Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.     

Recombination protein A gene,recA, fromSpirulina platensis IAM-M135

We report the cloning and sequencing of therecA gene fromSpirulina platensis. A genomic library ofSpirulina was constructed in pUC19 and screened by PCR using oligonucleotides corresponding to the conserved amino acid sequences ofAnabaena variabilis andSynechococcus RecA proteins. TheSpirulina recA gene consists of an open reading frame (ORF) of 1095 nucleotides encoding a protein (365 residues) which shares an identity of 79%, 70% and 57% with the RecA proteins ofAnabaena variabilis, Synechococcus andEscherichia coli respectively. TherecA gene is located close to one end of the clonedBglII fragment and has only 53 bp of 5 nucleotides. The isolation of this gene has implications for the development of gene transfer system(s) forSpirulina.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ⁻) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation. Project supported by the National Natural Science Foundation of China (Grant No. 39830010).     

Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation

The expression of theSRS2 gene, which encodes a DNA helicase involved in DNA repair inSaccharomyces cerevisiae, was studied using anSRS2-lacZ fusion integrated at the chromosomalSRS2 locus. It is shown here that this gene is expressed at a low level and is tightly regulated. It is cell-cycle regulated, with induction probably being coordinated with that of the DNA-synthesis genes, which are transcribed at the G1-S boundary. It is also induced by DNA-damaging agents, but only during the G2 phase of the cell cycle; this distinguishes it from a number of other repair genes, which are inducible throughout the cycle. During meiosis, the expression ofSRS2 rises at a time nearly coincident with commitment to recombination. Sincesrs2 null mutants are radiation sensitive essentially when treated in G1, the mitotic regulation pattern described here leads us to postulate that either secondary regulatory events limit Srs2 activity to G1 cells or Srs2 functions in a repair mechanism associated with replication.     

Genetic analysis of rec E activities in Bacillus subtilis

Summary ArecE mutant (recE6) ofBacillus subtilis was constructed by insertion of a selectable marker into therecE coding region. The insertional inactivation of therecE gene renders cells very sensitive to DNA damaging agents and severely impairs intermolecular recombination, but does not markedly affect plasmid interstrand annealing and intramolecular recombination. TherecE6 allele was then introduced into a set of DNA repair-deficient strains ofB. subtilis. The removal of DNA damage by therecF,addAaddB,recH,recL andrecP gene products is strictly dependent on an activerecE gene product (recE-dependent pathway). On the other hand, the increased sensitization to purine adducts in theuvrA42recE6 andpolA5recE6 strains suggests that such lethal lesions may be removed either by therecE-dependent or by therecE-independent pathway.     

Efficient transformation of the medicinal mushroomGanoderma lucidum

Ganoderma lucidum is a well-known and important medicinal mushroom, but its genetic modification has not been reported. We developed an efficient procedure for isolation and regeneration of protoplasts fromG. lucidum. To construct a vector for high-level expression of heterologous genes inG. lucidum, the 1.4-kb regulatory region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) was isolated from the genomic DNA ofLentinus edodes, and theGPD promoter was fused to the β-glucuronidase (GUS) and bialaphos resistance (bar) genes. Using the resulting construct, p301-bG1, an efficient transformation system based on electroporation was established forG. lucidum. GUS expression was observed among transformants conferring bialaphos resistance, indicating that theL. edodes GPD promoter can be used for expression of exogenous genes inG. lucidum. We also studied green fluorescent protein (GFP) as another reporter for transformation ofG. lucidum. TheL. edodes GPD promoter was fused respectively to theGFP andbar genes, and the resulting construct, p301-bg, was introduced intoG. lucidum. StableGFP expression in transformants was detectable by fluorescence microscopy, suggesting the suitability ofGFP as a reporter system in transformation of this mushroom. This is the first report of an efficient transformation system forG. lucidum using different reporters, paving the way for genetic modification of this famous medicinal mushroom.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^– strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.Deceased, December 15, 1995     

Strong evolutionary conservation of broadly expressed protein isoforms in the troponin I gene family and other vertebrate gene families

The sequences of the adenylate kinase gene (adk) and the RecA gene (recA) were determined from the same isolates ofNeisseria gonorrhoeae, N. meningitidis, N. lactamica, N. polysaccharea, N. cinerea, N. mucosa, N. pharyngis var.flava, N. flavescens, andN. animalis. The patterns of sequence divergence observed atadk andrecA were very different. Dendrograms constructed from therecA data using two different algorithms were statistically robust and were congruent with each other and with the relationships between the species previously proposed using other data. In contrast, the dendrograms derived from theadk data were noncongruent with each other, and with those from therecA data, and were statistically poorly supported. These results, along with the uniform distribution of pairwise sequence divergences between the species atadk, suggest there has been a history of interspecies recombination within theadk gene of the humanNeisseria species which has obscured the phylogenetic relationships between the species. This view was supported by Sawyer's runs test, and the Index of Association (I_A) between codons, which provided significant evidence for interspecies recombination between theadk genes from the humanNeisseria species, but no evidence of interspecies recombination between therecA sequences.     

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

<Emphasis Type="Italic"> Cis</Emphasis>-acting elements sufficient for induction of<Emphasis Type="Italic"> FDH1</Emphasis> expression by formate in the methylotrophic yeast<Emphasis Type="Italic"> Candida boidinii</Emphasis>

The FDH1 gene of Candida boidinii encodes an NAD⁺-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5 non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.Communicated by C. P. Hollenberg     

Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.     

The<Emphasis Type="Italic"> GAOLAOZHUANGREN2</Emphasis> gene is required for normal glucose response and development of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Recent studies of glucose (Glc) sensing and signaling have revealed that Glc acts as a critical signaling molecule in higher plants. Several Glc sensing-defective Arabidopsis mutants have been characterized in detail, and the corresponding genes encoding Glc-signaling proteins have been isolated. However, the full complexity of Glc signaling in higher plants is not yet fully understood. Here, we report the identification and characterization of a new Glc-insensitive mutant, gaolaozhuangren2 (glz2), which was isolated from transposon mutagenesis experiments in Arabidopsis. In addition to its insensitivity to Glc, the glz2 plant exhibits several developmental defects such as short stature with reduced apical dominance, short roots, small and dark-green leaves, late flowering and female sterility. Treatment with 4% Glc blocked expression of the OE33 gene in wild-type plants, whereas expression of this gene was unchanged in the glz2 mutant plants. Taken together, our results suggest that the GLZ2 gene is required for normal glucose response and development of Arabidopsis.Mingjie Chen and Xiaoxiang Xia contributed equally to this work.     

The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions

Summary We have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo⁺ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.     

Construction of an Azospirillum brasilense Sp7 recA mutant

Summary Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant. Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A. brasilense recA gene on a 1.2 kb DNA fragment. One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A. brasilense by marker exchange. The resulting A. brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5 regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.