Figure 1.
Predicted biosynthetic units of apratoxin A.
Carbon atoms are numerically labeled.
Figure 2.
Microscopy and mass spectrometry of Lyngbya bouillonii filaments.
(a) Light and fluorescent microscopy of a laboratory grown strain of L. bouillonii collected from Papua New Guinea. Brightfield (top) and 4′,6-diamidino-2-phenylindole (DAPI) stained and epi-fluorescent imaging (bottom) at 1000×. Cyanobacterial chlorophyll appears orange while filament sheath-associated bacterial DNA appears blue. (b) Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) of an intact L. bouillonii filament demonstrates biosynthetic production of apratoxin A (obs. [M+H]+ m/z 840).
Figure 3.
Scanning electron microscope image of a single L. bouillonii filament at 10,000× and 30,000× (inset) revealing heterotrophic bacterial growth on the sheath material of the cyanobacteria.
Figure 4.
Consecutive strategies used to isolate the putative apratoxin biosynthetic gene cluster from a complex microbial assemblage.
(a) Micromanipulation to isolate single cells used as template in multiple displacement amplification (MDA) and partial genome sequencing, and (b) metagenomic DNA from cultured non-axenic L. bouillonii used to create a fosmid library for subsequent PCR screening.
Figure 5.
In silico determined contig, 04978, containing the distinctive HCS- ECH1-ECH2 catalytic motif [2].
Specific primers were designed (arrows) and used to PCR screen the metagenomic fosmid library. Domain nomenclature is the same as in Fig. 6.
Figure 6.
Proposed functions of the biosynthetic proteins produced by the apr pathway.
Domain nomenclature is as follows: adapter region (AR), GCN5 acyltransferase (GNAT), glycine-N methyltransferase (GNMT), methyl transferase (MT), ketosynthase (KS), acyltransferase (AT), ketoreductase (KR), enoylreductase (ER), acyl carrier protein (ACP), HMG-CoA synthase (HCS), enoyl-CoA hydratase (ECH), condensation (C), adenylation (A), peptidyl carrier protein (PCP), cyclase (CY). The cluster has type I modular mixed polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) organization containing 12 open reading frames, including a PKS-type loading module and nine extension modules.
Table 1.
Deduced Functions of the Proteins in the apr Biosynthetic Gene Cluster.