Identification,Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50°C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.Protein secretion plays important roles in bacterial life, as many of the secreted proteins are involved in biological processes that are fundamental to the survival and environmental adaptations of the cells. Gram-negative bacteria have evolved a number of secretion systems that utilize different secretion apparatus and mechanisms (9). The classical autotransporter secretion pathway belongs to the type V secretion system (8, 12, 13). Compared to other types of secretion mechanisms, the autotransporter system is unique in that all the components that are required for protein translocation are contained within a single polypeptide. Structurally, autotransporters are characterized by three domains: (i) an N-terminal signal sequence that is recognized by the Sec translocon; (ii) a central passenger domain (or α-domain) that contains the effector molecule and is highly variable; and (iii) a C-terminal translocation domain (or β-/autotransporter domain) that is conserved in length (250 to 300 amino acids) but varies in primary structure (7, 12, 55). In most cases, the β-domain contains 12 antiparallel strands of 9 to 12 residues that, upon integration into the outer membrane, form a β-barrel conformation (27, 57). Another conserved feature of the β-domain is the presence at the C terminus of a sequence motif, (Y/V/I/F/W)-X-(F/W), that is characterized by alternating hydrophobic and hydrophilic residues and ends with either a tryptophan or a phenylalanine (12, 16, 27). The integrity of this end motif seems to be required for protein translocation, as deletion of certain residues in this sequence impairs protein secretion. The secretion process of the autotransporter is initiated by the signal sequence, which directs the translocation of the protein precursor across the inner membrane into the periplasmic space via the Sec system. Once inside the periplasm, the β-domain inserts into the outer membrane and adopts the structure of a β-barrel through which the passenger domain is translocated to the cell surface, where it may exist as a membrane-anchored protein covalently linked to the β-domain or be cleaved from the β-domain as a result of proteolysis (7).Since the discovery of the gonococcal immunoglobulin A1 protease (35), the first autotransporter, and especially with the advent of genome sequencing technology, autotransporters have been identified in many bacterial species (18, 19, 28, 33). Functions assigned to autotransporters are mostly associated with bacterial pathogenicity, which includes adhesion and invasion into host cells, biofilm formation, and cytotoxicity (11, 54). In the present study, we identified and analyzed an autotransporter, PfaI, from a pathogenic Pseudomonas fluorescens strain isolated from diseased fish. We found that, like many of the autotransporters identified in other pathogens, PfaI is a virulence factor that is involved in interactions with host cells and modulation of host immune responses via a protease effector. In addition, we found that the autotransporter property of PfaI could be exploited for the delivery and surface display of an immunoprotective antigen.     

一株联苯降解菌的特性及鉴定*

从华北油田污染土壤中筛选出一株能够以联苯为唯一碳源和能源生长的菌株。该菌生长的最适联苯浓度为0．2％～0．4％,在联苯浓度为0．1％的培养基中培养36h后降解率达99．8%。该菌还可以降解苯甲酸钠、邻苯二酚、间苯二酚、对苯二酚和多氯联苯Aroclorl221、Aroclorl242等芳香族化合物。通过16S rDNA基因序列分析鉴定该菌为嗜吡啶红球菌(Rhodococcus pyridinovorans)。     

一个甲烷氧化菌株的分离、鉴定及其特性研究

实验室自行设计方案分离多株以甲烷为唯一碳源的菌株, 对其中的1株QJ16进行了研究, 根据该菌株形态特征与16S rDNA序列的同源性分析, 证实该菌株是一个与其最近的甲基单胞菌属各成员都不相同的菌株。对该菌株的培养条件和利用甲烷的特性进行研究结果表明, 氮源以氯化铵和硝酸钾共同作用最好, 碳源以甲烷最佳, 最佳生长温度为30℃, 最佳生长pH为6~7; 在批式实验时菌株利用甲烷的最适pH为6.5左右, 微量元素Cu2+的浓度为15 mmol/ L。     

一个甲烷氧化菌株的分离、鉴定及其特性研究

实验室自行设计方案分离多株以甲烷为唯一碳源的菌株,对其中的1株QJ16进行了研究,根据该菌株形态特征与16S rDNA序列的同源性分析,证实该菌株是一个与其最近的甲基单胞菌属各成员都不相同的菌株.对该菌株的培养条件和利用甲烷的特性进行研究结果表明,氮源以氯化铵和硝酸钾共同作用最好,碳源以甲烷最佳,最佳生长温度为30℃,最佳生长pH为6～7;在批式实验时菌株利用甲烷的最适pH为6.5左右,微量元素Cu2 的浓度为15 μmol/L.     

Isolation and Characterization of a Pseudomonas fluorescens Strain Suppressive to Bipolaris maydis

Strain PJ0210, identified as Pseudomonas fluorescens, inhibited Bipolaris maydis both in vitro. The absence of detectable inhibitory substances assayed by a variety of methods indicated nutrient competition as the operative component of antagonism. This was further supported by observations of reduced inhibition by addition of glucose both in vitro and in vivo. Strain PJ0210 was adaptecd to the phyllosphere showing good survivability and colonizing ability both inplant house and field trials. Disease suppression was generally correlated with population sizes of strain PJ0210. Population sizes in the field were lower than those recorded in the plant house with a concomitant reduction in disease suppressive ability, of strain PJ0210.     

Isolation and Characterization of A Metal-resistant Pseudomonas Aeruginosa Strain

Summary The use of microorganisms to remove heavy metals from industrial effluent is an area of extensive research and development. Attempts have been made to isolate and characterize metal-resistant microorganisms from treated oil mill industry effluent wastewater samples. The metal-resistant organisms that showed values of minimum inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800 ppm level were screened. A potent metal-resistant organism, isolate BC15 from the wastewater samples was tentatively identified as Pseudomonas sp. Detailed analysis of morphological, biochemical and 16S rDNA sequence of the isolate revealed that it is closely related to Pseudomonas aeruginosa (94%). Pseudomonas BC15 was capable of absorbing 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100 mg of each heavy metal per liter. The multiple metal tolerance of this strain was also associated with resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin.     

Genome Sequence of Pseudomonas putida Strain SJTE-1, a Bacterium Capable of Degrading Estrogens and Persistent Organic Pollutants

Pseudomonas putida strain SJTE-1 can utilize 17β-estradiol and other environmental estrogens/toxicants, such as estrone, and naphthalene as sole carbon sources. We report the draft genome sequence of strain SJTE-1 (5,551,505 bp, with a GC content of 62.25%) and major findings from its annotation, which could provide insights into its biodegradation mechanisms.     

Genome Sequence of Pseudomonas aeruginosa Strain SJTD-1, a Bacterium Capable of Degrading Long-Chain Alkanes and Crude Oil

Pseudomonas aeruginosa strain SJTD-1 can utilize long-chain alkanes, diesel oil, and crude oil as sole carbon sources. We report the draft genome sequence of strain SJTD-1 (6,074,058 bp, with a GC content of 66.83%) and major findings from its annotation, which could provide insights into its petroleum biodegradation mechanism.     

Isolation and Characterization of a Pseudomonas Strain Producing Glutaryl-7-Aminocephalosporanic Acid Acylase

Several screening methods were developed for the selection of Pseudomonas strains capable of hydrolyzing glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid. An isolate exhibiting high acylase activity, designated BL072, was identified as a strain of Pseudomonas diminuta. It grew optimally at pH 7 to 8 and at a temperature of 32 to 40°C, but acylase activity was highest when the strain was grown at 28°C. Mutants of BL072 were generated by nitrosoguanidine treatment and screened for increased production of glutaryl-7-aminocephalosporanic acid acylase. A superior mutant gave a fourfold increase in acylase titer. The cell-associated acylase had similar activities against various glutaryl-cephems but had undetectable activity against cephalosporin C. This acylase may prove useful for the conversion of cephalosporin C to 7-aminocephalosporanic acid.     

Characterization of a Pseudomonas sp. Capable of Aniline Degradation in the Presence of Secondary Carbon Sources

Pseudomonas strain K1 is a gram-negative rod which grows aerobically on minimal media containing aniline with a doubling time of 2 h at 30°C. The half-saturation parameter for aniline metabolism by aniline-grown cells was 3.8 μmol · liter⁻¹. Concentrations of aniline as low as 50 nM were metabolized. Neither substituted anilines nor other aromatic compounds (other than aromatic amino acids) supported growth. Cells grew as fast on aniline as on nonaromatic substrates such as lactate. The aromatic ring was cleaved via the meta pathway. Catechol 2,3-oxygenase activity was induced by aniline, even in cultures containing alternative carbon sources such as lactate. Cultures grown on a mixture of aniline and lactate mineralized aniline in the presence of the second substrate. Lactate-grown cultures lacked catechol oxygenase activity, and resting cells from these cultures did not respire aniline. Resting cells from aniline-grown cultures exhibited high respiratory activity upon the addition of aniline or catechol, some activity with toluidine, and no activity after addition of a wide variety of other aromatic compounds, including dihydroxybenzylamine, chloroanilines, ethylanilines, aminophenols, aminobenzoates, and dihydroxybenzoates. Although substituted anilines were not metabolized, 3-or 4-chloroaniline did induce the enzymes for aniline oxidation.     

Detection and Characterization of Plasmids in Pseudomonas aeruginosa Strain PAO

Using a variety of techniques, it has been established that the sex factor FP2 has a density of 1.717 g/cm(3) (corresponding to a guanine plus cytosine [G + C] content of 58%) and a mean circular contour length of 28.5 +/- 0.6 mum (corresponding to a molecular weight of 59 x 10(6)). Another sex factor FP39 has a density of 1.719 g/cm(3) (corresponding to a G + C content of 60%) and a mean circular contour length of 26.5 +/- 0.5 mum (corresponding to a molecular weight of 55 x 10(6)). It appears that all, or nearly all, of the FP sex factor deoxyribonucleic acid occurs as covalent circular molecules under the conditions employed. In addition, these procedures have been used to demonstrate that strain PAO, a naturally occurring female (i.e., FP(-)) strain of Pseudomonas aeruginosa, harbors a number of cryptic plasmids having similar densities to the bulk of the cell deoxyribonucleic acid (1.726 g/cm(3)) and occurring as covalent circular molecules.     

卡马西平降解菌的筛选及降解特性研究

药品和个人护理品类污染物日益成为新兴污染物研究的重点, 药品卡马西平因具有多种药效被广泛使用, 在环境中频繁被检出, 且浓度较高, 不易去除, 通常作为环境中药品和个人护理品污染状况的指示化合物。本研究从某制药厂的污水处理厂中分离到一株细菌HY-7, 能以卡马西平为唯一碳源、氮源和能源生长, 通过生理生化以及16S rDNA、gyrB基因序列分析鉴定并命名为Acinetobacter sp. HY-7。该菌株生长和降解卡马西平的最适条件为25°C和pH 6.0, 经HPLC分析10 d内能将初始浓度为20 mg/L的卡马西平降解48%。菌株HY-7还能以邻苯二酚、吲哚、萘、蒽等芳香族化合物为唯一碳源生长。     

Isolation and Characterization of a Pseudomonas Strain That Restricts Growth of Various Phytopathogenic Fungi

The characterization of a novel Pseudomonas strain exhibiting antagonism towards many important corn fungal pathogens is presented. This strain was isolated from the caryopses of the grass Tripsacum dactyloides and was identified as Pseudomonas cepacia. The antagonistic activity is due to the production of an antifungal compound. The chromatographic properties of this partially purified compound isolated from growth medium differ from those reported previously for other pseudomonads. The suppression of the growth of economically important phytopathogens by this strain and by the partially purified compound indicates a potential biocontrol agent.     

1株用于甜瓜采后病害生物防治的假单胞菌株的分离及鉴定

甜瓜以营养丰富口味独特而深受世人青睐。同时,其采后病害高效、"绿色"防治药剂的缺乏也成为多年来困扰甜瓜生产的难题。本文采用直接生物测定的方法,从健康的甜瓜表面分离到1株有生防潜能的菌株X4。该菌株对引起甜瓜主要采后病害的粉红单端胞菌(Trichothecium roseum)、镰刀菌(Fusarium spp.)和链格孢菌(Alternaria alternata)的防治效果分别达到了90.6%、84.4%和67.4%;定殖实验显示,接菌4 d后该菌在瓜内数量可上升并稳定至1.1×10~6CFU/ml;通过16S rDNA测定、形态特征观察及生理生化测定结果表明该菌为Pseudomonas fluorescens biovarⅢ。     

Characterization of Fluorescent and Nonfluorescent Peptide Siderophores Produced by Pseudomonas syringae Strains and Their Potential Use in Strain Identification

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in β-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.     

Isolation,Characterization of a Strain Capable of Degrading Imazethapyr and Its Use in Degradation of the Herbicide in Soil

One strain of bacterium IM-4, capable of degrading imazethapyr (IMZT), was isolated from the IMZT-contaminated soil. The isolate was identified as Pseudomonas sp. according to its physiological characteristics, biochemical tests, and 16S rRNA gene phylogenetic analysis. This strain could utilize IMZT as the sole carbon and energy source. About 73.4% of the 50 mg l⁻¹ initially added IMZT was degraded after 7 days of inoculation with strain IM-4. This strain also showed the capability to degrade other imidazolinone herbicides such as imazapyr, imazapic, and imazamox. The inoculation strain IM-4 to soil treated with IMZT resulted in a higher degradation rate than in noninoculated soil regardless if the soil was sterilized or nonsterilized. Inoculation of strain IM-4 could also mitigate the phytotoxic effects of IMZT on the growth of maize.     

Identification and Characterization of a Gamma-Glutamyl Transpeptidase from a Thermo-Alcalophile Strain of Bacillus pumilus

A γ-glutamyltranspeptidase (GGT, E.C. 2.3.2.2) was isolated from a strain (A8) originating from Lake Bogoria (Kenya) and homologous with Bacillus pumilus. This GGT shows an optimal activity at pH 8.9 and 62°C. The enzyme is thermostable up to 43°C. The best reagent among the potential inhibitors was shown to be DON, which is an inhibitor highly specific for GGTs. Gly-Gly-Ala, Gly-Gly-Gly and Gly-Gly were identified as the best acceptors for the transpeptidation reactions catalyzed by the enzyme. The SDS-PAGE study revealed that the enzyme consists of two non-identical subunits (38,000 and 23,000). Only the large subunit was active when the enzyme was dissociated under denaturing conditions. The behavior of the native enzyme suggests that the active site of the large subunit is masked by the small subunit.     

Isolation and Characterization of a Hydrogen Sulfide-Removing Bacterium,Pseudomonas sp. Strain DO-1

Five microbial strains that removed hydrogen sulfide (H₂S) or methylmercaptan (CH₃SH) gas were newly isolated from soil samples. Strain DO-1, one of the isolates, was identified as a member of Pseudomonas sp., and it’s immobilized cells removed 1 or 10 ppm of H₂S gas within 2 hours. When strain DO-1 was cultured aerobically in a flask containing nutrient broth medium, the deodorizing activity increased, depending on the growth of the culture, and the maximum activity was obtained after 48 hours. Even though the immobilized cells were stored at 4 or 25°C in sealed bottles for 6 months, the deodorizing activity remained. Throughout this study, strain DO-1 removed H₂ S gas without preliminary feeding or exposure to sulfur com-pounds as growth substrates or inducers. These characteristics are advantageous for the deodorization of the malodorous gases surrounding us in daily life.