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).     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense

Magnetosome biomineralization and magnetotaxis in magnetotactic bacteria are controlled by numerous, mostly unknown gene functions that are predominantly encoded by several operons located within the genomic magnetosome island (MAI). Genetic analysis of magnetotactic bacteria has remained difficult and requires the development of novel tools. We established a Cre-lox-based deletion method which allows the excision of large genomic fragments in Magnetospirillum gryphiswaldense. Two conjugative suicide plasmids harboring lox sites that flanked the target region were subsequently inserted into the chromosome by homologous recombination, requiring only one single-crossover event, respectively, and resulting in a double cointegrate. Excision of the targeted chromosomal segment that included the inserted plasmids and their resistance markers was induced by trans expression of Cre recombinase, which leaves behind a scar of only a single loxP site. The Cre helper plasmid was then cured from the deletant strain by relief of antibiotic selection. We have used this method for the deletion of 16.3-kb, 61-kb, and 67.3-kb fragments from the genomic MAI, either in a single round or in subsequent rounds of deletion, covering a region of approximately 87 kb that comprises the mamAB, mms6, and mamGFDC operons. As expected, all mutants were Mag⁻ and some were Mot⁻; otherwise, they showed normal growth patterns, which indicates that the deleted region is not essential for viability in the laboratory. The method will facilitate future functional analysis of magnetosome genes and also can be utilized for large-scale genome engineering in magnetotactic bacteria.Magnetosomes are unique membrane-enveloped organelles that are formed by magnetotactic bacteria (MTB) for magnetic navigation (2, 37). The mechanism of magnetosome formation is within the focus of a multidisciplinary interest and has relevance for biotechnological applications (5). It has been recognized that the biomineralization of inorganic magnetite crystals and their assembly into highly ordered magnetosome chains are under strict genetic control. Recent studies combining proteomic and bioinformatic approaches suggested that the genetic determination of magnetosome formation is complex and may potentially involve 25 to 50 gene functions (15), with unknown numbers of accessory genes and those controlling signal transduction and motility to achieve effective magnetotaxis (8, 9, 12, 26, 27, 29). However, the functional characterization of these candidate genes has been lagging behind. This is due to technical difficulties and the lack of facile tools for genetic manipulation of MTB. Allelic replacement systems have been established for Magnetospirillum magneticum (18) and Magnetospirillum gryphiswaldense (39, 40), but so far, there are only few examples of these for magnetosome genes that were functionally characterized because of the tedious and cumbersome procedures required for mutant generation (11, 19, 28, 31-32). Most genes controlling magnetosome formation in these and other MTB are located within a genomic magnetosome island (MAI) (34), which is genetically instable during stationary growth (47) and more or less conserved in other MTB (12, 13, 35). Most known magnetosome genes are organized within several conserved operons, which are interspersed with large, poorly conserved genome sections of unknown functions that have been speculated to represent genetic junk irrelevant for magnetotaxis but to cause genetic instability by their high content of repeats and transposable elements (34, 47). Thus, for large-scale functional genome analysis and rearrangements of the MAI, there is a great need for additional and more efficient genetic methods.Artificial genome recombination systems have been described for a number of bacteria. Many of them are based on the Cre-loxP system of the P1 phage (42). The Cre-loxP recombination system is a simple two-component system that is recognized as a powerful genetic tool in a multitude of eukaryotic and prokaryotic organisms (4, 6, 48). The Cre protein belongs to the integrase family of site-specific recombinases and catalyzes reciprocal site-specific recombination of DNA at 34-bp loxP sites, resulting in either excision or inversion, depending on the parallel or antiparallel orientation of the loxP sites, respectively (21). It does not require any host cofactors or accessory proteins (7). Cre-lox deletion has several advantages over other methods, such as a high efficiency and the independency of the length of DNA located between the two lox sites. The utility of Cre-lox systems has been demonstrated in a wide variety of Gram-positive and Gram-negative bacteria (17, 22-23). In several studies, it was applied for the generation of large-scale deletions, as in for example, the Gram-positive Corynebacterium glutamicum (43-46) and Bacillus subtilis (49).In M. gryphiswaldense, the functionality of a Cre-loxP antibiotic marker recycling system (25) has been previously demonstrated by deletion of a single gene based on double-crossover insertion of two loxP sites, followed by subsequent Cre-mediated excision (31). In this study, we describe a novel strategy for Cre-loxP-mediated deletion of large genomic fragments which requires only two single crossovers. The system has been validated by the generation of three large deletions, two single and one combination within the MAI, which demonstrated that the total deleted region of approximately 87 kb is not essential for viability and growth in the laboratory.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

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.     

Exopolyphosphatases PPX1 and PPX2 from Corynebacterium glutamicum

Corynebacterium glutamicum accumulates up to 300 mM of inorganic polyphosphate (PolyP) in the cytosol or in granules. The gene products of cg0488 (ppx1) and cg1115 (ppx2) were shown to be active as exopolyphosphatases (PPX), as overexpression of either gene resulted in higher exopolyphosphatase activities in crude extracts and deletion of either gene with lower activities than those of the wild-type strain. PPX1 and PPX2 from C. glutamicum share only 25% identical amino acids and belong to different protein groups, which are distinct from enterobacterial, archaeal, and yeast exopolyphosphatases. In comparison to that in the wild type, more intracellular PolyP accumulated in the Δppx1 and Δppx2 deletion mutations but less when either ppx1 or ppx2 was overexpressed. When C. glutamicum was shifted from phosphate-rich to phosphate-limiting conditions, a growth advantage of the deletion mutants and a growth disadvantage of the overexpression strains compared to the wild type were observed. Growth experiments, exopolyphosphatase activities, and intracellular PolyP concentrations revealed PPX2 as being a major exopolyphosphatase from C. glutamicum. PPX2^His was purified to homogeneity and shown to be active as a monomer. The enzyme required Mg²⁺ or Mn²⁺ cations but was inhibited by millimolar concentrations of Mg²⁺, Mn²⁺, and Ca²⁺. PPX2 from C. glutamicum was active with short-chain polyphosphates, even accepting pyrophosphate, and was inhibited by nucleoside triphosphates.Inorganic polyphosphate (PolyP), a linear polymer made of up to hundreds of orthophosphate residues (P_i), has been found in all organisms tested for its presence (3, 4, 7, 12, 20, 22, 48). In nature''s phosphorus cycle, diatom-derived PolyP has recently been shown to be critically important for marine phosphorus sequestration (6). In cells, PolyP may function as a means of storage of phosphorus and/or energy, may substitute ATP in kinase reactions, and was shown to be important in response to many stresses. Mutants of Escherichia coli, Pseudomonas aeruginosa, Shigella spp., Salmonella spp., Vibrio cholerae, and Helicobacter pylori with a low PolyP content showed defects in environmental stress responses and/or virulence (2, 14, 17, 38). In amino acid-starved E. coli, PolyP accumulates and is bound by Lon protease, which degrades ribosomal proteins to liberate amino acids (23).The presence of PolyP granules is used as a diagnostic criterion to distinguish the pathogenic Corynebacterium diphtheriae from nonpathogenic corynebacteria, such as Corynebacterium glutamicum (54). However, these metachromatic granules have recently been shown to be present also in nonpathogenic C. glutamicum (33). When sufficient phosphate is available, C. glutamicum accumulates up to 300 mM of PolyP (24) either soluble in the cytosol or in volutin granules (18, 33). During growth of C. glutamicum on glucose, intracellular PolyP concentrations peaked in the early exponential growth phase and at the entry to stationary phase (18). Soluble PolyP prevailed in the stationary growth phase, while PolyP occurred in granules in the early exponential growth phase (18). C. glutamicum is widely used for the biotechnological production of about 2,200,000 tons of amino acids per year, mainly l-glutamate and l-lysine (50, 58), while the related Corynebacterium ammoniagenes is used for the production of the flavor-enhancing purine nucleotides IMP and XMP (30). As it is conceivable that engineering corynebacterial PolyP metabolism affects overproduction of amino acids or of the phosphorus-containing compounds IMP and XMP, the study of PolyP metabolism and the enzymes involved has recently received increasing attention.PolyP formation in C. glutamicum was shown to be stimulated by MgCl₂ (33), probably due to the magnesium dependence of PolyP synthesizing enzymes (27). In microorganisms, PolyP may be synthesized by PolyP kinases belonging to three distinct families (PPK1, PPK2, and PPK3; EC 2.7.4.1) from ATP or other nucleoside triphosphates (NTPs) in a reversible reaction (12). C. glutamicum possesses two PPK2 genes (ppk2A and ppk2B) (27). Purified PPK2B of C. glutamicum is active as a homotetramer and shows higher catalytic efficiency in the PolyP-forming direction than in the reverse direction, forming NTPs from PolyP. The intracellular PolyP content was increased by overexpression of ppk2B and decreased in the absence of PPK2B (27). Besides PPK2B, no other PolyP-dependent enzyme has been characterized in C. glutamicum, although the cg2091 gene product, a putative PolyP-dependent glucokinase (EC 2.7.1.63), was found to be associated with PolyP granules (33).Degradation of PolyP by hydrolysis may be catalyzed by exopolyphosphatases (PPX) (EC 3.6.1.11) and/or endopolyphosphatases (PPN) (EC 3.6.1.10) (1, 49). Exopolyphosphatases hydrolyze PolyP from the chain''s termini, liberating P_i. The C. glutamicum genome contains two genes encoding putative exopolyphosphatases (ppx1-cg0488 and ppx2-cg1115) (15), but their functions have not yet been characterized. The corresponding proteins are distinct from each other as they share only 25% identical amino acids. Both proteins show 25% amino acid identity to E. coli PPX (1), which possesses 200 additional C-terminal amino acids (56). Here, we have analyzed PolyP degradation in C. glutamicum and show that both cg0488 (ppx1) and cg1115 (ppx2) gene products are functional exopolyphosphatases. Growth experiments, determination of exopolyphosphatase activities, and intracellular PolyP concentrations in strains lacking or overexpressing these genes revealed that cg1115 (ppx2) encodes the major exopolyphosphatase of C. glutamicum, which was characterized enzymatically.     

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.     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability

A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.The production of biofuels from nonfood cellulosic biomass would benefit the economy, the environment, and national energy security (17, 32). The largest technological and economical obstacle is the release of soluble fermentable sugars at prices competitive with those from sugarcane or corn kernels (17, 31). One of the approaches is discovering new cellulases from cellulolytic microorganisms, followed by cellulase engineering for enhanced performance on pretreated solid substrates. However, cellulase engineering remains challenging because enzymatic cellulose hydrolysis is complicated, involving heterogeneous substrates (33, 37), different action mode cellulase components (18), synergy and/or competition among cellulase components (36, 37), and declining substrate reactivity over the course of conversion (11, 26). Directed enzyme evolution, independent of knowledge of the protein structure and the enzyme-substrate interactions (6, 34), has been conducted to generate endoglucanase mutants, such as enhanced activities on soluble substrates (14, 16, 22), prolonged thermostability (20), changed optimum pH (24, 28), or improved expression levels (21). Here, we cloned and characterized a family 5 glycoside hydrolase (Cel5A) from a cellulolytic bacterium, Clostridium phytofermentans ISDg (ATCC 700394) (29, 30), and engineered it for enhanced thermostability.     

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).