Integral Cytoplasmic Membrane Protein Research Articles

More than Rotating Flagella: Lipopolysaccharide as a Secondary Receptor for Flagellotropic Phage 7-7-1.

Bacteriophage 7-7-1, a member of the family Myoviridae, infects the soil bacterium Agrobacterium sp. strain H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify the secondary receptor(s) on the cell surface, we isolated motile, phage-resistant Agrobacterium sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary primed PCR and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyltransferase; AGROH133_13050, a UDP-glucose 4-epimerase; and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the last is predicted to be a relatively small (13.4-kDa) cytosolic membrane protein with up to four transmembrane helices. The phenotypes of the transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage-resistant mutants is also described. Given these findings, we propose a model for Agrobacterium sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down toward the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria.IMPORTANCE Flagellotropic bacteriophages belong to the tailed-phage order Caudovirales, the most abundant phages in the virome. While it is known that these viruses adhere to the bacterial flagellum and use flagellar rotation to reach the cell surface, their infection mechanisms are poorly understood. Characterizing flagellotropic-phage-host interactions is crucial to understanding how microbial communities are shaped. Using a transposon mutagenesis approach combined with a screen for motile, phage-resistant mutants, we identified lipopolysaccharides as the secondary cell surface receptor for phage 7-7-1. This is the first cell surface receptor identified for flagellotropic phages. One hypothetical membrane protein was also recognized as essential for infection. These new findings, together with previous results, culminated in an infection model for phage 7-7-1.

Phage lysis: Three steps, three choices, one outcome

The lysis of bacterial hosts by double-strand DNA bacteriophages, once thought to reflect merely the accumulation of sufficient lysozyme activity during the infection cycle, has been revealed to recently been revealed to be a carefully regulated and temporally scheduled process. For phages of Gramnegative hosts, there are three steps, corresponding to subversion of each of the three layers of the cell envelope: inner membrane, peptidoglycan, and outer membrane. The pathway is controlled at the level of the cytoplasmic membrane. In canonical lysis, a phage encoded protein, the holin, accumulates harmlessly in the cytoplasmic membrane until triggering at an allele-specific time to form micron-scale holes. This allows the soluble endolysin to escape from the cytoplasm to degrade the peptidoglycan. Recently a parallel pathway has been elucidated in which a different type of holin, the pinholin, which, instead of triggering to form large holes, triggers to form small, heptameric channels that serve to depolarize the membrane. Pinholins are associated with SAR endolysins, which accumulate in the periplasm as inactive, membrane-tethered enzymes. Pinholin triggering collapses the proton motive force, allowing the SAR endolysins to refold to an active form and attack the peptidoglycan. Surprisingly, a third step, the disruption of the outer membrane is also required. This is usually achieved by a spanin complex, consisting of a small outer membrane lipoprotein and an integral cytoplasmic membrane protein, designated as o-spanin and i-spanin, respectively. Without spanin function, lysis is blocked and progeny virions are trapped in dead spherical cells, suggesting that the outer membrane has considerable tensile strength. In addition to two-component spanins, there are some single-component spanins, or u-spanins, that have an N-terminal outer-membrane lipoprotein signal and a C-terminal transmembrane domain. A possible mechanism for spanin function to disrupt the outer membrane is to catalyze fusion of the inner and outer membranes.

MasABK Proteins Interact with Proteins of the Type IV Pilin System to Affect Social Motility of Myxococcus xanthus

Gliding motility is critical for normal development of spore-filled fruiting bodies in the soil bacterium Myxococcus xanthus. Mutations in mgl block motility and development but one mgl allele can be suppressed by a mutation in masK, the last gene in an operon adjacent to the mgl operon. Deletion of the entire 5.5 kb masABK operon crippled gliding and fruiting body development and decreased sporulation. Expression of pilAGHI, which encodes type IV pili (TFP) components essential for social (S) gliding, several cryptic pil genes, and a LuxR family protein were reduced significantly in the Δmas mutant while expression of the myxalamide operon was increased significantly. Localization and two-hybrid analysis suggest that the three Mas proteins form a membrane complex. MasA-PhoA fusions confirmed that MasA is an integral cytoplasmic membrane protein with a ≈100 amino acid periplasmic domain. Results from yeast two-hybrid assays showed that MasA interacts with the lipoprotein MasB and MasK, a protein kinase and that MasB and MasK interact with one another. Additionally, yeast two-hybrid analysis revealed a physical interaction between two gene products of the mas operon, MasA and MasB, and PilA. Deletion of mas may be accompanied by compensatory mutations since complementation of the Δmas social gliding and developmental defects required addition of both pilA and masABK.

Phage Shock Protein C (PspC) of Yersinia enterocolitica Is a Polytopic Membrane Protein with Implications for Regulation of the Psp Stress Response

Phage shock proteins B (PspB) and C (PspC) are integral cytoplasmic membrane proteins involved in inducing the Yersinia enterocolitica Psp stress response. A fundamental aspect of these proteins that has not been studied in depth is their membrane topologies. Various in silico analyses universally predict that PspB is a bitopic membrane protein with the C terminus inside. However, similar analyses yield conflicting predictions for PspC: a bitopic membrane protein with the C terminus inside, a bitopic membrane protein with the C terminus outside, or a polytopic protein with both termini inside. Previous studies of Escherichia coli PspB-LacZ and PspC-PhoA fusion proteins supported bitopic topologies, with the PspB C terminus inside and the PspC C terminus outside. Here we have used a series of independent approaches to determine the membrane topologies of PspB and PspC in Y. enterocolitica. Our data support the predicted arrangement of PspB, with its C terminus in the cytoplasm. In contrast, data from multiple independent approaches revealed that both termini of PspC are located in the cytoplasm. Additional experiments suggested that the C terminus of PspC might be the recognition site for the FtsH protease and an interaction interface with PspA, both of which would be compatible with its newly proposed cytoplasmic location. This unexpected arrangement of PspC allows a new model for events underlying activation of the Psp response, which is an excellent fit with observations from various previous studies.

The Spanin Complex Is Essential for Lambda Lysis

Phage lysis is a ubiquitous biological process, the most frequent cytocidal event in the biosphere. Lysis of Gram-negative hosts has been shown to require holins and endolysins, which attack the cytoplasmic membrane and peptidoglycan, respectively. Recently, a third class of lysis proteins, the spanins, was identified. The first spanins to be characterized were λ Rz and Rz1, an integral cytoplasmic membrane protein and an outer membrane lipoprotein, respectively. Previous work has shown that Rz and Rz1 form complexes that span the entire periplasm. Phase-contrast video microscopy was used to record the morphological changes involved in the lysis of induced λ lysogens carrying prophages with either the λ canonical holin-endolysin system or the phage 21 pinholin-signal anchor release (SAR) endolysin system. In the former, rod morphology persisted until the instant of an explosive polar rupture, immediately emptying the cell of its contents. In contrast, in pinholin-SAR endolysin lysis, the cell began to shorten and thicken uniformly, with the resultant rounded cell finally bursting. In both cases, lysis failed to occur in inductions of isogenic prophages carrying null mutations in the spanin genes. In both systems, instead of an envelope rupture, the induced cells were converted from a rod shape to a spherical form. A functional GFPΦRz chimera was shown to exhibit a punctate distribution when coexpressed with Rz1, despite the absence of endolysin function. A model is proposed in which the spanins carry out the essential step of disrupting the outer membrane, in a manner regulated by the state of the peptidoglycan layer.

Recent findings about the Yersinia enterocolitica phage shock protein response

The phage shock protein (Psp) system is a conserved extracytoplasmic stress response in bacteria that is essential for virulence of the human pathogen Yersinia enterocolitica. This article summarizes some recent findings about Y. enterocolitica Psp system function. Increased psp gene expression requires the transcription factor PspF, but under non-inducing conditions PspF is inhibited by an interaction with another protein, PspA, in the cytoplasm. A Psp-inducing stimulus causes PspA to relocate to the cytoplasmic membrane, freeing PspF to induce psp gene expression. This PspA relocation requires the integral cytoplasmic membrane proteins, PspB and PspC, which might sense an inducing trigger and sequester PspA by direct interaction. The subsequent induction of psp gene expression increases the PspA concentration, which also allows it to contact the membrane directly, perhaps for its physiological function. Mutational analysis of the PspB and PspC proteins has revealed that they both positively and negatively regulate psp gene expression and has also identified PspC domains associated with each function. We also compare the contrasting physiological roles of the Psp system in the virulence of Y. enterocolitica and Salmonella enterica sv. Typhimurium (S. Typhimurium). In S. Typhimurium, PspA maintains the proton motive force, which provides the energy needed to drive ion importers required for survival within macrophages. In contrast, in the extracellular pathogen Y. enterocolitica, PspB and PspC, but not PspA, are the Psp components needed for virulence. PspBC protect Y. enterocolitica from damage caused by the secretin component of its type 3 secretion system, an essential virulence factor.

The small protein CydX is required for function of cytochrome bd oxidase in Brucella abortus

A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3′ end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H2O2, decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.

Mutations in the ExbB Cytoplasmic Carboxy Terminus Prevent Energy-Dependent Interaction between the TonB and ExbD Periplasmic Domains

The TonB system of Gram-negative bacteria provides passage across the outer membrane (OM) diffusion barrier that otherwise limits access to large, scarce, or important nutrients. In Escherichia coli, the integral cytoplasmic membrane (CM) proteins TonB, ExbB, and ExbD couple the CM proton motive force (PMF) to active transport of iron-siderophore complexes and vitamin B(12) across the OM through high-affinity transporters. ExbB is an integral CM protein with three transmembrane domains. The majority of ExbB occupies the cytoplasm. Here, the importance of the cytoplasmic ExbB carboxy terminus (residues 195 to 244) was evaluated by cysteine scanning mutagenesis. D211C and some of the substitutions nearest the carboxy terminus spontaneously formed disulfide cross-links, even though the cytoplasm is a reducing environment. ExbB N196C and D211C substitutions were converted to Ala substitutions to stabilize them. Only N196A, D211A, A228C, and G244C substitutions significantly decreased ExbB activity. With the exception of ExbB(G244C), all of the substituted forms were dominant. Like wild-type ExbB, they all formed a formaldehyde cross-linked tetramer, as well as a tetramer cross-linked to an unidentified protein(s). In addition, they could be formaldehyde cross-linked to ExbD and TonB. Taken together, the data suggested that they assembled normally. Three of four ExbB mutants were defective in supporting both the PMF-dependent formaldehyde cross-link between the periplasmic domains of TonB and ExbD and the proteinase K-resistant conformation of TonB. Thus, mutations in a cytoplasmic region of ExbB prevented a periplasmic event and constituted evidence for signal transduction from cytoplasm to periplasm in the TonB system.

The TonB Dimeric Crystal Structures Do Not Exist In Vivo

The TonB system energizes transport of nutrients across the outer membrane of Escherichia coli using cytoplasmic membrane proton motive force (PMF) for energy. Integral cytoplasmic membrane proteins ExbB and ExbD appear to harvest PMF and transduce it to TonB. The carboxy terminus of TonB then physically interacts with outer membrane transporters to allow translocation of ligands into the periplasmic space. The structure of the TonB carboxy terminus (residues ~150 to 239) has been solved several times with similar results. Our previous results hinted that in vitro structures might not mimic the dimeric conformations that characterize TonB in vivo. To test structural predictions and to identify irreplaceable residues, the entire carboxy terminus of TonB was scanned with Cys substitutions. TonB I232C and N233C, predicted to efficiently form disulfide-linked dimers in the crystal structures, did not do so. In contrast, Cys substitutions positioned at large distances from one another in the crystal structures efficiently formed dimers. Cys scanning identified seven functionally important residues. However, no single residue was irreplaceable. The phenotypes conferred by changes of the seven residues depended on both the specific assay used and the residue substituted. All seven residues were synergistic with one another. The buried nature of the residues in the structures was also inconsistent with these properties. Taken together, these results indicate that the solved dimeric crystal structures of TonB do not exist. The most likely explanation for the aberrant structures is that they were obtained in the absence of the TonB transmembrane domain, ExbB, ExbD, and/or the PMF.

Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

Lipopolysaccharide (LPS) is a major component of the Gram-negative outer membrane and is an important virulence determinant. The O-antigen polysaccharide of the LPS molecule provides protection from host defenses, and the length of O-antigen chains plays a pivotal role. In the Wzy-dependent O-antigen biosynthesis pathway, the integral inner membrane protein Wzz determines the O-antigen chain length. How these proteins function is currently unknown, but the hypothesis includes activities such as a "molecular ruler" or a "molecular stopwatch," and other possibilities may exist. Wzz homologs are membrane proteins with two transmembrane helices that flank a large periplasmic domain. Recent x-ray crystallographic studies of the periplasmic portions of Wzz proteins found multiple oligomeric forms, with quaternary structures favoring the "molecular ruler" interpretation. Here, we have studied full-length Wzz proteins with the transmembrane portions embedded in lipid membranes. Using electron microscopy and image analysis we find a unique hexameric state rather than differing oligomeric forms. The data suggest that in vivo Wzz proteins determine O-antigen chain length via subtle structure-function relationships at the level of primary, secondary, or tertiary structure within the context of a hexameric complex.

Outer and Inner Membrane Proteins Compose an Arginine-Agmatine Exchange System inChlamydophila pneumoniae

Most chlamydial strains have a pyruvoyl-dependent decarboxylase protein that converts L-arginine to agmatine. However, chlamydiae do not produce arginine, so they must import it from their host. Chlamydophila pneumoniae has a gene cluster encoding a putative outer membrane porin (CPn1033 or aaxA), an arginine decarboxylase (CPn1032 or aaxB), and a putative cytoplasmic membrane transporter (CPn1031 or aaxC). The aaxC gene was expressed in Escherichia coli producing an integral cytoplasmic membrane protein that catalyzed the exchange of L-arginine for agmatine. Expression of the aaxA gene produced an outer membrane protein that enhanced the arginine uptake and decarboxylation activity of cells coexpressing aaxB and aaxC. This chlamydial arginine/agmatine exchange system complemented an E. coli mutant missing the native arginine-dependent acid resistance system. These cells survived extreme acid shock in the presence of L-arginine. Biochemical and evolutionary analysis showed the aaxABC genes evolved convergently with the enteric arginine degradation system, and they could have a different physiological role in chlamydial cells. The chlamydial system uniquely includes an outer membrane porin, and it is most active at a higher pH from 3 to 5. The chlamydial AaxC transporter was resistant to cadaverine, L-lysine and L-ornithine, which inhibit the E. coli AdiC antiporter.

His20Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain

The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His(20) is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser(16) and His(20), the resultant "all-Ala Ser(16) His(20)" TMD TonB retained 90% of wild-type iron transport activity. Ser(16)Ala in the context of a wild-type TonB TMD was fully active. In contrast, His(20)Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser(16) His(20) TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser(16) His(20) TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser(16) His(20) TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser(16) His(20) TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.

A new ferrous iron‐uptake transporter, EfeU (YcdN), from Escherichia coli

Escherichia coli possesses multiple routes for iron uptake. Here we present EfeU (YcdN), a novel iron acquisition system of E. coli strain Nissle 1917. Laboratory strains of E. coli such as K12 lack a functional (efeU) ycdN gene caused by a frameshift mutation. EfeU, a member of the oxidase-dependent iron transporters (OFeT), is a homologue of the iron permease Ftr1p from yeast. The ycdN gene is part of the ycdNOB tricistronic operon which is expressed in response to iron deprivation in a Fur-dependent manner. Expression of efeU resulted in improved growth of an E. coli mutant lacking all known iron-uptake systems and mediated increased iron uptake into cells. Furthermore, the presence of other divalent metal cations did not impair growth of strains expressing efeU. The EfeU protein functioned as ferrous iron permease in proteoliposomes in vitro. Topology analysis indicated that EfeU is an integral cytoplasmic membrane protein exhibiting seven transmembrane helices. Two REXXE motifs within transmembrane helices of OFeT family members are implicated in iron translocation. Site-directed mutagenesis of each REGLE motif of EfeU diminished iron uptake in vivo and growth yield. In vitro the EfeU variant protein with an altered first REGLE motif was impaired in iron permeation, whereas activity of the EfeU variant with a mutation in the second motif was similar to the wild-type protein.

RhuR, an extracytoplasmic function sigma factor activator, is essential for heme-dependent expression of the outer membrane heme and hemoprotein receptor of Bordetella avium.

Genes involved in iron (Fe) acquisition often are regulated in response to the local availability of Fe. In many bacteria, Fe-dependent responsiveness is mediated by Fur, a global Fe-dependent transcriptional repressor. Tighter regulatory control of Fur-responsive genes is afforded by incorporating additional regulators into Fur-dependent regulatory cascades. RhuI, a Fur-dependent extracytoplasmic function sigma factor of Bordetella avium, in response to the dual stimulation of Fe starvation and the presence of heme (or hemoproteins), regulates P(bhuR), a heme-responsive promoter which directs expression of the bhuRSTUV heme utilization operon. While BhuR, the outer membrane heme receptor, and RhuI have been shown to be indispensable for heme-dependent activation of P(bhuR), collateral components of the regulatory cascade have not been described. In this investigation, RhuR, an integral cytoplasmic membrane protein with homology to anti-sigma factors, is shown to be an essential activator of P(bhuR) expression. The functional domain of RhuR required for heme-dependent activation of P(bhuR) expression was mapped to the N-terminal 97 amino acids of the protein by use of a chimeric RhuR-BlaM fusion. Expression of the chimera in a rhuR mutant rendered P(bhuR) constitutive, thereby decoupling the promoter from heme dependency. Growth studies confirmed that B. avium requires RhuR for optimal utilization of hemoglobin, but not hemin, as a sole source of nutrient Fe. These data imply that B. avium expresses, in addition to the BhuR heme/hemoprotein utilization system, an alternative RhuR-independent heme utilization mechanism. A model is proposed in which RhuR is the functional bridge between BhuR and RhuI in a heme-dependent regulatory cascade.

Molecular Evolution of thedotAGene inLegionella pneumophila

The molecular evolution of dotA, which is related to the virulence of Legionella pneumophila, was investigated by comparing the sequences of 15 reference strains (serogroups 1 to 15). It was found that dotA has a complex mosaic structure. The whole dotA gene of Legionella pneumophila subsp. pneumophila serogroups 2, 6, and 12 has been transferred from Legionella pneumophila subsp. fraseri. A discrepancy was found between the trees inferred from the nucleotide and deduced amino acid sequences of dotA, which suggests that multiple hits, resulting in synonymous substitutions, have occurred. Gene phylogenies inferred from three different segments (the 5'-end region, the central, large periplasmic domain, and the 3'-end region) showed impressively dissimilar topologies. This was concordant with the sequence polymorphisms, indicating that each region has experienced an independent evolutionary history, and was evident even within the same domain of each strain. For example, the PP2 domain was found to have a heterogeneous structure, which led us hypothesize that the dotA gene of L. pneumophila may have originated from two or more different sources. Comparisons of synonymous and nonsynonymous substitutions demonstrated that the PP2 domain has been under strong selective pressure with respect to amino acid change. Split decomposition analysis also supported the intragenic recombination of dotA. Multiple recombinational exchange within the dotA gene, encoding an integral cytoplasmic membrane protein that is secreted, probably provided increased fitness in certain environmental niches, such as within a particular biofilm community or species of amoebae.

Features of Rhodobacter sphaeroides CcmFH.

In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or beta-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.

An Aeromonas salmonicida type IV pilin is required for virulence in rainbow trout Oncorhynchus mykiss.

Aeromonas salmonicida expresses a large number of proven and suspected virulence factors including bacterial surface proteins, extracellular degradative enzymes, and toxins. We report the isolation and characterization of a 4-gene cluster, tapABCD, from virulent A. salmonicida A450 that encodes proteins homologous to components required for type IV pilus biogenesis. One gene, tapA, encodes a protein with high homology to type IV pilus subunit proteins from many gram-negative bacterial pathogens, including Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio vulnificus. A survey of A. salmonicida isolates from a variety of sources shows that the tapA gene is as ubiquitous in this species as it is in other members of the Aeromonads. Immunoblotting experiments demonstrate that it is expressed in vitro and is antigenically conserved among the A. salmonicida strains tested. A mutant A. salmonicida strain defective in expression of TapA was constructed by allelic exchange and found to be slightly less pathogenic for juvenile Oncorhynchus mykiss (rainbow trout) than wild type when delivered by intraperitoneal injection. In addition, fish initially challenged with a high dose of wild type were slightly more resistant to rechallenge with wild type than those initially challenged with the tapA mutant strain, suggesting that presence of TapA contributes to immunity. Two of the other three genes identified, tapB and tapC, encode proteins with homology to factors known to be required for type IV pilus assembly in P. aeruginosa, but in an as yet unidentified manner. TapB is a member of the ABC-transporter family of proteins that contain characteristic nucleotide-binding regions, and which may provide energy for type IV pilus assembly through the hydrolysis of ATP. TapC homologs are integral cytoplasmic membrane proteins that may play a role in pilus anchoring or initiation of assembly. The fourth gene, tapD, encodes a product that shares homology with a family of proteins with a known biochemical function, namely, the type IV prepilin leader peptidases. These bifunctional enzymes proteolytically cleave the leader peptide from the pilin precursor (prepilin) and then N-methylate the newly exposed N-terminal amino acid prior to assembly of the subunits into the pilus structure. We demonstrate that A. salmonicida TapD is able to restore type IV pilus assembly and type II secretion in a P. aeruginosa strain carrying a mutation in its type IV peptidase gene, suggesting that it plays the same role in A. salmonicida.

Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE.

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.

Expression of the Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease.

ureI encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of Helicobacter pylori and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we produced H. pylori ureI deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determined ureI expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastric Helicobacter species. ureI was found to be present in the genome of the gastric Helicobacter species and absent in the nongastric Helicobacter species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacter species. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacter species. The ureI gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

A multifunctional ATP-binding cassette transporter system from Vibrio cholerae transports vibriobactin and enterobactin.

Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD, viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [(3)H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations in fepB, fepD, fepG, or fepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or in viuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores.