Sequence–function relationships in MalG, an inner membrane protein from the maltose transport system in Escherichia coli

The maIG gene encodes a hydrophobic cytoplasmic membrane protein which is required for the energy-dependent transport of maltose and maltodextrins in Escherichia coli. The MalG protein, together with MalF and MalK proteins, forms a multimeric complex in the membrane consisting of two MalK subunits for each MalF and MalG subunit. Fifteen mutations have been isolated in malG by random linker insertion mutagenesis. Two regions essential for maltose transport have been identified. In particular, a hydro philic region containing the peptidic motif EAA—G———I-LP, highly conserved among inner membrane proteins from binding protein-dependent transport systems, is essential for maltose transport. The results also show that several regions of MalG are not essential for function. A region (residues 30–50) encompassing the first predicted transmembrane segment and the first periplasmic loop in MalG may be modified extensively with little effect on maltose transport and no effect on the stability and the localization of the protein. A region located at the middle of the protein (residues 153–157) is not essential for the function of the protein. A region, essential for maltodextrin utilization but not for maltose transport, has been identified near the C-terminus of the protein.     

A topological model for the high-affinity nickel transporter of Alcaligenes eutrophus

The gene hoxN of Alcaligenes eutrophus encodes a membrane protein with a molecular mass of 33.1 kDa that mediates energy-dependent uptake of nickel ions. Based on the hydrophobicity of the HoxN protein five, six, or seven transmembrane segments were predicted, depending on the algorithm used for computer analysis. To distinguish between these possibilities varying segments of the amino-terminal end of the transporter were fused to the Escherichia coli enzymes aikaline phosphatase (PhoA) or β-galactosidase (LacZ). The enzymatic activity of 16 HoxN-PhoA and 15 HoxN-LacZ fusions was determined. On the assumption that PhoA fusions only exhibit high activity when fused to periplasmic domains of the target, while LacZ fusions are only active when oriented towards the cytoplasm, a two-dimensional model for the nickel transporter was developed. This model proposes that HoxN contains four periplasmic and four cytoplasmic regions, and seven transmembrane helices. The amino terminus is located in the cytoplasm, and the carboxyl terminus faces the periplasm.     

Topological Analysis of the Aerobic Membrane-Bound Formate Dehydrogenase of Escherichia coli

Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate, Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, α (FdoG), β (FdoH), and γ (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using blaM (β-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the αβ catalytic dimer is located in the cytoplasm, with a C-terminal anchor for β protruding into the periplasm. As expected, the γ subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the ³⁵S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.     

Topological and mutational analysis of KpsM, the hydrophobic component of the ABC-transporter involved in the export of polysialic acid in Escherichia coli K1

The 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E. coli K1, is divided into three functional regions. Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP-binding site. KpsM and KpsT belong to the ATP-binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using β-lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane-spanning regions, with the N- and C-terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV–SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space. In vivo cross-linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker-insertion and site-directed mutagenesis define the N-terminus, the first cytoplasmic loop, and the SV-SVI linker as regions that are important for the function of KpsM in K1 polymer transport.     

An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase

Pseudomonas glumae PG1 is able to secrete lipase into the extracellular medium. The lipase is produced as a precursor protein, with an N-terminal signal sequence. A second open reading frame (ORF) was found immediately downstream of the lipase structural gene, lip A, a situation found for the lipases of some other Pseudomonas species. Inactivation of this ORF resulted in a lipase-negative phenotype, indicating its importance in the production of active extracellular lipase. The ORF, lipB, potentially encodes a protein of 353-amtno-acid residues, having a hydrophobic N-terminal (amino acids 1 to 90) and a hydrophilic C-terminal part. As a first step in determining the role of LipB, its subcellular location was determined. The protein was found to fractionate with the inner membranes. The expression of fusions of lipB fragments with phoA revealed an N_in–C_out topology for the LipB protein, which was confirmed by protease accessibility studies on EDTA-permeabilized cells and on inverted inner membrane vesicles. These and other results indicate that most of the LipB polypeptide is located in the periplasm and anchored to the inner membrane by an an N-terminal transmembrane helix, located between amino acids 19 and 40.     

Topological analyses of the l-fucose-H+ symport protein, FucP, from Escherichia coli

The transport of l -fucose into Escherichia coli is mediated by the l -fucose-H⁺ symport protein (FucP). The fucP gene has been sequenced and encodes a hydrophobic protein that contains 438 amino acid residues, with a predicted M_r of 47773. The hydropathic profile of FucP indicates 10 to 12 hydrophobic regions that could span the membrane as α-helices. A 12-helix model with the N- and C-termini located in the cytoplasm was derived from the hydropathic profile and from application of the ‘positive inside’ rule. This model was tested using β-lactamase fusion technology. Analyses of 62 different FucP-β-lactamase fusions suggested that the FucP protein crosses the cytoplasmic membrane of E. coli 12 times, with the N- and C-termini in the cytoplasm. From measurements of [¹⁴C]-l -fucose uptake, it was deduced that the last putative transmembrane region must be complete for transport activity to be retained and that the four C-terminal residues were unnecessary for transport activity. Fourier transform analyses show that all the predicted helices contain a periodicity that enables hydrophobic/hydrophilic faces to be identified; these were particularly evident in putative helices 1, 3, 4, 5, 6, 10 and 11.     

The membrane topology of the Rhizobium meliloti C4-dicarboxylate permease (DctA) as derived from protein fusions with Escherichia coli K12 alkaline phosphatase (PhoA) and β-galactosidase (LacZ)

The Rhizobium meliloti dctA gene encodes the C₄-dicarboxylate permease which mediates uptake of C₄-dicarboxylates, both in free-living and symbiotic cells. Based on the hydrophobicity of the DctA protein, 12 putative membrane spanning regions were predicted. The membrane topology was further analysed by isolating in vivo fusions of DctA to Escherichia coli alkaline phosphatase (PhoA) and E. coli β-galactosidase (LacZ). Of 10 different fusions 7 indicated a periplasmic and 3 a cytoplasmic location of the corresponding region of the DctA protein. From these data a two-dimensional model of DctA was constructed which comprised twelve transmembrane α-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm. In addition, four conserved amino acid motifs present in many eukaryotic and prokaryotic transport proteins were observed.     

Subcellular sites for bacterial protein export

Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA‐YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the secretory protein pre‐AmyQ, were analysed using green fluorescent protein fusions, immunostaining and/or immunogold labelling techniques. It is shown that SecA, SecY and (pre‐)AmyQ are located at specific sites near and/or in the cytoplasmic membrane of Bacillus subtilis. The localization patterns of these proteins suggest that the Sec machinery is organized in spiral‐like structures along the cell, with most of the translocases organized in specific clusters along these structures. However, this localization appears to be independent of the helicoidal structures formed by the actin‐like cytoskeletal proteins, MreB or Mbl. Interestingly, the specific localization of SecA is dynamic, and depends on active translation. Moreover, reducing the phosphatidylglycerol phospholipids content in the bacterial membrane results in delocalization of SecA, suggesting the involvement of membrane phospholipids in the localization process. These data show for the first time that, in contrast to the recently reported uni‐ExPortal site in the coccoïd Streptococcus pyogenes, multiple sites dedicated to protein export are present in the cytoplasmic membrane of rod‐shaped B. subtilis.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain

The membrane topology of the Rhizobium meliloti 2011 ExoP protein involved in polymerization and export of succinoglycan was analysed by translational fusions of lacZ and phoA reporter genes to the exoP gene. Based on this analysis, the ExoP protein could be divided into an N-terminal domain mainly located in the periplasmic space and a C-terminal domain located in the cytoplasm. Whereas the C-terminal domain of ExoP is characterized by a potential nucleotide-binding motif, the N-terminal ExoP domain contains the sequence motif‘PX₂pX₄SPKX₁₁GXMXG₁′, which is also present in proteins involved in the determination of O-antigen chain length. R. meliloti strains carrying mutated exoP* genes, exclusively encoding the N-terminal ExoP domain, produced a reduced amount of succinoglycan. This reduction could be suppressed by a mutation in the regulatory gene exoR. The ratio of low-molecular-weight to high-molecular-weight succinoglycan was significantly increased in the exoP* mutant strain. In the exoP*lexoR mutant strain only low-molecular-weight succinoglycan could be detected. Based on sequence homologies and similar hydropathic profiles, the N-terminal domain of ExoP was proposed to be a member of a protein family thought to be involved in polysaccharide chain-length determination.     

Expression of the nmpC gene of Escherichia coli K-12 is modulated by external pH. Identification of cis-acting regulatory sequences involved in this regulation

Using a set of gene fusions generated with TnphoA, we previously identified the phmA locus, whose expression is modulated as a function of external pH (pHo). The phmA::phoA fusion was cloned and sequenced and the phmA locus was identified with the nmpC gene. This gene lies within the defective lambdoid prophage qsr′ and NmpC is an outer membrane protein which functions as a porin. We demonstrated that nmpC is sensitive to catabotite repression and dependent on the CRP—cAMP complex. However, cAMP is not a signal for the pHo-dependent expression of nmpC. By generating step deletions in the sequence 5′ to the nmpC coding region, we identified a DNA region in position —345 to —127 which is involved in nmpC repression, mainly during growth at acid pHo. Four regions with strong homologies and a very well-conserved organization of the functional sequence were found in the nmpC and ompF promoters. We propose that the negative regulation of nmpC during growth at low pHo might involve DNA looping of the nmpC promoter.     

LIGHT AND ELECTRON MICROSCOPIC STUDIES OF THE FUSION CELL IN ASTEROCOLAX GARDNERI SETCH. (RHODOPHYTA,CERAMIALES)12

The fusion cell in Asterocolax gardneri Setch, is a large, multinucleate, irregularly-shaped cell resulting from cytoplasmic fusions of haploid and diploid cells. Subsequent enlargement takes place by incorporating adjacent gonimoblast cells. The resultant cell consists of two parts—a central portion of isolated cytoplasm, surrounded by an electron dense cytoplasmic barrier, and the main component of the fusion cell cytoplasm surrounding the isolated cytoplasm. The fusion cell contains many nuclei, large quantities of floridean starch, endoplasmic reticulum, and vesicles, but few mitochondria, plastids and dictyosomes. The endoplasmic reticulum forms vesicles that apparently secrete large quantities of extracellular mucilage which surrounds the entire carposporophyte. The isolated cytoplasm also is multinucleate but lacks starch and a plasma membrane. Few plastids, ribosomes and mitochondria are found in this cytoplasm. However, numerous endoplasmic reticulum cisternae occur near the cytoplasmic barrier and they appear to secrete material for the barrier. In mature carposporophytes, all organelles in the isolated cytoplasm have degenerated.     

Genetic analysis of rye (Secale cereale L.) Genetics of male sterility of the G-type

Summary The genetics and relationships between the genes in rye located in the nucleus and cytoplasm of the male sterility of the G-type were investigated. A factor inducing male sterility was found in the cytoplasms or rye cv Schlägler alt and rye cv Norddeutscher Champagner. Monogenic inheritance was observed in linkage tests. Using primary trisomies of rye cv Esto, the nuclear gene ms1 was found to be located on chromosome 4R. Modifying genes, probably masked in normal cytoplasm but expressed in male-sterility-inducing cytoplasm together with gene ms1, were located on chromosomes 3R (ms2) and 6R (ms3). Mono-, di-, and trigenic inheritance types were found in backcross progenies of trisomies.     

Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy)

Lipopolysaccharides (LPS), particularly the O-antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O-antigen polymerase, an integral membrane protein, which polymerizes the O-antigen repeat units of the LPS. The wild-type rfc/wzy gene has no detectable ribosome-binding site (RBS) and four rare codons in the translation initiation region (TIR). Site-directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O-antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O-antigen chain length regulator (Rol/Wzz) on a low-copy-number plasmid. This suggests that the O-antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM-PREDICT indicated that Rfc/Wzy had 10–13 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of Rfc/Wzy to alkaline phosphatase and β-galactosidase. Rfc::PhoA fusion proteins near the amino-terminal end were detected by Coomassie blue staining and Western blotting using anti-PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino- and carboxy-termini are located on the cytoplasmic face of the membrane.     

Carbohydrate uptake genes inEscherichia coli are induced by carbon starvation

Escherichia coli produces several membrane-associated and periplasmic proteins in response to deprivation for a carbon source. The carbon starvation response involves a two- to fourfold, cAMP-dependent induction of operons involved in carbohydrate uptake and utilization, including thelac operon. Threecarbon-starvation-inducible (sci) gene fusions to aphoA reporter sequence were characterized. ThephoA-fusions werecya ⁺/crp ⁺-dependent and located in three previously characterized genes involved in high-affinity uptake of alternative carbon sources:mglB, encoding the periplasmic galactose binding protein;rbsB, encoding the periplasmic ribose binding protein; andlamB, encoding the maltodextrin-specific outer membrane porin.     

Independent evolutionary origins of functional polyamine biosynthetic enzyme fusions catalysing de novo diamine to triamine formation

We have identified gene fusions of polyamine biosynthetic enzymes S‐adenosylmethionine decarboxylase (AdoMetDC, speD) and aminopropyltransferase (speE) orthologues in diverse bacterial phyla. Both domains are functionally active and we demonstrate the novel de novo synthesis of the triamine spermidine from the diamine putrescine by fusion enzymes from β‐proteobacterium Delftia acidovorans and δ‐proteobacterium Syntrophus aciditrophicus, in a ΔspeDE gene deletion strain of Salmonella enterica sv. Typhimurium. Fusion proteins from marine α‐proteobacterium Candidatus Pelagibacter ubique, actinobacterium Nocardia farcinica, chlorobi species Chloroherpeton thalassium, and β‐proteobacterium D. acidovorans each produce a different profile of non‐native polyamines including sym‐norspermidine when expressed in Escherichia coli. The different aminopropyltransferase activities together with phylogenetic analysis confirm independent evolutionary origins for some fusions. Comparative genomic analysis strongly indicates that gene fusions arose by merger of adjacent open reading frames. Independent fusion events, and horizontal and vertical gene transfer contributed to the scattered phyletic distribution of the gene fusions. Surprisingly, expression of fusion genes in E. coli and S. Typhimurium revealed novel latent spermidine catabolic activity producing non‐native 1,3‐diaminopropane in these species. We have also identified fusions of polyamine biosynthetic enzymes agmatine deiminase and N‐carbamoylputrescine amidohydrolase in archaea, and of S‐adenosylmethionine decarboxylase and ornithine decarboxylase in the single‐celled green alga Micromonas.     

Ght Protein of Neisseria meningitidis Is Involved in the Regulation of Lipopolysaccharide Biosynthesis

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is responsible for the barrier function of this membrane. A ght mutant of Neisseria meningitidis that showed increased sensitivity to hydrophobic toxic compounds, suggesting a breach in this permeability barrier, was previously described. Here, we assessed whether this phenotype was possibly caused by a defect in LPS transport or synthesis. The total amount of LPS appeared to be drastically reduced in a ght mutant, but the residual LPS was still detected at the cell surface, suggesting that LPS transport was not impaired. The ght mutant was rapidly overgrown by pseudorevertants that produced normal levels of LPS. Genetic analysis of these pseudorevertants revealed that the lpxC gene, which encodes a key enzyme in LPS synthesis, was fused to the promoter of the upstream-located pilE gene, resulting in severe lpxC overexpression. Analysis of phoA and lacZ gene fusions indicated that Ght is an inner membrane protein with an N-terminal membrane anchor and its bulk located in the cytoplasm, where it could potentially interact with LpxC. Cell fractionation experiments indeed indicated that Ght tethers LpxC to the membrane. We suggest that Ght regulates LPS biosynthesis by affecting the activity of LpxC. Possibly, this mechanism acts in the previously observed feedback inhibition of LPS synthesis that occurs when LPS transport is hampered.     

Membrane topology of Escherichia coli prolipoprotein signal peptidase (signal peptidase II)

The lsp gene of Escherichia coli encodes the inner membrane enzyme, signal peptidase II (SPase II). SPase II is comprised of 164 amino acid residues and contains four hydrophobic domains. A series of lsp-phoA and lsp-lacZ gene fusions have been constructed in vitro to determine the topology of SPase II. The fusion junction for each of these gene fusions was determined by DNA sequencing. The lengths of the SPase II fragment in the fusions varied from 12 to 159 amino acid residues. Strains containing SPase II-PhoA fusions to the two predicted periplasmic loops exhibited higher levels of alkaline phosphatase activity than fusions to the predicted cytoplasmic domains. In contrast, SPase II-LacZ fusions at the cytoplasmic and the periplasmic domains of SPase II showed high and low levels of beta-galactosidase activity, respectively, a result opposite to those shown by SPase II-PhoA fusions located at precisely the same amino acid of SPase II. Taken together, these results strongly support the predicted model for SPase II topology, i.e. this enzyme spans the cytoplasmic membrane four times with both the amino and the carboxyl termini facing the cytoplasm.