Abstract
To adapt to an ever-changing environment, bacteria must constantly regulate cellular processes to survive. A key way in which bacteria can adapt to environmental pressures is by acquiring new genetic information through horizontal gene transfer (Rodriguez-Beltran et al., 2021). This genetic transfer is mediated by an array of mobile genetic elements, with the diverse family of large conjugative plasmids of particular interest to researchers studying the soil and rhizosphere microbiomes. Large conjugative plasmids are found in almost all bacterial genera and are typically present in low- or single copy numbers within a cell.
Harboring and maintaining a plasmid is expensive for the host cell and often comes with a measurable fitness cost (Brockhurst and Harrison, 2022; Harrison et al., 2016). Thus, the classical model of plasmid biology states that plasmids need to be maintained through selective pressure (Brockhurst and Harrison, 2022). This can take the form of positive selection, meaning the cells carrying the plasmid are at a distinct advantage to surrounding plasmid-free cells, as is the case with multidrug resistant plasmids and antimicrobial resistance (Buckner et al., 2018; Carattoli, 2011; Loftie-Eaton et al., 2016).
Alternatively, addiction mechanisms such as toxin–antitoxin systems ensure that loss of their carrier plasmid leads to cell death (Jurenas et al., 2022). In recent years, however, this model has begun to be challenged. Although a number of large conjugative plasmids have observable selection markers, many are maintained stably within microbial populations without discernible positive selection or addiction systems (Hall et al., 2015; Kottara et al., 2018; Wein et al., 2019).
Some plasmids have become so engrained within their host, and the behavioral changes they exert are so essential for their hosts' survival, that they are considered part of the core genetic material for those bacteria. Some of the most well studied are the pSym plasmids in legume-associated Rhizobia, which encode the genes responsible for nodule formation (Brom et al., 2000; Gamez-Reyes et al., 2017). A more extreme example of plasmid domestication is Vibrio cholerae chromosome II, which encodes large number of virulence factors without which V. cholerae would be unable to infect its host (Bruhn et al., 2018; Venkova and Chattoraj, 2011).
These are examples of the direct impact of plasmid carriage on their hosts through the expression of plasmid-encoded genes, however, behavioral changes can also be exerted through plasmid-chromosome crosstalk (PCC), in which chromosomal or plasmid-encoded genes are able to cross-regulate (Vial and Hommais, 2020).
The initial acquisition of a plasmid is a stressful situation for a cell, leading to a general upregulation of the host SOS response (Baharoglu et al., 2010; Hall et al., 2021) and differential expression of the lexA regulon (Hall et al., 2021). However, after acquisition, the carriage of a plasmid frequently leads to more widespread changes in the phenotypic profile of the host bacterium (Billane et al., 2021). Such changes may, in turn, manifest at the level of the wider microbial community. Several studies have reported plasmid carrying bacteria more readily forming biofilms and displaying reduced motility (Billane et al., 2021; Thompson et al., 2023), however, until recently the reasons and causes of this were poorly understood.
Environmental cues or changes in surrounding nutrients are frequently translated into behavioral changes through signaling and regulation pathways within the cell. Cellular regulatory networks are exquisitely tuned microbial systems and are able to rapidly alter transcriptional and translational profiles in response to environmental cues or stressors. However, a recent study has shown that plasmids are able to subvert these regulatory systems for their own gain, in effect completely rewiring the hosts' response to the environment (Billane et al., 2021; Thompson et al., 2023; Vial and Hommais, 2020). These subversions can be either local, controlling just one specific set of genes, or global, changing large parts of the transcriptome or proteome.
Similar to their chromosomal counterparts, plasmid-borne PCC regulators also react to signals from the environment to manipulate host behavior (Chu et al., 2023). Many PCC regulators specifically target the regulation of an individual gene or operon. An example of this is the acquisition of the Klebsiella pneumoniae virulence plasmid (KpVP) by K. pneumoniae strains, leading to hypervirulence (Chu et al., 2023). The acquisition of KpVP leads to the production of the hyper mucoid capsule and the repression of type 3 fimbriae production (Chu et al., 2023).
This lifestyle switch is controlled through the plasmid-encoded iroP gene, which cross regulates the type 3 fimbriae transcription in an iron-dependent manner (Chu et al., 2023). This leads in turn to the production of hypermucoid capsules in low iron conditions (Chu et al., 2023). This switch from a motile to a sessile lifestyle in response to environmental changes, mediated through an acquired regulator, is a common occurrence (Billane et al., 2021).
Highly targeted PCC also occurs in Acinetobacter baumannii, where a plasmid-encoded regulator is able to specifically repress the expression type VI secretion system (Di Venanzio et al., 2019; Weber et al., 2015). This repression allows for higher rates of plasmid conjugation (Di Venanzio et al., 2019) as well as increased survival in the presence of antibiotics (Weber et al., 2015). Specific plasmid-encoded regulators are also able to reprogram their hosts to increase pathogenesis or colonization. Carriage of a virulence plasmid encoding the virR and virS transcriptional regulators is essential for Rhodococcus equi infection and proliferation. These plasmid-borne genes prevent phagosomal maturation, as well as inducing wider changes to the host transcriptome (Coulson et al., 2015).
Rather than simply enabling positive/negative selection, plasmid maintenance in natural environments in fact represents a complex balance between enhancing host fitness and thus increasing vertical transmission, and enabling plasmid spread throughout the wider microbial community, that is, horizontal transmission (Hall et al., 2020). Responding to this, some PCC regulators go beyond targeted signaling interventions, subverting global regulatory systems to induce major adaptive shifts in the host transcriptome or proteome.
A recent example of this is the rsmA/csrA homologue rsmQ, encoded on a number of clinical and environmentally important plasmids (Thompson et al., 2023). RsmQ binds to a large number of intracellular mRNA targets and substantially alters the proteome of its host, Pseudomonas fluorescens. These changes primarily target cellular metabolism, chemotaxis, and motility, suggesting that RsmQ can broadly change the hosts' perception of its environment and its associated ecological responses. Recent study supports the importance of PCC regulators within the wider environment, with PCC-mediated behavioral changes manifesting in the plant rhizosphere (Bird et al., 2023; Thompson et al., 2023).
Bird et al. (2023) showed that the loss of rsmQ leads to significant alterations in the ability of plasmids to persist in the rhizosphere. Far from being confined to soil systems, rsmA/csrA homologues have been identified on a number of clinically relevant plasmids (Thompson et al., 2023) as well as integrative conjugative elements (Abbott et al., 2016), suggesting that the targeting of global regulators is a common strategy employed by plasmids.
The mechanisms by which plasmids manipulate host behavior are highly varied (Fig. 1), ranging from PCC regulators that control individual genes or operons to global regulators that alter their host's environmental perception and subsequently affect its propensity to colonize specific niches. In recent years, an increasing number of diverse PCC regulators have been characterized and in some instances shown to be essential for host survival. This number is likely to represent only a small fraction of the true number of PCC regulators present on large conjugative plasmids and working to circumvent host decision making.
Illustration summarising the ways in which plasmids and plasmid encoded regulators manipulate bacterial behaviour. Plasmid encoded regulators have been shown to 1. Increase conjugation (Di Venanzio et al., 2019) 2. Mucoid capsid formation (Chu et al., 2023) 3. Nutrient sensing and metabolism (Thompson et al., 2023) 4. Rhizosphere persistence (Hall et al., 2020) 5. The motile/sessile lifestyle switch (Billane et al., 2021, Thompson et al., 2023) 6. Root nodulation (Brom et al., 2000, Gamez-Reyes et al., 2017). Made using Biorender.
As plasmids and PCC regulators are widespread, understanding the roles of these global regulators in individual hosts, polymicrobial communities, and different environments is a major undertaking. This study will ultimately lead to a clearer insight into how bacteria function and evolve and is essential to fully understand bacteria–host interactions and the course of clinical infections.