Carbon and nitrogen metabolism in legume root nodules

The literature concerning the metabolism of carbon compounds during the reduction, assimilation and translocation of nitrogen in root nodules of leguminous plants is reviewed. The reduction of dinitrogen requires an energy source (ATP) and a reluctant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid or urcide synthesis during the assimilation of ammonia produced by the bacteria within the nodule tissue. Competition for photosynthates occurs between the bacteroids, nodule tissue and the various vegetative and reproductive sinks in the host plant. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolises between the various sites of utilization are only poorly understood. It is apparent that dinitrogen is reduced to ammonia in the bacteroids. Both fast- and slow-growing strains of Rhizobium possess the Entner-Doudoroff pathway of glucose catabolism, and some, if not all, enzymes of the Emden-Meyerhof pathway. Some bacterial cultures also metabolize carbon through the ketogluconate pathway but only the fast-growing strains of cultured rhizobia possess the key enzyme of the pentose phosphate pathway (6-phosphogluconate dehydrogenase). The host cells are thought to contain the complete Emden-Meyerhof pathway and tricarboxylic acid cycle, which provides the carbon skeletons for assimilation of the ammonia, formed by the bacteroids, into α-amino acids. A pathway of anapleurotic carbon conservation, operative in the host cells, synthesizes oxaloacetic acid through β-carboxylation of phosphoenol pyruvate. This process could be important in the recapture and assimilation of respired CO₂ in the rhizosphere. The main route of assimilation of ammonia produced by the bacteroids would appear to be via the glutamine synthetase-glutamate synthase pathway in the host cells. However, glutamate dehydrogenase may also be involved in ammonia assimilation. These enzymes also occur in in vitro cultures of Rhizobium and in bacteroids where they presumably participate in the synthesis of amino acids for growth of the bacteria or bacteroids. Nitrogen assimilated into glutamine or glutamate is exported from the nodules in a variety of forms, which include asparagine, glutamine, aspartate, homoserine and allantoates, in proportions which depend on the legume species. Studies on regulation of the overall process have focussed on expression of bacteroid genes and on the control of enzyme activity, at the level of nitrogenase and enzymes of nitrogen assimilation in particular. However, due to the wide range of experimental techniques, environmental conditions and plant species which have been used, no clear conclusions can yet be drawn. The pathways of carbon flow in nitrogen metabolism, particularly in relation to the synthesis of ureides and the regulation of carbon metabolism, remain key areas for future research in symbiotic nitrogen fixation.     

不同寄主植物西花蓟马内生细菌多样性分析

为探讨不同寄主植物对西花蓟马Frankliniella occidentalis内生细菌分布的影响,应用Illumina MiSeq第二代测序技术对取食不同寄主植物的西花蓟马内生细菌的16S rDNA V5~V7区域进行测定。结果显示,Alpha多样性分析发现,西花蓟马内生细菌在取食不同寄主植物后多样性表现为西葫芦Cucurbita pepo L. >菊花Dendranthema morifolium(Ramat.) Tzvelev >曼陀罗Datura stramonium L. >玫瑰Rosa rugosa Thunb. >牵牛花Pharibitis purpurea (L.) Voigt >辣椒Capsicum annuum L.;在门水平的菌群组成上,变形菌门在各样品中所占比例最高,是西花蓟马内生菌中的优势菌门,厚壁菌门为次优势菌菌门。在属水平上内生细菌也表现出较大的差异,其中假单胞菌属Pseudomonas和欧文氏菌属Erwinia在西花蓟马取食各寄主植物时均有分布,取食辣椒、玫瑰、曼陀罗时,优势菌属均为欧文氏菌属Erwinia,取食菊花和牵牛花时,优势菌属为立克次体Rickettsia,而取食西葫芦时,则为短波毛单胞菌属Brevundimonas;利用KEGG数据库,对内生细菌进行基因功能预测分析发现,内生细菌群富集了高丰度的代谢功能基因,且主要集中于碳水化合物代谢、氨基酸代谢以及能量代谢。综上所述取食不同寄主植物时西花蓟马内生细菌多样性与群落结构具有一定的差异,寄主植物可对西花蓟马内生细菌群落组成产生重要的影响;且各寄主植物上西花蓟马内生细菌基因功能主要集中在碳水化合物代谢、氨基酸代谢和能量代谢。     

植物内生菌促进宿主氮吸收与代谢研究进展

内生菌与植物共生能够提高宿主的氮吸收与氮代谢水平,这可能是由于内生菌在植物体内引发的多种效应的综合结果.植物内生菌能够通过促进植物根系发育和固氮作用为宿主植物提供更多的无机氮素;能够通过分泌多种胞外酶系如漆酶、蛋白水解酶等使宿主植物更好地利用有机氮素;能够提高宿主氮代谢关键酶如硝酸还原酶(NR)、谷氨酰胺合成酶(GS)等酶的活性;能够提高宿主植物激素水平和维生素含量从而促进宿主氮代谢;能够通过影响宿主植物氮代谢促进宿主植物分蘖、提高宿主植物叶绿素含量和光合速率等等.综述了国内外关于植物内生菌促进宿主氮代谢的相关报道,归纳了植物内生菌影响宿主氮素吸收与代谢的可能机制,并展望了关于植物内生菌促进宿主氮代谢机制方面的研究方向.     

Novel bacteriolytic activity associated with the style microflora of the mussel Mytilus edulis (L.)

Previous studies have shown that bacterioplankton are retained by the mussel Mytilus edulis (L.), and that lysozyme-like enzymes are associated with the crystalline style of the mussels. This study has demonstrated the presence of a lytic agent which is produced by bacteria associated with the crystalline style. The production of the agent is inhibited by chloramphenicol, and anaerobic growth conditions. The agent is heat-sensitive and nonfilterable. Attempts at the isolation of the agent in a cell-free extract have been unsuccessful. The presence of bacteria-like cells in the outer laminae of the crystalline style, coupled with the nonfilterable nature of the lytic agent, make it seem possible that these bacteria are responsible for lysis of gram-negative bacteria which are taken in with the food supply.     

Citrate metabolism in lactic acid bacteria

Abstract: Citrate metabolism plays an important role in many food fermentations involving lactic acid bacteria. Since citrate is a highly oxidized substrate, no reducing equivalents are produced during its degradation, resulting in the formation of metabolic end products other than lactic acid. Some of these end products, such as diacetyl and acetaldehyde, have very distinct aroma properties and contribute significantly to the quality of the fermented foods. In this review the metabolic pathways involved in product formation from citrate are described, the bioenergetic consequences of this metabolism for the lactic acid bacteria are discussed and detailed information on some key enzymes in the citrate metabolism is presented. The combined knowledge is used for devising strategies to avoid, control or improve product formation from citrate.     

乳酸菌素-安全、天然的食品防腐剂

乳酸菌素是一类由乳酸菌在代谢过程中通过核糖体合成机制产生的抗菌多肽或蛋白,胞外分泌,能够通过在细胞膜上形成孔道或抑制细胞壁合成来达到溶菌目的。主要从乳酸菌素在食品中应用的安全性、影响乳酸菌素生物合成的条件、乳酸菌素活性的影响因素、以及目前乳酸菌素在食品领域的应用等方面进行了评述,揭示了乳酸菌素广阔的市场应用前景,从而使乳酸菌素这种天然防腐剂能获得更好地开发和利用。     

Chemoautotrophic,sulfur-oxidizing symbiotic bacteria on marine nematodes: Morphological and biochemical characterization

The marine, free-living Stilbonematinae (Nematoda: Desmodorida) inhabit the oxygen sulfide chemocline in marine sands. They are characterized by an association with ectosymbiotic bacteria. According to their ultrastructure the bacteria are Gram-negative and form morphologically uniform coats that cover the entire body surface of the worms. They are arranged in host-genus or host-species specific patterns: cocci form multilayered sheaths, rods, and crescent- or filament-shaped bacteria form monolayers. The detection of enzymes associated with sulfur metabolism and of ribulose-1,5 bisphosphate carboxylase oxygenase, as well as elemental sulfur in the bacteria indicate a chemolithoautotrophic nature of the symbionts. Their reproductive patterns appear to optimize space utilization on the host surface: vertically standing rods divide by longitudinal fission, whereas other bacteria form non-septate filaments of up to 100 m length.     

A novel peptidoglycan D,L‐endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence

Bacteria remodel peptidoglycan structure in response to environmental changes. Many enzymes are involved in peptidoglycan metabolism; however, little is known about their responsiveness in a defined environment or the modes they assist bacteria to adapt to new niches. Here, we focused in peptidoglycan enzymes that intracellular bacterial pathogens use inside eukaryotic cells. We identified a peptidoglycan enzyme induced by Salmonella enterica serovar Typhimurium in fibroblasts and epithelial cells. This enzyme, which shows γ‐D‐glutamyl‐meso‐diaminopimelic acid D,L‐endopeptidase activity, is also produced by the pathogen in media with limited nutrients and in resting conditions. The enzyme, termed EcgA for e ndopeptidase responding to c essation of g rowth’, is encoded in a S. Typhimurium genomic island absent in Escherichia coli. EcgA production is strictly dependent on the virulence regulator PhoP in extra‐ and intracellular environments. Consistent to this regulation, a mutant lacking EcgA is attenuated in the mouse typhoid model. These findings suggest that specialised peptidoglycan enzymes, such as EcgA, might facilitate Salmonella adaptation to the intracellular lifestyle. Moreover, they indicate that readjustment of peptidoglycan metabolism inside the eukaryotic cell is essential for host colonisation.     

Responses of lactic acid bacteria to oxygen

Abstract A small number of flavoprotein oxidase enzymes are responsible for the direct interaction of lactic acid bacteria (LAB) with oxygen; hydrogen peroxide or water are produced in these reactions. In some cultures exposed to oxygen, hydrogen peroxide accumulates to inhibitory levels.
Through these oxidase enzymes and NADH peroxidase, O₂ and H₂O₂ can accept electrons from sugar metabolism, and thus have a sparing effect on the use of metabolic intermediates, such as pyruvate or acetaldehyde, as electron acceptors. Consequently, sugar metabolism in aerated cultures of LAB can be substantially different from that in unaerated cultures. Energy and biomass yields, end-products of sugar metabolism and the range of substrates which can be metabolised are affected.
Lactic acid bacteria exhibit an inducible oxidative stress response when exposed to sublethal levels of H₂O₂. This response protects them if they are subsequently exposed to lethal concentrations of H₂O₂. The effect appears to be related to other stress responses such as heat-shock and is similar, in some but not all respects, to that previously reported for enteric bacteria.     

Microbial starch-binding domain

Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.     

Metabolism of arachidonic acid to eicosanoids within the nucleus

The eicosanoids are a diverse family of molecules that have powerful effects on cell function. They are best known as intercellular messengers, having autocrine and paracrine effects following their secretion from the cells that synthesize them. Many of the eicosanoids are produced from one polyunsaturated fatty acid, arachidonic acid. The diversity of possible products that can be synthesized from arachidonic acid is due, in part to the variety of enzymes that can act on it. Over the past 15 years, studies have placed many, but not all, of these enzymes at or inside the nucleus. In some cases, the nuclear import or export of arachidonic acid-processing enzymes is highly regulated. Furthermore, nuclear receptors that are activated by specific eicosanoids are known to exist. Taken together, these findings indicate that the enzymatic conversion of arachidonic acid to specific signaling molecules can occur in the nucleus, that it is regulated, and that the synthesized products may act within the nucleus. The objectives of this commentary are to review what is known about the metabolism of arachidonic acid to eicosanoids within the nucleus and to point to important areas for future discovery.     

茎瘤固氮根瘤菌Azorhizobium caulinodans ORS571基因组分泌蛋白的生物信息学分析

为了获取茎瘤固氮根瘤菌(Azorhizobium caulinodans ORS571)的分泌蛋白,以便更深入地了解该菌的共生固氮作用,本研究采用SignalP、TMHMM、PSORTb、TargetP、LipoP、TatP和SecretomeP软件对该菌全部4717个蛋白序列进行分析预测。结果共识别了653个分泌蛋白,其中具有分泌型信号肽的蛋白54个,具有RR-motif型信号肽的蛋白1个,具有脂蛋白信号肽的蛋白2个和非经典分泌蛋白596个。该菌含信号肽分泌蛋白仅占全部蛋白的1.2%,低于其它固氮菌。在分泌蛋白中识别了核酸内切酶和核糖核酸酶等6个核酸酶。它们可能参与宿主植物遗传物质的降解,干扰宿主遗传代谢,进一步在宿主植物侵染过程中起到重要作用。此外还识别了超氧化物歧化酶、过氧化氢酶和谷胱甘肽S-转移酶等4个抗氧化酶。它们可能参与活性氧的清除以保护固氮酶,是该菌固氮过程的重要参与者。     

Control of Listeria superoxide dismutase by phosphorylation

Superoxide dismutases (SODs) are enzymes that protect organisms against superoxides and reactive oxygen species (ROS) produced during their active metabolism. ROS are major mediators of phagocytes microbicidal activity. Here we show that the cytoplasmic Listeria monocytogenes MnSOD is phosphorylated on serine and threonine residues and less active when bacteria reach the stationary phase. We also provide evidence that the most active nonphosphorylated form of MnSOD can be secreted via the SecA2 pathway in culture supernatants and in infected cells, where it becomes phosphorylated. A Deltasod deletion mutant is impaired in survival within macrophages and is dramatically attenuated in mice. Together, our results demonstrate that the capacity to counteract ROS is an essential component of L. monocytogenes virulence. This is the first example of a bacterial SOD post-translationally controlled by phosphorylation, suggesting a possible new host innate mechanism to counteract a virulence factor.     

Helminth anti-oxidant enzymes: a protective mechanism against host oxidants?

Highly reactive oxygen species potentially represent a powerful effector mechanism against parasites. They are produced during normal cellular metabolism, especially by activated phagocytes, and also by some anti parasitic drugs. From studies to date, all protozoan and helminth parasites appear to have one or more anti-oxidant enzymes able to scavenge or quench the reactive oxygen species, and there is strong evidence that such enzymes play a crucial role in protecting against the host response. This detailed review, which summarizes studies on the major anti-oxidant enzymes of helminths, clearly illustrates that methods to block or overcome anti-oxidant protection may be a fertile field in the search for improved ways to inhibit parasite survival.     

Pyridoxal phosphate-dependent histidine decarboxylases. Cloning, sequencing, and expression of genes from Klebsiella planticola and Enterobacter aerogenes and properties of the overexpressed enzymes

The hdc genes encoding the inducible pyridoxal-P-dependent histidine decarboxylase (HisDCase) of Klebsiella planticola and Enterobacter aerogenes were isolated, sequenced, and expressed in Escherichia coli under control of the lac promoter, and the overproduced enzymes were purified to homogeneity from the recombinant host. Formation of inclusion bodies during synthesis of the E. aerogenes enzyme was avoided by cooling the culture and inducing at 25 degrees C. The cloned enzymes were produced in amounts three to four times those present in the fully induced native hosts and were identical in properties to those isolated earlier (Guirard, B. M., and Snell, E. E. (1987) J. Bacteriol. 169, 3963-3968). The two enzymes showed 85% sequence identity and also showed 80% sequence identity with the previously sequenced (Vaaler, G. L., Brasch, M. A., and Snell, E. E. (1986) J. Biol. Chem. 261, 11010-11014) HisDCase of Morganella morganii. Nevertheless, antibodies to the M. morganii HisDCase do not cross-react with these enzymes suggesting that the regions of amino acid variations are located on the outer surface of the proteins. All three HisDCases are the same length (377 amino acid residues); encoded N-terminal methionine was completely removed in each case. These closely related pyridoxal-P enzymes show no sequence homology with the pyruvoyl-dependent HisDCases of Gram-positive bacteria.     

Lactic acid bacteria found in fermented fish in Thailand

Forty-seven strains of homofermentative rod-shaped and 5 heterofermentative sphere-shaped lactic acid bacteria were isolated from 4 kinds of fermented fish (pla-ra, pla-chom, kung-chom, and hoi-dong) in Thailand. These bacteria were separated into four groups by phenotypic and chemotaxonomic characteristics, including fluorometric DNA-DNA hybridization. Five strains (Group I) contained meso-diaminopimelic acid in the cell wall. Four strains were identified as Lactobacillus pentosus, and one strain was L. plantarum. Tested strains of this group produced DL-lactic acid. The rest of the rod-shaped bacteria, 23 strains (Group II) and 19 strains (Group III), lacked meso-diaminopimelic acid in the cell wall and were identified as L. farciminis and Lactobacillus species, respectively. The tested strains of these groups produced L-lactic acid. The amount of cellular fatty acids of C16:0 and C18:1, and the DNA base compositions were significant for differentiating the strains in Groups II and III. Five strains of cocci in chains (Group IV) produced gas from glucose. The tested strains of this group produced d-lactic acid. They were identified as a Leuconostoc species. The distribution of these bacteria in fermented fish in Thailand is discussed.     

Bile salt biotransformations by human intestinal bacteria

Secondary bile acids, produced solely by intestinal bacteria, can accumulate to high levels in the enterohepatic circulation of some individuals and may contribute to the pathogenesis of colon cancer, gallstones, and other gastrointestinal (GI) diseases. Bile salt hydrolysis and hydroxy group dehydrogenation reactions are carried out by a broad spectrum of intestinal anaerobic bacteria, whereas bile acid 7-dehydroxylation appears restricted to a limited number of intestinal anaerobes representing a small fraction of the total colonic flora. Microbial enzymes modifying bile salts differ between species with respect to pH optima, enzyme kinetics, substrate specificity, cellular location, and possibly physiological function. Crystallization, site-directed mutagenesis, and comparisons of protein secondary structure have provided insight into the mechanisms of several bile acid-biotransforming enzymatic reactions. Molecular cloning of genes encoding bile salt-modifying enzymes has facilitated the understanding of the genetic organization of these pathways and is a means of developing probes for the detection of bile salt-modifying bacteria. The potential exists for altering the bile acid pool by targeting key enzymes in the 7alpha/beta-dehydroxylation pathway through the development of pharmaceuticals or sequestering bile acids biologically in probiotic bacteria, which may result in their effective removal from the host after excretion.     

芽孢杆菌SC27胞外代谢产物的活性与成分分析

以从红树林土壤分离并经紫外线和亚硝基胍复合诱变获得的SC27突变菌株作为目标菌,对其胞外代谢产物的活性与成分进行分析。结果表明:芽孢杆菌SC27产生乳酸,产量为5.04 g/L;发酵液蛋白酶、淀粉酶和纤维素酶活力分别为1 316.6、513.3和176.2 U/mL,未检测出脂肪酶;胞外代谢产物对革兰氏阳性菌的抑菌活性强,且抑菌活性物质可耐受高温及木瓜蛋白酶、蛋白酶K和胰蛋白酶处理;发酵液二氯甲烷萃取物的主要化学成分及相对含量为二丁基羟基甲苯(10.28%)、二甲基二氧基硅烷(7.87%)、2,4-二叔丁基苯酚(2.92%)和2个未确定化合物(4.47%、2.36%)。     

Cloning and expression of cellulase and xylanase genes in Lactobacillus plantarum

Summary Eleven cellulase genes from Gram-positive bacteria were cloned in a Lactobacillus plantarum silage inoculum. Eight of these genes were expressed as active enzymes from their original promotors and translation signals. Where tested, the enzymes produced by transformed L.plantarum had the same temperature and pH optimum as enzymes produced in the original host, or in transformed Escherichia coli. Using chloramphenicol acetyltransferase as a cell-internal marker enzyme, it could be demonstrated that at least endoglucanase D from Clostridium thermocellum was actively secreted by transformed L. plantarum. In growing L. plantarum cultures, most of the enzymes were irreversibly inactivated when the pH decreased below 4.5. If the transformed strains were to be applied as an inoculum in silage, this pH inactivation might be useful in preventing overdigestion of the crop fibre. Offprint requests to: F. Michiels