Glucolipids and lipoteichoic acids affect the activity of SigI, an alternative sigma factor, and WalKR, an essential two-component system, in Bacillus subtilis.

In a Bacillus subtilis ugtP mutant lacking glucolipids, SigI was activated in the log phase, and the activation of SigI in the mutant was suppressed by the expression of native ugtP. By contrast, SigI was inhibited in a yfnI mutant lacking one of the lipoteichoic acid (LTA) synthase genes, and the inhibition was suppressed by the expression of yfnI. A series of mutation analyses of the sigI promoter revealed that the two WalR binding sites were involved in the increase of PsigI -lacZ activity in the ugtP mutant and decrease of the lacZ activity in the yfnI mutant. Transcription from the SigI recognition sequence was enhanced in the ugtP mutant, whereas yfnI disruption inhibited the transcription from the SigA recognition sequence in the sigI promoter. We found that not only SigI but also WalKR, the essential two-component system, was activated in the ugtP mutant and inhibited in the yfnI mutant. The walK mutants with activated WalR exhibited abnormal morphology, but this phenotype was suppressed by the addition of MgSO4 . We conclude that glucolipids and LTA are key compounds in the maintenance of normal cell surface structure in B. subtilis.

Role of the inner-membrane histidine kinase RcsC and outer-membrane lipoprotein RcsF in the activation of the Rcs phosphorelay signal transduction system in Escherichia coli.

The Rcs phosphorelay signal transduction system of Escherichia coli controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. The outer-membrane lipoprotein RcsF is an essential component of the Rcs system. Mislocalization of RcsF to the periplasm or the cytoplasmic membrane leads to high activation of the Rcs system, suggesting that RcsF functions by interacting with the cytoplasmic membrane component(s) of the system in activating the system. This is consistent with the result reported by Cho et al. (Cell159, 1652-1664, 2014) showing that RcsF interacts with the periplasmic domain (YrfFperi) of the inner-membrane protein YrfF (IgaA in Salmonella enterica serovar Typhimurium), which is a negative regulator of the Rcs system. In this study we show that RcsF also interacts with the periplasmic domain of the innermembrane-localized histidine kinase RcsC (RcsCperi). RcsCperi, which was secreted to the periplasm by fusion to maltose-binding protein, titrated RcsF's activating effect. A bimolecular fluorescence complementation experiment showed interaction of RcsF with RcsCperi, as well as with YrfFperi. We conclude that RcsF interacts with the periplasmically exposed region of RcsC, as well as with that of YrfF.

Septal membrane localization by C-terminal amphipathic α-helices of MinD in Bacillus subtilis mutant cells lacking MinJ or DivIVA.

The Min system, which inhibits assembly of the cytokinetic protein FtsZ, is largely responsible for positioning the division site in rod-shaped bacteria. It has been reported that MinJ, which bridges DivIVA and MinD, is targeted to the cell poles by an interaction with DivIVA, and that MinJ in turn recruits MinCD to the cell poles. MinC, however, is located primarily at active division sites at mid-cell when expressed from its native promoter. Surprisingly, we found that Bacillus subtilis MinD is located at nascent septal membranes and at an asymmetric site on lateral membranes between nascent septal membranes in filamentous cells lacking MinJ or DivIVA. Bacillus subtilis MinD has two amphipathic α-helices rich in basic amino acid residues at its C-terminus; one of these, named MTS1 here, is the counterpart of the membrane targeting sequence (MTS) in Escherichia coli MinD while the other, named MTS-like sequence (MTSL), is the nearest helix to MTS1. These amphipathic helices were located independently at nascent septal membranes in cells lacking MinJ or DivIVA, whereas elimination of the helices from the wild type protein reduced its localization considerably. MinD variants with altered MTS1 and MTSL, in which basic amino acid residues were replaced with proline or acidic residues, were not located at nascent septal membranes, indicating that the binding to the nascent septal membranes requires basic residues and a helical structure. The septal localization of MTSL, but not of MTS1, was dependent on host cell MinD. These results suggest that MinD is targeted to nascent septal membranes via its C-terminal amphipathic α-helices in B. subtilis cells lacking MinJ or DivIVA. Moreover, the diffuse distribution of MinD lacking both MTSs suggests that only a small fraction of MinD depends on MinJ for its localization to nascent septal membranes.

Suppression of abnormal morphology and extracytoplasmic function sigma activity in Bacillus subtilis ugtP mutant cells by expression of heterologous glucolipid synthases from Acholeplasma laidlawii.

Glucolipids in Bacillus subtilis are synthesized by UgtP processively transferring glucose from UDP-glucose to diacylglycerol. Here we conclude that the abnormal morphology of a ugtP mutant is caused by lack of glucolipids, since the same morphology arises after abolition of glucolipid production by disruption of pgcA and gtaB, which are involved in UDP-glucose synthesis. Conversely, expression of a monoglucosyldiacylglycerol (MGlcDG) produced by 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii (alMGS) almost completely suppressed the ugtP disruptant phenotype. Activation of extracytoplasmic function (ECF) sigmas (SigM, SigV, and SigX) in the ugtP mutant was decreased by alMGS expression, and was suppressed to low levels by MgSO4 addition. When alMGS and alDGS (A. laidlawii 1,2-diacylglycerol-3-glucose (1-2)-glucosyltransferase producing diglucosyldiacylglycerol (DGlcDG)) were simultaneously expressed, SigX activation was repressed to wild type level. These observations suggest that MGlcDG molecules are required for maintenance of B. subtilis cell shape and regulation of ECF sigmas, and DGlcDG regulates SigX activity.

Septal localization by membrane targeting sequences and a conserved sequence essential for activity at the COOH-terminus of Bacillus subtilis cardiolipin synthase.

The acidic phospholipid cardiolipin (CL) is localized on polar and septal membranes and plays an important physiological role in Bacillus subtilis cells. ClsA, the enzyme responsible for CL synthesis, is also localized on septal membranes. We found that GFP fusion proteins of the enzyme with NH2-terminal and internal deletions retained septal localization. However, derivatives with deletions starting from the COOH-terminus (Leu482) ceased to localize to the septum once the deletion passed the Ile residue at 448, indicating that the sequence responsible for septal localization is confined within a short distance from the COOH-terminus. Two sequences, Ile436-Leu450 and Leu466-Leu478, are predicted to individually form an amphipathic α-helix. This configuration is known as a membrane targeting sequence (MTS) and we therefore refer to them as MTS2 and MTS1, respectively. Either one has the ability to affect septal localization, and each of these sequences by itself localizes to the septum. Membrane association of the constructs of this enzyme containing the MTSs was verified by subcellular fractionation of the cells. CL synthesis, in contrast, was abolished after deleting just the last residue, Leu482, in the COOH-terminal four amino acid residue sequence, Ser-Pro-Ile-Leu, which is highly conserved among bacterial CL synthases.

Repression of the activities of two extracytoplasmic function σ factors, σM and σV, of Bacillus subtilis by glucolipids in Escherichia coli cells.

Extracytoplasmic function (ECF) σ factors respond to environmental stresses and regulate numerous genes required for adaptation. Under normal growth conditions, the ECF σ factors are sequestered by transmembrane anti-σ factor proteins, from which they are released under stress conditions. In Bacillus subtilis ugtP null mutant cells, which lack glucolipids, three of the seven ECF σ factors, σM, σV and σX, are activated. The Escherichia coli cell membrane does not contain glucolipids. When the genes for these three ECF σ and anti-σ factors were introduced into E. coli cells, expression of lacZ fused to the ECF σ factor-regulated promoters indicated ECF σ factor activity. Additional expression of the ugtP gene in these E. coli cells led to the synthesis of small amounts of glucolipids, and the activities of σM and σV were repressed, but the activity of σX was unaffected. It is likely that glucolipids directly influence anti-σM and anti-σV factors by stabilizing conformations that sequester the respective ECF σ factors.

The membrane: transertion as an organizing principle in membrane heterogeneity.

The bacterial membrane exhibits a significantly heterogeneous distribution of lipids and proteins. This heterogeneity results mainly from lipid-lipid, protein-protein, and lipid-protein associations which are orchestrated by the coupled transcription, translation and insertion of nascent proteins into and through membrane (transertion). Transertion is central not only to the individual assembly and disassembly of large physically linked groups of macromolecules (alias hyperstructures) but also to the interactions between these hyperstructures. We review here these interactions in the context of the processes in Bacillus subtilis and Escherichia coli of nutrient sensing, membrane synthesis, cytoskeletal dynamics, DNA replication, chromosome segregation, and cell division.

An essential enzyme for phospholipid synthesis associates with the Bacillus subtilis divisome.

The mechanism by which the membrane synthetic machinery might be co-organized with the cell-division architecture during the bacterial cell cycle remains to be investigated. We characterized a key enzyme of phospholipid and fatty acid synthesis in Bacillus subtilis, the acyl-acyl carrier protein phosphate acyltransferase (PlsX), and identified it as a component of the cell-division machinery. Comprehensive interaction analysis revealed that PlsX interacts with FtsA, the FtsZ-anchoring protein. PlsX mainly localized at the potential division site independent of FtsA and FtsZ and then colocalized with FtsA. By multidirectional approaches, we revealed that the Z-ring stabilizes the association of PlsX at the septum and pole. The localization of PlsX is also affected by the progression of DNA replication. PlsX is needed for cell division and its inactivation leads to aberrant Z-ring formation. We propose that PlsX localization is prior to Z-ring formation in the hierarchy of septum formation events and that PlsX is important for co-ordinating membrane synthesis with cell division in order to properly complete septum formation.

Importance of the proline-rich region for the regulatory function of RcsF, an outer membrane lipoprotein component of the Escherichia coli Rcs signal transduction system.

The outer membrane lipoprotein RcsF is an essential component of the Rcs phosphorelay signal transduction system in Escherichia coli. It senses stresses imposed on the cell envelope and conveys the information to histidine kinase RcsC in the cytoplasmic membrane. Mislocalization of RcsF to the periplasm, effected by fusing it to the periplasmic maltose-binding protein, or to the cytoplasmic membrane, brought about by changing the lipoprotein sorting signal, leads to high activation of the Rcs system, suggesting that RcsF functions as a ligand for RcsC in activating the system. Here, we focus on the proline-rich region (PRR) in the N-terminal half of RcsF, a region which also contains many basic amino acid residues. Deletion of the PRR in the mislocalized RcsF resulted in even higher activation of the Rcs system. The same deletion in wild-type RcsF lipoprotein that is correctly localized to the outer membrane, however, blocked activation of the system under stresses that normally should activate it. It is highly likely that the PRR plays an important role in the regulation of the function of RcsF in activating the Rcs system.

Induction of extracytoplasmic function sigma factors in Bacillus subtilis cells with defects in lipoteichoic acid synthesis.

Lipoteichoic acid (LTA) is an important cell envelope component of Gram-positive bacteria. Bacillus subtilis has four homologous genes for LTA synthesis: ltaS (yflE), yfnI, yqgS and yvgJ. The products LtaS (YflE), YfnI and YqgS are bona fide LTA synthetases, whereas YvgJ functions only as an LTA primase. To clarify whether defects in LTA on the cell envelope trigger extracytoplasmic function (ECF) sigma factors, mRNA levels of the autoregulated ECF sigma factors in cells with singly and multiply deleted alleles of the ltaS homologues were examined by real-time RT-PCR. This revealed that sigM and sigX were induced in cells with a null allele of ΔltaS and ΔyfnI, respectively, and that no ECF sigma factor was induced in cells with a single null allele of ΔyqgS or ΔyvgJ. In cells with double null alleles (ΔltaS and ΔyfnI), sigW and ylaC were induced in addition to sigM and sigX. Cells with triple null alleles (ΔltaS ΔyfnI and ΔyqgS) showed a pattern of induction similar to that of the double null. In cells with quadruple null alleles, sigV and sigY were newly induced. Cells with ΔltaS had approximately 1/4 the diglucosyldiacylglycerol and over 10 times the CDP-diacylglycerol of wild-type cells. Compensatory elevation of the mRNA level of other homologues was observed (in ΔltaS cells the level of yfnI was elevated; in ΔyfnI cells that of yqgS and yvgJ was elevated; both were even higher in ΔltaS ΔyfnI cells). In ΔltaS cells, the mRNA level of yfnI was corroborated to be regulated by σ(M), which is activated in the null mutant cells. In ΔyfnI cells, the mRNA levels of yqgS and yvgJ reverted to less than those of wild-type when a defective sigX allele was introduced. Since sigX was activated in cells with ΔyfnI, this suggests that the induction of yqgS and yvgJ is dependent on σ(X). The LTAs produced by the four ltaS homologues seem to play distinct physiological roles to maintain the full function of LTA on the B. subtilis cell envelope.

Activation of the Cpx phosphorelay signal transduction system in acidic phospholipid-deficient pgsA mutant cells of Escherichia coli.

The pgsA gene encodes the enzyme for the committed step in the synthesis of acidic phospholipids in Escherichia coli, and the pssA gene does the same for zwitterionic phospholipid. It has been reported that the Rcs and Cpx phosphorelay signal transduction systems are activated in pgsA- and pssA-defective mutants, respectively. In this study, we show that the Cpx system is activated also in a pgsA mutant, whereas the Rcs system was not activated in a pssA mutant. Lack of phosphatidylglycerol in pgsA mutants causes inadequate modification of lipoproteins, resulting in poor localization to the outer membrane. The outer membrane lipoprotein RcsF is necessary for the response of the Rcs system to various stimuli, and Rcs activation in pgsA mutants involves inner membrane mislocalization of this lipoprotein. The outer membrane lipoprotein NlpE, however, while necessary for the surface adhesion-induced Cpx response, was not involved in Cpx activation in the pgsA mutant.

Role of Slr1045 in environmental stress tolerance and lipid transport in the cyanobacterium Synechocystis sp. PCC6803.

ATP-binding cassette (ABC) transporter proteins mediate energy-dependent transport of substrates across cell membranes. Numerous ABC transporter-related genes have been found in the Synechocystis sp. PCC6803 genome by genome sequence analysis including H(+), iron, phosphate, polysaccharide, and CO(2) transport-related genes. The substrates of many other ABC transporters are still unknown. To identify ABC transporters involved in acid tolerance, deletion mutants of ABC transporter genes with unknown substrates were screened for acid stress sensitivities in low pH medium. It was found that cells expressing the deletion mutant of slr1045 were more sensitive to acid stress than the wild-type cells. Moreover, slr1045 expression in the wild-type cells was increased under acid stress. These results indicate that slr1045 is an essential gene for survival under acid stress. The mutant displayed high osmotic stress resistance and high/low temperature stress sensitivity. Considering the temperature-sensitive phenotype and homology to the organic solvent-resistant ABC system, we subsequently compared the lipid profiles of slr1045 mutant and wild-type cells by thin-layer chromatography. In acid stress conditions, the phosphatidylglycerol (PG) content in the slr1045 mutant cells was approximately 40% of that in the wild-type cells. Moreover, the addition of PG to the medium compensated for the growth deficiency of the slr1045 mutant cells under acid stress conditions. These data suggest that slr1045 plays a role in the stabilization of cell membranes in challenging environmental conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Expression and localization of two SecA homologs in the unicellular red alga Cyanidioschyzon merolae.

SecA is an ATP-driven motor for Sec translocase that participates in bacterial protein export and thylakoidal import in plants. We have reported that Cyanidioschyzon merolae, a unicellular red alga, possesses a nuclear-encoded secA(nuc) and a plastid-encoded secA(pt) gene. In this study we found that the amount of SecA(nuc) protein almost quadrupled at high temperature, whereas that of the SecA(pt) protein increased far less. We were also able to determine the localization of both SecAs to the chloroplast by immunofluorescence and immunoelectron microscopy. We suggest that SecA(nuc) has an important role in the chloroplast at high temperatures.

Exploring the relationship between lipoprotein mislocalization and activation of the Rcs signal transduction system in Escherichia coli.

The Rcs phosphorelay signal transduction system controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. We investigated the activation of the Rcs system in a pgsA null mutant of Escherichia coli, which completely lacks the major acidic phospholipids phosphatidylglycerol and cardiolipin. We found that the Rcs activation, and consequent thermosensitivity, were suppressed by overexpression of the lgt gene, encoding diacylglyceryltransferase, which catalyses the modification of prolipoproteins that is the first step in the maturation and localization process of lipoproteins, and is a prerequisite for the later steps. The outer-membrane lipoprotein RcsF is an essential component of Rcs signalling. This lipoprotein was poorly localized to the outer membrane in the pgsA null mutant, probably because of the absence of phosphatidylglycerol, the major donor of diacylglycerol in the Lgt reaction. Even in a pgsA(+) background, the Rcs system was activated when RcsF was mislocalized to the inner membrane by alteration of the residues at positions 2 and 3 of its mature form to inner-membrane retention signals, or when it was mislocalized to the periplasm by fusing the mature form to maltose-binding protein. These results suggest that RcsF functions as a ligand for RcsC in activating Rcs signalling. Mislocalized versions of RcsF still responded to mutations pgsA, mdoH and tolB, further activating the Rcs system, although the rfaP mutation barely caused activation. It seems that RcsF must be localized in the outer membrane to respond effectively to stimuli from outside the cell.

Characterization of the nuclear- and plastid-encoded secA-homologous genes in the unicellular red alga Cyanidioschyzon merolae.

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.

Fission yeast ATF/CREB family protein Atf21 plays important roles in production of normal spores.

Activating transcription factor/cAMP response element binding protein (ATF/CREB) family transcription factors play central roles in maintaining cellular homeostasis. They are activated in response to environmental stimuli, bind to CRE sequences in the promoters of stress-response genes and regulate transcription. Although ATF/CREB proteins are widely conserved among most eukaryotes, their characteristics are highly diverse. Here, we investigated the functions of a fission yeast ATF/CREB protein Atf21 to find out its unique properties. We show that Atf21 is dispensable for the adaptive response to several stresses such as nitrogen starvation and for meiotic events including nuclear divisions. However, spores derived from atf21Δ mutants are not as mature as wild-type ones and are unable to form colonies under nutrition-rich conditions. Furthermore, we demonstrate that the Atf21 protein, which is scarce in early meiosis, gradually accumulates as meiosis proceeds; it reaches maximum levels approximately 8 h after nitrogen starvation and is present during germination. These results suggest that Atf21 is expressed and functions long after nitrogen starvation. Given that other well-characterized fission yeast ATF/CREB proteins Atf1 and Pcr1 accumulate and function promptly upon exposure to environmental stresses, we propose that Atf21 is a distinct member of the ATF/CREB family in fission yeast.

Abnormal morphology of Bacillus subtilis ugtP mutant cells lacking glucolipids.

Bacillus subtilis Marburg 168 cells with disrupted ugtP, which encodes UDP-glucosyltransferase involved in glucolipid synthesis, were bent and distended. In the ugtP mutant cells, the extracytoplasmic function sigmas SigM, SigV and SigX, were found to be activated. Introduction of a disrupted allele of sigM into the ugtP strain caused even more abnormal morphology, with cells taking on a balloon-like shape; growth of these cells in LB medium was hampered by addition of 1.5% NaCl. Addition of MgSO4 or MnCl2 suppressed the abnormal morphology. In ugtP mutant cells the transcription of the mreB operon from an upstream promoter in maf (designated Pupstream mreB) and PmreBH was 4.3- and 2.3-fold higher, respectively, and localization of GFP-MreB was not in discrete dots (in an apparently helical pattern), but faint and in irregular clusters. GFP-MreB protein was reduced in the ugtP mutant cells. We suggest that glucolipids are important for MreB isoforms to take on the configuration that appears as discrete dots and plays a role in shaping cells into straight rods.

The Bacillus subtilis essential gene dgkB is dispensable in mutants with defective lipoteichoic acid synthesis.

The dgkB gene is essential for the growth of Bacillus subtilis. It encodes a diacylglycerol (DG) kinase that converts DG to phosphatidic acid to reintroduce it into the phospholipid synthesis pathway. Repression of the dgkB gene placed under a regulatable promoter causes accumulation of DG and leads to lethality. DG is formed as a byproduct of the synthesis of lipoteichoic acid (LTA), a polyanionic component of the cell envelope. B. subtilis synthesizes LTA by polymerizing the glycerophosphate moiety of phosphatidylglycerol (PG) onto a glucolipid membrane anchor, and releasing the DG moiety of PG. B. subtilis has four genes homologous to Staphylococcus aureus ltaS, which encodes LTA synthase. Disruption of either or both of two genes, yflE and yfnI, whose products show higher homology with S. aureus LtaS among the four homologues, suppressed the lethality caused by dgkB repression. In cells with dgkB repression, DG was accumulated to 43 ± 3% of total lipids, about three times the content of wild type cells (13 ± 1%). Disruption of yfnI in the dgkB-repressed cells reduced the DG content to 15 ± 2%, but yflE-disruption did not (42 ± 1%); this was probably due to efficient LTA synthesis by YfnI in the yflE-disrupted cells. Further introduction of a disrupted allele of ugtP, encoding glucolipid synthase that consumes DG as a substrate, partially lowered the colony forming capacity in strains with yflE-disruption. A disrupted dgkB allele was successfully introduced into strains disrupted for either or both of yflE and yfnI, indicating that the essential gene dgkB is dispensable in mutants defective in LTA synthesis.

Accumulation of sigmaS due to enhanced synthesis and decreased degradation in acidic phospholipid-deficient Escherichia coli cells.

The Escherichia coli pgsA3 mutation, which causes deficiency in acidic phospholipids, leads to a significant accumulation of sigma(S). This accumulation is partly accounted for by the higher transcription level of rpoS; however, it has also been suggested that the cells accumulate sigma(S) post-transcriptionally. We found that the level of the small regulatory RNA RprA, which is involved in the promotion of rpoS translation, is higher in pgsA3 cells than in pgsA(+) cells. Induction of altered rpoS mRNA that does not depend on RprA in pgsA(+) cells did not increase the level of sigma(S) to the high level observed in pgsA3 cells, suggesting post-translational sigma(S) accumulation in the latter. The mRNA levels of clpX and clpP, whose products form a ClpXP protease that degrades sigma(S), were much reduced in pgsA3 cells. Consistent with the reduced mRNA levels, the half-life of sigma(S) in pgsA3 cells was much longer than in pgsA(+) cells, indicating that downregulation of the degradation is a major cause for the high sigma(S) content. We show that the downregulation can be partially attributed to activated CpxAR in the mutant cells, which causes repression of rpoE on whose gene product the expression of clpPX depends.