Melphalan, a second chemical for which specific-locus mutation induction in the mouse is maximum in early spermatids.

Melphalan (MLP), a bifunctional alkylating agent structurally related to the highly mutagenic chemical chlorambucil (CHL), was found to induce high frequencies of specific-locus mutations in postspermatogonial germ cells of the mouse, and to be one of only a few chemicals that is also mutagenic in spermatogonial stem cells. Productivity patterns following MLP exposures resembled those that had been found for CHL. Mutation rates in successive male germ-cell stages were measured at three MLP-exposure levels in a total of 95,375 offspring. While the induced (experimental minus historical-control) mutation rate is relatively low in stem-cell spermatogonia (1.2 x 10(-5) per locus at a weighted-mean exposure of 7.3 mg/kg), it is about 5 times higher in poststem-cell stages overall, and peaks at 26.7 x 10(-5) per locus in early spermatids at a weighted-mean exposure of only 5.7 mg/kg. This "type-2 pattern" of mutation yield (Russell et al., 1990), i.e., peak sensitivity in early spermatids, has heretofore been found for only one other chemical, CHL. Mutation-rate data earlier reported for CHL (Russell et al., 1989) were augmented in the present study for comparison with MLP-induced rates. Because of the greater toxicity of MLP, average exposures used for this chemical were only about one-half of those for CHL. When MLP and CHL mutation rates are extrapolated to equimolar doses, they appear very similar for poststem-cell stages overall. However, in the case of CHL, a somewhat higher proportion of the mutations is induced in early spermatids than in the case of MLP.     

Effect of ssbA1 and lexC113 mutations on lambda prophage induction, bacteriophage growth, and cell survival.

Escherichia coli strains containing mutations (ssbA1 and lexC113) which affect single-strand deoxyribonucleic acid binding protein have been examined. Among the properties studied were: sensitivity to ultraviolet irradiation and methyl methane sulfonate, temperature sensitivity, induction of prophage lambda by ultraviolet light, temperature, and mitomycin C, and deoxyribonucleic acid synthesis. Strains containing the ssbA1 and lexC113 mutations differ significantly in several of these properties.     

DNA methylation in lysogens of pathogenic Burkholderia spp. requires prophage induction and is restricted to excised phage DNA

Burkholderia mallei-specific phage PhiE125 encodes DNA methyltransferases in both the lysogenic and replication modules within its genome. Characterization of DNA methylation in recombinant systems, specifically in PhiE125 lysogenic strains of B. mallei and Burkholderia thailandensis, revealed that, upon induction, cytosine methylation was targeted specifically to the phage episome but not the phage provirus or the host chromosome.     

Merosin promotes cell attachment and neurite outgrowth and is a component of the neurite-promoting factor of RN22 schwannoma cells.

The laminin-like protein merosin was purified from human placenta in intact form and as pepsin fragments and compared to laminin in heparin affinity chromatography and cell binding assays. Intact merosin and a small fragment of merosin comprising the last two repeats of the heavy chain g domain bind to heparin. Intact merosin and large pepsin fragments of merosin, but not the small C-terminal fragment, mediate the attachment and spreading of several types of cells and promote neurite outgrowth from neuronal cells similar to laminin and its corresponding fragments. Cells with various integrin-type receptors for laminin attached equally well to merosin and laminin, suggesting that several of the known laminin binding receptors also bind to merosin. Antibodies to the beta 1 subunit of integrins inhibited neurite outgrowth on merosin as well as on laminin, confirming the involvement of integrin-mediated interaction of cells with both merosin and laminin. Schwannoma cells, which have previously been shown to produce a laminin-like, neurite-promoting factor, synthesize merosin in vivo and in vitro as shown by protein and mRNA analysis. The results suggest that merosin, which is the more abundant basement membrane protein in the laminin family, has properties very similar to laminin despite differences in the structure of the heavy chain. Furthermore, merosin may be identical to or a component of the neurite-promoting factors previously reported from heart, muscle, and Schwann cells.     

Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis

The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.     

HadA is an atypical new multifunctional trimeric coiled-coil adhesin of Haemophilus influenzae biogroup aegyptius, which promotes entry into host cells

The Oca (Oligomeric coiled-coil adhesin) family is a subgroup of the bacterial trimeric autotransporter adhesins, which includes structurally related proteins, such as YadA of Yersinia enterocolitica and NadA of Neisseria meningitidis . In this study, we searched in silico for novel members of this family in bacterial genomes and identified HadA ( Haemophilus adhesin A), a trimeric autotransporter expressed only by Haemophilus influenzae biogroup aegyptius causing Brazilian purpuric fever (BPF), a fulminant septicemic disease of children. By comparative genomics and sequence analysis we predicted that the hadA gene is harboured on a mobile genetic element unique to BPF isolates. Biological analysis of HadA in the native background was limited because this organism is not amenable to genetic manipulation. Alternatively, we demonstrated that expression of HadA confers to a non-invasive Escherichia coli strain the ability to adhere to human cells and to extracellular matrix proteins and to induce in vitro bacterial aggregation and microcolony formation. Intriguingly, HadA is predicted to lack the typical N-terminal head domain of Oca proteins generally associated with cellular receptor binding. We propose here a structural model of the HadA coiled-coil stalk and show that the N-terminal region is still responsible of the binding activity and a KGD motif plays a role. Interestingly, HadA promotes bacterial entry into mammalian cells. Our results show a cytoskeleton re-arrangement and an involvement of clathrin in the HadA-mediated internalization. These data give new insights on the structure-function relationship of oligomeric coiled-coil adhesins and suggest a potential role of this protein in the pathogenesis of BPF.     

Effect of near-UV light on Escherichia coli in the presence of 8-methoxypsoralen: wavelength dependency of killing, induction of prophage, and mutation.

Wavelength dependency of photo-inactivation and photoinduced reverse mutation of Escherichia coli sensitized with 8-methoxypsoralen, and wavelength dependency of photoinduction of lambda prophage from the sensitized lysogen were measured in a range of 298 to 400 nm. The most efficient sensitization for these biological effects was observed between 320 and 340 nm. In the presence of 8-methyoxypsoralen, the induced mutation frequency per lethal hit was highest of 298 nm in the range examined and was gradually decreased with increasing wavelength to a minimum frequency at 345 nm. This finding may be a reflection of the production of more than one type of lesions which have different efficiencies for mutation compared with the killing efficiency.     

Shigella deliver an effector protein to trigger host microtubule destabilization,which promotes Rac1 activity and efficient bacterial internalization

Shigella deliver a subset of effectors into the host cell via the type III secretion system, that stimulate host cell signal pathways to modulate the actin dynamics required for invasion of epithelial cells. Here we show that one of the Shigella effectors, called VirA, can interact with tubulin to promote microtubule (MT) destabilization, and elicit protrusions of membrane ruffling. Under in vitro conditions, VirA inhibited polymerization of tubulin and stimulated MT destabilization. Upon microinjection of VirA into HeLa cells, a localized membrane ruffling was induced rapidly. Overexpression of VirA in host cells caused MT destruction and protruding membrane ruffles which were absent when VirA was co-expressed with a dominant-negative Rac1 mutant. Indeed, Shigella but not the virA mutant stimulated Rac1, including the formation of membrane ruffles in infected cells. Importantly, the MT structure beneath the protruding ruffling was destroyed. Furthermore, drug-induced MT growth in HeLa cells greatly enhanced the Shigella entry. These results indicate that VirA is a novel type of bacterial effector capable of inducing membrane ruffling through the stimulation of MT destabilization.     

Survival,prophage induction,and invasive properties of lysogenic Salmonella Typhimurium exposed to simulated gastrointestinal conditions

This study was designed to evaluate the viability, prophage induction, invasive ability, and relative gene expression in lysogenic Salmonella Typhimurium exposed to the simulated gastric juice (SGJ) at pH 2 (SGJ-2), 3 (SGJ-3), 4 (SGJ-4), and 5 (SGJ-5) for 30 min followed by 0.5 % bile salts for 2 h. The susceptibility of lysogenic S. Typhimurium increased with decreasing pH value and increasing bile salt concentration. The lysogenic S. Typhimurium cells were least susceptible to SGJ-4 and SGJ-5, showing <1 log reduction. The highest prophage induction was observed by 3.34 log PFU/ml in lysogenic S. Typhimurium at SGJ-3 in the presence of 0.5 % bile salts. The numbers of invading lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5 were 3.57, 3.73, and 4.15 log CFU/cm², respectively. Most genes (hilA, hilC, hilD, invA, invE, invF, and sirA) were down-regulated in lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5. This study provides useful information for understanding physiological changes of lysogenic S. Typhimurium in the simulated gastrointestinal conditions.     

Deletion of nudC, a nuclear migration gene of Aspergillus nidulans, causes morphological and cell wall abnormalities and is lethal.

Nuclear migration is required for normal development in both higher and lower eukaryotes. In fungi this process is mediated by cytoplasmic dynein. It is believed that this motor protein is anchored to the cell membrane and moves nuclei by capturing and pulling on spindle pole body microtubules. To date, four genes have been identified and shown to be required for this process in Aspergillus nidulans. The nudA and nudG genes, respectively, encode the heavy and light chains of cytoplasmic dynein, and the nudF and nudC gene products encode proteins of 49 and 22 kDa. The precise biochemical functions of the nudF and nudC genes have not yet been identified. In this report we further investigate NUDC protein function by deleting the nudC gene. Surprisingly, although deletion of nudA and nudF affect nuclear migration, deletion of nudC profoundly affected the morphology and composition of the cell wall. Spores of the strain deleted for nudC grew spherically and lysed. The thickness of the cell wall was increased in the deletion mutant and wall polymer composition was abnormal. This phenotype could be repressed by growth on osmotically buffered medium at low temperature. Similar, but less severe, effects were also noted in a strain depleted for NUDC by down-regulation. These results suggest a possible relationship between fungal cell wall biosynthesis and nuclear migration.     

The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to the Bacillus cereus linear plasmid pBClin15, has a prophage state

Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis. Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15. We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage. This state is common, as several B. thuringiensis strains release Bam35-related viruses.     

The E. coli divE mutation, which differentially inhibits synthesis of certain proteins, is in tRNASer1.

The temperature-sensitive divE mutant of Escherichia coli cannot synthesize certain membrane and cytoplasmic proteins at a non-permissive temperature. Growth of the mutant cells is arrested at a specific stage of the cell cycle when exposed to the non-permissive conditions, suggesting that the divE mutant possesses a defect in cell division control. From sequence determination of a cloned 1.35-kbp DNA fragment that complements the temperature-sensitive divE42 mutation, we characterized two genes in the segment ; one for tRNASer1 and the other for a 23 500 dalton protein. In parallel experiments we cloned the homologous 1.35-kbp DNA fragment from the divE42 mutant and determined its entire nucleotide sequence. Comparison of the two sequences showed that the mutation site is located not in the protein gene, but in the tRNA gene, where A10 is replaced by G10 in the D-stem. Lambda transducing phages carrying the subcloned tRNASer1 gene complemented the divE42 mutation, thereby confirming the conclusion obtained from sequence analyses of the fragments. This finding indicates that tRNASer1 is specifically involved in regulation of cell cycle-specific protein synthesis, coupled with an important step in the process of cell division, or that usage of serine tRNA is functionally specific for the biosynthesis of certain proteins.     

Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells

Neisseria meningitidis is a human pathogen, which is a major cause of sepsis and meningitis. The bacterium colonizes the upper respiratory tract of approximately 10% of humans where it lives as a commensal. On rare occasions, it crosses the epithelium and reaches the bloodstream causing sepsis. From the bloodstream it translocates the blood-brain barrier, causing meningitis. Although all strains have the potential to cause disease, a subset of them, which belongs to hypervirulent lineages, causes disease more frequently than others. Recently, we described NadA, a novel antigen of N. meningitidis, present in three of the four known hypervirulent lineages. Here we show that NadA is a novel bacterial invasin which, when expressed on the surface of Escherichia coli, promotes adhesion to and invasion into Chang epithelial cells. Deletion of the N-terminal globular domain of recombinant NadA or pronase treatment of human cells abrogated the adhesive phenotype. A hypervirulent strain of N. meningitidis where the nad A gene was inactivated had a reduced ability to adhere to and invade into epithelial cells in vitro. NadA is likely to improve the fitness of N. meningitidis contributing to the increased virulence of strains that belong to the hypervirulent lineages.     

An orbivirus of mosquitoes which induces CO2 sensitivity in mosquitoes and is lethal for rabbits.

An orbivirus, JKT-7400, isolated from Culex mosquitoes in Indonesia, replicated to a high titer and induced cytopathic effects in Aedes albopictus cell cultures. The virus produced lethal sensitivity to carbon dioxide in Culex and Aedes mosquitoes as well as in Drosophila melanogaster fruit flies but was not the agent of the hereditary sensitivity to carbon dioxide previously described for Culex quinquefasciatus. When injected intravenously in high doses, JKT-7400 virus was lethal for rabbits, apparently without replicating to a significant extent. It was not pathogenic for adult mice inoculated intravenously or for adult or suckling mice inoculated intracerebrally and intraperitoneally. Unlike an orbivirus isolated from Culex mosquitoes in China, JKT-7400 did not interfere with the replication of Japanese encephalitis virus in mosquitoes.     

tif-dependent induction of colicin E1, prophage lambda, and filamentation in Escherichia coli K-12.

To help understand how the tif-1 mutation of the recA gene of Escherichia coli confers adenine activability on the recA protein, we used the fact that cytidine plus guanosine inhibits induction of prophage lambda and cell filamentation in a tif-1 mutant, and that adenine reverses this inhibition. We varied the amount of adenine in agar plates containing a fixed amount of cytidine and scored for survivors of three different tif-dependent lethal induction processes. Much more adenine was required for cell killing when cytidine was present than when it was absent. Therefore adenine does not override cytidine inhibition, but instead appears to compete with it for a site of action which may be on the recA protein. The competition is not at the cell transport level. Our results lead to a model in which the tif form of the recA protein is an allosteric enzyme that binds both negative and positive modulators. By varying the adenine-cytidine ratio of the medium it is possible to control the degree of induction in a tif-1 cell. For the three different tif-dependent inductions studied here, least adenine was required for lambda induction and most for lethal filamentation, presumably reflecting requirements for different amounts of activated recA protein in each process. Varying the adenine-cytidine ratio revealed two stable intermediate stages in lambda induction, as well as a stage of colicin E1 induction in which the cells produced colicin without cell death. The rate of filament formation could be similarly controlled. Experiments with tif (ColE1, lambda) gave evidence of a competition between colicin repressor and lambda repressor for activated recA protein.     

double-time is identical to discs overgrown, which is required for cell survival, proliferation and growth arrest in Drosophila imaginal discs.

We have isolated the discs overgrown gene of Drosophila and shown that it encodes a homolog of the Casein kinase I(delta)/(epsilon) subfamily and is identical to the double-time gene. However, in contrast to the weak double-time alleles, which appear to affect only the circadian rhythm, discs overgrown alleles, including bona fide null alleles, show strong effects on cell survival and growth control in imaginal discs. Analysis of their phenotypes and molecular lesions suggests that the Discs overgrown protein is a crucial component in the mechanism that links cell survival during proliferation to growth arrest in imaginal discs. This work provides the first analysis in a multicellular organism of Casein kinase I(delta)/(epsilon) functions necessary for survival. Since the amino acid sequences and three-dimensional structures of Casein kinase I(delta)/(epsilon) enzymes are highly conserved, the results suggest that these proteins may also function in controlling cell growth and survival in other organisms.