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Pasteurellaceae ComE1 proteins combine the properties of fibronectin adhesins and DNA binding competence proteins.

Mullen LM, Bossé JT, Nair SP, Ward JM, Rycroft AN, Robertson G, Langford PR, Henderson B - PLoS ONE (2008)

Bottom Line: This binding to DNA is non sequence-specific and is confined to double-stranded DNA.We have cloned and expressed ComE1 proteins from five members of the Pasteurellaceae in order to further investigate the function(s) of these proteins.When expressed as recombinant GST-fusion proteins, all of the homologues bound both to fibronectin and to double-stranded DNA.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbial Diseases, UCL Eastman Dental Institute, University College London, London, United Kingdom. l.mullen@eastman.ucl.ac.uk

ABSTRACT
A novel fibronectin-binding protein from Pasteurella multocida (PM1665) that binds to the fibronectin type III(9-10) modules via two helix-hairpin-helix motifs has recently been described [1]. This protein shares homology with competence-related DNA-binding and uptake proteins (ComEA and ComE) from Gram-positive and Gram-negative bacteria. Here, we show that recombinant PM1665 (now designated ComE1) also binds to DNA through the same helix-hairpin-helix motifs required for fibronectin-binding. This binding to DNA is non sequence-specific and is confined to double-stranded DNA. We have cloned and expressed ComE1 proteins from five members of the Pasteurellaceae in order to further investigate the function(s) of these proteins. When expressed as recombinant GST-fusion proteins, all of the homologues bound both to fibronectin and to double-stranded DNA. Inactivation of the gene encoding the ComE1 homologue in Actinobacillus pleuropneumoniae indicates major roles for these proteins in at least two processes: natural transformation, and binding of bacteria to fibronectin.

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Comparison of the transformation frequency of wild-type A. pleuropneumoniae and ApcomE1.The effect of the presence of Fn on the transformation frequency of wild-type A. pleuropneumoniae was also tested. Bars with different letters are significantly different from each other (one-way ANOVA, P<0.001).
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pone-0003991-g009: Comparison of the transformation frequency of wild-type A. pleuropneumoniae and ApcomE1.The effect of the presence of Fn on the transformation frequency of wild-type A. pleuropneumoniae was also tested. Bars with different letters are significantly different from each other (one-way ANOVA, P<0.001).

Mentions: To determine the contribution of ComE1 to bacterial binding to Fn and DNA, the A. pleuropneumoniae comE1 gene was insertionally inactivated. This approach was taken for two reasons: firstly, the availability of a naturally transformable strain of A. pleuroneumoniae [17], that is genetically more tractable than P. multocida and secondly, the fact that A. pleuroneumoniae is naturally competent allows us to investigate a possible role for ComE1 in natural transformation in this bacterium. The comE1 gene was insertionally activated with a kanamycin gene (KanR) by alleleic replacement. The resultant mutant was verified by PCR (using primers 5′-ACAAGCGGTTTCACCCATTCGGGTTTCTACG-3′ and 5′-ACAAGCGGTGTAGTTTCAGTCGTAGGCGCTG-3′ anddesignated ApΔcomE1. Binding assays were used to compare the relative capacities of A. pleuropneumoniae HS143 and its isogenic ΔcomE1 mutant, in various stages of growth, to bind to Fn and DNA. The growth phase made a significant difference to the ability of wild-type A. pleuropneumoniae to bind to Fn, with far greater numbers of bacteria binding when the bacteria were grown to the stationary phase (Fig. 8c) compared with either early (Fig. 8a) or late exponential phases (Fig. 8b). Wild-type A. pleuropneumoniae in the early exponential or stationary phases bound to DNA, but the numbers of bacteria binding to DNA in these growth phases were always much lower than those binding to Fn (Fig. 8). The loss of comE1 resulted in a significant decrease (paired t-test; P<0.001) in the number of bacteria bound to Fn in all stages of growth. Complementation of ApΔcomE1 with the comE1 gene supplied on the plasmid pMIDG311 restored binding of ApΔcomE1 to Fn at levels similar to that observed for wild-type A. pleuropneumoniae (Fig. 8d). Thus, we could rule out polar effects as an explanation of the change in binding of the comE1 mutant. The effect of comE1 inactivation on bacterial competence was also tested. There was a 104-fold decrease in the ability of ApΔcomE1 to undergo natural transformation compared with wild-type A. pleuropneumoniae (Fig. 9). As ComE1 recombinant protein bound to both Fn and DNA, the effect of the presence of soluble Fn on DNA uptake was also tested. There was no significant effect (one-way ANOVA) of Fn (at concentrations of up to 300 µg/ml) on transformation frequency in wild-type A. pleuropneumoniae (Fig. 9). Attempts were made compare the transformation frequencies of ApΔcomE1 and the complemented mutant to that of wild-type A. pleuropneumoniae using donor DNA from a spontaneous streptomycin resistant mutant (Ap15StrR). However, for some reason the frequency of transformation of the wild-type strain HS143 to StrR was 4 log orders lower compared to the frequency of transformation to KanR or ChlR, and no transformants were detected with either the ApΔcomE1 or the complemented mutant using the StrR donor DNA.


Pasteurellaceae ComE1 proteins combine the properties of fibronectin adhesins and DNA binding competence proteins.

Mullen LM, Bossé JT, Nair SP, Ward JM, Rycroft AN, Robertson G, Langford PR, Henderson B - PLoS ONE (2008)

Comparison of the transformation frequency of wild-type A. pleuropneumoniae and ApcomE1.The effect of the presence of Fn on the transformation frequency of wild-type A. pleuropneumoniae was also tested. Bars with different letters are significantly different from each other (one-way ANOVA, P<0.001).
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Related In: Results  -  Collection

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pone-0003991-g009: Comparison of the transformation frequency of wild-type A. pleuropneumoniae and ApcomE1.The effect of the presence of Fn on the transformation frequency of wild-type A. pleuropneumoniae was also tested. Bars with different letters are significantly different from each other (one-way ANOVA, P<0.001).
Mentions: To determine the contribution of ComE1 to bacterial binding to Fn and DNA, the A. pleuropneumoniae comE1 gene was insertionally inactivated. This approach was taken for two reasons: firstly, the availability of a naturally transformable strain of A. pleuroneumoniae [17], that is genetically more tractable than P. multocida and secondly, the fact that A. pleuroneumoniae is naturally competent allows us to investigate a possible role for ComE1 in natural transformation in this bacterium. The comE1 gene was insertionally activated with a kanamycin gene (KanR) by alleleic replacement. The resultant mutant was verified by PCR (using primers 5′-ACAAGCGGTTTCACCCATTCGGGTTTCTACG-3′ and 5′-ACAAGCGGTGTAGTTTCAGTCGTAGGCGCTG-3′ anddesignated ApΔcomE1. Binding assays were used to compare the relative capacities of A. pleuropneumoniae HS143 and its isogenic ΔcomE1 mutant, in various stages of growth, to bind to Fn and DNA. The growth phase made a significant difference to the ability of wild-type A. pleuropneumoniae to bind to Fn, with far greater numbers of bacteria binding when the bacteria were grown to the stationary phase (Fig. 8c) compared with either early (Fig. 8a) or late exponential phases (Fig. 8b). Wild-type A. pleuropneumoniae in the early exponential or stationary phases bound to DNA, but the numbers of bacteria binding to DNA in these growth phases were always much lower than those binding to Fn (Fig. 8). The loss of comE1 resulted in a significant decrease (paired t-test; P<0.001) in the number of bacteria bound to Fn in all stages of growth. Complementation of ApΔcomE1 with the comE1 gene supplied on the plasmid pMIDG311 restored binding of ApΔcomE1 to Fn at levels similar to that observed for wild-type A. pleuropneumoniae (Fig. 8d). Thus, we could rule out polar effects as an explanation of the change in binding of the comE1 mutant. The effect of comE1 inactivation on bacterial competence was also tested. There was a 104-fold decrease in the ability of ApΔcomE1 to undergo natural transformation compared with wild-type A. pleuropneumoniae (Fig. 9). As ComE1 recombinant protein bound to both Fn and DNA, the effect of the presence of soluble Fn on DNA uptake was also tested. There was no significant effect (one-way ANOVA) of Fn (at concentrations of up to 300 µg/ml) on transformation frequency in wild-type A. pleuropneumoniae (Fig. 9). Attempts were made compare the transformation frequencies of ApΔcomE1 and the complemented mutant to that of wild-type A. pleuropneumoniae using donor DNA from a spontaneous streptomycin resistant mutant (Ap15StrR). However, for some reason the frequency of transformation of the wild-type strain HS143 to StrR was 4 log orders lower compared to the frequency of transformation to KanR or ChlR, and no transformants were detected with either the ApΔcomE1 or the complemented mutant using the StrR donor DNA.

Bottom Line: This binding to DNA is non sequence-specific and is confined to double-stranded DNA.We have cloned and expressed ComE1 proteins from five members of the Pasteurellaceae in order to further investigate the function(s) of these proteins.When expressed as recombinant GST-fusion proteins, all of the homologues bound both to fibronectin and to double-stranded DNA.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbial Diseases, UCL Eastman Dental Institute, University College London, London, United Kingdom. l.mullen@eastman.ucl.ac.uk

ABSTRACT
A novel fibronectin-binding protein from Pasteurella multocida (PM1665) that binds to the fibronectin type III(9-10) modules via two helix-hairpin-helix motifs has recently been described [1]. This protein shares homology with competence-related DNA-binding and uptake proteins (ComEA and ComE) from Gram-positive and Gram-negative bacteria. Here, we show that recombinant PM1665 (now designated ComE1) also binds to DNA through the same helix-hairpin-helix motifs required for fibronectin-binding. This binding to DNA is non sequence-specific and is confined to double-stranded DNA. We have cloned and expressed ComE1 proteins from five members of the Pasteurellaceae in order to further investigate the function(s) of these proteins. When expressed as recombinant GST-fusion proteins, all of the homologues bound both to fibronectin and to double-stranded DNA. Inactivation of the gene encoding the ComE1 homologue in Actinobacillus pleuropneumoniae indicates major roles for these proteins in at least two processes: natural transformation, and binding of bacteria to fibronectin.

Show MeSH
Related in: MedlinePlus