Long range PCR reveals the genetic cargo of IncP-1 plasmids in the complex microbial community of an on-farm biopurification system treating pesticide contaminated wastewater

Promiscuous plasmids like IncP-1 plasmids play an important role in the bacterial adaptation to pollution by acquiring and distributing xenobiotic catabolic genes. However, most information comes from isolates and the role of plasmids in governing community-wide bacterial adaptation to xenobiotics and other adaptive forces is not fully understood. Current information on the contribution of IncP-1 plasmids in community adaptation is limited because methods are lacking that directly isolate and identify the plasmid borne adaptive functions in whole-community DNA. In this study, we optimized long range PCR to directly access and identify the cargo carried by IncP-1 plasmids in environmental DNA. The DNA between the IncP-1 backbone genes trbP and traC , a main insertion site of adaptive trait determinants, is amplified and its content analysed by high-throughput sequencing. The method was applied to DNA of an on-farm biopurification system (BPS), treating pesticide contaminated wastewater, to examine whether horizontal gene exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. The cargo recovered from BPS community DNA, encoded catabolic but also resistance traits and various other (un)known functions. Unexpectedly, catabolic traits composed only a minor fraction of the cargo, indicating that the IncP-1 region between trbP and traC is not a major contributor to catabolic adaptation of the BPS microbiome. Instead, it contains a functionally diverse set of genes which either may assist biodegradation functions, be remnants of random gene recruitment, or confer other crucial functions for proliferation in the BPS environment. IMPORTANCE This study presents a long range PCR for direct and cultivation-independent access to the identity of the cargo of a major insertion hot spot of adaptive genes in IncP-1 plasmids and hence a new mobilome tool for understanding the role of IncP-1 plasmids in complex communities. The method was applied to DNA of an on-farm biopurification system (BPS) treating pesticide-contaminated wastewater, aiming at new insights on whether horizontal exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. Unexpectedly, catabolic functions represented a small fraction of the cargo genes while multiple other gene functions were recovered. These results show that the cargo of the target insertion hot spot in IncP-1 plasmids in a community, not necessarily relates to the main selective trait imposed on that community. Instead these functions might contribute to adaptation to unknown selective forces or represent remnants of random gene recruitment.

Conjugative transfer of naturally occurring plasmids in Mycolicibacterium sp.

Conjugation is considered the main horizontal gene transfer (HGT) mechanism in bacterial adaptation and evolution. In the Mycobacteriaceae family, Mycolicibacterium smegmatis has been used as the model organism for the conjugative transfer of hybrid plasmids. However, the natural conjugation process in any bacteria would involve the transfer of naturally occurring plasmids. Currently, there is a gap in this regard in relation to this abundant environmental genus of Mycobacteriaceae. Here, we performed conjugation experiments between wild Mycolicibacterium sp. strains involving naturally occurring plasmids (sizes of 21 and 274 kb), and interestingly, evidence of conjugative transfer was obtained. Thus, it is likely that conjugation occurs in Mycolicibacterium in the natural environment, representing a source of diversification and evolution in this genus of bacteria.

Biofilms can act as plasmid reserves in the absence of plasmid specific selection

Plasmids facilitate rapid bacterial adaptation by shuttling a wide variety of beneficial traits across microbial communities. However, under non-selective conditions, maintaining a plasmid can be costly to the host cell. Nonetheless, plasmids are ubiquitous in nature where bacteria adopt their dominant mode of life - biofilms. Here, we demonstrate that biofilms can act as spatiotemporal reserves for plasmids, allowing them to persist even under non-selective conditions. However, under these conditions, spatial stratification of plasmid-carrying cells may promote the dispersal of cells without plasmids, and biofilms may thus act as plasmid sinks.

Adaptive Response of Listeria monocytogenes to the Stress Factors in the Food Processing Environment

Listeria monocytogenes are Gram-positive, facultatively anaerobic, non-spore-forming bacteria that easily adapt to changing environmental conditions. The ability to grow at a wide range of temperatures, pH, and salinity determines the presence of the pathogen in water, sewage, soil, decaying vegetation, and animal feed. L. monocytogenes is an etiological factor of listeriosis, especially dangerous for the elderly, pregnant women, and newborns. The major source of L. monocytogenes for humans is food, including fresh and smoked products. Its high prevalence in food is associated with bacterial adaptation to the food processing environment (FPE). Since the number of listeriosis cases has been progressively increasing an efficient eradication of the pathogen from the FPE is crucial. Understanding the mechanisms of bacterial adaptation to environmental stress will significantly contribute to developing novel, effective methods of controlling L. monocytogenes in the food industry.

Viral transduction and the dynamics of bacterial adaptation

Viral infections can exert a large influence on their hosts' ecology by causing widespread mortality, but they also shape the evolutionary adaptation of hosts in a number of ways. A major pathway for viruses to do so is through the transfer of genetic material among individual hosts, a process known as transduction. While horizontal gene transfer is known as a major factor in prokaryotic macroevolution, its role in the microevolutionary adaptation of hosts populations is poorly known. By facilitating the transfer of beneficial alleles between host cells, transduction might facilitate and accelerate bacterial adaptation. Conversely, the risk of transferring deleterious alleles may hinder and slow it down. Here we resolve the effect of transduction on bacterial adaptation in a simple eco-evolutionary model for the combined dynamics of transduction and adaptive evolution of an ecological (resource-use) trait. The transfer of beneficial alleles by tranduction speeds up adaptation whereas the transfer of deleterious alleles causes strong stochastic fluctuations of the trait value around the adapted value. In contrast to the expected effect of recombination, which tends to oppose phenotypic diversification, viral transduction can increase host phenotypic diversity.

Genomic Analysis of Citrobacter Portucalensis Sb-2 Identifies a Phage Predation Driven Metalloid Resistance

Bacterial adaptation to extreme environments is often mediated by horizontal gene transfer (HGT). At the same time, phage mediated HGT for conferring bacterial arsenite and antimonite resistance has not been documented before. In this study, a highly arsenite and antimonite resistant bacterium, C. portucalensis strain Sb-2, was isolated and subsequent genome analysis showed that putative arsenite and antimonite resistance determinants were flanked or embedded by prophages. We predict these phage-mediated resistances play a significant role in maintaining genetic diversity within the genus of Citrobacter and are responsible for endowing the corresponding resistances to C. portucalensis strain Sb-2.

Apomorphine Targets the Pleiotropic Bacterial Regulator Hfq

Hfq is a bacterial regulator with key roles in gene expression. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, thanks to its binding to small regulatory noncoding RNAs. This property is of primary importance for bacterial adaptation and survival in hosts. Small RNAs and Hfq are, for instance, involved in the response to antibiotics. Previous work has shown that the E. coli Hfq C-terminal region (Hfq-CTR) self-assembles into an amyloid structure. It was also demonstrated that the green tea compound EpiGallo Catechin Gallate (EGCG) binds to Hfq-CTR amyloid fibrils and remodels them into nonamyloid structures. Thus, compounds that target the amyloid region of Hfq may be used as antibacterial agents. Here, we show that another compound that inhibits amyloid formation, apomorphine, may also serve as a new antibacterial. Our results provide an alternative in order to repurpose apomorphine, commonly used in the treatment of Parkinson’s disease, as an antibiotic to block bacterial adaptation to treat infections.