## 2 Results and Discussion
### Expression Analysis of EPS-Related Genes  
The EPS structure is a key element of a biofilm. After having listed all the components involved in the three main bacterial pathways of EPS assembly and export (Wzy-dependent, ABC transporter-dependent, and synthase-dependent) and those responsible for lipopolysaccharide export [10; 17; 19], their presence in the genome of _Halomicronema_ sp. was verified using NCBI and toid 2767773 ([23], [[https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA658956](https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA658956)]([https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA658956](https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA658956)), accessed on 10 July 2023). We focused on the last stages of EPS biosynthesis as they are the best-known and most conserved among the various bacterial species. Table S3 shows the proteins that were identified in _Halomicronema_ sp. As can be seen, most of the genes involved in these mechanisms encode for proteins characteristic of the Wzy-dependent pathway, suggesting that in _Halomicronema_ sp., the assembly and export of EPS most often proceed through this system, but the complete assembly pathway is not present. The same can be observed for the lipopolysaccharide export pathway.  
Halotolerance in cyanobacteria has been reviewed recently [33; 34], while salt acclimation in _Syntechocystis_ sp. has been reviewed previously [35]. Data from _Synchocystis_ sp showed that many of the factors involved in this response, such as the expression of the _gpp_S gene and the accumulation of glucosylglycerol [36], the sensory histidine kinases that transduce salt stress signals [37], and heat shock proteins [38]. In _Synchocystis_ sp., different stresses trigger similar stress-related genes [38]. In all these studies, information on EPS-related genes is missing.  
Gene expression of the identified EPS genes (Figure 3) was sensitive to the conditions tested. Specifically, _wzx_ genes, involved in transferring the lipid-bound oligosaccharides are repeat units to the periplasmic side of the plasma membrane [19; 39], displayed opposite behaviors: _wzx2_ was overexpressed in the conditions tested, while _wzx1_ was repressed. _wzy_, a gene that polymerases polysaccharides units, is mildly downregulated in all the conditions tested. _wza1_, which exports polymer across the outer membrane [18; 19], was over-expressed in most of the conditions tested except in the presence of EDTA, while _wza2_ was repressed by EDTA and at the lowest NaCl concentration. _wzt_, which transfers the polysaccharide that has been fully polymerized to the inner leaflet of the plasma membrane [19], was slightly induced, except at the highest concentrations of NaCl. _excd1_, whose function is still not clear, was over-expressed in all the conditions except when MgSO\({}_{4}\) was present in the growing medium. _wzy_, which polymerizes polysaccharide units, was repressed when NaCl or EDTA were present. In the previous section, it was reported that MgSO\({}_{4}\) was the only chemical that stimulated biofilm formation. This phenotype is supported also by gene expression. Specifically, Figure 3 shows that _wza2_ and _excd2_ have opposite expression profiles when MgSO\({}_{4}\) is present in the medium or when any of the other chemicals. Moreover, the expression of _wza1_ and _wzt_ is much higher when MgSO\({}_{4}\) is present compared to the other conditions. All the genes studied were modulated except for _excd1_ and _lptc_, which maintained constant expression levels. This genetic distribution in _Halomicronema_ sp. seems similar to what has been described in _Synchocystis_ sp. strain PCC 6803 [40]; however, in _Halomicronema_ sp., the _kpsM_ gene was not identified. This gene was reported as the only EPS-related gene that, when mutated, impairs the secretion of EPS [40].