Candida arabinofermentans, a new L-arabinose fermenting yeast

Candida arabinofermentans (type strain NRRL YB-2248, CBS 8468), a new yeast that ferments the pentose L-arabinose, is described. The three known strains of this new species were isolated from insect frass of pine and larch trees in the U.S. Phylogenetic analysis of nucleotide sequences from the D1/D2 domain of large subunit (26S) ribosomal DNA places C. arabinofermentans among the methanol-assimilating yeasts and most closely related to Candida ovalis. Strains of the new species produce 0.7-1.9 g/l ethanol from L-arabinose.     

Allelic Exchange in Actinomyces oris with mCherry Fluorescence Counterselection

Described here is a method for facile generation of markerless gene deletion mutants of Actinomyces oris. Homologous integration of a nonreplicative vector carrying a gene exchange cassette into the bacterial chromosome was selected for by using mCherry fluorescence and resistance to kanamycin. Completion of allelic replacement was counterselected for by using loss of fluorescence.Actinomyces oris (formerly Actinomyces naeslundii [3]) is Gram positive, facultatively anaerobic, and commonly found in the human oral cavity and plays a major role in the formation of oral biofilm or dental plaque. It is thought that adherence of A. oris to the tooth surface and its coaggregation with oral streptococci create an adhesive platform for subsequent colonization of bacteria in the plaque community (4). A. oris surface molecules such as fimbriae and pili have been shown previously to be required for the bacterial interactions with host tissues and other oral bacteria (7). However, the roles of fimbrial molecules or other surface proteins involved in these processes and their molecular assembly on the cell surface remain elusive. Lack of a facile gene disruption technology is the main reason for this obscurity.Conventional methods of genetic manipulation employing nonreplicative plasmids as delivery vectors in A. oris have been used to create gene disruption by allelic exchange, which allows insertion of a selectable marker (1, 8, 9). Often, this strategy generates polar mutations that affect downstream genes, and it is inadequate for multigene deletion because antibiotic markers for Actinomyces are scarce. To circumvent this problem, we successfully developed a method that utilizes a pUC19 derivative (namely, pHTT177) to generate a nonpolar, in-frame deletion of the sortase gene srtC2 (5). However, this system proved extremely laborious because the second homologous recombination (double-crossover) event leading to chromosomal excision and loss of the plasmid could not be efficiently selected for. Consequently, we explored fluorescence as a positive selection marker for A. oris, as described below.To generate a nonreplicative delivery vector for gene replacement with a counterselectable marker, we cloned the gene encoding the red fluorescent protein mCherry under the control of the constitutive promoter PrpsJ into pHTT177 by using EcoRI and NdeI sites (5) (Fig. ​(Fig.1).1). Initially, the mCherry sequence was amplified from plasmid pRSET-B-mCherry DNA (6) by using primers P1 (5′-GGCGGCTAGCATGGTGAGCAAGGGCGAGGAG-3′) and P2 (5′-GGCGCATATGCTACTACTTGTACAGCTCGTCCATG-3′), which contain NheI and NdeI sites (underlined), respectively. Primers P3 (5′-GGCGGAATTCCGCCCGAGCGCGGGGACCAGT-3′) and P4 (5′-GGCGGCTAGCGGCGCCTAACCTCTCTTGTACTTG-3′), containing EcoRI and NheI sites, were used to amplify the untranslated region of rpsJ from A. oris MG-1 chromosomal DNA (see gene identification no. ANA_0026 in the A. oris database at www.oralgen.lanl.gov). Both fragments were subcloned into pJRD215 at EcoRI and NdeI sites (2). The resulting vector, pCWU3, has a multiple-cloning site (MCS) containing EcoRI, SacI, KpnI, BamHI, XbaI, SalI, and HindIII sites for cloning purposes (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Construction of the nonreplicative delivery vector with red fluorescent mCherry protein as a counterselectable marker. The mCherry gene under the control of the A. oris rpsJ promoter was subcloned into the Escherichia coli/Actinomyces shuttle vector pJRD215 before being cloned into pHTT177, which is a derivative of pUC19. The resulting plasmid, pCWU3, has a kanamycin resistance cassette (kan^R) and an MCS containing EcoRI, SacI, KpnI, BamHI, XbaI, SalI, and HindIII sites.As a proof of concept, we utilized the vector pCWU3, created as described above, to generate an in-frame deletion of acaA (see gene identification no. ANA_0196 in the database at www.oralgen.lanl.gov), encoding a putative cell wall anchor protein (called Aca for actinomyces cell wall anchor). Primer sets P5/P6 (5′-GGCGGAATTCGCCGGAGGCGCCGTCGGGGAAG-3′/5′-GGCGGGTACCAGGATCTCCGTTAGACACGG-3′) and P7/P8 (5′-GGCGGGTACCCAGCGAGACTGCGACCAGCAG-3′/5′-GGCGTCTAGAGGTGGGCGTACTTCTGGTCCAT-3′) were used to amplify ∼1.0-kb sequences upstream and downstream, respectively, of acaA from A. oris MG-1 chromosomal DNA. The upstream DNA fragment was digested with EcoRI and KpnI, while the downstream fragment was digested with KpnI and XbaI (restriction enzyme sites are underlined); both fragments were ligated into pCWU3, which had been precut with EcoRI and XbaI. A. oris MG-1 was transformed with the resulting plasmid by electroporation (5), and kanamycin-resistant colonies representing integration of the plasmid into the bacterial chromosome were selected for their ability to grow on heart infusion (HI; Difco) agar plates supplemented with 50-μg ml⁻¹ kanamycin. These colonies were also examined for their fluorescence by using an Olympus XI71 inverted microscope equipped with a Hamamatsu charge-coupled device camera and a tetramethyl rhodamine isothiocyanate (TRITC) filter set (Fig. ​(Fig.22 A).Open in a separate windowFIG. 2.Analysis of a gene deletion in A. oris. (A) A. oris cells expressing mCherry under the control of the rpsJ promoter were viewed with an Olympus inverted microscope using a TRITC filter. (B and C) Selection of A. oris acaA deletion mutants was performed with a FluorChem Q imaging system (Alpha Innotech, CA). Fluorescent cells appeared green (pseudocolored) with the Cy3 setting, while nonfluorescent cells (potential mutants, indicated by the white arrow in panel C) were gray. An enlarged area (indicated by the white box in panel B) is shown in panel C. (D and E) Nonfluorescent bacteria were further examined for the absence of the acaA gene by PCR amplification (D) and Southern blot analysis (E). Bands of approximately 2.2 kb indicate acaA deletion, whereas bands of approximately 3.3 kb indicate a wild-type (WT) genotype (D). Samples from the parent strain MG-1 (WT) and size markers (M) are indicated, and the black arrow marks a 3.4-kb hybridized fragment.To select Actinomyces clones that had undergone the double-crossover event leading to chromosomal excision and loss of the plasmid, we inoculated a sample of bacteria carrying the integrated plasmid into HI broth overnight at 37°C. The bacterial culture was then serially passaged seven times with a 1:40 dilution in HI broth without antibiotics. Forty-microliter aliquots of the 10,000-fold-diluted final culture were plated onto HI agar plates. After 3 days of growth at 37°C, plates were screened for nonfluorescent colonies by using a FluorChem Q imaging system (Alpha Innotech, CA) with a Cy3 filter. The Cy3 filter was chosen because it produced brighter images than those produced by the Cy5 filter (data not shown) and the images were given with false green coloring (Fig. ​(Fig.2B).2B). Of approximately 16,000 colonies that were screened (a procedure taking less than 30 min), 11 showed no fluorescence (an example indicated by an arrow is shown in Fig. ​Fig.2C),2C), corresponding to a frequency of ∼7.0 × 10⁻⁴. This is consistent with the low frequency of homologous recombination in A. oris (5). Nonfluorescent colonies were also confirmed to be sensitive to kanamycin (data not shown), and their genomic DNA was extracted for PCR and Southern blot analyses. For PCR analysis, primers P5 and P8 were used. As shown in Fig. ​Fig.2D,2D, 8 of the 11 isolates generated amplicons of approximately 2.2 kb, which is indicative of acaA deletion, while the remaining 3 isolates generated the expected wild-type amplicons of approximately 3.3 kb. For further confirmation, DNA samples from three acaA mutants and the wild-type strain MG-1 were analyzed by Southern blotting using a 550-bp probe generated by primers 5′-AGTCTCCAACGCATCCGTCTC-3′ and 5′-GTGTCCCGAGACATTGGCCGTG-3′. Based on sequence analysis of acaA and surrounding genes, digestion by PstI will generate a 3.4-kb fragment for the wild type. As expected, the probe hybridized to the 3.4-kb DNA fragment, which was missing from the three mutant samples (Fig. ​(Fig.2E).2E). Thus, the lack of a hybridization signal for the three mutants further confirmed the absence of acaA in these mutants.In summary, we have developed a facile allelic exchange system for A. oris that reduces the laborious step of screening for a double-crossover event to less than 30 min. To our knowledge, this is the first report of an application that employs a fluorescent protein as a positive selectable marker for gene disruption in bacteria. Conceptually, this strategy can be applied for gene disruption in any system.     

Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells

Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection.     

A sodium zinc exchange mechanism is mediating extrusion of zinc in mammalian cells

Zinc influx, driven by a steep inward electrochemical gradient, plays a fundamental role in zinc signaling and in pathophysiologies linked to intracellular accumulation of toxic zinc. Yet, the cellular transport mechanisms that actively generate or maintain the transmembrane gradients are not well understood. We monitored Na+-dependent Zn2+ transport in HEK293 cells and cortical neurons, using fluorescent imaging. Treatment of the HEK293 cells with CaPO4 precipitates induced Na+-dependent Zn2+ extrusion, against a 500-fold transmembrane zinc gradient, or zinc influx upon reversal of Na+ gradient, thus indicating that Na+/Zn2+ exchange is catalyzing active Zn2+ transport. Depletion of intracellular ATP did not inhibit the Na+-dependent Zn2+ extrusion, consistent with a mechanism involving a secondary active transporter. Inhibitors of the Na+/Ca2+ exchanger failed to inhibit Na+-dependent Zn2+ efflux. In addition, zinc transport was unchanged in HEK293 cells heterologously expressing functional cardiac or neuronal Na+/Ca2+ exchangers, thus indicating that the Na+/Zn2+ exchange activity is not mediated by the Na+/Ca2+ exchanger. Sodium-dependent zinc exchange, facilitating the removal of intracellular zinc, was also monitored in neurons. To our knowledge, the Na+/Zn2+ exchanger described here is the first example of a mammalian transport mechanism capable of Na+-dependent active extrusion of zinc. Such mechanism is likely to play an important role, not only in generating the transmembrane zinc gradients, but also in protecting cells from the potentially toxic effects of permeation of this ion.     

Protein sorting to the cell wall envelope of Gram-positive bacteria

The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria.     

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.     

Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose

Five reactor systems (free cell batch, free cell continuous, entrapped cell immobilized, adsorbed cell packed bed, and cell recycle membrane reactors) were compared for ethanol production from xylose using Escherichia coli FBR5. In the free cell batch and free cell continuous reactors (continuous stirred tank reactor‐CSTR) productivities of 0.84 gL^?1 h^?1 and 1.77 gL^?1 h^?1 were achieved, respectively. A cell recycle membrane reactor resulted in the highest productivity of 55.56 gL^?1 h^?1, which is an increase of 66‐fold (e.g., 6614%) over the batch reactor. Calcium alginate gel CSTR resulted in a productivity of 2.04 gL^?1 h^?1 whereas adsorbed cell packed bed reactor resulted in a productivity of 4.39 gL^?1 h^?1. In the five reactor systems, ethanol concentrations ranged from 18.9 to 40.30 gL^?1 with metabolic yields from 0.44 to 0.51. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012