The S-layer biogenesis system of Synechocystis 6803: Role of Sll1180 and Sll1181 (E. coli HlyB and HlyD analogs) as type-I secretion components for Sll1951 export

Multiple secretion pathways are known for export of protein(s) forming the S-layer in bacteria. The unicellular model cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter S. 6803) also possesses a well-defined S-layer composed of Sll1951 protein. However, the mechanism of its secretion is not completely understood. In the present study, the putative T1SS (Type I secretion system) components, Sll1180 and Sll1181 [inner membrane ABC transporter and membrane fusion protein (MFP), respectively] were characterized for their role in Sll1951 secretion. The corresponding ORFs i.e. sll1180 and sll1181 were inactivated by insertion of a spectinomycin resistance gene. The viability of the homozygous mutants of both the genes indicated dispensability of the corresponding proteins under the experimental conditions. Interestingly, the culture supernatants of the mutants i.e. Δsll1180 and Δsll1181, lacked Sll1951 as observed on SDS-PAGE and confirmed by mass spectrometry. Immunofluorescence delineated a distinct outer ring of Sll1951 in S. 6803 cells only that was further iterated by transmission and scanning electron microscopy. The loss of S-layer imparted an aggregative phenotype to both the mutants. Surprisingly, Δsll1181 cells showed increased sensitivity to different antibiotics indicating a role in multidrug efflux. This is the first report establishing Sl1180 and Sll1181 proteins as partners of the previously characterized Slr1270, for Sll1951 secretion and thus S-layer biogenesis in S. 6803. Sll1181 (in conjunction with Slr1270) also acts as MFP in multidrug efflux along with a yet uncharacterized inner membrane protein.     

Involvement of lipopolysaccharide in the secretion of Escherichia coli α haemolysin and Erwinia chrysanthemi proteases

The presence of the α-haemolysin secretion genes sensitizes Escherichia coli to vancomycin, a glycopeptide antibiotic that is normally excluded from the Gram-negative envelope (owing to its large size) (M_r 1400). The selection of vancomycin mutants in strains carrying such genes was found to be a very powerful method for selecting non-haemolytic mutants. In this way, mutations in the known secretion genes, hlyB, hlyD and tolC, were obtained. However additional mutations mapped in genes rfaH and galU which are required for lipopolysaccharide (LPS) biosynthesis. Mutations in rfaH and galU strongly reduced α-haemolysin secretion as weli as the secretion of Erwinia chrysanthemi proteases in E. coli without affecting their synthesis. These mutations markedly lowered the content of TolC protein, required for haemolysin secretion and also of the PrtF protein necessary for protease secretion. These results raise the possibility that LPS is involved in the correct incorporation of the TolC and PrtF proteins into the cell envelope.     

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Outer membrane vesicles (OMVs) are extracellular vesicles released from the surface of Gram-negative bacteria, including Escherichia coli. Several gene-deficient mutants relating to envelope stress (nlpI and degP) and phospholipid accumulation in the outer leaflet of the outer membrane (mlaA and mlaE) increase OMV production. This study examined the combinatorial deletion of these genes in E. coli and its effect on OMV production. The nlpI and mlaE double-gene-knockout mutant (ΔmlaEΔnlpI) showed the highest OMV production. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based quantitative analysis showed that OMV production by strain ΔmlaEΔnlpI was ~30 times that by the wild-type (WT). In addition, to evaluate the protein secretion capacity of OMVs, a green fluorescent protein (GFP) fused with outer membrane protein W (OmpW) was expressed in OMVs. Western blot analysis showed that GFP secretion through OMVs reached 3.3 mg/L in the culture medium of strain ΔmlaEΔnlpI/gfp, 500 times that for the WT. Our approach using OMVs for extracellular protein secretion in E. coli is an entirely new concept compared with existing secretion systems.     

Enterohaemorrhagic Escherichia coli produces outer membrane vesicles as an active defence system against antimicrobial peptide LL‐37

Antimicrobial peptides (AMPs) are important components of the innate immune system. Enterohaemorrhagic Escherichia coli (EHEC), a food‐borne pathogen causing serious diarrheal diseases, must overcome attack by AMPs. Here, we show that resistance of EHEC against human cathelicidin LL‐37, a primary AMP, was enhanced by butyrate, which has been shown to act as a stimulant for the expression of virulence genes. The increase of resistance depended on the activation of the ompT gene, which encodes the outer membrane protease OmpT for LL‐37. The expression of the ompT gene was enhanced through the activation system for virulence genes. The increase in ompT expression did not result in an increase in OmpT protease in bacteria but in enhancement of the production of OmpT‐loaded outer membrane vesicles (OMVs), which primarily contributed to the increase in LL‐37‐resistance. Furthermore, a sublethal dosage of LL‐37 stimulated the production of OMVs. Finally, we showed that OMVs produced by OmpT‐positive strains protect the OmpT‐negative strain, which is susceptible to LL‐37 by itself more efficiently than OMVs from the ompT mutant. These results indicate that EHEC enhances the secretion of OmpT‐loaded OMVs in coordination with the activation of virulence genes during infection and blocks bacterial cell attack by LL‐37.     

Metabolic shutdown in Escherichia coli cells lacking the outer membrane channel TolC

The outer membrane channel TolC is a key component of multidrug efflux and type I secretion transporters in Escherichia coli. Mutational inactivation of TolC renders cells highly susceptible to antibiotics and leads to defects in secretion of protein toxins. Despite impairment of various transport functions, no growth defects were reported in cells lacking TolC. Unexpectedly, we found that the loss of TolC notably impairs cell division and growth in minimal glucose medium. The TolC‐dependent phenotype was further exacerbated by the loss of ygiB and ygiC genes expressed in the same operon as tolC and their homologues yjfM and yjfC located elsewhere on the chromosome. Our results show that this growth deficiency is caused by depletion of the critical metabolite NAD⁺ and high NADH/NAD⁺ ratios. The increased amounts of PspA and decreased rates of NADH oxidation in ΔtolC membranes indicated stress on the membrane and dissipation of a proton motive force. We conclude that inactivation of TolC triggers metabolic shutdown in E. coli cells grown in minimal glucose medium. The ΔtolC phenotype is partially rescued by YgiBC and YjfMC, which have parallel functions independent from TolC.     

Accumulation of periplasmic enterobactin impairs the growth and morphology of Escherichia coli tolC mutants

TolC is the outer membrane component of tripartite efflux pumps, which expel proteins, toxins and antimicrobial agents from Gram‐negative bacteria. Escherichia coli tolC mutants grow well and are slightly elongated in rich media but grow less well than wild‐type cells in minimal media. These phenotypes have no physiological explanation as yet. Here, we find that tolC mutants have highly aberrant shapes when grown in M9‐glucose medium but that adding iron restores wild‐type morphology. When starved for iron, E. coli tolC mutants synthesize but cannot secrete the siderophore enterobactin, which collects in the periplasm. tolC mutants unable to synthesize enterobactin display no growth or morphological defects, and adding exogenous enterobactin recreates these aberrations, implicating this compound as the causative agent. Cells unable to import enterobactin across the outer membrane grow normally, whereas cells that import enterobactin only to the periplasm become morphologically aberrant. Thus, tolC mutants grown in low iron conditions accumulate periplasmic enterobactin, which impairs bacterial morphology, possibly by sequestering iron and inhibiting an iron‐dependent reaction involved in cell division or peptidoglycan synthesis. The results also highlight the need to supply sufficient iron when studying TolC‐directed export or efflux, to eliminate extraneous physiological effects.     

Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria

For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 ? into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system. Electronic Publication     

Genetic assessment of the role of AcrB β‐hairpins in the assembly of the TolC–AcrAB multidrug efflux pump of Escherichia coli

The tripartite AcrAB–TolC multidrug efflux pump of Escherichia coli is the central conduit for cell‐toxic compounds and contributes to antibiotic resistance. While high‐resolution structures of all three proteins have been solved, much remains to be learned as to how the individual components come together to form a functional complex. In this study, we investigated the importance of the AcrB β‐hairpins belonging to the DN and DC subdomains, which are presumed to dock with TolC, in complex stability and activity of the complete pump. Our data show that the DN subdomain β‐hairpin residues play a more critical role in complex stability and activity than the DC subdomain hairpin residues. The failure of the AcrB DN β‐hairpin deletion mutant to engage with TolC leads to the drug hypersensitivity phenotype, which is reversed by compensatory alterations in the lipoyl and β‐barrel domains of AcrA. Moreover, AcrA and TolC mutants that induce TolC opening also reverse the drug hypersensitivity phenotype of the AcrB β‐hairpin mutants, indicating a failure by the AcrB mutant to interact and thus induce TolC opening on its own. Together, these data suggest that both AcrB β‐hairpins and AcrA act to stabilize the tripartite complex and induce TolC opening for drug expulsion.     

Campylobacter jejuni outer membrane vesicle‐associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E‐cadherin and occludin

Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram‐negative bacteria. Campylobacter jejuni produces OMVs that trigger IL‐8, IL‐6, hBD‐3 and TNF‐α responses from T84 intestinal epithelial cells and are cytotoxic to Caco‐2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E‐cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co‐incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E‐cadherin and occludin. The addition of 11168H OMVs to the co‐culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time‐dependent and dose‐dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells.     

Dissecting the Photoprotective Mechanism Encoded by the flv4‐2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization

In Synechocystis sp. PCC 6803, the flv4‐2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4‐2 operon mutants. In the ?sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ?sll0218‐flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ?sll0218 and ?sll0218‐flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218‐lacking mutants. The YFP‐tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA‐defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ?flv4‐2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting.     

On the role of TolC in multidrug efflux: the function and assembly of AcrAB–TolC tolerate significant depletion of intracellular TolC protein

TolC channel provides a route for the expelled drugs and toxins to cross the outer membrane of Escherichia coli. The puzzling feature of TolC structure is that the periplasmic entrance of the channel is closed by dense packing of 12 α‐helices. Efflux pumps exemplified by AcrAB are proposed to drive the opening of TolC channel. How interactions with AcrAB promote the close‐to‐open transition in TolC remains unclear. In this study, we investigated in vivo the functional and physical interactions of AcrAB with the closed TolC and its conformer opened by mutations in the periplasmic entrance. We found that the two conformers of TolC are readily distinguishable in vivo by characteristic drug susceptibility, thiol modification and proteolytic profiles. However, these profiles of TolC variants respond neither to the in vivo stoichiometry of AcrAB:TolC nor to the presence of vancomycin, which is used often to assess the permeability of TolC channel. We further found that the activity and assembly of AcrAB–TolC tolerates significant changes in amounts of TolC and that only a small fraction of intracellular TolC is likely used to support efflux needs of E. coli. Our findings explain why TolC is not a good target for inhibition of multidrug efflux.     

Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells

Cytolethal‐distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram‐negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella‐containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71‐CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71‐CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71‐CDT also secreted OMVs‐like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin‐loaded OMVs by bystander cells was dependent on dynamin‐1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.     

The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.     

Antibody analysis of the localisation, expression and stability of HlyD, the MFP component of the E. coli haemolysin translocator

HlyD has a single transmembrane domain (residues 59-80) and a large periplasmic domain, and is essential for the secretion of haemolysin from Escherichia coli. Using an antibody raised against HlyD, the protein was localised to the cell envelope by immunofluorescence and to the cytoplasmic membrane by sucrose gradient analysis. We have examined the stability of this protein in the presence and absence of other putative components of the translocator, HlyB and TolC. HlyD is normally highly stable but in the absence of TolC, the steady-state level of HlyD is greatly reduced and the protein has a half-life at 37° C of 36 min. In the absence of HlyB, HlyD is also unstable and specific degradation products are detected, which co-fractionate with the inner membrane, indicating in this case limited cleavage at specific sites. However, the effect of removing both HlyB and TolC is not additive. On the contrary, in the absence of both HlyB and TolC the half-life of HlyD is approximately 110 min. This result shows that in the presence of HlyB removal of TolC renders HlyD more unstable than it is in the absence of both HlyB and TolC. This suggests that the presence of HlyB induces a structural change in HlyD. In addition, HlyB itself appears to be less stable in the absence of HlyD. These results are consistent with an interaction between HlyD/TolC and HlyB/HlyD. A derivative of HlyD, HlyD22, lacking the 40 N-terminal residues of HlyD assembles into the inner membrane displaying the same stability with and without HlyB as wild type HlyD does. This N-terminal region therefore appears to play no role in stable localisation but is involved in secretion, since HlyD22 is completely secretion defective. Modification of the C-terminus on the other hand completely destabilised the molecule and HlyD was not detectable in the envelope. Secretion of active haemolysin is limited to a brief period during mid to late exponential phase. In contrast, HlyD is apparently synthesised constitutively throughout the growth phase, demonstrating that the production of this component of the translocator is not the limiting factor for growth phase-dependent secretion. Received: 10 July 1998 / Accepted: 19 October 1998     

Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes

The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane‐bound compartment termed Legionella‐containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small‐angle X‐ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co‐incubation experiments showed a dose‐ and time‐dependent binding of fluorophore‐labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4°C. Purified OMVs induced tumour necrosis factor‐α production in human macrophages at concentrations starting at 300 ng ml^?1. Experiments on HEK293‐TLR2 and TLR4/MD‐2 cell lines demonstrated a dominance of TLR2‐dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells.     

Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells

Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N‐terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW‐GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW‐GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW‐GFP into OMVs through the increased OmpW‐GFP expression on the ΔnlpI cells. Finally, the amount of OmpW‐GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW‐GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:51–57, 2018     

An iTRAQ characterisation of the role of TolC during electron transfer from Shewanella oneidensis MR‐1

Anodophilic bacteria have the ability to generate electricity in microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigated the anode‐specific responses of Shewanella oneidensis MR‐1, an exoelectroactive Gammaproteobacterium, using for the first time iTRAQ and 2D‐LC MS/MS driven membrane proteomics to compare protein abundances in S. oneidensis when generating power in MFCs, and growing in a continuous culture. The regulated dataset produced was enriched in membrane proteins. Proteins shown to be more abundant in anaerobic electroactive anodic cells included efflux pump TolC and an uncharacterised tetratricopeptide repeat (TPR) protein, whilst the TonB2 system and associated uncharacterised proteins such as TtpC2 and DUF3450 were more abundant in microaerobic planktonic cells. In order to validate the iTRAQ data, the functional role for TolC was examined using a δTolC knockout mutant of S. oneidensis. Possible roles for the uncharacterised proteins were identified using comparative bioinformatics. We demonstrate that employing an insoluble extracellular electron acceptor requires multiple proteins involved in cell surface properties. All MS and processed data are available via ProteomeXchange with identifier PXD004090.     

Multidisciplinary Evidences that Synechocystis PCC6803 Exopolysaccharides Operate in Cell Sedimentation and Protection against Salt and Metal Stresses

Little is known about the production of exopolysaccharides (EPS) in cyanobacteria, and there are no genetic and physiological evidences that EPS are involved in cell protection against the frequently encountered environmental stresses caused by salt and metals. We studied four presumptive EPS production genes, sll0923, sll1581, slr1875 and sll5052, in the model cyanobacterium Synechocystis PCC6803, which produces copious amounts of EPS attached to cells (CPS) and released in the culture medium (RPS) as shown here. We show that sll0923, sll1581, slr1875 and sll5052 are all dispensable to the growth of all corresponding single and double deletion mutants in absence of stress. Furthermore, we report that sll0923, sll1581 and slr1875 unambiguously operate in the production of both CPS and RPS. Both sll1581 and slr1875 are more important than sll0923 for CPS production, whereas the contrary is true for RPS production. We show that the most EPS-depleted mutant, doubly deleted for sll1581 and slr1875, lacks the EPS mantle that surrounds WT cells and sorbs iron in their vicinity. Using this mutant, we demonstrate for the first time that cyanobacterial EPS directly operate in cell protection against NaCl, CoCl₂, CdSO₄ and Fe-starvation. We believe that our EPS-depleted mutants will be useful tools to investigate the role of EPS in cell-to-cell aggregation, biofilm formation, biomineralization and tolerance to environmental stresses. We also suggest using the fast sedimenting mutants as biotechnological cell factories to facilitate the otherwise expensive harvest of the producer cell biomass and/or its separation from products excreted in the growth media.     

Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp. PCC 6803

The gene products of sll0337 and slr0081 in Synechocystis sp. PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively. Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions. A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin. In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting. Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon. Although there are three assignments for putative alkaline phosphatase genes in the Synechocystis sp. PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation. This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcus sp. PCC 7942 [Ray, J.M., Bhaya, D., Block, M.A. and Grossman, A.R. (1991) J. Bact. 173: 4297–4309]. An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcus sp. PCC 7942.