Streptococcus pneumoniae is one of the few species within the group of low-G ؉C gram-positive bacteria reported to contain no D-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for D-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, D-alanine-D-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released D-alanine from dltAproficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G؉C gram-positive bacteria, teichoic acids of S. pneumoniae contain D-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides.Teichoic acids (TAs) are polymers with a relatively wide structural diversity that are present at the surfaces of many gram-positive bacteria (18). The most common types of TAs are comprised of either polyglycerol phosphate or polyribitol phosphate chains of variable length that are substituted with glycosyl residues or D-alanyl esters, or both. TAs may be covalently linked to peptidoglycan (wall teichoic acids [WTAs]) or anchored in the cytoplasmic membrane by their glycolipid moiety (lipoteichoic acids [LTAs]). As major constituents of the surfaces of gram-positive bacteria, TAs have an impact on a number of important biological processes, such as autolysis (60), binding of cations (25) and surface-associated proteins (9, 29), adhesion (1, 61), biofilm formation (21), coaggregation (13), resistance to antimicrobial agents (50, 51), protein secretion (46), acid tolerance (7), stimulation of immune response (20, 41), and virulence (1, 14, 52). In most of these processes, the degree of D-alanylation of TAs has been shown to be of outstanding importance. Addition of D-alanine to TAs reduces the negative charge of the cell envelope, thereby influencing the binding and interaction of various compounds. Incorporation of D-alanine in LTAs is accomplished in a t...

The two-component signal-transducing system CiaRH of Streptococcus pneumoniae plays an important role during the development of beta-lactam resistance in laboratory mutants. We show here that a functional CiaRH system is required for survival under many different lysis-inducing conditions. Mutants with an activated CiaRH system were highly resistant to lysis induced by a wide variety of early and late cell wall inhibitors, such as cycloserine, bacitracin, and vancomycin, and were also less susceptible to these drugs. In contrast, loss-of-function CiaRH mutants were hypersusceptible to these drugs and were apparently unable to maintain a stationary growth phase in normal growth medium and under choline deprivation as well. Moreover, disruption of CiaR in penicillin-resistant mutants with an altered pbp2x gene encoding low-affinity PBP2x resulted in severe growth defects and rapid lysis. This phenotype was observed with pbp2x genes containing point mutations selected in the laboratory and with highly altered mosaic pbp2x genes from penicillin-resistant clinical isolates as well. This documents for the first time that PBP2x mutations required for development of beta-lactam resistance are functionally not neutral and are tolerated only in the presence of the CiaRH system. This might explain why cia mutations have not been observed in penicillin-resistant clinical isolates. The results document that the CiaRH system is required for maintenance of the stationary growth phase and for prevention of autolysis triggered under many different conditions, suggesting a major role for this system in ensuring cell wall integrity.

Rust fungi are plant parasites which colonise host tissue with an intercellular mycelium that forms haustoria within living plant cells. To identify genes expressed during biotrophic growth, EST sequencing was performed with a haustorium-speciWc cDNA library from Uromyces fabae. One thousand seventeen ESTs were generated, which assembled into 530 contigs. Several of the most frequently represented sequences in the EST database were identical to the in planta induced genes (PIGs) identiWed previously (Hahn, M., Mendgen, K., 1997. Characterisation of in planta-induced rust genes isolated from a haustorium-speciWc cDNA library, Mol. Plant-Microbe Interact. 10, 427-437). Virus-encoded sequences were identiWed, providing evidence for two novel RNA mycoviruses in U. fabae. Microarray hybridisation revealed many cDNAs that were signiWcantly activated in rust-infected leaves compared to germinated uredospores. Very strong in planta expression was found for two PIGs encoding putative metallothioneins. Furthermore, several genes involved in ribosome biogenesis and translation, glycolysis, amino acid metabolism, stress response, and detoxiWcation showed an increased expression in the parasitic mycelium. These data indicate a strong shift in gene expression in rust fungi between germination and the biotrophic stage of development. 

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