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A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus lig A gene. Orthologues of the putative S. aureus NAD + -dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli , and the enzyme was purified to near homogeneity. NAD + -dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of 32 P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD + -dependent DNA ligase from B. stearothermophilus , two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

A temperature-sensitive lethal mutant ofStaphylococcus aureuswas found to harbor a mutation in the uncharacterized two-component histidine kinase (HK)-response regulator (RR) pair encoded byyycFG; orthologues ofyycFGcould be identified in the genomes ofBacillus subtilisand other gram-positive bacteria. Sequence analysis of the mutant revealed a point mutation resulting in a nonconservative change (Glu to Lys) in the regulator domain of the RR at position 63. To confirm that this signal transduction system was essential, a disrupted copy of either the RR (yycF) or the HK (yycG) was constructed with a set of suicide vectors and used to generate tandem duplications in the chromosome. Resolution of the duplications, leaving an insertion in either theyycFor theyycGcoding region, was achieved only in the presence of an additional wild-type copy of the two open reading frames. Phenotypic characterization of the conditional lethal mutant showed that at permissive growth conditions, the mutant was hypersusceptible to macrolide and lincosamide antibiotics, even in the presence of theermBresistance determinant. Other mutant phenotypes, including hypersensitivity to unsaturated long-chain fatty acids and suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations, suggest that the role of the two-component system includes the proper regulation of bacterial cell wall or membrane composition. The effects of this point mutation are strongly bactericidal at the nonpermissive temperature, indicating that this pathway provides an excellent target for the identification of novel antibiotics.

A temperature-sensitive (ts) mutant of Salmonella typhimurium that accumulated unmodified murein prolipoprotein at 42 degrees C but not at 30 degrees C was identified. In vivo and in vitro studies of the biosynthesis of Braun's lipoprotein revealed that this mutant (SE5221) was defective in the glyceryl modification of prolipoprotein. The ts mutation was mapped to 60.6 min of the S. typhimurium chromosome and was linked to argA and cysH. A clone with a 1.4-kilobase S. typhimurium DNA insert that complemented the ts mutation and restored the prolipoprotein modification activity both in vivo and in vitro was isolated. DNA sequencing of the complementing region revealed an open reading frame encoding a protein with 291 amino acids lacking NH2-terminal signal sequence. This open reading frame is immediately 5' to the thyA gene and is allelic to umpA of Escherichia coli. Wild-type strains harboring the cloned gene exhibited elevated levels of prolipoprotein modification activity. At the non-permissive temperature, the mutation affected both growth and viability, and the mutant cells exhibited anomalous cell morphology. The ts phenotype was suppressed by the introduction of a lpp::Tn10 mutation. These results suggest that the cloned gene encodes prolipoprotein glyceryl transferase (lgt), and in the wild-type background, this prolipoprotein modification enzyme is essential for the growth and viability of S. typhimurium.

On screening 440 temperature-sensitive (ts) mutants of Salmonella typhimurium, a mutant strain SE5312 which accumulated apolipoprotein (ALP) at 42 degrees C was identified. In vitro assay of apolipoprotein N-acyltransferase activity indicated that the mutant cell envelope contained reduced activity as compared to the wild-type strain. Transduction with a Mud-P22 mapping set placed the ts mutation to 14-17 min region of the S. typhimurium chromosome. P22 transduction using transposon insertions in this region revealed a linkage of the ts mutation to cobD (6%), nag (8%), and corC68 (99%). The ts phenotype was complemented by a 2.3-kilobase EcoRI subclone derived from lambda-phage 170 of Kohara's bank of Escherichia coli. Restriction enzyme analysis of the cloned DNA revealed that this 2.3-kilobase EcoRI fragment included the copper transport (cutE) gene in E. coli. The mutant strain SE5312 was copper-sensitive at 30 degrees C, and the complementing clone conferred copper resistance and restored the ALP N-acyltransferase activity in the mutant cell. Wild-type strain of S. typhimurium harboring this clone exhibited elevated levels of ALP N-acyltransferase activity. These results suggest that the cloned gene encodes the ALP N-acyltransferase. Upon shift to the non-permissive temperature, the viability of the mutant cells decreased, and the mutant cells assumed anomalous morphology. Temperature-resistant revertants could be readily isolated, and a subset of tr revertants contained no detectable lipoprotein. A lpp::Tn10 derivative of the mutant SE5312 was also temperature-resistant. These observations suggest that ALP N-acyltransferase is essential for the growth and viability of S. typhimurium, and this requirement is decreased in the absence of major outer membrane lipoprotein.

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governingStaphylococcus aureuspathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300S. aureusinsertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important forS. aureusin vivo survival. Our findings extend the understanding of pathogenic mechanisms employed byS. aureusto ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by beta-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

The yycF1 (Ts) mutation in Staphylococcus aureus conferred hypersensitivity to macrolide-lincosamide-streptogramin B (MLS B ) antibiotics on strains either containing or lacking ermB . The overexpression of the S. aureus Ssa protein restored the yycF1 mutant to wild-type levels of susceptibility. Inactivation of ssa in an unmutagenized strain dramatically reduced ermB -based resistance. Conditional loss of function or expression of ssa in the yycF1 mutant is proposed to result in the observed hypersensitivity to MLS B antibiotics.

We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo , suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork.

The murB gene of Salmonella typhimurium was cloned and found to be 75% and 82% identical to the DNA and protein sequences, respectively, of the same gene in Escherichia coli. These identities are among the lowest recorded between the two bacteria. Nevertheless, wild-type S. typhimurium murB complemented the known temperature-sensitive E. coli mutant, and wild-type E. coli murB complemented three temperature-sensitive mutants of S. typhimurium. The 5S rRNA gene, rrfB, and the region between murB and rrfB were also cloned and sequenced. The rrfB gene of S. typhimurium differs from rrfB of E. coli in only 2 of 120 nt, but the region between murB and rrfB has diverged greatly and includes a sequence that elosely resembles a repetitive extragenic palindrome of the type normally associated with E. coli. Previous comparisons of gene divergence have suggested that the chromosomal mutation rate is lower in the vicinity of the origin of replication. However, the S. typhimurium murB gene, located 6 map minutes from the origin of replication, is highly substituted at synonymous sites and the sequence between murB and rrfB is significantly modified as well. Thus, murB is an exception to the general observation that genes near the origin of replication show less divergence than do genes elsewhere in the bacterial chromosome.

Two internal promoters in the his operon of Salmonella typhimurium have been precisely mapped genetically. The internal promoters are found in, or very close to, gene border regions in the his operon. The his operon was examined for the presence of additional internal promoters whose transcripts were sensitive to rho-mediated transcription termination and therefore had escaped detection. No new internal promoters were found. It is argued that the internal promoters described here are not likely to be fortuitous message start sites, but may play a physiologically important role in operon expression.

In bacteria there appear to be two distinct levels of DNA condensation: the organization of independently supercoiled domains of DNA (averaging 100 kbp in size) and the organization of shorter segments of DNA (60–120 bp) with abundant DNA-binding proteins.

We have isolated 440 mutants of Salmonella typhimurium that show temperature-sensitive growth on complex medium at 44 degrees. Approximately 16% of the mutations in these strains have been mapped to 17 chromosomal locations; two of these chromosomal locations seem to include several essential genes. Genetic analysis of the mutations suggests that the collection saturates the genes readily mutable to a ts lethal phenotype in S. typhimurium. Physiological characteristics of the ts lethal mutants were tested: 6% of the mutants can grow at high temperature under anaerobic conditions, 17% can grow when the medium includes 0.5 M KCl, and 9% of the mutants die after a 2-hr incubation at the nonpermissive temperature. Most ts lethal mutations in this collection probably affect genes required for growth at all temperatures (not merely during high temperature growth) since Tn10 insertions that cause a temperature-sensitive lethal phenotype are rare.