Effects of pH and phosphate on the production of struvite by Myxococcus xanthus

We studied the influence of pH and the phosphate content of the culture medium on the precipitation of struvite by Myxococcus xanthus, a bacterium that undergoes autolysis at the end of its exponential growth phase in liquid cultures. The best results were obtained with pH values between 7.2 and 8.0 and with a phosphate concentration of 10 mM. Our studies reveal for the first time that the precipitation of struvite always begins at the onset of autolysis and that culture conditions favoring the early occurrence of autolysis also enhance struvite production.     

黄色粘球菌发育阶段外膜蛋白表达的变化

【目的】黄色粘球菌是研究原核发育的一种模式生物,对其膜蛋白的研究仍然十分缺乏。【方法】利用6种预测软件,在黄色粘球菌的基因组中筛选编码外膜蛋白(OMP)的基因。根据报告基因lacZ,检测这些基因在营养性生长和发育阶段的表达。【结果】基于生物信息学分析,筛选出11个编码外膜蛋白的基因。其中2个基因(MXAN3106和MXAN3883)在发育阶段表达量上升,它们分别编码Secretin家族和Fimbrial usher protein (FUP)家族转运蛋白。其余9个基因在发育起始阶段表达量降低或保持较低水平,它们均编码TonB依赖型受体或外排蛋白。【结论】这些数据提示,黄色粘球菌由生长到发育的转换过程,伴随着膜蛋白表达的显著变化。     

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch

Abstract The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.     

Transcriptional regulation of genes required for antibiotic TA synthesis in Myxococcus xanthus

Abstract Regulatory mutants were isolated for six genes required for the production of antibiotic TA in Myxococcus xanthus , using Tn5lac as a promoter probe. Two of the regulatory mutants were closely linked to their corresponding Tn5lac insertions and three were separated by at least 40 kb from their original insertions. All of the β-galactosidase-overproducing mutants showed higher specific activities than their parent strains in three different media. The specific activity was higher throughout the growth cycle, starting earlier and finishing higher. In addition to helping to understand the mechanism of repression of TA genes, the mutants are potentially useful in the construction of antibiotic-overproducing strains.     

Natural variation of gliding motility in a centimetre-scale population of Myxococcus xanthus

A major challenge in microbial evolutionary ecology is to understand how fitness-related traits vary in natural populations of microorganisms at defined spatial scales and subsequently to identify the forces that maintain such variation. The Gram-negative soil bacterium Myxococcus xanthus is a model system for the study of gliding motility, which is driven by two complementary motility systems in this species and is central to its social lifestyle. We tested whether the ecological context of a centimetre-scale M. xanthus population allows the coexistence of diverse motility-related phenotypes. Swarming rates among 26 clones isolated at the centimetre scale were found to vary greatly in multiple laboratory environments. This variation appears to be motility-specific, as it is not explained by a correlated variation in intrinsic growth rate. In contrast to the common reference strain DK1622, most isolates swarmed faster on hard agar than on soft agar, highlighting the difficulty of inferring species characteristics from laboratory reference strains. These isolates also varied greatly in swarm morphology and in the effect of nutrient limitation on swarming rate. Our results show that diverse swarming phenotypes can coexist in a small-scale bacterial population.     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

Dual targeting of Myxococcus xanthus protoporphyrinogen oxidase into chloroplasts and mitochondria and high level oxyfluorfen resistance

Much attention has been paid to the signal sequences of eukaryotic protoporphyrinogen oxidases (protoxes); both the organelles targeted by protoxes and the role of protoxes in conferring resistance against protox‐inhibiting herbicides, such as oxyfluorfen, have been examined. However, there have been no reports on the translocation of prokaryotic protoxes. This study investigated the targeting ability of Myxococcus xanthus protox in vitro and in vivo. In an in vitro translocation assay using a dual import system, M. xanthus protein was detected in chloroplasts and mitochondria, suggesting that the M. xanthus protox protein was targeted into both organelles. In order to confirm the in vitro dual targeting ability of M. xanthus, we used a stable transgenic strategy to investigate dual targeting in vivo. In transgenic rice plants overexpressing M. xanthus protox, M. xanthus protox antibody cross‐reacted with proteins with predicted molecular masses of 50 kDa from both chloroplasts and mitochondria, and this in vivo transgene expression corresponded to a prominent increase in chloroplastic and mitochondrial protox activity. Seeds from the transgenic lines M4 and M7 germinated in solid Murashige and Skoog media of up to 500 µm of oxyfluorfen, whereas wild‐type seeds did not germinate in 1 µm . After 4‐week‐old‐rice plants were treated with oxyfluorfen for 3 d, lines M4 and M7 exhibited normal growth, whereas the wild‐type line was severely bleached and necrotized. The herbicidal resistance is attributed to the insignificant accumulation of photodynamic protoporphyrin IX in cytosol because the high chloroplastic and mitochondrial protox activity in oxyfluorfen‐treated transgenic lines, compared with that in oxyfluorfen‐treated and untreated wild‐type plants, metabolizes protoporphyrinogen IX to chlorophyll and heme. A practical application of the dual targeting of M. xanthus protox for obtaining outstanding resistance to peroxidizing herbicides is discussed.     

A Myxococcus xanthus rppA-mmrA double mutant exhibits reduced uptake of amino acids and tolerance of some antimicrobials

Myxococcus xanthus RppA and MmrA are homologous to methyl-accepting chemotaxis proteins (MCPs) and to multidrug transporters, respectively. We reported previously that rppA-mmrA double mutant exhibited reduced colony expansion, agglutination, and polysaccharide levels. We have demonstrated here that the rppA-mmrA mutant also exhibited reduced amino acid uptake. Furthermore, the double mutant appeared to be more susceptible to some antimicrobial agents, such as streptomycin, ethidium bromide and norfloxacin, than the wild-type. These phenotypes were not shown in the rppA or mmrA single mutant. These results indicate that M. xanthus RppA and MmrA are also involved in the uptake of amino acids and efflux of some antimicrobial agents.     

A Myxococcus xanthus CbpB containing two cAMP-binding domains is involved in temperature and osmotic tolerances

Our previous data indicated that a Myxococcus xanthus sensor-type adenylyl cyclase (CyaA) functions in signal transduction during osmotic stress. However, the cAMP-mediated signal transduction pathway in this bacterium was unknown. Here, we isolated a clone from a M. xanthus genomic DNA library using oligonucleotide probes designed based on the conserved cAMP-binding domains of the cAMP-dependent protein kinase (PKA) regulatory subunits. The clone contained two open-reading frames (ORFs), cbpA and cbpB, encoding hydrophilic proteins with one and two cAMP-binding domains, respectively. The CbpB exhibited partial primary structural similarity to PKA regulatory subunits. cbpA and cbpB mutants, generated by gene disruption, showed normal growth, development and spore germination. However, the cbpB mutant cultured under high- or low-temperature conditions exhibited a marked reduction in growth. cbpB mutant cells were also more sensitive to osmotic stress than wild-type cells. The cbpA mutant possessed normal resistance to such stress. The phenotype of cbpB mutant was similar to those of PKA regulatory subunit mutants of some eukaryotic microorganisms.     

Nonreductive Biomineralization of Uranium(VI) Phosphate Via Microbial Phosphatase Activity in Anaerobic Conditions

The remediation of uranium from soils and groundwater at Department of Energy (DOE) sites across the United States represents a major environmental issue, and bioremediation has exhibited great potential as a strategy to immobilize U in the subsurface. The bioreduction of U(VI) to insoluble U(IV) uraninite has been proposed to be an effective bioremediation process in anaerobic conditions. However, high concentrations of nitrate and low pH found in some contaminated areas have been shown to limit the efficiency of microbial reduction of uranium. In the present study, nonreductive uranium biomineralization promoted by microbial phosphatase activity was investigated in anaerobic conditions in the presence of high nitrate and low pH as an alternative approach to the bioreduction of U(VI). A facultative anaerobe, Rahnella sp. Y9602, isolated from soils at DOE's Oak Ridge Field Research Center (ORFRC), was able to respire anaerobically on nitrate as a terminal electron acceptor in the presence of glycerol-3-phosphate (G3P) as the sole carbon and phosphorus source and hydrolyzed sufficient phosphate to precipitate 95% total uranium after 120 hours in synthetic groundwater at pH 5.5. Synchrotron X-ray diffraction and X-ray absorption spectroscopy identified the mineral formed as chernikovite, a U(VI) autunite-type mineral. The results of this study suggest that in contaminated subsurfaces, such as at the ORFRC, where high concentrations of nitrate and low pH may limit uranium bioreduction, the biomineralization of U(VI) phosphate minerals may be a more attractive approach for in situ remediation providing that a source of organophosphate is supplied for bioremediation.     

An unusual beta-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthus

The antibiotic TA of Myxococcus xanthus is produced by a type-I polyketide synthase mechanism. Previous studies have indicated that TA genes are clustered within a 36-kb region. The chemical structure of TA indicates the need for several post-modification steps, which are introduced to form the final bioactive molecule. These include three C-methylations, an O-methylation and a specific hydroxylation. In this study, we describe the genetic analysis of taK, encoding a specific polyketide beta-ketoacyl:acyl carrier protein synthase, which contains an unusual beta-ketoacyl synthase and acyltransferase motifs and is likely to be involved in antibiotic TA post-modification. Functional analysis of this beta-ketoacyl:acyl carrier protein synthase by specific gene disruption suggests that it is essential for the production of an active TA molecule.     

粘球菌基因功能和表达情况分析质粒pZCY11的构建及其应用

【目的】旨在构建一个用于粘球菌基因插入失活、同时可通过报告基因分析插入位点基因表达情况的质粒载体,并应用该质粒对黄色粘球菌MXAN1334基因功能和表达情况进行分析。【方法】通过PCR、酶切和连接等方法构建质粒载体pZCY11。从黄色粘球菌DK1622基因组上PCR扩增MXAN1334基因内部部分片段,插入载体pZCY11上lacZ基因的上游,构建重组质粒pZCY13,将其转入DK1622菌株,获得MXAN1334基因插入失活突变株ZC16-18。【结果】基因功能和表达情况分析质粒载体pZCY11含有抗性标记基因aph、自杀性质粒复制子OriR6K和无启动子的报告基因lacZ。突变株ZC16-18在CTT软硬琼脂平板上菌落扩展结果显示,MXAN1334基因插入失活会造成黄色粘球菌S运动能力缺陷。通过X-gal检测突变株ZC16-18中β-半乳糖苷酶酶活,实验结果显示,含有X-gal的平板上培养的ZC16-18菌落呈现蓝色,表明MXAN1334基因能够表达;颜色呈现的时间分析结果显示,MXAN1334基因表达时间较早。【结论】构建的质粒载体pZCY11不仅能够对目的基因进行功能的分析,而且能够同时通过报告基因分析基因的表达情况,可简化粘球菌中基因功能及表达情况的研究。     

Accumulation of carotenoids in structural and regulatory mutants of the bacterium Myxococcus xanthus

Summary Accumulation of carotenoids in Myxococcus xanthus is absolutely dependent on illumination with blue light. We report the analysis of the carotenoids of dark- and light-grown cultures of the wild type and several previously characterized mutants. A carR mutant produces the same carotenoids in the dark as the wild type grown in the light. This agrees with previous evidence indicating that the carR gene codes for a general negative regulator of the system. A cis-dominant mutation in the gene carA causes constitutive expression of the light-inducible gene carB, which is linked to carA. In the dark, the carA mutant produces high levels of phytoene, the first C₄₀ colourless carotenoid precursor; in the light, it produces the same carotenoids as the wild type. Since a mutation in carB blocks accumulation of phytoene, we propose that carB, and probably other linked genes also controlled by carA, code for enzymes involved in the synthesis of phytoene. This is virtually the only carotene accumulated by strains mutated in the gene carC, which is unlinked to the others. Thus carC codes for phytoene dehydrogenase, the enzyme that converts phytoene into coloured carotenoids. The results presented here also provide evidence for control of carotenogenesis by an endproduct that is independent of the blue light effect.     

Transmission electron microscopic and X-ray absorption fine structure spectroscopic investigation of U repartition and speciation after accumulation in renal cells

After environmental contamination, U accumulates in the kidneys and in bones, where it causes visible damage. Recent in vitro data prove that the occurrence of citrate increases U bioavailability without changing its speciation. Two hypotheses can explain the role of citrate: it either modifies the U intracellular metabolization pathway, or it acts on the transport of U through cell membrane. To understand which mechanisms lead to increased bioavailability, we studied the speciation of U after accumulation in NRK-52E kidney cells. U speciation was first identified in various exposure media, containing citrate or not, in which U was supplied as U carbonate. The influence of serum proteins was analyzed in order to detect the formation of macromolecular complexes of U. Transmission electron microscopy (TEM) was employed to follow the evolution of the U species distribution among precipitated and soluble forms. Finally, extended X-ray absorption fine structure spectroscopy (EXAFS) enabled the precipitates observed to be identified as U-phosphate. It also demonstrated that the intracellular soluble form of U is U carbonate. These results suggest that citrate does not change U metabolization but rather plays a role in the intracellular accumulation pathway. U speciation inside cells was directly and clearly identified for the first time. These results elucidate the role of U speciation in terms of its bioavailability and consequent health effects.     

Green synthesis and characterization of Carica papaya leaf extract coated silver nanoparticles through X-ray diffraction,electron microscopy and evaluation of bactericidal properties

The evolution of nanotechnology and the production of nanomedicine from various sources had proven to be of intense value in the field of biomedicine. The smaller size of nanoparticles is gaining importance in research for the treatment of various diseases. Moreover the production of nanoparticles is eco-friendly and cost effective. In the present study silver nanoparticles were synthesized from Carica papaya leaf extract (CPL) and characterized for their size and shape using scanning electron microscopy and transmission electron microscopy, respectively. Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDS/EDX) and X-ray diffraction spectroscopy (XRD) were conducted to determine the concentration of metal ions, the shape of molecules. The bactericidal activity was evaluated using Luria Bertani broth cultures and the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using turbidimetry. The data analysis showed size of 50–250 nm spherical shaped nanoparticles. The turbidimetry analysis showed MIC and MBC was >25 μg/mL against both Gram positive and Gram negative bacteria in Luria Bertani broth cultures. In summary the synthesized silver nanoparticles from CPL showed acceptable size and shape of nanoparticles and effective bactericidal activity.     

Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus

Myxococcus xanthus cells harbor two motility machineries, type IV pili (Tfp) and the A-engine. During reversals, the two machineries switch polarity synchronously. We present a mechanism that synchronizes this polarity switching. We identify the required for motility response regulator (RomR) as essential for A-motility. RomR localizes in a bipolar, asymmetric pattern with a large cluster at the lagging cell pole. The large RomR cluster relocates to the new lagging pole in parallel with cell reversals. Dynamic RomR localization is essential for cell reversals, suggesting that RomR relocalization induces the polarity switching of the A-engine. The analysis of RomR mutants shows that the output domain targets RomR to the poles and the receiver domain is essential for dynamic localization. The small GTPase MglA establishes correct RomR polarity, and the Frz two-component system regulates dynamic RomR localization. FrzS localizes with Tfp at the leading pole and relocates in an Frz-dependent manner to the opposite pole during reversals; FrzS and RomR localize and oscillate independently. The Frz system synchronizes these oscillations and thus the synchronous polarity switching of the motility machineries.