Patterns of fungal communities among and within decaying logs, revealed by 454 sequencing.

Owing to previous methodological limitations, knowledge about the fine-scale distribution of fungal mycelia in decaying logs is limited. We investigated fungal communities in decaying Norway spruce logs at various spatial scales at two environmentally different locations in Sweden. On the basis of 454 pyrosequencing of the ITS2 region of rDNA, 1914 operational taxonomic units (OTUs) were detected in 353 samples. The communities differed significantly among logs, but the physical distance between logs was not found to have a significant effect on whether fungal communities had any resemblance to each other. Within a log, samples that were closer together generally had communities that showed more resemblance to each other than those that were further apart. OTUs characteristic for particular positions on the logs could be identified. In general, these OTUs did not overlap with the most abundant OTUs, and their ecological role was often unknown. Only a few OTUs were detected in the majority of logs, whereas numerous OTUs were rare and present in only one or a few logs. Wood-decaying Basidiomycetes were often represented by higher sequence reads in individual logs than Ascomycete OTUs, suggesting that Basidiomycete mycelia spread out more rapidly when established. OTU richness tended to increase with the decay stage of the sample; however, the known wood decayers were most abundant in less-decomposed samples. The fungi identified in the logs represented different ecological strategies. Our findings differ from previously published sporocarp studies, indicating that the highly abundant fruiting species may respond to environment in different ways than the rest of the fungal community.

Impact of T Cell Repertoire Diversity on Mortality Following Cord Blood Transplantation.

INTRODUCTION: Cord blood transplantation (CBT) recipients are at increased risk of mortality due to delayed immune recovery (IR). Prior studies in CBT patients have shown that recovery of absolute lymphocyte count is predictive of survival after transplant. However, there are no data on the association of T-cell receptor (TCR) and clinical outcomes after CBT. Here we retrospectively performed TCR beta chain sequencing on peripheral blood (PB) samples of 34 CBT patients. METHODS: All patients received a total body irradiation based conditioning regimen and cyclosporine and MMF were used for graft versus host disease (GvHD) prophylaxis. PB was collected pretransplant on days 28, 56, 80, 180, and 1-year posttransplant for retrospective analysis of IR utilizing high-throughput sequencing of TCRß rearrangements from genomic DNA extracted from PB mononuclear cells. To test the association between TCR repertoire diversity and patient outcomes, we conducted a permutation test on median TCR repertoire diversity for patients who died within the first year posttransplant versus those who survived. RESULTS: Median age was 27 (range 1-58 years) and most of the patients (n = 27) had acute leukemias. There were 15 deaths occurring between 34 to 335 days after transplant. Seven deaths were due to relapse. Rapid turnover of T cell clones was observed at each time point, with TCR repertoires stabilizing by 1-year posttransplant. TCR diversity values at day 100 for patients who died between 100 and 365 days posttransplant were significantly lower than those of the surviving patients (p = 0.01). CONCLUSIONS: Using a fast high-throughput TCR sequencing assay we have demonstrated that high TCR diversity is associated with better patient outcomes following CBT. Importantly, this assay is easily performed on posttransplant PB samples, even as early as day 28 posttransplant, making it an excellent candidate for early identification of patients at high risk of death.

A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA.

Direct evidence is presented for a conformational switch in 16S ribosomal RNA (rRNA) that affects tRNA binding to the ribosome and decoding of messenger RNA (mRNA). These data support the hypothesis that dynamic changes in rRNA structure occur during translation. The switch involves two alternating base-paired arrangements apparently facilitated by ribosomal proteins S5 and S12, and produces significant changes in the rRNA structure. Chemical probing shows reciprocal enhancements or protections at sites in 16S rRNA that are at or very near sites that were previously crosslinked to mRNA. These data indicate that the switch affects codon-anticodon arrangement and proper selection of tRNA at the ribosomal A site, and that the switch is a fundamental mechanism in all ribosomes.

The alpha subunit of initiation factor 2 is phosphorylated in vivo in the yeast Saccharomyces cerevisiae.

The phosphorylation state of the alpha subunit of initiation factor 2 (eIF-2 alpha) in Saccharomyces cerevisiae has been determined by two-dimensional gel electrophoresis and autoradiography of lysates from cultures grown under a variety of conditions. The alpha subunit was maintained in a phosphorylated state during logarithmic growth on fermentable and nonfermentable carbon sources, during starvation for an essential amino acid, during heat shock, during stationary phase, and during sporulation. Only when cells were starved for a carbon source for 2 h in 1 M sorbitol was eIF-2 alpha isolated in the nonphosphorylated state. This is in contrast with the studies in rabbit reticulocyte lysates, in which arrested protein synthesis was correlated with a relative increase in the extent of phosphorylation of eIF-2 alpha.

Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide.

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.

Cord blood transplantation: rewind to fast forward.

The utilization of cord blood as a source of stem cells for transplantation has decreased in recent years. Although cord blood transplantation (CBT) is an established practice for the treatment of adult and pediatric patients with hematological malignancies, the high acquisition cost of CB units along with high transplant-related mortality due to delayed hematopoietic recovery and immune reconstitution have contributed to the slowing in widespread adoption of CBT. Strategies aimed to enhance speed of engraftment and ongoing clinical trials are investigating ways to make CBT more widely available. Meanwhile, the recent clinical data suggest that the choice of CBT might be preferable for patients with pre-transplant minimal residual disease. We review here the background data on the utilization of CB for the treatment of hematological malignancies, and discuss the current challenges and future directions in the field of CBT.

Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA.

The epsilon enhancer element is a pyrimidine-rich sequence that increases expression of T7 gene 10 and a number of Escherichia coli mRNAs during initiation of translation and inhibits expression of the recF mRNA during elongation. Based on its complementarity to the 460 region of 16S rRNA, it has been proposed that epsilon exerts its enhancer activity by base pairing to this complementary rRNA sequence. We have tested this model of enhancer action by constructing mutations in the 460 region of 16S rRNA and examining expression of epsilon-containing CAT reporter genes and recF-lacZ fusions in strains expressing the mutant rRNAs. Replacement of the 460 E.coli stem-loop with that of Salmonella enterica serovar Typhimurium or a stem-loop containing a reversal of all 8 bp in the helical region produced fully functional rRNAs with no apparent effect on cell growth or expression of any epsilon-containing mRNA. Our experiments confirm the reported effects of the epsilon elements on gene expression but show that these effects are independent of the sequence of the 460 region of 16S rRNA, indicating that epsilon-rRNA base pairing does not occur.

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable.

U2449 is one of many invariant residues in the central loop of domain V of 23S rRNA, a region that constitutes part of the peptidyltransferase center of the ribosome. In Escherichia coli, this U is post-transcriptionally modified to dihydrouridine (D) and is the only D modification found in E.coli rRNAs. To analyze the role of this base and its modification in ribosomal function, all three base substitutions were constructed on a plasmid copy of the rrnB operon and assayed for their ability to support cell growth in a strain of E.coli lacking chromosomal rrn operons. Both purine substitution mutations were not viable. However, growth and antibiotic sensitivity of cells expressing only the mutant D2449C rRNA was indistinguishable from wild type. We conclude that while a pyrimidine is required at position 2449 for proper ribosomal function, the D modification is dispensable.

Effects of mutagenesis of C912 in the streptomycin binding region of Escherichia coli 16S ribosomal RNA.

Four different mutations were produced at position 912 of Escherichia coli 16S rRNA in the multicopy plasmid pKK3535. Cells transformed with the mutant plasmids were assayed for growth in steptomycin. The U912 mutant conferred low level streptomycin resistance as reported originally by Montandon and co-workers (EMBO J 1986; 5:3705-3708). The G912 mutant also gave low level resistance but, unlike U912, caused significant retardation in growth rate and tended to select for fast-growing revertants. The A912 mutant was without effect on growth rate or streptomycin sensitivity, while deletion of C912 was lethal. Cells with U912 were selected for increased streptomycin resistance (MIC up to 160 micrograms/ml) and then cured of the plasmid. The cured cells retained a higher level of streptomycin resistance (MIC: 80 micrograms/ml) than the original wild type strain (MIC: 10 micrograms/ml), but sequencing by reverse transcriptase showed no evidence of U912 in the cellular 16S rRNA. Thus, recombination of the plasmid-coded U912 mutation into host rrn operons was not the mechanism by which increased streptomycin resistance occurred. The plasmid with U912 was transformed into three different streptomycin-dependent strains to determine whether the rRNA mutation, which presumably alters streptomycin binding, was compatible with S12 mutations which require bound streptomycin in order to function properly. In one strain, no transformants could be isolated, indicating that the plasmid was lethal. The two other streptomycin-dependent strains were transformed, but ribosomes containing the mutant rRNA were non-functional.

Mutations in helix 34 of Escherichia coli 16 S ribosomal RNA have multiple effects on ribosome function and synthesis.

Helix 34 of E. coli 16 S rRNA (1046 to 1067 and 1189 to 1211) has been proposed to participate directly in the termination of translation at UGA stop codons. We have constructed mutations in this helix in plasmid-encoded rDNA to explore the specific functional roles of the sequence UCAUCA (1199 to 1204) and a secondary structure also involving positions 1054 and 1057-1058. The rRNA mutations were analyzed for their effects on in vivo translational accuracy (stop codon readthrough and frameshifting) as well as growth rate, ribosome synthesis and incorporation into polysomes. Mutations at positions 1054, 1057, 1058, 1199 and 1200 had significant effects on translational accuracy, causing non-specific readthrough of all three stop codons as well as enhanced +1 and -1 frameshifting. Mutations at 1202 and 1203, however, had no effect. The incorporation of deleterious mutant subunits into 70 S ribosomes and polysomes was severely reduced and was associated with a slower growth rate and increased synthesis of host-encoded ribosomes. These data support the proposal that helix 34 is an essential component of the decoding center of the 30 S ribosomal subunit and is not restricted in function to UGA-codon specific termination.

Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23 S ribosomal RNA.

Evidence is presented for the participation of the P loop (nucleotides G2250-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidyl transferase center. Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interaction with the CCA end of P site-bound tRNA. These mutations altered the pattern of reactivity of RNA to chemical probes in a structural subdomain encompassing the P loop and extending roughly from G2238 to A2433. Most of the effects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indicating that these mutations perturb the tertiary structure of this region of RNA. Changes in chemical modification were also seen in a subdomain composed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U2552-C2556), the latter a site of interaction with the CCA end of A site-bound tRNA. Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity. These results indicate that, in addition to its direct role in peptide bond formation, the P loop contributes to the tertiary structure of the peptidyl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites.

The involvement of two distinct regions of 23 S ribosomal RNA in tRNA selection.

The role of ribosomal RNA in maintaining the accuracy of translation has been investigated genetically by selecting for rRNA mutations that promoted frameshifting at a specific site in a reporter gene in Escherichia coli. Mutations were recovered in two different regions of 23 S rRNA and each promoted readthrough of stop codons as well as increasing the levels of frameshifting. The first group of mutations was in a small stem loop (the 1916 loop) in domain IV of 23 S rRNA. This stem-loop has been mapped to the subunit interface of the ribosome, close to the decoding center on the 30 S subunit. The second group of mutations was in helix 89, one of the helices emerging from the central loop of domain V. Helix 89 has been implicated in subunit-subunit interactions and peptidyltransferase activity, and it is proposed that mutations in helix 89 influence the accuracy of decoding by affecting the interaction of the CCA end of the tRNA with the peptidyltransferase center.

Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction.

Interactions within the decoding center of the 30 S ribosomal subunit have been investigated by constructing all 15 possible mutations at nucleotides C1402 and A1500 in helix 44 of 16 S rRNA. As expected, most of the mutations resulted in highly deleterious phenotypes, consistent with the high degree of conservation of this region and its functional importance. A total of seven mutants were viable under conditions where the mutant ribosomes comprised 100 % of the ribosomal pool. A suppressor mutation specific for the C1402U-A1500G mutant was isolated at position 1520 in helix 45 of 16 S rRNA. In addition, lack of dimethylation of A1518/A1519 caused by mutation of the ksgA methylase enhanced the deleterious effect of many of the 1402/1500 mutations. These data suggest that a higher-order interaction between helices 44 and 45 in 16 S rRNA is important for the proper functioning of the ribosome. This is consistent with the recent high-resolution crystal structures of the 30 S subunit, which show a tertiary interaction between the 1402/1500 region of helix 44 and the dimethyl A stem loop.

Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA.

We have used chemical modification to examine the conformation of 23 S rRNA in Escherichia coli ribosomes bearing erythromycin resistance mutations in ribosomal proteins L22 and L4. Changes in reactivity to chemical probes were observed at several nucleotide positions scattered throughout 23 S rRNA. The L4 mutation affects the reactivity of G799 and U1255 in domain II and that of A2572 in domain V. The L22 mutation influences modification in domain II at positions m5U747, G748, and A1268, as well as at A1614 in domain III and G2351 in domain V. The reactivity of A789 is weakly enhanced by both the L22 and L4 mutations. None of these nucleotide positions has previously been associated with macrolide antibiotic resistance. Interestingly, neither of the ribosomal protein mutations produces any detectable effects at or within the vicinity of A2058 in domain V, the site most frequently shown to confer macrolide resistance when altered by methylation or mutation. Thus, while L22 and L4 bind primarily to domain I of 23 S rRNA, erythromycin resistance mutations in these ribosomal proteins perturb the conformation of residues in domains II, III and V and affect the action of antibiotics known to interact with nucleotide residues in the peptidyl transferase center of domain V. These results support the hypothesis that ribosomal proteins interact with rRNA at multiple sites to establish its functionally active three-dimensional structure, and suggest that these antibiotic resistance mutations act by perturbing the conformation of rRNA.

Site-directed mutagenesis of Escherichia coli 23 S ribosomal RNA at position 1067 within the GTP hydrolysis centre.

Site-directed mutagenesis has been used to change, specifically, residue 1067 within 23 S ribosomal RNA of Escherichia coli. This nucleoside (adenosine in the wild-type sequence) lies within the GTPase centre of the larger ribosomal subunit and is normally the target for the methylase enzyme responsible for resistance to the antibiotic thiostrepton. The performance of the altered ribosomes was not impaired in cell-free protein synthesis nor in GTP hydrolysis assays (although the 3 mutant strains grew somewhat more slowly than wild-type) but their responses to thiostrepton did vary. Thus, ribosomes containing the A to C or A to U substitution at residue 1067 of 23 S rRNA were highly resistant to the drug, whereas the A to G substitution resulted in much lesser impairment of thiostrepton binding and the ribosomes remained substantially sensitive to the antibiotic. These data reinforce the hypothesis that thiostrepton binds to 23 S rRNA at a site that includes residue A1067. They also exclude any possibility that the insensitivity of eukaryotic ribosomes to the drug might be due solely to the substitution of G at the equivalent position within eukaryotic rRNA.

Effects of mutagenesis of a conserved base-paired site near the decoding region of Escherichia coli 16 S ribosomal RNA.

Eleven of 15 possible single and double mutations were constructed in a cloned copy of Escherichia coli 16 S rDNA at a base-paired site, 1409-1491. Expression of any of these mutations was detrimental to the growth of E. coli. Mutations that substituted unpaired purine bases were lethal in the system described. Otherwise, the degree of detrimental effect on growth-rate was not directly correlated with specific rRNA primary or secondary structures. Using reverse transcription of rRNA isolated from subunits or 70 S ribosomes, we were able to determine the amount of mutant rRNA used in translation. From these experiments, we found that the lethal mutations appeared to be selectively excluded from the pool of 70 S ribosomes following expression from a repressible plasmid. In contrast, a non-lethal mutation was present in subunits, ribosomes and polysomes in approximately equal amounts. Mutations that disrupted base-pairing were found to confer varying levels of resistance to nine aminoglycosides, including four neomycins, two kanamycins, gentamicin, apramycin and hygromycin. A high frequency of reversion from resistant and slow-growth phenotypes due to a host mutation was observed.

Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of Escherichia coli.

Three ribosomal RNA mutations conferring resistance to the antibiotic kasugamycin were isolated using a strain of Escherichia coli in which all of the rRNA is transcribed from a plasmid-encoded rrn operon. The mutations, A794G, G926A, and A1519C, mapped to universally conserved sites in the 16 S RNA gene. Site-directed mutagenesis studies showed that virtually all mutations at these three sites conferred kasugamycin resistance and had very slight effects on cell growth. It has been known for many years that the absence of post-transcriptional modification at A1519 and the adjacent A1518 in strains lacking a functional KsgA methylase produces a kasugamycin resistance phenotype. Mutations at A1519 conferred kasugamycin resistance and had minor effects on cell growth, whereas mutations at 1518 did not confer resistance and increased the doubling time of the cells dramatically. Expression of mutations at A1518/A1519 in a methylase deficient ksgA(-)strain had divergent effects on the phenotype of the rRNA mutants, suggesting that the base identity at either position does not affect methylation at the adjacent site. Residues A794 and G926 are protected from chemical modification by kasugamycin and tRNA, and have been implicated in the initiation of protein synthesis. Despite the universal conservation and functional importance of these residues, the results presented here show that the identity of the bases is not critical for ribosomal function.

Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus.

We have isolated spontaneous streptomycin-resistant, streptomycin-dependent and streptomycin-pseudo-dependent mutants of the thermophilic bacterium Thermus thermophilus IB-21. All mutant phenotypes were found to result from single amino acid substitutions located in the rpsL gene encoding ribosomal protein S12. Spontaneous suppressors of streptomycin dependence were also readily isolated. Thermus rpsL mutations were found to be very similar to rpsL mutations identified in mesophilic organisms. This similarity affords greater confidence in the utility of the crystal structures of Thermus ribosomes to interpret biochemical and genetic data obtained with Escherichia coli ribosomes. In the X-ray crystal structure of the T. thermophilus HB8 30 S subunit, the mutated residues are located in close proximity to one another and to helices 18, 27 and 44 of 16 S rRNA. X-ray crystallographic analysis of ribosomes from streptomycin-resistant, streptomycin-pseudo-dependent and streptomycin-dependent mutants described here is expected to reveal fundamental insights into the mechanism of tRNA selection, translocation, and conformational dynamics of the ribosome.

Mutations at position A960 of E. coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA.

The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA. Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Escherichia coli deficient in all seven chromosomal copies of the rRNA operon. Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself. These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation.

A mutation in an Escherichia coli ribosomal RNA operon that blocks the production of precursor 23 S ribosomal RNA by RNase III in vivo and in vitro.

We have isolated on a multicopy plasmid a mutant rrnB ribosomal RNA operon containing a 130 base-pair deletion immediately preceding the 23 S rRNA gene. The deletion shortens by just three base-pairs the 26 base-pair complementarity of the sequences that flank the 23 S rRNA gene, and which normally form an RNase III cleavage site in the rrnB primary transcript. Both in vivo and in vitro, cleavage at the altered RNase III site was almost completely abolished by the mutation. Our results therefore indicate that even a small perturbation of the double-stranded region normally recognized by RNase III strongly inhibits the action of the enzyme.