Generated March 28, 2024
Authors: Aaron Schirmer
Here we continue our exploration of novel genomes discovered in module 1. We will explore different approaches for examining the genome sequence and annotation data, investigating the best approaches for answering our question: Do benthic microbes have functioning circadian clocks?
v0.1 (13 Sept 2022): Drafting StRoNG Net Metagenome analysis Module 2
Previously, you assembled and annotated a metagenome of benthic microbes from which you derived novel strains of bacteria. Here, we will focus on interpreting those annotations to develop hypotheses about the presence of circadian clocks in these different microrganisms.
We'll first need to load the RAST assembly set and fasta file for your unknown genome you created in Part 1.
1) In the upper left hand panel, under "DATA", click the red "Add Data" button
2) A list of all the data contained in your narratives should pop up. Select the .RAST files for your GTDB-Bacteria.AssemblySet and bin.fasta extracted genome, and then click to add them into this Narrative.
In most living organisms, many biological, physiological, and behavioral processes oscillate with a daily rhythm. These circadian rhythms provide synchrony between an organism and its external environment, which then allows the organism to exploit temporal niches and adapt its physiology and behavior to changing environmental conditions. The presence of these circadian rhythms in nearly every living organism confers an adaptive advantage for survival on a planet that revolves on its axis once every 24 hours (Pittendrigh, 1960).
Circadian rhythms are generated in cells at the molecular level by interlocking feedback loops that produce an approximate 24-hour cycle. In cyanobacteria, three proteins (KiaA, KiaB, and KiaC) have been identified as essential clock components. These proteins regulate clock directed gene expression for almost the entire genome (Cohen and Golden, 2015). For more specifics on these proteins, their feedback loop, and their circadian regulation of gene expression, please watch the following videos.
Blue-green algae, or cyanobacteria, are microscopic organisms found naturally in water. These organisms live in all types of water including fresh, brackish (combined salt and fresh water), and marine water. These organisms use photosynthesis to make food, so knowing the time of day and when the sun is present or absent is critical for the survival of these organisms. Their circadian clock provides these bacteria with a mechanism to synchronize their internal physiology to the environmental light dark cycle (the sun). This synchronization directs oscillations in gene expression to maximize their photosynthetic potential during the day.
While Cyanobacteria have provided the best evidence for the presence of circadian rhythms in bacteria. Data suggests that other bacteria (both photosynthetic and non-photosynthetic) bacteria may have circadian rhythms and clocks as well (for a review of this, please see Sartor et. al. 2019). In this module we will use your constructed metagenome and explore your gene annotations to attempt to identify circadian clocks in the various species you have discovered.
Using the Protein-Protein BLAST apps below search for homologs of the Kia proteins (below) in your annotated metagenome assembly and answer the following questions.
IMPORTANT: Copy only the protein sequences. Adding the names will alter your BLAST search
BAD78522.1 circadian clock protein KaiA [Synechococcus elongatus PCC 6301] MLSQIAICIWVESTAILQDCQRALSADRYQLQVCESGEMLLEYAQTHRDQIDCLILVAANPSFRAVVQQL CFEGVVVPAIVVGDRDSEDPDEPAKEQLYHSAELHLGIHQLEQLPYQVDAALAEFLRLAPVEAMADHIML MGANHDPELSSQQRDLAQRLQERLGYLGVYYKRDPDRFLRNLPAYESQKLHQAMQTSYREIVLSYFSPNS NLNQSIDNFVNMAFFADVPVTKVVEIHMELMDEFAKKLRVEGRSEDILLDYRLTLIDVIAHLCEMYRRSI PRET
BAD78523.1 circadian clock protein KaiB [Synechococcus elongatus PCC 6301] MSPRKTYILKLYVAGNTPNSVRALKTLKNILEVEFQGVYALKVIDVLKNPQLAEEDKILATPTLAKVLPL PVRRIIGDLSDREKVLIGLDLLYGELQDSDDF
BAD78524.1 circadian clock protein KaiC [Synechococcus elongatus PCC 6301] MTSAEMTSPNNNSEHQAIAKMRTMIEGFDDISHGGLPIGRSTLVSGTSGTGKTLFSIQFLYNGIIEFDEP GVFVTFEETPQDIIKNARSFGWDLAKLVDEGKLFILDASPDPEGQEVVGGFDLSALIERINYAIQKYRAR RVSIDSVTSVFQQYDASSVVRRELFRLVARLKQIGATTVMTTERIEEYGPIARYGVEEFVSDNVVILRNV LEGERRRRTLEILKLRGTSHMKGEYPFTITDHGINIFPLGAMRLTQRSSNVRVSSGVVRLDEMCGGGFFK DSIILATGATGTGKTLLVSRFVENACANKERAILFAYEESRAQLLRNAYSWGMDFEEMERQNLLKIVCAY PESAGLEDHLQIIKSEINDFKPARIAIDSLSALARGVSNNAFRQFVIGVTGYAKQEEITGLFTNTSDQFM GAHSITDSHISTITDTIILLQYVEIRGEMSRAINVFKMRGSWHDKAIREFMISDKGPDIKDSFRNFERII SGSPTRITVDEKSELSRIVRGVQEKGPES
Q1) Did you find matches to these proteins in your metagenome? If so, in which strains?
Q2) Based on your species tree, what is the relationship between these strains? Are they closely related? What information could their relatedness provide you about their circadian clocks?
Q3) Based on the data available to this point, do you believe strains in the sample contain a functioning circadian clock? For full credit, please be sure to explain your reasoning for your answer.
Now do a similar set of BLAST searches using your novel strain
Q4) Are clock proteins present in this sample? Do your results suggest that this organism has circadian rhythms and/or clocks?
Q5) Does your answer to the previous question make sense given what we know about the origin of these organisms? Please explain your answer.
Below you will find an excerpt from Sartor et. al. 2019 on the possible timekeeping components in non-photosynthetic eubacteria:
“In addition to Kai homologs as possible clock components in non-photosynthetic Eubacteria, peroxiredoxin proteins may also be strong candidates. These proteins have circadian rhythms of their redox state in a wide variety of organisms, including in photosynthetic prokaryotic organisms like cyanobacteria and in the Archaea species Halobacterium salinarum [42]. These redox rhythms persisted in several species even when the previously defined major oscillator had been disabled. This led to the suggestion that the cycling of peroxiredoxin oxidative state is a separate but interconnected circadian clock in these organisms. Furthermore, the conserved nature of the peroxiredoxin proteins begs the question of whether the oscillation in their redox state might be a conserved oscillator that exists even in non-photosynthetic Eubacteria. According to Hall et al. [43], only Borrelia species lack evidence of the peroxiredoxin proteins. Thus, the examination of an entrained and a free-running rhythm of peroxiredoxin redox state in non-photosynthetic Eubacteria might provide further evidence of a circadian timekeeping mechanism in these organisms. Formally, and speculatively, these proteins might also be candidates of the oscillator itself.”
Based on this passage peroxiredoxin proteins may also be strong candidates. Below you will find the protein sequence for peroxiredoxin. Run a BLAST search using this sequence on your metagenome and selected novel strain.
QFZ93181.1 peroxiredoxin [Synechococcus elongatus PCC 11802] MPVSRRQLLVSSLLALPALVLAPRSAQALGGPQPPLGEAAPDFSLPTDDGRDRLSLADFRGQWLVLYFYP KDGTPGCTLEAQRFQQDQTAYAERNAQVVGVSADDVSSHSRFRENEGLSYPLLADVKGEVSKRYGSWLAP FSLRHTYIIDPEGVLRASFTAVRPVIHSKEVLAKLDELQAA
Q6) Is peroxiredoxin present in the metagenome? Is it present in your strain?
Q7) Does this change your answer to question #4? Please explain your reasoning.
Q8) Based on all of the available data, do you believe strains in the sample contain a functioning circadian clock? Please be sure to explain your reasoning for your answer.
Q9) In the strains where you have identified circadian proteins what experiments could you do to validate that these strains do have functioning circadian clocks? Given the set up outlined in these two modules what would limit you from conducting these experiments?
Q10) Some of the stains have some but not all of the circadian proteins present. Why would this be the case? What might this tell you about the proteins that are present? What about the proteins that are not present?
The possibility of circadian rhythms in non-photosynthetic Eubacteria are exciting. We suspect that the timekeeping abilities of such organisms are significant but that elucidating those abilities might require systems in which these microbes are in more complex environments than where they are typically cultured in a laboratory setting. The interaction between the timekeepers in these complex environments will be important to understand for basic scientific reasons, as well as for medical, agricultural and environmental reasons as well.