Biosynthesis and incorporation of an alkylproline-derivative (APD) precursor into complex natural products.

Covering: up to 2017This review covers the biosynthetic and evolutionary aspects of lincosamide antibiotics, antitumour pyrrolobenzodiazepines (PBDs) and the quorum-sensing molecule hormaomycin. These structurally and functionally diverse groups of complex natural products all incorporate rarely occurring 4-alkyl-l-proline derivatives (APDs) biosynthesized from l-tyrosine through an unusual specialized pathway catalysed by a common set of six proteins named Apd1-Apd6. We give an overview of APD formation, which involves unusual enzyme activities, and its incorporation, which is based either on nonribosomal peptide synthetase (PBDs, hormaomycin) or a unique hybrid ergothioneine-dependent condensation system followed by mycothiol-dependent sulphur atom incorporation (lincosamides). Furthermore, within the public databases, we identified 36 novel unannotated biosynthetic gene clusters that putatively encode the biosynthesis of APD compounds. Their products presumably include novel PBDs, but also novel classes of APD compounds, indicating an unprecedented potential for the diversity enhancement of these functionally versatile complex metabolites. In addition, phylogenetic analysis of known and novel gene clusters for the biosynthesis of APD compounds allowed us to infer novel evolutionary hypotheses: Apd3 methyltransferase originates from a duplication event in a hormaomycin biosynthetic gene cluster ancestor, while putative Apd5 isomerase is evolutionarily linked to PhzF protein from the biosynthesis of phenazines. Lastly, we summarize the achievements in preparing hybrid APD compounds by directing their biosynthesis, and we propose that the number of nature-like APD compounds could by multiplied by replacing l-proline residues in various groups of complex metabolites with APD, i.e. by imitating the natural process that occurs with lincosamides and PBDs, in which the replacement of l-proline for APD has proved to be an evolutionary successful concept.

Elucidation of salicylate attachment in celesticetin biosynthesis opens the door to create a library of more efficient hybrid lincosamide antibiotics.

Lincosamides, which are produced by streptomycetes, compose a small but clinically important class of antibiotics. The recent elucidation of the condensation and post-condensation biosynthetic steps of the lincosamides lincomycin and celesticetin revealed several unexpected reaction mechanisms. Here, we prepared recombinant proteins involved in the celesticetin biosynthetic pathway and used them for in vitro assays that were monitored by LC-MS. Our results elucidate the last biosynthetic step of celesticetin: the attachment of salicylic acid is catalyzed by the Ccb2 acyl-CoA ligase and the Ccb1 acyltransferase. Ccb1 belongs to the WS/DGAT protein family and, in contrast to the characterized members of the family, has unusual substrate specificity. To the best of our knowledge, Ccb1 is the first protein in this family that transfers a benzoyl derivative-CoA conjugate and is the first WS/DGAT protein involved in the biosynthesis of secondary metabolites. Furthermore, we exploited the relaxed substrate specificities of Ccb1 and Ccb2, as well as three additional upstream post-condensation biosynthetic proteins in the celesticetin pathway, and combined the lincomycin and the celesticetin biosynthetic pathways in vitro. In this way, we prepared a library of 150 novel hybrid lincosamides, including two unnatural chimeras of lincomycin and celesticetin, which were shown to have antibacterial properties more pronounced than clinically used lincomycin. These achievements may be considered a case study in applying knowledge about biosynthetic machinery to assemble a large number of compounds from originally a small group of natural products without the need for chemical synthesis.

Changes in the incidence of periodontal pathogens during long-term monitoring and after application of antibacterial drugs.

The incidence of potential periodontal pathogens (Aggregatibacter actinomycetemcomitans, formerly Actinobacillus actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia and Capnocytophaga ochracea) was monitored in patients with chronic periodontitis and in healthy control subjects. Two types of studies were carried out in which the composition of the bacterial communities in different niches of the same oral cavity ecosystem was investigated. Fluctuation or at least pronounced quantitative changes in the incidence of individual species in time were documented in the long-term study as well as after the local administration of antibacterial drug Chlo-Site or Metronidazole. Even within two weeks, a turnover of the monitored bacteria in separate niches of the oral biotope can be detected. A relatively high incidence of the tested periopathogens in the clinically healthy teeth of patients implies that even the "healthy" niches in the periodontal biotope function as a dynamic reservoir of periopathogenic microorganisms. This should be kept in mind when a local application of antibacterial compounds is used in the therapy of periodontal disease.

[Evaluation of screening for developmental dysplasia of the hip in the Liberec region in 1984-2005]. / Souborné zhodnocení screeningu vývojové dysplazie kycelního kloubu v regionu Liberec za období 1984-2005.

PURPOSE OF THE STUDY: To analyze our results of screening for developmental dysplasia of the hip (DDH) in the Liberec region between 1984 and 2005 MATERIAL AND METHODS: The study comprised 30,010 infants screened for DDH without any primary selection between 1984 and 2005.The infants were clinically examined on the basis of a three-step screening system (newborn, at 6 weeks and between 3 and 4 months of age) and by an imaging method. In the period from 1984 to 1991, this was plain radiography of the hip at 3 months of age, and a total of 12,944 infants were assessed. Between 1992 to 2005 ultrasonography (USG) was used at 6 weeks (or earlier when clinical signs were present) and again at 3 to 4 months of age, and a total of 17,066 infants were evaluated. In 16,678 of these infants, the sonogram obtained at 6 weeks was evaluated in detail by the Graf method. The presence of the epiphyseal nucleus of the proximal femur was assessed at 3 months, and from 2000 at 4 months of age. Attention was paid to the occurrence of DDH in general and findings of dislocation, both subluxation and luxation, of the hip, the proportion of right-side, left-side and bilateral findings, gender differences, the presence or absence of ossification of the femoral epiphyseal nucleus in infants between 3 and 4 months of age and, in the 1992-2005 period, the stratification of Graf types at 6 weeks. A question of whether the child's months of birth might have any relation to a possibility of DDH occurrence was also raised. RESULTS: Between 1984 and 1991, DDH was recorded in 10.4% of the infants, usually in a light form responding well to conservative treatment; subluxation was found in 1.99% and luxation in 0.6%. Between 1992 and 2005, DDH (mostly light forms) was recorded in 5.2%, subluxation in 0.5% and luxation in 0.2% of the infants. Right-side, left-side and bilateral findings were seen in 29%, 46% and 25% of the infants, respectively, and there were distinct differences between the genders. In an evaluated group of 453 infants with either subluxation or luxation, 86% of girls and 14% of boys were affected. The month of birth had no effect on DDH occurrence. Of the 9878 infants examined at 3 months, ossification of the femoral epiphyseal nucleus was found only in 37%, and of the 7188 infants examined at 4 months this was recorded in 64%. In a total of 33,356 hips (16,678 infants) evaluated at 6 weeks on the Graf scale, the following Graf types were recorded: Ia, 21%; Ib, 58%; IIa, 20%; IIc, 1%; D/IIIa, 0.19%; and IIIb/IV, 0.08%. DISCUSSION: The occurrence of DDH (formerly congenital dysplasia of the hip), including all forms/grades, is reported to be 3% to 5% in Central Europe. Our recent results obtained by the USG method are in agreement with this, as are our findings of its more frequent occurrence in girls (boys to girls, 1 to 6) and on the left side. Several reasons--medical, statistical or even sociodemographic--can be considered to explain the discrepancies in DDH detection rates obtained with radiography and ultrasonography, i.e., 10.5% vs 5.3%. For instance, the USG detection of DDH at 6 weeks of age initiates early treatment preventing progression of hip dysplasia. Better facilities available for home neonatal care and increased interest in general baby wellness may also play a role. CONCLUSIONS: Screening for developmental dysplasia of the hip is fully justified, because DDH prevalence is still high (500 subluxations and 200 luxations per 100,000 population). However, attention should also be paid to reducing factors and conditions that may adversely affect hip development from the baby's prenatal period to spontaneous walking.

Sequence analysis and heterologous expression of the lincomycin biosynthetic cluster of the type strain Streptomyces lincolnensis ATCC 25466.

A cosmid bearing an insert of 38 217 bp covering the gene cluster and its flanking regions of type strain Streptomyces lincolnensis ATCC 25466 was sequenced. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin (Lin) industrial strain S. lincolnensis 78-11. Analysis of the cluster-flanking regions revealed its localization within the genome of the ATCC 25466 strain. The cluster-bearing cosmid was integrated into the chromosome of Lin non-producing strains S. coelicolor CH 999 and S. coelicolor M 145. The modified strains heterologously produced Lin but the level dropped to approximately 1-3% of the production in the ATCC 25466 strain.

Deregulation of acetohydroxy-acid synthase: Loss of allosteric inhibition conferred by mutations in the catalytic subunit.

Acetohydroxy-acid synthases (AHAS) of two mutant strains Streptomyces cinnamonensis ACB-NLR-2 and BVR-18 were chosen for this study for their apparent activation by valine, which regularly acts as an allosteric inhibitor. Sequencing the ilvB genes coding for the AHAS catalytic subunit revealed two distant changes in the mutants, DeltaQ217 and E139A, respectively. Homology modeling was used to propose the structural changes caused by those mutations. In the mutant strain ACB-NLR-2 (resistant to 2-amino-3-chlorobutyrate and norleucine), deletion of Q217 affected a helix in ss-domain, distant from the active center. As no mutation was found in the regulatory subunit of this strain, DeltaQ217 in IlvB was supposed to be responsible for the observed valine activation, probably via changed properties on the proposed regulatory-catalytic subunit interface. In mutant strain BVR-18 (resistant to 2-oxobutyrate), substitution E139A occurred in a conservative loop near the active center. In vitro AHAS activity assay with the enzyme reconstituted from the wild-type regulatory and BVR-18 catalytic subunits proved that the substitution in the catalytic subunit led to the apparent activation of AHAS by valine. We suggest that the conservative loop participated in a conformational change transfer to the active center during the allosteric regulation.

Hybridization analysis and mapping of the celesticetin gene cluster revealed genes shared with lincomycin biosynthesis.

The first insight into celesticetin biosynthetic gene cluster of S. caelestis is presented. The genomic DNA of producing strain was digested, digoxigenin-labeled and hybridized with a set of probes designed according to S. lincolnensis gene sequences. Genes with high homology to the lincomycin biosynthetic genes coding for the predicted common parts of the pathway were identified in S. caelestis. Then, genomic DNA of S. caelestis treated by a multiple digestion was hybridized with five digoxigenin-labeled probes to construct a rough restriction map. Two consecutive islands formed by the genes with a putative function in biosynthesis of the shared saccharide moiety revealed an organization similar to the lincomycin biosynthetic gene cluster. The celesticetin cluster was mapped and essential information was obtained for subsequent steps, i.e. isolation and sequence analysis of the cluster.

In vitro activity of telithromycin and quinupristin/dalfopristin against methicillin-resistant coagulase-negative staphylococci with defined resistance genotypes.

We determined the activities of new antibiotics telithromycin (ketolide) and quinupristin/dalfopristin (streptogramins) against 88 macrolide and/or lincosamide resistant coagulase-negative staphylococci (CoNS) isolates with defined resistance gene status. Telithromycin susceptibility was determined only in erythromycin-sensitive isolates (15) indicating the same mechanisms of resistance. In contrast, all erythromycin-resistant isolates (73) were either constitutively resistant to telithromycin (13 isolates with constitutive erm genes) or demonstrated telithromycin D-shaped zone (60 isolates with inducible msr(A) and/or erm). However, the level of inducible resistance conferred by msr(A) (35 isolates) was borderline even after induction by erythromycin. No quinupristin/dalfopristin resistant isolate was observed if tested by disk-diffusion method (DDM) but 18 isolates were intermediate (MIC = 1-3 mg/L) and two isolates resistant (MIC = 8 mg/L) if tested by E-test. All these isolates were resistant to streptogramin A and harbored vga(A) gene (1 isolate) or vga(A)LC gene (19 isolates). MICs for quinupristin/dalfopristin were higher for isolates with combination of streptogramin A resistance and constitutive MLSB resistance (MIC = 3-8 mg/L in 4 isolates) than for streptogramin A-resistant isolates susceptible to streptogramin B (MIC = 0.5-2 mg/L in 16 isolates). In addition to S. haemolyticus, vga(A)LC was newly identified in S. epidermidis and S. warnerii indicating its widespread occurrence in CoNS. Misidentification of low-level resistant isolates by DDM may contribute to dissemination of streptogramin A resistance.

The prospects of advanced molecular methods in the detection of periodontal pathogens: hybridization in situ.

The paper summarizes the present state in the diagnostics of periodontal pathogens. Both the main advantages and drawbacks of the classic cultivation methods and those of the new DNA techniques are discussed. From the emerging methods of molecular diagnostics, the method of in situ hybridization is presented in more details. Its principle, various modifications of performance and possibilities of use are explained, including examples of its application in the detection of periodontal pathogens.

A new evolutionary variant of the streptogramin A resistance protein, Vga(A)LC, from Staphylococcus haemolyticus with shifted substrate specificity towards lincosamides.

We found a new variant of the streptogramin A resistance gene, vga(A)LC, in clinical isolates of Staphylococcus haemolyticus resistant to lincomycin and clindamycin but susceptible to erythromycin and in which no relevant lincosamide resistance gene was detected. The gene vga(A)LC, differing from the gene vga(A) at the protein level by seven amino acid substitutions, was present exclusively in S. haemolyticus strains resistant to both lincosamides and streptogramin A (LS(A) phenotype). Antibiotic resistance profiles of the ATP-binding cassette (ABC) proteins Vga(A)(LC) and Vga(A) in the antibiotic-susceptible host S. aureus RN4220 were compared. It was shown that Vga(A)LC conferred resistance to both lincosamides and streptogramin A, while Vga(A) conferred significant resistance to streptogramin A only. Detailed analysis of the seven amino acid substitutions, distinguishing the two related ABC proteins with different substrate specificities, identified the substrate-recognizing site: four clustered substitutions (L212S, G219V, A220T, and G226S) in the spacer between the two ATP-binding cassettes altered the substrate specificity and constituted the lincosamide-streptogramin A resistance phenotype. A transport experiment with radiolabeled lincomycin demonstrated that the mechanism of lincosamide resistance in S. haemolyticus was identical to that of the reported macrolide-streptogramin B resistance conferred by Msr(A).

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. METHODS AND RESULTS: Lincomycin production was monitored by thin-layer chromatography, proteins LmbJ and LmbIH were assayed in the cell-free extracts of S. lincolnensis by immunodetection. LmbJ occurred at stable level (2-4 mg x g(-1) of total proteins) for a long time period (36-96 h of cultivation) covering the whole production phase. This fairly corresponds to the catalytic function of the protein in the antibiotic biosynthesis (N-demethyllincomycin methyltransferase). On the contrary, LmbIH reached the detectable level (0.1 and 0.7 mg x g(-1)) just for a short period at 60-72 h. CONCLUSIONS: The absence of LmbIH protein at a detectable level during the major part of the antibiotic production phase casts doubt on its possible catalytic function. Rather a different connection with the final biosynthetic steps, e.g. regulatory, can be envisaged. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of a newly found putative regulatory gene was demonstrated during production of industrial antibiotic, lincomycin.

Spore-specific modification of DNA-dependent RNA polymerase alpha subunit in streptomycetes--a new model of transcription regulation.

At the very beginning of spore germination in streptomycetes the full-length alpha subunit of DNA-dependent RNA polymerase is shortened from its C-terminus. The C-terminal domain of the protein is required for binding of DNA and transcription regulators but its regulatory role in streptomycetes was not extensively studied. Comparison of the sequences of E. coli and S. coelicolor RNA polymerase alpha subunit (RNAP alpha) C-terminal domains reveals that the majority of amino acid residues responsible for the interaction with transcription regulators is conserved in both microorganisms. The spore specific modification of streptomycete RNAP alpha could thus have its regulatory role. The nature of the proteolytic enzyme, responsible for the RNAP alpha cleavage is discussed.

Chemical plume tracking. 1. Chemical information encoding.

It is shown experimentally that chemical information can be encoded and preserved in flowing liquid streams. It can be retrieved by chemical sensing arrays using correlation analysis. This finding is important for understanding of the mechanism of chemotaxis as practiced by some aquatic animals and also is a necessary prerequisite for construction of chemical plume tracking robots.

Chemical plume tracking. 2. Multiple-frequency modulation.

The purpose of this research is to estimate the effect of turbulent mixing on the information content in a chemically encoded signal, to investigate the effect of the presence of multiple encoded frequencies, and to evaluate the information contained in the higher harmonics of the coherence spectra. The virtual plume instrument is used to mimic the flow conditions and signal patterns in a real chemical plume. Two-frequency modulation experiments are performed using solenoid valves to introduce concentration plugs of a marker into the carrier flows at certain constant frequencies. In our experiments, the length of the delay elements and the dispersion were varied to mimic different characteristics of the turbulent plume. In addition, an artificial but uncorrelated white noise was added to the raw amperometric signals in order to simulate the "noisy" conditions existing in a real plume. Our experiments reveal that the introduced turbulence has only a marginal effect on the coherence spectra. Moreover, it is shown that when the second frequency is present in the plume, both fundamental frequencies can be unambiguously assigned. Higher harmonics in the coherence spectra have been found to depend on the distance from the source. These findings are important for understanding of the mechanism of chemotaxis and may also lead to the design of optimized search algorithms for chemical plume tracking robots.

Plume-tracking robots: a new application of chemical sensors.

Many animals have the ability to search for odor sources by tracking their plumes. Some of the key features of this search behavior have been successfully transferred to robot platforms, although the capabilities of animals are still beyond the current level of sensor technologies. The examples described in this paper are (1) incorporating into a wheeled robot the upwind surges and casting used by moths in tracking pheromone plumes, (2) extracting useful information from the response patterns of a chemical sensor array patterned after the spatially distributed chemoreceptors of some animals, and (3) mimicking the fanning behavior of silkworm moths to enhance the reception of chemical signals by drawing molecules from one direction. The achievements so far and current efforts are reviewed to illustrate the steps to be taken toward future development of this technology.

Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase.

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.

Headspace analysis of engine oil by gas chromatography/mass spectrometry.

This study establishes the rationale necessary for determining the time to change engine oil. This is based on identifying gaseous components in new and used automobile lubricants. Key compounds, so-called "signature", are separated and identified qualitatively by coupled gas chromatography/mass spectrometry. Volatile antioxidants at zero miles and fuel contaminants at low mileage are observed in the headspace of engine oil. Several oxidative degradation components have been positively identified in the used oil, which include the following: acetaldehyde, acetone, butanal, 2-propanol, acetic acid, 2-hexanol, benzoic acid, benzaldehyde, and 1-pentanol. This study strongly suggests that the status of lubricating oil can be determined by the analysis of the gas phase above the oil. Most importantly, it opens the possibility of performing conditional maintenance of the combustion engine based on information obtained from gas sensors.

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 T1dD protein family. Escherichia coli T1dD 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.

Two forms of DNA-dependent RNA polymerase alpha subunit in streptomycetes.

We demonstrated two different DNA-dependent RNA polymerase (RNAP) alpha subunits in spores of Streptomyces granaticolor with apparent molecular masses of 40 and 43 kDa. The 43-kDa subunit was also found in vegetative cells. These two proteins are highly similar to each other as well as to other bacterial RNAP alpha subunits. The 40-kDa subunit is shortened from its C-terminus, in the portion of the protein, required for binding of DNA and transcription regulators. The gene for RNAP alpha from S. granaticolor was cloned and sequenced and the corresponding protein was overproduced in Escherichia coli. In vitro experiments using purified RNAP alpha showed that the cell free extract from spores of S. granaticolor exhibits proteolytic activity responsible for the alpha subunit shortening, whereas that from vegetative cells does not. This modification of alpha subunit might point to a novel mechanism of transcriptional control in streptomycetes.