Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10–50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

tRNA 3 processing is one of the essential steps during tRNA maturation. The tRNA 3-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity.tRNA molecules are essential for protein synthesis, providing the amino acids during translation. They are not directly transcribed as functional molecules but as precursor RNAs, which require several processing steps to generate the functional tRNA molecule. Two of these processing steps are the removal of the additional 5Ј and 3Ј sequences of the tRNA. Although the removal of the additional 5Ј sequence (the 5Ј leader) is well understood (1), maturation of the tRNA 3Ј end is not as well studied, although a correctly generated tRNA 3Ј end is essential for the addition of the CCA triplet and thus for aminoacylation (2).It has been shown that in Escherichia coli, tRNA 3Ј maturation is a multistep process involving endo-as well as exonucleases, the final steps being performed by an exonuclease (3). In contrast, Bacillus subtilis employs an endonuclease, called tRNase 6 Z (EC 3.1.26.11), which cleaves CCA-less tRNA precursors directly 3Ј to the discriminator (4).Precursors, which do contain the CCA, are not processed by tRNase Z. Archaea and eukaryotes also use tRNase Z enzymes to process the tRNA 3Ј trailer in a single-step mechanism (5-8).The first tRNase Z, TRZ1, was isolated from Arabidopsis thaliana (5). Data base analyses showed that TRZ1 homologues are present in organisms from all three kingdoms, bacteria, archaea, and eukarya (Fig. 1). The tRNase Z family of proteins (also called Elac1/Elac2) can be divided into two subgroups: the short tRNase Z proteins (being 250 -350 amino acids long), tRNase Z S enzymes, and the long tRNase Z proteins (with 700 -950 amino acids), the tRNase Z L enzymes. Although the tRNase Z S proteins are present in all kingdoms, the tRNase Z L enzymes can only be found in eukarya. Both subgroups are part of the same protein family since the C-terminal part of the tRNase Z L proteins has high sequence similarity to the tRNase Z S enzymes. TRZ1 belongs to the family of metal-dependent ␤-lactamases (9), a group of met...

Streptococcus agalactiae (group B streptococcus [GBS]) causes neonatal sepsis, pneumonia, and meningitis, as well as infections of the bovine udder. The S. agalactiae hemolysin is regarded as an important virulence factor, and hemolysin expression is dependent on the cyl gene cluster. cylA and cylB encode the ATP binding and transmembrane domains of a typical ATP binding cassette (ABC) transporter. The deduced proteins contain the signature sequence of a multidrug resistance (MDR) transporter, and mutation of the genes results in a nonhemolytic and nonpigmented phenotype. To further elucidate the function of the putative transporter, nonpolar deletion mutants of cylA were constructed. These mutants are nonhemolytic and can be complemented by the transporter genes. Wild-type strain and nonhemolytic cylA and cylK deletion mutants were exposed to known substrates of MDR transporters. Mutation of cylA significantly impaired growth in the presence of daunorubicin, doxorubicin, and rhodamine 6G and resulted in a decreased export of doxorubicin from the cells. The mutation of cylK, a gene of unknown function located downstream from cylA, caused a loss of hemolysis but had no effect on the transport of MDR substrates. Furthermore, the hemolytic activity of the wild-type strain was inhibited by reserpine in a dose-dependent manner. We conclude that CylAB closely resembles an ABC-type MDR transporter and propose that the GBS hemolysin molecule represents a natural substrate of the transporter.