Herpes simplex virus type 1 virion‐derived US11 inhibits type 1 interferon‐induced protein kinase R phosphorylation

The herpes simplex virus type 1 (HSV‐1) VRTK^? strain that was previously isolated in our laboratory as an acyclovir‐resistant thymidine kinase (TK)‐deficient mutant, is more sensitive to type 1 interferon than is the parent strain VR3. The properties of this mutant were investigated to clarify the mechanism for its hyper‐sensitivity to interferon (IFN). It was found that: (i) IFN‐pretreated cells, but not those treated with IFN after adsorption, are hyper‐sensitive to IFN; (ii) the mutant cannot inhibit protein kinase R phosphorylation efficiently during the early stage of replication (2 hrs post‐infection); (iii) expression of US11 in infected cells and its incorporation into the virion is reduced in the mutant compared to the wild type, despite the fact that a similar degree of DNA synthesis occurs during replication of both strains and; (iv) over‐expression of wild‐type viral TK has no effect on the phenotype of the VRTK^? strain, indicating that the phenotype is induced by a mutation(s) that does not involve the TK gene. These results suggested that the presence of US11 in the virion, but not that expressed after infection, plays an important role in the escape function of HSV‐1 from the antiviral activity of type 1 IFN.

     

Mu-induced polarity in the Escherichia coli K-12 ent gene cluster: evidence for a gene (entG) involved in the biosynthesis of enterochelin.

A strain of Escherichia coli K-12 has been isolated that carries a Mu bacteriophage-induced mutation in the ent gene cluster. Nutritional tests together with examination of the compounds accumulated by the mutant strain indicated that the mutant was blocked both in the synthesis of 2,3-dihydroxy-benzoate and its subsequent conversion into enterochelin. Enzymic complementation assays of the mutant with several mutants each affected in one of the ent genes showed that the Mu-induced mutant was entA-, entB-, entC+, entD+, entE+, and entF+. Since the mutant produced the entD, entE, and entF gene products but was unable to produce enterochelin from 2,3-dihydroxybenzoate, it must therefore be affected in an additional protein concerned with this conversion. It is therefore postulated that the Mu-induced mutation affects a previously unrecognized gene, entG. Genetic experiments indicate that the mutation in strain AN462 which affects the three ent genes is the result of a single insertion of Mu in the ent gene cluster. This polarity mutant therefore provides evidence that three of the ent genes are part of an operon.     

Effect of mutations of Escherichia coli K-12 chromosome on the integration of bacteriophage Mu introduced into cells by infection or conjugation

We present the detailed research on the previously described Escherichia coli K-12 Mud- mutants with impaired development of bacteriophage Mu. The ability of Mu phage DNA to penetrate into mutant cells on infection was shown. If introduced into the cells or combined with mud mutation by recombination, the prophage may be induced, which results in phage Mu lythic development and phage burst from mutant cells. In the course of conjugative transfer into the mutant cells, within a DNA fragment of the lysogenic donor chromosome, MupAp1 prophage is not inherited by recombinants. At the same time, Mu prophage deficient in genes A and B, whose products are required for transposition, is inherited by the mutant with the usual frequency. These data enable us to conclude that the mud mutations disturb the stage of conservative transposition which is connected with the insertion of the Mu prophage into the chromosome, after excision from the linear DNA introduced into the cells via infection or conjugation.     

Anaerobiosis, formate, nitrate, and pyrA are involved in the regulation of formate hydrogenlyase in Salmonella typhimurium.

Three groups of mutants defective in the fermentative production of gas were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). One group consisted of strains which lacked hydrogenase. The mutation site for this group was located in the vicinity of the known hyd gene. A second group consisted of mutants which lacked the formate dehyrogenase associated with hydrogenase. The mutation site was located in four of them. It was not in the vicinity of the previously described fhlD gene but was instead located at 93 min on the Salmonella map. The third mutant group, which consisted of strains that produced gas in triple sugar iron agar but not in nutrient agar supplemented with glucose, appeared to be pyrA mutants. The insertion site was located in the vicinity of pyrA , and they required arginine and pyrimidines for growth. Expression of the lac operon in the hyd mutants was induced by anaerobiosis. It was only slightly increased by the addition of formate under anaerobic conditions and slightly decreased by the addition of nitrate. Nitrate had no effect in an hyd ::Mu d strain that also carried a chlC::Tn10 insertion. Full expression of the lac operon in the fhl mutants required both formate and anaerobic conditions. The presence of nitrate in addition to formate resulted in activities about half those obtained in its absence, even in the fhl ::Mu d chlC::Tn10 double mutant. In the absence of formate, nitrate reduced expression only in the fhl ::Mu d single mutants. Expression of the lac operon among the pyrA mutants was repressed by arginine and cytosine and also by anaerobiosis. An explanation for the involvement of pyrA in aerobic and anaerobic energy metabolism is proposed.     

Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes

Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.     

Isolation and characteristics of mutation pfm affecting the penetration of phage Mu into Escherichia coli K-12 cells

A mutant of Escherichia coli K-12 strain with the destroyed process of establishment of lysogenic state for phage Mu in the course of zygotic induction has been obtained. The mutation revealed, designated pfm (penetration factor for Mu), interferes with adsorption of phage Mu to the surface of E. coli K-12 cells. On the basis of data obtained, there is every reason to believe that the phage Mu DNA connection with the membrane components of the bacterial cell provides optimizing condition for the primary integrative transposition of phage Mu at the stage of Mu DNA introduction into the cell.     

Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae.

Two dominant uninducible mutant alleles in the gal80 locus were identified. The GAL80s-1 and GAL80s-2 mutants showed novel phenotypes in response to the newly isolated GAL81-1 mutant allele, a dominant constitutive mutation linked to the gal4 locus; the GAL80s-1 GAL81-1 strain was inducible and the GAL80s-2 GAL81-1 strain was uninducible. Many galactose positive revertants from the GAL80s-2 GAL81-1 strain were isolated. It was proved that each revertant was due to a secondary mutation either in the gal80 or GAL81 locus, whereas revertants due to mutation at the supposed controlling site for the structural gene cluster of the galactose-pathway enzymes have not been isolated.     

Mutation of waaC, encoding heptosyltransferase I in Campylobacter jejuni 81-176, affects the structure of both lipooligosaccharide and capsular carbohydrate

Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first L-glycero-D-manno-heptose residue to 3-deoxy-D-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.     

The flhDC gene affects motility and biofilm formation in Yersinia pseudotuberculosis

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mu- tated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIII?flhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regula- tory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.     

Thermo-inducible expression of cloned early genes of bacteriophage Mu.

An EcoRI fragment, containing approx. 5100 base pairs (bp) of the immunity-end of bacteriophage Mu, was inserted into the multicopy plasmid pMB9 by in vitro recombination. The expression of early Mu genes, located on the cloned fragment, is thermo-inducible because of the presence of the ts mutation in gene c. The isolation of a transformant harbouring the recombinant plasmid, pGP1, was possible only when expression of Mu genes was prevented. pGP1 can be maintained at 28 degrees C at high copy number, but at 42 degrees C the pGP1 containing cells are killed due to the expression of the kil gene of Mu. The following Mu genes are present on pGP1: the ner gene, the integration and replication genes A and B, the cim gene, and the kil gene. pGP1 containing cells do not show Gam and Sot activity at 42 degrees C, therefore the leftmost EcoRI site on the Mu DNA is located between genes kil and gam or sot, or within the gam or sot gene.     

铜绿假单胞菌新基因PA0058插入失活对氨基糖苷类抗生素耐药性的影响

【目的】铜绿假单胞菌是一种重要的条件致病菌,临床上常引起难治性和顽固性感染,随着各种抗生素的广泛使用,该菌对多种抗生素呈现耐药性,研究其耐药性机理有着重要意义。【方法】以一株临床分离株Pseudomonas aeruginosa PA68作为出发菌株,应用人工Mu转座技术构建突变文库并从中筛选得到一株对链霉素抗性明显增强的菌株M122,并对突变株M122进行测序分析及表型检测。通过Southern杂交实验证实转座子是否为单拷贝插入,对突变株M122的基因表达谱与野生型PA68菌株进行对比分析。【结果】确定了Mu转座子在M122基因组上为单拷贝插入,插入位点为基因PA0058的第214 bp处。对M122进行表型检测,发现其对多种氨基糖苷类抗生素的耐药性均得到增强,通过转入携带完整基因PA0058的表达质粒可以使突变株M122的耐药性有所降低,利用同源重组的方法,在模式菌株P.aeruginosa PAK中进行PA0058基因敲除,得到的敲除株具有链霉素耐药性升高的表型。基因PA0058的缺失引起多种基因表达水平改变,尤其是katB、ahpC、ahpF等抗氧化酶基因转录表达显著增高。【结论】首次发现铜绿假单胞菌PA0058基因的插入失活提高了细菌对氨基糖苷类抗生素的耐药性,且导致突变株M122中抗氧化酶基因转录表达水平的上调。     

华癸根瘤菌外膜孔蛋白编码基因MCHK_1326突变株的构建与共生固氮表型分析

[目的]Mesorh izob ium huakuii 7653R的MCHK _1326基因编码一种外膜孔蛋白,可能参与根瘤菌侵染宿主植物以及结瘤固氮过程,本研究旨在探索该基因在共生固氮中的功能.[方法]生物信息学分析MCHK _1326蛋白的结构特征及生物学功能,启动子原位表达技术检测MCHK_1326共生时空表达特...