Evolution of novel metabolic pathways for the degradation of chloroaromatic compounds

Chlorobenzenes are substrates not easily metabolized by existing bacteria in the environment. Specific strains, however, have been isolated from polluted environments or in laboratory selection procedures that use chlorobenzenes as their sole carbon and energy source. Genetic analysis indicated that these bacteria have acquired a novel combination of previously existing genes. One of these gene clusters contains the genes for an aromatic ring dioxy-genase and a dihydrodiol dehydrogenase. The other contains the genes for a chlorocatechol oxidative pathway. Comparison of such gene clusters with those from other aromatics degrading bacteria reveals that this process of recombining or assembly of existing genetic material must have occurred in many of them. Similarities of gene functions between pathways suggest that incorporation of existing genetic material has been the most important mechanism of expanding a metabolic pathway. Only in a few cases a horizontal expansion, that is acqui sition of gene functions to accomodate a wider range of substrates which are then all transformed in one central pathway, is observed on the genetic level. Evidence is presented indicating that the assembly process may trigger a faster divergence of nearby gene sequences. Further fine-tuning, for example by developing a proper regulation, is then the next step in the adaptation.     

RT-TGGE as a guide for the successful isolation of phosphonoacetate degrading bacteria

AIMS: Use of molecular techniques for the isolation of bacteria capable of phosphonoacetate mineralization as carbon, phosphorus and energy source. METHODS AND RESULTS: RNA extracts obtained at three different stages of an enrichment selecting for phosphonoacetate degrading bacteria were reverse transcribed using 16S rRNA-specific primers, amplified and analysed by temperature gradient gel electrophoresis (TGGE). This information was used to devise a strategy for the isolation of members of the enrichment that were otherwise difficult to obtain in pure culture. We were able to pull out, in total, four out of the six main microbial cultures that were detected by TGGE. Two of the isolates belonging to Mycobacterium and Agromyces genera were for the first time shown to grow in the presence of phosphonoacetate as sole carbon, phosphorus and energy source releasing almost equimolar levels of inorganic phosphate into the culture medium, and they were shown to exhibit phosphonoacetate hydrolase activity in vitro. CONCLUSIONS: The ubiquity of pseudomonad in degradation processes is more likely a consequence of our ignorance of bacterial requirements and physiology, rather than their possession of unique metabolic properties. SIGNIFICANCE AND IMPACT OF THE STUDY: RT-TGGE analysis can be used to guide the successful isolation of micro-organisms difficult to obtain by culture-dependent methods alone.     

工程菌种子制备方法对rEPA表达量影响的研究

由重组E.colirPE553D所表达的基因缺失突变脱毒的重组铜绿假单胞菌外毒素A(rEPA),目前大量以载体蛋白用于多种细菌多糖蛋白结合疫苗研究。rEPA的表达量受众多因素的影响,种子的制备方式就是重要因素之一。本文用长期冷冻保存的和新鲜提取的质粒分别转化宿主菌E.coliBL21(λDE3)后制备种子,并将它们和冻干保存的E.colirPE553D在相同条件下培养和诱导,经SDS-PAGE分析表明长期保存的质粒转化宿主后的重组工程菌生长速度较慢,但rEPA的表达量和新鲜质粒转化制备的工程菌基本相同,而冻干保存的工程菌E.colirPE553D几乎丧失了表达rEPA的能力。     

Induction of apoptosis in macrophage cell line,J774, by the cell-free supernatant from Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell-free supernatants from P. aeruginosa (Pa) IID1117 (elastase-positive and protease-positive) and Pa IID1130 (elastase-positive and protease-negative). After 6 hr of incubation with the cell-free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5+/-11%), but not Pa IID1130 (96.4+/-1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2+/-1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase-3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell-free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.     

Bioremediation of Textile Azo Dyes by Aerobic Bacterial Consortium Aerobic Degradation of Selected Azo Dyes by Bacterial Consortium

An aerobic bacterial consortium consisting of two isolated strains (BF1, BF2) and a strain of Pseudomonas putida (MTCC1194) was developed for the aerobic degradation of a mixture of textile azodyes and individual azodyes at alkaline pH (9-10.5) and salinity (0.9-3.68 g/l) at ambient temperature (28 +/- 2 degrees C). The degradation efficiency of the strains in different media (mineral media and in the Simulated textile effluent (STE)) and at different dye concentrations were studied. The presence of a H2O2 independent oxidase-laccase (26.5 IU/ml) was found in the culture filtrate of the organism BF2. The analysis of the degraded products by TLC and HPLC, after the microbial treatment of the dyes showed the absence of amines and the presence of low molecular weight oxidative degradation products. The enzymes present in the crude supernatant was found to be reusable for the dye degradation.     

Phosphorus composition and release in sediment bacteria of the genus Pseudomonas during aerobic and anaerobic conditions

A substantial amount of sediment phosphorus can be bound in bacterial biomass. In this study the fractional composition of phosphorus in the bacteria Pseudomonas was determined by sequential extraction with ammonium chloride, sodium hydroxide and hydrochloric acid according to the scheme of Hieltjes & Lijklema (1980). Both non-labelled and ³²P-labelled bacteria were used for fractionation. Up to 80% of the bacterial phosphorus was found in the NaOH-nRP fraction, which is in agreement with the results of Hupfer & Uhlman (1992) for Acinetobacter and activated sludge obtained with the sequential extraction scheme of Psenner et al. (1985). A significant correlation was found between bacterial biomass and the amount of phosphorus retained in the NaOH-nRP fraction when sediments were fractionated. Additional experiments with ³²P-labelled Pseudomonas in sediment-water systems were performed in order to follow bacterial release of phosphorus under aerobic and anaerobic conditions. These studies did not sustain the hypothesis that anaerobic conditions lead to rapid release of phosphorus from bacterial cells.     

Antibacterial properties of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide-specific monoclonal antibody (MAb) in a murine thigh infection model: combined effects of MAb and ceftazidime

A murine monoclonal antibody (MAb) specific for the Pseudomonas aeruginosa immunotype 1 (It-1) lipopolysaccharide (LPS) O-side chain was evaluated in terms of its in vitro bactericidal opsonophagocytic activity and in vivo bacterial killing in a mouse thigh infection model. An immunoglobulin (Ig) G2a MAb Ld3-2F2, specific for It-1 LPS, mediated in vitro complement-dependent opsonophagocytic killing at a concentration of 10 microg/ml. MAb-mediated, complement-dependent killing also occurred in the absence of neutrophils at serum concentrations in excess of 20%. A remarkable synergy was observed in opsonophagocytic assays between MAb Ld3-2F2 (0.5 microg/ml) and ceftazidime (1/4 MIC). The administration of MAb Ld3-2F2 at a level of 1 microg resulted in a significant decrease in the number of bacteria in the thigh muscles of normal mice, while 100 microg of the same MAb was required for one log of reduction in the number of bacteria at the same site in neutropenic mice. The combined therapy with MAb Ld3-2F2 and ceftazidime provided a significant reduction in the density of bacteria in the thigh muscle at 9 hr post-infection in normal and neutropenic mice as compared with those after treatment alone or with no treatment (P< 0.01). These favorable in vitro and in vivo interactions of an LPS-specific IgG MAb and ceftazidime strongly support their potential for use in therapy, combined with an LPS-reactive MAb and parenteral antipseudomonas beta-lactam antibiotics in the therapy of systemic Pseudomonas infections in normal and neutropenic hosts.     

Effect of sulbactam on anti-pseudomonal activity of beta-lactam antibiotics in cells producing various levels of the MexAB-OprM efflux pump and beta-lactamase

The beta-lactamase inhibitor, sulbactam, was tested for beta-lactamase inhibitory activity in Pseudomonas aeruginosa cells producing various levels of both the MexAB-OprM efflux pump and beta-lactamase. We found that sulbactam lowered the MICs of cefoperazone and piperacillin by inhibiting the beta-lactamase 8-fold in the cell producing a constitutively high level of AmpC-type beta-lactamase and a wild-type level of MexAB-OprM pump compared with that without sulbactam. The MICs of cefoperazone and piperacillin in the cell producing a constitutively high level of both the efflux pump and beta-lactamase under the presence of sulbactam were 8 and 4 times, respectively, lower than that without sulbactam. The MICs of sulbactam in the cell producing a constitutively high and a wild-type level of the efflux pump were 16- and 8-fold higher, respectively, than that in the mutant lacking the efflux pump. We concluded that sulbactam exerts potent beta-lactamase inhibitory activity in the cell producing a high level of efflux pump, in spite of the fact that sulbactam serves as a substrate of the MexAB-OprM pump. Increasing amounts of sulbactam over the weight of beta-lactams further strengthen the effect of beta-lactam antibiotics.     

Evaluation of the biofilm-forming ability and genetic typing for clinical isolates of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus-based PCR

Biofilm formation is an important phenotype associated with chronic Pseudomonas aeruginosa infections. In the present study, a total of 48 P. aeruginosa strains isolated from clinical specimens were examined for their biofilm-forming ability using a microtiter plate method. The different biofilm-forming abilities were demonstrated among the strains; however, most strains formed a larger biofilm than strain PAO1, a reference strain. The genetic typing was also carried out by enterobacterial repetitive intergenic consensus-based polymerase chain reaction. Although they were divided into five groups (A to E), most of the strains showing the higher biofilm-forming ability were found to be in groups D and E, suggesting a significant relationship between the biofilm-forming ability and the genetic group.     

EFFECT OF METAL TOLERANT PLANT GROWTH PROMOTING BACTERIA ON GROWTH AND METAL ACCUMULATION IN ZEA MAYS PLANTS GROWN IN FLY ASH AMENDED SOIL

The present study was undertaken to examine the effect of the application of fly ash (FA) into Garden soil (GS), with and without inoculation of plant growth promoting bacteria (PGPB), on the growth and metal uptake by Zea mays plants. Three FA tolerant PGPB strains, Pseudomonas sp. PS5, PS14, and Bacillus sp. BC29 were isolated from FA contaminated soils and assessed for their plant growth promoting features on the Z. mays plants. All three strains were also examined for their ability to solubilize phosphate and to produce Indole Acetic Acid (IAA), siderophores, and hydrogencynide acid (HCN) production. Although inoculation of all strains significantly enhanced the growth of plants at both the concentration of FA but maximum growth was observed in plants inoculated with BC29 and PS14 at low level (25%) of FA concentration. The experimental results explored the plant growth promoting features of selected strains which not only enhanced growth and biomass of plants but also protected them from toxicity of FA.