Hi fellow scientists,
I am researching a gene in Enterococcus faecalis, classified in two groups: 1) Penicillin-sensitive/ampicillin-sensitive (Control) and; 2) Penicillin-resistant/ampicillin-sensitive (Experimental).
I have performed qPCR experiments for both the copy-number of the gene and for its expression.
So far, I have found my experimental group, on average, has 5 times the amount of copies of the target gene, when compared to the average of the control group. That is indeed what we were expecting to find.
However, when testing the expression of this gene, we found that there is a disappointing 1.5-fold difference between these groups. Shouldn't these numbers be proportional to each other? Or should I speculate about other aspects of gene expression such as silencing when writing my discussion?
Is the 1.5-fold difference still as significant?
Thanks in advance.
Hi Luiza,
Inducible structural genes can be regulated positive or negative, despite the copy number of them. Of course that a high copy number should imply a higher expression rate, but if it is a constitutive gene.
I think gene regulation would be a more equilibrated strategy for bacteria to synthesize more mRNA and proteins required to cope a external signal. They have a polycistronic genes and the polyribbosomes can translate faster. Moreover, transcription factors and correct catalysts are required.
You are not explaining clearly your experiment details, but I can assume you are testing a fixed level of antibiotics.
This example is very blunt, but I hope you get my point. If you apply 1 mM of antibiotics, the bacteria produces 1 mM of enzymes to neutralize them.
Of course, transcription and translation are up or down regulated, so if the bacteria produces 0.5 of RNAm, from this molecules it could translate to 1.5 of proteins, despite of having 5 of DNA.
That is, if the antibiotics activate the resistant genes, it is logical that it will synthesize just the appropriate amount of mRNA and/or translate the right amount of enzymes required for deactivating the antibiotic.
Another hypothesis why your findings does not make sense, could be that not all the genes have a promoter or are silenced.
I hope this helps.
Hi Luiza,
Inducible structural genes can be regulated positive or negative, despite the copy number of them. Of course that a high copy number should imply a higher expression rate, but if it is a constitutive gene.
I think gene regulation would be a more equilibrated strategy for bacteria to synthesize more mRNA and proteins required to cope a external signal. They have a polycistronic genes and the polyribbosomes can translate faster. Moreover, transcription factors and correct catalysts are required.
You are not explaining clearly your experiment details, but I can assume you are testing a fixed level of antibiotics.
This example is very blunt, but I hope you get my point. If you apply 1 mM of antibiotics, the bacteria produces 1 mM of enzymes to neutralize them.
Of course, transcription and translation are up or down regulated, so if the bacteria produces 0.5 of RNAm, from this molecules it could translate to 1.5 of proteins, despite of having 5 of DNA.
That is, if the antibiotics activate the resistant genes, it is logical that it will synthesize just the appropriate amount of mRNA and/or translate the right amount of enzymes required for deactivating the antibiotic.
Another hypothesis why your findings does not make sense, could be that not all the genes have a promoter or are silenced.
I hope this helps.
In most bacteria, ampicillin and penicillin resistance is due to the production of beta lactamases and/or reduced affinity of the proteins that the antibiotics bind to.
Mechanisms of resistance to imipenem and ampicillin in Enterococcus faecalis.
Ono S, Muratani T, Matsumoto T.
Antimicrob Agents Chemother. 2005 Jul;49(7):2954-8.
PMID: 15980374
Treatment of experimental endocarditis caused by a beta-lactamase-producing strain of Enterococcus faecalis with high-level resistance to gentamicin.
Hindes RG, Willey SH, Eliopoulos GM, Rice LB, Eliopoulos CT, Murray BE, Moellering RC Jr.
Antimicrob Agents Chemother. 1989 Jul;33(7):1019-22.
PMID: 2506803
Infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis.
Wells VD, Wong ES, Murray BE, Coudron PE, Williams DS, Markowitz SM.
Ann Intern Med. 1992 Feb 15;116(4):285-92.
PMID: 1733382
Overproducton of antibiotic export proteins (eflux pumps) is usually described as a mechanism of multi-drug resistance.
Article Mechanisms of antibiotic resistance in enterococci
Overproduction of a messenger RNA is not always necessary to produce more protein action. As noted by Julio above, the efficiency of translation can affect protein production, and changes in affinity or efficiency of the proteins can make a large difference too.
Antibiotic-resistant enterococci: the mechanisms and dynamics of drug introduction and resistance.
Shepard BD, Gilmore MS.
Microbes Infect. 2002 Feb;4(2):215-24. Review.
PMID: 11880055
Horizontal gene transfer-emerging multidrug resistance in hospital bacteria.
Dzidic S, Bedeković V.
Acta Pharmacol Sin. 2003 Jun;24(6):519-26. Review.
PMID: 12791177
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