Exploring biochemistry using metabolic pathways in bacteria:
Genome Reduction
Since the first bacterial genome was sequenced in 1995, we have come a long way in
our understanding of these organisms. We now have an understanding of the
average size of a genome
[approximately 4 million base
pairs(Mbp)] and the number of
genes (approximately 3000
genes). Since 2006, scientists
have found that there are bacteria
with extremely small genomes.
Many, but not all of these bacteria
are symbionts or pathogens that
live within a host organism[1].
Originally, the smallest bacterial
genome was thought to be
Mycoplasma genitalium, weighing
in at 0.58 Mbp with 475 genes.
We now know that M. genitalium
is at the larger end of the reduced
genome size spectrum, with many
genomes considerable smaller
(see figure on right, from McCutcheon and Moran[1] that shows the relative sizes of
some of these within a M. genitalium genome). The smallest is Candidatus
Tremblaya princeps, which has an extremely reduced genome of 0.13 Mbp.
What are the impacts of having an extremely small genome? We will explore this
question in PATRIC by examining the genomes and comparing them to a “normal”
sized bacterium, using metabolic pathways to search for differences and similarities.
For this exercise we will use the table below.
Genome ID Organism
1423.136 Bacillus subtilis ATCC 13952
243273.25 Mycoplasma genitalium G37
272947.5 Rickettsia prowazekii str. Madrid E
392021.5 Rickettsia rickettsii str. 'Sheila Smith'
325775.3 Coxiella endosymbiont of
Amblyomma americanum C904
372461.17 Buchnera aphidicola BCc
Genome
Size
(Mbp)
3.87
0.58
1.11
1.25
0.656
0.42
Protein
Coding
Genes Lifestyle
4033
Free-living
542
924
1567
Pathogenic
577
375
Symbionts
Buchnera aphidicola (Cinara
tujafilina)
Wigglesworthia glossinidia
36870.6 endosymbiont of Glossina brevipalpis
261317.3
36868.4 Wigglesworthia glossinidia
endosymbiont of Glossina morsitans
444179.5 Candidatus Sulcia muelleri GWSS
871271.4 Candidatus Zinderia insecticola CARI
Candidatus Carsonella ruddii HT
1202539.3
isolate Thao2000
387662.1 Candidatus Carsonella ruddii PV
573658.4 Candidatus Hodgkinia cicadicola
Candidatus Hodgkinia cicadicola
573234.4
Dsem
Candidatus Tremblaya phenacola
1266371.3
PAVE
891398.4 Candidatus Tremblaya princeps PCIT
Candidatus Tremblaya princeps
1053648.4 PCVAL
0.44
410
0.7
665
0.71
0.24
0.2
663
277
304
0.15
0.15
0.15
174
179
373
0.14
177
0.17
0.13
205
233
0.13
235
with
reduced
genomes
Symbionts
with
extremely
reduced
genomes
Creating genome groups
1. Login to the PATRIC website so that you can use your workspace in the
downstream analysis.
2. On the PATRIC homepage (patricbrc.org), open the Tools tab at the top of the
page.
3. When the tab opens to reveal the box listing the tools, click on Genome
Finder (highlighted in dark blue below).
4. This will open the landing page for the Genome Finder tool. As you are
logged into the website, any genome groups you have created will be visible
in the box that you can see under “Select Organism(s)”. We will ignore those
groups as we will be generating our own.
5. In the box below “Enter Keyword” enter the genome IDs for free-living
bacteria in the table above (Hint: You can cut and paste directly from the
table), then click the Search button.
6. This will take you to the results page for the Genome Finder too.. On the left
side you will see a dynamic filter that we won’t use in this experiment, and on
the right side you will see a table that lists the best results of your search.
7. Pay close attention to the genomes that were returned. One of those is not
from the list that we provided.
8. Select the genomes that match those from the table, then click on the “Add
Genomes” next to the folder icon in the Workspace header.
9. This will open up a pop-up window that allows you to save the group.
10. Select the “Create New Group” option.
11. Name the group and click “Save to Workspace”. Now that data is saved and
you can use a number of tools to explore it.
Assignment
Create genome groups for the three categories below. Make sure that you are
getting the genomes from the table into your groups. There might be some extras
that you will have to weed out.



Pathogenic
Symbionts with Reduced Genomes
Symbionts with Extremely Reduced Genomes
Comparing pathways using the Comparative Pathway Tool
1. Open the Tools tab at the top of the page.
2. When the tab opens to reveal the box listing the tools, click on Comparative
Pathway Tool (highlighted in dark blue below).
3. This will take you to the landing page for the Comparative Pathway View
tool.
4. If you have a lot of genome groups, you will have to scroll through the Search
box to find your genome group of interest. For this example, find the genome
group you created that had the free living bacterial genomes (Bacillus subtilis
and Mycoplasma genitalium) and click the check box in front of it.
5. Under Enter keyword, you will need to click the Search button.
6. This will return the pathway summary page that summarizes all the
information for specific pathways scoped to the genomes in your specific
group (in this case the two genomes from free-living bacteria).
7. To look more specifically at how the data is summarized, choose the first
pathway on the list (in this case, the pathway called Ascorbate and aldarate
metabolism) by clicking on the pathway name, which is a hyperlink.
8. The page that returns is the Pathway page. It has two parts, a summary table
on the left that shows the enzyme commission (E.C.) numbers that have been
assigned to each gene. These numbers denote a specific metabolic function
that a specific gene has.
9. The summary table also has other data. You can look more closely at the
column heads by grabbing the edge of the column box that contains the title
and moving it to the right. Text that is in bold (2.7.1.69 below) indicates that
all genomes in the group have at least one gene that has that particular EC
number
10. On the right you can see a pathway map that has the gene data mapped to it.
The particular pathway maps we use in PATRIC were designed by the Kyoto
Encyclopedia of Genes and Genomes (KEGG) group.
11. You’ll notice that there are boxes with numbers in them, and some of them
have different colors. Boxes that are not colored (white) indicate that genes
with this particular functionality are absent from the genome group you are
examining. Boxes that are olive green colored indicated that some, but not all
to the genomes in your particular selection, have a gene in their genome with
this particular function. Boxes that are colored bright green mean that all the
genomes in your selection have at least one gene with this particular
function.
12. Tools at the top of the pathway map allow you to save the pathway summary
to your computer, or expand the entire pathway map for easier viewing.
13. One of the problems with a pathway summary map is that its often difficult
for researchers to see which organism has the specific enzyme, and which is
missing it. To see a summary of this type of information, find the Heatmap
tab that is located next to the KEGG tab at the upper left and click on that.
14. This will take you to a data summary in a heatmap format. The legend is on
the right, and the summary is on the left. The heatmap has the genomes on
the x axis, and the genes on the y axis. B. subtilis has a lot of genes involved in
this pathway, and M. genitalium has only one. Remember that M. genitalium
is free-living, but has a much smaller genome than B. subtilis.
15. Scrolling over a specific cell with provide information about the gene in the
blue band above the genome names.
16. To see more details about a specific gene, you can double click on a
particular cell. In the picture above that would be the orange cell to the right
of the gene name. This will generate a pop-up box that provides you with a
number of choices.
17. Click the Show Proteins button.
18. This will take you to another pathway summary table that will tell you the
other pathways this particular gene is involved in.
Comparing pathways across different genome groups
1. Open the Tools tab at the top of the page.
2. When the tab opens to reveal the box listing the tools, click on Comparative
Pathway Tool (highlighted in dark blue below).
3. This will take you to the landing page for the Comparative Pathway View
tool.
4. Select the free-living group and the group of symbionts with reduced
genomes in the box below “Select organism(s).”
5. In this example, let’s define a specific family earlier in the process. Enter
Glycolysis in the text box. Glycolysis is a metabolic pathway that converts
glucose to pyruvate, and it is supposed to be conserved across nearly all
organisms. The fact that it is so widely shared suggests that this is an ancient
pathway that evolved very early. Click on the Select button.
6. This will take you to a table that has Glycolysis as the only entry.
7. Click on the name Glycolysis/ Gluconeogenesis
8. This will take you to the pathway number. Note from the numbers with the
bold font (and also the bright green boxes) that there are a number of
genomes that all have genes with specific EC numbers, but there are also a lot
that appear to be specific, or limited to, certain genomes.
9. Click on the Heatmap tab.
10. This will show the gene present and absence for both genome groups.
11. You can move the order of the genomes in the table by clicking on the
genome header with the genome you’re interested in, and then drag it to the
area of the heatmap you would like to see it. Be careful not to release the
click until you have moved the column, or it will generate the download popup box.
Before
A er
12. Refer to the table at the beginning of this document and move the genomes in
order of their size from smallest to largest. Sometimes this can reveal some
interesting patterns.
Assignment: Answer the following questions using
the PATRIC website.
1. Return to the Compare Pathway tool and select all the genome groups you
created for this exercise (free-living, pathogenic, symbionts with reduced
genomes, symbionts with extremely reduced genomes). Enter Glycolysis into
the Keyword text box and click select.
a. Arrange the genomes in order of their size. What patterns do you see?
b. What is happening with the extremely reduced genomes? If all
organisms are supposed to be able to perform glycolysis, what do you
think is happening with these bacteria.
2. Advanced question: You will need to create two new genome groups, one
with all the complete genomes found in Brucella, and another from all the
complete genomes found in Bartonella (Hint: You may have to look at the
previous classes, like the one that looked at antibiotic resistance, to remind
yourself how to do that). These two bacteria are related to each other, but
each cause different types of disease and have different lifestyles.
a. Using the Comparative Pathway tool, look at the Citrate Cycle for both
of these groups. What are the genes that all the genomes share? Are
there specific patterns of presence or absence that you see are specific
to each of these genera? Locate those genes that are unique on the
KEGG map so that you can see the strategy of each genus.
b. Generate a multiple sequence alignment for the all the genes that have
an EC number of 1.1.1.42 across those two groups. Examine the gene
tree. How are the genes clustering together? Do you see any distinct
changes in the alignment that are specific to a certain genus?
3. Compare the pathogenic group that you created earlier with the Brucella and
Bartonella genome groups. If you were to look only at the Citrate Cycle,
whom do the Rickettsia more closely resemble?
References
1.
McCutcheon, J.P. and N.A. Moran, Extreme genome reduction in symbiotic
bacteria. Nat Rev Microbiol, 2012. 10(1): p. 13-26.