Genome sequences are available for tens of thousands of microbes. For most of these microbes, little is known about their physiology other than the condition under which they were isolated. If the microbe was isolated using a complex substrate, such as yeast extract, then nothing is known about its nutritional requirements.
These low-confidence candidates are shown on the GapMind website because they may be useful for filling gaps in amino acid biosynthesis pathways. In four cases, we are confident that the protein is involved in the pathway, but we cannot predict its precise activity. Four proteins were similar to both prephenate dehydrogenases and arogenate dehydrogenases, and the genetic data confirmed that they were important for fitness in minimal media unless a mixture of amino acids is added. This confirms these proteins are involved in amino acid biosynthesis, but we still do not know whether they act on prephenate, arogenate, or both. In the updated GapMind, these proteins (and their homologs) are considered good candidates for either activity. It appears that most members of the phylum Bacteroidetes synthesize arginine via succinylated intermediates.
First, at the beginning of the pathway, no candidates for N-acetylglutamate synthase (ArgA or ArgJ) were identified. Given the confidence levels for the candidates, GapMind computes confidence levels for steps and pathways and finds the highest-confidence pathway for synthesizing each amino acid. The confidence of a step is the highest confidence of any candidate for that step. Steps are considered low confidence even if they have no candidates at all.
Gather with friends and family
- We also examined the microbe with the most gaps, which was the hyperthermophilic archaeon Pyrolobus fumarii 1A.
- For high-confidence candidates, GapMind requires 40% identity to a characterized protein with 80% coverage or a match to a curated family with 80% coverage.
- GapMind automatically joins these proteins together based on the nonoverlapping alignments of two pieces to the same characterized protein (Fig. 2B).
- We chose 40% identity as a threshold, because more distantly related enzymes often have different substrates (18).
- Specifically, we will focus on whether a microbe can synthesize the 20 standard amino acids.
- We found evidence for two poorly studied pathways and confirmed dozens of candidates that were divergent from previously characterized proteins.
We thank Valentine V. Trotter for prepublication access to genetic data for Desulfovibrio vulgaris Hildenborough. These certificates can be issued for lbl.gov and nersc.gov. If you have questions or need a certificate for another domain, please contact email protected. Now, researchers in France and Germany have taken the technology one lbl komodo step further. They have found a way to gather up and move around atoms in bunches.
On the GapMind website, split candidates are marked with an asterisk. If the two parts of the split are adjacent, it is often ambiguous whether the protein-coding gene is actually split, disrupted by a genuine frameshift, or disrupted by a frameshift error in the genome sequence. Besides the amino acids that are represented in GapMind, L.
Because of this, GapMind implicitly assumes that all intermediates in central metabolism are available. This is likely to be true if the microbe contains most of the amino acid biosynthesis pathways, but it might not be true for microbes that have many auxotrophies, such as Lactobacillus helveticus CNRZ 32. GapMind also assumes that other amino acids are available, but if this is not likely to be the case, it should be obvious from GapMind’s results.
Expanding GapMind’s database using genetic data.
On 27 December 2015, President Joko Widodo inaugurated a new modern terminal at the airport. The larger terminal could provide passenger services for up to 1.5 million passengers per year, compared with the capacity of the old terminal of around 150,000 passengers per annum. The new infrastructure was thus expected to encourage a marked increase in the number of tourists coming to the island of Flores and its surroundings. This section collects any data citations, data availability statements, or supplementary materials included in this article. The Web-based interface relies on the common gateway interface library (CGI.pm).
Associated Data
For 96 of the 148 microbes, the set included another microbe from the same family (as classified by GTDB). We focused on the gaps that were low-confidence steps and that were confirmed by analyzing the six-frame translation. The 96 microbes had 118 such gaps, and 96 of these steps (81%) were also gaps in another microbe from the same family.
The best path for an amino acid is the one that gives the best score. If two paths have the same score, then GapMind considers a secondary score that gives weights of −2, −0.1, and +1 to low-, medium-, and high-confidence steps. If there is still a tie, then GapMind chooses the longer path.
- This suggests that the two pathways are genetically redundant.
- In 69 of these 70 cases, the predicted pathway was the Bacteroides-type pathway.
- First, many bacteria do not use the standard biosynthetic pathways from Escherichia coli or Bacillus subtilis that are described in textbooks.
- Both genes are important for fitness under some conditions (Fig. 4B), but there are no experiments where both genes had fitness values under −1 (which corresponds to a 2-fold reduction in the abundance of mutant strains).
- For heteromeric enzymes, each subunit is treated as a separate step.
- Now, researchers in France and Germany have taken the technology one step further.
FIG 4.
Thus, HSERO_RS20920 is a high-confidence candidate for both AroB and AroL. When analyzing a new genome, GapMind uses usearch with global alignment (16) to quickly find proteins in the new genome that are at least 50% identical to the marker genes and with alignment coverage of at least 70%. GapMind only searches for the top 20 hits (-maxaccepts 20 -maxrejects 20).
Like a minuscule vacuum cleaner, it can easily slide over a copper surface, sucking up loose copper atoms. When scientists want to study atoms one at a time, however, they can use special, highly sensitive microscopes to see them. Using these tools, called scanning tunneling microscopes (STMs), researchers can also move individual atoms around.
For instance, if a diverged candidate for a step is identified, then it is labeled as medium confidence; if this step is part of the most likely pathway, then it will be highlighted. The user can examine the results and decide if the pathway is likely to be present or not. We tried to include all known pathways for amino acid biosynthesis that begin with intermediates in central metabolism and that occur in bacteria or archaea. Because most free-living bacteria and archaea can probably make all 20 standard amino acids (3), we also allow pathways to use other amino acids as starting points. For example, many microorganisms synthesize cysteine from serine and sulfide. In our analysis, we found that the gaps in amino acid biosynthesis pathways were often conserved between related organisms.
If these requirements are violated, then GapMind issues a warning. We chose not to give amino acids as requirements (such as a serine requirement for cysteine biosynthesis), because GapMind already shows if an amino acid might be required for growth. However, we do use requirements to define dependencies on intermediates. For example, some organisms form cysteine from phosphoserine instead of from serine; if this pathway is on the best path, then GapMind will check if serA and serC are present. As another example, GapMind will issue a warning if the organism is predicted to synthesize methionine from cysteine (transsulfuration) and also cysteine from methionine (reverse transsulfuration).
GapMind’s matching of terms to curated descriptions is case-insensitive, and each match must begin and end at word boundaries. For some steps, we also identified specific sequences (by UniProt identifier) that are known to perform the step but are not curated in the databases that GapMind relies on. We identified 99 such sequences, mostly by using the fitness data but also from the literature. Helveticus requires lysine for growth (35), the biosynthetic pathway appears to be complete except for the acetyl-diaminopimelate aminotransferase DapX; GapMind identified a medium-confidence candidate for DapX.
