattern that ensures each parcel of DNA is replicated separately. Throughout Figure 2, Cori refers to the origin of replication for the chromosome, while SymAori and SymBori represent the origins of replication for the two megaplasmids.
Replication origins of the S. meliloti chromosome and megaplasmids pSymA and pSymB traced through time and space during replication. Source
Figure 2A shows labeled origins associated with the chromosome, SymA, and SymB megaplasmids using fluorescence microscopy. Each panel represents a five minute interval, and the origins clearly move throughout the dividing cells. As the authors point out, all origins follow roughly the same spatial pattern, beginning with a single origin at the original pole, then the appearance of a second origin that moves to the new pole. In 2B, the movement of these origins is traced through time using a graphical interpretation. The similar patterns of movement are slightly displaced temporally. Figure 2C further explores the different timings of replication, showing that the megaplasmids and chromosome move through the cell cycle at a unique pace. In 2D, a graphic depicts the combination of spatial and temporal factors controlling the origins of the chromosome and megaplasmids. The temporal progression of cell replication can be seen in the succession of the seven drawings; in each snapshot, the origins of replication of the chromosome and megaplasmids are at different points in their journeys as they trace out similar paths from the poles to the center and back.
Following their exploration of the temporal and spatial dynamics of the chromosome and megaplasmid origins, Frage et al. located possible genes and proteins responsible for this replication control. Further, the authors determine that altering the activity of DnaN unsynches the patterns of replication between the chromosome and megaplasmids.
By organizing how different components initiate replication, Frage et al. provide a framework for understanding how bacteria with multi-part genomes replicate accurately and quickly and confirm that S. meliloti operates in a similar manner to other bacterial multi-part genomes. For S. meliloti, knowledge of replication and the cell cycle contributes to our understanding of the important symbiosis between rhizobia and legumes. Without the presence of its megaplasmids, and the ability to control when and where the megaplasmids replicate, S. meliloti would be unable to provide nitrogen to its plant hosts in the form of ammonia, and the mutualistic symbiosis would be impossible. Because legumes provide us with an important source of food, knowledge of how to control and optimize the relationship between plant and bacteria has the potential to increase our agricultural production. Without the symbiosis between legumes and S. meliloti, the plants’ only source of nitrogen would be what is available in the soil, which can be inconsistent based on environmental conditions. This symbiosis allows for a steady source of ammonia from the nitrogen gas universally present in the atmosphere. Next time you are faced with meiosis or mitosis, take a moment to remember the different ways organisms have adapted to pass their genome along and the complexities of the microbial world.
About the author:
Claire Glover ‘21 is a biology and chemistry double major from Andover, Massachusetts. She loves hiking, backpacking, and learning new things about the natural world around her. After Mount Holyoke, Claire hopes to attend veterinary school and work with wildlife. In her free time, Claire is part of the soccer and rowing teams, and loves spending time with her family and her dog Merlin. She has recently started gardening, and is excited to keep her crops happy and full of nitrogen.
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