Natural transformation of Gallibacterium anatis

Abstract

Gallibacterium anatis is a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function in G. anatis, we have established methods for transformation and targeted mutagenesis. The genus Gallibacterium belongs to the Pasteurellaceae, a group with several naturally transformable members, including Haemophilus influenzae. Bioinformatics analysis identified G. anatis homologs of the H. influenzae competence genes, and natural competence was induced in G. anatis by the procedure established for H. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies with G. anatis chromosomal DNA and with linearized plasmid DNA carrying G. anatis sequences. Both DNA types integrated into the G. anatis chromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10(-4) transformants CFU(-1). Transformation was also efficient with circular plasmid containing no G. anatis DNA; this resulted in the establishment of a self-replicating plasmid. Nine diverse G. anatis strains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. The G. anatis genome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, but G. anatis did preferentially take up its own DNA over that of Escherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation of G. anatis.

Fig 1

G. anatis competence regulon. (a) Sequence logo of all predicted G. anatis CRP-S sites (based on sites identified upstream of comA, comF, comE1, pilA, comN, rec2, pilF2, comM, dprA, radC, ssb, and ligA). (b) Organization of comABCDEF genes in G. anatis, A. pleuropneumoniae, and H. influenzae.

Fig 2

Effect of DNA concentration on transformation frequency. G. anatis 12656-12 was transformed with various concentrations of chromosomal Nal^r DNA, and the transformation frequency (transformants CFU⁻¹) was calculated. The averages and standard deviations of three independent experiments are shown.

Fig 3

Transformation frequencies in various G. anatis strains. Competence-induced cells were transformed with 0.5 μg ml⁻¹ chromosomal DNA from strain 12656-12; Nal^r DNA was used except for strains 4895 and 07990. These strains have reduced sensitivity to nalidixic acid and were transformed with chromosomal DNA from 12656-12 ΔgtxA (Km^r). Transformation frequency (transformants CFU⁻¹) was calculated, and the averages and standard deviations of a minimum of three independent experiments are shown.

Fig 4

Transformation competition experiments. Competence-induced G. anatis 12656-12 was transformed with chromosomal DNA from 12656-12 Nal^r alone or mixed with various concentrations of competing DNA: chromosomal DNA from E. coli DH5α or unmarked chromosomal DNA from G. anatis 12656-12 as indicated. C_x/C_o, ratio of competing DNA to G. anatis 12656-12 Nal^r DNA; T_o/T_x, ratio of transformation frequency in the absence of competing DNA to transformation frequency in the presence of competing DNA. The averages and standard deviations of three independent experiments are shown. Numbers above each data point represent P values from comparisons (paired one-tailed Student's t test) of the average relative transformation frequencies with E. coli DNA as competing DNA and G. anatis DNA as competing DNA.

Fig 5

Sequence logos of positions flanking Hin-type USS cores (a) or Apl-type USS cores (b). The height of the base at each position is a relative indicator of consensus strength at that position. The gray rectangles show the position of the USS cores. The light blue shading highlights known informative positions flanking USS cores, and the grey shading shows the Apl-specific extended USS. H.i., H. influenzae; A.p., A. pleuropneumoniae; E.c., E. coli; G.a., G. anatis.

Fig 6

Genomic 9-mer spectra. (a to c) Genomic 9-mer spectra for G. anatis (a), H. influenzae (b), and A. pleuropneumoniae (c). Plots zoom in on the abundant 9-mers, while the insets show the full spectra. x axes indicate the number of occurrences of each specific 9-mer word, while the y axes indicate the number of words with that many occurrences. (d) Plot of the density per kb of each 9-mer in G. anatis against the density in E. coli. Blue ovals show 9-mers with the indicated properties. For USS cores, several 9-mers are circled, because of the reverse complements and those shifted by a single base.