Novel Bacterial Artificial Chromosome Vector pUvBBAC for Use in Studies of the Functional Genomics of Listeria spp

Abstract

Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC؉vgc1 and pUvBBAC؉vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics.With the rapid increase in the number of completed microbial genome sequences, interest in the manipulation and functional characterization of whole genomes is being revolutionized. Methods and technologies that permit the cloning and manipulation of large chromosomal regions, such as the genomic islands which impart either virulence or novel metabolic properties to pathogenic and other microorganisms, are rapidly becoming valuable tools to study in detail properties encoded within these regions (41).The development of bacterial artificial chromosomes (BACs) has provided an important genetic tool for the cloning and mapping of complex genomes. Different cloning vectors based on low-copy-number replicons either from the bacterial F plasmid (45) or bacteriophage P1 (23, 49) have been developed. These provide a powerful resource in molecular biology, because they allow the cloning of several tens to hundreds of kilobases of contiguous DNA sequences (55) and are stable and easy to handle (31). Their particular use is in the study of functional genome segments that are otherwise too large to be cloned into other more conventional vectors. BAC-based libraries of genomic DNA from numerous viruses (1, 4, 8, 29-31, 42, 43) and plant (9, 15, 27, 32, 47, 53), animal (10, 34, 50, 55), and fungal (20, 39, 54) species have been generated and are now established technologies in large-scale sequencing projects. Classical and molecular genetic techniques are being used in conjunction with BAC recombinants for the introduction of reporter systems into mammalian organisms, the in vivo complementation of mutations, and in vivo and in vitro reverse genetic technologies that introduce point mutations (25), targeted deletions, or new sequence elements into BAC vectors (21). More recently, in vivo recombination ("recombineering") (37) technology employing either the bacterial recA mutant or the bacteriophage red gam mutant recombination has been used for the dir...

Galleria mellonella as a Model System for Studying Listeria Pathogenesis

“…Fifty microliters of each dilution was plated out on BHI agar plates and incubated at 37°C for 24 h, and the bacterial CFU were used to calculate the inoculum injected. Cultures of L. innocua harboring the pUvBBAC vector containing the vgc1 locus from L. monocytogenes EGD-e were grown in the presence of 5 g/ml erythromycin and 5 g/ml kanamycin (27). The Escherichia coli host for plasmid constructions was INV␣FЈ.…”

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

“…In order to overcome the inherent limitation of expressing genes in E. coli , the most frequent surrogate host for metagenomic libraries, a number of shuttle or broad host range vectors have been constructed with differing degrees of success in allowing the transfer and screening of the metagenomic library to different host bacteria678910111213. Functional screening of metagenomic libraries using these vectors have shown that different positive clones can be obtained depending on the host bacteria used for the screening14.…”

Tools for Functional Postgenomic Analysis ofListeria monocytogenes

“…3B). The increase in competence of the sequenced strains made it easier to obtain transformants with larger plasmids and should enable new techniques, such as co-plasmid transformation, direct cloning of toxic genes, and genomic library construction, to be used directly for L. monocytogenes (32).…”