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Chrysomycin A (CA), a promising antibiotic agent, usually coexists with two analog chrysomycins B (CB) and C (CC) produced by several wild-type (WT) Streptomyces strains. With the aim to increase CA production, UV mutagenesis-based breeding had been employed on a marine-derived strain Streptomyces sp. 891 in our previous study and afforded an improved strain 891-B6 with enhanced CA yield. By comparative transcriptome analysis, significant differences in chrysomycin BGC-related gene expression between the WT strain 891 and the mutant strain 891-B6 were unveiled in the current study. Among 25 up-regulated genes in mutant 891-B6, chryA, chryB, chryC, chryF, chryG, chryK, chryP, and chryQ, responsible for the biosynthesis of benzonaphthopyranone aglycone, and chryD, chryE, and chryU in charge of production of its deoxyglycoside, were characterized. Furthermore, the expression of genes chryOII, chryOIII, and chryOIV responsible for the formation of 8-vinyl in CA from 8-ethyl in CB were greatly enhanced in strain 891-B6. These findings provide molecular mechanisms for increased yield of CA and decreased yield of CB for mutant 891-B6, which has potential application in industrial CA production.

Global warming poses new challenges for insects to adapt to higher temperatures. Trehalose is the main blood sugar in insects and plays an important role in energy metabolism and stress resistance. The transmembrane transport of trehalose mainly depends on the trehalose transporter (TRET1). Plutella xylostella (L.) is a worldwide agricultural pest; however, the effects of the trehalose transport mechanism and trehalose distribution in tissues on the development, reproduction and temperature adaptation of P. xylostella have yet to be reported. In this study, PxTret1-like was cloned and analyzed regarding its expression pattern. It was found that the expression of PxTret1-like was affected by ambient temperature. The knockout mutation of PxTret1-like was generated using a CRISPR/Cas9 system by targeted knockout. The trehalose content and trehalase activity of mutant P. xylostella increased at different developmental stages. The trehalose content increased in the fat body of the fourth-instar P. xylostella, and decreased in the hemolymph, and there was no significant change in glucose in the fat body and hemolymph. Mutant strains of P. xylostella showed a significantly reduced survival rate, fecundity and ability to withstand extreme temperatures. The results showed that PxTret1-like could affect the development, reproduction and temperature adaptability of P. xylostella by regulating the trehalose content in the fat body and hemolymph.

Helicobacter pylori and Epstein-Barr virus (EBV) are two well-known contributors to cancer and can establish lifelong persistent infection in the host. This leads to chronic inflammation, which also contributes to development of cancer. Association with H. pylori increases the risk of gastric carcinoma, and coexistence with EBV enhances proliferation of infected cells. Further, H. pylori-EBV coinfection causes chronic inflammation in pediatric patients. We have established an H. pylori-EBV coinfection model system using human gastric epithelial cells. We showed that H. pylori infection can increase the oncogenic phenotype of EBV-infected cells and that the cytotoxin-associated gene (CagA) protein encoded by H. pylori stimulated EBV-mediated cell proliferation in this coinfection model system. This led to increased expression of DNA methyl transferases (DNMTs), which reprogrammed cellular transcriptional profiles, including those of tumor suppressor genes (TSGs), through hypermethylation. These findings provide new insights into a molecular mechanism whereby cooperativity between two oncogenic agents leads to enhanced oncogenic activity of gastric cancer cells., IMPORTANCE We have studied the cooperativity between H. pylori and EBV, two known oncogenic agents. This led to an enhanced oncogenic phenotype in gastric epithelial cells. We now demonstrate that EBV-driven epigenetic modifications are enhanced in the presence of H. pylori, more specifically, in the presence of its CagA secretory antigen. This results in increased proliferation of the infected gastric cells. Our findings now elucidate a molecular mechanism whereby expression of cellular DNA methyl transferases is induced influencing infection by EBV. Hypermethylation of the regulatory genomic regions of tumor suppressor genes results in their silencing. This drastically affects the expression of cell cycle, apoptosis, and DNA repair genes, which dysregulates their associated processes, and promotion of the oncogenic phenotype.

Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.

A multiblocked mutant strain (ΔAHBA and Δasm12, asm21) of Actinosynnema pretiosum, the producer of the highly toxic maytansinoid ansamitocin, has been used for the mutasynthetic production of new proansamitocin derivatives. The use of mutant strains that are blocked in the biosynthesis of an early building block as well as in the expression of two tailoring enzymes broadens the scope of chemo‐biosynthetic access to new maytansinoids. Remarkably, a ring‐expanded macrolactone derived from ansamitocin was created for the first time., Make it larger! A new variant of the chemo‐biosynthetic concept of mutasynthesis using a multiblocked strain of the ansamitocin producer A. pretiosum yielded new macrolactam proansamitocins as well as the first ring‐expanded maytansin macrolactone.

Spontaneous mutations are important players in evolution. Nevertheless, there is a paucity of information about the mutagenic processes operating in most bacterial species. In this work, we implemented two forward mutational markers for studies in Caulobacter crescentus. We confirmed previous results in which A:T → G:C transitions are the most prevalent type of spontaneous base substitutions in this organism, although there is considerable deviation from this trend in one of the loci analyzed. We also investigated the role of dinB and imuC, encoding error-prone DNA polymerases, in spontaneous mutagenesis in this GC-rich organism. Both dinB and imuC mutant strains show comparable mutation rates to the parental strain. Nevertheless, both strains show differences in the base substitution patterns, and the dinB mutant strain shows a striking reduction in the number of spontaneous -1 deletions and an increase in C:G → T:A transitions in both assays.

In this announcement, we present the complete annotated genome sequence of an Escherichia coli MC4100 mutant strain, BE104. This strain has several methionine sulfoxide reductase gene deletions, making it ideal for studying enzymes that alter the redox state of methionine.

A combination of microbial strain improvement and statistical optimization is investigated to maximize pneumocandin B0 production from Glarea lozoyensis ATCC 74030. Atmospheric and room temperature plasma (ARTP) was used to enhance G. lozoyensis ATCC 74030 in pneumocandin B0 yield. Mutant strain G. lozoyensis Q1 exhibited 1.39-fold increase in pneumocandin B0 production to 1134 mg/L when compared with the parent strain (810 mg/L). Further, the optimized medium provided another 1.65-fold in final pneumocandin B0 concentration to 1873 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively pneumocandin B0 production from G. lozoyensis and could be a tool to improve other secondary metabolites production by fungal strains.

The aim of this work was to study the random mutagenesis of Clostridium butyricum strain. A high 1,3-PD tolerant mutant strain, designated as C. butyricum YP855, was developed from the wild strain C. butyricum XYB11, using combined chemical (NTG, N-methyl-N′-nitro-N-nitrosoguanidine,) and plasma-based mutagenesis (ARTP, atmospheric and room temperature plasma). The YP855 showed a maximum tolerance of 85 g/L to 1,3-PD (up to 30.8% increase) when compared with the tolerance exhibited by the wild strain. Under the optimum conditions as established by the response surface methodology (RSM), the mutant strain produced 37.20 g/L of 1,3-PD, which is 29.48% higher than the concentration obtained from the wild strain (28.73 g/L). This research would offer information for further development of the biosynthesis of 1,3-PD by the mutant strain of C. butyricum.

We report a draft genome sequence of Streptomyces xinghaiensis ( fradiae ) OlgR, which is resistant to oligomycin A. This mutant strain is derived from S. xinghaiensis OlgR2.100, which is resistant to (33 S )-azido-33-deoxyoligomycin A.

Caenorhabditis elegans postembryonic development consists of four discrete larval stages separated by molts. Typically, the speed of progression through these larval stages is investigated by visual inspection of the molting process. Here, we describe an automated method to monitor the timing of these discrete phases of C. elegans maturation, from the first larval stage through adulthood, using bioluminescence. The method was validated with a lin-42 mutant strain that shows delayed development relative to wild-type animals and with a daf-2 mutant that shows an extended second larval stage. This new method is inherently high throughput and will finally allow dissecting the molecular machinery governing the speed of the developmental clock, which has so far been hampered by the lack of a method suitable for genetic screens., Our work is supported by the Ludwig-Maximilians-University Munich and the Friederich-Bauer-Stiftung (M.M., M.O., and M.G.); the European Research Council (ERC-2011-StG-281691) and the Spanish Ministerio de Economía y Competitividad (BFU2012-35509) (M.A.-S.); and a Marie-Curie Intra-European Fellowship (FP7-PEOPLE-2013-IEF/GA Nr: 627263) (to M.O. and M.A.-S.).

A series of novel N-arylmethyl substituted piperidine-linked aniline derivatives were designed, synthesized and evaluated for their anti-HIV activity in MT-4 cells. All the new compounds showed moderate to potent activities against wild-type (wt) HIV-1 with an EC50 range from 0.022 to 2.1 μM. Among them, compound 5a6 (EC50 = 0.022 ± 0.0091 μM, SI >10,770) was confirmed to be the most potent and selective inhibitor, which proved more potent than DDI and DLV in a cell-based assay against wt HIV-1, and more efficient than NVP in an RT (reverse transcriptase) assay. Besides, it is worth noting that compound 7a1 retained moderate inhibitory activity (EC50 = 4.8 ± 0.95 μM) against the HIV-1 double RT mutant strain (K103N/Y181C). The preliminary structure–activity relationship was discussed and rationalized by molecular simulation.

wild type strain W3110; in the YS1234 strain defective in ynbD, yeiU, ybjG, and pgpB; in the YS1235 strain defective in ynbD, yeiU, ybjG, and bacA; and in the YS1245 strain defective in ynbD, yeiU, pgpB, and bacA were 78.0% ± 3.5%, 74.7% ± 1.2%, 68.4% ± 6.8%, and 64.0% ± 7.5%, respectively. The quadruple mutant strain that possessed the wild-type bacA showed almost identical UP level as the wild type strain W3110, indicating that sufficient level of UPP phosphatse activity was conferred by a bacA product without the products of ynbD, yeiU, ybjG, and pgpB. However, the mutants defective in bacA showed a slight but significant (p < 0.01) decrease in the UP level.

Staphylococci are Gram-positive spherical bacteria of enormous clinical and biotechnological relevance. Staphylococcus aureus has been extensively studied as a model pathogen. A plethora of methods and molecular tools has been developed for genetic modification of at least ten different staphylococcal species to date. Here we review recent developments of various genetic tools and molecular methods for staphylococcal research, which include reporter systems and vectors for controllable gene expression, gene inactivation, gene essentiality testing, chromosomal integration and transposon delivery. It is furthermore illustrated how mutant strain construction by homologous or site-specific recombination benefits from sophisticated counterselection methods. The underlying genetic components have been shown to operate in wild-type staphylococci or modified chassis strains. Finally, possible future developments in the field of applied Staphylococcus genetics are highlighted.

Foodborne infections caused by Salmonella enterica serovars are a significant problem worldwide. Presented here is the genome sequence of the nontyphoidal S. enterica serovar Typhimurium mutant strain NC983, a potential vaccine candidate.

Data on involvement of nitric oxide in apoptosis are contradictory. The balance between anti- and proapoptotic activities of nitric oxide depends on many factors, including its concentration in a tissue and interactions with other regulators of apoptosis. This paper describes the results of a series of experiments on the effect of nitric oxide donors and inhibitors as well as dNOS1 and dNOS4 transgenes on the apoptosis on drosophila Lobe RSV mutant strain and wild-type strain Oregon R. It has been shown that a high nitric oxide content in cells is able to inhibit antiapoptotic effect of HSP70 and stimulate apoptosis, possibly, via the grim-mediated apoptotic pathway. Moreover, long-term action of a high nitric oxide concentration during the entire development more efficiently stimulates the proapoptotic genes as compared with short-term action of this agent.

Taxa included in the Aspergillus niger aggregate are difficult to distinguish by phenotypic characterization. In this work, the effect of gentian violet on the growth of the N and T RFLP types of A. niger aggregate strains has been investigated. In total, 105 strains from different sources and origins, including reference cultures and field isolates were studied. Type N and T RFLP patterns, ochratoxin A production and the effect of different concentrations of gentian violet on the growth were determined in these strains. Forty nine strains belonged to the N type and 56 strains to the T type. Sixteen out of the 105 strains assayed were OTA producers. All the OTA-producing species belonged to the RFLP type N and none of the T type strains was able to produce OTA. Approximately 90% of the N type strains grew in the presence of 25 ppm of gentian violet. Only five N type strains did not grow on this medium. One of these strains was A. niger ATCC 22343, a well documented induced mutant strain and the remaining four strains belonged to the new species A. brasiliensis. On the contrary, all the T type strains failed to grow on this medium after 3 days of incubation (sensitivity 89.79%; specificity 100%). The use of growth in gentian violet as an additional character for classification and identification purposes in this taxonomic group may be useful because no phenotypic methods have yet been found that can distinguish between these species.

We report a draft genome sequence of Streptomyces fradiae olg1-1, a mutant strain derived from the model object S. fradiae ATCC 19609, which is resistant to nitrone-oligomycin and has a mutation in the DNA-binding domain of a transcriptional regulator PadR.

Inducamide A (Ia), B (Ib) and C (II) are shown to not exhibit significant antibacterial activity, but (II) reveals modest cytotoxicity.

We report the elucidation of the complete genome of the Neurospora crassa (Shear and Dodge) strain FGSC 73, a mat-a , trp-3 mutant strain. The genome sequence around the idiotypic mating type locus represents the only publicly available sequence for a mat-a strain. 40.42 Megabases are assembled into 358 scaffolds carrying 11,978 gene models.

It is well known that Escherichia coli executes chemotactic motion in response to chemical cues by modulating the flagellar motor bias alone. However, previous studies have reported the possibility of variation in run speed in the presence of attractants although it is unclear whether bacteria can deliberately modulate their swimming speeds in response to environmental cues or if the motor speeds are hardwired. By studying the detailed motion of cells in a uniform concentration of glucose and its non-metabolizable analogue, we show that changing concentrations may be accompanied by variation in the swimming speed. For a fixed run duration, cells exposed to the attractants achieved a higher peak-swimming speed after a tumble compared with that in plain motility buffer. Our experiments using the mutant strain lacking the Trg sensor show no change in swimming speed with varying concentrations of the non-metabolizable analogue, suggesting that sensing may play a role in the observed variation of swimming speed.

A mutant strain of Pseudomonas putida (LU2201) expressing a novel ethylphenylacetate (EPA) esterase was isolated after mutagenesis of P. putida LU6456. The DNA fragment conferring the EPA+ phenotype was cloned and characterized. It included the structural genes of the 2,3-butanediol catabolic pathway, including acoC, which catalyzes the acetyl transfer step (i.e., dihydrolipoamide acetyltransferase). The acoC gene product from LU2201 was expressed in E. coli and shown to be responsible for the novel EPA-esterase activity. Biochemical characterization revealed that the enzyme still retained the natural dihydrolipoamide acetyltransferase activity. Sequence comparison with the corresponding wild-type gene indicated a single-base pair change leading to a I238V replacement in the respective protein. Biochemical characterization of wild-type AcoC enzyme showed that it too was able to catalyze EPA hydrolysis despite the apparent absence of this activity in cells grown in the presence of EPA. Unlike the wild type, EPA serves as an inducer in the mutant strain. Inverse PCR was used to clone the putative regulator acoR from the aco operon in both wild type and mutant strains. Sequence analyses indicated an S14R exchange in the AcoR from the P. putida mutant. This mutation was located in the N-terminal region responsible for regulatory functions, presumably by interaction with signal molecules. This may account for the ability of EPA to induce expression of acoC only in the mutant strain.Key words: Pseudomonas putida, ethylphenylacetate esterase, acetoin cleaving system, acoC gene, dihydrolipoamide acetyl-transferase, E2 component, acoR gene.

Members of the Mycobacterium tuberculosis group synthesize a family of long-chain fatty acids, mycolic acids, which are located in the cell envelope. These include the non-oxygenated α-mycolic acid and the oxygenated keto- and methoxymycolic acids. The function in bacterial virulence, if any, of these various types of mycolic acids is unknown. We have constructed a mutant strain of M. tuberculosis with an inactivated hma (cmaA, mma4) gene; this mutant strain no longer synthesizes oxygenated mycolic acids, has profound alterations in its envelope permeability and is attenuated in mice.

Two new avemectin analogues 1 and 2 belonging to 'b' series of components were isolated from a ∆aveCDE mutant Streptomyces avermectinius strain. Their structures were elucidated by detailed analysis of NMR and MS spectroscopic data. Compounds 1 and 2 displayed moderate cytotoxicities against B16, MG-63, and Saos-2 cell lines in an in vitro assay.

The open reading frame at 86.7 min on the Escherichia coli chromosome, “ yigC ,” complemented a ubiD mutant strain, AN66, indicating that yigC is the ubiD gene. The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database.

Formylation of the initiator methionyl-tRNA, catalyzed by methionyl-tRNA formyltransferase, has long been regarded as essential for initiation of protein synthesis in eubacteria. Here, we show that this process is, in fact, dispensable in Pseudomonas aeruginosa. Disruption of the chromosomal methionyl-tRNA formyltransferase gene in P. aeruginosa resulted only in a moderate decrease in the rate of cell growth, whereas in Escherichia coli cell growth was severely impaired. The ability of the P. aeruginosa mutant strain to grow was not due to an additional copy of the methionyl-tRNA formyltransferase gene or to N-acylation of the methionyl moiety by a group other than formyl. These results indicate that P. aeruginosa can carry out formylation-independent initiation of protein synthesis, using the nonformylated methionyl-tRNA. Therefore, the dogma that eubacteria require formylation of the initiator methionyl-tRNA for initiation of protein synthesis may have been an invalid generalization of results obtained with E. coli.

A mutant strain of Salmonella typhimurium, SJW46, has flagellar filaments supercoiled in the same form as the wild-type strain, SJW1103, and swims normally. However, its flagellar filaments are mechanically unstable and show anomalous behaviors of polymorphism. Flagellin from SJW46 has a large central deletion from Ala204 to Lys292 of SJW1103 flagellin, which has been thought to be located in the outer surface of the filament. Since the filament structure is determined by intersubunit interactions of the terminal regions in the densely packed core of the filament, no serious involvement of the deleted portion was expected in the filament stability and polymorphism. In order to locate the deleted portion and to understand the underlying mechanism of these anomalous characteristics, we carried out structure analysis of the L-type straight filament reconstituted from a mutant flagellin of SJW46 (SJW46S) and compared the structure with that of the SJW1660 filament, which is also the L-type but composed of flagellin with no deletion. The deleted portion was identified as the outermost subdomain, and the structure in the core region showed no appreciable differences. The structure revealed the previously identified folding of flagellin in further detail, and the significance of intersubunit interactions between outer domains, which are present in the SJW1660 filament but absent in the SJW46 filament. This suggests that these contacts have a significant contribution to the filament stability and polymorphic behavior, despite the fact that the contacting surface area occupies only a minor portion of the whole intersubunit interactions.

Natural avermectins (AVEs) share a 6,6-spiroketal moiety with an exclusive R configuration at the C21 spirocyclic junction. Herein, we report the characterization of nine AVE-like spiroketals of two types (C21 S and R) in a mutant strain that lacks spirocyclase activity. Comparative analysis of their structures facilitated evaluation of the effect of stereochemistry on endogenous biotransformations and biological activities of the spiroketals.

The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.

An assay was developed which identifies individual bacterial tumbles and so allows rapid, quantitative measurements of tumble frequency in free-swimming bacteria. Tumble frequency is modulated by cells to enable chemotaxis. Mutations in the chemotaxis signal transduction pathway typically have phenotypes of altered tumble frequency. The purpose of this assay is to quantitatively measure steady-state tumble frequency to enable comparisons of mutant strain phenotypes. It was developed using Escherichia coli but should be applicable to other species with a peritrichous flagellation pattern, such as Salmonella typhimurium. Tumbles are defined by a combination of the parameters rate of change of direction and swimming speed, with a rapid change of direction defining the beginning of a tumble and increased swimming speed defining the end. These parameters have previously been shown to be correlated with tumbles in general but not used to identify discrete tumble events. The computer assay was validated by comparing its results with manual observations by eye. The assay was intended to be most sensitive to swimming patterns similar to wild type so as to resolve subtle changes which would result from partial-function mutations. It quantitatively detects extreme behavioral phenotypes as well and can be modified to increase resolution at either extreme if necessary.

Mutations in Salmonella typhimurium fliG, fliM and fliN give rise either to non-flagellate, non-motile or non-chemotactic mutant bacteria. The FliG, FliM and FliN proteins form part of recently characterized extended flagellar basal structures, and have been postulated to form a mutually interacting structural complex. We have examined basal body preparations from non-motile or non-chemotactic fliG, fliM and fliN mutant strains by electron microscopy and immunoblot gel analysis. Most flagellar preparations isolated from the non-motile mutants lacked FliM, but contained FliG. The basal bodies lacked the belled morphology characteristic of the wild-type structures, but had protrusions which could be labelled with anti-FliG. Non-motile mutant preparations severely depleted of FliG but containing FliM were also obtained. These preparations contained extended, belled flagellar structures that were labelled with anti-FliM. Thus, FliM is part of the shell of the extended structures responsible for the belled morphology, while FliG may be part of the inner substructure. The extended basal structures from a FliG temperature-sensitive mutant strain rapidly lost FliM, as well as FliG, upon a shift to a non-permissive temperature, implying interaction between the FliG- and FliM-containing substructures. In dramatic contrast to non-motile mutants, extended basal structures isolated from non-chemotactic mutants were indistinguishable from wild-type structures. This difference may reflect the energetics of the different protein-protein interactions operative during torque generation and the switching of rotation sense.

Caenorhabditis elegans (C. elegans) is an excellent model organism for the study of aging and longevity. In this work, we presented a microfluidic approach for the evaluation of longevity in C. elegans under stress. The microfluidic device integrated multiple microvalves with parallel channels, which enabled the long-term culture and flexible manipulation of C. elegans in real-time. The utility of the device was demonstrated by characterizing the lifespan, mobility behavior and fluorescence expression of oxidative stress in mutant strain CL2166 simultaneously at single animal resolution. A certain dose of polydatin was found to enable the extension of mean lifespan of CL2166 for the first time, and the prolonged longevity activity was possibly mediated by the protective response to oxidative stress, indicating the promising role of polydatin involved in aging process. The device is simple to operate, easy for real-time imaging and multiparatemer evaluations in parallel, providing the powerful platform for drug evaluation/screening in highthroughput format.

The gangliosides in the livers of various inbred strains of rats and hepatoma of LEC rats were purified and analyzed by thin-layer chromatography. The patterns of ganglioside distribution in these rat livers were classified into three phenotypes depending on the strain, that is, a-type (ACI, LEA, LEW, BUF), b-type (WKAH, SHR/SP), and LEC type, which are characterized by dominance of a- or b-series of gangliosides, or a variation of a-type, respectively. A sex difference was also recognized in the molar ratio of GM3 which was much higher in males (60-75%) than in females (33-56%) except in LEC rats. In addition, the content of a-series gangliosides was lower and the content of b-series gangliosides was higher in a-type male rats than in a-type female rats. The opposite was true in b-type rats. LEC rats were an exception, characterized by no sex difference and a quite low content of b-series gangliosides. The LEC rat is a mutant strain that spontaneously develops fulminant hepatitis around 14 to 20 weeks of age and hepatoma at 1 to 1.5 years old. The gangliosides of the hepatoma were characterized by the appearance of the newly synthesized gangliosides, fucosyl-GM1 and alpha-galactosyl alpha-fucosyl GM1 (BGM1). In particular, BGM1 ganglioside accumulated in the hepatoma of female rats.

Polyacrylamide gel electrophoresis profiles of various cell surface lipopolysaccharides (LPS) obtained from fast-growing strains and plasmidless mutant strains of Rhizobium ‘hedysari’, R. leguminosarum and from slow-growing Bradyrhizobium japonicum strains have been compared. Correlations were obtained by using serological techniques. Two of the fast-growing strains and one slow-growing strain employed showed several LPS with slow mobility and a few others characterized by fast mobility in the gel. Other fast-growing rhizobium strains and all the other slow-growing bradyrhizobia tested had bands widely distributed in the gels. Location of bands, in these rhizobia, did not correlate with rapidity of growth of the strains. Induced antibiotic resistance did not affect LPS composition for either the fast- or slow-growing rhizobia. The LPS composition was apparently affected by the loss of plasmids. It appears to be associated with the lack of some bands in the region ranging from 30–50 kDa. The LPS composition was also affected by the mutation in the fast mobility region. The mutant strain lacked a low molecular mass band compared with the parental strain. [ 1 H]-NMR spectra of LPS extracts from the latter strain also revealed a signal that is missing from the spectrum of the LPS of mutant strain.

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all-trans retinal restored both photoresponses in a carotenoid-deficient mutant strain of Chlamydomonas reinhardtii in a dose-dependent manner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All-trans retinal was more effective than 11-cis retinal to regenerate both photobehavioral responses. Analogs having locked 11-cis configurations and a phenyl ring in the side chain also induced photoresponses, although at concentrations more than two orders of magnitude higher than all-trans retinal. According to the present assay method, the responses were hardly detectable in cells incubated with retinal analogs in which the 13-ene was locked in either its trans or cis configuration. The results strongly suggest that the isomerization of the 13–14 double bond is important for photobehavioral signal transduction and that a single retinal-dependent photoreceptor controls both phototactic and photophobic responses.

We used wild-type UTEX481; SF33, a shortened-filament mutant strain that shows normal complementary chromatic adaptation pigmentation responses; and FdBk14, an RcaE-deficient strain that lacks light-dependent pigmentation responses, to investigate the molecular basis of the photoregulation of cellular morphology in the cyanobacterium Fremyella diplosiphon . Detailed microscopic and biochemical analyses indicate that RcaE is required for the photoregulation of cell and filament morphologies of F. diplosiphon in response to red and green light.

Haemophilus influenzae Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type H. influenzae and whose phage recombination is 8 times lower was isolated. The affected gene, comM , is induced during competence development in wild-type H. influenzae but not in RJ248.

The fate of exogenous short-chain analogues of phosphatidylethanolamine and phosphatidylserine was studied in a deep-rough derivative of E. coli mutant strain AD93 that cannot synthesize phosphatidylethanolamine de novo. Using mass spectrometry, it was shown that dicaproyl(di 6:0)-phosphatidylethanolamine is extensively remodeled, eventually adopting the phosphatidylethanolamine species profile of the parental wild-type strain of AD93. Dicaproyl-phosphatidylserine was decarboxylated to form phosphatidylethanolamine, and yielded a species profile, which strongly resembled that of the introduced phosphatidylethanolamine. This demonstrates transport of phosphatidylserine to the cytosolic leaflet of the inner membrane. The changes of the species profile of phosphatidylethanolamine indicate that the short-chain phospholipids are most likely remodeled via two consecutive acyl chain substitutions, and at least part of this remodeling involves transport to the inner membrane.

Motivation:Predicting the outcome of specific experiments (such as the growth of a particular mutant strain in a particular medium) has the potential to allow researchers to devote resources to experiments with higher expected numbers of ‘hits’. Results: We use decision trees to predict phenotypes associated with Saccharomyces cerevisiae genes on the basis of Gene Ontology (GO) functional annotations from the Saccharomyces Genome Database (SGD) and other phenotypic annotations from the Yeast Phenotype Catalog at the Munich Information Center for Protein Sequences (MIPS). We assess the methodology in three ways: (1) we use cross-validation on the phenotypic annotations listed in MIPS, and show ROC curves indicating the tradeoff between true-positive rate and false-positive rate; (2) we do a literature-search for 100 of the predicted gene-phenotype associations that are not listed in MIPS, and find evidence for 43 of them; (3) we use deletion strains to experimentally assess 61 predicted gene-phenotype associations not listed in MIPS; significantly more of these deletion strains show abnormal growth than would be expected by chance. Contact: fritz_roth@hms.harvard.edu Supplementary Information: Complete results are available at http://llama.med.harvard.edu/~king/pheno.html Keywords: decision trees; phenotype; gene function *To whom correspondence should be addressed.

A sensitive procedure for the determination of nitrate within the 1- to 50-microM range is described. The method is based on the photoreduction of nitrate to nitrite by whole cells of a nitrite reductase-less mutant strain of the unicellular cyanobacterium Synechococcus R2. Cell suspensions of this cyanobacterium retain high levels of nitrate photoreduction activity for at least 2 months when maintained in the presence of dimethyl sulfoxide at -70 degrees C.

A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfection with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 amplicon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-I amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-titer rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.

Lipopolysaccharide (LPS) antigens of A. brasilense strains Sp245, Sp7 and their variants with an altered plasmid content were investigated. SDS electrophoresis and a set of immunoelectrophoretic methods with monospecific antibodies to the LPS of the parent strains were used. A. brasilense Sp245 harbours plasmids of 85 (p85), 120 (p120) and 130 (p130) MDa and several plasmids of more than 300 MDa. A. brasilense Sp7 (wild, R-form) harbours plasmids of 90 (p90) and 115 (p115) MDa. Spontaneous morphological R-S dissociation of Sp7-R was related to disappearance of p115 in the plasmid profile (Matveev et al., 1987). The antibodies to the LPS were obtained as described earlier (Matora et al., 1995).

Autonomously replicating sequence binding factor 1 (ABF1) has been implicated in the control of a variety of gene expressions in Saccharomyces cerevisiae. In this paper evidence is presented that ABF1 is involved in the glucose-dependent expression of the TDH3 gene which encodes glyceraldehyde-3-phosphate dehydrogenase. ABF1 binds to consensus sites located between -420 and -250, and between +77 and +200, and acts as a transactivator in an orientation-independent manner on both upstream and downstream sites. TDH3-lacZ fusions having an ABF1 consensus motif showed glucose-dependent expression of TDH3, whereas in the abf1 mutant strain JCA35 glucose-dependent expression disappeared. These findings suggest that ABF1 functions as a glucose-dependent transactivator for the expression of the TDH3 gene.

Background PRV Fa is common in China and causes most of the pseudorabies in the pig industry. A PRV SA215 strain with deleted gE, gI, and TK genes was constructed to develop a commercial attenuated live vaccine. However, the physicochemical properties, growth pattern, penetration kinetics, and morphogenesis of the PRV SA215 and its parental PRV Fa strain are unclear. Results A series of experiments were conducted to characterize both strains and provide more information. PRV Fa and PRV SA215 were found to have similar penetration patterns, with about 5 min half-time of penetration. The SA215 strain exhibited a slight delay in entry compared with PRV Fa. In the one-step growth test, the titers of the SA215 strain were first detected at 8 h, rapidly increased, and peaked at 12 h. A plateau was formed between 12-36 h of culturing. PRV SA215 showed delayed replication and approximately 10-30-fold lower titers during 0-16 h of culturing compared with the PRV-Fa strain. After 16 h, the PRV Fa titers dramatically decreased, whereas those of PRV SA215 were prolonged to 36 h and reached a titer value equal to that of PRV Fa and then decreased. Both strains were sensitive to both heat and acid-alkali treatments; however, PRV Fa was relatively more stable to heat treatment than PRV SA215. Both strains could propagate in the cultures with pH values from 5.0 to 9.0. Cultures with pH below 3.0 or above 11.0 were fatal to both strains. Both strains had considerable resistance to freeze-thawing treatments. Morphogenetic investigations showed that typical phases in the maturation pathway were observed in the PRV Fa-infected PK15 cells, whereas secondary envelopment was not observed in the PRV SA215 strain. Instead, capsid aggregations with concomitants of electrodense materials were observed. Conclusions These results suggest that PRV SA215 is a promising strain for vaccine development

Chronic irradiation (accumulated dose 0.6–0.8 Gy) was shown to change the life span in male Drosophila melanogaster.Death was retarded in wild-type strains and accelerated in mutant strains defective in DNA repair and displaying a higher sensitivity to induction of apoptosis.