Abstract
The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.
Highlights
The resistance-nodulation-cell division (RND) efflux transporters of cyanobacteria are largely unknown
To identify such transporters in cyanobacteria, we performed a global search for RND-like efflux pumps in Gram-negative bacteria, including Anabaena sp
The Complexity of the RND/membrane fusion proteins (MFPs) System in Anabaena—We dissected the network of RND family and MFP proteins in Anabaena sp. likely to be involved in the formation of tripartite efflux pumps with the TolC-like outer membrane channel HgdD
Summary
The RND efflux transporters of cyanobacteria are largely unknown. Results: Six RNDs with different functionality and mutant phenotypes exist in Anabaena sp. TolC (tolerance to colicin E1) is a versatile outer membrane protein involved in the secretion of numerous substances across the cell wall of Gram-negative bacteria [1,2,3] Among these substances are proteins [4], antibiotics [2, 5], siderophores (6 – 8), and lipids, which in the case of some cyanobacteria are required for the formation of the heterocyst-specific cell wall (e.g. see Ref. 9). In the best understood system (iii), TolC is the outer membrane factor for different superfamilies of multidrug efflux pumps, such as the resistance-nodulation-cell division type (RND), ABC transporter superfamily, or the major facilitator superfamily (MFS) (Fig. 1) We propose its annotation as AcrB, especially because All3143 is able to partially complement the E. coli ⌬acrB mutant
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