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Editor: Li Xinyu, She Ni Zhuoga, Wang Jiaying, Wei Tao (College of Food, South China Agricultural University) Enterococcus is one of the main causes of multidrug-resistant nosocomial infections.
It is known for its robustness to the environment and extends to inherent antibiotic resistance.
In order to identify genes that confer this unique attribute, Michael's team used Tn-seq technology to fully explore the genome of Enterococcus faecalis strain MMH594, looking for genes that are important for growth in nutrient-containing media and have low-level antibiotic attack.
As expected, the large core of genes shared with other bacteria for DNA replication, expression, and central metabolism is not tolerant to transposon destruction.
However, genetic identification is very important for Enterococcus faecalis.
After similar tests, it can be seen that they have no influence or no importance on the adaptability of Staphylococcus aureus and Streptococcus pneumoniae.
In addition, 217 genes have been identified that, when challenged by sub-MIC antibiotic levels, exhibit reduced tolerance to transposon destruction, including those previously proven to contribute to intrinsic resistance, as well as other previous genes.
There is no gene that confers this effect.
E.
Stool is one of the few Gram-positive bacteria that has proved to have a functional Entner-Doudoroff carbon metabolism pathway in experimental experiments, which helps other microorganisms to resist stress.
Through functional genomics and network analysis, Michael's team defined the abnormal structure of this pathway in Enterococcus faecalis and evaluated its importance.
These methods also identify toxin-antitoxin and related systems.
Finally, Michael’s team identified genes that are not present in the closest non-enterococcal relative, vagococci.
These genes have important contributions to adaptability with and without antibiotics, thereby enhancing the unique biology of enterococci.
understanding.Enterococcus is the main cause of antibiotic-resistant infections spread in hospitals.
The inherent resistance of these organisms allows them to survive disinfection operations and then proliferate in the gastrointestinal tract of patients treated with antibiotics.
The purpose of this research is to determine its unusually strong underlying genetic basis.
By using functional genomics methods, Michael's team determined the universally significant characteristics and pathways of enterococci's survival and growth, thereby distinguishing them from closely related pathogens and their original species.
We further identified the unique characteristics that enabled them to survive the antibiotic challenge, revealed a large set of genes that contribute to intrinsic antibiotic resistance, and a small set of unique and important genes that are rare outside of Enterococcus.
Michael's team constructed a highly complex sailor transposon inserted into Enterococcus faecalis MMH594, which lasted more than 3 years due to multi-drug resistant strains.
Before exploring the role and contribution of the genes on which Enterococcus faecalis depends, Michael's team first assessed the integrity of the library.
In order to ensure the strongest possible data set and test reproducibility, 10 different cultures of Sailor were independently inoculated with transposon inserts and cultured in nutrient-rich Mueller-Hinton broth for 12 generations on different days.
From the number of sequence reads for each transposon linker, the relative abundance of various transposon insertions in the population can be inferred.
Michael's team used the procedure originally used for the transposon insertion sequencing (Tn-seq) study of Staphylococcus aureus, and improved the analysis process to map the insertion map to the genes in the closed genome of Enterococcus faecalis MMH594.
A new closed genome sequence of this strain was recently determined from a population that has not been archived since 1985.
The analysis identified 38,366 sailor inserts at different locations and produced highly reproducible results between cultures.
A further source of insertion bias was observed, which stems from the nature of bacterial chromosome replication.
With evidence of multiple insertions of almost all structural genes that will tolerate the destruction of transposon insertion and still produce cells capable of growing and dividing, Michael's team next sought to identify those codes that have unique contributions to the fitness of Enterococcus faecalis The function of the gene.
In order to normalize the number of insertions and normalize the reads of the insertion connection to the gene size, the Michael team calculated a variant of the previously used D value (dVal) indicator.
In addition, excluding the first 10% and last 10% insertions of the reading frame, Michael's team scored the insertions that occurred within the central 80% of each gene.
Inferring from dVal, the relative abundance of MMH594 gene mutation in the mutant population is shown in Figure 1.
Figure 1 The promotion from a low dVal-based candidacy to a "critical" or "important" category requires additional verification.
First, Michael's team used rigorous statistical permutation tests to evaluate transposon insertions relative to the local genomic environment.
However, only 36 indel genes were found in the chromosomes centered at the end of replication in the quartile of the chromosome, accounting for 33% of the frequency of the quartile centered at the origin of replication, and the genes of the former were 29% higher on average.
%, which indicates that the observed replication bias and other possible causes have a high false negative rate.
In order to account for the skew of the local environment due to replication bias and necessary gene clustering, Michael's team performed a second statistical evaluation of neighboring genes with low dVal in general.
This identified an additional 56 genes in the terminal quartile, but still showed limited sensitivity for verifying genes that are intolerant to disruption.
To determine other possible false negatives, Michael's team used a third method, comparing the Tn-seq results with studies of two related low G + C gram-positive pathogens, Streptococcus pneumoniae and Staphylococcus aureus.
In those studies, genes with highly conserved orthologs were classified as necessary or possibly necessary, and then external verification was considered.
Through this multi-layer orthogonal method, 349 genes verified by at least one additional standard are classified as "Fitness Critical", and 224 genes are similarly classified as "Fitness Important".
The structure and function analysis of Figure 2 shows that the dimer of HEPN family protein has RNase activity against mRNA, and the actual toxin HEPN family protein and antitoxin nucleoside acyltransferase form an octagon.
This once again proves the robustness of Tn-seq analysis, which is used to identify features related to negative selection.
With regard to almost all known results about the intrinsic resistance of Enterococcus faecalis to cephalosporins, other β-lactic acids, circulating peptides and daptomycin, Tn-seq provides information on the intrinsic resistance of genes to this species A powerful tool.
In addition to supporting known relationships, the internally highly competitive nature of Tn-seq analysis also provides additional insights into the relative penalties associated with the construction of various types of mutants, identifying those mutations whose second suppressor may easily exceed the mutant population body.
In addition to identifying functions of unique importance to enterococci, Michael's team also revealed a long-standing mystery of how enterococci and possibly related organisms can metabolize glucose through the Entner-Doudoroff shunt without ED enzymes.
Acid salt.
Finally, many hypothetical protein-coding genes are associated with phenotypes to provide insights into function and establish a priority basis for attempts to genetically define the unique properties of Enterococcus.
Although many of the identified genes have been verified through direct experiments in the literature, and experiments have done their best to supplement methods and extract new insights from the generated data, thereby limiting and balancing false positives and false negatives, many identified genes The role and importance of it remains to be examined.
Link to the paper: https://mbio.
asm.
org/content/11/6/e02962-20#sec-1 Top 101 in 2019 hot articles.
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.
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It is known for its robustness to the environment and extends to inherent antibiotic resistance.
In order to identify genes that confer this unique attribute, Michael's team used Tn-seq technology to fully explore the genome of Enterococcus faecalis strain MMH594, looking for genes that are important for growth in nutrient-containing media and have low-level antibiotic attack.
As expected, the large core of genes shared with other bacteria for DNA replication, expression, and central metabolism is not tolerant to transposon destruction.
However, genetic identification is very important for Enterococcus faecalis.
After similar tests, it can be seen that they have no influence or no importance on the adaptability of Staphylococcus aureus and Streptococcus pneumoniae.
In addition, 217 genes have been identified that, when challenged by sub-MIC antibiotic levels, exhibit reduced tolerance to transposon destruction, including those previously proven to contribute to intrinsic resistance, as well as other previous genes.
There is no gene that confers this effect.
E.
Stool is one of the few Gram-positive bacteria that has proved to have a functional Entner-Doudoroff carbon metabolism pathway in experimental experiments, which helps other microorganisms to resist stress.
Through functional genomics and network analysis, Michael's team defined the abnormal structure of this pathway in Enterococcus faecalis and evaluated its importance.
These methods also identify toxin-antitoxin and related systems.
Finally, Michael’s team identified genes that are not present in the closest non-enterococcal relative, vagococci.
These genes have important contributions to adaptability with and without antibiotics, thereby enhancing the unique biology of enterococci.
understanding.Enterococcus is the main cause of antibiotic-resistant infections spread in hospitals.
The inherent resistance of these organisms allows them to survive disinfection operations and then proliferate in the gastrointestinal tract of patients treated with antibiotics.
The purpose of this research is to determine its unusually strong underlying genetic basis.
By using functional genomics methods, Michael's team determined the universally significant characteristics and pathways of enterococci's survival and growth, thereby distinguishing them from closely related pathogens and their original species.
We further identified the unique characteristics that enabled them to survive the antibiotic challenge, revealed a large set of genes that contribute to intrinsic antibiotic resistance, and a small set of unique and important genes that are rare outside of Enterococcus.
Michael's team constructed a highly complex sailor transposon inserted into Enterococcus faecalis MMH594, which lasted more than 3 years due to multi-drug resistant strains.
Before exploring the role and contribution of the genes on which Enterococcus faecalis depends, Michael's team first assessed the integrity of the library.
In order to ensure the strongest possible data set and test reproducibility, 10 different cultures of Sailor were independently inoculated with transposon inserts and cultured in nutrient-rich Mueller-Hinton broth for 12 generations on different days.
From the number of sequence reads for each transposon linker, the relative abundance of various transposon insertions in the population can be inferred.
Michael's team used the procedure originally used for the transposon insertion sequencing (Tn-seq) study of Staphylococcus aureus, and improved the analysis process to map the insertion map to the genes in the closed genome of Enterococcus faecalis MMH594.
A new closed genome sequence of this strain was recently determined from a population that has not been archived since 1985.
The analysis identified 38,366 sailor inserts at different locations and produced highly reproducible results between cultures.
A further source of insertion bias was observed, which stems from the nature of bacterial chromosome replication.
With evidence of multiple insertions of almost all structural genes that will tolerate the destruction of transposon insertion and still produce cells capable of growing and dividing, Michael's team next sought to identify those codes that have unique contributions to the fitness of Enterococcus faecalis The function of the gene.
In order to normalize the number of insertions and normalize the reads of the insertion connection to the gene size, the Michael team calculated a variant of the previously used D value (dVal) indicator.
In addition, excluding the first 10% and last 10% insertions of the reading frame, Michael's team scored the insertions that occurred within the central 80% of each gene.
Inferring from dVal, the relative abundance of MMH594 gene mutation in the mutant population is shown in Figure 1.
Figure 1 The promotion from a low dVal-based candidacy to a "critical" or "important" category requires additional verification.
First, Michael's team used rigorous statistical permutation tests to evaluate transposon insertions relative to the local genomic environment.
However, only 36 indel genes were found in the chromosomes centered at the end of replication in the quartile of the chromosome, accounting for 33% of the frequency of the quartile centered at the origin of replication, and the genes of the former were 29% higher on average.
%, which indicates that the observed replication bias and other possible causes have a high false negative rate.
In order to account for the skew of the local environment due to replication bias and necessary gene clustering, Michael's team performed a second statistical evaluation of neighboring genes with low dVal in general.
This identified an additional 56 genes in the terminal quartile, but still showed limited sensitivity for verifying genes that are intolerant to disruption.
To determine other possible false negatives, Michael's team used a third method, comparing the Tn-seq results with studies of two related low G + C gram-positive pathogens, Streptococcus pneumoniae and Staphylococcus aureus.
In those studies, genes with highly conserved orthologs were classified as necessary or possibly necessary, and then external verification was considered.
Through this multi-layer orthogonal method, 349 genes verified by at least one additional standard are classified as "Fitness Critical", and 224 genes are similarly classified as "Fitness Important".
The structure and function analysis of Figure 2 shows that the dimer of HEPN family protein has RNase activity against mRNA, and the actual toxin HEPN family protein and antitoxin nucleoside acyltransferase form an octagon.
This once again proves the robustness of Tn-seq analysis, which is used to identify features related to negative selection.
With regard to almost all known results about the intrinsic resistance of Enterococcus faecalis to cephalosporins, other β-lactic acids, circulating peptides and daptomycin, Tn-seq provides information on the intrinsic resistance of genes to this species A powerful tool.
In addition to supporting known relationships, the internally highly competitive nature of Tn-seq analysis also provides additional insights into the relative penalties associated with the construction of various types of mutants, identifying those mutations whose second suppressor may easily exceed the mutant population body.
In addition to identifying functions of unique importance to enterococci, Michael's team also revealed a long-standing mystery of how enterococci and possibly related organisms can metabolize glucose through the Entner-Doudoroff shunt without ED enzymes.
Acid salt.
Finally, many hypothetical protein-coding genes are associated with phenotypes to provide insights into function and establish a priority basis for attempts to genetically define the unique properties of Enterococcus.
Although many of the identified genes have been verified through direct experiments in the literature, and experiments have done their best to supplement methods and extract new insights from the generated data, thereby limiting and balancing false positives and false negatives, many identified genes The role and importance of it remains to be examined.
Link to the paper: https://mbio.
asm.
org/content/11/6/e02962-20#sec-1 Top 101 in 2019 hot articles.
The big cow who reversed type 2 diabetes posted another article: Type 2 diabetes is a simple disease.
Lose weight or reverse the condition! 2.
Just now, Science released the top ten scientific breakthroughs in 2019! 3.
Science is heavy! Broccoli "awakens" anti-tumor genes 4.
Readers' tears! "The Lancet" published a Chinese medical doctor's "Family Letter" in Chinese: A Letter to Father 5.
"Science" is heavy! Your "honey", my "arsenic.
.
.
" 6.
Drink alcohol, especially those who are still blushing, or face a higher risk of dementia 7.
Nature is heavy! The first completely synthetic creature that completely changed the DNA code was born 8.
This is not a big hazelnut! Science has published a new type of oral insulin, or it will replace traditional injections 9.
Science's anti-balding tips: start with no hair loss.
.
.
10.
Can changing sperm speed affect the sex of the offspring?
