Genetically Engineered Erwinia carotovora: Survival, Intraspecific Competition, and Effects upon Selected Bacterial Genera

Environmental use of genetically engineered microorganisms has raised concerns about potential ecological impact. This research evaluated the survival, competitiveness, and effects upon selected bacterial genera of wild-type and genetically engineered Erwinia carotovora subsp. carotovora to ascertain if differences between the wild-type and genetically engineered strains exist in soil microcosms. The engineered strain contained a chromosomally inserted gene for kanamycin resistance. No significant differences in survival in nonsterile soil over 2 months or in the competitiveness of either strain were observed when the strains were added concurrently to microcosms. For reasons that remain unclear, the engineered strain did survive longer in sterilized soil. The effects of both strains on total bacteria, Pseudomonas and Staphylococcus strains, and actinomycetes were observed. While some apparent differences were observed, they were not statistically significant. A better understanding of the microbial ecology of engineered bacteria, especially pathogens genetically altered for use as biological control agents, is essential before commercial applications can be accomplished.     

Probabilistic Models to Describe the Dynamics of Migrating Microbial Communities

In all but the most sterile environments bacteria will reside in fluid being transported through conduits and some of these will attach and grow as biofilms on the conduit walls. The concentration and diversity of bacteria in the fluid at the point of delivery will be a mix of those when it entered the conduit and those that have become entrained into the flow due to seeding from biofilms. Examples include fluids through conduits such as drinking water pipe networks, endotracheal tubes, catheters and ventilation systems. Here we present two probabilistic models to describe changes in the composition of bulk fluid microbial communities as they are transported through a conduit whilst exposed to biofilm communities. The first (discrete) model simulates absolute numbers of individual cells, whereas the other (continuous) model simulates the relative abundance of taxa in the bulk fluid. The discrete model is founded on a birth-death process whereby the community changes one individual at a time and the numbers of cells in the system can vary. The continuous model is a stochastic differential equation derived from the discrete model and can also accommodate changes in the carrying capacity of the bulk fluid. These models provide a novel Lagrangian framework to investigate and predict the dynamics of migrating microbial communities. In this paper we compare the two models, discuss their merits, possible applications and present simulation results in the context of drinking water distribution systems. Our results provide novel insight into the effects of stochastic dynamics on the composition of non-stationary microbial communities that are exposed to biofilms and provides a new avenue for modelling microbial dynamics in systems where fluids are being transported.     

改造肠道微生物在疾病诊断与治疗中的应用

肠道微生物是近年来新兴的热门研究领域,它与人类疾病健康存在着密切的关系。伴随高通量测序技术的发展,研究者们发现了肠道微生物在疾病的诊断与治疗中的潜力。合成生物学通过设计编辑工具以及反馈回路,可以构建具有诊断疾病或者靶向治疗疾病的肠道微生物工程菌株。这些工程菌能够对环境进行感知、计算和反馈。本文概述了改造后的肠道微生物在疾病诊断与治疗中的应用,同时阐述了目前改造后肠道微生物的临床应用现状,并对"工具短缺"以及目前改造后肠道微生物所存在的安全性等问题进行了讨论。     

Lipopeptide surfactants: Production,recovery and pore forming capacity

Lipopeptides are microbial surface active compounds produced by a wide variety of bacteria, fungi and yeast. They are characterized by highly structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Surfactin, iturin and fengycin of Bacillus subtilis are among the most studied lipopeptides. This review will present the main factors encountering lipopeptides production along with the techniques developed for their extraction and purification. Moreover, we will discuss their ability to form pores and destabilize biological membrane permitting their use as antimicrobial, hemolytic and antitumor agents. These open great potential applications in biomediacal, pharmaceutic and agriculture fields.     

Current advances in molecular methods for detection of nitrite-dependent anaerobic methane oxidizing bacteria in natural environments

Nitrite-dependent anaerobic methane oxidation (n-damo) process uniquely links microbial nitrogen and carbon cycles. Research on n-damo bacteria progresses quickly with experimental evidences through enrichment cultures. Polymerase chain reaction (PCR)-based methods for detecting them in various natural ecosystems and engineered systems play a very important role in the discovery of their distribution, abundance, and biodiversity in the ecosystems. Important characteristics of n-damo enrichments were obtained and their key significance in microbial nitrogen and carbon cycles was investigated. The molecular methods currently used in detecting n-damo bacteria were comprehensively reviewed and discussed for their strengths and limitations in applications with a wide range of samples. The pmoA gene-based PCR primers for n-damo bacterial detection were evaluated and, in particular, several incorrectly stated PCR primer nucleotide sequences in the published papers were also pointed out to allow correct applications of the PCR primers in current and future investigations. Furthermore, this review also offers the future perspectives of n-damo bacteria based on current information and methods available for a better acquisition of new knowledge about this group of bacteria.     

Manufacturing of recombinant therapeutic proteins in microbial systems

Recombinant therapeutic proteins have gained enormous importance for clinical applications. The first recombinant products have been produced in E. coli more than 20 years ago. Although with the advent of antibody-based therapeutics mammalian expression systems have experienced a major boost, microbial expression systems continue to be widely used in industry. Their intrinsic advantages, such as rapid growth, high yields and ease of manipulation, make them the premier choice for expression of non-glycosylated peptides and proteins. Innovative product classes such as antibody fragments or alternative binding molecules will further expand the use of microbial systems. Even more, novel, engineered production hosts and integrated technology platforms hold enormous potential for future applications. This review summarizes current applications and trends for development, production and analytical characterization of recombinant therapeutic proteins in microbial systems.     

Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation

In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications.     

Engineering promiscuity of chloramphenicol acetyltransferase for microbial designer ester biosynthesis

Robust and efficient enzymes are essential modules for metabolic engineering and synthetic biology strategies across biological systems to engineer whole-cell biocatalysts. By condensing an acyl-CoA and an alcohol, alcohol acyltransferases (AATs) can serve as interchangeable metabolic modules for microbial biosynthesis of a diverse class of ester molecules with broad applications as flavors, fragrances, solvents, and drop-in biofuels. However, the current lack of robust and efficient AATs significantly limits their compatibility with heterologous precursor pathways and microbial hosts. Through bioprospecting and rational protein engineering, we identified and engineered promiscuity of chloramphenicol acetyltransferases (CATs) from mesophilic prokaryotes to function as robust and efficient AATs compatible with at least 21 alcohol and 8 acyl-CoA substrates for microbial biosynthesis of linear, branched, saturated, unsaturated and/or aromatic esters. By plugging the best engineered CAT (CATec3 Y20F) into the gram-negative mesophilic bacterium Escherichia coli, we demonstrated that the recombinant strain could effectively convert various alcohols into desirable esters, for instance, achieving a titer of 13.9 g/L isoamyl acetate with 95% conversion by fed-batch fermentation. The recombinant E. coli was also capable of simulating the ester profile of roses with high conversion (>97%) and titer (>1 g/L) from fermentable sugars at 37 °C. Likewise, a recombinant gram-positive, cellulolytic, thermophilic bacterium Clostridium thermocellum harboring CATec3 Y20F could produce many of these esters from recalcitrant cellulosic biomass at elevated temperatures (>50 °C) due to the engineered enzyme's remarkable thermostability. Overall, the engineered CATs can serve as a robust and efficient platform for designer ester biosynthesis from renewable and sustainable feedstocks.     

Dielectrophoresis-Based Discrimination of Bacteria at the Strain Level Based on Their Surface Properties

Insulator-based dielectrophoresis can be used to manipulate biological particles, but has thus far found limited practical applications due to low sensitivity. We present linear sweep three-dimensional insulator-based dielectrophoresis as a considerably more sensitive approach for strain-level discrimination bacteria. In this work, linear sweep three-dimensional insulator-based dielectrophoresis was performed on Pseudomonas aeruginosa PA14 along with six isogenic mutants as well as Streptococcus mitis SF100 and PS344. Strain-level discrimination was achieved between these clinically important pathogens with applied electric fields below 10 V/mm. This low voltage, high sensitivity technique has potential applications in clinical diagnostics as well as microbial physiology research.     

Genetically Engineered Erwinia carotovora in Aquatic Microcosms: Survival and Effects on Functional Groups of Indigenous Bacteria

The survival of genetically engineered Erwinia carotovora L-864, with a kanamycin resistance gene inserted in its chromosome, was monitored in the water and sediment of aquatic microcosms. The density of genetically engineered and wild-type E. carotovora strains declined at the same rate, falling in 32 days below the level of detection by viable counts. We examined the impact of the addition of genetically engineered and wild-type strains on indigenous bacteria belonging to specific functional groups important in nutrient cycling. For up to 16 days, the densities of total and proteolytic bacteria were significantly higher (P < 0.05) in microcosms inoculated with genetically engineered or wild-type E. carotovora, but by 32 days after inoculation, they had decreased to densities similar to those in control microcosms. Inoculation of genetically engineered or wild-type E. carotovora had no apparent effect on the density of amylolytic and pectolytic bacteria in water and sediment. Genetically engineered and wild-type E. carotovora did not have significantly different effects on the densities of specific functional groups of indigenous bacteria (P > 0.05).     

Impact of microbial proteases on biotechnological industries

The demand of enzymes in industrial sectors is increasing rapidly due to their economical and ecological advantages. Micro-organisms produce different types of extracellular enzymes for maintaining their own metabolism, defense, and normal physiological condition. Among several enzymes, proteases have gained special attention in industrial sectors. Several sources of extracellular enzymes are reported by various researchers, but enzymes obtain from microbial sources have high demand in industries due to lower cost, high production rate, availability, stability, and diversity. Among micro-organism, bacteria and fungi are reported to be good sources of different types of proteases such as alkaline protease, cysteine protease, aspartate protease, and metallo protease. In this review, we have summarized the available information about the sources of bacterial and fungal proteases, their purification strategies and their temperature and pH optima. Due to huge competition, companies are trying to reduce their manufacturing cost and that’s why microbial sources of enzymes are important. However, genetically engineered strains or engineered proteases have much more importance over natural isolates/protease in industries due to higher production rate and other advantages. Here we have also summarized the important applications of protease in different industries such as, paper mill, starch degrading sector, food processing factories, and detergent making companies.     

微生物胞外电子传递效率的合成生物学强化

电活性微生物(产电微生物和亲电微生物)通过与外界环境进行双向电子和能量传递来实现多种微生物电催化过程(包括微生物燃料电池、微生物电解电池、微生物电催化等),从而实现在环境、能源领域的广泛应用,并为开发有效且可持续性生产新能源或大宗精细化学品的工艺提供了新机会。但是,电活性微生物的胞外电子传递效率比较低,这已经成为限制微生物电催化系统在工业应用中的主要瓶颈。以下综述了近年来利用合成生物学改造电活性微生物的相关研究成果,阐明了合成生物学如何用于打破电活性微生物胞外电子传递途径低效率的瓶颈,从而实现电活性微生物与环境的高效电子传递和能量交换,推动电活性微生物电催化系统的实用化进程。     

New methods for selective isolation of bacterial DNA from human clinical specimens

Separation of bacterial DNA from human DNA in clinical samples may have an important impact on downstream applications, involving microbial diagnostic systems. We evaluated two commercially available reagents (MolYsis^®, Molzym GmbH & Co. KG, Bremen and Pureprove^®, SIRS-Lab GmbH, Jena, both Germany) for their potential to isolate and purify bacterial DNA from human DNA. We chose oral samples, which usually contain very high amounts of both human and bacterial cells. Three different DNA preparations each were made from eight caries and eight periodontal specimens using the two reagents above and a conventional DNA extraction strategy as reference. Based on target-specific real-time-quantitative PCR assays we compared the reduction of human DNA versus loss of bacterial DNA. Human DNA was monitored by targeting the β-2-microglobulin gene, while bacteria were monitored by targeting 16S rDNA (total bacteria and Porphyromonas gingivalis) or the glycosyltransferase gene (Streptococcus mutans).We found that in most cases at least 90% of human DNA could successfully be removed, with complete removal in eight of 16 cases using MolYsis, and two (of 16) cases using Pureprove. Conversely, detection of bacterial DNA was possible in all cases with a recovery rate generally ranging from 35% to 50%. In conclusion, both strategies have the potential to reduce background interference from the host DNA which may be of remarkable value for nucleic-acid based microbial diagnostic systems.     

Synthetic biology in multicellular organisms: Opportunities in nematodes

Synthetic biology has mainly focused on introducing new or altered functionality in single cell systems: primarily bacteria, yeast, or mammalian cells. Here, we describe the extension of synthetic biology to nematodes, in particular the well-studied model organism Caenorhabditis elegans, as a convenient platform for developing applications in a multicellular setting. We review transgenesis techniques for nematodes, as well as the application of synthetic biology principles to construct nematode gene switches and genetic devices to control motility. Finally, we discuss potential applications of engineered nematodes.     

Model‐based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions

The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi‐aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large‐scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major milestone on the way to industrial bioprocesses. As a starting point, we focus on aerobic growth due to higher growth rates and more facile process control under this condition, with the option to extend the process by an anaerobic production phase. Process design and optimization were supported by an unstructured computational process model, based on mixed‐substrate kinetics. Key parameters for growth and process control were determined in shake‐flask experiments or estimated by simulation studies. For fed‐batch cultivation, a computer‐controlled exponential feed algorithm in combination with a pH‐stat element was implemented. As a result, a maximal cell density of 59 g cell dry weight (CDW) L^?1 was obtained, representing so far not attainable cell densities for photosynthetic bacteria. The applied exponential fed‐batch methodology therefore enters a range which is commonly employed for industrial applications with microbial cells. The biochemical analysis of high cell density cultures revealed metabolic imbalances, such as the accumulation and excretion of tetrapyrrole intermediates of the bacteriochlorophyll biosynthetic pathway. Biotechnol. Bioeng. 2010. 105: 729–739. © 2009 Wiley Periodicals, Inc.     

Bacterial Community Survey of Solenopsis invicta Buren (Red imported fire Ant) Colonies in the Presence and Absence of Solenopsis invicta Virus (SINV)

Insect bacterial symbionts contribute to many essential biological functions of their hosts and can also influence host fecundity and fitness. The physiological contribution symbionts provide can aid in immune response and xenobiotic detoxification. Both of these immune factors can directly impact strategies aimed at managing insect populations. One biological control strategy that shows promise in insects is the use of single-stranded RNA viruses within the group Dicistroviridae. The Solenopsis invicta Virus (SINV; Dicistroviridae), a ssRNA virus, has been proposed as a potential biological control agent for the urban pest S. invicta Buren or red imported fire ant (RIFA). SINV has been shown to be prevalent in RIFA populations of Texas and Florida; however, mortality is associated with high viral load. In other insect microbe systems, presence of particular bacteria induced resistance against Dicistrovirus. If this type of relationship is present in the RIFA–SINV system, their bacterial community could reduce the effectiveness of SINV as a biological control system. The advantage of 454 pyro-sequencing is that it enables classification of unculturable bacteria. This study examines the bacterial community in brood, workers, and reproductive cast members from colonies with and without SINV infection. Manipulation of the bacterial community may alter virus infection and replication within the mid-gut. Understanding the differences in the microbial community of ant colonies may provide insights that will refine current efforts designing control strategies for this important urban pest.     

抗辐射菌Deinococcus radiodurans的多样性

抗辐射菌Deinococcus radiodurans是一种对电离辐射和其他DNA损伤因子具有极强抵抗能力的细菌,是研究DNA损伤与修复的模式生物。综述了国内外在抗辐射菌研究上取得的最新研究成果,从生存环境、对DNA损伤因子的抗性、抗性机理及其损伤修复关联基因等方面报道了抗辐射菌的多样性,并探讨了该细菌高效正确的DNA损伤修复机理的相关研究成果在生命科学、农业、环境修复及医学等领域的应用前景。     

A phylogenetic analysis of the phylum Fibrobacteres

Members of the phylum Fibrobacteres are highly efficient cellulolytic bacteria, best known for their role in rumen function and as potential sources of novel enzymes for bioenergy applications. Despite being key members of ruminants and other digestive microbial communities, our knowledge of this phylum remains incomplete, as much of our understanding is focused on two recognized species, Fibrobacter succinogenes and F. intestinalis. As a result, we lack insights regarding the environmental niche, host range, and phylogenetic organization of this phylum. Here, we analyzed over 1000 16S rRNA Fibrobacteres sequences available from public databases to establish a phylogenetic framework for this phylum. We identify both species- and genus-level clades that are suggestive of previously unknown taxonomic relationships between Fibrobacteres in addition to their putative lifestyles as host-associated or free-living. Our results shed light on this poorly understood phylum and will be useful for elucidating the function, distribution, and diversity of these bacteria in their niches.