PubMed · 5089018
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A Storaas. 1971-06-10. [Dosage experiments with ampicillin].. https://pubmed.ncbi.nlm.nih.gov/5089018/
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The ddcA gene from Streptomyces fradiae, which is located adjacent to the left edge of the tylosin biosynthetic cluster, has been cloned and sequenced. DNA sequence analysis revealed an ORF of 1194 bp that encodes a product of 42.6 kDa. This protein showed significant similarity to the extracellular endopeptidase with beta-lactamase activity encoded by the adp gene from Bacillus cereus and to PBPs (DD-carboxypeptidases and DD-endopeptidases) and beta-lactamases. Moreover, it contains three characteristic motifs conserved in PBPs and beta-lactamases, including an essential serine residue in the active centre and a putative leader peptide. Heterologous expression of the ddcA gene in Streptomyces lividans demonstrated the presence in the transformants of an extracellular beta-lactamase active against penicillin G, ampicillin and the chromogenic cephalosporin nitrocefin.
S. gallolyticus, formerly known as S. bovis is known since 1988 as a facultative pathogen of racing pigeons. Important clinical signs include acute mortality, inability to fly, lameness, weight loss and slimy green diarrhea. A pathognomonic sign at post mortem examination is the presence of well circumscribed areas of necrosis in the pectoral muscle. Furthermore tenosynovitis of the supracoracoid muscle and arthritis of the knee, shoulder and hock can be observed. In one study S. gallolyticus septicaemia was diagnosed in 10% of necropsied pigeons. Since S. gallolyticus was also isolated from nearly 40% of clinical healthy pigeons it is regarded as a facultative pathogen. Various biotypes, serotypes and culture supernatant phenotypes can be distinguished. Supernatant phenotypes are identified on the basis of the presence of either a T1, T2 or T3 protein triplet and the presence or absence of an extracellular A protein. S. gallolyticus strains with A protein are highly virulent, while strains with only T3 or T2 protein are of moderately or low virulence respectively. Fimbriae are only seen in highly virulent and some of the moderately virulent strains. Possible virulence factors include survival in macrophages, adhesion to cells and toxin production. Infection with serotype 1 and 2 induces some degree of protection against re-infection with serotype 1, which offers perspectives for the development of a vaccine. Experimentally ampicillin, doxycycline and erythromycin have shown therapeutic effects. For the treatment of clinical cases the use of ampicillin is advocated, together with hygienic measures, such as the use of grid floors and avoiding overcrowding.
To establish an experimental system to directly observe molecular evolution, a DNA fragment that confers ampicillin resistance on Escherichia coli was cloned from an archaeal genomic DNA. The activity of this clone was enhanced by 50 rounds of directed evolution by using DNA shuffling. Analysis of the evolved DNA fragments shows that two genetic regions have coevolved: One region, which has no obvious ORF, is essential for the activity, whereas the other, which appears to encode a protein, is not essential but enhances the activity of the former region. Analysis of the evolutionary intermediates shows that negative mutations are effectively removed while beneficial mutations accumulate and illustrates how a protein has evolved over the course of the evolution experiments. Although the mechanism of the activity remains unclear, the evolved DNA fragments also confer resistance to other drugs that inhibit bacterial cell-wall synthesis. The present system would serve as an experimental model to study evolutionary dynamics in the laboratory and provide the concept of screening natural libraries to obtain starting materials for directed evolution.