Plasmid-mediated horizontal gene mobilisation: Insights from two lactococcal conjugative plasmids.

The distinct conjugation machineries encoded by plasmids pNP40 and pUC11B represent the most prevalent plasmid transfer systems among lactococcal strains. In the current study, we identified genetic determinants that underpin pNP40- and pUC11B-mediated, high-frequency mobilisation of other, non-conjugative plasmids. The mobilisation frequencies of the smaller, non-conjugative plasmids and the minimal sequences required for their mobilisation were determined, owing to the determination of the oriT sequences of both pNP40 and pUC11B, which allowed the identification of similar sequences in some of the non-conjugative plasmids that were shown to promote their mobilisation. Furthermore, the auxiliary gene mobC, two distinct functional homologues of which are present in several plasmids harboured by the pNP40- and pUC11B-carrying host strains, was observed to confer a high-frequency mobilisation phenotype. These findings provide mechanistic insights into how lactococcal conjugative plasmids achieve conjugation and promote mobilisation of non-conjugative plasmids. Ultimately, these insights would be harnessed to optimise conjugation and mobilisation strategies for the rapid and predictable development of robust and technologically improved strains.

Discovering genetic determinants for cell-to-cell adhesion in two prevalent conjugative lactococcal plasmids.

Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by traAd and trsAd, respectively. Despite their significant sequence divergence, TraAd and TrsAd represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in traAd or trsAd, respectively, were shown to act as efficient donors provided the corresponding recipient expresses either traAd or trsAd, thus demonstrating in trans reciprocal complementarity of these proteins in conjugation systems.

Transcriptional control of two distinct lactococcal plasmid-encoded conjugation systems.

Lactococcal conjugative plasmids are poorly characterized compared to those harbored by numerous other Gram-positive bacteria, despite their significance in dairy fermentations and starter culture development. Furthermore, the transcriptional landscape of these lactococcal conjugation systems and their regulation have not been studied in any detail. Lactococcal plasmids pNP40 and pUC11B possess two genetically distinct and prevalent conjugation systems. Here, we describe the detailed transcriptional analysis of the pNP40 and pUC11B conjugation-associated gene clusters, revealing three and five promoters, respectively, for which the corresponding transcriptional start sites were identified. Regulation of several of these promoters, and therefore conjugation, is shown to involve the individual or concerted activities of the corresponding relaxase and transcriptional repressor(s) encoded by each conjugative plasmid. This work highlights how the conjugative potential of these systems may be unlocked, with significant implications for the starter culture and food fermentation industry.

Delineation of a lactococcal conjugation system reveals a restriction-modification evasion system.

Plasmid pUC11B is a 49.3-kb plasmid harboured by the fermented meat isolate Lactococcus lactis subsp. lactis UC11. Among other features, pUC11B encodes a pMRC01-like conjugation system and tetracycline-resistance. In this study, we demonstrate that this plasmid can be conjugated at high frequencies to recipient strains. Mutational analysis of the 22 genes encompassing the presumed pUC11B conjugation cluster revealed the presence of several genes with essential conjugation functions, as well as a gene, trsR, encoding a putative transcriptional repressor of this conjugation cluster. Furthermore, plasmid pUC11B encodes an anti-restriction protein, TrsAR, which facilitates higher conjugation frequencies when pUC11B is transferred into recipient strains containing Type II or Type III RM systems. These findings demonstrate how RM mechanisms can be circumvented when they act as a biological barrier for conjugation events.

Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis.

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.

Heterologous biosynthesis and characterization of a glycocin from a thermophilic bacterium.

The genome of the thermophilic bacterium, Aeribacillus pallidus 8, encodes the bacteriocin pallidocin. It belongs to the small class of glycocins and is posttranslationally modified, containing an S-linked glucose on a specific Cys residue. In this study, the pallidocin biosynthetic machinery is cloned and expressed in Escherichia coli to achieve its full biosynthesis and modification. It targets other thermophilic bacteria with potent activity, demonstrated by a low minimum inhibitory concentration (MIC) value. Moreover, the characterized biosynthetic machinery is employed to produce two other glycopeptides Hyp1 and Hyp2. Pallidocin and Hyp1 exhibit antibacterial activity against closely related thermophilic bacteria and some Bacillus sp. strains. Thus, heterologous expression of a glycocin biosynthetic gene cluster including an S-glycosyltransferase provides a good tool for production of hypothetical glycocins encoded by various bacterial genomes and allows rapid in vivo screening.

Major gene-regulatory mechanisms operating in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis.

Post-translationally modified peptides commonly display antimicrobial activity, but can also aid the development of bacterial colonies, giving a competitive advantage in the ecological niche. The production of post-translationally modified peptides by bacteria is a complex and energetically costly process that is strictly orchestrated in the cell. The onset of peptide production is linked to the different enzymes that take part during maturation, the transporters and the immunity determinants (if required). Thus, the population can make optimal use of available resources and obtain the benefits of production at an advantageous moment during growth, avoiding toxicity to itself. The timing and level of expression of the different operons is controlled by diverse (complex) regulatory pathways in response to environmental changes, stress or master regulators during specific growth transition phases. In this review, we highlight the basic principles and mechanisms of regulation of expression of post-translationally modified peptides and the relationship with the overall culture developmental processes and/or cellular differentiation. We also discuss the biotechnological consequences derived from the understanding of regulatory networks involved in the biosynthesis of these natural products.

Prospects of In vivo Incorporation of Non-canonical Amino Acids for the Chemical Diversification of Antimicrobial Peptides.

The incorporation of non-canonical amino acids (ncAA) is an elegant way for the chemical diversification of recombinantly produced antimicrobial peptides (AMPs). Residue- and site-specific installation methods in several bacterial production hosts hold great promise for the generation of new-to-nature AMPs, and can contribute to tackle the ongoing emergence of antibiotic resistance in pathogens. Especially from a pharmacological point of view, desirable improvements span pH and protease resistance, solubility, oral availability and circulation half-life. Although the primary focus of this report is on ribosomally synthesized and post-translationally modified peptides (RiPPs), we have included selected cases of peptides produced by solid phase peptide synthesis to comparatively show the potential and impact of ncAA introduction. Generally speaking, the introduction of ncAAs in recombinant AMPs delivers novel levels of chemical diversification. Cotranslationally incorporated, they can take part in AMP biogenesis either through direction interaction with elements of the post-translational modification (PTM) machinery or as untargeted sites with unique physicochemical properties and chemical handles for further modification. Together with genetic libraries, genome mining and processing by PTM machineries, ncAAs present not a mere addition to this process, but a highly diverse pool of building blocks to significantly broaden the chemical space of this valuable class of molecules. This perspective summarizes new developments of ncAA containing peptides. Challenges to be resolved in order to reach large-scale pharmaceutical production of these promising compounds and prospects for future developments are discussed.

Draft Genome Sequences of Two Geobacillus Species Strains, Isolated from Oil Wells and Surface Soil above Oil Pools.

Here, we present the draft genome sequences of two Geobacillus species strains isolated from oil wells and surface soil above oil pools in Lithuania.

Archaeal viruses multiply: temporal screening in a solar saltern.

Hypersaline environments around the world are dominated by archaea and their viruses. To date, very little is known about these viruses and their interaction with the host strains when compared to bacterial and eukaryotic viruses. We performed the first culture-dependent temporal screening of haloarchaeal viruses and their hosts in the saltern of Samut Sakhon, Thailand, during two subsequent years (2009, 2010). Altogether we obtained 36 haloarchaeal virus isolates and 36 archaeal strains, significantly increasing the number of known archaeal virus isolates. Interestingly, the morphological distribution of our temporal isolates (head-tailed, pleomorphic, and icosahedral membrane-containing viruses) was similar to the outcome of our previous spatial survey supporting the observations of a global resemblance of halophilic microorganisms and their viruses. Myoviruses represented the most abundant virus morphotype with strikingly broad host ranges. The other viral morphotypes (siphoviruses, as well as pleomorphic and icosahedral internal membrane-containing viruses) were more host-specific. We also identified a group of Halorubrum strains highly susceptible to numerous different viruses (up to 26). This high virus sensitivity, the abundance of broad host range viruses, and the maintenance of infectivity over a period of one year suggest constant interplay of halophilic microorganisms and their viruses within an extreme environment.

Clinical isolates of Pseudomonas aeruginosa from superficial skin infections have different physiological patterns.

Pseudomonas aeruginosa are known to have a wide physiological potential allowing them to constantly populate diverse environments leading to severe infections of humans such as septicemia, leg ulcers, and burn wounds. We set out to probe physiological characteristics of P. aeruginosa isolates from diabetic leg ulcers collected from Helsinki metropolitan area. A total of 61 clinical isolates were obtained. Detailed phenotypic (physiological) characteristics [outer membrane (OM) permeability, membrane voltage, and activity of multidrug resistance pumps] were determined in several growth phases leading to the division of the analyzed set of P. aeruginosa strains into five distinct clusters including cells with similar physiological properties. In addition, their antibiotic resistance patterns and genetic heterogeneity were determined. Multiple isolates from the same patient were genetically very closely related and belonged to the same phenotypic cluster. However, genetically close isolates from different patients expressed very different phenotypic properties. The characteristics of infected patients seem to determine the growth environments for microorganisms that adapt by changing their physiological and/or genetic properties.

Increasing the success rate of lantibiotic drug discovery by Synthetic Biology.

INTRODUCTION: Lantibiotics are post-translationally modified antimicrobial peptides produced by bacteria from diverse environments that exhibit an activity against pathogenic bacteria comparable to that of medically used antibiotics. The actual need for new antimicrobials in therapeutics has placed them in the pipeline of antibiotic research, due not only to their high antimicrobial activity but also to the fact that they are directed to novel targets. AREAS COVERED: This review covers the different approaches traditionally used in bacteriocin discovery, based on the isolation of bacteria from different habitats and determining their inhibitory spectrum against a set of relevant strains. It also elaborates on more recent approaches covering organic synthesis and semi-synthesis of lantibiotics, genomic and proteomic approaches and the application of Synthetic Biology to the field of antimicrobial drug discovery. EXPERT OPINION: Lantibiotics show a great potential in fulfilling the requirements for new antimicrobials. Culture-dependent techniques are still applied to lantibiotic discovery producing successful results that can be furthered by employing high-throughput screening techniques and peptidogenomics. The necessity of culturing bacteria and growing them in specific conditions for lantibiotic expression, can hamper the discovery rate, especially in exotic or unculturable bacteria. Thus, a combination of genome mining procedures, to detect novel lantibiotic-related sequences, with heterologous production systems and high-throughput screening, offers a promising strategy. Furthermore, the characterization of the mechanism of action of many lantibiotics, and the development of "plug and play" peptide biosynthesis systems, offers the possibility of initiating the rational design of non-natural lantibiotics based on structure-activity relationships.

Assessment of the activity of RND-type multidrug efflux pumps in Pseudomonas aeruginosa using tetraphenylphosphonium ions.

Multidrug resistance (MDR) pumps are one of the major causes of antibiotic resistance in Pseudomonas aeruginosa. Thus, fast and reliable methods are needed to assay the efficiency of MDR pumps in these bacteria. In this study, it was demonstrated that a membrane voltage (Deltapsi) indicator tetraphenylphosphonium (TPP(+)) in combination with the efflux pump inhibitor phenylalanyl-arginyl-beta-naphthylamide can be used to monitor the activity of resistance-nodulation-cell division (RND)-type efflux pumps in P. aeruginosa. By controlling the outer membrane permeability and Deltapsi, electrochemical measurements of RND pump activity in real time were performed. It was demonstrated that the composition of the medium, the presence of nutrients and the level of aeration affect the efficiency of the TPP(+)-extruding activity of P. aeruginosa, urging the standardisation of experimental conditions to obtain quantitative and comparative results.