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Antibiotic activity of polyketide products derived from combinatorial biosynthesis: implications for directed evolution.

A library of over 100 polyketides, generated via combinatorial cloning of genes encoding subunits of aromatic polyketide synthases, was screened for molecules capable of inhibiting the growth of gram-positive bacteria. A total of 26 polyketides, with varying levels of antibiotic activity in filter-disk assays, were purified. Most bioactive polyketides were produced as relatively minor compounds (< 1 mg/l), although two major anthraquinones, with yields in the range of 10-100 mg/l, were also identified and structurally characterized. When tested against Bacillus subtilis 168 beta, they were found to cause a 50% reduction in colony-forming units at concentrations of 20 and 300 micrograms/ml, respectively. We speculate that many of the minor (and possibly more potent) bioactive polyketides are synthesized via nonspecific enzymatic modifications of shunt products derived from engineered polyketide synthase pathways. If so, then these 'fortuitous' pathways should be amenable to further rationally guided manipulation. Our results support the notion that combinatorial biosynthesis can be used to generate novel, biologically active molecules. They also point to the feasibility of designing mutagenesis selection experiments aimed at the directed evolution of organic molecules with desirable pharmaceutical properties.

Anti-Bacterial Agents↗

An automated annotation tool for genomic DNA sequences using GeneScan and BLAST.

Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.

Algorithms↗

Regulation of IgA synthesis at mucosal surfaces.

Immunoglobulin A is the main element of the humoral immune response that has been selected through evolution, together with innate mucosal defences, to provide protection against microbial antigens at mucosal surfaces. IgA responses are initiated in organized inductive structures, such as Peyer's patches and nasal-associated lymphoid tissues, as well as diffuse effector tissues, such as gut lamina propria and nasal mucosa. Hypermutated secretory IgAs play a critical role in regulating the composition of the intestinal microflora. Dysregulation of gut homeostasis in IgA-deficient gut causes a continuous activation of the immune cells and induces inflammatory processes leading to lymphoneogenesis. Recent advances in this field include new insights into the role of IgA in the maintenance of gut homeostasis and the proposal of an updated model for the induction of IgA responses in the gut.

Bacteria↗

The influence of single and multiple applications of pyrene on the evolution of pyrene catabolism in soil.

The influence of pyrene added in a single application (0, 50, 100 and 200 mg kg(-1)) was investigated in multiple applications (1 x 50, 2 x 50 and 4 x 50 mg kg(-1)) on the evolution of catabolic activity in a pristine pasture soil. The microbial community's ability to degrade pyrene was assessed at 0, 4, 8 and 12 weeks by the mineralization of added 14C-pyrene. Significant mineralization (>5%) of added 14C-pyrene only occurred after 4 weeks soil-pyrene contact time in most of the pyrene-amended soils. Pyrene-amended soils showed statistically significantly shorter (P<0.05) lag times compared to the control soil after 8 and 12 weeks soil-pyrene contact time. Further, the rates of degradation increased in the presence of pyrene, peaking at 8 weeks. In terms of the overall extents of pyrene mineralization, there were statistically significant increases (P<0.05) between 4 and 8 weeks, with little difference between 8 and 12 weeks, with the general trend that an increase in pyrene concentration resulted in higher levels of mineralization. Increasing the concentration and number of pyrene additions can have a significant impact on the adaptation of the soil microflora to degrade pyrene over time.

Adaptation, Physiological↗

Change of pneumococcal resistance to antibiotics in adults between 1995 and 1997: a study in eight French counties.

The main object of this study was to describe the evolution of antibiotic resistance in pneumococci from adults, in eight French counties of France between 1995 and 1997. Despite the high and increasing prevalence (23-35%) of pneumococci with diminished susceptibility to penicillin G (PSDP), resistance to amoxycillin (0.8-0.5%) and to cefotaxime (0-0.3%) was rare in both 1995 and 1997 respectively. The percentage of pneumococci resistant to penicillin G (PRP, minimum inhibitory concentration >1 mg/l) remained stable between the two periods. PSDP showed increased resistance to macrolides (30-41%), to cotrimoxazole (28-34%) and to tetracycline (19-25%). These figures are lower than those obtained over the same periods and the same regions in children. The distribution of PSDP serotypes isolated in adults was the same as that seen in children: by descending order serotypes 23, 14, 9 and 6. This study by the Regional Pneumococcal Observatories confirms the high prevalence and the main characteristics of antibiotic resistance among pneumococci with variations in levels of resistance with the age of patients, with the site of sampling and from one Observatory to another.

Adolescent↗

The superbugs: evolution, dissemination and fitness.

Since the introduction of antibiotics, bacteria have not only evolved elegant resistance mechanisms to thwart their effect, but have also evolved ways in which to disseminate themselves or their resistance genes to other susceptible bacteria. During the past few years, research has revealed not only how such resistance mechanisms have been able to evolve and to rapidly disseminate, but also how bacteria have, in some cases, been able to adapt to this new burden of resistance with little or no cost to their fitness. Such adaptations make the control of these superbugs all the more difficult.

Anti-Bacterial Agents↗

Oligosaccharide recognition signals and defence reactions in marine plant-microbe interactions.

Recent findings on the involvement of oligosaccharide signals in pathogen recognition and defence reactions in marine algae shine a new light on the ecology of their interactions with associated microorganisms. Since the marine environment encompasses lineages that have diverged a long time ago from the terrestrial phyla, these results suggest that cell-cell recognition pathways typical of terrestrial plants appeared very early in the evolution of eukaryotes. Production of oligosaccharides from marine algae using microbial recombinant polysaccharidases is also of industrial interest as plants can be protected from infections by preincubation in the presence of appropriate signals that mimic the attacks by pathogens.

Bacteria↗

The costs and benefits of killer toxin production by the yeast Pichia kluyveri.

Numerous yeast species in many genera are able to produce and excrete extracellular toxic proteins (mycocins) that can kill other specific sensitive yeasts. Natural distributions of killer yeasts suggest that they may be important in maintaining community composition and provide a benefit to the toxin producing cells. The fact that not all yeasts are killers and that polymorphisms exist within some killer species suggests there may be a cost associated with killer toxin production. This study focuses on the costs and benefits associated with toxin production by the yeast Pichia kluyveri. Strains differing in their ability to kill were obtained by tetrad dissection. One parent strain produced spores that exhibited a trade-off between killing ability and intrinsic growth rate. A killer clone from this strain was able to maintain a higher proportion of cells than a non-killer when grown with the same sensitive yeast under laboratory-simulated natural conditions. On the other hand, when grown with a yeast not sensitive to Pichia kluyveri toxin, the non-killer maintained a higher proportion of the total community than did the killer clone. The data support the hypothesis that there are both costs and benefits to producing killer toxin, and based on this, selection may favor different phenotypes in different conditions.

Animals↗

Population genomics of drug resistance in Candida albicans.

We followed adaptation in experimental microbial populations to inhibitory concentrations of an antimicrobial drug. The evolution of drug resistance was accompanied in all cases by changes in gene expression that persisted in the absence of the drug; the new patterns of gene expression were constitutive. The changes in gene expression occurred in four replicate populations of the pathogenic fungus Candida albicans during 330 generations of evolution in the presence of the antifungal drug fluconazole. Genome-wide expression profiling of over 5,000 ORFs identified 301 whose expression was significantly modulated. Cluster analysis identified three distinct patterns of gene expression underlying adaptation to the drug. One pattern was unique to one population and included up-regulation of the multidrug ATP-binding cassette transporter gene, CDR2. A second pattern occurred at a late stage of adaptation in three populations; for two of these populations profiled earlier in their evolution, a different pattern was observed at an early stage of adaptation. The succession of early- and late-stage patterns of gene expression, both of which include up-regulation of the multidrug major facilitator transporter gene, MDR1, must represent a common program of adaptation to this antifungal drug. The three patterns of gene expression were also identified in fluconazole-resistant clinical isolates, providing further evidence that these patterns represent common programs of adaptation to fluconazole.

ATP-Binding Cassette Transporters↗

Degradation of pyrimethanil in soil: influence of light, oxygen, and microbial activity.

The research was carried out in order to verify the influence that light, oxygen, and microbial activity have on the degradability of pyrimethanil (PYR) in soil. The products of degradation were also identified and their evolution in time evaluated. The results indicate that the molecule is more persistent in the absence of light, oxygen, and microbial activity. The order of importance of these three factors is as follows: light < microbial activity < oxygen. The following products of degradation were identified: (1) benzoic acid, (2) cis,cis-muconic acid, (3) hydroxyl-4,6-dimethyl-2-pirimidinamine, (4) N'-ethyl-N-hydroxyformamidine, and (5) 4,6-dimethyl-2-piridinamine, which appeared different from those reported in literature for the degradation of PYR in abiotic conditions. This result suggests that the degradation in soil is mainly biotic.

Adsorption↗

Mal3, the fission yeast homologue of the human APC-interacting protein EB-1 is required for microtubule integrity and the maintenance of cell form.

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3+. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3+ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3+ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3+ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.

Adenomatous Polyposis Coli↗

Host innate defenses in the lung: the role of cytokines.

PURPOSE OF REVIEW: The lung has a unique relationship with the environment. Through evolution the lung has developed strategies to defend itself from microbial invasion. As we encounter increasing multidrug-resistant microorganisms, we need to further our knowledge of innate defense systems in order to design novel strategies to deal with these microbes without inducing over-exuberant inflammation and lung injury. RECENT FINDINGS: The development of lung innate immunity requires microbial molecular pattern recognition by the recently described Toll like receptors, the release of early response cytokines that further activate the 'master switch', nuclear factor-kappaB, leading to amplified host defense to invading microbes. A balance of Type 1 and Type 2 cytokines modulates the intensity of innate immunity. Cytokines/chemokines orchestrate the polarization and transition of innate to adaptive immunity. SUMMARY: The elucidation of the pathways involved in innate immunity and factors controlling the transition to adaptive immunity will improve our understanding of the host response to infection and improve our ability to design new therapies for the treatment of infectious disease.

Chemokines↗

Origin of tetracycline efflux proteins: conclusions from nucleotide sequence analysis.

The sequences of six tetracycline efflux proteins and three transport proteins which have some resemblance to them were compared. The tetracycline efflux proteins fall into three families: (i) those encoded by pBR322, RP1, and Tn10 (Escherichia coli); (ii) pT181 (Staphylococcus aureus) and pTHT15 (Bacillus subtilis); and (iii) tet347 (Streptomyces rimosus). There is global sequence homology within each of the first two families, but there is none between the families. The pT181/pTHT15 family shares close homology with the N-terminal half of the methylenomycin A efflux protein (Streptomyces coelicor), while tet347 resembles the C-terminal half. Portions of the N-terminal half of the Tn10-encoded protein show significant resemblance to portions in the N-terminal half of the pT181/pTHT15 family, but this sometimes occurs among transport proteins which do not have a common substrate. Tetracycline efflux proteins, therefore, appear to have arisen on at least two, or possibly three, separate occasions, probably from other transport proteins.

Amino Acid Sequence↗

Adaptive radiation in microbial microcosms.

It has often been argued that evolutionary diversification is the result of divergent natural selection for specialization on alternative resources. I provide a comprehensive review of experiments that examine the ecology and genetics of resource specialization and adaptive radiation in microbial microcosms. In these experiments, resource heterogeneity generates divergent selection for specialization on alternative resources. At a molecular level, the evolution of specialization is generally attributable to mutations that de-regulate the expression of existing biosynthetic and catabolic pathways. Trade-offs are associated with the evolution of resource specialization, but these trade-offs are often not the result of antagonistic pleiotropy. Replicate adaptive radiations result in the evolution of a similar assemblage of specialists, but the genetic basis of specialization differs in replicate radiations. The implications of microbial selection experiments for evolutionary theory are discussed and future directions of research are proposed.

Adaptation, Biological↗