A protease-mediated switch regulates the growth of magnetosome organelles in Magnetospirillum magneticum

Magnetosomes are lipid-bound organelles that direct the biomineralization of magnetic nanoparticles in magnetotactic bacteria. Magnetosome membranes are not uniform in size and can grow in a biomineralization-dependent manner. However, the underlying mechanisms of magnetosome membrane growth regulation remain unclear. Using cryoelectron tomography, we systematically examined mutants with defects at various stages of magnetosome formation to identify factors involved in controlling membrane growth. We found that a conserved serine protease, MamE, plays a key role in magnetosome membrane growth regulation. When the protease activity of MamE is disrupted, magnetosome membrane growth is restricted, which, in turn, limits the size of the magnetite particles. Consistent with this finding, the upstream regulators of MamE protease activity, MamO and MamM, are also required for magnetosome membrane growth. We then used a combination of candidate and comparative proteomics approaches to identify Mms6 and MamD as two MamE substrates. Mms6 does not appear to participate in magnetosome membrane growth. However, in the absence of MamD, magnetosome membranes grow to a larger size than the wild type. Furthermore, when the cleavage of MamD by MamE protease is blocked, magnetosome membrane growth and biomineralization are severely inhibited, phenocopying the MamE protease-inactive mutant. We therefore propose that the growth of magnetosome membranes is controlled by a protease-mediated switch through processing of MamD. Overall, our work shows that, like many eukaryotic systems, bacteria control the growth and size of biominerals by manipulating the physical properties of intracellular organelles.

Biomineralization is a common phenomenon across the tree of life; one type is a biologically controlled mineral production process that is often initiated within intracellular membrane-bound organelles or vesicle-like structures (1, 2). For instance, matrix vesicles serve as initial sites for mineral formation in the growth plate and most other vertebrate mineralization tissues (3). Vesicles also play a central role in the formation of calcitic spicules in sea urchins (4), extracellular calcitic plates in marine coccolithophores (5), and the silica-based cell walls of diatoms (6). Compartmentalization within a membrane is believed to provide an isolated microenvironment and a template for efficient nucleation, growth, and shaping of minerals.In contrast to the multiple examples of eukaryotic biomineralization noted here, little is known regarding the diversity and dynamics of bacterial biomineralization at the molecular and cellular level. Production of magnetic minerals within magnetosome organelles of magnetotactic bacteria (MTB) stands as one of the best-studied examples of biomineralization in bacteria. MTB are a diverse group of gram-negative bacteria often found near the oxic-anoxic transition zone of aquatic environments (7). Magnetic nanoparticles (magnetite or greigite) mineralized by MTB are generally 35 to 120 nm in length, a size range that yields a single, stable magnetic moment (8). Magnetosomes are typically arranged into one or multiple chains that function as a complete magnetic unit, enabling MTB to navigate along geomagnetic field lines and efficiently find the oxic-anoxic transition zone in a process termed magneto-aerotaxis (9).Biomineralization compartments generally contain a specific cohort of proteins that play critical roles during organelle formation and mineralization. Proteins involved in magnetosome biogenesis are normally encoded from a genomic region called the magnetosome gene island (MAI) (SI Appendix, Fig. S1A). Many magnetosome‐associated membrane (Mam) and magnetic particle membrane‐specific (Mms) proteins are associated with magnetosomes (10–12). The genes encoding the Mam and Mms proteins are organized into four clusters (mamAB, mamGFDC, mms6, and mamXY) in the model Magnetosprillum species and are necessary and sufficient for magnetosome formation (13–17) (Fig. 1A). Analyses of deletion mutants have been used to assign roles for individual genes in one of four distinct stages of magnetosome biogenesis in the model organism Magnetospirillum magneticum AMB-1 (AMB-1): 1) empty membrane invagination (mamI, -L, -Q, and -B), 2) chain alignment (mamK, -J, and -Y), 3) crystal nucleation (mamM, -N, and -O), and 4) crystal maturation (other genes within the four clusters) (13, 14, 18) (Fig. 1A).Open in a separate windowFig. 1.The essential genes and the process of magnetosome production. (A) Schematic depicting the four key magnetosome gene clusters of AMB-1. A total of 10 genes were tested in this study for magnetosome membrane growth regulation: genes involved in crystal initiation (mamM, -N, and -O) are marked in orange, and genes involved in crystal maturation (mamE, -P, -A, -S, -T, -D, and mms6) are marked in blue. Based on previous work, mmsF is known to not be involved in magnetosome membrane growth (8). (B) Model of the biomineralization-dependent magnetosome membrane growth based on Cornejo et al. 2016 (19). OM, outer membrane. IM, inner membrane.The resulting stepwise model outlines a set of processes that are seemingly distinct from one another. However, examination of the dynamics of magnetosome formation has revealed that magnetosome membrane growth is closely linked to the progression of biomineralization. Within a given AMB-1 cell, the magnetosome chain consists of some empty magnetosome membranes (EMMs) as well as the crystal-containing magnetosome membranes (CMMs) that provide the dipole moment necessary for orientation in magnetic fields. Cornejo et al. showed that at steady state, the diameter of the magnetosome lumen ranges from 20 to 80 nm (volume of about 4,189 to 268,083 nm³), yet no EMMs grow beyond 55 nm (volume of about 65,450 nm³) (19). Accordingly, when biomineralization is disrupted by limiting iron availability, only EMMs are produced and their growth stalls at about 55 nm, implying the existence of a checkpoint for membrane growth (Fig. 1B). Upon iron addition, membranes that have initiated biomineralization (CMMs) grow larger than this limit, implying that active biomineralization is needed for further membrane growth (19) leading to a linear relationship between the size of the growing crystals and the surrounding membranes (Fig. 1B). One possible explanation for these observations is that the growing mineral pushes against the membrane and drives its expansion. However, a mutant missing MmsF, a late-stage biomineralization protein, makes small magnetite crystals and still produces membranes as large as the wild-type (WT) parent in a biomineralization-dependent manner (19). These observations imply that magnetosome membrane growth is tightly regulated to create an optimal environment for crystal nucleation, which triggers the second membrane growth stage for crystal maturation (19).The discovery of biomineralization-dependent magnetosome membrane growth provides a lens to examine the function of magnetosome proteins. One hypothesis holds that regulated growth of the magnetosome membrane allows for proper accumulation of iron to high concentrations to initiate nucleation and growth of magnetic particles. Thus, factors known to influence the growth and geometry of magnetite crystals may actually do so by regulating the physical properties of the magnetosome membrane. Here, we explored this possibility by using whole cell cryoelectron tomography (cryo-ET) to directly measure the sizes of magnetosome membranes in a series of mutants with known defects in crystal production. MamE belongs to a highly conserved high temperature requirement A (HtrA) family of trypsin-like serine proteases, whose activity is required for crystal maturation with an unknown mechanism (20). Here, we find that the catalytic activity of MamE plays a central role in the progression of magnetosome membrane growth. MamE proteolytically processes itself and two other biomineralization factors, MamO and MamP (21). MamO is required for MamE protease activation (22), and we find it acts as an upstream regulator of MamE for magnetosome membrane growth. MamP is not involved in magnetosome membrane growth. We also identified MamD, a protein that binds tightly to magnetite and was previously thought to promote crystal maturation (23, 24), as a direct substrate of MamE and showed that MamD is in fact a negative regulator of biomineralization. Our results indicate that MamE activates membrane remodeling by relieving MamD’s inhibition on the size of the magnetosome lumen and demonstrate how spatial restructuring of an organelle can regulate its biochemical output.     

Desulfovibrio magneticus RS-1 contains an iron- and phosphorus-rich organelle distinct from its bullet-shaped magnetosomes

Intracellular magnetite crystal formation by magnetotactic bacteria has emerged as a powerful model for investigating the cellular and molecular mechanisms of biomineralization, a process common to all branches of life. Although magnetotactic bacteria are phylogenetically diverse and their crystals morphologically diverse, studies to date have focused on a few, closely related species with similar crystal habits. Here, we investigate the process of magnetite biomineralization in Desulfovibrio magneticus sp. RS-1, the only reported species of cultured magnetotactic bacteria that is outside of the α-Proteobacteria and that forms bullet-shaped crystals. Using a variety of high-resolution imaging and analytical tools, we show that RS-1 cells form amorphous, noncrystalline granules containing iron and phosphorus before forming magnetite crystals. Using NanoSIMS (dynamic secondary ion mass spectroscopy), we show that the iron-phosphorus granules and the magnetite crystals are likely formed through separate cellular processes. Analysis of the cellular ultrastructure of RS-1 using cryo-ultramicrotomy, cryo-electron tomography, and tomography of ultrathin sections reveals that the magnetite crystals are not surrounded by membranes but that the iron-phosphorus granules are surrounded by membranous compartments. The varied cellular paths for the formation of these two minerals lead us to suggest that the iron-phosphorus granules constitute a distinct bacterial organelle.     

Rapid magnetosome formation shown by real-time x-ray magnetic circular dichroism

Magnetosomes are magnetite nanoparticles formed by biomineralization within magnetotactic bacteria. Although there have been numerous genetic and proteomic studies of the magnetosome-formation process, there have been only limited and inconclusive studies of mineral-phase evolution during the formation process, and no real-time studies of such processes have yet been performed. Thus, suggested formation mechanisms still need substantiating with data. Here we report the examination of the magnetosome material throughout the formation process in a real-time in vivo study of Magnetospirillum gryphiswaldense, strain MSR-1. Transmission EM and x-ray absorption spectroscopy studies reveal that full-sized magnetosomes are seen 15 min after formation is initiated. These immature magnetosomes contain a surface layer of the nonmagnetic iron oxide-phase hematite. Mature magnetite is found after another 15 min, concurrent with a dramatic increase in magnetization. This rapid formation result is contrary to previously reported studies and discounts the previously proposed slow, multistep formation mechanisms. Thus, we conclude that the biomineralization of magnetite occurs rapidly in magnetotactic bacteria on a similar time scale to high-temperature chemical precipitation reactions, and we suggest that this finding is caused by a biological catalysis of the process.     

Architecture of a flagellar apparatus in the fast-swimming magnetotactic bacterium MO-1

The bacterial flagellum is a motility organelle that consists of a rotary motor and a helical propeller. The flagella usually work individually or by forming a loose bundle to produce thrust. However, the flagellar apparatus of marine bacterium MO-1 is a tight bundle of seven flagellar filaments enveloped in a sheath, and it has been a mystery as to how the flagella rotate smoothly in coordination. Here we have used electron cryotomography to visualize the 3D architecture of the sheathed flagella. The seven filaments are enveloped with 24 fibrils in the sheath, and their basal bodies are arranged in an intertwined hexagonal array similar to the thick and thin filaments of vertebrate skeletal muscles. This complex and exquisite architecture strongly suggests that the fibrils counter-rotate between flagella in direct contact to minimize the friction of high-speed rotation of individual flagella in the tight bundle within the sheath to enable MO-1 cells to swim at about 300 µm/s.     

In situ magnetic identification of giant,needle-shaped magnetofossils in Paleocene–Eocene Thermal Maximum sediments

Near-shore marine sediments deposited during the Paleocene–Eocene Thermal Maximum at Wilson Lake, NJ, contain abundant conventional and giant magnetofossils. We find that giant, needle-shaped magnetofossils from Wilson Lake produce distinct magnetic signatures in low-noise, high-resolution first-order reversal curve (FORC) measurements. These magnetic measurements on bulk sediment samples identify the presence of giant, needle-shaped magnetofossils. Our results are supported by micromagnetic simulations of giant needle morphologies measured from transmission electron micrographs of magnetic extracts from Wilson Lake sediments. These simulations underscore the single-domain characteristics and the large magnetic coercivity associated with the extreme crystal elongation of giant needles. Giant magnetofossils have so far only been identified in sediments deposited during global hyperthermal events and therefore may serve as magnetic biomarkers of environmental disturbances. Our results show that FORC measurements are a nondestructive method for identifying giant magnetofossil assemblages in bulk sediments, which will help test their ecology and significance with respect to environmental change.

The Paleocene–Eocene Thermal Maximum (PETM; ∼56 Ma) is a geologically rapid global warming event with many characteristics that make it an analog for the Anthropocene (1, 2). These characteristics include rapid warming of the sea surface and atmosphere, increased seasonality of precipitation and temperature, and biological extinctions (1). The PETM is identified globally by a −3‰ carbon isotope excursion (CIE) in bulk marine carbonate and is characterized by three stratigraphic intervals: 1) a preonset excursion, 2) the main CIE, and 3) a recovery toward baseline δ¹³C levels (1). The main CIE interval is further subdivided into the CIE onset and CIE core, which correspond to the first ∼6 to 10 kyr and 100 to 200 kyr of the PETM (1, 3, 4). The Wilson Lake A (WL-A) core from Wilson Lake, NJ, contains a continental shelf section of the PETM within the Marlboro Clay and, within the Marlboro Clay, an expanded and nearly complete record of the CIE onset and CIE core (1, 5). Several near-shore and offshore cores complement the WL-A record and enable a broader understanding of how Paleogene coastal ecosystems responded to the rapid onset of global hyperthermal conditions (5–8). The New Jersey continental shelf experienced an overall rapid influx of clay, mineralization of iron oxides, dinoflagellate blooms, and benthic foraminifera species turnover coincident with the CIE onset (5, 9, 10).Abundant conventional and giant magnetofossils, the fossil remains of magnetotactic bacteria and other iron-biomineralizing microorganisms, were identified in several New Jersey cores. These magnetofossils are interpreted to be the predominant source of the PETM magnetic enhancement of these cores (7, 11–14), although alternative sources have been suggested (15–18). Giant magnetofossils have so far only been identified in sediments from the PETM and the Middle Eocene Climatic Optimum, leading to the interpretation that they are unique to hyperthermal events (6, 7, 11–14). For example, Chang et al. (6) suggest that giant magnetofossils are linked to oceanic deoxygenation during the PETM.Previous studies, which used micromagnetic simulations, electron holography, or both, suggest that giant magnetofossils have distinct magnetic properties (6, 11, 14, 18, 19). These interpretations are limited, however, by assumptions regarding crystal arrangement, spacing, and magnetic domain structure, and they lack independent confirmation of defining characteristics. Additionally, some of these methods have been applied to only a few giant magnetofossil morphologies.Here we show independent, physical evidence of the magnetic signature of giant magnetofossils in situ (i.e., not in extracts) using low-noise, high-resolution first-order reversal curves (FORCs). FORC routines measure the response of all magnetic particles, including giant magnetofossils, within a bulk sediment sample. We also present micromagnetic simulations of giant needle-shaped crystals whose morphologies were characterized with transmission electron microscopy (TEM) of magnetic extracts. Our results show that giant, needle-shaped magnetofossils produce a high-coercivity component distinct from conventional magnetofossils that is identified using a specific FORC measurement protocol. We argue that these are definitive magnetic signatures of giant magnetofossils, which further supports the interpretation that the magnetic enhancement of the WL-A core has a biogenic origin. The link between giant magnetofossils, hyperthermal events, and oceanic deoxygenation makes the magnetic signature of giant needles a powerful tool for identifying giant magnetofossil assemblages and, by extension, testing their ecological significance in the context of global change events in the geologic record.     

Visualization and structural analysis of the bacterial magnetic organelle magnetosome using atomic force microscopy

The unique ability of magnetotactic bacteria to navigate along a geomagnetic field is accomplished with the help of prokaryotic organelles, magnetosomes. The magnetosomes have well-ordered chain-like structures, comprising membrane-enveloped, nano-sized magnetic crystals, and various types of specifically associated proteins. In this study, we applied atomic force microscopy (AFM) to investigate the spatial configuration of isolated magnetosomes from Magnetospirillum magneticum AMB-1 in near-native buffer conditions. AFM observation revealed organic material with a ∼7-nm thickness surrounding a magnetite crystal. Small globular proteins, identified as magnetosome-associated protein MamA, were distributed on the mica surface around the magnetosome. Immuno-labeling with AFM showed that MamA is located on the magnetosome surface. In vitro experiments showed that MamA proteins interact with each other and form a high molecular mass complex. These findings suggest that magnetosomes are covered with MamA oligomers in near-native environments. Furthermore, nanodissection revealed that magnetosomes are built with heterogeneous structures that comprise the organic layer. This study provides important clues to the supramolecular architecture of the bacterial organelle, the magnetosome, and insight into the function of the proteins localized in the organelle.     

血清和胸水铜、锌、铁检测对结核性和癌性胸水的鉴别诊断价值

目的通过检测结核性和癌性胸水患者血清及胸水中铜、锌、铁水平,探讨其对鉴别良恶性胸水的意义。方法采用火焰原子吸收分光光度法(FAA S)检测32例结核性胸水和32例癌性胸水患者血清及胸水铜、锌、铁水平。结果癌性胸水组血清和胸水铜含量及铜/锌比值显著高于结核性胸水组,而血清和胸水锌含量显著降低(P<0.01)。癌性胸水组血清铁含量显著降低,而胸水铁含量显著高于结核性胸水组(P<0.01)。结论同时检测血清及胸水中铜、锌、铁水平对鉴别良恶性胸水有重要价值。     

中缅树鼩常见病原菌的分离鉴定

目的调查中缅树鼩常见病原细菌携带情况。方法通过病原体分离培养、系列生化试验、血清学鉴定和核糖体16s rDNA分型技术对树鼩携带的细菌进行分离鉴定。结果从树鼩肠道和体表分离到金黄色葡萄球菌、表皮葡萄球菌、链球菌、绿脓杆菌、变形杆菌、大肠埃希菌、沙雷菌,未分离到沙门氏菌、志贺菌、克雷伯菌和产气杆菌。结论树鼩携带多种病原菌,建立树鼩病原菌的检测方法是树鼩实验动物化和质量监测的重要内容。     

颈动脉粥样硬化斑块中牙周可疑致病菌的检测

目的:检测颈动脉粥样硬化斑块中的主要牙周可疑致病菌。方法:选择颈动脉粥样硬化内膜剥脱术的病例,收集术中分离的含有颈动脉粥样硬化斑块的标本15例。提取斑块的组织DNA,分别用5种牙周可疑致病菌的特异引物采取聚合酶链式反应(PCR),扩增16S rDNA片段来鉴定细菌种类。结果:15例患者颈动脉粥样硬化斑块中4例样本检测到了具核梭杆菌(Fusobacterium nucleatum,Fn),2例样本检测到了牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)。其中1例同时检测到了Fn和Pg。未检测到伴放线聚生杆菌(Aggregatibacter actinom ycetemcomitans,Aa),福赛坦菌(Tannerella for-sythia,Tf),中间普氏菌(Prevotella intermedia,Pi)。结论:颈动脉粥样硬化的发生发展可能与牙周可疑致病菌有一定相关性。     

微量元素硒与萎缩性胃炎关系的研究

为研究萎缩性胃炎及肠上皮化生与胃癌的关系，我们对42例胃癌，38例萎缩性胃炎和38例浅表性胃炎的血浆，尿液和胃液中Se含量进行了检测。结果提示：（1）萎缩性胃炎与胃癌的关系密切，特别是重度萎缩性胃炎和伴肠化者与胃癌尤为密切，应严密观察和随访行病理检查，对胃部的早期发现有一定帮助。（2）随胃炎加重，Se含量炎加重，Se含量呈阶梯状变化，对临床治疗和防止胃炎进一步发展有一定的指导意义；（3）适量补硒在胃癌防治上具有一定意义。     

The frequency of live bacteria in gallstones

Background

Septic complications reported from stones and concretions lost in the peritoneal cavity following laparoscopic cholecys-tectomy reflect the infective potential of gallstones. Although bacteria have been demonstrated in the core of gallstones by scanning electron microscopy and molecular genetic techniques, gallstone culture is the only conclusive proof of whether such bacteria are viable and can cause infection.

Methods

Gallstones retrieved from patients undergoing cholecystect-omy were decontaminated after surface cleaning with alcohol, and each core was scooped for culture.

Results

In this study organisms were cultured from the cores of gallstones in 81% of cases of cholelithiasis and 77% of cases of gallbladder carcinoma, irrespective of stone type and size. Both enteric (57.5%) and non-enteric (20%) organisms were isolated in cholelithiasis, whereas only enteric bacteria could be cultured from cases of gallbladder carcinoma. Long thought to be a causative agent, Salmonella organisms were detected in none of the 14 cases of gallbladder carcinoma.

Conclusion

Contrary to the popular belief that bacteria inside gallstones are dead, gallstones from most patients contain live bacteria with the potential to cause infective complications.     

雌二醇对大鼠血压和血浆微量元素的影响

用补充雌二醇方法探讨雌二醇对wistar大鼠血压及血浆微量元素的影响。结果表明，大剂量补充雌二醇可以诱发高血压，大鼠血浆中铜,镁、钙离子含量显著升高（P＜0.05）；而铁、锌离子无显著改变（P＜0．05）。提示微量元素参与雌二醇诱发高血压的过程。     

Adhesion of anaerobic bacteria to platelet containers

Anaerobic Propionibacterium acnes and Staphylococcus saccharolyticus are frequently isolated during platelet screening with anaerobic culture methods. Although neither P. acnes nor S. saccharolyticus proliferates during platelet storage, both species survive well in this environment. This study was aimed at determining whether strains of P. acnes and/or S. saccharolyticus form surface‐attached bacterial cell aggregates, known as biofilms, under platelet storage conditions. We report that these organisms are able to adhere to the inner surface of platelet containers in tight interaction with activated platelets.     

专性厌氧菌污染血液1例

<正>我站在常规血液产品质量控制抽检过程中,发现1例专性厌氧菌造成的血液污染,现报告如下。1病例资料2013年4月11日采集全血400ml,当日下午分离制备为去白细胞悬浮红细胞和新鲜冰冻血浆。4月17日质管科随机从成品库血液中选取4袋去白细胞悬浮红细胞(含上述血液),采用无菌接管机从原袋中留取约15~20ml血液到空转移袋,作为质检血样。于当日16:47样本分别接种于需氧培     

革兰阴性细菌Ⅳ型菌毛的致病机制研究进展

革兰阴性杆菌是临床上能引起严重感染的重要菌群，其毒力因子之-Ⅳ型菌毛的作用正越来越受到关注。Ⅳ型菌毛分布于革兰阴性细菌的表面，由多基因编码各种结构性和功能性菌毛蛋白，在革兰阴性细菌的致病过程中发挥作用。Ⅳ型菌毛有重要的黏附功能，介导细菌和宿主上皮细胞的识别、接触；而且其特有的表面运动、帮助细菌生物膜的形成及转化、噬菌转导和免疫调节等作用均构成了其独特且重要的毒力作用。     

革兰阴性菌肺部感染病例回顾性分析

目的回顾性分析多重耐药（MDR）革兰阴性菌和非MDR革兰阴性菌肺部感染的差异,探讨MDR细菌对患者的影响,给临床医师选择用药提供参考。方法收集我院2012年1月至2012年12月合格呼吸道标本、血液、胸腔积液等临床标本中分离出的MDR革兰阴性菌和非MDR革兰阴性菌,根据临床资料将导致肺部感染的病例纳入研究,分析MDR组和非MDR组之间的差异。结果MDR革兰阴性菌和非MDR革兰阴性菌科室分布不同。MDR革兰阴性菌和非MDR革兰阴性菌在住院天数、血清白蛋白、日均住院费用、先期使用抗生素、先期使用碳青霉烯类抗生素、病死率之间的差异有统计学意义（P〈0．05）,在年龄、血红蛋白、感染时间之间的差异无统计学意义（P〉0．05）。结论MDR革兰阴性菌感染是临床抗生素选择面临的挑战,增加患者病死率,增加住院费用,我们需要高度重视。     

Terpene synthases are widely distributed in bacteria

Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes.Some 50,000 terpenoid metabolites, including monoterpenes, sesquiterpenes, and diterpenes representing nearly 400 distinct structural families, have been isolated from both terrestrial and marine plants, liverworts, and fungi. In contrast, only a relatively minor fraction of these widely occurring metabolites has been identified in prokaryotes. The first study of bacterial terpenes grew out of an investigation of the characteristic odor of freshly plowed soil reported in 1891 by Berthelot and André (1). Berthelot and André noted that a volatile substance apparently responsible for the typical earthy odor of soil could be extracted from soil by steam distillation. Their attempts to assign a structure to the isolated odor constituent failed;, however, when the neutral alcohol resisted oxidative degradation or other conventional chemical modification. The first modern studies of volatile bacterial terpenes were carried out some 75 years later by Gerber and Lechevalier (2) and Gerber (3–7), who speculated that the characteristic odor of cultures of Actinomycetales microorganisms, which are widely distributed in soil, might be caused by volatile terpenes. In addition to determining the structure of Berthelot’s geosmin, shown to be a C₁₂ degraded sesquiterpene alcohol (and giving it its name, which means earth odor) (2, 3), Gerber (4) also isolated and determined the structures of the methylated monoterpene 2-methylisoborneol as well as several other cyclic sesquiterpenes produced by streptomycetes (5–7). In subsequent years, numerous volatile terpenes have been detected in streptomycetes (8–16). The three most commonly detected streptomycetes terpenoids, geosmin, and 2-methylisoborneol and the tricyclic α,β-unsaturated ketone albaflavenone (Fig. 1) are well-known as volatile odoriferous microbial metabolites. The two terpene alcohols are, in fact, the most frequently found secondary metabolites in actinomycetes (8, 11, 17), filamentous Cyanobacteria (18–20), and Myxobacteria (21), and they are also produced by a small number of fungi (22–24). The production of 2-methylisoborneol is associated with a characteristic scent, whereas albaflavenone, which was first isolated from cultures of a highly odoriferous Streptomyces albidoflavus species, is best described as earthy and camphor-like (25).Open in a separate windowFig. 1.The structures of the major known terpenes produced by bacteria.Cyclic monoterpene, sesquiterpene, and diterpene hydrocarbons and alcohols are formed by variations of a universal cyclization mechanism that is initiated by enzyme-catalyzed ionization of the universal acyclic precursors geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) to form the corresponding allylic cations. These parental branched, linear isoprenoid precursors are themselves synthesized by mechanistically related electrophilic condensations of the 5-carbon building blocks dimethylallyl diphosphate and isopentenyl diphosphate. The several thousand known or suspected terpene synthases from plants and fungi have a strongly conserved level of overall amino acid sequence similarity, thus making possible the application of local alignment methods, such as the widely used BLAST algorithm, for the discovery of genes encoding presumptive terpene synthases from plant and fungal sources. Despite the relatively high level of overall sequence conservation, however, assignment of the actual biosynthetic cyclization product of each fungal or plant terpene synthase has remained beyond the reach of available bioinformatic methods. The discovery and biochemical characterization of bacterial terpene synthases represent an even greater challenge, because unlike the plant and fungal enzymes, bacterial terpene synthases not only exhibit no significant overall amino acid sequence similarity to those from plants and fungi but typically display relatively low levels of mutual sequence similarity. To address this challenge, we recently described the successful application of an alternative genome mining strategy for the discovery of previously unidentified bacterial terpene synthases based on the use of hidden Markov models (HMMs) and protein families database (Pfam) searching methods (26). These initial efforts identified a large number of previously unrecognized bacterial terpene synthase candidates, including the discovery of the previously unidentified synthase for the methylated monoterpene 2-methylisoborneol, and led to the heterologous expression of the relevant genes that produce 2-methylisoborneol and 2-methylenebornane from 2-methylgeranyl diphosphate (27). We subsequently refined and expanded the set of HMM parameters using as a reference set exclusively the group of newly predicted bacterial terpene synthases in distinction to the original HMM model (PF03936), which had been based on plant terpene synthases. Using these newly refined parameters, we then succeeded in identifying a previously unrecognized ortholog of 2-methylisoborneol synthase in the cyanobacterium Pseudanabaena limnetica str. Castaic Lake (28). Application of this second generation HMM model allowed, in total, the discovery of 140 predicted terpene synthases of bacterial origin.We now report the development of a third generation HMM model trained by the previously identified 140 bacterial terpene synthases that has expanded the number of predicted bacterial terpene synthases to 262 from within the most complete set of predicted proteins incorporated in the most recent collection of public databases and in-house draft genome sequences of streptomycete microorganisms. Among the newly identified gene sequences, a subset selected by phylogenetic analysis has been expressed in a specially engineered heterologous Streptomyces host, and the resultant terpenes have been identified and structurally characterized.     

Increasing frequency of Gram-positive bacteria in spontaneous bacterial peritonitis.

AIM: To evaluate the characteristics and possible recent changes of the microbial causes of spontaneous bacterial peritonitis (SBP) in cirrhotic patients. METHODS: We retrospectively evaluated 42 cirrhotic patients with positive ascitic fluid culture and without evidence of secondary peritonitis who were admitted consecutively to our Department between 1998 and 2002. RESULTS: Twenty (48%) of 42 patients with positive ascitic fluid culture were diagnosed during 1998-1999 (period A) and the remaining 22 (52%) patients during 2000-2002 (period B). Gram-negative bacteria were the cause of SBP in 15 (75%) of the 20 patients during period A and in only nine (41%) of the 22 patients during period B (P=0.026). SBP patients with Gram-positive bacteria compared with those with Gram-negative bacteria were less frequently in Child class C (P=0.058) and had significantly higher ascitic fluid protein (P=0.014) and albumin concentrations (P=0.009) and lower ascitic fluid neutrophil count (P=0.008). Resistance to quinolones was detected significantly more frequently in the isolated Gram-positive than Gram-negative bacteria (P<0.001). CONCLUSION: Culture-positive SBP in cirrhotic patients are caused more frequently by Gram-positive bacteria during the recent years, which are, in their vast majority, resistant to quinolones.