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.

FIG. 1.

Map of the novel hybrid BAC vector pUvBBAC. This derivative of the second-generation BAC cloning vector pBeloBAC11 replicates in both E. coli and a broad range of gram-positive hosts. Recombinant clones are selectable with both chloramphenicol and erythromycin. Regions deriving from pBeloBAC11 are displayed in light gray. Dark gray regions are from pSOG7. The origin, replication, and partition functions of the mini-F derivate are indicated as ori-2; repE; and sopA, sopB and sopC, respectively. ResD is a putative resolvase of the F plasmid also known as protein D. The vector harbors a 5′-end truncated version of ResD. Additional features include the cos and loxP sites, which are required for packaging lambda particles if desired; the loxP site includes the cleavage site for the Cre recombinase. Selection markers in the E. coli host include the chloramphenicol acetylase gene (cat) and the lacZα gene for insert screening by alpha-complementation. The replication and copy number functions derived from pIP501 are indicated as repR and copR, respectively; the selection marker ermC encoding a methylase gene is also indicated.

FIG. 2.

PFGE of NotI-digested BAC DNA obtained from the L. monocytogenes EGD-e pUvBBAC library. Insert sizes range from 68 to 178 kb. Lanes 1 and 20 contain the low-range PFG marker (New England Biolabs), while the lambda ladder PFG marker (New England Biolabs) is depicted in lanes 2 and 19.

FIG. 3.

(A) Mapping and visualization of pUvBBAC recombinants on the L. monocytogenes EGD-e chromosome using GenomeViz (16). The first (outer) circle represents the scale in megabases, starting with the origin of replication at position 0. The second and third circles show the distribution of coding sequences of the leading and lagging strands and indicate the classification of the clusters of orthologous groups (COG) for each gene and the distribution of COG classes within the genome. The color scheme and categories are according to the convention of the COG database. The chromosomal localization of the mapped pUvBBAC inserts are indicated as thin purple lines. The inner circle indicates the deviations of the GC content averages, with values greater than zero in red and less than zero in blue. (B) Alignment of the chromosomal DNA inserts in recombinants harboring vgc1 and vgc2 to the genome of L. monocytogenes EGD-e. The exact positions of genomic fragments derived from DNA sequencing of either end of the inserted DNA are indicated. All of the known open reading frames within this region are depicted as individual bars.

FIG. 4.

Demonstration of virulence gene expression and detection of hemolysis for recombinant L. innocua strains. (A) Bacterial culture supernatants and cell wall proteins were separated by SDS-PAGE and further analyzed by immunoblotting. (B) Culture supernatants were probed with an anti-Hly antibody, and the fraction containing cell surface proteins was used for the detection of ActA. Hemolytic activity was determined on blood agar. Lanes: 1, L. monocytogenes EGD-e; 2, L. innocua; 3, L. innocua(pUvBBAC); 4, L. innocua(pUvBBAC+vgc1); 5, L. innocua(pUvBBAC+vgc2). The experiment was repeated three times independently, and one representative immunoblot is shown here.

FIG. 5.

Intracellular growth rates of L. monocytogenes EGD-e and recombinant L. innocua strains in J774 macrophage. Bacteria were incubated with macrophage at a multiplicity of infection of 10 for 20 min, after which gentamicin (20 μg/ml) was added and bacterial growth monitored at 1, 2, 4, and 8 h postinfection.

FIG. 6.

Intracellular actin accumulation and motility of L. monocytogenes EGD-e (A), L. innocua(pUvBBAC+vgc1) (B) or L. innocua(pUvBBAC+vgc2) (C) 4 h after the infection of J774 macrophage cells. Listeriae were detected with monoclonal antibody N81/N111 against ActA and visualized with a Cy3-labeled secondary antibody. Actin filaments of the host cell were stained with Oregon Green 488-conjugated phalloidin.