## 3 Physiochemical Determinants in _L. pneumoniae_ Biofilm Formation and Colonization  
Attachment is the process of one object adhering to another, and when producing surface associated biofilms, attachment of _L. pneumoniae_ serves as an initial crucial step, whether it is on biotic or abiotic surfaces. Once bacteria are attached to a substratum and a biofilm is formed, the process of spreading and persisting within a new area is defined as colonization. Although _L. pneumoniae_ can often be found attached to various surfaces in the environment, colonization of existing biofilms in addition to attachment to abiotic substrates is determined by a wide variety of parameters (Figure 3) [35]. One important factor that governs the adherence of _L. pneumoniae_ in anthropogenic water systems is the composition of the surface material to which the bacteria are adhering [36]. _L. pneumoniae_ can adhere well to several different plastics that are commonly used in plumbing, whereas copper inhibits its  
Figure 2: _A. castellani_ infected with _L. pneumoniae_ expressing green fluorescent protein. Scale bar represents 10 μm. attachment [36, 37, 38]. It remains unclear however whether this is due to differences in surface and _L. pneumoniae_ interactions or because different plumbing materials select for different pioneering species, which establishes the initial biofilm and _L. pneumoniae_ colonizes afterwards. **Figure 3.**_L. pneumoniae_ (denoted by Lpn and shown in orange) replicate within environmental protozoa. Uptake within protozoan hosts is promoted by the presence of other amoebae and bacterial species such as _P. aeruginosa_ **(top left)** and by environmental cues such as quorum sensing (green hexagons, **top right**). Environmental cues can also influence changes in _L. pneumoniae_ cell metabolism that favour biofilm production and colonization, which may occur following replication within protozoa or independently of protozoa infection **(middle)**. Other microbial species such as _P. aeruginosa_ (Lpn antagonistic microbial species, brown) can inhibit _L. pneumoniae_ colonization (**bottom left**). The presence of other microorganisms such as _K. pneumoniae_ alleviates the inhibitory effect of _P. aeruginosa_ (Lpn permissive microbial species, blue) and allows _L. pneumoniae_ to be incorporated within biofilms. _L. pneumoniae_ produces a surfactant (brown circles), which is toxic to other _Legionella_ species (red), and may therefore prevent incorporation of these bacteria within biofilms. Physio-chemical parameters such as divalent cations (grey circles) can favour _L. pneumoniae_ biofilm colonization while other factors such as the presence of nanoparticles and copper (blue circles) can hinder _L. pneumoniae_ colonization. Cations are implicated in the attachment of bacteria to different substrata, and can contribute to biofouling [39]. Similarly, both calcium and magnesium were demonstrated to facilitate the attachment of _L. pneumophila_ to abiotic surfaces [40]. Elevated zinc, magnesium, and manganese levels are correlated with increased _L. pneumophila_ contamination and zinc increases the ability of _L. pneumophila_ to bind to host cells such as human lung epithelial cells, suggesting that cations may increase the attachment of _L. pneumophila_ to biotic surfaces in addition to abiotic substrata [41, 42, 43]. Interestingly as it pertains to the cation dependent attachment of _L. pneumophila_, an orthologue of the _Pseudomonas fluorescens_ calcium-dependent cyclic-diGMP regulated protease LapG was identified in _L. pneumophila_. LapG regulates biofilm formation of _Pseudomonas fluorescens_ by cleaving the surface adhesin LapA required for biofilm formation [44, 45]. In addition to the presence of cations, the availability of carbon favours the colonization of biofilms with _L. pneumophila_, presumably because it provides nutrients for the bacteria to replicate [46].