Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa

Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf 4 i nhibition t oxin and Rorf0727 be named PfiA for Pf iT a ntitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5′-AATTC N₅GTTAA -3′) overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.     

Three of the seven bphC genes of Rhodococcus erythropolis TA421, isolated from a termite ecosystem, are located on an indigenous plasmid associated with biphenyl degradation.

Rhodococcus erythropolis TA421, a polychlorinated biphenyl and biphenyl degrader isolated from a termite ecosystem, has seven bphC genes expressing 2,3-dihydroxybiphenyl dioxygenase activity. R. erythropolis TA421 harbored a large and probably linear plasmid on which three (bphC2, bphC3, and bphC4) of the seven bphC genes were located. A non-biphenyl-degrading mutant, designated strain TA422, was obtained spontaneously from R. erythropolis TA421. TA422 lacked the plasmid, suggesting that the three bphC genes were involved in the degradation of biphenyl. Southern blot analyses showed that R. erythropolis TA421 and Rhodococcus globerulus P6 have a similar set of bphC genes and that the genes for biphenyl catabolism are located on plasmids of different sizes. These results indicated that the genes encoding the biphenyl catabolic pathway in Rhodococcus strains are borne on plasmids.     

trans-Dominant Interference with Human Immunodeficiency Virus Type 1 Replication and Transmission in CD4+ Cells by an Envelope Double Mutant

We previously reported that a human immunodeficiency virus type 1 (HIV-1) envelope (Env) mutant with the whole cytoplasmic domain deleted, denoted mutant TC, is able to dominantly interfere with wild-type (wt) virus infectivity. In the present study, the feasibility of developing a dominant negative mutant-based genetic anti-HIV strategy targeting the gp41 cytoplasmic domain was investigated. Mutants TC and 427,TC, a TC derivative with a Trp-to-Ser substitution introduced into residue 427 in the CD4-binding site, and a series of mutants with deletions in the cytoplasmic domain, effectively trans-dominantly interfered with wt Env-mediated viral infectivity, as demonstrated by an env trans-complementation assay. The syncytium formation-defective 427, TC double mutant not only inhibited heterologous LAV and ELI Env-mediated viral infectivity but also interfered with syncytium formation and infectivity mediated by the Env proteins of the two primary isolates 92BR and 92US. Stable HeLa-CD4-LTR-beta-gal clones that harbored Tat-controlled expression cassettes encoding the control DeltaKS, which had a deletion in the env gene, wt, or mutant env gene were generated. Viral transmission mediated by laboratory-adapted T-cell-tropic HXB2 and NL4-3 viruses was greatly reduced in the TC and 427,TC transfectants compared to that observed in the control DeltaKS and wt transfectants. Viral replication caused by HXB2 and NL4-3 viruses and by macrophage-tropic ConB and ADA-GG viruses was delayed or reduced in human CD4(+) T cells transfected with the 427,TC env construct compared to that observed in cells transfected with the control DeltaKS or TC env construct. The lack of significant interference by TC mutant was due neither to the lack of TC env gene integration into host DNA nor to the lack of TC Env expression upon Tat induction. These results indicate that this 427,TC Env double mutant has a role in the development of trans-dominant mutant-based genetic anti-HIV strategies.     

Observation of EGFP-visualized nuclei and distribution of vacuoles in Aspergillus oryzae arpA null mutant

The arpA gene encoding Arp1 (actin-related protein) was previously cloned and characterized from Aspergillus oryzae. Phenotypes of the arpA null mutant indicate its requirement for normal nuclear distribution and morphology of conidiophores. In this study, we further characterized the function of the arpA gene in distribution of organelles. For further analysis of nuclear migration in living cells, an expression system consisting of a fusion protein of Aspergillus nidulans histone H2B and EGFP (H2B::EGFP) was used. This demonstrated diminished hyphal-tip growth rate and inefficient nuclear transport to apical regions in the arpA null mutant. Expression of H2B::EGFP also revealed an increase in the nuclear number of each conidium in the arpA null mutant, implicating a role for the arpA gene in controlling the nuclear movement into conidia. Furthermore, staining of vacuoles of the arpA null mutant with CMAC (cell tracker blue) suggested that the arpA gene is required for proper vacuolar distribution in addition to its role in normal nuclear distribution.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

A type 2C protein phosphatase FgPtc3 is involved in cell wall integrity, lipid metabolism, and virulence in Fusarium graminearum

Type 2C protein phosphatases (PP2Cs) play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8) exhibited reduced aerial hyphae formation and deoxynivalenol (DON) production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.     

ORF18-disrupted mutant of Comamonas testosteroni TA441 accumulates significant amounts of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and its derivatives after incubation with steroids

In a steroid degradation gene cluster of Comamonas testosteroni TA441 consisting of ORF18, 17 and tesIHA2A1DEFG, ORF18 was implicated in encoding a CoA-transferase by database searches, but the matching substrate was not clear. In this study, ORF18 was shown to be necessary for conversion of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, a product of hydrolysis of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid in steroid degradation by TA441. The ORF18-disrupted mutant accumulates 7-hydroxy-9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 7,12-dihydroxy-9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid when incubated with chenodeoxycholic acid and cholic acid, respectively.     

Small GTP-binding protein TC10 differentially regulates two distinct populations of filamentous actin in 3T3L1 adipocytes

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (DeltaN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/DeltaVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.     

Functional characterization of the P1059L mutation in the inositol 1,4,5-trisphosphate receptor type 1 identified in a Japanese SCA15 family

Spinocerebellar ataxia type 15 (SCA15) is a group of human neurodegenerative disorders characterized by a slowly progressing pure cerebellar ataxia. The inositol 1,4,5-trisphosphate (IP₃) receptor type 1 (IP₃R1) is an intracellular IP₃-induced Ca²⁺ release channel that was recently identified as a causative gene for SCA15. In most case studies, a heterozygous deletion of the IP₃R1 gene was identified. However, one Japanese SCA15 family was found to have a Pro to Leu (P1059L) substitution in IP₃R1. To investigate the effect of the P1059L mutation, we analyzed the channel properties of the mutant human IP₃R1 by expressing it in an IP₃R-deficient B lymphocyte cell line. The P1059L mutant was a functional Ca²⁺ release channel with a twofold higher IP₃ binding affinity compared to wild-type IP₃R1. The cooperative dependence of the Ca²⁺ release activity of the mutant on IP₃ concentration was reduced, but both wild-type and mutant receptors produced similar B cell receptor-induced Ca²⁺ signals. These results demonstrate that the Ca²⁺ release properties of IP₃R1 are largely unaffected by the P1059L mutation.     

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.     

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes.     

Amino acid transport and metabolism in mycobacteria: cloning, interruption, and characterization of an L-Arginine/gamma-aminobutyric acid permease in Mycobacterium bovis BCG

Genes encoding L-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter from Mycobacterium bovis BCG and the effects of its deletion on L-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to the rocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using (14)C-labeled substrates. Greatly reduced uptake of L-arginine and gamma-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow), L-[(14)C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenous L-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. L-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.     

PssO, a unique extracellular protein important for exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii

Synthesis and secretion of polysaccharides by Gram-negative bacteria are a result of a concerted action of enzymatic and channel-forming proteins localized in different compartments of the cell. The presented work comprises functional characterization of PssO protein encoded within the previously identified, chromosomal exopolysaccharide (EPS) biosynthesis region (Pss-I) of symbiotic bacterium Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). pssO gene localization between pssN and pssP genes encoding proteins engaged in exopolysaccharide synthesis and transport, suggested its role in EPS synthesis and/or secretion. RtTA1 pssO deletion mutant and the PssO protein overproducing strains were constructed. The mutant strain was EPS-deficient, however, this mutation was not complemented. The PssO-overproducing strain was characterized by increase in EPS secretion. Subcellular fractionation, pssO-phoA/lacZ translational fusion analyses and immunolocalisation of PssO on RtTA1 cell surface by electron microscopy demonstrated that PssO is secreted to the extracellular medium and remains attached to the cell. Western blotting analysis revealed the presence of immunologically related proteins within the species R. leguminosarum bv. trifolii, bv. viciae and Rhizobium etli. The secondary structure of PssO-His(6), as determined by FTIR spectroscopy, consists of at least 32% alpha-helical and 12% beta-sheet structures. A putative function of PssO in EPS synthesis and/or transport is discussed in the context of its cellular localization and the phenotypes of the deletion mutant and pssO-overexpressing strain.     

Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2

The most frequent disorder of glycosylation is due to mutations in the gene encoding phosphomannomutase2 (PMM2-CDG). For this disease, which is autosomal and recessive, there is no cure at present. Most patients are composite heterozygous and carry one allele encoding an inactive mutant, R141H, and one encoding a hypomorphic mutant. Phosphomannomutase2 is a dimer. We reproduced composite heterozygosity in vitro by mixing R141H either with the wild type protein or the most common hypomorphic mutant F119L and compared the quaternary structure, the activity and the stability of the heterodimeric enzymes. We demonstrated that the activity of R141H/F119L heterodimers in vitro, which reproduces the protein found in patients, has the same activity of wild type/R141H, which reproduces the protein found in healthy carriers. On the other hand the stability of R141H/F119L appears to be reduced both in vitro and in vivo. These findings suggest that a therapy designed to enhance protein stability such as those based on pharmacological chaperones or modulation of proteostasis could be beneficial for PMM2-CDG patients carrying R141H/F119L genotype as well as for other genotypes where protein stability rather than specific activity is affected by mutations.     

Genetic Analysis of a Pyocin-Resistant Lipooligosaccharide (LOS) Mutant of Haemophilus ducreyi: Restoration of Full-Length LOS Restores Pyocin Sensitivity

DNA sequence and Southern blot analyses were used to determine the genetic defect of a Haemophilus ducreyi pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R chromosome containing the suspected mutation was amplified, and sequence analysis detected a 3,189-bp deletion. This deletion resulted in the loss of the entire waaQ gene, another open reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of H. influenzae, the gene encoding an argininosuccinate synthase homolog, and a change in the 3' sequence of the lgtF gene. Southern blot analysis confirmed that no genomic rearrangements had occurred. Isogenic LOS mutants and the respective complemented mutants were evaluated for susceptibility to pyocin C. The mutants expressing truncated LOS were resistant to lysis by pyocin C, and complementation restored sensitivity to the pyocin. We conclude that HD35000R is defective in both glycosyltransferase genes and that pyocin resistance is due to truncation of the full-length LOS molecule.     

Simultaneous detection of multiple mutations in epidermal growth factor receptor based on fluorescence quenching of quantum dots

We have developed a simultaneous detection method for two common mutations in the epidermal growth factor receptor gene based on the fluorescence quenching phenomenon caused by aggregation of CdSe quantum dots. For detection of the in-frame deletion in exon 19 and the L858R point mutation in exon 21, water-soluble CdSe quantum dots with two sizes were functionalized using four different types of probe oligonucleotides. Addition of target oligonucleotides with the deletion mutation in exon 19 into the suspensions caused crosslinking-induced aggregation of green-emitting quantum dots, followed by the fluorescence quenching while that with the L858R point mutation resulted in aggregation of yellow-emitting quantum dots. In addition, targets with both deletion and point mutations caused aggregation of both green- and yellow-emitting quantum dots. This method allows a simultaneous detection of mutations in exon 19 and 21 of EGFR gene in a single experiment. We found that minimum mutant concentration that could be detected by this method was as low as 2% for deletion mutation, and 5% for point mutation. PCR products of EGFR gene were also used to confirm that our method could be used to detect mutation in amplified DNA fragments.     

炭疽芽孢杆菌A16R株eag基因缺失突变株构建

【目的】构建炭疽芽孢杆菌A16R株eag基因缺失突变株, 为研究eag基因的功能奠定了基础。【方法】本研究以我国人用炭疽杆菌活疫苗A16R株中eag基因为目的缺失基因,根据炭疽芽孢杆菌Ames株基因组序列,利用软件设计了扩增上下游同源臂以及抗性基因引物,构建了重组质粒,将该重组质粒电击转入炭疽杆菌A16R感受态细胞中,利用同源重组原理筛选到炭疽杆菌A16R株eag基因缺失突变株。在分子水平及蛋白质组学方面对基因缺失突变株进行验证。【结果】成功构建了重组质粒,经同源重组后获得eag基因缺失突变株。PCR鉴定表明目的基因已经丢失;SDS PAGE表明野生株与突变株在93 KDa处有差异蛋白条带,经质谱鉴定分析该条带为目的基因所表达的EA1蛋白;双向电泳结果显示突变株与野生株比较明显缺失3个蛋白点,经质谱分析后确定这3个点都是EA1蛋白。【结论】成功获得炭疽芽孢杆菌A16R株eag基因缺失突变株,为深入研究eag基因的功能奠定了基础,同时也为炭疽芽孢杆菌重要基因功能的研究建立了一个良好的技术平台。