Characterisation of siderophore producing marine bacteria

Abstract

Marine bacteria are diverse and highly abundant in the oceans. Under iron-depleted conditions, marine bacteria produce an iron chelator called a siderophore to scavenge for iron. Very little is known about siderophore producing bacteria in the oceans. However, as all marine bacteria are competing for the essential iron molecule, most marine bacteria should produce siderophores.

The aims of this thesis were to obtain pure cultures of marine bacteria sampled on a recent international GeoTraces (TAN1109) research cruise and characterise these isolates by phenotypic and molecular methods such as ERIC-PCR, GTG-PCR and restriction enzyme digestion of 16S-23S ISR PCR products. Bacteria were screened for siderophore production by growing pure isolates on CAS agar. Isolates were further dereplicated and identified using 16S rRNA gene sequencing and phylogenetic analysis.

Of 188 isolates, 178 were Gram-negative and 10 were Gram-positive. Of all isolates, 179 showed clearing and/or halo formation when grown on CAS agar. For molecular analysis, 91 isolates were chosen based on their ability to form clearings and halos on CAS agar. These included 89 isolates with medium to strong clearing intensities (early and late) as well as 2 with large halo formations only. Of the chosen representatives, three isolates were Gram-positive.

GTG-PCR typed 68 of the 91 isolates into 22 clusters (similarity of 70%). ERIC-PCR typed 76 of the 91 isolates into 18 clusters (similarity of 70%). TaqI digestion of 16S-23S-PCR products placed 33 of the 91 isolates into 18 clusters (similarity of 70%). The 16S rRNA gene sequence analysis showed three of the isolates clustered within the class of Bacilli, two clustered within the alpha sub-group of the proteobacteria, and 11 within the gamma sub-group of the proteobacteria.

16S rRNA sequencing of 16 chosen representatives identified four siderophore producing marine bacterial genera across all samples. Sampled isolates showed over 90% sequence similarities to Marinobacter algicola, Halomonas meridiana, Ruegeria scottomollicae, and Bacillus subtilis. MMO3035 is potentially a new Gram-positive siderophore producing marine bacteria belonging to the genus Bacillus.

In summary, fingerprint data of ERIC-PCR, GTG-PCR and TaqI digestion of 16S-23S-PCR products suggested that siderophore producing marine bacteria are more phylogenetically diverse and widespread in the environment than current literature indicates. However, 16S rRNA sequencing identified only four siderophore producing marine bacterial genera. This study contributes new research findings to the field of marine microbial ecology and highlights the importance of siderophore producing bacteria in the ocean environment.