Identification of LytSR-regulated genes from Staphylococcus aureus.

In this report, the characterization of a Staphylococcus aureus operon containing two LytSR-regulated genes, lrgA and lrgB, is described. Sequence and mutagenesis studies of these genes suggest that lrgA encodes a murein hydrolase exporter similar to bacteriophage holin proteins while lrgB may encode a protein having murein hydrolase activity.     

Two distinct regions in Staphylococcus aureus GatCAB guarantee accurate tRNA recognition

In many prokaryotes the biosynthesis of the amide aminoacyl-tRNAs, Gln-tRNA^Gln and Asn-tRNA^Asn, proceeds by an indirect route in which mischarged Glu-tRNA^Gln or Asp-tRNA^Asn is amidated to the correct aminoacyl-tRNA catalyzed by a tRNA-dependent amidotransferase (AdT). Two types of AdTs exist: bacteria, archaea and organelles possess heterotrimeric GatCAB, while heterodimeric GatDE occurs exclusively in archaea. Bacterial GatCAB and GatDE recognize the first base pair of the acceptor stem and the D-loop of their tRNA substrates, while archaeal GatCAB recognizes the tertiary core of the tRNA, but not the first base pair. Here, we present the crystal structure of the full-length Staphylococcus aureus GatCAB. Its GatB tail domain possesses a conserved Lys rich motif that is situated close to the variable loop in a GatCAB:tRNA^Gln docking model. This motif is also conserved in the tail domain of archaeal GatCAB, suggesting this basic region may recognize the tRNA variable loop to discriminate Asp-tRNA^Asn from Asp-tRNA^Asp in archaea. Furthermore, we identified a 3₁₀ turn in GatB that permits the bacterial GatCAB to distinguish a U1–A72 base pair from a G1–C72 pair; the absence of this element in archaeal GatCAB enables the latter enzyme to recognize aminoacyl-tRNAs with G1–C72 base pairs.     

Genetic characterization of staphopain genes in Staphylococcus aureus

Staphylococcus aureus , a leading cause of bacterial infections in humans, is endowed with a wealth of virulence factors that contribute to the disease process. Several extracellular proteolytic enzymes, including cysteine proteinases referred to as the staphopains (staphopain A, encoded by the scpA gene, and staphopain B, encoded by sspB ), have proposed roles for staphylococcal virulence. Here we present data regarding the distribution, copy number and genetic variability of the genes encoding the staphopains in a large number of S. aureus strains. The polymorphism of the scpA and sspB genes in three laboratory strains and 126 clinical isolates was analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Both genes were detected in all isolates by PCR amplification and, based on the PCR-RFLP patterns, classified as four types for scpA and six types for sspB . Those with the most divergent patterns were subjected to DNA sequencing and compared with genomic sequence data for the seven available strains of S. aureus . Southern blot analysis of the scpA and sspB sequences indicates that they are strongly conserved as single-copy genes in the genome of each S. aureus strain investigated. Taken together, these data suggest that the staphopains have important housekeeping and/or virulence functions, and therefore may constitute an interesting target for the development of therapeutic inhibitors for the treatment of staphylococcal diseases.     

Rapid method for the identification of essential genes in Staphylococcus aureus.

A strategy based on a vector host-dependent for autonomous replication, pSA3182, was utilized both for the rapid screening for Staphylococcus aureus genes essential for cell viability and for the introduction of specific polarity-neutral deletions in nonessential genes. The results obtained support the use of pSA3182 for both purposes.     

Occurrence of adhesin genes in coagulase-negative Staphylococcus aureus strains

Sixty fenotypically coagulase-negative Staphylococcus aureus strains were screened for the presence of six adhesion genes. The strains were isolated from varied clinical samples and varied patients in 16 medical centers, in majority from the region of Gdansk. Multiplex PCR in two primer sets was used for detection of the following genes: bbp (bone binding protein), cna (collagen binding protein), ebp (elastin binding protein)and fnbB (fibronectin B binding protein), fib (fibrinogen bindng protein) and clfA (clunmping factor A). More than half (57%) of the examined population harbored four genes: fnbB,fib, cna i clfA. All of the strains were found to be clfA positive and 90% of them were positive for fnbB, 90% for fib and 67% for cna. All of these genes were significantly more common in MRSA than in MSSA. The particular genes were occurred in strains derived from varied clinical samples.     

Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA

Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.     

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA^Asn. The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA^Asn. S. aureus thus encodes both the direct and indirect routes for Asn-tRNA^Asn formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.     

Alpha-toxin of Staphylococcus aureus.

Alpha-toxin, the major cytotoxic agent elaborated by Staphylococcus aureus, was the first bacterial exotoxin to be identified as a pore former. The protein is secreted as a single-chain, water-soluble molecule of Mr 33,000. At low concentrations (less than 100 nM), the toxin binds to as yet unidentified, high-affinity acceptor sites that have been detected on a variety of cells including rabbit erythrocytes, human platelets, monocytes and endothelial cells. At high concentrations, the toxin additionally binds via nonspecific absorption to lipid bilayers; it can thus damage both cells lacking significant numbers of the acceptor and protein-free artificial lipid bilayers. Membrane damage occurs in both cases after membrane-bound toxin molecules collide via lateral diffusion to form ring-structured hexamers. The latter insert spontaneously into the lipid bilayer to form discrete transmembrane pores of effective diameter 1 to 2 nm. A hypothetical model is advanced in which the pore is lined by amphiphilic beta-sheets, one surface of which interacts with lipids whereas the other repels apolar membrane constitutents to force open an aqueous passage. The detrimental effects of alpha-toxin are due not only to the death of susceptible targets, but also to the presence of secondary cellular reactions that can be triggered via Ca2+ influx through the pores. Well-studied phenomena include the stimulation of arachidonic acid metabolism, triggering of granule exocytosis, and contractile dysfunction. Such processes cause profound long-range disturbances such as development of pulmonary edema and promotion of blood coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.     

Compilation of tRNA sequences and sequences of tRNA genes.

Sequences of 3279 sequences of tRNA genes and tRNAs published up to December 1996 are included in the compilation. Alignment of the sequences, which is most compatible with the tRNA phylogeny and known three-dimensional structures of tRNA, is used. Sequences and references are available under http://www.uni-bayreuth. de/departments/biochemie/trna/