Utilization of nitrogen compounds and ammonia assimilation by chromatiaceae

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH₄Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina.Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrate in the above organisms grown on NH₄Cl.     

Oxic and anoxic growth of a new Citrobacter species on amino acids

A facultatively anaerobic bacterium, strain P-88, was enriched selectively under dual limitation by glutamate and oxygen in a chemostat. The new strain is a gram-negative motile rod. The mol% guanine plus cytosine of the DNA is 51.4±0.6 mol%. The organism grows on citrate as a sole source of carbon and energy, does not form acetoin, does not induce lysine decarboxylase and was thus classified as a species of the genus Citrobacter. A remarkable characteristic of the new isolate is its ability to grow on several amino acids with either a respiratory or a fermentative type of metabolism. Under strictly anoxic conditions glutamate was fermented to acetate, H₂, CO₂ and ammonia. Asparagine, aspartate and serine could also be fermented. Furthermore, all type strains of the genus Citrobacter were shown to have the same fermentative abilities. Based on enzyme activities determined in cell-free extracts a combination of the methylaspartate pathway and the mixed acid fermentation of Enterobacteriaceae is proposed to explain the glutamate fermentation pattern observed in cultures of strain P-88. Analysis of the growth of strain P-88 in continuous culture with various degrees of oxygen supply, demonstrated that the bacterium can rapidly switch between oxic and anoxic metabolism. Cultures of strain P-88 grown under oxygen limitation simultaneously respire and ferment glutamate, suggesting that the organism is particularly well adapted to growth in microoxic environments.     

Comparative genomics of <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> with emphasis on strains from meat

Lactobacillus sakei is a lactic acid bacterium important in food microbiology mainly due to its ability to ferment and preserve meat. The genome sequence of L. sakei strain 23K has revealed specialized metabolic capacities that reflect the bacterium’s adaption to meat products, and that differentiate it from other LAB. An extensive genomic diversity analysis was conducted to elucidate the core features of the species, and to provide a better comprehension of niche adaptation of the organism. Here, we describe the genomic comparison of 18 strains of L. sakei originating mainly from processed meat against the 23K strain by comparative genome hybridization. Pulsed field gel electrophoresis was used to estimate the genome sizes of the strains, which varied from 1.880 to 2.175 Mb, and the 23K genome was among the smallest. Consequently, a large part of the genome of this strain belongs to a common gene pool invariant in this species. The majority of genes important in adaption to meat products, the ability to flexibly use meat components, and robustness during meat processing and storage were conserved, such as genes involved in nucleoside scavenging, catabolism of arginine, and the ability to cope with changing redox and oxygen levels, which is indicative of the role these genes play in niche specialization within the L. sakei species. Moreover, an additional set of sequenced L. sakei genes beyond the 23K genome was present on the microarray used, and it was demonstrated that all the strains carry remnants of or complete bacteriocin operons. The genomic divergence corresponded mainly to five regions in the 23K genome, which showed features consistent with horizontal gene transfer. Carbohydrate-fermentation profiles of the strains were evaluated in light of the CGH data, and for most substrates, the genotypes were consistent with the phenotypes. We have demonstrated a highly conserved organization of the L. sakei genomes investigated, and the 23K strain is a suitable model organism to study core features of the L. sakei species.     

Correlation of genomic and physiological traits of thermoanaerobacter species with biofuel yields

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B₁₂ biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.     

Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches

Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro‐intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low‐resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum‐marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.     

Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination

Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain’s performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches.     

Improved production of propionic acid using genome shuffling

Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. Production of other organic acids reduces propionic acid yield and productivity, pointing to by‐products gene‐knockout strategies as a logical solution to increase yield. However, removing by‐product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand improved strains. In this work, we use genome shuffling on two wild type strains to generate a better propionic acid producing strain. Using next generation sequencing, we mapped the genomic changes leading to the improved phenotype. The best strain produced 25% more propionic acid than the wild type strain. Sequencing of the strains showed that genomic changes were restricted to single point mutations and gene duplications in well‐conserved regions in the genomes. Such results confirm the involvement of gene conversion in genome shuffling as opposed to long genomic insertions.     

蜡状芽胞杆菌群代谢途径的分析和比较

微生物基因组测序和高通量分析方法获得了大量的数据和信息,利用这些信息研究代谢网络成为当前的一个新热点。文章在比较和分析重构代谢网络不同方法的基础上,利用蜡状芽胞杆菌群中已测序的9株蜡状芽胞杆菌、6株炭疽芽胞杆菌、6株苏云金芽胞杆菌基因组,对它们的碳水化合物代谢途径、氨基酸代谢途径和能量代谢途径进行比较与分析,找出它们的共性和特性。这3种菌都存在必需的糖酵解、三羧酸循环、丙氨酸代谢、组氨酸代谢及能量代谢等途径;同时它们还存在特殊的代谢途径,蜡状芽胞杆菌对单糖的利用率较高;炭疽芽胞杆菌的氨基酸降解和转运途径较丰富;苏云金芽胞杆菌中存在催化谷氨酸转化的代谢旁路等。代谢途径的分析为深入研究它们的食物毒素、炭疽毒素和杀虫毒素提供了新思路。     

Proteins involved in difference of sorbitol fermentation rates of the toxigenic and nontoxigenic Vibrio cholerae El Tor strains revealed by comparative proteome analysis

Background  

The nontoxigenic V. cholerae El Tor strains ferment sorbitol faster than the toxigenic strains, hence fast-fermenting and slow-fermenting strains are defined by sorbitol fermentation test. This test has been used for more than 40 years in cholera surveillance and strain analysis in China. Understanding of the mechanisms of sorbitol metabolism of the toxigenic and nontoxigenic strains may help to explore the genome and metabolism divergence in these strains. Here we used comparative proteomic analysis to find the proteins which may be involved in such metabolic difference.     

Effect of Lineage-Specific Metabolic Traits of Lactobacillus reuteri on Sourdough Microbial Ecology

This study determined the effects of specific metabolic traits of Lactobacillus reuteri on its competitiveness in sourdoughs. The competitiveness of lactobacilli in sourdough generally depends on their growth rate; acid resistance additionally contributes to competitiveness in sourdoughs with long fermentation times. Glycerol metabolism via glycerol dehydratase (gupCDE) accelerates growth by the regeneration of reduced cofactors; glutamate metabolism via glutamate decarboxylase (gadB) increases acid resistance by generating a proton motive force. Glycerol and glutamate metabolisms are lineage-specific traits in L. reuteri; therefore, this study employed glycerol dehydratase-positive sourdough isolates of human-adapted L. reuteri lineage I, glutamate decarboxylase-positive strains of rodent-adapted L. reuteri lineage II, as well as mutants with deletions in gadB or gupCDE. The competitivenesses of the strains were quantified by inoculation of wheat and sorghum sourdoughs with defined strains, followed by propagation of doughs with a 10% inoculum and 12-h or 72-h fermentation cycles. Lineage I L. reuteri strains dominated sourdoughs propagated with 12-h fermentation cycles; lineage II L. reuteri strains dominated sourdoughs propagated with 72-h fermentation cycles. L. reuteri 100-23ΔgadB was outcompeted by its wild-type strain in sourdoughs fermented with 72-h fermentation cycles; L. reuteri FUA3400ΔgupCDE was outcompeted by its wild-type strain in sourdoughs fermented with both 12-h and 72-h fermentation cycles. Competition experiments with isogenic pairs of strains resulted in a constant rate of strain displacement of the less competitive mutant strain. In conclusion, lineage-specific traits of L. reuteri determine the competitiveness of this species in sourdough fermentations.     

Enzymes of ammonia assimilation and their regulation inAzospirillum lipoferum strain D-2

Azospirillum lipoferum strain D-2 possesses the following enzymes for the assimilation of N₂ and NH ₄ ⁺ : nitrogenase, glutamine synthetase, NADPH-dependent glutamate synthase, NADH-/NADPH-dependent glutamate dehydrogenase, and NADH-dependent alanine dehydrogenase. Nitrogenase and glutamine synthetase are repressed, whereas glutamate dehydrogenase and alanine dehydrogenase are induced by NH ₄ ⁺ . Glutamine synthetase activity is modulated by both repression and depression and also by adenylylation.     

海南地区与环渤海湾美人鱼发光杆菌美人鱼亚种水产动物分离株的表型与遗传特征分析

美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp.damselae,PDD)是一种广泛分布于海洋环境内的重要病原菌,可导致多种海洋生物患病死亡,近年来在我国不同养殖区域内均有发现和报道.[目的]通过生理代谢表型、毒力基因分布和分子遗传分析,系统比较我国海南地区和环渤海湾不同株系PDD...     

Integrated genomics and phenotype microarray analysis of Saccharomyces cerevisiae industrial strains for rice wine fermentation and recombinant protein production

The industrial potential of Saccharomyces cerevisiae has extended beyond its traditional use in fermentation to various applications, including recombinant protein production. Herein, comparative genomics was performed with three industrial S. cerevisiae strains and revealed a heterozygous diploid genome for the 98-5 and KSD-YC strains (exploited for rice wine fermentation) and a haploid genome for strain Y2805 (used for recombinant protein production). Phylogenomic analysis indicated that Y2805 was closely associated with the reference strain S288C, whereas KSD-YC and 98-5 were grouped with Asian and European wine strains, respectively. Particularly, a single nucleotide polymorphism (SNP) in FDC1, involved in the biosynthesis of 4-vinylguaiacol (4-VG, a phenolic compound with a clove-like aroma), was found in KSD-YC, consistent with its lack of 4-VG production. Phenotype microarray (PM) analysis showed that KSD-YC and 98-5 displayed broader substrate utilization than S288C and Y2805. The SNPs detected by genome comparison were mapped to the genes responsible for the observed phenotypic differences. In addition, detailed information on the structural organization of Y2805 selection markers was validated by Sanger sequencing. Integrated genomics and PM analysis elucidated the evolutionary history and genetic diversity of industrial S. cerevisiae strains, providing a platform to improve fermentation processes and genetic manipulation.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-alanine by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli W was genetically engineered to produce l-alanine as the primary fermentation product from sugars by replacing the native d-lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native d-lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of l-alanine to d-alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l⁻¹ glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g⁻¹ glucose) with a chiral purity greater than 99.5% l-alanine. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

一株冠突散囊菌的分离鉴定、基因组成及其枇杷花发酵特性分析

【背景】冠突散囊菌LYEC03是从陕西泾阳茯砖茶中分离得到的主要发酵菌株。【目的】研究冠突散囊菌LYEC03菌株的基因组信息及其发酵枇杷花产品的特性,从分子水平阐明冠突散囊菌的发酵机制。【方法】应用形态和显微形态观察、ITS序列鉴定、重测序及框架图测序对所分离的菌株LYEC03进行鉴定和基因组信息解析,采用所分离的菌株LYEC03发酵枇杷花,研究冠突散囊菌对枇杷花主要功效成分及抗氧化性能的影响。【结果】陕西泾阳茯砖茶中分离得到的主要发酵菌株LYEC03确定为冠突散囊菌。菌株LYEC03与参考基因组覆盖率高,含有丰富的纤维素酶、蛋白酶、氧化酶和脂肪酶相关基因;基因组相关整体变异较小,其中假设蛋白、碳水化合物激酶、纤维素酶家族糖基水解酶、位点2蛋白酶家族蛋白、多酚氧化酶和谷胱甘肽过氧化物酶等与菌株生长、能量代谢调节、产纤维酶、产蛋白酶和产氧化酶相关的基因发生了变异。菌株LYEC03基因组序列长度30 623 602 bp、GC含量51.70%、编码13 033个基因、编码基因占比55.74%,参与了碳水化合物代谢、氨基酸代谢、外源生物降解代谢、能量代谢、脂质代谢与萜类化合物和聚酮类化合物的...     

Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Acid protease is essential for degradation of proteins during soy sauce fermentation. To breed more suitable koji molds with high activity of acid protease, interspecific genome recombination between A. oryzae and A. niger was performed. Through stabilization with d-camphor and haploidization with benomyl, several stable fusants with higher activity of acid protease were obtained, showing different degrees of improvement in acid protease activity compared with the parental strain A. oryzae. In addition, analyses of mycelial morphology, expression profiles of extracellular proteins, esterase isoenzyme profiles, and random amplified polymorphic DNA (RAPD) were applied to identify the fusants through their phenotypic and genetic relationships. Morphology analysis of the mycelial shape of fusants indicated a phenotype intermediate between A. oryzae and A. niger. The profiles of extracellular proteins and esterase isoenzyme electrophoresis showed the occurrence of genome recombination during or after protoplast fusion. The dendrogram constructed from RAPD data revealed great heterogeneity, and genetic dissimilarity indices showed there were considerable differences between the fusants and their parental strains. This investigation suggests that genome recombination is a powerful tool for improvement of food-grade industrial strains. Furthermore, the presented strain improvement procedure will be applicable for widespread use for other industrial strains.     

酸性旱地红壤无机氮同化菌株筛选及其全基因组分析

微生物执行的无机氮同化作用可固定施入土壤后未被作物直接吸收的化学氮肥,有效减少化学氮肥损失、降低环境氮素污染风险。土壤无机氮同化作用不是由大量冗余微生物共同执行的,而是由一小部分功能微生物优先执行。【目的】对酸性旱地红壤中的优势无机氮同化细菌进行富集、菌株分离鉴定及全基因组测序,并明确菌株在土壤中的氮同化能力,为酸性土壤化学氮肥应用及其转化过程研究提供菌株资源和理论依据。【方法】在酸性旱地红壤中添加KNO3或(NH4)2SO4作为无机氮源,以葡萄糖作为碳源,在好氧条件下进行富集预培养,采用稀释分离法筛选出优势无机氮同化细菌菌株;将菌株回接至土壤中从而验证其无机氮同化能力,并通过全基因组测序分析菌株的氮素代谢途径及相关功能基因。【结果】酸性旱地红壤经富集预培养一周后,优势无机氮同化微生物的16SrRNA基因相对丰度从0.20%–0.94%增长至20.2%–30.2%;分离筛选后得到的3株优势无机氮同化细菌菌株,鉴定为伯克霍尔德氏菌(Burkholderia sp.) M6-3、索状芽孢杆菌(Bacillus funiculus) M2-4和节杆菌(Arthrobacter sp.) M7...