Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), of herpes simplex virus is required for viral DNA synthesis and normal regulation of viral gene expression. Previous genetic analysis has indicated that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of acting independently as a nuclear localization signal. In this study, we constructed a mutant virus (n11SV) in which the carboxyl-terminal 28 residues of ICP8 were replaced by the simian virus 40 large-T-antigen nuclear localization signal. The n11SV ICP8 localized into the nucleus and bound to single-stranded DNA in vitro as tightly as wild-type ICP8 did but was defective for viral DNA synthesis and viral growth in Vero cells. Two mutant ICP8 proteins (TL4 and TL5) containing amino-terminal alterations could complement the n11SV mutant but not ICP8 gene deletion mutants. Cell lines expressing TL4 and TL5 ICP8 were isolated, and in these cells, complementation of n11SV was observed at the levels of both viral DNA replication and viral growth. Therefore, complementation between n11SV ICP8 and TL4 or TL5 ICP8 reconstituted wild-type ICP8 functions. Our results demonstrate that (i) the carboxyl-terminal 28 residues of ICP8 are required for a function(s) involved in viral DNA replication, (ii) this function can be supplied in trans by another mutant ICP8, and (iii) ICP8 has multiple domains possessing different functions, and at least some of these functions can complement in trans.     

Regulation of Klebsiella pneumoniae hut operons by oxygen.

We investigated the regulation of genes concerned with nitrogen metabolism by oxygen in the facultative anaerobe Klebsiella pneumoniae. We found oxygen to be required for the expression of the hut operons; the effect of O2 on the glutamine synthetase and urease was less pronounced than on the hut operons. Glutamine synthetase was transiently repressed during the transition from an aerobic to an anaerobic environment. Regulation of hut by O2 suppressed the effect of nitrogen limitation on the expression of these genes.     

Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.     

Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.     

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae.

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.     

Metabolism of acrylonitrile by Klebsiella pneumoniae

A gram-negative rod-shaped bacterium capable of utilizing acrylonitrile as the sole source of nitrogen was isolated from industrial sewage and identified as Klebsiella pneumoniae. The isolate was capable of utilizing aliphatic nitriles containing 1 to 5 carbon atoms or benzonitrile as the sole source of nitrogen and either acetamide or propionamide as the sole source of both carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae was capable of hydrolyzing 6.15 mmol of acrylonitrile to 5.15 mmol of acrylamide within 24 h. The acrylamide was hydrolyzed to 1.0 mmol of acrylic acid within 72 h. Another metabolite of acrylonitrile metabolism was ammonia, which reached a maximum concentration of 3.69 mM within 48 h. Nitrile hydratase and amidase, the two hydrolytic enzymes responsible for the sequential metabolism of nitrile compounds, were induced by acrylonitrile. The optimum temperature for nitrile hydratase activity was 55°C and that for amidase was 40°C; both enzymes had pH optima of 8.0.Abbreviations PBM phosphate buffered medium - GC gas chromatography - GC/MS gas chromatography/mass spectrometry     

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.     

Reduction of fensulfothion to fensulfothion sulfide by Klebsiella pneumoniae.

A cell suspension of Klebsiella pneumoniae converted the organophosphorus pesticide fensulfothion to a product that was shown by chemical oxidation, gas-liquid chromatography, infrared spectrophotometry, and mass spectrometry to be fensulfothion sulfide. Further alteration of this metabolite was not noted.     

Reduction of fensulfothion to fensulfothion sulfide by Klebsiella pneumoniae.

A cell suspension of Klebsiella pneumoniae converted the organophosphorus pesticide fensulfothion to a product that was shown by chemical oxidation, gas-liquid chromatography, infrared spectrophotometry, and mass spectrometry to be fensulfothion sulfide. Further alteration of this metabolite was not noted.     

采用RAPD分析臭鼻克雷伯菌临床菌株的基因型

臭鼻克雷伯菌是重要的条件致病菌 ,常引起老人及婴幼儿重症护理病房的交叉感染及爆发流行 ,对其进行流行病学监测有重要的医学意义。用随机引物聚合酶链式反应 (RAPD)对 37株臭鼻克雷伯菌临床菌株基因组DNA进行分型 ,分析扩增产物的指纹图谱。实验结果表明 ,37株臭鼻克雷伯菌RAPD后可出现不同的指纹图谱 ,根据特异泳带C ,J ,F ,Q的有无 ,可将其分 7型 (I～VII)。RAPD可用于检测臭鼻克雷伯菌基因组DNA的异质性 ,是医院内感染分子流行病学研究的好方法。     

Improved process for exopolysaccharide production by Klebsiella pneumoniae sp. pneumoniae by a fed-batch strategy

Exopolysaccharide (EPS) was produced by Klebsiella pneumoniae K63 grown in fed-batch cultures using different procedures of the supply of carbon or nitrogen (N) source, or both. Cultures grown with excess of glucose and limitation or exhaustion of N produced 54.8 and 47.4 g(EPS) l(-1), respectively. These cultures also led to an accumulation of 'overflow' metabolites representing more than 16% of carbon conversion. The consistency indexes ( K ) obtained to the end of the cultures, characteristic of the rheological property of the biopolymer, were 16.4 Pa s(n) for N deficiency and 5.2 Pa s(n) for N limitation conditions. The simultaneous limitation of glucose and N decreased the excretion of co-metabolites (6.4% of carbon conversion) and the EPS production (18.1 g(EPS) l(-1)), while improving the quality of the polysaccharide, characterized by the highest K of 126.2 Pa s(n) and the highest pseudoplasticity degree (flow behaviour index, n=0.2).     

Temperature sensitivity of the regulation of nitrogenase synthesis by Klebsiella pneumoniae.

Temperature sensitivity of the regulatory protein coded by nifA prevents the organism from utilizing N2 at 37 degrees C. The purpling of 6-cyanopurine, a function of nifA expression, also is thermolabile.     

Intragenic complementation between Escherichia coli trp repressors with different defects in the tryptophan-binding pocket.

Site-directed mutagenesis of the trpR gene (encoding the trp repressor, TrpR) was used to replace Gly85 with tryptophan (Trp or W), in order to place Trp near its normal location in the L-tryptophan(L-W)-binding pocket. The resulting mutant protein (G85W) exhibits weak, but significant repressor activity in vivo that is independent of the presence of L-W in the media. This mutant negatively complements the chromosomal wild type (wt), but does not negatively complement either the wt or the super-repressor, E49K, when any of these alleles is expressed on a multicopy plasmid. Activity of the mutant repressor, G85W, when produced in vivo together with T44M, approaches that of the wt repressor. This result presumably reflects complementation between the two mutant polypeptides. Similar results are obtained when G85R or G85K are combined with T44M in vivo, but not when G85W is replaced by G85E. The level of repression is dependent on the presence of L-W in the media. The TrpR with two mutations altering both Gly85 (G85W, G85R, G85E or G85K) and Thr44 (T44M) has no repressor activity. These results suggest a type of site-specific intragenic complementation where only certain alterations at Gly85 complement T44M. In this study, a positive charge or an indole ring appears to be required for the observed intragenic complementation.     

Nitrogenase of Klebsiella pneumoniae nifV mutants.

The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore the ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (0.031 matm) CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.     

The capsular network of Klebsiella pneumoniae.

Attempts at improving chemical fixation for electron-microscopic observation of the capsule of Klebsiella pneumoniae were made. The capsule was preserved by using alcian blue - lanthanum and tris-(1-aziridinyl) phosphine oxide (TAPO) - aldehyde - osmium procedures. Despite the different retention of the overall capsular material and minor variations in morphological details, in both cases the interpretation of ultrastructural patterns suggested that the capsule be composed of a meshed network of thin polysaccharide fibrils radiating from the cell wall. This organization is in keeping with all recognized chemical properties of bacterial polysaccharide capsules or, at least, does not contradict them. Moreover, an effective preservation of bacterial structures other than capsule has been obtained, mostly in specimens fixed by the TAPO-aldehyde-osmium method, a fact which gives further reliability to the technical approach used for capsule visualization.     

Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

Two hundred and thirty-five Nif- strains of Klebsiella pneumoniae were characterized by two-dimensional polyacrylamide gel electrophoresis. Forty-two of these strains were tested further by in vitro acetylene reduction assays. By these techniques, nine nif-coded polypeptides were identified, and eight of these were assigned to specific nif genes. Nitrogenase component I required nifK and nifD, which coded for the beta and alpha subunits, and nifB, -E, and -N were required for the iron-molybdenum cofactor, which is a part of the active site of nitrogenase. nifH coded for the structural protein of component II, and nifM and nifS products seemed to be necessary for the synthesis of an active component II. There were two genes, nifF and nifJ, that were required for N2 fixation in vivo but not for N2 fixation in vitro. There were at least two cases (nifE and nifN, nifK and nifD) of two proteins that seemed to require each other for stability in vivo. Regulation of N2 fixation is apparently complex, and this is reflected by the assignment of regulatory functions to the gene products of nifA, nifL, nifK, nifD, nifH, and NIFJ.