PhoA Fusion Research Articles

Pkg2, a novel transmembrane protein Ser/Thr kinase of Streptomyces granaticolor.

A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.

Expression of an 18 kDa::PhoA fusion protein in Mycobacteriumspp.

Recent advances with mycobacterial vectors hold promise for the development of recombinant mycobacterial vaccines. Production of heterologous proteins by mycobacteria can elicit strong cellular and humoral immune responses. Importantly, expression of proteins at the surface of Mycobacterium spp. results in significant humoral responses as compared to those against cytoplasmic proteins. We have developed pCR7, a plasmid vector that expresses the M . leprae 18 kDa antigen fused in-frame to E. coli alkaline phosphatase (PhoA). The fusion sequence is flanked by insertion sequence (IS900) elements, allowing stable integration into the mycobacterial chromosome. A 59-kDa protein, the predicted size of the fusion product, was detectable by immunoblotting with monoclonal antibody to PhoA and to the 18 kDa antigen. M. smegmatis and M. vaccae transformed with pCR7 exhibited alkaline phosphatase (PhoA) activity, indicating transport of the heterologous protein across the mycobacterial membrane. pCR7 transformants: (a) had a single copy of the gene construct, (b) varied in the level of PhoA activity and (c) were cultivated stably in the absence of antibiotic pressure. Furthermore, production of the 18 kDa::PhoA fusion protein in pCR7 transformants was significantly enhanced during intracellular incubation in J774 macrophage monolayers. Thus, pCR7 may offer several advantages as a recombinant vaccine vector. Target antigens can be expressed in-frame with the 18 kDa::PhoA construct. Such recombinant Mycobacterium spp. would express the target antigen at the mycobacterial surface, co-express the immunostimulatory M. leprae 18 kDa sequences, and allow enhanced production of target antigens in vivo. Importantly, production of heterologous proteins could be verified by screening for PhoA activity, providing a potential alternative to antibiotic selection.

FtsE(Ts) affects translocation of K+-pump proteins into the cytoplasmic membrane of Escherichia coli.

The ftsE(Ts) mutation of Escherichia coli causes defects in cell division and cell growth. We expressed alkaline phosphatase (PhoA) fusion proteins of KdpA, Kup, and TrkH, all of which proved functional in vivo as K+ ion pumps, in the mutant cells. During growth at 41 degrees C, these proteins were progressively lost from the membrane fraction. The reduction in the abundance of these proteins inversely correlated with cell growth, but the preformed proteins in the membrane were stable at 41 degrees C, indicating that the molecules synthesized at the permissive temperature were diluted in a growth-dependent manner at a high temperature. Pulse-chase experiments showed that KdpA-PhoA was synthesized, but the synthesized protein did not translocate into the membrane of the ftsE(Ts) cells at 41 degrees C and degraded very rapidly. The loss of KdpA-PhoA from the membrane fractions of ftsE(Ts) cells was suppressed by a multicopy plasmid carrying the ftsE+ gene. While cell growth stopped when the abundance of these proteins decreased 15-fold, the addition of a high concentration of K+ ions specifically alleviated the growth defect of ftsE(Ts) cells but not cell division, and the cells elongated more than 100-fold. We conclude that one of the causes of growth cessation in the ftsE(Ts) mutants is a defect in the translocation of K+-pump proteins into the cytoplasmic membrane.

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum.

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180(T)) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum.

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 beta-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.

Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus.

The agr P2 operon in Staphylococcus aureus codes for the elements of a density-sensing cassette made up of a typical two-component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post-translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC, to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr+ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N-terminal region of AgrC is replaced by the Escherichia coli maltose-binding protein is also autophosphorylated in response to stimulation by agr+ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two-component signal system in S. aureus, and that the ligand-binding site of AgrC is probably located in the third extracellular loop of the protein.

Topological study of Vibrio alginolyticus NhaB Na +/H + antiporter using gene fusions in Escherichia coli cells

NhaB, an Na +/H + antiporter, of Vibrio alginolyticus is a 528-amino-acid protein. Hydropathy profile-based computer analysis predicted that the NhaB might contain up to 13 membrane-spanning domains. To examine this hypothesis, we applied the phoA fusion method to the cloned nhaB gene. Eighteen plasmid-borne nhaB–phoA fusion genes were constructed in Escherichia coli cells and the alkaline phosphatase activity and expression level of the fusion proteins analyzed. These results and the results obtained with additional constructs indicated that V. alginolyticus NhaB has a unique topology consisting of nine transmembrane segments with the N-terminus in the cytoplasm and the C-terminus in the periplasm.

Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins.

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins. To study the topology of the Agrobacterium T-DNA transfer proteins, we constructed a transposon, Tn3phoA. The transposon mobilizes into plasmids at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein topology directly in the bacterium of interest. For studies on the DNA transfer proteins, an Agrobacterium strain deficient in phoA under our experimental conditions was constructed by chemical mutagenesis. A plasmid containing virB and virD4 was used as a target for mutagenesis. Twenty-eight unique phoA-positive clones that mapped to eight virB genes were isolated. Multiple insertions throughout VirB1, VirB5, VirB7, VirB9 and VirB10 indicated that these proteins primarily face the periplasm. Insertions in VirB2, VirB6 and VirB8 allowed the identification of their periplasmic domains. No insertions were found in VirB3, VirB4 and VirB11. These proteins either lack or have a short periplasmic domain. No insertions mapped to VirD4 either. To study VirD4 topology, targeted phoA fusions and random lacZ fusions were constructed. Analysis of the fusion proteins indicated that VirD4 contains a single periplasmic domain near the N-terminus, and most of the protein lies in the cytoplasm. A hypothetical model for the T-DNA transport pore is presented.

Periplasmic copper-zinc superoxide dismutase protects Haemophilus ducreyi from exogenous superoxide.

Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease implicated in increased heterosexual transmission of HIV. As part of an effort to identify H. ducreyi gene products involved in virulence and pathogenesis, we created random TnphoA insertion mutations in an H. ducreyi 35000 library cloned in Escherichia coli. Inserts encoding exported or secreted PhoA fusion proteins were characterized by DNA sequencing. One such clone encoded a Cu-Zn superoxide dismutase (SOD) enzyme. The Cu-Zn SOD was periplasmic in H. ducreyi and accounted for most of the detectable SOD activity in whole-cell lysates of H. ducreyi grown in vitro. To investigate the function of the Cu-Zn SOD, we created a Cu-Zn SOD-deficient H. ducreyi strain by inserting a cat cassette into the sodC gene. The wild-type and Cu-Zn SOD null mutant strains were equally resistant to excess cytoplasmic superoxide induced by paraquat, demonstrating that the Cu-Zn SOD did not function in the detoxification of cytoplasmic superoxide. However, the Cu-Zn SOD null strain was significantly more susceptible to killing by extracellular superoxide than the wild type. This result suggests that the H. ducreyi Cu-Zn SOD may play a role in bacterial defence against oxidative killing by host immune cells during infection.

Identification, genomic organization, and analysis of the group III capsular polysaccharide genes kpsD, kpsM, kpsT, and kpsE from an extraintestinal isolate of Escherichia coli (CP9, O4/K54/H5).

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes kpsDMTE. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although kps(K54)DMTE are significantly divergent from their group II homologs at both the DNA and protein levels phoA fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps(K54)M and Kps(K54)T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps(K54)E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated kpsM (kpsM(truncated) and its 5' noncoding regulatory sequence were identified. In contrast to the complete kps(K54)M, this region was highly homologous to the group II kpsM. Fifty-three base pairs 3' from the end of kpsM(truncated) was a sequence 75% homologous to the 39-bp inverted repeat in the IS110 insertion element from Streptomyces coelicolor. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS110-mediated horizontal transfer.

Differential levels of specific cytochrome c biogenesis proteins in response to oxygen: analysis of the ccl operon in Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus synthesizes c-type cytochromes under a variety of growth conditions. For example, under aerobic growth, c-type cytochromes are synthesized as part of an electron transport pathway, using oxygen as the terminal electron acceptor. Anaerobically in the light, R. capsulatus requires cytochrome bc1 and other c-type cytochromes for the photosynthetic electron transport pathway. It is shown here that the ccl1 and ccl2 genes of R. capsulatus are required for the synthesis of all c-type cytochromes, including the cytochrome c' protein of unknown function but of structural similarity to cytochrome b562. Polar and nonpolar mutations constructed in each gene demonstrated that the ccl12 genes form an operon. Expression of the ccl12 genes was examined by using lacZ and phoA fusions as translational reporters. Primer extension analysis was used to determine transcriptional control and the start site of the ccl12 promoter. Finally, antiserum to the Ccl2 protein was used to quantitate levels of Ccl2 under six different growth conditions. The Ccl2 protein is present at 20-fold-higher levels under conditions where oxygen is present. In contrast, other cytochromes c biogenesis proteins, HelA and HelX, previously shown to be part of an helABCDX operon, are at relatively similar levels under these six growth conditions. This discovery is discussed in terms of the physiology and evolution of cytochromes c biogenesis, with particular attention to oxidative environments.

Topology of eukaryotic multispanning transmembrane proteins: use of LacZ fusions for the localization of cytoplasmic domains in COS.M6 cells.

In Escherichia coli, the topology of inner membrane proteins can be studied conveniently with the alkaline phosphatase/beta-galactosidase (PhoA/LacZ) gene fusion system. PhoA is enzymatically active only when fused to external domains, LacZ when fused to cytoplasmic domains. In eukaryotic cells, only time consuming methods exist to study the topology of membrane proteins. We have extended in the first systematic study the PhoA/LacZ gene fusion system originally developed for E.coli for use in eukaryotic COS.M6 cells. We have fused PhoA and LacZ to the putative external and cytoplasmic loops of rat aquaporin 2 (AQP2), for which a model with six transmembrane domains was proposed previously. The fusion proteins were expressed in E.coli and COS.M6 cells and immunoblot analyses and enzyme activity assays were performed to localize the protein domains in both cell types. The data obtained in E.coli correlated mostly with the predictions of the six transmembrane domain model. However, two fusions were found to exhibit both high PhoA and high LacZ activity, thereby complicating the construction of a complete AQP2 model. In COS.M6 cells, the PhoA fusions were inactive. In contrast, the LacZ fusions succeeded and showed an activity pattern in complete agreement with the predictions of the six transmembrane domain model. Therefore, LacZ fusions can localize cytoplasmic loops in COS.M6 cells by means of a simple enzymatic assay with high reliability and may be used in future studies to develop topological models of other eukaryotic membrane proteins in their authentic cell systems.

Posttranslational control of the algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN).

Pseudomonas aeruginosa strains associated with cystic fibrosis are often mucoid due to the copious production of alginate, an exopolysaccharide and virulence factor. Alginate gene expression is transcriptionally controlled by a gene cluster at 68 min on the chromosome: algT (algU)-mucA-mucB (algN)-mucC (algM)-mucD (algY). The algT gene encodes a 22-kDa alternative sigma factor (sigma22) that autoregulates its own promoter (PalgT) as well as the promoters of algR, algB, and algD. The other genes in the algT cluster appear to regulate the expression or activity of sigma22. The goal of this study was to better understand the functional interactions between sigma22 and its antagonist regulators during alginate production. Nonmucoid strain PAO1 was made to overproduce alginate (indicating high algD promoter activity) through increasing sigma22 in the cell by introducing a plasmid clone containing algT from mucA22(Def) strain FRD1. However, the bacterial cells remained nonmucoid if the transcriptionally coupled mucB on the clone remained intact. This suggested that a stoichiometric relationship between sigma22 and MucB may be required to control sigma factor activity. When the transcription and translational initiation of algT were measured with lacZ fusions, alginate production correlated with only about a 1.2- to 1.7-fold increase in algT-lacZ activity, respectively. An algR-lacZ transcriptional fusion showed a 2.8-fold increase in transcription with alginate production under the same conditions. A Western blot analysis of total cell extracts showed that sigma22 was approximately 10-fold higher in strains that overproduced alginate, even though algT expression increased less than 2-fold. This suggested that a post-transcriptional mechanism may exist to destabilize sigma22 in order to control certain sigma22-dependent promoters like algD. By Western blotting and phoA fusion analyses, the MucB antagonist of sigma22 was found to localize to the periplasm of the cell. Similar experiments suggest that MucA localizes to the inner membrane via one transmembrane domain with amino- and carboxy-terminal domains in the cytoplasm and periplasm, respectively. These data were used to propose a model in which MucB-MucA-sigma22 interact via an inner membrane complex that controls the stability of sigma22 protein in order to control alginate biosynthesis.

Promoter strength influences phase variation of neisserial opa genes.

The opa multigene family of Neisseria gonorrhoeae encodes 11 related outer-membrane proteins which phase vary in vitro and in vivo. Illegitimate recombination within direct pentameric DNA repeats, encoding the signal-peptide region of pre-Opas, leads to switches in expression states. Despite the conserved nature of the variation mechanism, specific genes are expressed at high frequencies in the transition from Opa- to Opa+. The genes which are expressed at elevated frequencies differ from the rest of the family with respect to promoter structure, based on sequence comparisons between the opa genes of strain MS11mk. We have analysed transcription of the opa gene family of N. gonorrhoeae MS11mk, focussing on the different promoters found among the 11 genes to determine whether increased levels of expression are associated with increased phase-variation rates. Primer extension and Northern blotting was used to assess the levels of transcription of three representative opa genes (opaA, B and C) in 'on' and 'off' states. Full-length opa mRNA was detected primarily in strains expressing the homologous gene. Truncated opa mRNA was constitutively expressed from all opa genes regardless of their expression state. Quantitative comparisons in N. gonorrhoeae were complicated by the simultaneous expression of all 11 genes and the cross-reactivity of mRNA probes. Expression levels from the individual promoters were therefore assessed by creating transcriptional and translational lacZ fusions to each of the representative opa promoters which lacked the DNA repeats responsible for variation. The expression levels were compared to the phase-variation rates of translational opa::phoA fusions containing the same promoters in addition to the corresponding coding repeat regions. A strong correlation was found between expression levels from the different promoters and the variation rates at which 'on' variants appeared from an 'off' population (i.e. opaA > opaB > opaC). These results provide an explanation for the favoured expression of specific Opa proteins and indicate that expression of opa genes may be regulated at the level of transcription.

Membrane Topology of Kch, a Putative K+ Channel from Escherichia coli

We have mapped the topology of the C-terminal half of the putative potassium channel protein Kch in the inner membrane of Escherichia coli using PhoA fusions. Our results are consistent with the widely assumed six-transmembrane helix model for eukaryotic voltage-gated ion channels and place both ends of the proposed channel-lining P-segment on the periplasmic side of the inner membrane. The rather hydrophobic P-segment is found to translocate only slowly across the inner membrane and seems to be near a threshold for stop-transfer function.

Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.

We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.

Cytochrome c550 expression in Paracoccus denitrificans strongly depends on growth condition: identification of promoter region for cycA by transcription start analysis.

The periplasmic cytochrome c550 content of Paracoccus denitrificans has been shown by immunological detection to be strongly dependent on the mode of growth. Cells grown under anaerobic, denitrifying conditions or methylotrophically in the presence of oxygen contained substantially more cytochrome c550 than cells grown aerobically on multicarbon substrates. A similar pattern was observed when expression of the cycA gene (encoding cytochrome c550), was monitored using an Escherichia coli alkaline phosphatase gene (phoA) fusion as a reporter of cycA promoter activity. The increase in cycA expression observed during growth on C1 substrates was substantially diminished if succinate was also present. These results reveal that expression of cycA is subject to multiple regulatory controls and suggest that cytochrome c550 has a general role in electron transfer to periplasmic reductases required for anaerobic denitrifying growth and from dehydrogenases required for aerobic growth on C1 compounds. Two major transcriptional initiation start points for the cycA gene have been identified.

Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli.

The Escherichia coli ProU system is a member of the ATP-binding cassette (ABC) superfamily of transporters. ProU consists of three components (ProV, ProW, and ProX) and functions as a high-affinity, binding protein-dependent transport system for the osmoprotectants glycine betaine and proline betaine. The ProW protein is the integral inner membrane component of the ProU system. Its hydropathy profile predicts seven transmembrane spans and a hydrophilic amino terminus of approximately 100 residues, and it suggests the presence of an amphiphilic alpha-helix (L-61 to F-97) in close proximity to the first strongly hydrophobic segment of ProW. We have studied the membrane topology of the ProW protein by the phoA and lacZ gene fusion approach. A collection of 10 different proW-phoA fusions with alkaline phosphatase activity and 8 different proW-lacZ fusions with beta-galactosidase activity were isolated in vivo after TnphoAB and TnlacZ mutagenesis of a plasmid-encoded proW gene. The recovery of both enzymatically active ProW-PhoA and ProW-LacZ hybrid proteins indicates that segments of ProW are exposed on both sides of the cytoplasmic membrane. To compare the enzymatic activities of each of the indicator proteins joined at a particular site in ProW, we switched the phoA and lacZ reporter genes in vitro in each of the originally in vivo-isolated gene fusions. A mirror-like pattern in the enzyme activity of the resulting new ProW-PhoA and ProW-LacZ hybrid proteins emerged, thus providing positive signals for the location of both periplasmic and cytoplasmic domains in ProW. The protease kallikrein digests the amino-terminal tail of a ProW-LacZ hybrid protein in spheroplasts, suggesting that the amino terminus of ProW is located on the periplasmic side of the cytoplasmic membrane. From these data, a two-dimensional model for ProW was constructed; this model consists of seven transmembrane alpha-helices and an unusual amino-terminal tail of approximately 100 amino acid residues that protrudes into the periplasmic space.

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II.

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.

A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 produces pyoverdine under iron-limiting conditions. A Tn5 mutant, 2G11, produced lower amounts of different pyoverdine forms and was unable to grow under iron limitation caused by ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA) or zinc. This mutant was complemented by a 9.6 kb HindIII-BamHI DNA fragment that contained eight contiguous open reading frames (ORFs cytA to cytH). The proteins possibly encoded by this polycistronic gene cluster were all similar to the products of cytochrome c biogenesis genes from, amongst others, Rhodobacter capsulatus and Bradyrhizobium japonicum, not only in terms of amino acid sequence, but also in the overall hydropathy index of these proteins. By TnphoA mutagenesis and site-specific gene replacement it was found that the first three ORFs (cytA to cytC) were essential for cytochrome c production while only the product of cytA was needed for normal pyoverdine production. The presence of a putative haem-binding site in the CytA protein (WGSWWVWD) was confirmed. From analysis of a constructed phoA fusion, a periplasmic location was found for this motif. The ability of the cytA gene to restore both cytochrome c and pyoverdine production suggests the involvement of this particular gene both in haem and in pyoverdine transport in P. fluorescens.