Elsevier

Anaerobe

Volume 22, August 2013, Pages 82-89
Anaerobe

Molecular biology, genetics and biotechnology
Identification and characterization of toxin–antitoxin systems in strains of Lactobacillus rhamnosus isolated from humans

https://doi.org/10.1016/j.anaerobe.2013.05.007Get rights and content

Highlights

  • Six putative toxin–antitoxin systems (TASs) were identified in L. rhamnosus genome.

  • PCR revealed all the TASs in 15 L. rhamnosus strains isolated from humans.

  • Strains demonstrated gene and genome polymorphisms of TASs.

  • Cloning of 3 toxin genes inhibited E. coli growth.

Abstract

The toxin–antitoxin gene systems (TASs) are present in the genomes of the overwhelming majority of bacteria and archaea. These systems are involved in various cellular regulatory processes (including stress response), and have not been previously investigated in Lactobacilli. We identified 6 putative TASs with toxins belonging to the MazE and RelE superfamilies (PemK1-А1Lrh, PemK2-А2Lrh, PemK3-RelB2Lrh, RelE1Lrh, RelB3-RelE3Lrh, and YefM-YoeBLrh) in the genomes of annotated strains of Lactobacillus rhamnosus. PCR analyses revealed that all systems were found in the genomes of 15 strains of L. rhamnosus isolated from humans in central Russia. These strains were highly heterogeneous with respect to the presence of TASs, as well as their nucleotide and amino acid sequences. In three cases, the relE1 genes contained IS3 elements. TAS heterogeneity may be used to reveal inter-genus differences between strains. Cloning of the toxin genes of 3 TASs inhibited Escherichia coli growth, thus confirming their functionality. Cell growth arrest caused by expression of the toxin genes could be reverted by the expression of a cognate antitoxins. Transcription of toxin–antitoxin loci in L. rhamnosus was shown by RT-PCR.

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.

Section snippets

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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