Evaluation of two alternative metabolic pathways for creatinine degradation in microorganisms

The microbial decomposition of creatinine was found to proceed mainly via N-methylhydantoin or creatine as the first degradation product. Either N-methylhydantoin or urea or both were detected as metabolites derived from creatinine in various microorganisms, and creatinine deiminase and creatinine amidohydrolase activities were detected concomitantly. N-Methylhydantoin hydrolase and N-carbamoylsarcosine amidohydrolase were found to be formed inducibly in the presence of creatinine or N-methylhydantoin. Three microorganisms which decompose creatinine in different ways were screened from soil. Pseudomonas putida 77 rapidly metabolized creatinine solely via N-methylhydantoin. Degradation of creatinine proceeded with both creatine and N-methylhydantoin as the first degradation products at the same time in Pseudomonas sp. H21. Pseudomonas sp. 0114 was found to metabolize creatinine mainly via creatine and to also metabolize N-methylhydantoin. Changes in the metabolites of creatinine during a cultivation or enzyme reaction were found to be closely related to the enzyme activities of interest which are regulated by creatinine or N-methylhydantoin in different ways depending on the microbial strain.     

Pyrimidine,purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of the glnLG and purR genes in the regulation of codA expression

Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.This paper is dedicated to Professor John Ingraham, Department of Bacteriology, University of California, Davis, on the occasion of his retirement, in recognition of his many contributions in the field of bacterial growth and metabolism     

Methylated cytosine at Dcm (CC T A GG) sites in Escherichia coli: Possible function and evolutionary implications

The frequency and distribution of methylated cytosine (5-MeC) at CC _T ^A GG (Dcm sites) in 49 E. coli DNA loci (207,530 bp) were determined. Principal observations of this analysis were: (1) Dcm frequency was higher than expected from random occurrence but lower than calculated with Markov chain analysis; (2) CCTGG sites were found more frequently in coding than in noncoding regions, while the opposite was true for CCAGG sites; (3) Dcm site distribution does not exhibit any identifiably regular pattern on the chromosome; (4) Dcm sites at oriC are probably not important for accurate initiation of DNA replication; (5) 5-MeC in codons was more frequently found in first than in second and third positions; (6) there are probably few genes in which the mutation rate is determined mainly by DNA methylation. It is proposed that the function of Dcm methylase is to protect chromosomal DNA from restriction-enzyme EcoRII. The Dcm methylation contribution to determine frequency of oligonucleotides, mutation rate, and recombination level, and thus evolution of the E. coli genome, could be interpreted as a consequence of the acquisition of this methylation.Correspondence to: M.C. Gómez-Eichelmann     

Inheritance of biodegradation plasmids in the cells of homo- and heterologous hosts

A systematic analysis of the inheritance of D plasmids of the IncP-9 group (α-, β-, γ-, δ-, ?-, ζ-, η-, and θ-subgroups), IncP-7, as well as of those of undefined systematic affiliation in the cells of homologous (Pseudomonas putida) and heterologous (Escherichia coli) hosts was performed for the first time. For this purpose, mini-Tn5 transposons determining resistance to kanamycin (or streptomycin) were introduced into all the D plasmids under study. It has been established that all IncP-9 plasmids can be transmitted to the cells of a heterologous host E. coli (with the exception of plasmid pSVS15 from gq-subgroup). IncP-7 plasmids and those of undefined systematic affiliation do not possess this property and can be transmitted and stably inherited only in P. putida. The distinctive feature of most IncP-9 plasmids (α-, β-, δ-, ?-, and ζ-subgroups) is strict dependence of their inheritance on the temperature factor. At 37°C, the plasmids of δ-, ζ-, and θ-subgroups are unstable in P. putida cells, while in E. coli nearly all plasmids of this systematic group are unstable. The exceptions are the plasmids of η- and γ-subgroups. Inheritance of these plasmids does not depend on temperature. At 28°C and 37°C, the η plasmid is not maintained stably (inheritance stability is 2%), while the γ-plasmid has almost 100% stability.     

Degradation of 2-chloroethanol by wild type and mutants of Pseudomonas putida US2

A strain of Pseudomonas putida was isolated that was able to degrade 2-chloroethanol. The degradation proceeded via 2-chloroacetaldehyde and chloroacetate to glycolate. In crude extracts the enzymes for this degradation pathway could be detected. All enzymes proved to be inducible. The dehalogenase that catalyzed the dehalogenation of chloroacetate to glycolate was further characterized. It consisted of a single polypeptide chain with a molecular mass of 28 kDa. After induction the dehalogenase was expressed at a high level. In a mutant resistant to high concentrations of 2-chloroethanol the dehalogenase was no longer expressed. The mechanism of resistance seemed to be due to the inability to convert chloroacetate and export of this compound out of the cell.Non-standard abbreviations CEO 2-chloroethanol - DCPIP 2,6-dichlorophenolindophenol - FPLC fast protein liquid chromatography - PAGE polyacrylamide gelelectrophoresis - PES phenazine ethosulfate - PMS phenazine methosulfate - PQQ pyrroloquinoline quinone     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 191–197.Original Russian Text Copyright © 2005 by Deryabin, Polyakov.     

Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose after batch culture, thereby giving hsBLyS at 3.7 g l⁻¹ and a productivity of 0.11 g l⁻¹ h⁻¹. Revisions requested 1 September 2005 and 11 November 2005; Revisions received 7 November 2005 and 4 January 2006     

Possible Reasons for Difference in Sensitivity to Oxygen of Two <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains

In preliminary experiments it was found that Escherichia coli strains AB1157 and KS400 are different in their abilities to grow under various oxygen levels in cultivation medium: the first strain does not grow under high oxygen conditions, unlike the second one. To investigate whether the damage to cellular components due to production of reactive oxygen species (ROS) was responsible for this difference, the intensity of free radical oxidation of proteins and lipids as well as the activities of selected antioxidant and associated enzymes (superoxide dismutase, catalase, peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase) were compared in the two strains. The level of thiobarbituric acid-reactive substances was 1.8–2.5-fold higher in AB1157 than in KS400, but the concentration of carbonyl proteins was lower in the AB1157 strain. In both strains growth under higher oxygen levels resulted in higher superoxide dismutase and peroxidase activities in both exponential and stationary phases. Overall, the activities of antioxidant enzymes were always higher in the KS400 strain than in AB1157. The results for both lipid and protein oxidative damage and antioxidant enzyme activities suggest that the differences in oxygen tolerance between these two strains may be due to their different abilities to cope with ROS.__________Translated from Biokhimiya, Vol. 70, No. 4, 2005, pp. 514–522.Original Russian Text Copyright © 2005 by Semchyshyn, Lushchak, Storey.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

Toroidal nucleoids in Escherichia coli exposed to chloramphenicol

The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms ("cm-nucleoids") that have been described as ring- or sphere-shaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed.     

Role of the intracellular proteolysis in the production of the periplasmic penicillin amidase in Escherichia coli

The yield of periplasmic enzyme, penicillin amidase (PA), from E. coli ATCC 11105 is regulated at the post-translational level by two competitive processes-intracellular proteolysis of newly synthesised pre-pro-PA (ppPA) in the cytoplasm and membrane transport and maturation of ppPA. Intracellular proteolysis results in a significant loss in the yield of active PA. Immunochemical analysis were used to study the influence of the cultivation temperature, phenylacetic acid, and glucose on the proteolysis of the ppPA in the cytoplasm. An increase in glucose concentrations or temperature during the cultivation resulted in a significant loss of PA activity due to the increased rate of intracellular proteolysis of ppPA. Addition of phenylacetic acid reduced the intracellular proteolysis of ppPA and, as a consequence, increased the PA production. Taken together, these data explain a new aspect of regulation of active PA production in E. coli.     

Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli

Multi-drug-resistant bacteria can cause serious infections that are extremely difficult to treat. Bacterial efflux pumps are known to contribute to multi-drug resistance and, thus, constitute a promising target for novel antibacterial agents. Resazurin is widely used to monitor bacterial growth because resazurin is reduced to the fluorescent resorufin by live cells. We have shown by flow cytometric analysis and by accumulation studies with wild type and efflux deficient strains that resazurin is a substrate of efflux pumps in Escherichia coli and Pseudomonas aeruginosa. Our investigations showed that the conversion rate of resazurin to resorufin is affected by efflux pumps. This finding was used to design an assay useful to detect efflux pump activity and to find potential efflux-pump inhibitors in a microtiter plate format. Mefloquine was detected as efflux-pump inhibitor when a panel of selected chemical compounds was tested for assay validation purposes.     

Induction of defense responses in tomato against Pseudomonas syringae pv. tomato by regulating the stress ethylene level with Methylobacterium oryzae CBMB20 containing 1-aminocyclopropane-1-carboxylate deaminase

This study aimed to examine the induction of defense responses in tomato elicited by Methylobacterium oryzae CBMB20 as a consequence of reduced stress ethylene level possibly through its ACC deaminase activity. Significantly increased activities of pathogenesis-related (PR) proteins and defense enzymes such as β-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were noted in M. oryzae CBMB20 pretreated and challenged with Pseudomonas syringae pv. tomato (Pst) compared to either control or M. oryzae-treated tomato plants in both growth chamber and greenhouse conditions. Increased PR proteins and defense enzyme activities were correlated with the reduction of stress ethylene level. M. oryzae CBMB20 reduced the stress ethylene level about 27% and 55% when challenged with Pst, in growth chamber and greenhouse on day 7 respectively and the effect was comparable to that of the chemical ethylene biosynthesis inhibitor AVG, L-α-(2-aminoethoxyvinyl)-glycine hydrochloride. As a consequence of reduced stress ethylene level and its effect on defense response in crop plants, the disease severity was reduced 26% in M. oryzae CBMB20-treated plants challenged with pathogen. Therefore, inoculation of M. oryzae CBMB20 would induce the defense enzymes and contribute to the enhanced resistance of tomato plants against the pathogen Pst.     

Resistance to trifluoroperazine, a calmodulin inhibitor, maps to the fabD locus in Escherichia coli

A mutant, tfpA1, resistant to the calmodulin inhibitor trifluoroperazine (TFP) at 30°C, was isolated in Escherichia coli. The mutant showed a reduced growth rate at 30°C and was temperature sensitive (ts) at 42°C for growth, forming short filaments. The mutation was mapped to the 24 min region of the chromosome and the gene was cloned by complementation of the is defect. Subsequent subcloning, complementation analysis, marker rescue mapping and sequencing, identified tfpA as fabD, encoding the 35 kDa, malonyl-coenzyme A transacylase (MCT) enzyme, required for the initial step in the elongation cycle for fatty acid biosynthesis. Resistance to TFP may result from altered permeability of the cell envelope, although the mutant remained sensitive to other calmodulin inhibitors and to other antibacterial agents. Alternatively, resistance may be more indirect, resulting from alterations in intracellular Ca⁺⁺ levels which affect the activity of the TFP target in some way.     

A two-step fermentation process for efficient production of penta-N-acetyl-chitopentaose in recombinant Escherichia coli

The nodC gene from Mesorhizobium loti was cloned into E. coli, leading to production of chitin oligosaccharides (COs)—mainly penta-N-acetyl-chitopentaose. A two-step fermentation procedure was then developed which gave 930 mg CO/L with a productivity of 37 mg/l·h.     

在大肠杆菌中拓展合成途径合成异胡椒醇北大核心

天然异胡椒醇作为一种植物来源香料,具有很高的经济价值。目前国内外还未见利用大肠杆菌工程菌株转化生产异胡椒醇的报道。【目的】构建工程菌,采用生物合成方法获得天然香料异胡椒醇。【方法】对来自胡椒薄荷的细胞色素P450家族的柠檬烯-3-羟化酶(LIM3H)基因PM2进行改造,截短LIM3H的N端疏水区,分别添加17α短肽或2B1短肽增加其亲水性,采用CO差示光谱法检测LIM3H活性;将N端疏水区截短后的来自拟南芥(Arabidopsis thaliana)的NADPH-细胞色素P450还原酶基因(trATR)和修饰后LIM3H基因(Modi-LIM3H)融合后表达,最终以柠檬烯为底物进行全细胞转化。【结果】3种N端修饰LIM3H基因均检测到LIM3H表达和异胡椒醇生成,其中17α短肽修饰后的蛋白表达量最高,异胡椒醇产量达到1.94 mg/L。【结论】本研究在大肠杆菌中增加外源LIM3H与ATR,成功催化柠檬烯生成异胡椒醇,为天然异胡椒醇的生产提供了新的方法。     

Effect of ogt expression on mutation induction by methyl-, ethyl- and propylmethanesulphonate in Escherichia coli K12 strains

We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents. We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O⁶-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives. Thus an ogt::kan^r disruption mutation was introduced into the parental ogt ⁺ bacteria, and the ogt::kan^r mutation was then eliminated by cotransduction of ogt ⁺ with the closely linked Tet ^r marker (zcj::Tn10). The (ogt-fnr) deletion or ogt::kan^r disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to l-arabinose resistance (Ara¹). Furthermore, the number of Ara^r mutants increased linearly with dose, unlike the case in ogt ⁺ bacteria, which had a threshold dose below which no mutants accumulated. Differences in mutability were even greater with propyl methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt mutant strains and also methylmethanesulphonate mutagenesis in ada ^– bacteria. A sample of AB 1157 obtained from the E. coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes. Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E. coli K12 strain being used is advisable.     

调控大肠杆菌胞内ATP和NADH水平促进琥珀酸生产

琥珀酸作为一种重要的C4平台化合物,广泛应用于食品、化学、医药等领域。利用大肠杆菌(Escherichia coli)发酵生产琥珀酸受胞内辅因子不平衡的影响,存在产率低、生产强度低、副产物多等问题。为此,对不同氧气条件下琥珀酸产量和化学计量学分析发现,微厌氧条件下E.coli FMME-N-26高效积累琥珀酸需要借助三羧酸循环(tricarboxylic acid cycle,TCA)为还原性三羧酸途径(reductive tricarboxylic acid pathway,r-TCA)提供足够的ATP和NADH。通过减少ATP消耗、强化ATP合成、阻断NADH竞争途径和构建NADH回补路径等代谢工程策略,组合调控胞内ATP与NADH含量,获得工程菌株E.coli FW-17。通过发酵条件优化,菌株E.coli FW-17在5 L发酵罐能积累139.52 g/L琥珀酸,比出发菌株提高了17.81%,乙酸浓度为1.40 g/L,降低了67.59%。进一步在1000 L发酵罐中进行放大实验,琥珀酸产量和乙酸浓度分别为140.2 g/L和1.38 g/L。