Genomic distribution and functions of uptake signal sequences in Actinobacillus actinomycetemcomitans

Abstract

Actinobacillus actinomycetemcomitans is naturally competent for transformation, with a transformation system similar to that of Haemophilus influenzae that preferentially takes up DNA bearing uptake signal sequences (USS) with the same 9-base USS core. This study examined the function of the extended 29-base USS, which comprises a highly conserved 1st region (containing the 9-base core) and 2nd and 3rd semi-conserved AT-rich regions, in transformation of A. actinomycetemcomitans. Transformation frequency was not affected by either location (in middle or at 59 end) or quantity (one or two) of USS in donor DNA. Relative transformation efficiencies (in comparison to the positive control) were 28-67 % for linear DNA with single-base mutations in the USS 1st region, and 47 % and 73 %, respectively, for linear DNA with USS that contained either a non-consensus 2nd or a non-consensus 3rd region. Plasmids with a stand-alone 1st or a stand-alone 2nd-3rd region exhibited 21 % and 6 % relative transformation efficiencies, respectively. It was also noted that A. actinomycetemcomitans and H. influenzae were similar in the frequencies and distribution patterns of USS in their genomes. In conclusion, all three regions of the extended 29-base USS are required for optimum transformation in A. actinomycetemcomitans.

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…influenzae-like transformation genes occur in the genome of A. actinomycetemcomitans (Wang et al, 2006;Cameron and Redfield, 2006;MK Bhattacharjee and DH Figurski, unpublished results), which is consistent with the ability of this organism to undergo natural transformation. In A. actinomycetemcomitans, genes also involved in the formation of a type IV pilus-like system, distinct from those that are responsible for Flp fibrils, are thought to be required for transformation (Wang et al, 2003).…”

“…We were the first to report that the A. actinomycetemcomitans genome has numerous copies of a sequence identical to the 9-bp core USS of H. influenzae, and we predicted that the sequence is part of a USS, important to transformation in A. actinomycetemcomitans (Thomson et al, 1999). Experimental evidence indicates that the function of the sequence is indeed in transformation, as predicted (Wang et al, 2002(Wang et al, , 2006this work). Other studies have been concerned with the presence of USSs in the genomes of the Pasteurellaceae and the functional structure, origin, lineage, and evolutionary significance of the USSs (Bakkali et al, 2004;Redfield et al, 2006;Wang et al, 2006).…”

“…High transformation efficiency also requires a nearby USS, and it works in the polylinker of the plasmid suicide vector that we constructed. A USS has been shown to be important for transformation in A. actinomycetemcomitans (Wang et al, 2002(Wang et al, , 2006. The use of linearized replicative plasmid DNA, circular suicide plasmid DNA, or genomic DNA readily yields transformants that have undergone allelic exchange with the chromosome.…”

Genome Dynamics of Short Oligonucleotides: The Example of Bacterial DNA Uptake Enhancing Sequences

“…Among these, Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences, called uptake signal sequences (USS) and DNA uptake sequences (DUS), respectively [3]–[9]. Both USSs and DUSs are overrepresented in their respective genomes and, while only one DUS (5′-GCCGTCTGAA) has been described [10]–[13], USS was reported in two slightly different versions: (i) 5′-AAGTGCGGT in Haemophilus influenzae [13]–[15], Actinobacillus actinomycetemcomitans [6], [9], [15], Haemophilus somnus and Pasteurella multocida [15], Mannheimia succiniciproducens [16] and, probably Haemophilus parasuis [16], [17] and (ii) 5′-ACAAGCGGTC in Mannheimia haemolytica [16], [18] and Actinobacillus pleuropneumoniae [16]. Bacterial competence for natural transformation is best characterized in Bacillus subtilis (Bacillaceae), H. influenzae (Pasteurellaceae), Neisseria gonorrhoeae (Neisseriaceae) and Streptococcus pneumoniae (Streptococcaceae) (see [1], [2], [19]–[21]).…”

Development of a Markerless Knockout Method for Actinobacillus succinogenes

“…They do so with membrane-bound DNA uptake machinery that specifically recognizes and binds a conserved uptake signal sequence (USS) (15). Low transformation efficiencies of A. actinomycetemcomitans were observed with DNA fragments not containing a USS (16). All Pasteurellaceae genomes sequenced contain USS repeats, in numbers ranging from 41 to 1,760 (1,690 in A. succinogenes) (17), even though not all these species are naturally competent (12).…”