JoAnn Hoskins

Genome of the Bacterium Streptococcus pneumoniae Strain R6

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.

The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid sequence of PBP 2a from strain 27r revealed a hydrophobic region at the amino terminus that possessed characteristics of an uncleaved signal peptide such as those found in type II integral membrane proteins. Site-specific mutagenesis was used to modify the strain 27r mecA gene to permit removal of the region encoding the putative transmembrane region (amino acids 2 to 22). When it was expressed in E. coli, the modified mecA gene from strain 27r encoded a water-soluble form of PBP 2a that was detectable in the cytoplasm of transformants. The water-soluble form of PBP 2a protein from S. aureus 27r retained the same binding efficiency for beta-lactam antibiotics as the unmodified membrane-bound PBP 2a from S. aureus 27r. Images

Identification and Characterization of the Penicillin-Binding Protein 2a of Streptococcus pneumoniae and Its Possible Role in Resistance to β-Lactam Antibiotics

ABSTRACT To further understand the role of penicillin-binding protein 2a (PBP 2a) of Streptococcus pneumoniae in penicillin resistance, we confirmed the identity of the protein as PBP 2a. The PBP 2a protein migrated electrophoretically to a position corresponding to that of PBP 2x, PBP 2a, and PBP 2b of S. pneumoniae and was absent in a pbp2ainsertional mutant of S. pneumoniae. We found that the affinities of PBP 2a for penicillins were lower than for cephalosporins and a carbapenem. When compared with other S. pneumoniae PBPs, PBP 2a exhibited lower affinities for β-lactam antibiotics, especially penicillins. Therefore, PBP 2a is a low-affinity PBP for β-lactam antibiotics in S. pneumoniae.

Cloning and characterization of femA and femB from Staphylococcus epidermidis

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.

Nucleotide sequence of the gene encoding yeast C-8 sterol isomerase ☆

The ERG2 gene encoding the Saccharomyces cerevisiae C-8 sterol isomerase, an enzyme involved in plant, animal, and fungal sterol biosynthesis was sequenced. A large open reading frame comprising 222 amino acids was observed.

Genetic Manipulation of the β-lactam Antibiotic Biosynthetic Pathway

Penicillins and cephalosoporins are members of the large group of sulfur-containing β-lactam antibiotics. Biosynthesis of the naturally occurring β-lactams, penicillin G, and cephalosporin C is illustrated in Figure 3.1. The key structural similarity of these heterocyclic compounds is the four-membered β-lactam ring (illustrated in the inset in Fig. 3.1), which is fused to a five-membered thiazolidine ring in penicillin G or a six-membered dihydrothiazine ring in cephalosporin C. Because of this structural similarity, the mode of action of all β-lactam antibiotics is the same: they interfer with bacterial cell wall synthesis and cause death by cell lysis. Penicillin G has been modified chemically to form many other clinically useful antibiotics that extended the spectrum of activity and, in some cases, provided resistance to penicillinases encoded by resistant pathogens. Although naturally resistant to penicillinases, cephalosporin C is not used clinically. However, chemical modifications of cephalosporin C have produced a variety of clinically useful agents, further extending the activity spectrum of β-lactam compounds.