Insertional Inactivation of Genes Responsible for the d -Alanylation of Lipoteichoic Acid in Streptococcus gordonii DL1 (Challis) Affects Intrageneric Coaggregations

Abstract

Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis ofStreptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous todltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encodedd-alanine-d-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting anermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion indltA exhibited a loss of d-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137–4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that d-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.

d‐Alanyl ester depletion of teichoic acids in Lactobacillus reuteri 100‐23 results in impaired colonization of the mouse gastrointestinal tract

“…The dlt mutant strain showed only marginal differences in growth characteristics in standard MRS medium. In addition, unlike dlt mutants of L. plantarum, Streptococcus mutants, and S. gordonii (Clemans et al, 1999;Boyd et al, 2000;Palumbo et al, 2006), morphological abnormalities such as the formation of pleomorphs were not observed. Increased autolysis, which is commonly observed for dlt mutants (Wecke et al, 1996;Steen et al, 2005;Palumbo et al, 2006), could not be detected for the dlt mutant of L. reuteri during stationary-phase growth.…”

“…D-Alanyl esters of WTA, which have a lower content of D-alanine, are derived from those of D-alanyl-LTA (Perego et al, 1995;Neuhaus and Baddiley, 2003). Studies of dlt mutants have shown that D-alanyl ester depletion of TA affects a variety of phenotypes of Gram-positive bacteria including biofilm formation (Gross et al, 2001), adherence (Abachin et al, 2002;Kristian et al, 2005), co-aggregation (Clemans et al, 1999), acid tolerance (Boyd et al, 2000;Kristian et al, 2005), autolysis (Wecke et al, 1996;Kristian et al, 2005;Steen et al, 2005;Palumbo et al, 2006), resistance to cationic peptides including defensins (Peschel et al, 1999;Poyart et al, 2003;Kristian et al, 2005;Kovács et al, 2006), and virulence (Abachin et al, 2002;Collins et al, 2002;Poyart et al, 2003). Because of their increased negative cell surface charge, dlt mutants bind positively charged compounds such as autolysins and antimicrobial peptides (defensins) more avidly, resulting in increased cell lysis and sensitivity respectively (Wecke et al, 1996;Peschel et al, 1999;Poyart et al, 2003;Kristian et al, 2005;Steen et al, 2005;Kovács et al, 2006;Palumbo et al, 2006).…”

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

“…The role of DltA in the esterification of D-alanine onto DltC has been demonstrated with genetics or in vitro activity for homologs from many organisms, e.g. L. casei (37,38,44), B. subtilis (39,63), Lactobacillus rhamnosus (45,58), S. aureus (12), Streptococcus mutans (15,24,73), Streptococcus gordonii (29), S. pneumoniae (5), and Bacillus cereus (40). Although studies in many of these organisms clearly link DltC to the DLT pathway, the fate of D-Ala-DltC is not clear.…”

“…Fluorescence polarization with S. aureus cells also showed an impact of D-alanylation on cell membrane fluidity (14). The change in charge of the cell wall from neutralization of polyanionic teichoic acids influences many cellular growth and homeostasis properties, including autolysis regulation (13, 18 -23), growth at low pH (8,10,13,(22)(23)(24)(25), metal ion binding (8,26,27), protein secretion (28), bacterial coaggregation (29), and biofilm formation (6,23,30). Furthermore, the D-alanine modification is important for pathogenesis, providing resistance to cationic antimicrobial peptides as well as antibacterial proteins induced during infection (12,31,32) (Table S1).…”

Attenuated virulence of Streptococcus agalactiae deficient in D‐alanyl‐lipoteichoic acid is due to an increased susceptibility to defensins and phagocytic cells

“…The D -alanylation of LTA allows Gram-positive bacteria to modulate their surface charge, regulate ligand binding and control the electromechanical properties of the cell wall. Genetic studies of the biosynthesis of LTA in various Gram-positive bacteria have shown that the incorporation of D-Ala residues requires the activity of four gene products (DltA-D), which are encoded by the dlt operon (Perego et al ., 1995;Neuhaus et al ., 1996;Clemans et al ., 1999;Peschel et al ., 1999;Boyd et al ., 2000;Debabov et al ., 2000;Poyart et al ., 2001a;Abachin et al ., 2002). Inactivation of any genes within this operon results in the complete absence of D-Ala ester in the LTA and these D-Ala-deficient LTA mutants were found to exhibit a variety of phenotypic changes that could be attributed to the resulting charge modification of their cell surface.…”