Identification and Characterization of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase from Bacillus megaterium

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.Polyhydroxyalkanoates (PHAs) are a group of polyesters that are produced by numerous bacteria as carbon and energy storage materials in response to nutritional stress (13, 27, 29). Poly(3-hydroxybutyrate) (PHB) is the most common and intensively studied PHA. Intracellular native PHB (nPHB) granules are composed of a hydrophobic PHB core and a surface layer consisting of proteins and phospholipids (13). The PHB of intracellular nPHB granules is in an amorphous state. When intracellular nPHB granules are exposed to extracellular environments due to cell death and lysis, the amorphous PHB is transformed into a denatured semicrystalline state. nPHB granules subjected to physical damage or solvent extraction to remove the surface layer can also crystallize into denatured PHB (dPHB) (13, 15). Artificial PHB (aPHB) granules, in which PHB is in an amorphous state, can be prepared from semicrystalline dPHB and detergents (1, 11, 23, 31).Various extracellular PHB depolymerases (PhaZs) that are secreted by many PHB-degrading bacteria have been demonstrated to specifically degrade dPHB (13, 14, 37). One exception is that PhaZ7, an extracellular PHB depolymerase secreted by Paucimonas lemoignei, displays unusual substrate specificity for amorphous PHB, with 3-hydroxybutyrate (3HB) oligomers as the main products of enzymatic hydrolysis (7). PhaZ7 exhibits no enzymatic activity toward dPHB. So far, a growing number of intracellular PHB depolymerases have been characterized. The intracellular PHB depolymerase PhaZa1 of Ralstonia eutropha (also called Cupriavidus necator) H16 has recently been established to be especially important for the intracellular mobilization of accumulated PHB (42). The main in vitro hydrolytic products of PhaZa1 degradation of amorphous aPHB are 3HB oligomers (31). PhaZd1, another intracellular PHB depolymerase of R. eutropha H16, shows no significant amino acid similarity to PhaZa1. The in vitro hydrolytic products of PhaZd1 degradation of amorphous aPHB are also 3HB oligomers. A 3HB monomer is rarely detected as a hydrolytic product (1). The intracellular PHB depolymerase PhaZ of Paracoccus denitrificans was reported previously to degrade protease-treated nPHB granules in vitro, with the release of 3HB dimers and oligomers as the main hydrolytic products (6). Recently, we have identified a novel intracellular PHB depolymerase from Bacillus thuringiensis serovar “israelensis” (39). The B. thuringiensis PhaZ shows no significant amino acid similarity to any known PHB depolymerase. This PhaZ has strong amorphous PHB-hydrolyzing activity and can release a considerable amount of 3HB monomers by the hydrolysis of trypsin-treated nPHB granules (39). It is of note that purified PhaZd1 from R. eutropha, PhaZ from P. denitrificans, and PhaZ from B. thuringiensis need pretreatment of nPHB granules with protease to remove surface proteins for PHB degradation (1, 6, 39). They show only very little or no activity toward nPHB granules without trypsin pretreatment. It has been demonstrated previously that these intracellular PHB depolymerases cannot hydrolyze dPHB (1, 31, 39).(R)-3HB, a biotechnologically valuable chiral compound, has been widely used for syntheses of antibiotics, vitamins, and pheromones (3, 30, 38). One way to produce (R)-3HB is heterologous coexpression of a PHB synthetic operon and a gene encoding an amorphous PHB-degrading PhaZ in Escherichia coli (3, 18, 25, 33, 38). A common problem encountered by this method is that oligomeric and dimeric forms of 3HB often constitute a major portion of the products of enzymatic hydrolysis, thus requiring further hydrolysis by 3HB oligomer hydrolase or heating under alkaline conditions to generate 3HB monomers (3, 18, 25, 33).Bacillus megaterium genes involved in the biosynthesis of nPHB granules have been cloned from strain ATCC 11561 and characterized previously (19, 21, 22). A gene encoding the extracellular PHB depolymerase PhaZ from B. megaterium was recently cloned from strain N-18-25-9 (34). However, little is known about B. megaterium genes involved in the intracellular mobilization of PHB. In this study, we have identified in B. megaterium ATCC 11561 an intracellular PHB depolymerase that could rapidly degrade nPHB granules in vitro without the need for trypsin pretreatment of the nPHB granules. Moreover, almost all the in vitro hydrolytic products released from the degradation of amorphous PHB by this PhaZ were 3HB monomers. This PhaZ could also hydrolyze dPHB with the generation of 3HB monomers. Thus, it appears to be a novel intracellular PHB depolymerase and may have promising potential for biotechnological application in the production of enantiomerically pure (R)-3HB monomers.     

Effects of Aeration on the Synthesis of Poly(3-Hydroxybutyrate) from Glycerol and Glucose in Recombinant Escherichia coli

Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose.Polyhydroxyalkanoates (PHAs), accumulated as intracellular granules by many bacteria under unfavorable conditions (5, 8), are carbon and energy reserves and also act as electron sinks, enhancing the fitness of bacteria and contributing to redox balance (9, 11, 19). PHAs have thermoplastic properties, are totally biodegradable by microorganisms present in most environments, and can be produced from different renewable carbon sources (8).Poly(3-hydroxybutyrate) (PHB) is the best known PHA, and its accumulation in recombinant Escherichia coli from several carbon sources has been studied (1, 13). In the last few years, increasing production of biodiesel has caused a sharp fall in the cost of its main by-product, glycerol (22). Its use for microbial PHA synthesis has been analyzed for natural PHA producers, such as Methylobacterium rhodesianum, Cupriavidus necator (formerly called Ralstonia eutropha) (3), several Pseudomonas strains (22), the recently described bacterium Zobellella denitrificans (7), and a Bacillus sp. (18), among others. Glycerol has also been used for PHB synthesis in recombinant E. coli (12, 15). PHAs obtained from glycerol were reported to have a significantly lower molecular weight than polymer synthesized from other substrates, such as glucose or lactose (10, 23).Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes that are involved in granule formation and/or have regulatory functions, such as phasins, granule-associated proteins that have been shown to enhance polymer synthesis and the number and size of PHA granules (17, 24). The phasin PhaP has been shown to exert a beneficial effect on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of strain K24KP, a recombinant E. coli that carries phaBAC and phaP of Azotobacter sp. FA8 (6).Because the redox state of the cells is known to affect the synthesis of PHB (1, 4, 14), the present study investigates the behavior of this recombinant strain under different aeration conditions, by using two substrates, glucose and glycerol, with different oxidation states.     

Comparison of the Complete Genome Sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes are dominant microbial phyla widely distributed in diverse ecosystems on the planet (10, 13, 20, 23, 33, 40, 51). Metagenomic analyses of the microbial landscape inhabiting various mammalian environments, notably the human gastrointestinal tract (GIT) and skin, have specifically identified Actinobacteria as an important and occasionally dominant phylum (18, 21, 33). Among the members of the large, diverse, and dynamic microbial community residing in the human GIT, Bifidobacterium is a dominant genus considered beneficial to humans and includes probiotic strains (live microorganisms which, when administered in adequate amounts, confer a health benefit on the host) (11). The population of bifidobacteria in the human intestine varies over time. Following vaginal delivery, the GIT of healthy newborns is typically colonized by bifidobacteria, especially in breast-fed infants, during the first few days of life (12). Interindividual variation, however, is remarkable in the human infant intestinal flora (41), and dominant genera are not always consistent across metagenomic analyses of the human gut flora (18, 30, 33, 41). Over time, the infant intestinal ecosystem becomes more complex as the diet becomes more diverse, with bifidobacteria typically remaining dominant until weaning (30).Bifidobacterium animalis subsp. lactis is a gram-positive lactic acid bacterium commonly found in the guts of healthy humans and has been identified in the infant gut biota, particularly in ileal, fecal, and mucosal samples (52, 56). Some strains of B. animalis subsp. lactis are able to survive in the GIT, to adhere to human epithelial cells in vitro, to modify fecal flora, to modulate the host immune response, or to prevent microbial gastroenteritis and colitis (4, 15, 20, 40, 52, 56). Additionally, B. animalis subsp. lactis has been reported to utilize nondigestible oligosaccharides, which may contribute to the organism''s ability to compete in the human gut. Carbohydrates resistant to enzymatic degradation and not absorbed in the upper intestinal tract are a primary source of energy for microbes residing in the large intestine. The benefits associated with probiotic strains of B. animalis subsp. lactis have resulted in their inclusion in the human diet via formulation into a large array of dietary supplements and foods, including dairy products such as yogurt. Deciphering the complete genome sequences of such microbes will provide additional insight into the genetic basis for survival and residence in the human gut, notably with regard to the ability to survive gastric passage and utilize available nutrients. Also, these genomes provide reference sequences for ongoing metagenomic analyses of the human environment, including the gut metagenome.Bifidobacterium animalis subsp. lactis is the most common bifidobacterium utilized as a probiotic in commercial dairy products in North America and Europe (22, 38). However, despite this commercial and probiotic significance, strain-level differentiation of B. animalis subsp. lactis strains has been hindered by the high genetic similarity of these organisms, as determined by pulsed-field gel electrophoresis and other nucleic acid-based techniques (6, 55, 56), and the lack of available genomic sequence information. The genome sequence of strain BB-12 (17) is not currently publicly available, and only a draft genome sequence in 28 contigs is available for strain HN019 (GenBank project 28807). The complete B. animalis subsp. lactis genome for strain AD011 (28) was only recently (2009) published. While this was an important first step, a single genome does not allow identification of unique targets for strain differentiation or comparative analyses within the subspecies.The objectives of this study were to determine the complete genome sequences of two B. animalis subsp. lactis strains, the type strain and a widely used commercial strain, to provide insights into the functionality of this species and into species identification and strain specialization.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Bacterial Polyester Inclusions Engineered To Display Vaccine Candidate Antigens for Use as a Novel Class of Safe and Efficient Vaccine Delivery Agents

Bioengineered bacterial polyester inclusions have the potential to be used as a vaccine delivery system. The biopolyester beads were engineered to display a fusion protein of the polyester synthase PhaC and the two key antigens involved in immune response to the infectious agent that causes tuberculosis, Mycobacterium tuberculosis, notably antigen 85A (Ag85A) and the 6-kDa early secreted antigenic target (ESAT-6) from Mycobacterium tuberculosis. Polyester beads displaying the respective fusion protein at a high density were successfully produced (henceforth called Ag85A-ESAT-6 beads) by recombinant Escherichia coli. The ability of the Ag85A-ESAT-6 beads to enhance mouse immunity to the displayed antigens was investigated. The beads were not toxic to the animals, as determined by weight gain and absence of lesions at the inoculation site in immunized animals. In vivo injection of the Ag85A-ESAT-6 beads in mice induced significant humoral and cell-mediated immune responses to both Ag85A and ESAT-6. Vaccination with Ag85A-ESAT-6 beads was efficient at stimulating immunity on their own, and this ability was enhanced by administration of the beads in an oil-in-water emulsion. In addition, vaccination with the Ag85A-ESAT-6 beads induced significantly stronger humoral and cell-mediated immune responses than vaccination with an equivalent dose of the fusion protein Ag85A-ESAT-6 alone. The immune response induced by the beads was of a mixed Th1/Th2 nature, as assessed from the induction of the cytokine gamma interferon (Th1 immune response) and increased levels of immunoglobulin G1 (Th2 immune response). Hence, engineered biopolyester beads displaying foreign antigens represent a new class of versatile, safe, and biocompatible vaccines.Bioengineered nano-/microstructures manufactured by microorganisms are becoming increasingly attractive because of their functional properties suitable for applications in various fields, particularly the medical sciences (9, 25, 29). Biopolyester beads comprising polyhydroxyalkanoate (PHA) are produced as intracellular inclusions by a wide range of bacteria and archaea when a carbon source is available in excess (30). PHA synthesis requires the key enzyme, polyester synthase, to catalyze the stereoselective polymerization of (R)-3-hydroxyacyl-coenzyme A to PHA. Self-assembly of polyester chains results in the formation of polymer granules with a hydrophobic core, and the PHA synthase protein remains covalently attached at the surface (28). These spherical granules range in size from 50 to 300 nm and accumulate in the intracellular space (34).Such biopolyester beads can be engineered to display the PHA synthase protein and its fusion partners on the surface at a high density (24). There have been recent examples where biopolyester beads were specifically engineered, produced in bacteria, and then harvested for their potential applications as life science tools. For example, biopolyester beads have been produced which display the immunoglobulin G (IgG) binding domain ZZ from protein A (6) for use as an alternative to protein A latex beads for a variety of diagnostic tests. Another study produced beads which displayed green fluorescent protein to enable tracking following in vivo administration (23). Beads have been developed with covalently attached enzymes, suggesting an application in immobilization and stabilization of biocatalysts (22). Recently, biopolyester beads have been produced which display immobilized antibody single-chain fragments as well as multiple binding functions, including the binding of inorganic compounds (4, 11, 14).Our interest in these biopolyester beads is to explore their properties for use as vaccine delivery agents. Potential advantages associated with using these beads as vaccine delivery agents include their size, versatility, and inherent biocompatibility with living tissues. Particles smaller than 2 μm in size are readily phagocytosed by macrophages and dendritic cells (20), suggesting the value of using nano-/microsized particles as vaccine delivery systems. The concept of using nano-/microparticles for delivering vaccines has already been explored; for example, biodegradable biocompatible polyesters polylactide and poly-d,l-lactide-co-glycolic acid have been used as vaccine delivery systems (31) or carriers of adjuvant systems (15). Employing PHA beads for delivery of vaccines may present additional advantages, such as low cost, ease of production, and mode of surface functionalization. Novel vaccines are required for a variety of infectious diseases, including tuberculosis, for which no truly efficacious vaccine has yet been designed (16). A number of antigens have been considered for developing new tuberculosis vaccines (3, 19, 33). Early secreted antigenic target 6-kDa protein (ESAT-6) is found in Mycobacterium bovis and Mycobacterium tuberculosis but not in the vaccine strain Mycobacterium bovis BCG (12). This antigen is recognized immunologically in tuberculosis-infected humans (27), cattle (26), and mice (5). The Ag85 complex is composed of three homologous proteins, Ag85A, Ag85B, and Ag85C (1). Ag85A has been used in a number of immunization studies and has been shown to elicit an immune response and, in some cases, enhanced protection (10, 13).This paper describes the development and microbial production of bioengineered biopolyester beads displaying on their surfaces a functional antigen comprising a fusion protein of polyester synthase, Ag85A, and ESAT-6 and subsequent evaluation of antigen-specific immune responses in immunized mice.     

Expression of Active Recombinant Human Tissue-Type Plasminogen Activator by Using In Vivo Polyhydroxybutyrate Granule Display

Recombinant human tissue plasminogen activator (rPA) is a truncated version of tissue plasminogen activator (tPA), which contains nine disulfide bonds and is prone to forming inactive inclusion bodies when expressed in bacteria. To obtain functional rPA expression, we displayed the rPA on the surface of polyhydroxybutyrate (PHB) granules using phasin as the affinity tag. rPA was fused to the N terminus of the phasin protein with a thrombin cleavage site as the linker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis showed that rPA fusion was successfully displayed on the surface of PHB granules. An activity assay indicated that the rPA fusion is active. The in vivo surface display strategy for functional rPA expression in Escherichia coli is distinct for its efficient folding and easier purification and may be expanded to the expression of other eukaryotic proteins with complex conformation.Tissue plasminogen activator (tPA) derives from a fibrinolytic system of blood vessel endothelial cells, activates plasminogen to form plasmin, and is an effective drug for thrombolytic therapy. Native tPA is composed of 527 amino acid residues with five structural domains and 17 disulfide bonds (19). Recombinant human tissue plasminogen activator (rPA) is a variant version of tPA with nine disulfide bonds, consisting of kringle 2 and serine protease domain (12). rPA was confirmed to possess enhanced capability for thrombolysis compared with that of tPA. Therefore, rPA is more beneficial for the treatment of acute myocardial infarction (17, 26, 28).Heterologous expression of tPA as well as rPA in Escherichia coli often results in the formation of the insoluble aggregates known as inclusion bodies due to the multidisulfide bonds (3). The refolding of the inclusion bodies in vitro is a long and difficult task, especially for proteins with complex conformation and multiple disulfide bonds. In order to obtain directly the functional rPA from recombinant E. coli, many approaches have been utilized: expressing the rPA gene in E. coli trxB gor ahpC* mutant strains, of which the cytoplasm is highly oxidized; fusing the rPA gene with gpIII of ΦM13 and linking to the OmpA signal sequence, through which rPA is secreted into the medium; exploiting the novel twin-arginine translocation (Tat) pathway to obtain active rPA in the periplasmic space based on its inherent properties; and cosecreting of rPA with chaperones and adding low-molecular-size medium additives to promote the formation of disulfide bonds (6, 11, 15, 25). However, the successful expression of rPA in its soluble or active form gives rise to another task: separation and purification of soluble active rPA from large amounts of other proteins in cytoplasm or medium.Normal protein purification typically involves several chromatographic steps. Each step can be costly and time-consuming (4). The development of simple and reliable methods for protein purification, which can be applied to arbitrary products, is therefore an important goal in bioseparation technology developments. One method that was recently developed is the addition of an affinity tag sequence to the target protein gene (13). It was demonstrated that heterologous proteins can be displayed actively on the surface of biopolyester granules in E. coli by fusing to the polyhydroxyalkanoate (PHA) synthase (PhaC), which serves as an affinity tag of PHA granules (21). PHA granules are carbon inclusions produced intracellularly by bacteria for coping with changing, often oligotrophic environments (1). These inclusions are composed of a hydrophobic polyester core and hydrophilic phospholipid membrane with many embedded proteins (24). Besides PhaC, phasins (namely PhaPs) are the main proteins tightly attached to the surface of polyhydroxybutyrate (PHB) granules, which can stabilize and prevent coalescence of separate PHB granules (22). Due to the inherent properties, PhaP has been used as the affinity tag in vivo to display recombinant proteins on the surface of PHB granules (5).In this study, we fused the rPA gene to the N terminus of phaP. A thrombin cleavage site was introduced between them to release rPA from PHB granules. The fusion gene was then expressed in engineered E. coli, which was conferred with the PHB production pathway by cloning the PHB biosynthesis genes. We confirmed that recombinant rPA fusion was able to be actively expressed in vivo on the surface of PHB granules.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.