Molecular biology, genetics and biotechnologyIdentification and characterization of toxin–antitoxin systems in strains of Lactobacillus rhamnosus isolated from humans
Introduction
Lactobacillus rhamnosus is a widespread species that inhabits various ecological niches: plants, food (often as a part of starter cultures of dairy products), and the gastrointestinal and urogenital flora of humans [1]. It can also be used for industrial production of lactic acid [2]. L. rhamnosus strains presented in the human body inhibit the propagation of pathogenic bacteria, contribute to the digestive process, and participate in the formation of innate and acquired immunity [3]; for this reason, they are a part of many probiotic drugs [4]. L. rhamnosus, together with Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus zeae, constitutes a separate systematic group of Lactobacillus [5]. The properties of L. rhamnosus strains differ significantly not only in the presence/absence of individual genes and gene systems but also in the mechanisms of their regulation. Toxin–antitoxin systems (TASs) play a significant role in the regulation of gene activity.
TASs were originally found on low copy number plasmids [6]. They were considered to cause death of cells that had lost plasmids (so called post-segregation killing) and thereby contributed to the conservation of plasmids in a population of bacterial cells. Recently, similar systems have been found to be part of chromosomes in the vast majority of bacteria and archaea [7], [8], [9], [10], [11]. The number of TASs in the genomes of microorganisms varies and is occasionally very large. At least 36 toxin genes have been detected in Escherichia coli K-12 genome [12], and more than 88 were detected in Mycobacterium tuberculosis [13]. TASs are not essential elements of the bacterial genome but rather belong to its flexible part. ТАSs usually consist of two components – toxin and antitoxin. 5 types of TASs have been identified [14], [15], [16]. The antitoxin may be a protein or an untranslatable RNA, while the toxin is a small protein. TASs of type II are the most abundant and well studied and are the subject of the present study. Both toxins and antitoxins are small proteins. The toxin and antitoxin genes are located near each other and form an operon. Toxins usually suppress cell proliferation, less likely they cause cell death; antitoxins interact with toxins and block their activity. Toxins can target mRNA, ribosomes, DNA gyrase, cytoskeletal proteins, and cell wall synthesis. Antitoxins are less stable than toxins. Under stress conditions antitoxins are digested by stress-induced proteases to release free toxins from the TA complex, leading to growth arrest or cell death [14], [17]. On the basis of the structure and functions of type II TASs, 12 superfamilies of toxins and 20 superfamilies of antitoxins have been distinguished [11]. It has been established that TASs participate in the stabilization of bacterial genomes and in regulation of programmed cell death, response to stressful environmental conditions, cell transition to a persistent state, biofilm formation, quorum sensing. TASs can be a part of general regulatory network of bacterial cells [18], [19], [20], [21], [22], [23], [24], [25]. Information on TASs is available on web servers at http://genoweb.univ-rennes1.fr./duals/RASTA-Bacteria/ [26] and http://bioinfo-mml.sjtu.edu.cn/TADB/ [10].
TASs were identified in silico in the chromosomal [7], [9], [10], [22] and plasmid [27], [28], [29] DNA of some Lactobacillus species. Several TASs have been identified in L. rhamnosus [10]. However, the properties of TASs of lactobacilli have not been studied.
The objective of this work was the identification of type II TASs in the genomes of sequenced L. rhamnosus strains from GenBank, and determination of the presence, functionality and polymorphism of these TASs in genomes of 15 L. rhamnosus strains isolated from individuals that reside in the central region of Russian Federation.
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Bacterial strains and plasmids
The bacterial strains and plasmids used in this work are denoted in Tables 1 and 2. L. rhamnosus strains were isolated from the guts, mouths and vaginas of healthy people inhabiting the central region of Russian Federation (Department of Genetics, State Medical Academy, Tver, Russian Federation). The L. rhamnosus strain 421-2 was isolated in the Laboratory of Microbial Ecology, Gabrichevski Institute of Epidemiology and Microbiology, Moscow, Russian Federation. Identification of Lactobacillus
Identification of TASs in annotated genomes of L. rhamnosus
By May 2013, the number of L. rhamnosus strains for which sequencing was brought to the stage “scaffolds” was equal to 12 (http://www.ncbi.nlm.nih.gov/genome/913). These strains were ATCC 21052, HN001, CASL, Lc705, ATCC 8530, LMS2-1, R0011, MTCC 5462, LRHMDP2, LRHMDP3 and two strains GG from diverse origin. In the strain MTCC 5462, the location of the scaffolds was such that we could not obtain accurate data on TAS structure; therefore, 11 genomes of 10 strains were taken for analysis. We were
Discussion
Five putative TASs have been identified in the genome of L. rhamnosus (http://bioinfo-mml.sjtu.edu.cn/TADB/). The toxin of one TAS belonged to the PemK family, while the proteins of the other systems were not assigned to any of the families. We found 6 different putative TASs in the genomes of L. rhamnosus strains from GenBank; one of them (PemK1-А1Lrh) coincided with TAS from TADB, and the 5 other TASs (PemK2-А2Lrh, PemK3-RelB2Lrh, RelE1Lrh, RelB3-RelE3Lrh, and YefM-YoeBLrh) were newly
Funding
Federal program of research and development in priority areas of science and technology (Russian Federation), 2013-1.2-14-512-0011.
Acknowledgments
We thank A.A. Shtil for preparing the manuscript.
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