Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors

In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: α₂βγδ (γ-AChR) or α₂βδ (-AChR). After birth, γ-AChRs are replaced by -AChRs (γ/-switch). The two receptors have different conductances and open times. During perinatal period, the long open time γ-AChRs generate random myofiber action potentials from uniquantal miniature end-plate potentials (mEPPs). -AChRs are suitable for strong adult muscle activities. Since the effect of the γ/-switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate γ-subunit-deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included -subunits, and muscle fibers had multiple neuromuscular junctions. Both pre- and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for γ-AChRs in mediating synaptically-induced action potential activity critical for neuromuscular development.     

Casein kinase I δ/ɛ phosphorylates topoisomerase IIα at serine-1106 and modulates DNA cleavage activity

We previously reported that phosphorylation of topoisomerase (topo) IIα at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Iδ and/or CKI, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II–DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II–DNA cleavable complex formation. Since, IC261 specifically targets the Ca²⁺-regulated isozymes, CKIδ and CKI, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIα and α′ did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIδ/ homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIα, was enhanced following expression of human CKI. Down-regulation of CKIδ and CKI also led to reduced formation of etoposide stabilized topo II–DNA cleavable complex. These results provide strong support for an essential role of CKIδ/ in phosphorylating Ser-1106 in human topo IIα and in regulating enzyme function.     

Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts

Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) β, ζ or η. Base excision repair was also proficient without Polβ. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polα (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Polδ (pol3-1) or Pol (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Polδ and Pol, respectively. Deleting the polymerase catalytic domain of Pol (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Pol, but not by purified yeast Polη. These results suggest that efficient repair synthesis of yeast NER requires both Polδ and Pol in vitro, and that the low fidelity Polη is not accessible to repair synthesis during NER.     

MDM2 interacts with the C-terminus of the catalytic subunit of DNA polymerase epsilon

MDM2 is induced by p53 in response to cellular insults such as DNA damage and can have effects upon the cell cycle that are independent or downstream of p53. We used a yeast two-hybrid screen to identify proteins that bind to MDM2 and which therefore might be involved in these effects. We found that MDM2 can bind to the C-terminus of the catalytic subunit of DNA polymerase (DNA pol ), to a region that is known to be essential in yeast. In an in vitro system we confirmed that MDM2 could bind to the homologous regions of both mouse and human DNA pol and to full-length human DNA pol . DNA pol co-immunoprecipitated with MDM2 from transfected H1299 cells and also from a HeLa cell nuclear extract. We show here that the DNA pol -interacting domain of MDM2 is located between amino acids 50 and 166. Our studies provide evidence that MDM2 interacts with a region of DNA pol that plays a critical role in the function of DNA pol .     

Lysine-2,3-Aminomutase and β-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of Nɛ-Acetyl-β-Lysine and Growth at High Salinity

The compatible solute N-acetyl-β-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of N-acetyl-β-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and β-lysine acetyltransferase (ablB), which are assumed to catalyze N-acetyl-β-lysine formation from α-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei Gö1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei Gö1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Δabl mutants of M. maripaludis no longer produced N-acetyl-β-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for N-acetyl-β-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.

     

Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds

Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors () of −1.5 and −3.9‰, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic (_intrinsic) were calculated. A comparison of _intrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average if no fractionation factor is available for single compounds.     

Host-Symbiont Relationships in Hydrothermal Vent Gastropods of the Genus Alviniconcha from the Southwest Pacific

Hydrothermal vent gastropods of the genus Alviniconcha are unique among metazoans in their ability to derive their nutrition from chemoautotrophic γ- and -proteobacterial endosymbionts. Although host-symbiont relationships in Alviniconcha gastropods from the Central Indian Ridge in the Indian Ocean and the Mariana Trough in the Western Pacific have been studied extensively, host-symbiont relationships in Alviniconcha gastropods from the Southwest Pacific remain largely unknown. Phylogenetic analysis using mitochondrial cytochrome c oxidase subunit I gene sequences of host gastropods from the Manus, North Fiji, and Lau Back-Arc Basins in the Southwest Pacific has revealed a new host lineage in a Alviniconcha gastropod from the Lau Basin and the occurrence of the host lineage Alviniconcha sp. type 2 in the Manus Basin. Based on 16S rRNA gene sequences of bacterial endosymbionts, two γ-proteobacterial lineages and one -proteobacterial lineage were identified in the present study. The carbon isotopic compositions of the biomass and fatty acids of the gastropod tissues suggest that the γ- and -proteobacterial endosymbionts mediate the Calvin-Benson cycle and the reductive tricarboxylic acid cycle, respectively, for their chemoautotrophic growth. Coupling of the host and symbiont lineages from the three Southwest Pacific basins revealed that each of the Alviniconcha lineages harbors different bacterial endosymbionts belonging to either the γ- or -Proteobacteria. The host specificity exhibited in symbiont selection provides support for the recognition of each of the host lineages as a distinct species. The results from the present study also suggest the possibility that Alviniconcha sp. types 1 and 2 separately inhabit hydrothermal vent sites approximately 120 m apart in the North Fiji Basin and 500 m apart in the Manus Basin.     

Phylogenetic Diversity of Bacterial and Archaeal Communities in the Anoxic Zone of the Cariaco Basin

Microbial community samples were collected from the anoxic zone of the Cariaco Basin at depths of 320, 500, and 1,310 m on a November 1996 cruise and were used to construct 16S ribosomal DNA libraries. Of 60 nonchimeric sequences in the 320-m library, 56 belonged to the subdivision of the Proteobacteria (-Proteobacteria) and 53 were closely related to ectosymbionts of Rimicaris exoculata and Alvinella pompejana, which are referred to here as epsilon symbiont relatives (ESR). The 500-m library contained sequences affiliated with the fibrobacteria, the Flexibacter-Cytophaga-Bacteroides division, the division Verrucomicrobia, the division Proteobacteria, and the OP3 candidate division. The Proteobacteria included members of the γ, δ, and new candidate subdivisions, and γ-proteobacterial sequences were dominant (25.6%) among the proteobacterial sequences. As in the 320-m library, the majority of the -proteobacteria belonged to the ESR group. The genus Fibrobacter and its relatives were the second largest group in the library (23.6%), followed by the δ-proteobacteria and the -proteobacteria. The 1,310-m library had the greatest diversity; 59 nonchimeric clones in the library contained 30 unique sequences belonging to the planctomycetes, the fibrobacteria, the Flexibacter-Cytophaga-Bacteroides division, the Proteobacteria, and the OP3 and OP8 candidate divisions. The proteobacteria included members of new candidate subdivisions and the β, γ, δ, and -subdivisions. ESR sequences were still present in the 1,310-m library but in a much lower proportion (8.5%). One archaeal sequence was present in the 500-m library (2% of all microorganisms in the library), and eight archaeal sequences were present in the 1,310-m library (13.6%). All archaeal sequences fell into two groups; two clones in the 1,310-m library belonged to the kingdom Crenarchaeota and the remaining sequences in both libraries belonged to the kingdom Euryarchaeota. The latter group appears to be related to the Eel-TA1f2 sequence, which belongs to an archaeon suggested to be able to oxidize methane anaerobically. Based on phylogenetic inferences and measurements of dark CO₂ fixation, we hypothesized that (i) the ESR are autotrophic anaerobic sulfide oxidizers, (ii) sulfate reduction and fermentative metabolism may be carried out by a large number of bacteria in the 500- and 1,310-m libraries, and (iii) members of the Euryarchaeota found in relatively large numbers in the 1,310-m library may be involved in anaerobic methane oxidation. Overall, the composition of microbial communities from the Cariaco Basin resembles the compositions of communities from several anaerobic sediments, supporting the hypothesis that the Cariaco Basin water column is similar to anaerobic sediments.     

Examination of the Role of DNA Polymerase Proofreading in the Mutator Effect of Miscoding tRNAs

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, , is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutD alleles. In the present work, we extended the study of specific mutD alleles and constructed 16 altered mutD genes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes . We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the subunit playing a major role in the mutator effect of misreading tRNAs.     

Carbon Isotope Fractionation during Acetoclastic Methanogenesis by Methanosaeta concilii in Culture and a Lake Sediment

The isotope enrichment factors () in Methanosaeta concilii and in a lake sediment, where acetate was consumed only by Methanosaeta spp., were clearly less negative than the usually observed for Methanosarcina spp. The fraction of methane produced from acetate in the sediment, as determined by using stable isotope signatures, was 10 to 15% lower when the appropriate of Methanosaeta spp. was used.     

Structure and function of the fourth subunit (Dpb4p) of DNA polymerase epsilon in Saccharomyces cerevisiae

DNA polymerase (Pol) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS–PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Pol. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Δdpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Δdpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Δdpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Pol in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S.cerevisiae Pol is conserved in eukaryotes.     

Fc{epsilon}RI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane

The aggregation of high affinity IgE receptors (Fc receptor I [FcRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule–plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.     

Drosophila and vertebrate casein kinase Idelta exhibits evolutionary conservation of circadian function

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase I/δ (CKI/δ) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI short-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKI/δ. Moreover, expression of either Drosophila DBT or the vertebrate CKIδ kinase carrying the Drosophila dbt^S or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKIδ carrying the dbt^L mutation does not lengthen circadian rhythms, while Drosophila DBT^L does. Different effects of the dbt^S and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKIδ and of the functions affected by their period-shortening mutations.     

Common Elements Regulating Gene Expression in Temperate and Lytic Bacteriophages of Lactococcus Species

A phage-inducible middle promoter (P_15A10) from the lytic, lactococcal bacteriophage 31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (>95%) to the right cohesive ends of two temperate phages of the P335 species, r1t and LC3. Sequencing upstream and downstream of P_15A10 showed that the high degree of homology between 31 and r1t continued beyond the phage promoter. With the exception of one extra open reading frame in 31, the sequences were highly homologous (95 to 98%) between nucleotides 13448 and 16320 of the published r1t sequence. By use of a β-galactosidase (β-Gal) gene under the control of a smaller, more tightly regulated region within the P_15A10 promoter, P_566–888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage r1t induced the P_566–888 promoter, as determined from an increase in β-Gal activity. Hybridization of nine other lactococcal strains with ³²P-labeled P_566–888 showed that the Lactococcus lactis strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter. Mitomycin C induced the resident prophages in all these strains and concurrently induced the P_566–888 promoter, as determined from an increase in β-Gal activity. DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of r1t. In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P_566–888 from the lytic phage 31. These results point to a conserved mechanism in the regulation of gene expression between the lytic phage 31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.     

Activation of Protein Kinase C Triggers Its Ubiquitination and Degradation

Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms α, δ, and in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC δ. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC α, δ, and were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC α could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway.     

Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000

RSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the RSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5′-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the RSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A RSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these RSA1 genes as 5′ TGTTGT-(X)₁₃-ACAACA. The genomic sequence similarity between RSA1 and related phages 52237 and CTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. RSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3′ portion of the arginine tRNA(CCG) gene. In the light of the RSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a RSA1-related prophage (designated RSX). RSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. RSX ORFs shared very high amino acid identity with their RSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.     

BDCA2/FcɛRIγ Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells

Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses. Human plasmacytoid dendritic cells (pDCs) uniquely express blood dendritic cell antigen 2 (BDCA2) protein, a C-type lectin lacking an identifiable signaling motif. We demonstrate here that BDCA2 forms a complex with the transmembrane adapter FcRIγ. Through pathway analysis, we identified a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of the B cell receptor (BCR), which is distinct from the system involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of the BCR-like cascade, which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands. Therefore, by associating with FcRIγ, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.     

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous γ-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

Cell wall yield properties of growing tissue : evaluation by in vivo stress relaxation

Growing pea stem tissue, when isolated from an external supply of water, undegoes stress relaxation because of continued loosening of the cell wall. A theoretical analysis is presented to show that such stress relaxation should result in an exponential decrease in turgor pressure down to the yield threshold (Y), with a rate constant given by ε where is the metabolically maintained irreversible extensibility of the cell wall and ε is the volumetric elastic modulus of the cell. This theory represents a new method to determine in growing tissues.

Stress relaxation was measured in pea (Pisum sativus L.) stem segments using the pressure microprobe technique. From the rate of stress relaxation, of segments pretreated with water was calculated to be 0.08 per megapascal per hour while that of auxin-pretreated tissue was 0.24 per megapascal per hour. These values agreed closely with estimates of made by a steady-state technique. The yield threshold (0.29 megapascal) was not affected by auxin. Technical difficulties with measuring by stress relaxation may arise due to an internal water reserve or due to changes in subsequent to excision. These difficulties are discussed and evaluated.

A theoretical analysis is also presented to show that the tissue hydraulic conductance may be estimated from the T_½ of tissue swelling. Experimentally, pea stems had a swelling T_½ of 2.0 minutes, corresponding to a relative hydraulic conductance of about 2.0 per megapascal per hour. This value is at least 8 times larger than . From these data and from computer modeling, it appears that the radial gradient in water potential which sustains water uptake in growing pea segments is small (0.04 megapascal). This means that hydraulic conductance does not substantially restrict growth. The results also demonstrate that the stimulation of growth by auxin can be entirely accounted for by the change in .