Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene ...transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.
Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity ...determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.
Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights ...into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed.
Abstract
Conjugation systems are present on many plasmids as well as on chromosomally integrated elements. Conjugation, which is a major route by which bacteria exchange genetic material, is a ...complex and energy-consuming process. Hence, a shared feature of conjugation systems is that expression of the genes involved is strictly controlled in such a way that conjugation is kept in a default ‘OFF’ state and that the process is switched on only under conditions that favor the transfer of the conjugative element into a recipient cell. However, there is a remarkable diversity in the way by which conjugation genes present on different transferable elements are regulated. Here, we review these diverse regulatory circuits on the basis of several prototypes with a special focus on the recently discovered regulation of the conjugation genes present on the native Bacillus subtilis plasmid pLS20.
Bacterial conjugation or mating is a process of genetic transfer that involves cell-to-cell contact. This process is a complex and energy consuming process which is strictly regulated. In this minireview, different regulatory circuits have been compared with recently discovered quorum sensing-mediated regulation of conjugation in spore forming Bacilli.
Graphical Abstract Figure.
Bacterial conjugation or mating is a process of genetic transfer that involves cell-to-cell contact. This process is a complex and energy consuming process which is strictly regulated. In this minireview, different regulatory circuits have been compared with recently discovered quorum sensing-mediated regulation of conjugation in spore forming Bacilli.
Geobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the ...resident mobile plasmids from Bacillus subtilis to G. kaustophilus and transferred long segments of chromosome from one cell to another between B. subtilis.
In this study, we applied mobilization of the B. subtilis chromosome mediated by pLS20catΔoriT to transform G. kaustophilus. We constructed a gene cassette to be integrated into G. kaustophilus and designed it within the B. subtilis chromosome. The pLS20catΔoriT-mediated conjugation successfully transferred the gene cassette from the B. subtilis chromosome into the G. kaustophilus allowing for the desired genetic transformation.
This transformation approach described here will provide a new tool to facilitate the flexible genetic manipulation of G. kaustophilus.
Bacterial genomes are folded and organized into compact yet dynamic structures, called nucleoids. Nucleoid orchestration involves many factors at multiple length scales, such as nucleoid-associated ...proteins and liquid-liquid phase separation, and has to be compatible with replication and transcription. Possibly, genome organization plays an intrinsic role in transcription regulation, in addition to classical transcription factors. In this review, we provide arguments supporting this view using the Gram-positive bacterium Bacillus subtilis as a model. Proteins BsSMC, HBsu and Rok all impact the structure of the B. subtilis chromosome. Particularly for Rok, there is compelling evidence that it combines its structural function with a role as global gene regulator. Many studies describe either function of Rok, but rarely both are addressed at the same time. Here, we review both sides of the coin and integrate them into one model. Rok forms unusually stable DNA-DNA bridges and this ability likely underlies its repressive effect on transcription by either preventing RNA polymerase from binding to DNA or trapping it inside DNA loops. Partner proteins are needed to change or relieve Rok-mediated gene repression. Lastly, we investigate which features characterize H-NS-like proteins, a family that, at present, lacks a clear definition.
Abstract
Transcriptional regulation allows adaptive and coordinated gene expression, and is essential for life. Processive antitermination systems alter the transcription elongation complex to allow ...the RNA polymerase to read through multiple terminators in an operon. Here, we describe the discovery of a novel bipartite antitermination system that is widespread among conjugative elements from Gram-positive bacteria, which we named conAn. This system is composed of a large RNA element that exerts antitermination, and a protein that functions as a processivity factor. Besides allowing coordinated expression of very long operons, we show that these systems allow differential expression of genes within an operon, and probably contribute to strict regulation of the conjugation genes by minimizing the effects of spurious transcription. Mechanistic features of the conAn system are likely to decisively influence its host range, with important implications for the spread of antibiotic resistance and virulence genes.
Graphical Abstract
Graphical Abstract
A two-component antiterminator system located at the beginning of the pLS20 conjugation operon contributes to proper expression of the conjugation genes and allows differential gene expression of subsets of genes.
Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and ...virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium
allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation.
Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of
encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation.
Transcriptional regulation usually requires the action of several proteins that either repress or activate a promotor of an open reading frame. These proteins can counteract each other, thus allowing ...tight regulation of the transcription of the corresponding genes, where tight repression is often linked to DNA looping or cross‐linking. Here, the tetramerization domain of the bacterial gene repressor Rco from Bacillus subtilis plasmid pLS20 (RcopLS20) has been identified and its structure is shown to share high similarity to the tetramerization domain of the well known p53 family of human tumor suppressors, despite lacking clear sequence homology. In RcopLS20, this tetramerization domain is responsible for inducing DNA looping, a process that involves multiple tetramers. In accordance, it is shown that RcopLS20 can form octamers. This domain was named TetDloop and its occurrence was identified in other Bacillus species. The TetDloop fold was also found in the structure of a transcriptional repressor from Salmonella phage SPC32H. It is proposed that the TetDloop fold has evolved through divergent evolution and that the TetDloop originates from a common ancestor predating the occurrence of multicellular life.
The tetramerization domain of the bacterial gene repressor Rco from Bacillus subtilis plasmid pLS20 was identified. The structure shows high similarity to the tetramerization domain of the p53 family of human tumor suppressors, despite having low sequence homology.
Some Bacillus-infecting bacteriophages use a peptide-based communication system, termed arbitrium, to coordinate the lysis-lysogeny decision. In this system, the phage produces AimP peptide during ...the lytic cycle. Once internalized by the host cell, AimP binds to the transcription factor AimR, reducing aimX expression and promoting lysogeny. Although these systems are present in a variety of mobile genetic elements, their role in the phage life cycle has only been characterized in phage phi3T during phage infection. Here, using the B. subtilis SPβ prophage, we show that the arbitrium system is also required for normal prophage induction. Deletion of the aimP gene increased phage reproduction, although the aimR deletion significantly reduced the number of phage particles produced after prophage induction. Moreover, our results indicated that AimR is involved in a complex network of regulation and brought forward two new players in the SPβ lysis-lysogeny decision system, YopN and the phage repressor YopR. Importantly, these proteins are encoded in an operon, the function of which is conserved across all SPβ-like phages encoding the arbitrium system. Finally, we obtained mutant phages in the arbitrium system, which behaved almost identically to the wild-type (WT) phage, indicating that the arbitrium system is not essential in the laboratory but is likely beneficial for phage fitness in nature. In support of this, by possessing a functional arbitrium system, the SPβ phage can optimize production of infective particles while also preserving the number of cells that survive after prophage induction, a strategy that increases phage persistence in nature.
Display omitted
•The arbitrium system controls prophage induction in B. subtilis•An operon downstream of the arbitrium system is involved in controlling lysogeny•The operon is functionally conserved in SPβ-like phages encoding arbitrium systems•YopR acts as the phage repressor in SPβ
Bacillus subtilis phages from the SPβ family use the arbitrium system to communicate during infection of the host. Brady et al. show that this system is also required for induction of the resident prophage after activation of the host SOS response and identify a key operon involved in the control of the lytic/lysogenic cycle.
PDF
