Jaroslav Spizek

Lincosamides: Chemical Structure, Biosynthesis, Mechanism of Action, Resistance, and Applications

Publisher Summary Natural lincosamides are produced by several Streptomyces species, mainly by Streptomyces lincolnensis, S. roseolus, and S. caelestis and by Micromonospora halophytica. Lincosamides constitute a relatively small group of antibiotics with a chemical structure consisting of amino acid and sugar moieties. Their mechanism of action is via inhibition of protein synthesis in sensitive micro-organisms. Lincosamides have an unusual antimicrobial spectrum, being active against only Gram-positive and not Gram-negative aerobic bacteria. They exhibit a significant antibiotic activity against some anaerobic bacteria. They are used therapeutically, especially in cases where synergistic effects of a mixed anaerobic and aerobic microflora are anticipated. Lincomycin and clindamycin are also useful alternatives to penicillin and its derivatives in the treatment of upper respiratory tract infections in patients with allergy to penicillin. The chapter also discusses the major route of resistance to lincosamides. Furthermore, with the current knowledge of gene clusters specifying biosynthesis of lincomycin and the related antibiotic celesticetin, production of new derivatives of lincosamides by genetic engineering can be easily imagined, resulting in production of new derivatives of lincosamides.

The genes lmbB1 and lmbB2 of Streptomyces lincolnensis encode enzymes involved in the conversion of l-tyrosine to propylproline during the biosynthesis of the antibiotic lincomycin A

Abstract The genes lmbA,B1,B2 in the lincomycin A production gene cluster of Streptomyces lincolnensis were shown to form a common transcription unit with the promoter located directly upstream of lmbA. The proteins LmbB1 (mol. mass, 18 kDa) and LmbB2 (mol. mass 34 kDa), when over-produced together in Escherichia coli, brought about enzyme activities for the specific conversion of both l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA) to a yellow-colored product. The LmbB1 protein alone catalyzed the conversion of l-DOPA, but not of l-tyrosine. The purified LmbB1 protein showed a Km for l-DOPA of 258.3 μM. The l-tyrosine converting activity could not been demonstrated in vitro. The preliminary interpretation of these data suggests that the protein LmbB1 is an l-DOPA extradiol-cleaving 2,3-dioxygenase and that the protein LmbB2, either alone or in accord with LmbB1, represents an l-tyrosine 3-hydroxylase. This sequence of putative oxidation reactions on l-tyrosine seems to represent a new pathway different from the ones catalyzed by mammalian l-tyrosine hydroxylases or the wide-spread tyrosinases. The protein LmbA seemed not to be involved in this process. The labile, yellow-colored product from l-DOPA could not be converted to a picolinic acid derivative [3-(2-carboxy-5-pyridyl)alanine] in the presence of ammonia. Therefore, it probably is not a derivative of a cis,cis-3-hydroxymuconic acid semialdehyde; instead, its speculative structure represents a heterocyclic precursor of the propylhygric acid moiety of lincomycin A.

Pharmacologically Active Sulfur-Containing Compounds

In addition to sulfur-containing compounds that form essential and indispensable constituents of all living cells, there is a plethora of less wide-spread sulfur-containing compounds with much less common and often unexpected prop- erties, chemical structures and biological activities. Some of these compounds have long been known as efficient antibi- otics. This review surveys these less known compounds with two and more sulfur atoms in their molecule. In particular, it deals with natural heterocyclic compounds isolated, e.g., from bacteria, fungi, lichens, plants, and animals but also from unusual and exotic sources such as sea invertebrates (coelenterates, sponges, tunicates, sea worms), etc. Compounds such as calicheamicins, thiazole-related compounds and epipolythiodioxopiperazines may serve as examples. Because of their vast variety, individual compounds are grouped according to their chemical structure (only compounds whose chemical structure has been identified are included). These compounds affect a huge array of organisms from microorganisms to man, and the pharmacological activities they exert range from antiviral, antibacterial, antifungal, insecticidal or antiproto- zoan effects to regulation of gene expression, cytoskeleton disruption, immunomodulation, plant growth stimulation or in- hibition, pro- or antioxidant action, effects on selected enzymes, receptors and signal transduction pathways, etc. The ac- tivities are however often known only sketchily, and this paucity of data, and the fact that the activities were tested on a diverse spectrum of test organisms, cell lines, etc., is of necessity reflected in our survey. Two seemingly trivial but im- portant points deserve attention: (a) compounds with widely different structures and from widely different sources have often very similar pharmacological effects; (b) a slight modification in the molecule may bring vast changes in the bio- logical effects of the compound, in terms of quality, quantity and targeting. The purpose of the review was to show both the unique and common features of these compounds and to point out their potential usefulness for mankind.

Prevalence of Resistance Mechanisms against Macrolides and Lincosamides in Methicillin-Resistant Coagulase-Negative Staphylococci in the Czech Republic and Occurrence of an Undefined Mechanism of Resistance to Lincosamides

ABSTRACT High occurrence of the non-macrolide-lincosamide-streptogramin B resistance genes msrA (53%) and linA/linA′ (30%) was found among 98 methicillin-resistant coagulase-negative staphylococci additionally resistant to macrolides and/or lincosamides. The gene msrA predominated in Staphylococcus haemolyticus (43 of 62 isolates). In Staphylococcus epidermidis, it was present in 7 of 27 isolates. A novel mechanism of resistance to lincosamides appears to be present in 10 genetically related isolates of S. haemolyticus in the absence of ermA, ermC, msrA, and linA/linA′.

Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.

Pilot-plant cultivation of Streptomyces griseus producing homologues of nonactin by precursor-directed biosynthesis and their identification by LC/MS-ESI

Precursor-directed biogenetic approach was used to produce a range of nonactin homologues in a 50 l fermentor cultivation of strain Streptomyces griseus 34/249 obtained by UV mutagenesis. The production medium contained sodium propionate, isobutyrate and isovalerate as individual precursors, and 10 g l−1 Diaion HP20 styrene-divinylbenzene resin that maintains suitable precursor concentration by reversibly adsorbing and releasing it. The produced nonactin homologues were separated on two C18 reversed-phase liquid chromatography columns in series and analyzed by MS with ESI source in the positive ion mode. Formation of doubly charged ions was suppressed by an excess of Na+ ions throughout the process. The production of the homologues increased up to day 5 and then it leveled off. Cultivations with individual precursors yielded a total of 18 nonactin homologues whose spectrum depended on the precursor used. The total production of the homologues was lowered but their spectrum was shifted to higher-molecular-weight compounds.

Putative lmbI and lmbH genes form a single lmbIH ORF in Streptomyces lincolnensis type strain ATCC 25466

The lincomycin-production gene cluster of the industrial overproduction strain Streptomyces lincolnensis 78-11 has been sequenced (Peschke et al. 1995) and twenty-seven putative open reading frames with biosynthetic or regulatory functions (lmb genes) identified. Two distinct hypothetical genes, lmbI and lmbH, were found downstream of the lmbJ gene, coding for LmbJ protein, which is believed to participate in the last lincomycin biosynthetic step, i.e. conversion of N-demethyllincomycin (NDL) to lincomycin. In the present study, we demonstrate the presence of a single larger open reading frame, called lmbIH, in the lincomycin low-production type strain Streptomyces lincolnensis ATCC 25466, instead of two smaller lmbI and lmbH genes. The product, LmbIH, is a protein of an unknown function and is homologous with the TldD protein family. Escherichia coli TldD protein was previously shown to be involved in the control of DNA gyrase by LetD protein. Moreover, our experiments indicate co-regulation of lmbJ and lmbIH expression. This translation coupling probably reflects an eight nucleotide overlap between the lmbJ and lmbIH genes, as well as the lack of a Shine-Dalgarno sequence upstream of the lmbIH gene.

Memories of a great man, Ivan Málek.

Ivan Málek was a great scientist and an inspiration to Czech scientists and to microbiologists and biochemical engineers around the world. He brought Czech microbiology into the world of science and medicine. His devotion to continuous fermentation was recognized throughout the world. Despite the German occupation of Czechoslovakia during World War II, Málek worked on autovaccination therapy and was able to produce small amounts of penicillin, which was used to treat ill patients. He was a prolific writer of microbiological textbooks including Microbiology and Epidemiology (1946), Laboratory Methods for Bacteriological Diagnosis (1948), Fight of Modern Science Against Microorganisms (1951) and General Medical Microbiology (1953). It was in 1945 that, as a microbiologist and physician, he was appointed Lecturer at Charles University. A year after that, he went to Connaught Laboratories at the University of Toronto to study methods of penicillin production. This led to the establishment in 1949 of the first Czechoslovak penicillin factory in Roztoky u Prahy, a suburb of Prague. After his return from Canada, Málek lectured and taught more than 1,500 students studying medicine or nursing. In 1947, he established the country’s first school of medical laboratory technology. Málek became Associate Professor in 1948 and published his paper on variability of bacteria. In that same year, he taught and helped organize research at the new School of Medicine in Hradec Kralove. He also continued his work on continuous fermentation and returned to an early interest in mycobacteria. When the country formed an Academy of Sciences (ASCR) made up of research institutes in natural and social sciences in 1950, Málek was called upon to become the Director of the Central Institute of Biology in Prague and the Chairman of the Czechoslovak Society for Microbiology. He was soon elected to the ASCR and appointed as Chairman of the Central Institute of Biology. He helped found the journal Folia Microbiologica in 1956 and became the first Director of the Institute of Microbiology which was spun off from the Central Institute of Biology. This new institute, which soon became famous around the world for its first-rate research, was established in a new building in Krc, a district of Prague. He continued his work on continuous fermentation and established the importance of the ‘‘physiologic state’’ of microbiological populations. He was instrumental in the annual sending of ten or more Institute scientists to various laboratories in the USA and Western Europe. This is the way that many of us became familiar with excellent Czech scientists in our laboratories and those of our colleagues. Málek also arranged for foreign scientists to do their sabbatical leave research at the Institute of Microbiology. He was always willing to help the Institute’s scientists and technicians to overcome their difficult problems of life and existence. Quite often he served as an umbrella, kindly covering their more or less frequent acts of misconduct against an oppressive regime. Of great interest to Málek was the setting up of international meetings. After organizing the First International Symposium on the Continuous Cultivation of Microorganisms in Prague in 1958, the Proceedings were published by the ASCR and became well known around the world. This was followed by later Symposia in Prague in 1962, Porton Down, UK in 1966, Prague in 1968, and Oxford in A. L. Demain (&) Charles A. Dana Research Institute for Scientists Emeriti (RISE), Drew University, Madison, NJ 07940, USA e-mail: ademain@drew.edu

LmbJ and LmbIH protein levels correlate with lincomycin production in Streptomyces lincolnensis.

Aims:  To demonstrate the expression of two overlapping genes lmbJ and lmbIH in Streptomyces lincolnensis and to document LmbJ and LmbIH protein levels during the lincomycin production phase. To analyse presumable function of the LmbIH protein.