Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5'-Phosphate Production in E. coli.

Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous 'replacer' gene rescues lethality caused by inactivation of a 'target' gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5'-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.

Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy.

BACKGROUND: Transrectal ultrasound-guided (TRUS) prostate biopsy is a very common procedure that is generally considered relatively safe. However, severe sepsis can occur after TRUS prostate biopsies, with Escherichia coli being the predominant causative agent. A common perception is that the bacteria that cause post-TRUS prostate biopsy infections originate in the urinary tract, but this view has not been adequately tested. Yet other authors believe on the basis of indirect evidence that the pathogens are introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa. METHODS: We compared E. coli isolates from male patients with bacteremic urinary tract infection (B-UTI) to isolates of patients with post prostate biopsy sepsis (PPBS), in terms of their sequence types, determined by multi-locus sequence typing (MLST) and their virulence markers. RESULTS: B-UTI isolates were much richer in virulence genes than were PPBS isolates, supporting the hypothesis that E. coli causing PPBS derive directly from the rectum. Sequence type 131 (ST131) strains and related strain from the ST131 were common (>30%) among the E. coli isolates from PPBS patients as well as from B-UTI patients and all these strains expressed extended spectrum beta-lactamases. CONCLUSIONS: Our finding supports the hypothesis that E. coli causing PPBS derive directly from the rectum, bypassing the urinary tract, and therefore do not require many of the virulence capabilities necessary for an E. coli strain that must persist in the urinary tract. In light of the increasing prevalence of highly resistant E. coli strains, a new approach for prevention of PPBS is urgently required.

["Do two walk together unless they have agreed to do so?"--Combining conventional and complementary medicine in the treatment of gastroparesis].

Gastroparesis is a chronic disorder of abnormal gastric motility causing considerable suffering. We describe two cases of gastroparesis which were treated by methods which were not part of routine conventional therapy, but which, nevertheless, led to significant clinical improvement. In the first case, the patient suffered from gastroparesis following a vagal injury while undergoing a lung transplant. During his illness a mega-bezoar formed, a well-described complication of gastroparesis. After conservative measures failed, and in order to avoid a surgical intervention that carried considerable risk under the circumstances, a successful trial consisting of imbibing large amounts of "Coca Cola" and acupuncture was initiated. The bezoar dissolved completely and considerable improvement of the patient's gastric motility was achieved. In the second case, the patient was a young woman suffering from idiopathic gastroparesis, which responded well to treatment with tricyclic antidepressants. Due to her intention to become pregnant, this treatment was discontinued and she was admitted to receive homeopathic treatment. After two failed attempts in finding the right homeopathic remedy to replace the conventional treatment, the third remedy (Sepia) used brought dramatic improvement in her general condition and her dyspepsia. Today, over two years after her successful homeopathic treatment, she is in the middle of her second pregnancy, without any treatment and free of complaints. These two cases are examples of simple solutions for seemingly complicated and complex conditions alleviated by an integration of conventional and complementary/alternative medicine.

The contribution of common rpsL mutations in Escherichia coli to sensitivity to ribosome targeting antibiotics.

Point mutations in the rpsL gene encoding ribosomal protein S12 can generate resistance to streptomycin, resulting in rapid emergence of resistance to this antibiotic during treatment. In this work, we demonstrate that while spontaneous rpsL mutants in Escherichia coli are resistant to streptomycin, they are more sensitive to the ribosome-targeting antibiotics chloramphenicol, tetracycline and erythromycin. Moreover, combinations of these antibiotics, even in low concentrations were enough to achieve complete growth inhibition of both wild type and rpsL mutant strains. Thus, combining ribosome-targeting drugs can be used as a new treatment strategy that may be effective against streptomycin-resistant ribosome mutants.

Coenzyme B12 controls transcription of the Streptomyces class Ia ribonucleotide reductase nrdABS operon via a riboswitch mechanism.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomycetes contain genes coding for two RNRs. The class Ia RNR is oxygen dependent, and the class II RNR is oxygen independent and requires coenzyme B12. Either RNR is sufficient for vegetative growth. We show here that the Streptomyces coelicolor M145 nrdABS genes encoding the class Ia RNR are regulated by coenzyme B12. The 5'-untranslated region of nrdABS contains a 123-nucleotide B12 riboswitch. Similar B12 riboswitches are present in the corresponding regions of eight other S. coelicolor genes. The effect of B12 on growth and nrdABS transcription was examined in a mutant in which the nrdJ gene, encoding the class II RNR, was deleted. B12 concentrations of just 1 mug/liter completely inhibited growth of the NrdJ mutant strain. Likewise, B12 significantly reduced nrdABS transcription. To further explore the mechanism of B12 repression, we isolated in the nrdJ deletion strain mutants that are insensitive to B12 inhibition of growth. Two classes of mutations were found to map to the B12 riboswitch. Both conferred resistance to B12 inhibition of nrdABS transcription and are likely to affect B12 binding. These results establish that B12 regulates overall RNR expression in reciprocal ways, by riboswitch regulation of the class Ia RNR nrdABS genes and by serving as a cofactor for the class II RNR.

Alternative oxygen-dependent and oxygen-independent ribonucleotide reductases in Streptomyces: cross-regulation and physiological role in response to oxygen limitation.

Ribonucleotide reductases (RNRs) catalyse the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs. We show here that the Streptomyces coelicolor M145 nrdAB genes encoding an oxygen-dependent class I RNR are co-transcribed with nrdS, which encodes an AraC-like regulatory protein. Likewise, the class II oxygen-independent RNR nrdJ gene forms an operon with a likely regulatory gene, nrdR, which encodes a protein possessing an ATP-cone domain like those present in the allosteric activity site of many class Ia RNRs. Deletions in nrdB and nrdJ had no discernible effect on growth individually, but abolition of both RNR systems, using hydroxyurea to inactivate the class Ia RNR (NrdAB) in the nrdJ deletion mutant, was lethal, establishing that S. coelicolor possesses just two functional RNR systems. The class II RNR (NrdJ) may function to provide a pool of deoxyribonucleotide precursors for DNA repair during oxygen limitation and/or for immediate growth after restoration of oxygen, as the nrdJ mutant was slower in growth recovery than the nrdB mutant or the parent strain. The class Ia and class II RNR genes show complex regulation. The nrdRJ genes were transcribed some five- to sixfold higher than the nrdABS genes in vegetative growth, but when nrdJ was deleted, nrdABS transcription was upregulated by 13-fold. In a reciprocal experiment, deletion of nrdB had little effect on nrdRJ transcription. Deletion of nrdR caused a dramatic increase in transcription of nrdJ and to a less extent nrdABS, whereas disruption of cobN, a gene required for synthesis of coenzyme B12 a cofactor for the class II RNR, caused similar upregulation of transcription of nrdRJ and nrdABS. In contrast, deletion of nrdS had no detectable effect on transcription of either set of RNR genes. These results establish the existence of control mechanisms that sense and regulate overall RNR gene expression.

Diminished dendritic cell interleukin 10 production in atopic children.

BACKGROUND: Diminished interleukin 10 (IL-10) and/or IL-12 production may contribute to the pathogenesis of asthma and atopy. Dendritic cells (DCs) produce these cytokines and have been implicated in the pathogenesis of these disorders. OBJECTIVE: To determine whether DC IL-10 and/or IL-12 production is diminished in children aged 6 to 12 years with allergic rhinitis (AR) and with or without asthma. METHODS: Monocyte-derived DCs were isolated from 20 subjects without AR or asthma (group 1), 20 subjects with AR without asthma (group 2), and 20 subjects with AR and asthma (group 3). Asthma was defined as a history of physician-diagnosed disease, and AR was defined as a positive history and positive puncture skin test responses (wheal > or = 5 mm) to relevant inhalant allergens. DCs were stimulated with either lipopolysaccharide (LPS) or diluent and cultured for 24 hours. Supernatants were assayed for IL-10 and IL-12 levels by enzyme-linked immunosorbent assay. RESULTS: DC IL-10 production was diminished in groups 2 and 3 compared with group 1. Median LPS-induced IL-10 levels were 11.0 pg/mL in group 1, 6.1 pg/mL in group 2, and 1.5 pg/mL in group 3. The frequencies of subjects with detectable IL-10 levels were 85%, 20%, and 20% in groups 1, 2 and 3, respectively. Median LPS-induced IL-12 levels were similar in all groups. CONCLUSIONS: These data support the hypothesis that atopic subjects have an intrinsic inability to up-regulate DC IL-10 production. Future studies in this area could lead to a better understanding of the pathogenesis of atopy.

Transcriptional regulation of the Staphylococcus aureus thioredoxin and thioredoxin reductase genes in response to oxygen and disulfide stress.

In this report we describe the cloning, organization, and promoter analysis of the Staphylococcus aureus thioredoxin (trxA) and thioredoxin reductase (trxB) genes and their transcription in response to changes in oxygen concentration and to oxidative stress compounds. Northern analysis showed that the S. aureus trxA and trxB genes were transcribed equally well in aerobic and anaerobic conditions. Several oxidative stress compounds were found to rapidly induce transcription of the trxA and trxB genes. The most pronounced effects were seen with diamide, a thiol-specific oxidant that promotes disulfide bond formation; menadione, a redox cycling agent; and tau-butyl hydroperoxide, an organic peroxide. In each case the induction was independent of the general stress sigma factor sigma(B). These studies show that the S. aureus trxA and trxB genes are upregulated following exposure to these oxidative stress agents, resulting in increased disulfide bond formation. In contrast, no effect of hydrogen peroxide on induction of the trxA and trxB genes was seen. We also show that the S. aureus thioredoxin reductase appears to be essential for growth. This observation, coupled with structural differences between the bacterial and mammalian thioredoxin reductases, suggests that it may serve as a target for the development of new antimicrobials.

Streptomyces spp. contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth.

Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genus Streptomyces. One gene, nrdJ, encoded an oligomeric protein comprising four identical subunits each with a molecular mass of approximately 108 kDa. The activity of this protein depended on the presence of 5'-deoxyadenosylcobalamine (coenzyme B12), establishing it as a class II RNR. The Streptomyces clavuligerus nrdJ gene was cloned, using internal peptide sequences from the purified protein, and was found to encode a polypeptide of 961 aa. Molecular phylogenetic analysis showed that the S. clavuligerus class II RNR shares significant similarity with most other bacterial and archaeal class II RNRs. Two other genes, nrdA and nrdB, were initially identified in the Streptomyces coelicolor genome database in unannotated ORFs as encoding a class Ia RNR. Southern analysis demonstrated that the nrdAB genes were present in different Streptomyces spp. The S. coelicolor nrdAB genes were cloned and expressed in Escherichia coli, and the recombinant proteins were shown to represent a class I RNR. It was shown, using quantitative real-time PCR, that the S. clavuligerus class Ia and class II RNR genes were differentially transcribed during vegetative growth. The copy number of the class II nrdJ transcripts was approximately constant throughout the exponential phase of vegetative growth (3-5x10(5) copies per 400 ng total RNA after reverse transcription). In contrast, the copy number of the class Ia nrdAB transcripts was some 10- to 20-fold less than that of nrdJ in the early-exponential growth phase (2.8x10(4) copies), and decreased markedly at the mid-exponential (4x10(3) copies) and late-exponential phases (1.1x10(3) copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.