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Gaithersburg, MD, United States

Lu Z.,Kennesaw State University | Altermann E.,Agresearch Ltd. | Breidt F.,North Carolina State University | Breidt F.,U.S. Department of Agriculture | Kozyavkin S.,Fidelity Systems Inc.
Applied and Environmental Microbiology | Year: 2010

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB). Leuconostoc mesenteroides initiates fermentation. As fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phages infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage Φ1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses showed that Φ1-A4 is a unique lytic phage with a large-scale genome inversion (∼30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Φ1-A4 clusters with several Lactococcus phages. To our knowledge, Φ1-A4 is the first genetically characterized L. mesenteroides phage. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 175.56K | Year: 2009

DESCRIPTION (provided by applicant): A novel approach is needed to overcome the deficiencies of protein production technology and ensure the produced enzymes fold into physiologically effective three-dimensional conformations. We discovered that incorporation of novel archaeal chaperones into a host-vector system and into in vitro enzymatic reactions helps both to recover proteins from inclusion bodies and to keep enzymes operationally active in vitro. This finding opens a way for novel protein folding technology both in vivo and in vitro. We propose to develop new host-vector systems, reagents and protocols based on archaeal chaperones for protein folding in vivo and in vitro. Archaeal chaperones exhibit folding activity for a broader range of substrates because archaeal species have evolved to occupy extreme ecological niches, which pose particular challenges to the problem of protein folding. In Phase 1, we will construct several host-vector systems to demonstrate the feasibility of our approach to produce aggregation-prone proteins in their native conformations with the help of archaeal chaperones. We will also develop simple assays to accelerate technology development, streamline problem management and enable quality control for new products and protocols. A robust protein production and folding technology plays a pivotal role in biotechnology innovation. It simplifies the multiple tasks of proteomics and encourages engineering new proteins for healthcare, environmental, and industrial purposes. PUBLIC HEALTH RELEVANCE: A robust and versatile protein folding technology will impact the healthcare related RandD programs at multiple levels. Most importantly, it will facilitate high-throughput production and purification of medically important proteins. It will also bring to a new level our ability to recover and analyze effective proteins in physiological three-dimensional conformations in vitro.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2003

DESCRIPTION (provided by applicant): The main objective of this SBIR Phase I application is to develop a generic approach for construction by methods of gene engineering hybrid DNA metabolizing enzymes with enhanced processivity. Particularly, a set of processive thermostable DNA polymerases, capable of extensive rapid replication, efficient recycling between substrate molecules and suitable for both LA-PCR and most challenging DNA sequencing applications will be developed. A number of plasmid constructs will be made and the encoding chimeric proteins will be expressed in E. coli, purified and assayed for enhanced processivity, i.e for capability to extend a primer for 1 kb or more prior to dissociating from a template, and for significantly higher affinity to a primer-template comparing to current commercial thermostable polymerases. Such improvement in basic enzyme properties will enhance both molecular biology research at universities and boost industrial genome research as well. The availability of a specialized enzyme for long PCR and advanced genomic sequencing will enhance diagnostic procedures for cancer, genetic defects and pathogens.

Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 143.40K | Year: 2005

DESCRIPTION (provided by applicant): The goal of this project is to develop a general method of improving the stability and durability of enzymes used in DNA and RNA amplification, sequencing, restriction analysis and ligation. The enzymes to be improved include DNA polymerases, topoisomerases, reverse transcriptases and ligases. In the event this goal can be achieved, broad impacts may result in the areas of clinical, forensic and basic science that rely on DNA sequencing, amplification and RT-PCR processes. To do this, extrinsic stabilization and refolding of proteins mediated by chaperones from hyperthermophiles will be adapted for incorporation into reaction mixtures with DNA modifying enzymes.

Agency: Department of Energy | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 725.00K | Year: 2001

60647 The shotgun DNA sequencing method is in wide use for the acquisition of draft data, but it is not followed by equally productive finishing technology. High throughput technology is needed to transform the art of finishing genome projects into a streamlined production operation. This project will develop advanced DNA sequencing reagents and protocols that are 100 to 1,000 times more efficient than current standards and apply them for the direct sequencing of Bacterial Artificial Chromosome (BAC) and genomic DNA templates. This will be accomplished by designing a model to optimize overall workflow in the high throughput environment and test it on ongoing genomic projects. Phase I demonstrated the feasibility of increasing the yield of sequencing reactions. 10,000 Fimers were synthesized and applied for direct sequencing of BAC and microbial genomic DNA. The results showed that the direct sequencing approach was suitable for further development and implementation into large scale projects. Phase II will incorporate new enzymatic and chemical tools for more efficient sequencing of microbial genomic DNA, and overall workflow will be optimized for the high throughput environment. Commercial Applications And Other Benefits as described by the awardee: Rapid, cost effective finishing of genome projects should fulfill unmet demand in academic and government projects, as well as the booming biotechnology industry. It should free genomics centers and companies from unproductive work and accelerate the overall rate of data acquisition.

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