Regulation of expression of nif and hut operons in Klebsiella pneumoniae by glnA linked genes of Escherichia coli

Summary Recombinant DNA plasmids containing inserts from the glnA region of Escherichia coli were used to study the expression of gln, hut, and nif operons in a regulation defective mutant (Gln^–Hut^–Nif^–) of Klebsiella pneumoniae, KP5060. Genes adjacent to the C-terminal end of glnA on the E. coli chromosome were able to derepress hut and nif operons in K. pneumoniae in the absence of glnA product. However, complete derepression of nif operons required inclusion of the segment adjacent to the N-terminal end of the glnA region of the E. coli chromosome along with the C-terminal end segment. In the absence of functional glnA, such a fully derepressed strain expressed nif and hut constitutively indicating a role for the catalytic activity of glutamine synthetase in repression of the genes under nitrogen control.     

Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes

When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged (a) by the incorporation of [¹⁴C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, (b) by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incroporation of [¹⁴C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and (c) by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogeneously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn²⁺, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe²⁺ (or Ni²⁺ together with ATP (or other nucleoside di- and triphosphates. After 40–60 h dialysis Fe³⁺ together with NADH (but not ATP) are required for glutamine synthetase inactivation. The results suggest that accelerated protein degradation in cells exposed to nitrogen-limited conditions reflects the differential destruction of some proteins, including aspartokinases I and III, in order to sustain the biosynthesis of others such as glutamine synthetase. The loss of glutamine synthetase activity in cell-free extracts is likely mediated in part by mixed-function oxidation systems and could represent a ‘marking’ step in protein turnover.     

Mapping of the sor genes for l-sorbose degradation in the chromosome of Klebsiella pneumoniae

Summary A series of mutants was isolated in Klebsiella pneumoniae strain 1033, among them mutants unable to grown on l-sorbose. Different R' plasmids carrying the sor genes and other surrounding chromosomal genes were also isolated. Each plasmid contained the structural genes sorA for an Enzyme II of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system, sorD for a d-glucitol 6-phosphate dehydrogenase, sorE for an l-sorbose 1-phosphate reductase, and the corresponding regulator gene sorR. These structural genes are coordinately expressed and inducible by l-sorbose. Cis-dominant and pleiotropic mutations rendering the expression of the sor genes constitutive or eliminating it were isolated. Complementation of a series of mutations in Escherichia coli K12 and K. pneumoniae by various R' and F' plasmids and by P1 transduction in K. pneumoniae located the sor genes within the following gene sequence: rbs rha pfkA metB ppc argH ilv btuB rpoB metA ace sor pgi malB uvrA. The rbs-ilv gene loci tightly linked in E. coli K12 at 84 min, are separated in the map of K. pneumoniae 1033 and located at 86 and 89 min, respectively.     

Regulation of 3-hydroxypropionaldehyde accumulation in Klebsiella pneumoniae by overexpression of dhaT and dhaD genes

3-Hydroxypropionaldehyde (3-HPA), an important intermediary metabolite of 1,3-propanediol (PDO) production, would be toxic to the cell growth and led to the abnormal cessation of the fermentation process. In this study, the dhaD gene encoding glycerol dehydrogenase (GDH) and dhaT gene encoding 1,3-propanediol oxidoreductase (PDOR) were overexpressed in Klebsiella pneumoniae ACCC 10082 to decrease the 3-HPA accumulation and increase the coenzyme NADH supply. By the construction of pTD plasmid, GDH and PDOR were both overexpressed and their enzyme activities were increased by 2.6- and 3.2-fold, respectively. The enzyme activity ratio of PDOR/GDHt (glycerol dehydratase) also was increased. On the other hand, NADH production was enhanced and the ratio of NADH/NAD⁺ exceeded 1 after the inducement of IPTG for the constructed strain. The two factors enhanced the transformation of 3-HPA to PDO. In the batch and fed-batch fermentation by the constructed strain, the peak of 3-HPA accumulation reduced by 52.2% and 33.3%, respectively, compared with the control. The PDO concentration and yield reached 59.2 g/L and 0.48 mol/mol, respectively. Furthermore, the fed-batch fermentation process appeared easier to be regulated. This work is considered helpful for the further understanding on the PDO metabolic mechanism of K. pneumoniae and also useful for the PDO fermentation in a large-scale bioreactor.     

酿酒酵母组氨酸转运蛋白Hip1p的泛素化水平对组氨酸利用的影响

【目的】为了研究泛素化对组氨酸转运及调控的影响。【方法】应用泛素化位点预测及定点突变等技术手段,Hip1p的3个潜在的泛素化位点K30、K42和K52被突变。这些突变的Hip1p被克隆到泛素化检测质粒中,检测泛素化位点突变对Hip1p的泛素化水平的影响。同时这些突变对细胞生长及组氨酸利用的影响也进一步做了检测。【结果】Hip1p的3个赖氨酸位点K30、K42和K52突变能有效降低其泛素化水平。同时,双重突变对其泛素化水平有明显的协同作用,并进一步影响了细胞生长和组氨酸利用。【结论】泛素化水平调控能有效调节组氨酸代谢,引起细胞对组氨酸利用的改变,为进一步研究氨基酸转运蛋白的调控机制提供了重要的依据。     

野生鸟类分离的多重耐药肺炎克雷伯氏菌(Klebsiella pneumoniae)耐药基因分子进化特征研究

【目的】调查野生鸟类携带菌的耐药状况,探索其在细菌耐药性传播过程中的作用。【方法】从野生鸟类石鸡、绯胸鹦鹉、太阳锥尾鹦鹉和黑领椋鸟的新鲜粪便分离4株Klebsiella pneumoniae,采用微量肉汤稀释法评估其多重耐药表型,并利用全基因组测序技术和细菌全因组关联分析、比较基因组学方法对分离株进行分子溯源,系统解析其携带的多重耐药质粒或基因与其宿主、同源质粒间的关联。【结果】4株肺炎克雷伯菌的耐药谱各不相同,来自石鸡样本的分离株S90-2对9种药物耐受,绯胸鹦鹉样本分离株S141对3种药物耐受,太阳锥尾鹦鹉分离株M911-1仅耐受氨苄西林,黑领椋鸟的样本分离株S130-1对所使用的14种药物完全敏感。S90-2属于ST629型,携带bla_CTX-M-14、fosA6、aac(3)-Iid和bla_SHV-11为主的30个耐药基因和携带1个耐药性质粒pS90-2.3 (IncR型)。S141属于ST1662型,携带fosA5、bla_SHV-217等27个耐药基因,1个质粒pS141.1 [IncFIB(K)(pCAV1099-114)/repB型]仅携带耐药基因adeF。M911-1为新ST类型,携带bla_SHV-1、fosA6等共计27个耐药基因,其质粒pM911-1.1携带了3个耐药基因。S130-1属于ST3753型,携带bla_SHV-11、fosA6等27个耐药基因,pS130-1 [IncFIB(K)型]则仅携带一个耐药基因tet(A)。质粒比对表明,质粒pS90-2.3携带的耐药基因片段源自不同的肠杆菌科菌株染色体或质粒。pS90-2.3的同源质粒主要来自人类宿主菌,且主要在中国分布,这些质粒主要细菌宿主为K. pneumoniae和Escherichia coli,且ST11型K. pneumoniae分离株为重要宿主菌。【结论】本研究中来自野生鸟类的多重耐药K. pneumoniae,其耐药基因主要来自质粒,质粒耐药基因主要由转座子、插入序列、整合子和前噬菌体等可移动元件介导,这些多重耐药质粒与人类的宿主菌密切相关。     

Cloning of the histidine, pyrimidine and cysteine genes of Azospirillum brasilense: Expression of pyrimidine and three clustered histidine genes in Escherichia coli

Summary A gene bank from Azospirillum brasilense, Sp6 strain, was constructed in Escherichia coli in plasmid pRK290 and was used to identify Azospirillum genes. Clones carrying the his1, his2, pyr and cys1 genes were identified by genetic complementation and the expression of A. brasilense his1, his2 and pyr genes in E. coli was demonstrated. By E. coli complementation experiments, a cluster of three genes for the histidine biosynthetic pathway in A. brasilense has also been found, suggesting the existence of an operon-like unit.     

Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12

Summary The genes xy1A and xy1B were cloned together with their promoter region from the chromosome of Klehsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xy1A encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M_r of 49 793). The gene xy1B encodes the enzyme xylulokinase (XK or Xy1B) with a calculated M, of 51 783 (483 amino acids). The two genes successfully complemented xy1 mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylA _Kp 5 upstream region in high copy number (but lacking an active xy1B gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5 upstream regions of xy1A on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.     

一株产酸克雷伯氏菌发酵培养基优化

为提高产酸克雷伯氏菌(Klebsiella oxytoca)的发酵水平,通过单因素优化,研究培养条件、碳源、氮源、无机盐对产酸克雷伯氏菌活菌量的影响,利用响应面分析法对影响产酸克雷伯氏菌活菌量的关键因子进行优化,30 L发酵罐进行扩大培养。得出最佳配方（质量分数）：黄豆饼粉2.19%,玉米粉1.0%,蔗糖1.10%,硫酸铵0.06%,玉米浆干粉0.5%,蛋白胨0.05%,硫酸镁0.04%,磷酸二氢钾0.02%,氯化钠0.080%,硫酸锰0.03%,pH 7.0~7.4。活菌量稳定在2.0×10¹⁰  cfu/mL以上,能够满足生产要求,为产酸克雷伯氏菌作为微生物菌肥的应用提供参考。     

噬菌体治疗肺炎克雷伯菌感染的研究进展

随着细菌的进化以及部分抗生素的滥用，耐药细菌的感染已成为21世纪主要的公共卫生挑战之一。其中，耐药肺炎克雷伯菌(Klebsiella pneumoniae)问题尤为突出。噬菌体在治疗耐药细菌感染引起的疾病方面展现出一定的潜力及独特优势，但目前噬菌体治疗尚缺乏统一的临床指导规范。虽然临床上有少数将噬菌体用于治疗肺炎克雷伯菌感染的成功案例，但多数情况下是采用噬菌体配合抗生素疗法，噬菌体在其中的作用仍不明确。本文综合评述国内外研究数据，回顾与噬菌体治疗肺炎克雷伯菌感染相关的数个重点问题，包括噬菌体的特性以及影响其疗效的因素，旨在为肺炎克雷伯菌和其他耐药细菌的噬菌体治疗提供参考。     

Positive and negative regulation of expression of the l-sorbose (sor) operon by SorC in Klebsiella pneumoniae

Summary In Klebsiella pneumoniae the gene products involved in the degradation of the ketose l-sorbose are encoded in the sor operon. It comprises, besides structural genes for uptake and catabolism, a promoter-proximal gene sorC, encoding a protein SorC of M_r 40 kDa, for which no enzymatic function has been detected. All sor genes are coordinately expressed and inducible by l-sorbose. Polar insertions and frameshift mutations in sorC cause a pleiotropic negative effect on the expression of all other sor genes. This defect is complemented in trans by the wild-type sorC ⁺ allele for frameshift mutations, but not for polar insertions. A single promoter for all sor genes, for which SorC is the activator, thus seems to be located in front of sorC. The repressor activity of SorC was demonstrated by complementation of constitutive sorC alleles with a sorC ⁺ allele leading to inducible expression of all sor genes, including sorC, which, as visualized by the use of a series of lacZ fusions, thus autoregulates its expression, both as an activator and a repressor.     

重组肺炎克雷伯氏菌转化甘油为聚3-羟基丙酸

聚3-羟基丙酸[Poly(3-hydroxypropionate),P3HP]是一种生物可降解及生物相容的新型聚羟基脂肪酸酯。目前已鉴定的生物均不能天然合成P3HP。采用PCR克隆鼠伤寒沙门氏菌的丙醛脱氢酶(Pdu P)基因及罗尔斯通氏菌的聚羟基脂肪酸酯合成酶(Pha C)基因,构建共表达载体,转化肺炎克雷伯氏菌后获得两株重组菌。以甘油为唯一碳源进行摇瓶发酵,pdu P和pha C共用tac启动子的工程菌K.p(p ET-tac-pdu P-pha C)产生0.054 g/L的P3HP,而pdu P和pha C各自独用tac启动子的工程菌K.p(p ET-tac-pdu P-tac-pha C)产生0.091 g/L的P3HP。     

Transfer and expression of Klebsiella nif genes in Alcaligenes faecalis, a nitrogen-fixing bacterium associated with rice root

Plasmid pRD1 carrying Klebsiella nif genes was found to be transferable by conjugation from Escherichia coli JC5466 (pRD1) to Alcaligenes faecalis A-15 at a frequency of 5 X 10(-4). Nitrogenase activity of four A-15 (pRD1) strains tested was found to be higher than that of their parent A-15, as determined by the acetylene reduction assay. A-15-1 was a Nif- mutant derived from A-15. After mating with JC5466 (pRD1), the nitrogenase activity was restored. PRD1 was stable in A. faecalis and could be transferred to E. coli JC5466-1 by conjugation. The fact that Klebsiella nif genes carried in pRD1 can be expressed in A. faecalis makes it possible to use pRD1 as a tool for genetic analysis and genetic engineering of the nitrogen fixation system in A. faecalis.     

Deletion mutants of nitrogen fixation in Klebsiella pneumoniae: Mapping of a cluster of nif genes essential for nitrogenase activity

Deletions of the nitrogen fixation (nif) region of the Klebsiella genome were isolated by selecting for resistance to virulent phages whose resistance loci are adjacent to nif. The extent of the various deletions was monitored by assaying several different enzymes or gene products coded for by this segment of DNA. Three classes of deletion mutants were detected: (1) gluconate-6-phosphate dehydrogenase minus (gnd^?), histidine minus but histidinol dehydrogenase plus (his^?, his D⁺), nitrogenase plus (nif⁺), shikimate utilization plus (shu⁺); (2) gnd^?, his D^?, nif^?, shu⁺; (3) gnd^?, his D^?, nif^?, shu^?. From these studies we conclude that the cluster of nif genes essential for nitrogenase activity is located on the genetic linkage map of Klebsiella between his and shu; the gene order in this region in thus phage-resistance locus (rfb?), gnd, his operon, nif, shu. Genetic analysis substantiates the finding that the nif cluster is located proximally to the operator end of the his operon.     

Genetic analysis of the bchC and bchA genes of Rhodobacter sphaeroides

Summary This study has identified by sequence analysis a single gene in the bchC locus of Rhodobacter sphaeroides and three genes, designated bchX, Y and Z, in the bchA locus, which was previously thought to contain only a single gene. All four genes may reside within the same operon and are transcribed in the order bchC-X-Y-Z. Complementation analysis of eight transposon insertion mutants within these genes suggests that bchX, Y and Z are essential for the reduction of 2-devinyl-2hydroxyethyl chlorophyllide a and that bchC encodes the 2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase. Similarity between the putative BchX protein and dinitrogenase reductase proteins suggests that BchX may also be a reductase, supplying electrons for reduction of 2-devinyl-2-hydroxyethyl chlorophyllide a.     

Genetic and physical mapping of the lateral suppressor (ls) locus in tomato

Tomato plants homozygous for the recessive lateral suppressor (ls) mutation show a number of phenotypic abnormalities among which the lack of lateral meristem initiation during vegetative growth and the absence of petals on the flower are the most prominent. As a first step towards the isolation of the Ls gene by means of map-based cloning, we have determined its position on the restriction fragment length polymorphism (RFLP) map of tomato. RFLP analysis of 527 F2 plants segregating for the ls allele allowed us to define an interval of 0.8 cM in which the Ls gene is located. Analysis of the physical distance between the two flanking RFLP markers by pulsed field gel electrophoresis revealed that they lie no further than 375 kb apart. Knowledge of the physical distance together with the availability of a tomato yeast artificial chromosome (YAC) library, makes it feasible to isolate the Ls gene by a map-based cloning approach.