## 4 Regulation of _L. pneumoniae_ Endogenous Factors that May Influence Biofilm Colonization  
For _L. pneumoniae_, as well as for other microorganisms, biofilm formation is an environmental response that can promote survival. _L. pneumoniae_, like many other microorganisms, responds to environmental cues, which can greatly influence biofilm formation and or colonization (Figure 3). One important environmental prompt is iron, which has important roles in the growth of many organisms, and can influence _L. pneumoniae_ replication [66]. The addition of lactoferrin, an iron chelator, can directly kill _L. pneumoniae_ demonstrating the importance of iron in _L. pneumoniae_ viability [67]. Furthermore, bacterial ferrous iron transport promotes the intracellular replication of _L. pneumoniae_ in protozoa, which may influence multispecies biofilm colonization [68]. Iron is also required for the production of melanin and it is believed that deletion of the _IbtA_ and _IbtB_ genes, which encode iron siderophores prevent growth within aquatic biofilms [66]. Interestingly, although iron is essential for biofilm formation, high iron concentrations can inhibit biofilm formation, yet to date the reasons for this are unknown [8]. The ability of bacteria to monitor and respond to cell density is known as quorum sensing and it is a crucial process during biofilm production. Among quorum sensing molecules, \(\alpha\)-hydroxy ketones (AHKs) have been identified in _L. pneumoniae_, and are similar to the AHKs produced by _Vibrio cholera_[69, 70]. Although there is currently no evidence that AHKs regulate _L. pneumoniae_ biofilm production directly, these molecules regulate a wide variety of traits that may influence _L. pneumoniae_ biofilm production/colonization indirectly, including virulence (infection of protozoan hosts in multispecies biofilms), extracellular filament production, and sedimentation through the _lqs_ gene cluster, which encodes for the AHK synthase LqsA, the AHK sensor LqsS and the response regulator LqsR [58, 71, 72]. In addition to the products of this gene cluster, an orphan sensor kinase named LqsT regulates competence, a process that is correlated to biofilm formation in other species [59]. The second-messenger molecule cyclic di-GMP (c-di-GMP) is also an important signaling system that allows bacteria to respond to environmental changes [73]_L. pneumoniae_ has 22 predicted genes related to c-di-GMP production, degradation and/or recognition [74]. One of these genes, _lpg1057_, was found to encode an enzyme responsible for the production of cyclic di-GMP which promotes biofilm formation, and is the only c-di-GMP related gene to date found to directly influence monospecies biofilm production of _L. pneumoniae_[75]. In response to amino acid starvation, the alarmone guanosine tetraphosphate (ppGpp) can also regulate _L. pneumoniae_ gene expression [76]. Although the ppGpp system is mainly linked to the regulation of virulence related traits, this system may indirectly affect environmental biofilm colonization by influencing _L. pneumoniae_amoeba interactions. In addition, sensitivity to ppGpp signaling requires the sigma factor RpoS [77]. RpoS in turn influences LqsR expression, suggesting that virulence related traits regulated by AHKs require multiple environmental signals [78]. In parallel with the ppGpp-RpoS regulation of virulence, downstream is the two-component system LetA/LetS [79]. The LetA/LetS system relieves the repression of virulence related genes by the RNA binding protein CsrA [80]. Despite the initially suspected roles of these transcriptional regulators in surface attachment and biofilm formation, none of the mutants lacking _rpoS_, _letA_ or _csrA_ were affected in biofilm formation in the _L. pneumoniae_ strain JR32 [26]. Of the known _L.