КОНСЕРВАТИВНОСТЬ ПОСЛЕДОВАТЕЛЬНОСТЕЙ ГЕНОВ ПОМПЫ МНОЖЕСТВЕННОЙ ЛЕКАРСТВЕННОЙ УСТОЙЧИВОСТИ ACRAB-TOLC ESCHERICHIA COLI КАК ПРИЗНАК ВОВЛЕЧЕННОСТИ В ПЕРМАНЕНТНУЮ «УБОРКУ» БАКТЕРИАЛЬНОЙ КЛЕТКИ

32 The gram-negative gammaproteobacterium Escherichia coli (E. coli) was first discovered by Theodor Escherich in the stool samples of healthy individuals in 1885 [1]. E. coli naturally inhabits the lower intestines of warm-blooded species and is an important object of research. Four strains of E. coli, including K-12, B, W and С, are now used as model organisms. Strain K-12 was first isolated at Stanford university in 1922 [2]. Strain B was described by d’Herelle at the Pasteur Institute in Paris in 1918 [3]. The other 2 strains are less common. Strain C was discovered by Margaret Lieb in 1951 [4, 5], and strain W was originally reported by Selman Waksman in 1943 [6]. Strains comprising groups K-12 and B are the most widespread and best known. Laboratory strains have “evolved” to lose some of their properties, such as the ability to form biofilms on abiotic surfaces, and therefore can be advantageously used in research studies, especially for the discovery of novel antibiotics [7]. М. В. Каракозова , П. А. Назаров КОНСЕРВАТИВНОСТЬ ПОСЛЕДОВАТЕЛЬНОСТЕЙ ГЕНОВ ПОМПЫ МНОЖЕСТВЕННОЙ ЛЕКАРСТВЕННОЙ УСТОЙЧИВОСТИ ACRAB-TOLC ESCHERICHIA COLI КАК ПРИЗНАК ВОВЛЕЧЕННОСТИ В ПЕРМАНЕНТНУЮ «УБОРКУ» БАКТЕРИАЛЬНОЙ КЛЕТКИ

The pressure of both natural and artificial selection existing in laboratories has produced numerous derivatives of K-12 and B that are now used all over the world (Table 1).Among the derivatives of strain B are BL21 and BL21(DE3); DH5α, JM109, W3110, XL-1 Blue, and MG1655 are examples of strain K-12 derivatives.
Discovery of novel antibiotics or their effective alternatives is a pressing challenge.One of the most promising areas of research is identification of multidrug resistance (MDR) pump inhibitors.MDR pumps are responsible for removing antibiotics from the bacterial cell.Studies of deletion mutants with knocked-out genes coding for MDR pumps demonstrate that minimum effective inhibitory concentrations of antibiotics in their case are several times lower than usual [8].This may help to reduce both treatment costs and the toxic effect of antibiotic therapies on the patient.Although effects of MDR pumps on antibacterial agents are actively studied, there is an extensive list of objective factors preventing cross-study comparisons, such as different genetic backgrounds of the strains.Even for such closely related strains as W3110 and MG1655 [9], the number of differences at genomic sites can be over 200, impeding comparison.Because bacterial resistance to drugs depends on the presence or absence of efflux pumps, we hypothesized that E. coli strains with identical sequences of MDR pumps might have comparable or equal resistance.To check this supposition, we selected the AcrAB-TolC pump.We aimed to compare sequences of AcrA, AcrB and TolC proteins obtained from different laboratory strains of E. coli and to study the associations between drug resistance and possible mutations if such were present in a sequence.

Selecting a reference sequence
When selecting a reference sequence, we bore in mind a large number of deletion mutants in E. coli K-12 BW25113.It is a parent strain for the Keio collection, which comprises E. coli strains with 3,985 deletions (of 4,288 total E. coli genes) [10].Sequence AIN30961.1 was selected as a reference sequence for AcrA; AIN30960.1,as a reference sequence for AcrB, and AIN33386.1,as a reference sequence for TolC.

Sequence alignment
Sequences were analyzed using a standard local alignment tool NCBI BLASTp, which allows comparison of multiple alignments [11], and the STRING database [12].Visual representation of the results was done in NCBI MSA Viewer [13].Each protein sequence was aligned against its reference sequence.

RESULTS
It is known that bacterial resistance can be a product of: 1) accumulation of resistance genes in plasmids; 2) increased expression of genes coding for MDR pumps; 3) gene duplication; 4) accumulation of mutations [14,15].Increased expression and accumulation of mutations in the genes coding for MDR pumps can result in single nucleotide polymorphisms (SNPs) in the amino acid sequences of proteins.Therefore, bacterial resistance can be predicted by sequence analysis.
Bacterial genes are subdivided into housekeeping genes, which support vital functions of the cell, and contingency genes, which play an important role in bacterial adaptation to the changing environment.Housekeeping genes usually have a low mutation rate, while contingency genes tend to demonstrate a high mutation rate [16].It is believed that genes coding for multidrug efflux pumps are contingency genes; therefore, the proteins they encode are expected to have variable primary structures.Because laboratory strains are usually subject to the pressure of natural selection induced by various biocides and mutagens, the strains that have been cultured in world laboratories for over 100 years, as well as their derivatives, might be different in terms of their amino acid polymorphisms.The strains compared in this work originate from different countries and continents (Table 1), so we can infer the presence of mutations in one of the AcrAB-TolCencoding genes.
Considering the fact that E. coli mutation rate is ~1×10 -3 per genome per generation [17] or even higher (3-4×10 -3 per genome per generation) [18], we hypothesize that the AcrAB-TolC pump sequence is conserved.Given the same sequence coverage for all studied proteins (397 amino acid residues for AcrA, 1049 amino acid residues for AcrB and 493 amino acid residues for TolC), the sequence identity was 100%.

DISCUSSION
According to the currently existing classification, strains from group B and K-12 belong to phylogroup A [19], which may explain the similarity of amino acid sequences between all three proteins but not their identity.Our findings allow us to conclude the presence of a consensus sequence of a highly conserved AcrAB-TolC ensemble.Thus, the selected protein reference sequences AcrA (AIN30961.1 for AcrA, AIN30960.1 for AcrB and AIN33386.1 for TolC, respectively) are consensus for the studied E. coli strains.
The discovered sequences are consensus for all representatives of group A and possibly other phylogroups, including B1, B2, D, and E, which can facilitate normalization of sequences against their consensus counterparts.
The absence of point mutations in the genes coding for protein components of the AcrAB-TolC pump in all studied strains is indicative of the strict selection control, as is the case with housekeeping genes.Such control is particularly important for the major multidrug efflux pump of E. coli (AcrAB-TolC) responsible for removing benzalkonium chloride, ethidium bromide, indole, hexane, antibiotics (erythromycin, ciprofloxacin, etc.), rhodamine, berberine and also triphenylphosphonium and its derivatives from the cell [20][21].
It would be wrong to see genes coding for MDR pumps as responsible for biocide resistance only.They have a role in bacterial colonization and persistence [22], so it is not limited to defense against antibiotics.It appears that proteins produced by MDR pump-encoding genes routinely protect bacterial cells from various biotic and abiotic agents and can be regarded as housekeeping genes engaged in permanent cell "cleaning", unlike contingency genes that get involved only at certain times.

CONCLUSION
Our findings suggest a unique role of the AcrAB-TolC multidrug resistance pump in E. coli.The protein sequence of AcrAB-TolC has turned to be surprisingly conserved.This provides a fresh look at AcrAB-TolC from a different angle: this pump ensures permanent protection against aggressive environment, determines bacterial resistance to antibiotics or their alternatives and even ensures bacterial survival.

Table 2 .
Accession numbers for the stored protein sequences of acrA, acrB and tolC genes