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.     

Expression of luminescence in Photobacterium phosphoreum: Na+ regulation of in vivo luminescence appearance

In Photobacterium phosphoreum strain 496, growth and luminescence in a complex medium are optimal with 3% NaCl. However, in the same medium with 1% NaCl growth is similar, but the development of bioluminescence does not occur. In cells grown to mid or late-log phase in 1% NaCl, light emission can be triggered by the addition of NaCl, but the time required for its appearance is quite long, at least 30–45 min. The synthesis of m-RNA and protein are required for the development of luminescence, but the long time interval suggests that some intermediate steps are required. The time required is not less in conditioned 3% NaCl medium.     

Assessing the influence of the entomopathogenic nematode Heterorhabditis baujardi LPP7 (Rhabiditina) on embryogenesis and hatching of the plant-parasitic nematode Meloidogyne mayaguensis (Tylenchina)

Two assays were conducted to assess the influence of infective juveniles (IJs) of Heterorhabditis baujardi LPP7 on the embryogenesis and hatching of Meloidogyne mayaguensis. In the first assay, eggs were incubated in water alone or in the presence of infective juveniles, and completion of embryogenesis was evaluated 14 days later. In the second assay, unhatched second-stage juveniles were incubated in distilled water alone or in the presence of infective juveniles. Cumulative hatching was compared at various time intervals. Embryogenesis was not affected, whereas second-stage juveniles hatching was delayed probably because of the eggs permeability to noxious metabolites released by Photorhabdus luminescens, which is the bacterial symbiont of H. baujardi.     

kpsE和kpsD双基因缺失显著降低肠道外致病性大肠杆菌的毒力

【目的】探究荚膜对肠道外致病性大肠杆菌致病作用的影响。【方法】选取负责荚膜多糖转运的基因kpsE和kpsD,利用λRed重组系统构建APEC E058和UPEC U17荚膜缺失株E058ΔkpsED和U17ΔkpsED,并通过一系列的体内及体外试验对其生物学特性及致病性进行研究。【结果】双基因缺失株的生长速度较野生株没有明显差异,但缺失株抗血清补体杀菌能力和抗鸡巨噬细胞HD-11细胞吞噬能力显著下降。1日龄雏鸡LD50致病性试验结果显示,缺失株E058ΔkpsED和U17ΔkpsED对鸡失去致病力,而回复株毒力恢复至野生株水平;35日龄SPF鸡体内动态分布和竞争试验显示ΔkpsED缺失株在鸡体内定殖能力和竞争性生长能力显著下降,表明kpsED双基因的缺失能显著降低APEC E058和UPEC U17的致病力。【结论】荚膜与肠道外致病性大肠杆菌的致病性相关,是其重要的毒力因子。     

Changes in the composition of anionic membrane phospholipids influence protein secretion and cell envelope biogenesis in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD102 and HDL11 with controlled synthesis of the anionic phospholipids, phosphatidylglycerol and cardiolipin. Inactivation of the pgsA gene responsible for the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. An increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of the outer membrane lipopolysaccharides and lipopolyproteins, which determine its barrier properties. The results obtained show that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between protein secretion and biogenesis of cell envelope components.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 179–184.Original Russian Text Copyright © 2005 by Anisimova, Badyakina, Vasileva, Nesmeyanova.     

A novel transposon-based method for elimination of large bacterial plasmids

Elimination or modification of large plasmids of bacteria is often an essential step in functional analysis of these replicons. However, the conventional plasmid-curing procedures such as ethidium bromide and heat treatment are insufficient in many cases. For instance, curing of the large virulence plasmid of Salmonella Enteritidis 2,102 has failed when these treatments were applied. To overcome the difficulties, a two-step transposon-based curing method has been developed. First, a Tn10-based transposable unit carrying a Km(R) marker gene and the joined IS30 ends transposes from a replication deficient conjugative plasmid into the target replicon. Then, the inducible IS30 transposase, using the highly reactive joined IS30 ends, mediates deletions or gives rise to the loss of the target plasmid. The efficiency of the method has been monitored by the frequency of Km(S) colonies after induction of IS30 transposase, and it was shown that the Km(S) phenotype often accompanied the complete loss of the virulence plasmid or the formation of deletion derivatives. The procedure has been successfully applied also in removing the large virulence plasmid from enterotoxigenic Escherichia coli (ETEC O147), suggesting that the transposon-based method can be a useful tool for eliminating native plasmids in several bacteria.     

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli.     

和厚朴酚对大肠埃希氏菌生物被膜形成的抑制机制

【目的】研究不同浓度的和厚朴酚(honokiol)抑制大肠埃希菌(Escherichia coli)的供试菌株10389生物被膜(biofilm,BF)形成的作用机制。【方法】用氯化三苯基四氮唑比色法(TTC)和四唑盐减低法(XTT)测定honokiol抑制E.coli10389生物被膜形成的药物最低抑菌浓度(MIC)和最低杀菌浓度(MBC)及其抑制作用与时间的关系;通过qRT-PCR法检测不同浓度的honokiol对E. coli 10389生物被膜形成基因和群体感应系统相关基因表达量的影响;通过生物发光法和qRT-PCR法检测亚-MIC honokiol对E. coli 10389呋喃糖基硼酸二酯(AI-2)及其调控的与生物被膜形成相关的下游基因表达量的影响。【结果】Honokiol能抑制E.coli10389生物被膜的形成,但不同浓度的honokiol抑制E. coli 10389 BF形成的作用机制不同。其中,与对照组相比,MIC的honokiol能使E. coli 10389 BF形成相关基因编码毒素(hha)和细菌酸性调节因子(ari R) mRNA的表达量显著提高,抗毒素...     

The influence of local plant growth conditions on non-fastidious bacterial contamination of meristem-tips ofHydrangea culturedin vitro

A high incidence ofin vitro bacterial contamination (69%) has been detected in meristem-tip explants ofHydrangea from widely differing locations in Ireland and the UK. The bacteria were characterised by API 20E biochemical test kits and by fatty acid profile analysis. The results obtained from the different methods were compatible and anomalies were explicable in terms of the limitations of the respective methods. The majority of the isolates were environmental or animal-associated bacteria with clusters ofEnterobacter isolates in Dublin, and ofEscherichia coli in the main Cork location. A cluster of Pseudomonads was detected in the Derby (UK) plants. The main association was between the location and the contaminant clusters. The main finding was that the nature of organic soil amendments may influence inoculum for the contamination of plants and the conclusion was that fertilisation with organic materials should be avoided in the preparation of plants for micropropagation.     

Development of a bioassay reagent usingPhotobacterium phosphoreum as a test for the detection of aquatic toxicants

Lyophilized cells ofPhotobacterium phosphoreum, rehydrated in 2% (w/v) NaCl in 0.022M KH₂PO₄ at pH 7.0, were used for developing an assay to test the acute toxicity of organic and inorganic compounds. The standardized assay gave good reproducibility of results with 11 organic and four inorganic compounds. Results were compared with reported data obtained with other test organisms and are within their sensitivity ranges. Environmental screening of wastes from oil and petrochemical industries is discussed.     

Iron control of the Vibrio fischeri luminescence system in Escherichia coli

Iron influences liminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB ⁺ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICD ABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di (o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB ⁺ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of -galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8–9 fold each for tonB, 2–3 fold each for tonB ⁺) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.     

Implication of a critical residue (Glu175) in structure and function of bacterial luciferase

Structural properties of a bacterial luciferase mutant, evolved by random mutagenesis, have been investigated. Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. By random mutagenesis, one of the mutants generated by a single mutation on LuxA at position 175 (E175G) resulted in the "slow decay" Xenorhabdus luminescens luciferase was converted into a luciferase with a significantly more rapid decay rate [Hosseinkhani, S., Szittner, R. and Meighen, E.A. (2005) Biochemical Journal 385, 575-580]. A single mutation (E175G), in a loop that connects alpha helix 5 and beta sheet 5 brought about changes in the kinetic and structural properties of the enzyme. Enhancement of tryptophan fluorescence was observed with a lower degree of fluorescence quenching by acrylamide upon mutation. Near- and far-UV circular dichroism spectra of the native and mutant forms suggested formation of an intermediate structure, further supported by 8-anilino-1-naphthalene-sulphonic acid (ANS) fluorescence which indicated lower exposure of hydrophobic residues as a result of mutation. Fluorescence quenching studies utilizing acrylamide indicated a more accessible fluor for the native form. Thus, the E175G point mutation appears to change the enzymatic decay rate by inducing a substantial tertiary structural change, without a large effect on secondary structural elements, as revealed by Fourier transform IR spectroscopy. Overall, the mutation caused structural changes that go beyond the simple change in orientation of Glu175.     

New developments in the understanding of the cation diffusion facilitator family

Cation diffusion facilitator (CDF) proteins are a phylogenetically ubiquitous family of intermembrane transporters generally believed to play a role in the homeostasis of a wide range divalent metal cations. CDFs are found in a host of membranes, including the bacterial cell membrane, the vacuolar membrane of both plants and yeast, and the golgi apparatus of animals. As such, they are potentially useful in the engineering of hyperaccumulative phytoremediation systems. While not yet sufficient for reliable biotechnological manipulation, characterization of this family is proceeding briskly. Experimental data suggests that CDFs are generally homodimers that use proton antiport to drive substrate translocation across a membrane. This translocation of both substrate and protons is likely mediated by a combination of histidines, aspartates, and glutamates. Functional data has suggested that CDFs are not limited to metal homeostasis roles, as some appear to be determinants in the operation of high-volume metal resistance systems, and others may facilitate cation-donation as a means of signal transduction. This review seeks to give an overview of the data prompting these conclusions, while presenting additional data whose interpretation is still contentious.     

Two new subspecies of Photorhabdus luminescens, isolated from Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae): Photorhabdus luminescens subsp. kayaii subsp. nov. and Photorhabdus luminescens subsp. thracensis subsp. nov

Bacterial isolates from nematodes from Turkish soil samples were initially characterized by molecular methods and seven members of the genus Photorhabdus identified to the species level, using riboprint analyses and metabolic properties. Strain 07-5 (DSM 15195) was highly related to the type strain of Photorhabdus luminescens subsp. laumondii DSM 15139T, and was regarded a strain of this subspecies. Strains 1121T (DSM 15194T), 68-3 (DSM 15198) and 47-10 (DSM 15197) formed one, strain 39-8T (DSM 15199T), 39-7 (DSM 15196) and 01-12 (DSM 15193) formed a second cluster that branched intermediate the three subspecies of Photorhabdus luminescens. Based upon moderate 16S rRNA gene sequence similarities and differences in metabolic properties among themselves and with type strains of the three subspecies we consider the two clusters to represent two new subspecies of Photorhabdus luminescens for which the names Photorhabdus luminescens subsp. kayaii, type strain 1121T (DSM 15194T, NCIMB 13951T), and Photorhabdus luminescens subsp. thracensis subsp. nov., type strain 39-8T (DSM 15199T, NCIMB 13952T) are proposed.     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.     

不同浓度外源乙酸钠对耐乙酸大肠埃希菌DA19代谢和关键酶酶活的影响

为研究外源乙酸钠对大肠埃希菌DA19生长代谢的影响,将该菌株在氮源限制基本培养基及添加不同浓度乙酸钠的氮源限制基本培养基中连续培养,测定稳态时生长代谢参数和胞内关键酶酶活。与MN培养基相比,葡萄糖比消耗速率和延胡索酸比生成速率随外源乙酸钠质量浓度增加而逐渐下降,丙酮酸比生成速率则随外源乙酸钠质量浓度增加而明显增加,而乙酸比生成速率则明显降低(除9 g/L乙酸钠外)。磷酸果糖激酶、异柠檬酸脱氢酶、异柠檬酸裂解酶、苹果酸脱氢酶、磷酸烯醇式丙酮酸羧化酶和乙酸激酶酶活随外源乙酸钠质量浓度增加而呈先下降后上升的趋势,而6-磷酸葡萄糖脱氢酶则随着外源乙酸钠质量浓度增加而逐渐降低。为了应对外源乙酸钠压力,大肠埃希菌DA19的生长代谢和中心代谢途径酶活都发生了明显改变。     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.