Diversity of the abundant pKLC102/PAGI-2 family of genomic islands in Pseudomonas aeruginosa

The known genomic islands of Pseudomonas aeruginosa clone C strains are integrated into tRNA(Lys) (pKLC102) or tRNA(Gly) (PAGI-2 and PAGI-3) genes and differ from their core genomes by distinctive tetranucleotide usage patterns. pKLC102 and the related island PAPI-1 from P. aeruginosa PA14 were spontaneously mobilized from their host chromosomes at frequencies of 10% and 0.3%, making pKLC102 the most mobile genomic island known with a copy number of 30 episomal circular pKLC102 molecules per cell. The incidence of islands of the pKLC102/PAGI-2 type was investigated in 71 unrelated P. aeruginosa strains from diverse habitats and geographic origins. pKLC102- and PAGI-2-like islands were identified in 50 and 31 strains, respectively, and 15 and 10 subtypes were differentiated by hybridization on pKLC102 and PAGI-2 macroarrays. The diversity of PAGI-2-type islands was mainly caused by one large block of strain-specific genes, whereas the diversity of pKLC102-type islands was primarily generated by subtype-specific combination of gene cassettes. Chromosomal loss of PAGI-2 could be documented in sequential P. aeruginosa isolates from individuals with cystic fibrosis. PAGI-2 was present in most tested Cupriavidus metallidurans and Cupriavidus campinensis isolates from polluted environments, demonstrating the spread of PAGI-2 across habitats and species barriers. The pKLC102/PAGI-2 family is prevalent in numerous beta- and gammaproteobacteria and is characterized by high asymmetry of the cDNA strands. This evolutionarily ancient family of genomic islands retained its oligonucleotide signature during horizontal spread within and among taxa.     

Complete Genome Sequence of the Wild-Type Commensal Escherichia coli Strain SE15, Belonging to Phylogenetic Group B2

Escherichia coli SE15 (O150:H5) is a human commensal bacterium recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B2, which includes the majority of extraintestinal pathogenic E. coli. Here, we report the finished and annotated genome sequence of this organism.The complete genome sequence of Escherichia coli SE15 was determined using a combination of 2-kb and 40-kb Sanger libraries and 454 pyrosequencing. We generated 57,600 sequences (ABI 3730xl sequencers) and three sequencing runs (GS20 sequencers). The 454 pyrosequencing reads were first assembled using the Newbler assembler software (4). A hybrid assembly of 454 and Sanger reads was performed using the Phred-Phrap-Consed program (1). Remaining gaps between contigs were closed by direct sequencing of clones. Prediction and annotation of protein-coding genes were performed as described previously (6).The genome of E. coli SE15 consists of a circular 4,717,338-bp chromosome containing 4,338 predicted protein-coding genes and a 122-kb plasmid (pSE15) encoding 150 protein-coding genes. From the multilocus sequence typing analysis based on the nucleotide sequences of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA), SE15 was found to belong to E. coli reference collection group B2. In the chromosome, two prophage regions and seven integrative elements are found. Of the predicted protein-coding genes, we could assign 2,883 (64%) to known functions, 1,528 (34%) as conserved hypothetical genes and 77 (2%) as novel hypothetical genes. Of the predicted protein-coding genes on the chromosome, 3,735 (86%) are common to three uropathogenic E. coli (UPEC) genomes (CFT073, UTI89, and 536) and 263 (6%) are not identified in any of the three UPEC genomes. The 263 genes include 7 genes for the phosphoenolpyruvate:sugar phosphotransferase system involved in the uptake of carbohydrates, reflecting the adaptation of SE15 to a commensal lifestyle in the intestinal tract. pSE15 shares 121 genes (81%) with a 114-kb plasmid (GenBank accession no. {type:entrez-nucleotide,attrs:{text:CP000244,term_id:91075696,term_text:CP000244}}CP000244) of UPEC UTI89, indicating that both plasmids are derived from the same origin.The chromosome contains six large segments (LSs; >30 kb) designated LSs I to VI, three of which overlap one prophage region and two integrative elements. Each of the six LSs is located at the same locus as at least one of the pathogenicity islands (PAIs) or other insertion regions in the three UPEC genomes. LS II (ECSF_1824 to ECSF_1835) and three PAIs (PAI IV_UTI89, PAI IV₅₃₆, and HPI_CFT073) are located at the same loci in each chromosome and share the ybt operon encoding the yersiniabactin iron acquisition system, indicating that the ancestral E. coli of group B2 strains may have acquired the ybt genes. LS III (ECSF_1852 to ECSF_1897), PAI VI_UTI89, PAI VI₅₃₆, and PAI_CFT073-asnW are located at the same loci in each chromosome. The three PAIs contain the pks island encoding multiple nonribosomal peptide synthases and polyketide synthases, whereas LS III in SE15 completely lacks the pks island. The commensal E. coli strain ED1a also lacks the pks island (8), but the commensal E. coli strain Nissle 1917 has the pks island (5). These data suggest that the presence of the pks island may not be common among intestinal commensal strains in group B2. LS V (ECSF_2770 to ECSF_2794) is almost identical to PAI V_UTI89, which contains the genes cluster for a type II secretion system (gsp), group II capsule synthesis (kps), and polysialic acid synthesis (neu). The neu operon between the kpsFEDUCS and kpsMT genes in PAI V_UTI89 is responsible for K1 capsule biosynthesis, and this region between the kpsFEDUCS and kpsMT genes is highly variable in E. coli (9). The corresponding region (ECSF_2777 to ECSF_2781) in LS V encodes genes different from those in the neu operon in PAI V_UTI89; differs from the corresponding regions of the CFT073 (K2 serotype), 536 (K15 serotype), and APEC O1 (K1 serotype) strains; and shows no homology with any sequence in public databases.SE15 lacks many virulence-related genes, whereas UPEC encodes virulence-related factors, including fimbrial adhesins, toxins, capsule, and serum resistance and iron uptake systems. The three UPEC strains have the genes encoding P fimbriae (pap), S fimbriae (sfa/foc), Auf fimbriae (auf), and type 1 fimbriae (fim), whereas SE15 contains only the fim genes and lacked the pap, sfa/foc, and auf genes. Amino acid replacements in FimH located at the tip of type 1 fimbriae produce a shift from a commensal-associated trimannose binding phenotype to a urinary tract infection-associated monomannose binding phenotype (7). The other sequenced B2 strains (three UPEC strains, APEC O1, LF82, and ED1a) have Ser-70 and Asn-78 residues in FimH, whereas SE15 has Asn-70 and Ser-78 residues that are conserved in intestinal E. coli strains. Of the seven chaperon-usher fimbrial operons in SE15, six (fim, yad, yde, yeh, yfc, and yqi) are conserved in the three UPEC genomes. The one remaining fimbrial operon (ECSF_0163 to ECSF_0166) is specific to SE15. The GC content (42%) of this 5-kb fimbrial region is lower than the average GC content (51%) of the chromosome. UPEC strains contain a greater number of iron acquisition systems than do commensal strains, which may be a consequence of their adaptation to the iron-limiting urinary tract environment (3). SE15 also contains iron uptake system genes encoding siderophore enterobactin, siderophore yersiniabactin, iron transporter (sit), and heme (chu) systems but lacks genes for siderophore salmochelin, siderophore aerobactin, and novel siderophore (ireA), which are encoded by PAIs of UPEC strains. Furthermore, SE15 lacks genes encoding alpha-hemolysin and cytotoxic necrotizing factor, which are known toxins encoded by PAIs of UPEC strains.It has been pointed out that extraintestinal pathogenic E. coli (ExPEC) virulence factors identified in commensal strains of group B2 may facilitate colonization of the human gut and thus act as fitness factors for commensal E. coli stains (2). SE15 contains fewer known ExPEC virulence-associated genes than other known commensal strains (ED1a and Nissle 1917) in group B2, suggesting that ExPEC virulence-related genes in the SE15 genome may be necessary for this commensal microorganism to colonize the human gut.     

Conjugative Transfer between Escherichia coli and Leptospira spp. as a New Genetic Tool

Our understanding of leptospiral pathogenesis, which remains poorly understood, depends on reliable genetic tools for functional analysis of genes in pathogenic strains. In this study, we report the first demonstration of conjugation between Escherichia coli and Leptospira spp. by using RP4 derivative conjugative plasmids. The DNA transfer described here was due to authentic conjugation, as shown by the requirement for cell-to-cell contact and the resistance of DNA transfers to the addition of DNase I. Transposition via conjugation of a plasmid delivering Himar1 yielded frequencies ranging from 1 × 10⁻⁶ to 8.5 × 10⁻⁸ transconjugants/recipient cell in the saprophyte L. biflexa and the pathogen L. interrogans, respectively. Analysis of mutants indicated that transposition occurs randomly, and at single sites in the genome of these strains, allowing the utilization of this system to generate libraries of transposon mutants.     

Characterization of a New CAMP Factor Carried by an Integrative and Conjugative Element in Streptococcus agalactiae and Spreading in Streptococci

Genetic exchanges between Streptococci occur frequently and contribute to their genome diversification. Most of sequenced streptococcal genomes carry multiple mobile genetic elements including Integrative and Conjugative Elements (ICEs) that play a major role in these horizontal gene transfers. In addition to genes involved in their mobility and regulation, ICEs also carry genes that can confer selective advantages to bacteria. Numerous elements have been described in S. agalactiae especially those integrated at the 3′ end of a tRNA^Lys encoding gene. In strain 515 of S. agalactiae, an invasive neonate human pathogen, the ICE (called 515_tRNA^Lys) is functional and carries different putative virulence genes including one encoding a putative new CAMP factor in addition to the one previously described. This work demonstrated the functionality of this CAMP factor (CAMP factor II) in Lactococcus lactis but also in pathogenic strains of veterinary origin. The search for co-hemolytic factors in a collection of field strains revealed their presence in S. uberis, S. dysgalactiae, but also for the first time in S. equisimilis and S. bovis. Sequencing of these genes revealed the prevalence of a species-specific factor in S. uberis strains (Uberis factor) and the presence of a CAMP factor II encoding gene in S. bovis and S. equisimilis. Furthermore, most of the CAMP factor II positive strains also carried an element integrated in the tRNA^Lys gene. This work thus describes a CAMP factor that is carried by a mobile genetic element and has spread to different streptococcal species.     

Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that ICEBs1 affects the subcellular position of the replisome.Integrative and conjugative elements (also known as conjugative transposons) and conjugative plasmids are key elements in horizontal gene transfer and are capable of mediating their own transfer from donor to recipient cells. ICEBs1 is an integrative and conjugative element found in some Bacillus subtilis strains. Where found, ICEBs1 is integrated into the leucine tRNA gene trnS-leu2 (Fig. ​(Fig.1)1) (7, 14, 21).Open in a separate windowFIG. 1.Genetic map of ICEBs1. conE (formerly yddE), regulatory genes (gray arrows), and genes required for integration, excision, and nicking (hatched arrows) are indicated. The number of transmembrane (TM) segments for each protein predicted by cPSORTdb (46) is indicated below each gene. Other topology programs yield similar but not identical predictions.ICEBs1 gene expression, excision, and potential mating are induced by activation of RecA during the SOS response following DNA damage (7). In addition, ICEBs1 is induced by increased production or activation of the ICEBs1-encoded regulatory protein RapI. Production and activity of RapI are indicative of the presence of potential mating partners that do not contain a copy of ICEBs1 (7). Under inducing conditions, the ICEBs1 repressor ImmR (6) is inactivated by proteolytic cleavage mediated by the antirepressor and protease ImmA (12). Most ICEBs1 genes then become highly expressed (7). One of these genes (xis) encodes an excisionase, which in combination with the element''s integrase causes efficient excision and formation of a double-stranded circle (7, 38). The circular form is nicked at the origin of transfer, oriT, by a DNA relaxase, the product of nicK (39). Under appropriate conditions, ICEBs1 can then be transferred by mating into B. subtilis and other species, including the pathogens Listeria monocytogenes and Bacillus anthracis (7). Once transferred to a recipient, ICEBs1 can be stably integrated into the genome at its attachment site in trnS-leu2 by the ICEBs1-encoded integrase (38).In contrast to what is known about ICEBs1 genes and proteins involved in excision, integration, and gene regulation, less is known about the components that make up gram-positive organisms'' mating machinery, defined as the conjugation proteins involved in DNA transfer (18, 24). The well-characterized mating machinery of gram-negative organisms can serve as a preliminary model (15, 16, 37, 48). Gram-negative organisms'' mating machinery is a type IV secretion system composed of at least eight conserved proteins that span the cell envelope. For example, the conjugation apparatus of the Agrobacterium tumefaciens Ti plasmid (pTi) is composed of 11 proteins (VirB1 through VirB11), including the ATPase VirB4 (16). VirB4 family members interact with several components of their cognate secretion systems and may energize machine assembly and/or substrate transfer (16, 48). The secretion substrate is targeted to the conjugation machinery by a coupling protein. Coupling proteins, such as VirD4 of pTi, interact with a protein attached to the end of the DNA substrate and couple the substrate to other components of the conjugation machinery. Coupling proteins might also energize the translocation of DNA through the machinery. Both VirB4 and VirD4 belong to the large HerA/FtsK superfamily of ATPases (29). Two other characterized members of this superfamily are the chromosome-partitioning proteins FtsK and SpoIIIE (29), which are ATP-dependent DNA pumps (reviewed in reference 2).Some of the proteins encoded by the conjugative elements of gram-positive organisms are homologous to components of the conjugation machinery from gram-negative organisms (1, 9, 14, 29), indicating that some aspects of conjugative DNA transfer may be similar in gram-positive and gram-negative organisms. For example, ConE (formerly YddE) of ICEBs1 has sequence similarities to VirB4 (29). YdcQ may be the ICEBs1-encoded coupling protein, as it is phylogenetically related to other coupling proteins (29, 44). Despite some similarities, the cell envelopes and many of the genes encoding the conjugation machinery are different between gram-positive and gram-negative organisms, indicating that there are likely to be significant structural and mechanistic differences as well.To begin to define the conjugation machinery of ICEBs1 and to understand spatial aspects of conjugation, we examined the function and subcellular location of ConE of ICEBs1. Our results indicate that ConE is likely a crucial ATPase component of the ICEBs1 conjugation machinery. We found that ConE and excised ICEBs1 DNA were located at or near the cell poles. We propose that the conjugation machinery is likely located at the cell poles and that mating might occur from a donor cell pole.     

Conjugative properties of the R factors found in 2 different strains of Escherichia coli]

The transfer frequency of R124-17, RI, RI-19 and RP4 factors as dependent on the origin of the donor strain was studied. The transfer frequencies of these factors from E. coli W strains are much lower than those from the strains of E. coli K12. The effect is connected neither with the repression of the tra-genes, nor with the restriction enzymes activity against the alien DNA in the recipient bacteria.     

Draft Genome Sequence of a Novel Bacterial Strain,LSJC7, Belonging to the Family Enterobacteriaceae with Dual Resistance to Arsenic and Tetracycline

Strain LSJC7, with dual resistance to arsenic and tetracycline, was isolated from an antimony tailing in China. Its 16S rRNA gene sequence has the highest similarity to that of Enterobacter cloacae subsp. dissolvens LMG 2683^T (97.02%). Here we present the approximately 4.6-Mbp draft genome sequence of strain LSJC7.     

Characterization of the Integration and Modular Excision of the Integrative Conjugative Element PAISt in Streptomyces turgidiscabies Car8

PAISt is a large genomic island located in the chromosome of the plant pathogen Streptomyces turgidiscabies Car8. The island carries clustered virulence genes, transfers to other Streptomyces species, and integrates by site-specific recombination at the 8 bp palindrome TTCATGAA. The palindrome is located at the 3′ end of the bacitracin resistance gene (bacA). We demonstrate that PAISt is able to excise in modules by recombination of one internal and two flanking palindromic direct repeats. The gene intSt located at the 3( end of PAISt encodes a tyrosine recombinase. Site-specific recombination activity of intSt was tested and confirmed by heterologous expression in Streptomyces coelicolor. Comparative analysis of PAISt homologues in Streptomyces scabies 87–22 and Streptomyces acidiscabies 84–104 indicates that these islands have been fixed by sequence erosion of intSt and the recombination sites.     

大肠杆菌原核增强子样序列的克隆及其结构与功能的研究

利用氯霉素乙酰转移酶(cat)以及β-半乳糖苷酶(lacZ)基因作为报告基因,从大肠杆菌MC1061株染色体基因组中克隆到3个原核增强子样序列——MC2,MC8,MC9,这3个片段均具有正反向增强活性,对β-半乳糖苷酶基因的增强活性(正向)在2～5.5倍之间。采用体内转录,RNA Dot blot杂交的方法对MC8的功能进行了研究,结果表明,MC8片段对于基因表达的调控发生在转录水平上。用核酸外切酶III末端缺失的方法对MC8的功能区进行了定位。结果显示,MC8的功能区位于距其正向克隆5′端450～950bp长约500bp的区段内。在450～600bp以及840～950bp区段内至少分别含有一个功能位点。序列分析的结果表明,MC8功能区有3个AT丰富区,其中2个分别位于450～600bp以及840～950bp区段内。     

AmiE,a Novel N-Acylhomoserine Lactone Acylase Belonging to the Amidase Family,from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24

Many Gram-negative bacteria use N-acyl-l-homoserine lactones (AHLs) as quorum-sensing signal molecules. We have reported that Acinetobacter strains isolated from activated sludge have AHL-degrading activity. In this study, we cloned the amiE gene as an AHL-degradative gene from the genomic library of Acinetobacter sp. strain Ooi24. High-performance liquid chromatography analysis revealed that AmiE functions as an AHL acylase, which hydrolyzes the amide bond of AHL. AmiE showed a high level of degrading activity against AHLs with long acyl chains but no activity against AHLs with acyl chains shorter than eight carbons. AmiE showed homology with a member of the amidases (EC 3.5.1.4) but not with any known AHL acylase enzymes. An amino acid sequence of AmiE from Ooi24 showed greater than 99% identities with uncharacterized proteins from Acinetobacter ursingii CIP 107286 and Acinetobacter sp. strain CIP 102129, but it was not found in the draft or complete genome sequences of other Acinetobacter strains. The presence of transposase-like genes around the amiE genes of these three Acinetobacter strains suggests that amiE is transferred by a putative transposon. Furthermore, the expression of AmiE in Pseudomonas aeruginosa PAO1 reduced AHL accumulation and elastase activity, which were regulated by AHL-mediated quorum sensing.     

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso''s in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.     

Draft Genome Sequence of Escherichia coli AI27, a Porcine Isolate Belonging to Phylogenetic Group B1

Escherichia coli AI27 is a putatively commensal strain isolated from feces of a pig. Here we report the draft genome sequence of E. coli AI27. This is the first porcine strain in the phylogenetic group B1 whose genome sequence has been determined.     

Replication and Active Partition of Integrative and Conjugative Elements (ICEs) of the SXT/R391 Family: The Line between ICEs and Conjugative Plasmids Is Getting Thinner

Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.     

TGF-β抑制型结合蛋白FLRG的研究进展

FLRG于1998年在白血病人B细胞中发现,与卵泡抑素(FS)高度同源,因而归属于FS家族。FLRG主要与TGF-β超家族成员结合产生负调节作用,其表达调控与多系统生理作用密切相关。此外,近来研究发现其可能与子宫内膜癌、胰岛素抵抗、代谢综合征等有重要作用。本文综述了FLRG的发现、结构特点、细胞内信号转导途径,组织分布、表达调控和生物学作用等方面的研究现状,并指出目前的研究热点集中于FLRG与TGF-β超家族成员之间以及与卵泡抑素之间的相互作用,希望为疾病的诊断和治疗提供新的途径。     

Discovery of a Eukaryotic Pyrroloquinoline Quinone-Dependent Oxidoreductase Belonging to a New Auxiliary Activity Family in the Database of Carbohydrate-Active Enzymes

Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes.     

Latheo, a New Gene Involved in Associative Learning and Memory in Drosophila Melanogaster, Identified from P Element Mutagenesis

Genetic dissection of learning and memory in Drosophila has been limited by the existence of ethyl methanesulfonate (EMS)-induced mutations in only a small number of X-linked genes. To remedy this shortcoming, we have begun a P element mutagenesis to screen for autosomal mutations that disrupt associative learning and/or memory. The generation of "P-tagged" mutant alleles will expedite molecular cloning of these new genes. Here, we describe a behavior-genetic characterization of latheoP1, a recessive, hypomorphic mutation of an essential gene. latheoP1 flies perform poorly in olfactory avoidance conditioning experiments. This performance deficit could not be attributed to abnormal olfactory acuity or shock reactivity-two task-relevant "peripheral" behaviors which are used during classical conditioning. Thus, the latheoP1 mutation appears to affect learning/memory specifically. Consistent with chromosomal in situ localization of the P element insertion, deficiencies of the 49F region of the second chromosome failed to complement the behavioral effect of the latheoP1 mutation. Further complementation analyses between latheoP1 and lethal alleles, produced by excision of the latheoP1 insert or by EMS or gamma-rays, in the 49F region mapped the latheo mutation to one vital complementation group. Flies heterozygous for latheoP1 and one of two EMS lethal alleles or one lethal excision allele also show the behavioral deficits, thereby demonstrating that the behavioral and lethal phenotypes co-map to the same locus.     

肠毒素性大肠杆菌CS3纤毛抗原在痢疾菌中的表达及其免疫效果研究

首先通过体内外重组的方法,构建了福氏2a痢疾菌T32asd基因缺陷的突变体FaD,作为抗原载体菌;同时,构建包含asd基因的表达质粒pYX102,与FaD一起,构成宿主-载体平衡致死系统,用于在没有抗生素条件选择的情况下,稳定表达克隆在表达质粒上的外源抗原基因.将肠毒素性大肠杆菌的CS3菌毛抗原基因克隆至pYX102,构建成重组表达质粒pYX103,ELISA检测结果证实CS3在痢疾菌中可以很好地表达.免疫小鼠后可诱生相应的抗体,虽然口服免疫和注射免疫产生的CS3抗体效价有一定差别,但对痢疾菌的毒株攻击均可提供较好保护.该结果为细菌性腹泻疫苗的研制提供了候选株.