Association of Missense Mutations in Epoxyalkane Coenzyme M Transferase with Adaptation of Mycobacterium sp. Strain JS623 to Growth on Vinyl Chloride

Vinyl chloride (VC) is a toxic groundwater pollutant associated with plastic manufacture and chlorinated solvent use. Aerobic bacteria that grow on VC as a carbon and energy source can evolve in the laboratory from bacteria that grow on ethene, but the genetic changes involved are unknown. We investigated VC adaptation in two variants (JS623-E and JS623-T) of the ethene-oxidizing Mycobacterium strain JS623. Missense mutations in the EtnE gene developed at two positions (W243 and R257) in cultures exposed to VC but not in cultures maintained on ethene. Epoxyalkane-coenzyme M transferase (EaCoMT) activities in cell extracts of JS623-E and JS623-T (150 and 645 nmol/min/mg protein, respectively) were higher than that of wild-type JS623 (74 nmol/min/mg protein), and in both variant cultures epoxyethane no longer accumulated during growth on ethene. The heterologous expression of two variant etnE alleles (W243G [etnE1] and R257L [etnE2]) from strain JS623 in Mycobacterium smegmatis showed that they had 42 to 59% higher activities than the wild type. Recombinant JS623 cultures containing mutant EtnE genes cloned in the vector pMV261 adapted to growth on VC more rapidly than the wild-type JS623 strain, with incubation times of 60 days (wild type), 1 day (pMVetnE1), and 35 days (pMVetnE2). The JS623(pMVetnE) culture did not adapt to VC after more than 60 days of incubation. Adaptation to VC in strain JS623 is consistently associated with two particular missense mutations in the etnE gene that lead to higher EaCoMT activity. This is the first report to pinpoint a genetic change associated with the transition from cometabolic to growth-linked VC oxidation in bacteria.Bacteria that biodegrade pollutants are useful for the cleanup of contaminated sites (i.e., bioremediation) and are interesting as models of evolutionary processes (21, 38, 40). Understanding the molecular genetic and evolutionary basis of biodegradation processes allows improved monitoring and predictions of bacterial activities in situ (39) and promises the development of improved strains and enzymes with increased specific activity (3), increased substrate affinity (16), extended substrate range (3, 16, 21, 37), extended inducer range (30, 31), or constitutive expression (39). Missense mutations in catabolic enzymes or regulatory proteins commonly lead to these changes (43), although other important mechanisms include duplication, deletion, and inversion (38-40).Vinyl chloride (VC) is a common groundwater pollutant (35) and known human carcinogen (24), and it poses a health risk to exposed populations. Although trace amounts (e.g., parts per trillion) of VC have been detected in uncontaminated soil (23), higher concentrations are found only associated with human industry, particularly the manufacture of polyvinylchloride (PVC) plastic and the chlorinated solvents trichloroethene (TCE) and perchloroethene (PCE) (4). Aerobic bacteria that grow on VC as a sole carbon and energy source are diverse, including strains of Mycobacterium (8, 17, 18), Nocardioides (8), Pseudomonas (11, 41, 42), Ochrobactrum (11), and Ralstonia (13, 33). The relative ease of the isolation of VC assimilators from chlorinated ethene-contaminated sites suggests that such bacteria are influential in the natural attenuation of VC, but this interpretation is complicated by the fact that VC-assimilating bacteria are closely related to ethene-assimilating bacteria (8-10, 29) and cannot yet be distinguished from them by molecular tests.The VC and ethene pathway and genes are homologous to some extent with the propene assimilation pathway and genes in Xanthobacter Py2 and Gordonia B-276. The comparison of the genomes of the VC-assimilating Nocardioides JS614 and the propene-assimilating Xanthobacter Py2 indicates that growth on alkenes requires about 20 kb of alkene/epoxide catabolic genes and approximately 7 kb of coenzyme M (CoM) biosynthesis genes. The oxidation of VC and ethene is initiated by an alkene monooxygenase (AkMO; EtnABCD) (8-10, 29), which yields epoxyethane from ethene and chlorooxirane from VC (8, 17). An epoxyalkane-coenzyme M transferase (EaCoMT) enzyme, EtnE, acts upon these reactive, toxic, and mutagenic epoxides (2, 19), converting them to hydroxyalkyl-CoM derivatives. The remainder of the VC/ethene pathway is unclear. The JS614 genome indicates further homology with propene oxidizers, in that a reductase/carboxylase and SDR family dehydrogenase are present, but that other aspects of the VC/ethene pathway gene cluster are unique (e.g., the presence of a semialdehyde dehydrogenase [5] and a disulfide reductase-like gene [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_008697","term_id":"119714007","term_text":"NC_008697"}}NC_008697]).The EtnE enzyme and the homologous XecA enzyme that acts on epoxypropane in Xanthobacter Py2 and Gordonia B-276 (9, 10, 12, 29) are unusual in their requirement for CoM as a cofactor. The C2- and C3-alkene oxidizers are the only Eubacteria known to biosynthesize and require CoM, which is otherwise found only in methanogenic Archaea. The XecA protein of Py2 has been purified and shown to be a Zn-dependent enzyme (1, 14, 26, 44). Based on sequence homology and the presence of the Cys-X-His-X_n-Cys motif (see Fig. S1 in the supplemental material), the EtnE enzymes also are likely to be Zn-dependent enzymes. Heterologous expression systems for XecA and EtnE have been developed (9, 25), but no crystal structures are available yet for EaCoMT from any source.Pure cultures of ethene-assimilating bacteria are capable of spontaneously adapting to growth on VC as a carbon source (22, 42), but the molecular basis of this phenomenon is not clear. This knowledge gap confounds the development of molecular probes specific for VC-assimilating bacteria. Pseudomonas aeruginosa strain DL1 shifted from cometabolism to growth on VC after more than 40 days of incubation (42), while Mycobacterium strains JS622, JS623, JS624, and JS625 took between 55 and 476 days to adapt to VC (22). The VC-adapted phenotype in Mycobacterium strains was not lost after growth in nonselective medium, suggesting a genetic change rather than a physiological adaptation (22).Here, we tested the hypothesis that mutations in the alkene/epoxide catabolic genes are responsible for VC adaptation. This was done by sequencing EtnEABCD genes in fosmid clones from cultures before and after VC adaptation, by sequencing etnE PCR products at different time points during VC adaptation, and by examining the EtnE enzyme activity in VC-adapted strains and recombinant strains carrying evolved etnE alleles.     

An Extreme Thermophile,Thermus thermophilus,Is a Polyploid Bacterium

An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid organism. As is the case for D. radiodurans in the radiation environment, the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. Polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology.The extreme thermophile Thermus thermophilus is a Gram-negative aerobic bacterium that can grow at temperatures ranging from 50°C to 82°C (33, 34). The genome sequences of two strains, HB27 and HB8, are available (13; see also http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=530). The genome of the HB27 strain consists of a chromosome (1.89 Mb) and a megaplasmid (0.23 Mb), while that of the HB8 strain includes a plasmid (9.3 kb) coupled with a chromosome (1.85 Mb) and a megaplasmid (0.26 Mb) (13; see also the NCBI website [above]). This organism has attracted attention as one of the model organisms for genetic manipulation, structural genomics, and systems biology (9, 44). In the case of the HB8 strain, the Structural and Functional Whole-Cell Project for T. thermophilus HB8, which aims to understand the mechanisms of all the biological phenomena occurring in the HB8 cell by investigating the cellular components at the atomic level on the basis of their three-dimensional (3-D) structures, is in progress (44). In addition to the stability and ease of crystallization of thermophilic proteins, natural competency and an established genetic engineering system add value to T. thermophilus HB8 as a model organism (12, 14, 23, 44). Thermostabilized resistances against antibiotics such as kanamycin (Km), hygromycin (Hm), and bleomycin (Bm), which were developed by directed evolution, have also encouraged the system (5, 6, 16, 29; Y. Koyama, unpublished data).However, we had been puzzled about several gene disruptions in T. thermophilus HB8 that resulted from replacement with the drug resistance gene. Even if drug-resistant transformants were obtained, the target gene of the transformants had not often been deleted. The target gene, probably an essential gene, seemed to coexist with the drug resistance gene. A similar phenomenon has been reported in the deletion of the recJ gene in Deinococcus radiodurans (7). Repeated observation of this phenomenon suggested that T. thermophilus HB8 might possess multiple genomic copies. Many bacteria, including the most-studied bacteria Escherichia coli and Bacillus subtilis, essentially carry a single genomic copy per cell and are genetically haploid organisms (3, 10, 42, 43). On the other hand, several bacteria have been proposed to be polyploid, harboring multiple genomic copies per cell. They include Buchnera species (21, 22), Blattabacterium species (24), Epulopiscium species (1, 4), Borrelia hermsii (20), Azotobacter vinelandii (28, 35), Neisseria gonorrhoeae (41), D. radiodurans (11, 27), a few Lactococcus lactis laboratory strains (26), and many cyanobacteria (2, 25, 37). In particular, D. radiodurans, an extremely radioresistant bacterium, has been suggested to be closely related to the genus Thermus by comparative genomic analysis (13, 32). The radioresistant bacterium carries four genome copies per cell in the stationary phase and up to 10 copies per cell during exponential growth (11, 27). In contrast with this well-known polyploidy of D. radiodurans, no report on the genomic copy number of Thermus has been done, in spite of the attention it has received as a model organism. Therefore, in this paper, the potential polyploidy and the genomic copy number were first studied in T. thermophilus HB8.     

Complete Genome Sequence of the Ethanol Producer Zymomonas mobilis NCIMB 11163

Zymomonas mobilis is an ethanol-producing alphaproteobacterium currently considered a major candidate organism for bioethanol production. Here we report the finished and annotated genome sequence of Z. mobilis subsp. mobilis strain NCIMB 11163, a British ale-infecting isolate. This is the first Z. mobilis strain whose genome, chromosomal and plasmid, is presented in its entirety.Zymomonas mobilis is a bacterium vigorously studied as a platform organism for bioethanol production in North America and other parts of the world. Z. mobilis converts sugars such as glucose or sucrose into ethanol and carbon dioxide to almost theoretical yields and to rates higher than those of yeasts (17). Genetically engineered strains that ferment pentoses in addition to naturally utilized hexoses also hold great promise for use in lignocellulosic biomass degradations (5, 22). Besides ethanol, Z. mobilis can produce other high-value chemicals such as sorbitol, levan, or phenylacetylcarbinol and has attracted interest for its unusual membrane steroid content (11). Lastly, Zymomonas is regarded as a safe organism and is even used for medicinal purposes (12, 20), which further facilitates its employment in large-scale biotechnological endeavors.The chromosomal sequence of the Z. mobilis subsp. mobilis industrial strain ATCC 31821 (ZM4) was recently published (19). Here we announce the first entire genome sequence of a Z. mobilis subsp. mobilis strain, that of the United Kingdom-originating strain NCIMB 11163 (B70) (20). Total DNA from NCIMB 11163 (16) was used for whole-genome shotgun sequencing at the U.S. DOE Joint Genome Institute. For this, an 8.7-kb DNA library and 454 and Solexa reads were used (http://www.jgi.doe.gov). Draft assemblies were based on 8,551 Sanger reads and 454 pyrosequencing to 20× coverage, whereas the Phred/Phrap/Consed software package was used for sequence assembly and quality assessment (6, 7, 9; http://www.phrap.com). After the shotgun stage, reads were assembled with parallel Phrap (High Performance Software, LLC), and misassemblies were corrected with Dupfinisher (10) or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). A total of 144 primer walk reactions, five transposon bomb libraries, 53 PCR end reads, and two PCR shatter libraries were necessary to close gaps, resolve repetitive regions, and raise the quality of the finished sequence. The completed genome sequence of NCIMB 11163 was based on 11,048 reads, with an error rate of less than 6 bp out of 100,000 bp.Open reading frame prediction and annotation were performed using Prodigal (http://compbio.ornl.gov/prodigal/) and BLAST (1); tRNAscan-SE and RNAmmer (14, 15) were used for tRNA and rRNA recognition, respectively. Functional assignment of genes was performed by searching translated open reading frames against sequences in the SPTR (TrEMBL) (2), Pfam (8), TIGRFAMs (18), COG (21), and KEGG (13) databases.Z. mobilis NCIMB 11163 contains a single, circular chromosome of 2,124,771 bp and three plasmids, p11163_1, p11163_2, and p11163_3 of 53,380 bp, 40,818 bp, and 4,551 bp, respectively. The overall GC content of the chromosome is 46.83%, whereas those of the plasmids are 42.32%, 43.80%, and 36.37%, respectively. The entire genome of NCIMB 11163 contains 1,884 protein-encoding genes and 51 tRNA and nine rRNA genes, which are chromosomally located.The chromosome of NCIMB 11163 is 68,355 bp larger than that of ZM4 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_006526","term_id":"283856168","term_text":"NC_006526"}}NC_006526) (19) and colinear at its largest part with that of ZM4 (genome structure comparisons were performed using ACT) (3). It bears several unique regions, among which are two genomic islands of ca. 25 and 79 kb, with no detectable nucleotide homology to same-species sequences and high regional similarity to chromosomal stretches of Paracoccus denitrificans PD1222 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000489.1","term_id":"119372524","term_text":"CP000489.1"}}CP000489.1), Xanthobacter autotrophicus Py2 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000781.1","term_id":"154158043","term_text":"CP000781.1"}}CP000781.1), and Gluconacetobacter diazotrophicus PAl 5 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP001189.1","term_id":"209529865","term_text":"CP001189.1"}}CP001189.1). Genome plasticity in NCIMB 11163 is further indicated by the presence of a type IV secretion system on the 79-kb island, syntenous to the Agrobacterium tumefaciens Ti (IncRh1) conjugal trb system (4), and also by multiple transposase and phage-related genes.In plasmids, housekeeping genes implicated in replication, active partitioning, and plasmid addiction are recognized, as well as genes involved in metabolism, transport, regulation, transposition, and DNA modification. Most notably, p11163_1 bears an arsenical resistance operon inserted in a type II secretion locus, whereas p11163_2, otherwise homologous to the 41-kb ZM4 plasmid (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY057845","term_id":"26983909","term_text":"AY057845"}}AY057845), harbors a unique ca. 12-kb CRISPR insertion that interrupts nucleotide colinearity with the aforementioned replicon.     

In Silico Prediction of Horizontal Gene Transfer Events in Lactobacillus bulgaricus and Streptococcus thermophilus Reveals Protocooperation in Yogurt Manufacturing

Lactobacillus bulgaricus and Streptococcus thermophilus, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both L. bulgaricus and S. thermophilus. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from S. thermophilus to L. bulgaricus, and the gene cluster cbs-cblB(cglB)-cysE for the metabolism of sulfur-containing amino acids, transferred from L. bulgaricus or Lactobacillus helveticus to S. thermophilus. The HGT event for the cbs-cblB(cglB)-cysE gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) and Streptococcus thermophilus have been used in starter cultures for yogurt manufacturing for thousands of years. Both species are known to stably coexist in a milk environment and interact beneficially. This so-called protocooperation, previously defined as biochemical mutualism, involves the exchange of metabolites and/or stimulatory factors (38). Examples of biochemical protocooperation between L. bulgaricus and S. thermophilus include the action of cell wall-bound proteases, produced by L. bulgaricus strains, and formate, required for growth of L. bulgaricus and supplied by S. thermophilus (6, 7). An overview of the interactions between the two yogurt bacteria, including the exchange of CO₂, pyruvate, folate, etc., can be found in a recently published review by Sieuwerts et al. (43). Putative genetic mechanisms underlying protocooperation, however, so far have not been studied in detail.The genomes of two L. bulgaricus strains and three S. thermophilus strains, all used in yogurt manufacturing, have been fully sequenced (3, 32, 33, 34, 39, 44, 46). The available genomic information could provide new insights into the genetic aspects of protocooperation between L. bulgaricus and S. thermophilus through the identification of putative horizontal gene transfer (HGT) events at the genome scale. HGT can be defined as the exchange of genetic material between phylogenetically unrelated organisms (23). It is considered to be a major factor in the process of environmental adaptation, for both individual species and entire microbial populations. Especially HGT events between two species existing in the same niche can reflect their interrelated activities and dependencies (13, 17). Nicolas et al. (36) predicted HGT events between Lactobacillus acidophilus and Lactobacillus johnsonii by analyzing 401 phylogenetic trees, also including the genes of L. bulgaricus. Several HGT events have been predicted in the S. thermophilus strains CNRZ1066 and LMG 18311 (3, 10, 18) as well as in L. bulgaricus ATCC 11842 (46). Moreover, a core genome of S. thermophilus and possibly acquired genes were identified by a comparative genome hybridization study of 47 strains (40).In this study, we describe an in-depth bioinformatics analysis in which we combined gene composition (GC content and dinucleotide composition) and gene transfer mechanism-associated features. Thus, we predicted horizontally transferred genes and gene clusters in the five sequenced L. bulgaricus and S. thermophilus genomes, with a focus on niche-specific genes and genes required for bacterial growth. Identification of HGT events led to a list of putative transferred genes, some of which could be important for bacterial protocooperation and the adaptation to their environment. The evolution and function of the transferred gene cluster cbs-cblB(cglB)-cysE (originally called cysM2-metB2-cysE2 in S. thermophilus), involved in the metabolism of sulfur-containing amino acids, were analyzed in detail. On the basis of our analysis, it can be concluded that both species probably agonistically coevolved at the genetic level to optimize their combined growth in a milk environment and that protocooperation thus includes both biochemical and genetic aspects.     

Discovery and Characterization of Cadherin Domains in Saccharophagus degradans 2-40

Saccharophagus degradans strain 2-40 is a prominent member of newly discovered group of marine and estuarine bacteria that recycle complex polysaccharides. The S. degradans 2-40 genome codes for 15 extraordinary long polypeptides, ranging from 274 to 1,600 kDa. Five of these contain at least 52 cadherin (CA) and cadherin-like (CADG) domains, the types of which were reported to bind calcium ions and mediate protein/protein interactions in metazoan systems. In order to evaluate adhesive features of these domains, recombinant CA doublet domains (two neighboring domains) from CabC (Sde_3323) and recombinant CADG doublet domains from CabD (Sde_0798) were examined qualitatively and quantitatively for homophilic and heterophilic interactions. In addition, CA and CADG doublet domains were tested for adhesion to the surface of S. degradans 2-40. Results showed obvious homophilic and heterophilic, calcium ion-dependent interactions between CA and CADG doublet domains. Likewise, CA and CADG doublet domains adhered to the S. degradans 2-40 surface of cells that were grown on xylan from birch wood or pectin, respectively, as a sole carbon source. This research shows for the first time that bacterial cadherin homophilic and heterophilic interactions may be similar in their nature to cadherin domains from metazoan lineages. We hypothesize that S. degradans 2-40 cadherin and cadherin-like multiple domains contribute to protein-protein interactions that may mediate cell-cell contact in the marine environment.Saccharophagus degradans strain 2-40 is the first free-living marine bacterium demonstrated to be capable of degrading cellulose of algal origin and higher plant material (29, 31). S. degradans 2-40 was isolated from decaying salt marsh cord grass, Spartina alterniflora, in the Chesapeake Bay watershed (3, 12). It is a pleomorphic, Gram-negative, aerobic, motile gammaproteobacterium, uniquely degrading at least 10 different complex polysaccharides, including agar, chitin, alginic acid, cellulose, β-glucan, laminarin, pectin, pullulan, starch, and xylan (9, 10, 15, 16, 30). These enzymatic capabilities suggest that the bacterium S. degradans may have a significant role in the marine carbon cycle, mediating the degradation of complex polysaccharides from plants, algae, and invertebrates (30, 31).The S. degradans 2-40 genome has been sequenced (http://genome.jgi-psf.org/finished_microbes/micde/micde.home.html; GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"CP000282","term_id":"89949249","term_text":"CP000282"}}CP000282 and {"type":"entrez-nucleotide","attrs":{"text":"NC_007912","term_id":"90019649","term_text":"NC_007912"}}NC_007912). It has 4,008 genes in a single replicon consisting of 5.06 Mb. The genome codes for 15 polypeptides longer than 2,000 amino acids (aa), ranging from 274 to 1,600 kDa. Each contains multiple domains and motifs that are reported to bind calcium ions and mediate protein-protein interactions (30, 31). They are acidic, pI 3.5 to 4.9, and have a secretion signal; cadherin (CA) and cadherin-like (CADG) domains are prevalent.Cadherins are a superfamily of transmembrane glycoproteins that mediate, Ca²⁺-dependent cell-cell adhesion in all solid tissues throughout the animal kingdom (25). At the molecular level, homotypic adhesion between cells arises from homophilic interactions between cadherin extracellular domains repeated in tandem (18, 19). Each of these domains, consisting of approximately 110 amino acids, forms a β-sandwich with Greek-key folding topology. The cadherin domains are mostly distributed in the metazoan lineage. According to the SMART database (accessed July 2009), there are 23,340 CA domains in 3,673 proteins, and of these 3,481 (94.7%) are metazoan proteins and only 186 (5.06%) are bacterial proteins (six other proteins with CA domains belong to Archaea).Genomic analysis showed that CA domains and CADG domains are not uncommon in bacteria (7). Focusing on the proteins Sde_3323 and Sde_0798, we tested the notion that they would interact as in metazoans, binding via calcium-dependent homophilic and heterophilic interactions. We also examined the possibility that they would directly interact with the S. degradans 2-40 cell surface, playing a role in protein-protein interactions and cellular aggregation.     

Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis

Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.The movement of water across cell membranes has long been thought to occur by free diffusion through the lipid bilayer. However, the discovery of the membrane protein CHIP28 in red blood cells has suggested the involvement of protein channels (29), and it is now well established that transmembrane water permeability is facilitated by aquaporins (AQPs), water channel proteins that are found in bacteria, fungi, plants, and animals (1, 7, 13, 24). AQPs contain six transmembrane α-helices and five connecting loops, and both the N and C termini are located in the cytosol. The monomers assemble into tetrameric complexes, with each monomer forming an individual water channel (11, 14, 24, 33). Apart from the exceptions of AQP11 and AQP12 from mice, as described by K. Ishibashi (15), AQPs have two signature Asn-Pro-Ala motifs, which are located in the second intracellular and the fifth extracellular loops, B and E.While 13 different AQPs have been identified in mammals (16), more than 33 AQP homologues have been discovered in plants (6, 17, 30). Plant AQPs fall into four subclasses: (i) the plasma membrane (PM) intrinsic proteins (PIPs), which are localized in the PM; (ii) the tonoplast intrinsic proteins (TIPs), which are localized in the vacuolar membranes; (iii) the nodulin-26-like intrinsic proteins; and (iv) the small basic intrinsic proteins (24). In Arabidopsis and maize, there are 13 PIPs, which can be divided further into two subfamilies, PIP1 and PIP2 (6, 17).The functions and mechanisms of regulation of plant AQPs have been extensively investigated (7, 13, 18, 24). There have been several reports on the water channel activity (WCA) of specific AQPs and their regulation by protein phosphorylation (3, 4, 8, 12, 18, 25, 32, 33). It has been shown that the WCA of the PIP2 member SoPIP2;1 from spinach is regulated by phosphorylation at two Ser residues (19, 33).The physiologically interesting temperature-dependent opening and closing of tulip (Tulipa gesneriana) petals occur concomitantly with water transport and are regulated by reversible phosphorylation of an undefined PIP (4, 5). Recently, four PIP homologues were isolated from tulip petals, and their WCAs have been analyzed by heterologous expression in Xenopus laevis oocytes (3). It has been shown that the tulip PIP TgPIP2;2 (DDBJ/EMBL/GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB305617","term_id":"196259707","term_text":"AB305617"}}AB305617) is ubiquitously expressed in all organs of the tulip and that TgPIP2;2 is the most likely of the TgPIP homologues to be modulated by the reversible phosphorylation that regulates transcellular water transport and mediates petal opening and closing (3, 4). However, while the members of the PIP2 subfamily are characterized as water channels (6), TgPIP2;1 (DDBJ/EMBL/GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB305616","term_id":"196259705","term_text":"AB305616"}}AB305616) shows no significant WCA in the oocyte expression system (3). There is growing interest in research on AQPs due to their crucial roles in the physiology of plants and animals (1, 16, 21-24, 26-28, 36). The assay of AQP channel activity is usually performed using either a X. laevis oocyte expression system (29) or a stopped-flow light-scattering spectrophotometer (35), both of which are not widely available. Furthermore, the complexity of these methods and requirement of expertise limit their high-throughput applications. In contrast, a Pichia pastoris expression system is simple to use, inexpensive, and feasible and can be used in high-throughput applications. Although a P. pastoris expression system has been shown to assay the WCA of a TIP (9), extensive research is necessary with other AQPs such as PIPs or AQPs present in intragranular membranes to establish whether this assay system can be used to characterize a water channel and study its regulation mechanisms. With this in view, in the study reported herein, TgPIP2;1 and TgPIP2;2 have been heterologously expressed in P. pastoris, and their WCAs have been assayed. The effects of several factors, such as osmolarity, pH, and inhibitors of protein kinases (PKs) and protein phosphatases (PPs), on the WCA of the recombinant P. pastoris have been investigated. Based on the results, we demonstrate that the P. pastoris heterologous expression system can be used to rapidly characterize PIP channels, to monitor the effects of mutations, and to score the effects of inhibitors and abiotic factors.     

Genome-Scale Model of Streptococcus thermophilus LMG18311 for Metabolic Comparison of Lactic Acid Bacteria

In this report, we describe the amino acid metabolism and amino acid dependency of the dairy bacterium Streptococcus thermophilus LMG18311 and compare them with those of two other characterized lactic acid bacteria, Lactococcus lactis and Lactobacillus plantarum. Through the construction of a genome-scale metabolic model of S. thermophilus, the metabolic differences between the three bacteria were visualized by direct projection on a metabolic map. The comparative analysis revealed the minimal amino acid auxotrophy (only histidine and methionine or cysteine) of S. thermophilus LMG18311 and the broad variety of volatiles produced from amino acids compared to the other two bacteria. It also revealed the limited number of pyruvate branches, forcing this strain to use the homofermentative metabolism for growth optimization. In addition, some industrially relevant features could be identified in S. thermophilus, such as the unique pathway for acetaldehyde (yogurt flavor) production and the absence of a complete pentose phosphate pathway.Lactic acid bacteria (LAB) are of great importance in the food industry because of their lactic acid production and their characteristic impact (e.g., texture, flavor) on the final product (19). LAB, as fastidious organisms, require a complex medium (such as milk) and are dependent on their proteolytic system for their supply of essential amino acids (34). Amino acids are not only the building blocks for proteins and peptides, but they also serve as precursors for many other biomolecules (1). Amino acids are also important for the final flavor of a product. Most amino acids do not directly influence the product flavor, but they will contribute indirectly to it because they are precursors of aromatic compounds (36). The conversion of amino acids to flavor compounds is initiated mainly by amino acid transamination, which uses an α-keto acid as an amino group acceptor for the aminotransferases (27). The presence (or absence) of the α-keto acid either by endogenous production or by addition to the medium is an important factor in flavor formation (13). The α-keto acids are decarboxylated into aldehydes, which are the precursors of other flavor compounds such as alcohols, esters, and carboxylic acids (27). A large variation in flavor formation between strains and species is observed. Different studies have reported this biodiversity (25, 27, 32, 33); van Hylckama Vlieg et al. studied, for instance, the difference between dairy and nondairy lactococcal strains, since the latter group has some unique flavor-forming activities (33).Amino acid catabolism and anabolism are complex processes, and thus, metabolic models will be helpful for their understanding. Genome-scale metabolic models provide an overview of all metabolic conversions in an organism based on its genome sequence and make it possible to visualize different metabolic pathways, such as amino acid metabolism. These models can be used to understand the metabolism and can then be applied for a directed study of functionality. For Lactobacillus plantarum and Lactococcus lactis, such genome-scale models have already been developed (18, 29); the construction of such a model for Streptococcus thermophilus LMG18311 is described in this paper. The characterization of the genome sequence of this S. thermophilus strain has revealed the presence of a large number of incomplete or truncated genes. These so-called pseudogenes amount to 10% of the total genes, and most of them relate to carbohydrate metabolism, transport, and regulation (2, 11). S. thermophilus is an important starter for the dairy industry. It is used in combination with Lactobacillus delbrueckii subsp. bulgaricus for the production of yogurt. It is also used for the manufacture of cheeses in which high cooking temperatures are applied (11). The objective of this paper is to study the metabolism of S. thermophilus with the use of genome-scale models and experimental data in a comparative way. This comparison with other LAB may reveal important differences. This study showed the simple primary metabolism and the extensive amino acid metabolism of S. thermophilus.     

The DR1 and DR6 First Exons of Human Herpesvirus 6A Are Not Required for Virus Replication in Culture and Are Deleted in Virus Stocks That Replicate Well in T-Cell Lines

Human herpesvirus 6A (HHV-6A) and HHV-6B are lymphotropic viruses which replicate in cultured activated cord blood mononuclear cells (CBMCs) and in T-cell lines. Viral genomes are composed of 143-kb unique (U) sequences flanked by ∼8- to 10-kb left and right direct repeats, DR_L and DR_R. We have recently cloned HHV-6A (U1102) into bacterial artificial chromosome (BAC) vectors, employing DNA replicative intermediates. Surprisingly, HHV-6A BACs and their parental DNAs were found to contain short ∼2.7-kb DRs. To test whether DR shortening occurred during passaging in CBMCs or in the SupT1 T-cell line, we compared packaged DNAs from various passages. Restriction enzymes, PCR, and sequencing analyses have shown the following. (i) Early (1992) viral preparations from CBMCs contained ∼8-kb DRs. (ii) Viruses currently propagated in SupT1 cells contained ∼2.7-kb DRs. (iii) The deletion spans positions 60 to 5545 in DR_L, including genes encoded by DR1 through the first exon of DR6. The pac-2-pac-1 packaging signals, the DR7 open reading frame (ORF), and the DR6 second exon were not deleted. (iv) The DR_R sequence was similarly shortened by 5.4 kb. (v) The DR1 through DR6 first exon sequences were deleted from the entire HHV-6A BACs, revealing that they were not translocated into other genome locations. (vi) When virus initially cultured in CBMCs was passaged in SupT1 cells no DR shortening occurred. (vii) Viral stocks possessing short DRs replicated efficiently, revealing the plasticity of herpesvirus genomes. We conclude that the DR deletion occurred once, producing virus with advantageous growth “conquering” the population. The DR1 gene and the first DR6 exon are not required for propagation in culture.Human herpesvirus 6 (HHV-6) is a member of the Betaherpesvirus subfamily, as recently reviewed (46). The virus can enter hematopoietic cells, including T cells, B cells, natural killer (NK) cells, monocytes, and dendritic cells (DCs), as well as nonhematopoietic cells, as reviewed in references 8, 17, and 46. In culture, the virus replicates in activated peripheral blood lymphocytes (PBLs), cord blood mononuclear cells (CBMCs), and in T-cell lines (1, 17, 46). HHV-6 isolates fall into two distinct classes designated as HHV-6A and HHV-6B variants. The two variants can be distinguished by their restriction enzyme patterns, antigenicity, DNA sequences, and disease association (1, 36, 46). HHV-6B is the causative agent of roseola infantum, a prevalent children''s disease characterized by high fever and skin rash (47). In rare cases, the virus exhibits neurotropism and has been found in children experiencing convulsions up to lethal encephalitis (1, 21, 46, 48).HHV-6B reactivation from latency was found to occur in patients receiving immunosuppressive treatment in bone marrow and other transplantations. This was associated with febrile illness, delayed transplant engraftment, and neurological involvement, up to lethal encephalitis (5, 13, 34, 46). HHV-6A has thus far no clear disease association, although several studies have suggested central nervous system (CNS) tropism, including aggravation of symptoms in patients with multiple sclerosis (MS) (6, 14, 33, 41).HHV-6A and HHV-6B share general genomic architecture. The unit-length DNA molecules are approximately 160 kb, composed of a 143-kb unique (U) segment flanked by left and right direct repeats (DR_L and DR_R, respectively) (19, 24, 27, 46). The DRs are of sizes 8 to 10 kb in different viral isolates (2, 19, 24, 46). In both the HHV-6A and HHV-6B genomes, the herpesvirus conserved cleavage/packaging signals pac-1 and pac-2 (9, 15, 17) are located at the left and the right termini of the DRs (17, 19, 46). The PubMed sequence for the U1102 strain (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_001664","term_id":"1344462938","term_text":"NC_001664"}}NC_001664) starts with the pac-1 signal at positions 1 to 56, followed by multiple copies of perfect and imperfect telomere-like sequences, up to position 418. It was suggested that the telomeric repeats may have originated from host cell chromosomal telomeres (43). Additionally, the DR encodes several open reading frames (ORFs), four of which are dealt with in our paper: (i) the spliced DR1 at positions 501 to 759 and 843 to 2653; (ii) DR5 at positions 3738 to 4164; (iii) the spliced DR6 at positions 4725 to 5028 and 5837 to 6720; and (iv) an ORF of DR7, at positions 5629 to 6720, partially overlapping the DR6 gene (20). Hollsberg and coworkers (37) have recently found that the homologous gene in HHV-6B encodes a nuclear protein that forms a complex with viral DNA processivity factor p41. Gompels and coworkers have also shown that DR1 and DR6 are partly homologous to the human cytomegalovirus (HCMV) US22 gene family. Both have a CXC motif: DR1 with homology to the HCMV US26 gene and DR6 with homology to the HCMV US22 gene (20). The map continues with reiterated perfect hexanucleotide telomeric sequences (GGGTAA)_n at positions 7655 to 8008 (19, 43). The number of telomeric repeats was found to vary in different viral strains (2, 43). The DR terminates with the pac-2 signal.We have recently cloned the intact HHV-6A genome into bacterial artificial chromosomes (BACs), by direct cloning of unit-length DNA produced from circular or head-to-tail replication intermediates into modified BAC vectors containing the green fluorescent protein (GFP) marker and ampicillin-puromycin (Amp-Puro) selection cassette (3). Surprisingly, the HHV-6A BAC clones as well as the parental HHV-6A (U1102) propagated in our laboratory in SupT1 cells were found to contain DRs of ∼2.7 kb instead of the expected ∼8- to 10-kb DRs, as in the early publications (19, 24, 27, 46) and in the PubMed sequence. This has raised the following questions. When did the deleted DRs arise? What was the detailed structure of deleted DRs?HHV-6 was discovered by Gallo and colleagues in 1986 (35), and viral isolates were obtained in multiple laboratories from AIDS patients, patients with lymphoproliferative disorders, and patients with roseola infantum (12, 26, 42, 45, 47). The isolates were propagated initially in activated PBLs and CBMCs and then in continuous T-cell lines, including HSB-2, J-JHAN, SupT1, Molt-3, and MT-4 (11, 45). The U1102 strain isolated by Downing and colleagues (12) was contributed to our laboratory by Robert Honess and was propagated first in activated PBLs and CBMCs (11, 18, 36, 45) and then in J-JHAN and SupT1 T cells (4, 30). To answer the question with regard to the origin of the short DRs and their structure, we have compared earlier viral HHV-6A passages with the currently propagated virus and the HHV-6A BAC clones. We describe here the detailed structure of the DR_L and DR_R of the “new” virus, containing the short ∼2.7-kb DR. We show that the deletion contained the left multiple repeats of telomere-like sequences and the ORFs from DR1 up to the DR6 first exon. Review of viral passaging since 1992 indicated that the deletion occurred spontaneously. The deleted viruses were stably and efficiently propagated in SupT1 T cells, indicating that the DR1 and DR6 first exons are not essential for virus in vitro replication.     

VP23R of Infectious Spleen and Kidney Necrosis Virus Mediates Formation of Virus-Mock Basement Membrane To Provide Attaching Sites for Lymphatic Endothelial Cells

Putative open reading frames (ORFs) encoding laminin-like proteins are found in all members of the genus Megalocytivirus, family Iridoviridae. This is the first study that identified the VP23R protein encoded by ORF23R of the infectious spleen and kidney necrosis virus (ISKNV), a member of these genes of megalocytiviruses. The VP23R mRNA covering the ISKNV genomic coordinates 19547 to 22273 was transcribed ahead of the major capsid protein. Immunofluorescence analysis demonstrated that VP23R was expressed on the plasma membrane of the ISKNV-infected cells and could not be a viral envelope protein. Residues 292 to 576 of VP23R are homologous to the laminin γ1III2-6 fragment, which covers the nidogen-binding site. An immunoprecipitation assay showed that VP23R could interact with nidogen-1, and immunohistochemistry showed that nidogen-1 was localized on the outer membrane of the infected cells. Electron microscopy showed that a virus-mock basement membrane (VMBM) was formed on the surface of the infected cells and a layer of endothelial cells (ECs) was attached to the VMBM. The VMBM contained VP23R and nidogen-1 but not collagen IV. The attached ECs were identified as lymphatic endothelial cells (LECs), which have unique feature of overlapping intercellular junctions and can be stained by immunohistochemistry using an antibody against a specific lymphatic marker, Prox-1. Such infection signs have never been described in viruses. Elucidating the functions of LECs attached to the surface of the infected cells may be useful for studies on the pathogenic mechanisms of megalocytiviruses and may also be important for studies on lymphangiogenesis and basement membrane functions.Basement membrane (BM), a dense and sheetlike structure that is always associated with cells, is a very important specialized form of extracellular matrix (31, 67). BMs mediate tissue compartmentalization and provide structural support to the epithelium, endothelium, peripheral nerve axons, fat cells, and muscle cells, as well as structural and functional foundations of the vasculature (25, 31, 52). BM is also an important regulator of cell behaviors, such as adhesion, migration, proliferation, and differentiation. BMs are highly cross-linked and insoluble materials. They are highly complex and are made up of more than 50 known components (31, 54). Although the molecular composition of BMs is unique in each tissue, their basic structures are similar. Even if many more isoforms exist in different species, the major BM proteins and their receptors are conserved from Caenorhabditis elegans to mammals. BM consists of a layer of laminin polymer, a layer of type IV collagen network, and the nidogen protein, which acts as a cross-linker of these two networks. Other BM components, such as perlecan and fibulin, interact with the laminin polymer and the type IV collagen network to organize a functional BM on the basolateral aspect of the cells (31, 45, 52).The components of BM are able to self-assemble and form a sheetlike structure, and laminin is the key molecule in this process (50). Laminin protein consists of three different chains (α, β, and γ), which comprise a cross-shaped molecular structure with three short amino-terminal arms and a long carboxyl-terminal triple-helical arm (58, 68). The three short arms of this cross-shaped structure can interact with each other in the presence of calcium. Through the binding of globular G domain at the carboxyl-terminal end of the α chain to the cell receptors (e.g., integrins and dystroglycans), laminin self-assembles into polygonal lattices on cell surfaces. This process initiates BM self-assembly (15, 21, 25, 38, 65, 66). To date, 17 laminin isoforms have been observed in different tissues (51). Among them, laminin-1, the crux of early embryonic BM assembly, has been well studied. Laminin-1 consists of α1, β1, and γ1 chains and can interact with nidogen-1 with high affinity through a laminin-type epidermal growth factor-like (LE) module, γ1III4, within the domain III of the γ1 chain (1, 42). The heptapeptide “NIDPNAV” within the γ1III4 motif of laminin-1 is essential for the interaction between laminin-1 and nidogen-1 (41, 46). Blocking the interactions between laminin-1 and nidogen-1 leads to the disruption of BMs. This indicates that the formation of laminin/nidogen complex is essential for BM assembly and stability (30, 61). Nidogen-1, also called entactin-1, is a dumbbell-shaped sulfated 150-kDa glycoprotein consisted of three domains (G1, G2, and G3) (12). By interacting with collagen IV through its G2 domain and binding with laminin γ1 chain through its G3 domain, nidogen-1 bridges the layers of the laminin network and the collagen IV network to construct the fundamental structure of BMs (48). Collagen IV is a triple-helical trimer composed of three α chains. Through the hexamer formation of the carboxyl-terminal globular non-collagenous-1 (NC1) domain of each α chain, two collagen IV proteins assemble into a dimer. Dimers of collagen IV connect with each other via their amino-terminal 7S domains and self-assemble into a network (24, 27, 31, 32). Six kinds of α chains of collagen IV have been identified in mammals. Among them, α1 and α2 chains are the most abundant forms of collagen IV found in all BMs (19, 23). They commonly form a collagen IV molecule with a α1 and α2 ratio of 2:1 (31, 35).Iridoviruses infect invertebrates and poikilothermic vertebrates, including insects, fish, amphibians, and reptiles. These viruses are a group of icosahedral cytoplasmic DNA viruses with circularly permuted and terminally redundant DNA genomes (6, 8, 9, 10, 57, 62). The family Iridoviridae has been subdivided into five genera: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystisvirus, and Megalocystivirus (7). The genus Megalocystivirus, characterized by the ability to cause swelling of the infected cells, is one group of the most harmful viruses to cultured fish (7, 26, 29). Infectious spleen and kidney necrosis virus (ISKNV), the causative agent of a disease that causes high mortality rates in farmed mandarin fish, Siniperca chuatsi, and large-mouth bass, Micropterus salmoides, is regarded as the type species of Megalocystivirus (7). Similar to infection caused by other members of the Megalocystivirus, fish ISKNV infection is characterized by cell hypertrophy in the spleen, kidney, cranial connective tissue, and endocardium (16, 17). Aside from mandarin fish and large-mouth bass, ISKNV-like virus can also be detected in the tissues of more than 60 marine and freshwater fishes (14, 28, 59, 64). The entire genome of ISKNV has been sequenced, and the organization of open reading frames (ORFs) of ISKNV was analyzed by using DNASTAR Omiga 2.0 and Genescan (18). The ISKNV genome is about 110 kbp and contains 125 putative ORFs (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF371960","term_id":"19773610","term_text":"AF371960"}}AF371960).Putative ORFs, encoding viral proteins containing a fragment homologous to laminin and a putative transmembrane fragment, were found in all of the sequenced genomes of the members of Megalocystivirus. These ORFs include ORF23R of ISKNV (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAL98747","term_id":"19773633","term_text":"AAL98747"}}AAL98747), laminin-like protein gene of olive flounder iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAT76907","term_id":"50404491","term_text":"AAT76907"}}AAT76907), ORF2 of sea perch iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAV51313","term_id":"55418187","term_text":"AAV51313"}}AAV51313), predicted laminin-type epidermal growth factor-like protein of large yellow croaker iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ABI32391","term_id":"113200502","term_text":"ABI32391"}}ABI32391), an unknown gene of red sea bream iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAQ07956","term_id":"33324323","term_text":"AAQ07956"}}AAQ07956), ORF2 of rock bream iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAN86692","term_id":"60458808","term_text":"AAN86692"}}AAN86692), and laminin-type epidermal growth factor-like protein of orange-spotted grouper iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAX82335","term_id":"62421215","term_text":"AAX82335"}}AAX82335). These putative proteins are highly homologous to each other in amino acid sequence (65 to 99% identity). However, the functions of these proteins have never been identified. This is the first study to identify that the VP23R protein encoded by ORF23R of ISKNV is a plasma membrane-localized viral protein. In addition, we discovered a new function of VP23R in a unique pathological phenomenon of virus infection: the attachment of lymphatic endothelial cells (LECs) to the infected cells. Nidogen-1 assisted VP23R in the construction of a BM-like structure, providing an attachment site for LECs. This unique pathological phenomenon has never been found in viruses and is an attractive direction for studies of pathogenic mechanisms of megalocystiviruses. Moreover, studies on the unique profiles of the virus-mock BM can help us learn more about the functions of BM components and the mechanisms of lymphangiogenesis.     

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).     

Draft Genome Sequences of Two Neisseria meningitidis Serogroup C Clinical Isolates

Neisseria meningitidis is a human-specific pathogen known for its capability to cause sepsis and meningitis. Here we report the availability of 2 draft genome sequences obtained from patients infected during the same epidemic outbreak. Both bacterial isolates belong to serogroup C, but their genome sequences show local and remarkable differences compared with each other or with the reference genome of strain FAM18.Neisseria meningitidis is found as a commensal organism of the human nasopharynx in 8 to 25% of the adult population (9), but sporadically, it is able to cross the mucosa and reach the bloodstream, causing severe septicemia and meningitis. Even though the reasons triggering these pathogenic outbreaks are not well understood, several factors related either to the host or the bacterium have been proposed 3, 8).So far, complete genome sequences for N. meningitidis serogroups A (strain Z2491 [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AL157959","term_id":"30407145","term_text":"AL157959"}}AL157959]) (4), B (strain MC58 [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE002098","term_id":"66731897","term_text":"AE002098"}}AE002098]) (10), and C (strains FAM18, 8013, and 053442 [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM421808","term_id":"120865607","term_text":"AM421808"}}AM421808, {"type":"entrez-nucleotide","attrs":{"text":"FM999788","term_id":"261391559","term_text":"FM999788"}}FM999788, and {"type":"entrez-nucleotide","attrs":{"text":"CP000381","term_id":"161594571","term_text":"CP000381"}}CP000381, respectively]) (1, 5, 6) have been reported, together with the unencapsulated strain α14 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM889136","term_id":"254667570","term_text":"AM889136"}}AM889136) (7). Here we announce the availability of 2 draft genome sequences for N. meningitidis serogroup C, strains K1207 and S0108, isolated from the same epidemic cluster which occurred in the Veneto region in northern Italy during the 2007-2008 winter (2).The genomes were sequenced using 454 pyrosequencing (Roche), combining shotgun and 30-kb paired-end strategies, according to the manufacturer''s recommendations. The coverage was nearly 27×, and assemblies were performed with Newbler. We obtained 223 and 226 contigs for the 2 genomes, which were finally mapped in 17 and 16 scaffolds, respectively. From both samples, we also isolated a 7-kb plasmid, whose sequence was nearly identical to that of pJS-B, already available in GenBank (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_004758","term_id":"30250494","term_text":"NC_004758"}}NC_004758).The first analysis was performed by comparing sequences of the two isolates with the most similar complete genome available, strain FAM18. This analysis showed that the genome lengths were almost identical (about 2.2 Mb) and GC contents were comparable (51.91% in both isolates versus 51.62% of strain FAM18). Then, to identify potential differences in coding sequence content, the contigs obtained for both isolates were aligned with those for strain FAM18 using MEGABLAST (11) and LASTZ tools, which showed that in the genomes of the two N. meningitidis isolates, several genes were missing or nonfunctional because of the presence of insertions or deletions. For example, a couple of FAM18 outer membrane proteins (NMC0214 and NMC0215) were completely missing in both genomes, due to a 3-kb deletion, and no homologues were present in other genomic regions.Sequences that did not map on the genome of strain FAM18 were investigated by performing a BLAST analysis on a nonredundant database. Interestingly, besides genes or partial genes belonging to the other completely sequenced N. meningitidis serogroup C strain 053442, the genomes of our isolates contained coding sequences from N. meningitidis serogroups A and B, from other Neisseria species, such as N. gonorrhoeae, N. cinerea, and N. mucosa, and even from other bacterial species, such as cobyrinic acid ac-diamide synthase from Shewanella baltica, attesting once more to the great capability of horizontal gene transfer, which is peculiar to this microorganism.A detailed report of our two isolates will be included in a future publication, with the results of a full comparative analysis between the genomes.     

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.     

Agrobacterium tumefaciens Type IV Secretion Protein VirB3 Is an Inner Membrane Protein and Requires VirB4, VirB7, and VirB8 for Stabilization

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.Bacteria use type IV secretion (T4S) to deliver macromolecules to prokaryotes and eukaryotes (12). Animal and human pathogens deliver proteins to their eukaryotic hosts to affect cellular processes causing disease. The plant-pathogenic bacterium Agrobacterium tumefaciens delivers both proteins and DNA to plants and other eukaryotes. DNA delivered by Agrobacterium directs constitutive synthesis of phytohormones in a transformed plant cell, promoting cancerous growth (56). The Ptl toxin of Bordetella pertussis modifies G proteins by ADP-ribosylation, affecting intracellular cell signaling, and CagA of Helicobacter pylori disrupts epithelial cell polarity by inhibiting PAR1 kinase activity (37, 44, 47). T4S is ancestrally related to bacterial conjugation, a mechanism used by bacteria for interbacterial plasmid transfer, enabling them to acquire novel genes for antibiotic resistance, degradation of organic molecules, toxin production, and other virulence traits (29).The VirD4/VirB family of proteins, found conserved in many alphaproteobacteria, mediates T4S (12). The Ti plasmid-encoded Agrobacterium T4S system requires VirD4 and 11 VirB proteins, VirB1 to VirB11, for efficient DNA transfer (7, 54). The membrane and membrane-associated VirB proteins assemble a macromolecular structure at the cell membrane to promote substrate transfer (12). The octopine Ti plasmid pTiA6NC-encoded VirB6 to VirB11 proteins assemble the T4S apparatus at a cell pole (34, 35, 39). The VirD4 coupling protein targets the VirE2 substrate protein to the cell pole (4). A recent study found that the nopaline Ti plasmid pTiC58 T4S system (T4SS) and its substrates form a helical array around the cell circumference (1). Structural studies using Escherichia coli conjugative plasmid pKM101-encoded VirB homologues showed that TraN (VirB7), TraO (VirB9), and TraF (VirB10) form the core complex and that TraF forms a channel at the outer membrane (11, 23). The Agrobacterium VirB proteins assemble a T-pilus, an appendage composed primarily of VirB2, with VirB5 and VirB7 as its minor constituents (38, 40, 41, 48, 50, 55). VirB3, a homolog of the pilin-like TraL protein encoded in E. coli plasmids, is postulated to function in T-pilus assembly (52). Three ATP-utilizing proteins, VirB4, VirB11, and VirD4, supply energy for substrate translocation (3, 9, 34).The membrane topology of all the VirB proteins, except for VirB3, was determined by analyses of random phoA insertion mutants, targeted phoA fusions, and targeted bla fusions (6, 14, 15, 21, 22, 31, 35, 53). phoA and bla, which encode alkaline phosphatase and β-lactamase, respectively, serve as excellent markers for periplasmic proteins, as they are enzymatically active only when targeted to the cell periplasm (8, 30). Green fluorescent protein (GFP) is an ideal cytoplasmic marker because it fluoresces only when located in the cytoplasm (19, 20). When GFP is targeted to the periplasm through fusion with a membrane-spanning domain (MSD), it fails to fold properly and does not fluoresce.The prevailing view, based on in silico analysis, is that VirB3 is a bitopic membrane protein with a periplasmic C terminus. No phoA-positive insertions in virB3, however, were identified in two random mutagenesis studies of the virB operon (6, 15). The small size of VirB3, a polypeptide of 108 amino acids (aa), could be a contributing factor to the negative findings. Yet several PhoA-positive insertions in two smaller VirB proteins, VirB2 (74-aa mature peptide) and VirB7 (41-aa mature peptide), were successfully obtained in both studies. Therefore, the negative findings may also be indicative of the presence of a small periplasmic domain in VirB3. Biochemical studies showed that the nopaline Ti plasmid pTiC58-encoded VirB3 protein (pTiC58 VirB3) associates with the bacterial outer membrane, while VirB2 associates with both the inner and outer membranes (52). The pTiC58 VirB4 protein is required for localization of VirB3 to the outer membrane (33). VirB4 is also required for VirB3 stability (33, 55). A low level of VirB3 accumulated in a nonpolar pTiC58 virB6 deletion mutant; however, addition of virB6 in trans did not restore the level of the protein, even though it restored tumorigenicity (27). VirB3 participates in the formation of protein complexes with the T-pilus proteins VirB2 and VirB5 (55).Homologues of VirB3 are found in many alphaproteobacteria with a T4SS. While most VirB3 homologues are small proteins, several recently identified homologues are fusions of VirB3 and the immediate downstream protein VirB4 (5, 10, 24). These fusion homologs, which include Actinobacillus MagB03 (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAG24434","term_id":"10880920","term_text":"AAG24434"}}AAG24434), Campylobacter CmgB3/4 ({"type":"entrez-protein","attrs":{"text":"EAQ71805","term_id":"87248841","term_text":"EAQ71805"}}EAQ71805), Yersinia pseudotuberculosis TriC ({"type":"entrez-protein","attrs":{"text":"CAF25448","term_id":"51591645","term_text":"CAF25448"}}CAF25448), Citrobacter koseri PilX3-4 ({"type":"entrez-protein","attrs":{"text":"ABV12046","term_id":"157082368","term_text":"ABV12046"}}ABV12046), and Klebsiella pneumoniae PilX3-4 ({"type":"entrez-protein","attrs":{"text":"BAF49490","term_id":"134048868","term_text":"BAF49490"}}BAF49490), have VirB3 at the N terminus and VirB4 at the C terminus. Agrobacterium VirB4 is an integral membrane protein with a cytoplasmic N terminus (14). Its homologues are expected to have a similar topology. The prevailing view that pTi VirB3 has a periplasmic C terminus is inconsistent with the cytoplasmic location of the N terminus of VirB4 in the VirB3-VirB4 fusion protein homologues.In this study, we report the membrane topology of Agrobacterium VirB3 and demonstrate that the C terminus of the protein resides in the cytoplasm. We also demonstrate that VirB3 is an inner membrane protein, not an outer membrane protein as previously reported (52). The octopine Ti plasmid pTiA6NC VirB4 protein does not affect membrane localization of VirB3 but does stabilize VirB3. VirB4, however, is not sufficient for pTiA6NC VirB3 stabilization. Two additional proteins, VirB7 and VirB8, are required for the stabilization of pTiA6NC VirB3.     

Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.The application of ammonium-based fertilizers has been shown to immediately reduce the uptake of methane in a number of diverse ecological systems (3, 5, 7, 8, 11-13, 16, 27, 28), due likely to competitive inhibition of methane monooxygenase enzymes by ammonia and production of nitrite (1). Longer-term inhibition of methane uptake by ammonium has been attributed to changes in methanotrophic community composition, often favoring activity and/or growth of type I Gammaproteobacteria methanotrophs (i.e., Gammaproteobacteria methane-oxidizing bacteria [gamma-MOB]) over type II Alphaproteobacteria methanotrophs (alpha-MOB) (19-23, 25, 26, 30). It has been argued previously that gamma-MOB likely thrive in the presence of high N loads because they rapidly assimilate N and synthesize ribosomes whereas alpha-MOB thrive best under conditions of N limitation and low oxygen levels (10, 21, 23).Findings from studies with rice paddies indicate that N fertilization stimulates methane oxidation through ammonium acting as a nutrient, not as an inhibitor (2). Therefore, the actual effect of ammonium on growth and activity of methanotrophs depends largely on how much ammonia-N is used for assimilation versus cometabolism. Many methanotrophs can also oxidize ammonia into nitrite via hydroxylamine (24, 29). Nitrite was shown previously to inhibit methane consumption by cultivated methanotrophs and by organisms in soils through an uncharacterized mechanism (9, 17, 24), although nitrite inhibits purified formate dehydrogenase from Methylosinus trichosporium OB3b (15). Together, the data from these studies show that ammonium and nitrite have significant effects on methanotroph activity and community composition and reveal the complexity of ammonia as both a nutrient and a competitive inhibitor. The present study demonstrates the differential influences of high ammonium or nitrite loads on the competitive fitness of a gamma-MOB versus an alpha-MOB strain.