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A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor-binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent secondary infection by the same or a closely related phage and nonproductive phage adsorption to lysed cell fragments, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA–Llp structure reveals that Llp is periplasmic and binds to a nonnative conformation of the plug of FhuA, causing the inward folding of two extracellular loops via “reverse” allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of Escherichia coli by phage T5 and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.

Designing bacterial superpowers Biological systems read all 64 triplet codons in DNA to encode the synthesis of proteins composed of 20 canonical amino acids. Robertson et al. created cells that do not read several codons and showed that this confers complete resistance to viruses, which normally rely on the host cell's ability to read all the codons in the viral genome to reproduce (see the Perspective by Jewel and Chatterjee). The authors reassigned each codon to several noncanonical amino acids (ncAAs). This advance enables the efficient synthesis of proteins containing three distinct ncAAs and the encoded synthesis of entirely noncanonical polymers and macrocycles. Science , abg3029, this issue p. 1057 ; see also abi9892, p. 1040

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca2+-dependent l-alanyl-d-glutamate peptidase, although the mode of Ca2+ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111–130) in Ca2+ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca2+-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca2+ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active “open” conformation. The possible mechanism of Ca2+ regulation of EndoT5 activity during bacteriophage T5’s life cycle through the Ca2+ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.

When studying viruses, the most prevalent aspects that come to mind are their structural and functional features, but this leaves in the shadows a quite universal characteristic: their mass. Even if approximations can be derived from size and density measurements, the multi MDa to GDa mass range, featuring a majority of viruses, has so far remained largely unexplored. Recently, nano-electromechanical resonator-based mass spectrometry (NEMS-MS) has demonstrated the ability to measure the mass of intact DNA filled viral capsids in excess of 100 MDa. However, multiple factors have to be taken in consideration when performing NEMS-MS measurements. In this article, phenomena influencing NEMS-MS mass estimates are listed and discussed, including some particle's extraneous physical properties (size, aspect ratio, stiffness), and the influence of frequency noise and device fabrication defects. These factors being accounted for, we could begin to notice subtler effects linked with (e.g.) particle desolvation as a function of operating parameters. Graphical abstract.

Numerous physical phase plates (PP) for phase-contrast enhancement in transmission electron microscopy (TEM) have been proposed and studied with the hole-free or Volta PP having a high impact and interest in recent years. This study is concerned with comparative TEM image simulations considering realistic descriptions of various PP approaches and samples from three different fields of application covering a large range of object sizes. The simulated images provide an illustrative characterization of the typical image appearance and common artifacts of the different PPs and the influence of simulation parameters especially important for PP simulations. A quantitative contrast analysis shows the superior phase-shifting properties of the hole-free phase plate for biological applications and the benefits of adjustable phase plates. The application of PPs in high-resolution TEM imaging, especially of weak-phase objects such as (atomically thin) 2D materials, is shown to increase image interpretability. The software with graphical user interface written and used for the presented simulations is available for free usage., S.H. and R.A. acknowledge funding by German Research Foundation (DFG project He 7675/1-1) and by the Spanish MICINN (PID2019-104739GB-100/AEI/10.13039/501100011033) and from the European Union H2020 program “ESTEEM3” (Grant number 823717) and European Union H2020 “Graphene Flagship” CORE 3 (Grant number 881603).

CRISPR–Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR–Cas systems against different viruses should vary. Here, we created a collection of Escherichia coli strains with type I-E CRISPR–Cas system targeting various positions in the genomes of bacteriophages λ, T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR–Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR–Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release.

The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. However, the T4 transcriptome is not significantly affected by a motB knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of motB impacts host gene expression. We find that motB is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold, either of which could provide MotB&rsquo, s ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne motB up-regulates 75 host genes, no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for Tevenvirinae and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.

Bridging the mass gap Viruses and many large biomolecule complexes are in a mass range that is challenging to measure with conventional mass spectrometry methods. Nanomechanical resonators can determine masses of impacting molecules, but separation methods often lose too much of the sample to be efficient. Dominguez-Medina et al. used an aerodynamic lens that improved separation and focusing of nebulized molecules with increasing mass. The mass of both filled and empty viral capsids was determined with an array of 20 nanoresonators. Science , this issue p. 918

Bacteriophage T7 capsid protein 10B has previously been proposed to arise by a translational frameshift near the 3' end of the capsid gene 10A coding sequence, adding an additional 53 amino acid residues to the carboxyl-terminal end of the protein. Here we show by peptide mapping experiments as well as by direct partial sequence analysis of an overlapping "junction" peptide, that 10B is in fact related to 10A by a -1 switch in reading frame in a narrow region near the carboxy terminus of 10A. Peptide mapping experiments demonstrate that 10A and 10B have the same amino terminus as well as virtually identical methionine-labeled peptide maps. However, the predicted unique carboxyl-terminal peptide from 10B was also identified. An overlapping peptide was isolated from 10B which spans the junction region in which the proposed translational frameshift is thought to occur. Partial sequencing of this junction peptide confirms a -1 frameshift within the last few codons of 10A.

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction–modification (R–M) and CRISPR–Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR–Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR–Cas systems by lowering the affinity of the Cascade and Cas9–crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR–Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.

A continuous-flow UV reactor operating at 254 nm wave-length was used to investigate inactivation of microorganisms including bacteriophage in coconut water, a highly opaque liquid food. UV-C inactivation kinetics of two surrogate viruses (MS2, T1UV) and three bacteria (E. coli ATCC 25922, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes ATCC 19115) in buffer and coconut water were investigated (D10 values ranging from 2.82 to 4.54 mJ·cm− 2). A series of known UV-C doses were delivered to the samples. Inactivation levels of all organisms were linearly proportional to UV-C dose (r2 > 0.97). At the highest dose of 30 mJ·cm− 2, the three pathogenic organisms were inactivated by > 5 log10 (p 0.97). Models for predicting log reduction as a function of UV-C irradiation dose were found to be significant (p Industrial relevance This research paper provides scientific evidence of the potential benefits of UV-C irradiation in inactivating bacterial and viral surrogates at commercially relevant doses of 0–120 mJ·cm− 2. The irradiated coconut water showed no cytotoxic effects on normal intestinal and healthy mice liver cells. UV-C irradiation is an attractive food preservation technology and offers opportunities for horticultural and food processing industries to meet the growing demand from consumers for healthier and safe food products. This study would provide technical support for commercialization of UV-C treatment of beverages.

A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2-35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.

We re-examined data from the classic Luria-Delbruck fluctuation experiment, which is often credited with establishing a Darwinian basis for evolution. We argue that, for the Lamarckian model of evolution to be ruled out by the experiment, the experiment must favor pure Darwinian evolution over both the Lamarckian model and a model that allows both Darwinian and Lamarckian mechanisms. Analysis of the combined model was not performed in the original 1943 paper. The Luria-Delbruck paper also did not consider the possibility of neither model fitting the experiment. Using Bayesian model selection, we find that the Luria-Delbruck experiment, indeed, favors the Darwinian evolution over purely Lamarckian. However, our analysis does not rule out the combined model, and hence cannot rule out Lamarckian contributions to the evolutionary dynamics., Comment: 13 pages, 5 figures. Presented at the 10th q-bio Conference, July 27-30, 2016, Nashville, TN

Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms.

Bacteriophage T5 has a 120 kb double-stranded linear DNA genome encoding most of the genes required for its own replication. This lytic bacteriophage has a burst size of ∼500 new phage particles per infected cell, demonstrating that it is able to turn each infected bacterium into a highly efficient DNA manufacturing machine. To begin to understand DNA replication in this prodigious bacteriophage, we have characterized a putative helicase encoded by gene D2. We show that bacteriophage T5 D2 protein is the first viral helicase to be described with bipolar DNA unwinding activities that require the same core catalytic residues for unwinding in either direction. However, unwinding of partially single- and double-stranded DNA test substrates in the 3′–5′ direction is more robust and can be distinguished from the 5′–3′ activity by a number of features including helicase complex stability, salt sensitivity and the length of single-stranded DNA overhang required for initiation of helicase action. The presence of D2 in an early gene cluster, the identification of a putative helix-turn-helix DNA-binding motif outside the helicase core and homology with known eukaryotic and prokaryotic replication initiators suggest an involvement for this unusual helicase in DNA replication initiation.

The genomic diversity of 99 T4-like coliphages was investigated by sequencing an equimolar mixture with Illumina technology and screening them against different databases for horizontal gene transfer and undesired genes. A 9-phage cocktail was given to 15 healthy adults from Bangladesh at a dose of 3×109 and 3×107 plaque-forming units and placebo respectively. Phages were detected in 64% of the stool samples when subjects were treated with higher titer phage, compared to 30% and 28% with lower-titer phage and placebo, respectively. No Escherichia coli was present in initial stool samples, and no amplification of phage was observed. One percent of the administered oral phage was recovered from the feces. No adverse events were observed by self-report, clinical examination, or from laboratory tests for liver, kidney, and hematology function. No impact of oral phage was seen on the fecal microbiota composition with respect to bacterial 16S rRNA from stool.

Conformation wheels, directly relating the amino acid sequence to the local torsion angles in a protein molecule, are presented for cytochromes c, c2, c550, and c551 and for lysozymes from hen egg-white and T4 bacteriophage. The circular plots for the cytochrome molecules aid in visualizing the common three-dimensional folding (“cytochrome fold”) observed in this family of proteins. Conformation wheels for lysozymes from two different species reveal the characteristic differences in their folding patterns. These novel plots are also useful in storing and comparing the several sets of crystallographic data reported for lysozyme.

Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models. Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology. Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies. Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications., British Journal of Cancer, 101 (4), ISSN:0007-0920, ISSN:1532-1827

The induction of rapid lysis mutants in bacteriophage T4D was used for measuring the mutagenic effect of the hair dye, 1,4 diaminoanthraquinone (DAQ), and of two solvents, dimethylsulfoxide and ethanol. Three concentrations of DAQ employed, 12.8, 30.5 and 61 μg/ml, were found to significantly increase the mutation frequency compared to the control. Other doses of DAQ, both lower and higher, had no effect on the mutation frequency. The solvent, dimethylsulfoxide, was found to significantly increase the mutation frequency at high concentrations (111.1 and 130.4 μl/ml). Ethanol had no mutagenic effect at doses ranging from 47.6–130.4 μl/ml.

Mosaic genome design, considered evidence of horizontal gene transfer, is prominent in T-even phage tail fiber genes involved in host recognition. The possibility of direct gene transfer was assessed through superinfection with two virulent phages T2 and PP01, which caused host recognition shift. Two recombinant phages designated as TPr03 and TPr04 were isolated. PCR-restriction fragment length polymorphism analysis and sequence analysis suggested that 18% of the TPr03 and 38% of the TPr04 genome derived from PP01. Both isolates showed host ranges identical to PP01. The results suggested the possibility of generating various recombinant phages by intentional dual infections and of the occasional occurrence in nature of generation of phage showing new characteristics through superinfection, followed by the genomic recombination.

Bacterial virus T3 undergoes host-controlled modification which is not based on "classical" processes of DNA modification and restriction. The adsorption and thus the growth of T3 on Escherichia coli W cells (E. coli K12 derivative) decisively depends on the host strain on which the virus was previously propagated. Depending on the modification conferred to the virus by its last host, its efficiency of plating (e.o.p.) on E. coli W varies by six orders of magnitude between 10(-7) and 10(-1). This does not reflect the appearance of T3 host-range mutants, but a fully reversible modification of genotypically unchanged T3 wild-type phage. The behaviour of T3 in the described host system constitutes a second case of so-called non-classical modification and restriction (KRUGER et al. 1977, Molec. gen. Genet. 153, 107-110) of bacteriophages. Non-classical modification (protein modification) is additive to and independent of DNA modification and restriction as demonstrated with the ocr- phage T3/R7. - Furthermore, our results suggest that the adsorption specificity of T3 is determined by at least two independent genetic factors; in both of these factors T3 differs from T7.

Anew Escherichia coli phage, named Rtp, was isolated and shown to be closely related to phage T1. Electron microscopy revealed that phage Rtp has a morphologically unique tail tip consisting of four leaf-like structures arranged in a rosette, whereas phage T1 has thinner, flexible leaves that thicken toward the ends. In contrast to T1, Rtp did not require FhuA and TonB for infection. The 46.2-kb genome of phage Rtp encodes 75 open reading frames, 47 of which are homologous to phage T1 genes. Like phage T1, phage Rtp encodes a large number of small genes at the genome termini that exhibit no sequence similarity to known genes. Six predicted genes larger than 300 nucleotides in the highly homologous region of Rtp are not found in T1. Two predicted HNH endonucleases are encoded at positions different from those in phage T1. The sequence similarity of rtp37 , - 38 , - 39 , - 41 , - 42 , and - 43 to equally arranged genes of lambdoid phages suggests a common tail assembly initiation complex. Protein Rtp43 is homologous to the λ J protein, which determines λ host specificity. Since the two proteins differ most in the C-proximal area, where the binding site to the LamB receptor resides in the J protein, we propose that Rtp43 contributes to Rtp host specificity. Lipoproteins similar to the predicted lipoprotein Rtp45 are found in a number of phages (encoded by cor genes) in which they prevent superinfection by inactivating the receptors. We propose that, similar to the proposed function of the phage T5 lipoprotein, Rtp45 prevents inactivation of Rtp by adsorption to its receptor during cells lysis. Rtp52 is a putative transcriptional regulator, for which 10 conserved inverted repeats were identified upstream of genes in the Rtp genome. In contrast, the much larger E. coli genome has only one such repeat sequence.

A protein-synthesizing system in vitro, programmed with bacteriophage T7 DNA as template, changed the specificity of gene expression in the course of incubation as a result of newly synthesized T7 early proteins. The system mimics largely the situation in vivo on both the transcriptional and the translational levels, i.e. early gene expression is turned off shortly after late synthesis has been started. These results suggest that the switch from early to late expression does not necessarily require changes in the cellular environment nor is it dependent on the presence of membranes. The main part in this process is played by the phage-dependent RNA polymerase (gene 1 product), whose activity appears 8-10 min after start of incubation. When its activity is reduced by inhibitors or creation of non-optimal conditions, the system is not able to manage the early--late switch.

The rate of formation of dinucleoside tetraphosphate, pppApU, from ATP and UTP by RNA polymerase on the A1 promoter of the mutant D111 of bacteriophage T7 is distinctly and specifically reduced not only by the third template-directed nucleotide, CTP, but also by CMP. The inhibitory effect of CMP is not changed when the enzyme contains prebound rifampicin. The synthesis of pppApU is also strongly reduced after preincubation of the enzyme with RNA. This inhibitory effect of RNA is, however, distinctly diminished by rifampicin bound to the enzyme prior to the addition of RNA. On the other hand RNA can suppress the specific binding of the antibiotic to the RNA polymerase subassembly alpha 2 beta.

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2–4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5–7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.

Functionalization of bacterial cell surfaces has the potential to introduce new activities by chemical modification. Here we show that a bacteriophage-receptor complex can be used to functionalize the surface of two Gram-negative proteobacteria, Escherichia coli and Ralstonia eutropha with CdSe/ZnS nanoparticles. This work highlights the potential for using microbe-phage interactions to generate new functions on living cells.

Bacteriophage T4 DNA topoisomerase gene 60 contains a 50 nucleotide untranslated region within the coding sequence of its mRNA. Translational bypass of this sequence by elongating ribosomes has been postulated for the mode of synthesis of an 18 kd polypeptide specified by the split coding segments. Ribosome bypass of the untranslated region also occurs when a segment of gene 60 is fused to lacZ and expressed in E. coli. The efficiency of bypass in these gene 60-lacZ fusions approaches 100%. Here, mutations that delete, insert, or substitute nucleotides from gene 60-lacZ fusions are examined. Essential features necessary for high level gap bypass emerging from this analysis are a cis-acting nascent peptide sequence, a short duplication bordering the gap, and a stop codon contained in a stem-loop structure at the 5' junction of the gap.

The nucleotide sequence corresponding to 13–19.5% of the bacteriophage T5 genome in early region C was determined (GenBank AY140897 ). One of the five major single-stranded interruptions (nicks) of bacteriophage T5 DNA was identified at 18.5%. The sequenced region was annotated and the putative functions of some open reading frames were proposed by comparison with databases. The dnk gene, encoding a deoxyribonucleoside monophosphate kinase, was identified using a previously defined N-terminal amino acid sequence. The gene was cloned and expressed in Escherichia coli , the enzyme was purified to homogeneity with high yield using two alternative methods, and the recombinant deoxyribonucleoside monophosphate kinase was found to have the same activity and specificity as the native enzyme.

Abortive initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative initiation parameters that describe abortive initiation and promoter escape; these parameters are the abortive rate, the productive rate, the abortive:productive ratio, the abortive probability, and the maximum size of abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher abortive yield, a higher abortive probability, a longer abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of abortive initiation.

Tag7/PGRP, a recently characterized antimicrobial protein, is conserved from insects to mammals. Recently its involvement in Toll signalling in Drosophila was demonstrated. A number of genes representing a new family homologous to PGRP were identified in Drosophila and human. Here we describe a splicing pattern of the tagL gene, mouse member of tag7/PGRP family. Some of the identified splice variants lacked characteristics for the family T phage lysozyme homology domain (also known as PGRP domain). Accordingly to the predicted transmembrane domains, mouse TagL may be secreted as inducible proteins or retained on intracellular membranes. All detected splice variant isoforms of TagL bound Gram-positive, Gram-negative bacteria and peptidoglycan. This binding did not depend on the presence of T phage lysozyme homology domain but was associated with the C-terminal portion of the polypeptides. Thus, this variety of isoforms of a single gene may play a role in circulating bacteria recognition in mammals.

We examined the bactericidal and virucidal effectiveness of a denture cleaner that uses ozone (ozone concentration, 10 ppm) against methicillin-resistant Staphylococcus aureus (MRSA) and T1 phage, respectively. In the bactericidal activity test, with the ozone supply turned on, the number of bacteria was 3.1 x 10(3) CFU/mL at the beginning of the experiment, fell to 1.0 x 10(0) CFU/mL 10 min later, and was 1.0 x 10(0) CFU/mL or less afterwards. In contrast, when the ozone supply was cut off (air bubble only), the number of bacteria was 3.4 x 10(3) CFU/mL at the beginning of the experiment, and had fallen to 3.0 x 10(3) CFU/mL 60 min later (no statistically significant difference). In the virucidal activity test, the number of phages was 1.2 x 10(6) PFU/mL before ozone treatment, fell to about 1/10 of that number 10 min later, and was 6.1 x 10(0) PFU/mL 40 min later. These results indicate that the use of ozone in this denture cleaner is effective against MRSA and viruses.

Bacteriophages (phages) modify microbial communities by lysing hosts, transferring genetic material, and effecting lysogenic conversion. To understand how natural communities are affected it is important to develop predictive models. Here we consider how variation between models—in eclipse period, latent period, adsorption constant, burst size, the handling of differences in host quantity and host quality, and in modeling strategy—can affect predictions. First we compare two published models of phage growth, which differ primarily in terms of how they model the kinetics of phage adsorption; one is a computer simulation and the other is an explicit calculation. At higher host quantities (∼108cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (107cells/ml), the computer simulation continues to closely predict phage growth rates, but the explicit model does not. Next we concentrate on predictions of latent-period optima. A latent-period optimum is the latent period that maximizes the population growth of a specific phage growing in the presence of a specific quantity and quality of host cells. Both models predict similar latent-period optima at higher host densities (e.g., 17 min at 108cells/ml). At lower host densities, however, the computer simulation predicts latent-period optima that are much shorter than those suggested by explicit calculations (e.g., 90 versus 1,250 min at 105cells/ml). Finally, we consider the impact of host quality on phage latent-period evolution. By taking care to differentiate latent-period phenotypic plasticity from latent-period evolution, we argue that the impact of host quality on phage latent-period evolution may be relatively small.

The fatal bacterial infection caused by inhalation of the Bacillus anthracis spores results from the synthesis of protein toxins-protective antigen (PA), lethal factor (LF), and edema factor (EF)--by the bacterium. PA is the target-cell binding protein and is common to the two effector molecules, LF and EF, which exert their toxic effects once they are translocated to the cytosol by PA. PA is the major component of vaccines against anthrax since it confers protective immunity. The large-scale production of recombinant protein-based anthrax vaccines requires overexpression of the PA protein. We have constitutively expressed the protective antigen protein in E. coli DH5alpha strain. We have found no increase in degradation of PA when the protein is constitutively expressed and no plasmid instability was observed inside the expressing cells. We have also scaled up the expression by bioprocess optimization using batch culture technique in a fermentor. The protein was purified using metal-chelate affinity chromatography. Approximately 125 mg of recombinant protective antigen (rPA) protein was obtained per liter of batch culture. It was found to be biologically and functionally fully active in comparison to PA protein from Bacillus anthracis. This is the first report of constitutive overexpression of protective antigen gene in E. coli.

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.

That two distinct kinds of substances—the d'Hérelle substances and the genes—should both possess this most remarkable property of heritable variation or “mutability,” each working by a totally different mechanism, is quite conceivable, considering the complexity of protoplasm, yet it would seem a curious coincidence indeed. It would open up the possibility of two totally different kinds of life, working by different mechanisms. On the other hand, if these d'Hérelle bodies were really genes, fundamentally like our chromosome genes, they would give us an utterly new angle from which to attack the gene problem. They are filterable, to some extent isolable, can be handled in test-tubes, and their properties, as shown by their effects on the bacteria, can then be studied after treatment. It would be very rash to call these bodies genes, and yet at present we must confess that there is no distinction known between the genes and them. Hence we cannot categorically deny that perhaps we may be able to grind genes in a mortar and cook them in a beaker after all. Must we geneticists become bacteriologists, physiological chemists, and physicists, simultaneously with being zoologists and botanists? Let us hope so. H. J. Muller (1922, pp. 48–49)

A versatile expression vector utilizing a promoter of coliphage T5, P(N25) (Gentz and Bujard, 1985. J. Bacteriol. 164, 70-77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78-89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (delta1-284, G665D) in Escherichia coli as well as in A. tumefaciens. The signal transduction proteins VirA (delta1-284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P(N25) driven expression overrides the complex transcriptional regulation present with the native promoters. This expression system will enable a more detailed analysis of the activation events in signal transduction in A. tumefaciens, and we expect it to be useful in other prokaryotes.

Genetic and biochemical studies have indicated that mismatch repair proteins can interact with recombination intermediates. In this study, gel shift assays and electron microscopic analysis were used to show that the Saccharomyces cerevisiae MSH2/6 complex binds to Holliday junctions and has an affinity and specificity for them that is at least as high as it has as for mispaired bases. Under equilibrium binding conditions, the MSH2/6 complex had a K d of binding to Holliday junctions of 0.5 nm. The MSH2/6 complex enhanced the cleavage of Holliday junctions by T4 endonuclease VII and T7 endonuclease I. This is consistent with the view that the MSH2/6 complex can function in both mismatch repair and the resolution of recombination intermediates as predicted by genetic studies.

Bacteriophage T5 5'-->3' exonuclease is a member of a family of sequence related 5'-nucleases which play an essential role in DNA replication. The 5'-nucleases have both exonucleolytic and structure-specific endo-nucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of a structure-specific single cleavage assay for this enzyme which uses a 5'-overhanging hairpin substrate. The products of DNA hydrolysis are characterised by mass spectrometry. The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'-->3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 10(15). The catalytic assay has been extended to three mutants of T5 5'-->3' exonuclease, K83A, K196A and K215A. Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency. All three lysines contribute to ground state binding of the substrate. In addition, K83 plays a significant role in the chemical reaction catalysed by this enzyme. Possible roles for mutated lysine residues are discussed.

Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea. In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time [Kleinschmidt, J. H., and Tamm, L. K. (1999) Biochemistry 38, 4996-5005]. Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilayers via three structurally distinct membrane-bound folding intermediates. To take full advantage of the TDFQ technique and to further dissect the folding pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein. All mutants refolded in vivo and in vitro and, as judged by SDS-PAGE, trypsin fragmentation, and Trp fluorescence, their refolded state was indistinguishable from the native state of OmpA. TDFQ analysis of the translocation across the lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30 degrees C, all Trps started from a far distance from the bilayer center and then gradually approached a distance of approximately 10 A from the bilayer center. In a narrow temperature range between 30 and 35 degrees C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center. When monitored at 40 degrees C, which resolved the last steps of OmpA refolding, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 A from the center after refolding was complete. In contrast Trp-7 approached the 10 A distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer. The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four beta-hairpins of the eight-stranded beta-barrel of OmpA were very similar to one another. This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate provide strong evidence for a synchronous translocation of all four beta-hairpins of OmpA across the lipid bilayer and suggest that OmpA inserts and folds into lipid bilayers by a concerted mechanism.