The outer membrane TolC-like channel HgdD is part of tripartite resistance-nodulation-cell division (RND) efflux systems conferring multiple-drug resistance in the Cyanobacterium Anabaena sp. PCC7120

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.

Keywords: Antibiotic Resistance; Cyanobacteria; Membrane; Metabolite Transport; Multidrug Transporters; Nitrosative Stress; RND; TolC.

FIGURE 1.

Identification of RND and HlyD family proteins of Anabaena sp. PCC 7120. A, a general model of the detoxification efflux pathway in Gram-negative bacteria. Small cytotoxic substances can cross the outer membrane (OM), often facilitated by porins, and accumulate in the periplasm (PP). Upon accumulation in the periplasm, cytotoxic substances can cross the plasma membrane (PM) and enter the cytosol (CYT). Single-component inner membrane transporters (MFS- and SMR-type) catalyze the electrochemical gradient-dependent clearing of the cytoplasm by transporting cytotoxins across the inner membrane into the periplasm, from which these can be extruded across the outer membrane in a RND- and TolC-dependent manner. (Rate constants are indicated; for explanation, see Table 5.) A clustering of the RND (B) or the HlyD (C) family proteins was created with CLANS. Each dot represents a protein. The proteins are grouped by their pairwise sequence similarity determined by all-versus-all BLASTs. The filled red circles highlight proteins with experimentally determined functions. The filled green circles highlight seven proteins of the Anabaena sp. proteome that cluster with RND transporters (B) and 22 that cluster with HlyD family proteins (C) (Table 4). The large, empty circles mark those clusters that contain experimentally characterized proteins. The colors of these circles correspond to the font colors of the RND (B) and HlyD (C) family transporter names given below the respective clustering.

FIGURE 2.

Secondary structure prediction of Alr4267. A, the amino acid sequence generated by in silico translation from the upstream ATG (green) and from the start codon according to the annotated reading frame of alr4256 (red). B, the amino acid sequence of Alr4267 as deposited at CyanoBase (78) was used for prediction of TMHs using TMHMM (dashed red line). The amino acid sequence translated from the alternative start codon as shown in A yields the highly conserved transmembrane organization of RND family transporter (green line). The number of the proposed TMH is given. Note that the score for the second predicted TMH at the N terminus is below threshold.

FIGURE 3.

Expression analysis of putative RND family genes. A, growth curve of Anabaena sp. to define early (1 day), late exponential (3 days) and stationary growth phase (9 days). Growth is expressed as a natural logarithm of the ratio of the cell density at the indicated times and at time 0. B, RT-PCR analysis of hgdD and RND gene transcript abundance on RNA isolated from wild-type Anabaena sp. at the indicated growth phase (lanes 1–3). Lane 4, RT-PCR in the absence of the reverse transcriptase; lane 5, PCR on isolated genomic DNA (gDNA). The transcript abundance of the constitutive rnpB was analyzed as an internal standard for normalization of total RNA concentration. C, RT-PCR analysis of hgdD and RND transcript abundance (lanes 1–7) on RNA isolated from wild-type cells grown to late exponential phase (3 days) under either deprivation of iron (−Fe) or the addition of 0.25 μm erythromycin (+Ery) or ethidium bromide (+EB). As a control, PCR on isolated genomic DNA is shown in the bottom lane. D, the transcript of the constitutive rnpB was analyzed as an internal standard for normalization of total RNA concentration. Error bars, S.D.

FIGURE 4.

Generation of RND mutants and growth analysis. A, derivative plasmids of pCSV3, which contain a homologous region of alr1656, all3143, alr4267, or all7631 (left), were used to generate single insertion mutants. Segregation was confirmed by PCR analysis on genomic DNA from wild-type (lane 1) or mutant strains (lanes 2–4) using the indicated primers with depicted binding site and orientation. B, RT-PCR analysis of hgdD (alr2887), rnpB, and RND gene transcript abundance on cDNA isolated from the indicated strains at exponential growth phase (day 3). Lane 7, PCR on genomic DNA isolated from wild-type Anabaena sp. C, antibiotic resistance of Anabaena sp. (strain CSR10) and deletion strains. Growth in the presence of ethidium bromide (1 μg ml⁻¹), erythromycin (10 ng ml⁻¹), roxithromycin (30 ng ml⁻¹), tylosin (100 ng ml⁻¹), neomycin (1 μg ml⁻¹), FeACi (0.1 mm), and CuSO₄ (5 μm) and in the absence of iron and copper (−Fe/−Cu) was analyzed on solid BG11 medium containing streptomycin (Sp) and spectinomycin (Sm) (6 μg ml⁻¹ each) as selective antibiotic. Cells of an early exponential growth phase culture were adjusted to OD₇₅₀ and diluted 1-, 10-, and 100-fold, and 5 μl of the cell suspensions were spotted onto the BG11 agar plates with the indicated additives. Shown is the 10-fold dilution after 7 days of incubation at constant light (30 μmol cm⁻² s⁻¹).

FIGURE 5.

Analysis of RND mutants during nitrogen step down. A, measurement of Chl and Phy content of whole cells after the indicated growth period and condition (top panel). Arrows, those strains and conditions that exhibit substantial differences in chlorophyll a and phycocyanin content with respect to CSR10 (57) used as control strain. B, 1 ml of cells with OD₇₅₀ = 1 were placed in a culture dish for visual inspection of filament color. Arrows indicate those strains and conditions with differences in filament color with respect to CSR10. The indicated differences are representative of three independent biological replicas. Shown are bright field light microscopic (BF) and autofluorescence (AUF) representations of Anabaena filaments after 7 days of nitrogen deprivation (BG11₀) (C) and 3 days of recovery in nitrate-containing (BG11) media under oxic conditions (D). The position of (in the case of I-hgdD partially) differentiated heterocysts can be easily recognized by the gap in the autofluorescence due to disassembly of photosystem II.

FIGURE 6.

Quantitative analysis of double and triple heterocyst formation. CSR10, AFS-I-alr1656, and AFS-I-all3143 were grown in BG11₀, and images were taken as described in the legend to Fig. 4. Heterocysts were counted (100%) and classified as being single standing in the filament (1), forming a cluster of two heterocysts (2), or forming an even larger cluster (3). The average and S.D. values (error bars) of multiple analyzed images and cultures are shown.

FIGURE 7.

Analysis of the lipid content in RND mutants. Thin layer chromatography analysis of the lipid content from whole filaments of the indicated strains is shown. Arrows indicate significant changes in the abundance of individual lipids of mutant strains with respect to the wild type. PG, phosphatidylglycerol; SQDG, sulfoquinovosyl diacylglycerol; DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; HGL1, heterocyst-specific glycolipid 1; HGL2, heterocyst-specific glycolipid 2.

FIGURE 8.

Analysis of ethidium efflux by RND mutants. The transport of ethidium at low (2.5 μm) and high (10 μm) concentrations of ethidium bromide (EB) was analyzed by monitoring the intercalation of ethidium into nucleic acids in whole cells over time. Wild type and I-hgdD are shown as controls. Kinetic parameters (Table 5) were calculated by consecutive reaction kinetics, as established previously (5).

FIGURE 9.

Complementation of E. coli ΔtolC and ΔacrAB by hgdD and all3144-all3143. A, intercalation of ethidium in E. coli wild-type cells (BW25113) and derived deletion mutant strains ΔtolC and ΔacrAB is shown in arbitrary fluorescence units (AFU) under constant settings for direct comparison. CCCP was used as uncoupler of the plasma membrane potential of E. coli wild-type strain. B, intercalation of ethidium in E. coli ΔtolC strain with transiently expressed E. coli tolC or Anabaena sp. tolC-like hgdD under the control of the tetracycline promoter. C, intercalation of ethidium in E. coli ΔacrAB strain with transiently expressed E. coli acrAB_His or Anabaena sp. all3144-all3143 gene cluster under the control of the tetracycline promoter. D, intercalation of ethidium in E. coli wild-type cells (BW25113) in the presence of indicated concentrations of CCCP. The signal given on the y axis in A–D is represented in arbitrary fluorescence units. E, immunoblot of AcrB expression in ΔtolC (top) and ΔarcAB (bottom) strains.