Bacterial genome size as an indicator of bacterial resistance
Bacteroidetes selected in this study - belonging to the Algoriphagus genera - exhibited a smaller genome compared to Firmicutes (Table 2). Similarly, the number of rRNA, tRNA, and proteins was lower in the Bacteroidetes species. Among Firmicutes, H. dabanensis displayed the smaller genome with 1297 and 1203 fewer proteins than B. megaterium and P. glucanolyticus, respectively. Noteworthy, UV filters resistant species for both Firmicutes and Bacteroidetes held the largest genome. A correlation between the appearance of resistance mechanisms and the genome size has already been discussed earlier28. Similarly, a high number of tRNA has been correlated with temperature resistance29.
Table 2
Bacterial genomes structures. Asterisks (*) indicate UV filters resistant bacteria.
Species | chromosome | plasmid | size (Mb) | GC% | Genes | rRNA | tRNA | Proteins |
Algoriphagus mannitolivorans* | 1 | 0 | 4.15 | 42.7 | 3636 | 7 | 39 | 3564 |
Algoriphagus ornithinivorans | 1 | 0 | 4.09 | 39.5 | 3,557 | 5 | 35 | 3,500 |
Paenibacillus glucanolyticus* | 1 | 0 | 5.9 | 49.2 | 5,380 | 24 | 74 | 5,174 |
Halobacillus dabanensis | 1 | 0 | 4.14 | 41.7 | 4,135 | 18 | 67 | 3,971 |
Bacillus megaterium* | 1 | 6 | 5.34 | 38.1 | 5,541 | 41 | 123 | 5,268 |
A. mannitolivorans and A. ornithinivorans shared 2823 proteins and displayed 34 and 28 unique proteins, respectively (Fig. 1A). Among these proteins, A. mannitolivorans displayed 2 protein counts included in the "cellular aromatic compound metabolic process" cluster, identified as the 4,5-DOPA dioxygenase extradiol, an enzyme involved in betalain biosynthesis. P. glucanolyticus and H. dabanensis shared 1719 proteins (Fig. 1B). Interestingly, P. glucanolyticus showed a fewer number of unique proteins with 586 against 797. It is noteworthy to underline that the Orthovenn tool only considers proteins included in clusters, which could explain why P. glucanolyticus had fewer unique proteins even though it had a greater genome. B. megaterium and H. dabanensis shared 2157 proteins and exhibited 455 and 359 unique proteins, respectively (Fig. 1C).
Genomic analysis of H. dabanensis and B. megaterium revealed relevant resistance features
The cell wall constitutes the first barrier against xenobiotics. Therefore, genes classified in this functional category were compared in this study. B. megaterium displayed 14 unique genes belonging to the cell wall category while H. dabanensis had only one. Among them, the subsystems “Rhamnose containing glycans” and “sialic acid metabolism” were the most represented. Rhamnose is a six-carbon deoxy hexose, mostly incorporated in cell wall anchored polysaccharides, glycoproteins, and the capsule of many bacteria30. It is essential for cell viability, environmental adaptation, and biofilm formation31,32. The presence of genes involved in rhamnose metabolism and the gene coding for the capsular polysaccharide biosynthesis protein strongly suggests that B. megaterium holds a capsule on its surface (see Fig. 2 for a schematic representation). While phospholipid bilayers are hypothetically permeable to lipophilic compounds such as homosalate, the capsule, made of a polysaccharides layer could prevent these compounds from entering the cell. Sialic acids are nine carbon sugar acids derivatives mainly incorporated into glycan chains. Common on the surface of pathogenic bacteria, they provide camouflage from the immune system by imitating eukaryotic structures.
Both species harbored genes involved in bacitracin response. B. megaterium possessed multiple stress response genes such as cadmium-transporting ATPase, multicopper oxidase and multidrug resistance transporter (Bcr/CflA family), the cytoplasmic copper homeostasis protein CutC, the mercuric resistance operon regulatory protein, and the membrane fusion protein of RND family multidrug efflux pump, known to confer resistance against multiple hydrophobic substrates33. Several gene copies belonging to the choline and betaine uptake and biosynthesis subsystem were observed in the genome of B. megaterium, including four choline specific ABC transporter. Choline is a precursor of glycine betaine, known as a potent osmoprotectant34. Furthermore, B. megaterium presented genes coding for flavodoxin, a small soluble protein belonging to the non-enzymatic antioxidant molecules, known to confer resistance against herbicides to soil bacteria35 (Fig. 2). Among the genes categorized in the regulation and cell signaling category, the aromatic hydrocarbon utilization transcriptional regulator CatR (LysR family) was the single unique gene belonging to B. megaterium. CatR regulates the expression of catechol and phenol degradation pathway, that has an aromatic structure (benzene) common to with UV filters36. Once the substrate binds to CatR, the promoter regulating the transcription of the catabolic genes is activated and the substrate is degraded into tricarboxylic acid intermediates36 (Fig. 2).
Comparison of H. dabanensis and P. glucanolyticus genomes highlighted cellular components involved in oxidative stress response
Compared to the UV filter sensitive H. dabanensis, P. glucanolyticus harbored more genes coding for proteins involved in pyridoxine (vitamin B6), cobalamin (vitamin B12), and thiamin (vitamin B1) biosynthesis. Vitamins, such as vitamin B12 allows tolerance to oxidative stress in bacteria belonging to the Nitrospira phylum37. Similarly to B. megaterium, P. glucanolyticus coded for Hyaluronan synthase and all genes belonging to the Rhamnose containing glycans subsystem, involved in capsule synthesis - a structure known to mediate antibiotic resistance38. A total of 11 unique genes categorized in subsystem related to antibiotic and metal stress response were identified in the genomes of P. glucanolyticus. Among them, the Multidrug resistance transporter (Bcr/CflA family), reported being involved in bicyclomycin, fosfomycin, kanamycin, and sulfathiazole resistance39. Transcriptomic analyses highlighted that this transporter was overexpressed (4.35 fold change) in the presence of toluene40 in Pseudomonas putida, hence revealing that this gene could play a pivotal role in xenobiotic resistance.
Both bacteria harbored DNA repair systems such as Rec, SOS response, and mutL-mutS system. P. glucanolytics expressed the ADA regulatory protein, involved in 6-O-Methylguanine demethylation, a mechanism known to bypass G:C to A:T due to guanine methylation (Fig. 2). P. glucanolyticus genome contained multiple genes involved in response against oxidative stress such as superoxide dismutase, HtrA protease/chaperone protein, Glutaredoxin, and the outer membrane stress sensor protease DegS. HtrA protease/chaperone protein was shown to be involved in high temperature and oxidative stress tolerance in Campylobacter jejuni2. Mutation induced lack of glutathione-glutaredoxin in a Rhodobacter strain resulted in decreased growth rates and high sensitivity to oxidative stress42. In addition, proteomic analyses revealed that glutaredoxin was up-regulated in Rhodobacter sp. exposed to artificial UVB treatment43. Overall, we can postulate that among Firmicutes, both P. glucanolyticus, and B. megaterium were better fitted to cope with the potentially harmful effect of xenobiotics, including UV filters, than H. dabanensis.
Comparison of A. ornithinivorans and A. mannitolivorans revealed fewer genomic variability but different phenotypes
The UV filter resistant A. mannitolivorans showed a chromosomic Doc/Phd toxin-antitoxin system, known to help cells maintaining genome integrity over generations, control cell growth, and face environmental stresses, by entering into persistent states44. Interestingly, A. mannitolivorans genome harbored the gene coding for the antitoxin ParD without the corresponding DNA gyrase targeting toxin, ParE. Therefore, we could hypothesize that ParD can compete for other DNA gyrase binding molecules. DNA repair systems were similar between the two species. However, A. ornithinivorans showed 5 genes belonging to the CRISPR system, involved in bacterial prophage immunity.
A. ornithinivorans and A. mannitolivorans showed genes involved in the resistance against Beta-lactam, Bacitracin, Fosfomycin, fluoroquinolones, and metal such as cadmium, cobalt, and copper. Genes coding for multiple multidrug resistance efflux pumps were present in both species. Minor differences were observed between genes classified into the cell wall and capsule category. No genes involved in xenobiotics resistance were identified in the two strains. As discussed earlier, these two species displayed only a few unique proteins (Fig. 1A). Overall, these observations suggested that only minor genomic differences could explain different phenotypes.