The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions

Genetic instability in Streptomyces species often involves large deletions sometimes accompanied by DNA amplification. Two such systems in Streptomyces lividans 66 involve the production of mutants sensitive to chloramphenicol and the production of mutants resistant to the galactose analogue 2-deoxygalactose, respectively. Overlapping cosmids were isolated that span the ca. 1 Mb region between the two amplifiable regions. The structure of the region was confirmed by restriction mapping using the rarely cutting enzymes AseI, BfrI and DraI and pulsed-field gel electrophoresis. The region contains a non-clonable gap flanked by inverted repeats; the structure is consistent with the presence of a physical gap, i.e. a linear chromosome.     

Atrial Fibrillation-Linked Germline GJA5/Connexin40 Mutants Showed an Increased Hemichannel Function

Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40) have been linked to lone atrial fibrillation. Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting atrial fibrillation. However, the effects of the atrial fibrillation-linked Cx40 mutants on hemichannel function have not been studied. Here we investigated two atrial fibrillation-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing either one of these two mutants displayed prominent propidium iodide-uptake distinct from cells expressing wild-type Cx40 or other atrial fibrillation-linked Cx40 mutants, I75F, L229M, and Q49X. Propidium iodide-uptake was sensitive to [Ca²⁺]_o and the hemichannel blockers, carbenoxolone, flufenamic acid and mefloquine, but was not affected by the pannexin 1 channel blocking agent, probenecid, indicating that uptake is most likely mediated via connexin hemichannels. A gain-of-hemichannel function in these two atrial fibrillation-linked Cx40 mutants may provide a novel mechanism underlying the etiology of atrial fibrillation.     

A cluster of cell division genes maps to the terC region of the chromosome of Escherichia coli K-12

Thirty-nine cell division mutants were isolated in Escherichia coli K-12 and were mapped in the terminus region of the chromosome, between 33.5 and 36 min. They were obtained by two different approaches involving specific mutagenesis of the terC region. The mutants could be divided into eight classes (I to VIII) based on their map position and phenotype at the restrictive temperature, and constitute a new cell division gene cluster.     

Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae

A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap. Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks. However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.     

Essential residues for DNA binding activity of ManR fromAnabaena sp. PCC 7120

Cyanobacterial ManR is a member of the OmpR family of response regulator that regulates the expression of themntABC andmntH in response to Mn²⁺ signals. Single-alanine substitutions of I204, L207 and R208 residues of the ManR, which constituted the DNA recognition helix, were obtained by the overlap extension method of PCR. EMSA was used to detect the complexes of the proteins of ManR mutants I204A, L207A, R208A, and the DNA fragment of promoter region of themntH gene fromAnabaena sp. PCC 7120. Results showed the formation of the complexes of the proteins of ManR mutants and DNA could not be detected, indicating that the mutagenesis of the residues I204, L207 and R208 in the ManR HTH domain could lead to the elimination of DNA binding activity of the ManR. Homologous analysis showed that residues I204, L207 and R208 of the ManR are also conservative in the αhelix 3 region of effector domain of other proteins of OmpR/PhoB subfamily, indicating that they are essential residues for DNA binding activity. No significant alteration between wild type and mutant proteins was detected by Far-UV CD spectra at the secondary structure level.     

Cloning and Mapping of a Novel Nodulation Region from Bradyrhizobium japonicum by Genetic Complementation of a Deletion Mutant

The phenotypes of a set of Bradyrhizobium japonicum 110 mutants with large deletions in the region of symbiotic gene cluster I were tested. The majority of the mutants showed a delayed nodulation on soybean and, by mixed-infection experiments, were found to be strongly reduced in their competitiveness. Phenotypic comparison of mutants with different deletion endpoints allowed a preliminary localization of two genomic regions, called nod-1 and nod-2, which were required for normal nodulation on soybean. Loss of nod-1 was found to result in a Nod⁻ phenotype on cowpea, mung bean, and siratro. A recombinant cosmid was identified which fully restored nodulation ability of a mutant lacking nod-1. Using Tn5-containing derivatives and subclones of this cosmid for complementation, we delimited the nod-1 region to a DNA segment of 3.1 to 3.5 kilobase pairs.     

Specific Amino Acid Substitutions in the Proline-Rich Motif of the Rhizobium meliloti ExoP Protein Result in Enhanced Production of Low-Molecular-Weight Succinoglycan at the Expense of High-Molecular-Weight Succinoglycan

The production of the acidic exopolysaccharide succinoglycan (EPS I) by Rhizobium meliloti exoP* mutants expressing an ExoP protein lacking its C-terminal cytoplasmic domain and by mutants characterized by specific amino acid substitutions in the proline-rich motif (RX₄PX₂PX₄SPKX₉IXGXMXGXG) located from positions 443 to 476 of the ExoP protein was analyzed. The absence of the C-terminal cytoplasmic ExoP domain (positions 484 to 786) and the substitution of both arginine₄₄₃ by isoleucine₄₄₃ and proline₄₅₇ by serine₄₅₇ within the proline-rich motif resulted in enhanced production of low-molecular-weight (LMW) EPS I at the expense of high-molecular-weight (HMW) EPS I. The ratios of HMW to LMW EPS I of the wild type and mutant strains increased with osmolarity.     

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background

Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase.

Methodology/Principal Findings

To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage.

Conclusions/Significance

This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.     

Efficient gene targeted random mutagenesis in genetically stable Escherichia coli strains

We describe a method to generate in vivo collections of mutants orders of magnitude larger than previously possible. The method favors accumulation of mutations in the target gene, rather than in the host chromosome. This is achieved by propagating the target gene on a plasmid, in Escherichia coli cells, within the region preferentially replicated by DNA polymerase I (Pol I), which replicates only a minor fraction of the chromosome. Mutagenesis is enhanced by a conjunction of a Pol I variant that has a low replication fidelity and the absence of the mutHLS system that corrects replication errors. The method was tested with two reporter genes, encoding lactose repressor or lipase. The proportion of mutants in the collection was estimated to reach 1% after one cycle of growth and 10% upon prolonged cell cultivation, resulting in collections of 10¹²–10¹³ mutants per liter of cell culture. The extended cultivation did not affect growth properties of the cells. We suggest that our method is well suited for generating protein variants too rare to be present in the collections established by methods used previously and for isolating the genes that encode such variants by submitting the cells of the collections to appropriate selection protocols.     

Tandem duplications resulting from recombination between ribosomal RNA genes in Escherichia coli.

A set of Escherichia coli K12 mutants, which carry a tandem duplication of the glyT purD region, have been analyzed. Three types of duplications have occurred repeatedly, and we show that they were generated by recombination between the ribosomal RNA gene, rrnE, which lies to one side of the glyT purD region and one of threerrn genes which occur as direct repetitions on the other side of this region. Characterization of these duplication mutants has involved the isolation of the duplicated material in the form of a DNA circle. Class I duplications, which extend from rrnE to rrnE, are 39,500 base-pairs long, class II duplications, which extend from rrnA to rrnE, are 164,000 base-pairs long, and class III duplications, which extend from rrnC to rrnE, are 258,000 base-pairs long.     

Mutations altering the cleavage specificity of a homing endonuclease

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.     

The synaptonemal complex central region modulates crossover pathways and feedback control of meiotic double-strand break formation

The synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. In budding yeast, Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. During SC elongation, the SUMOylated SC component Ecm11 and the Ecm11-interacting protein Gmc2 facilitate the polymerization of Zip1, an SC central region component. Through physical recombination, cytological, and genetic analyses, we found that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies on a short chromosome (chromosome III) were reduced, whereas CO and non-crossover frequencies on a long chromosome (chromosome VII) were elevated. Further, in ecm11 and gmc2 mutants, more double-strand breaks (DSBs) were formed on a long chromosome during late prophase I, implying that the Ecm11–Gmc2 (EG) complex is involved in the homeostatic regulation of DSB formation. The EG complex may participate in joint molecule (JM) processing and/or double-Holliday junction resolution for ZMM-dependent CO-designated recombination. Absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role for the EG complex in suppressing ZMM-independent recombination. Our results suggest that the SC central region functions as a compartment for sequestering recombination-associated proteins to regulate meiosis specificity during recombination.     

Lambda CIN-1, a New Mutation Which Enhances Lysogenization by Bacteriophage Lambda, and the Genetic Structure of the Lambda CY Region

Seven lambda cy mutants have been mapped within a small region located approximately halfway between the rightward boundary of the imm⁴³⁴ region and the lambda cII gene. The seven mutants lie at four sites separated by a total distance of about 12 nucleotide pairs, as estimated from recombination frequencies. Six of the seven mutants lie on the right side of the cy fine structure map, spanning a total distance of about 3–5 nucleotide pairs. Lying approximately 11–21 nucleotide pairs to the left of the leftmost cy mutant is a newly described mutation called cin-1, for c independent. The cin-1 mutation allows some lysogenization when coupled with any cy, cII or cIII mutant, but not when coupled with a defective cI gene. The cin-1 mutation, like cy mutants, has a cis-dominant action upon the cI gene in mixed infections. The observation that λimm⁴³⁴ cin-1 cy2001 lysogenizes efficiently, but not λimm⁴³⁴ cin-1 cy2001 cII68 nor any other λimm⁴³⁴ cin-1 cy derivative, is interpreted to mean that all of the cy mutants on the right side of the cy fine structure map inactivate a binding site for cII/cIII function, but that cy2001, the single mutant on the left side of the cy fine structure map, does not inactivate that binding site.     

Trifluoroleucine resistance and regulation of α-isopropyl malate synthase in Saccharomyces cerevisiae

Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level. The gene responsible for the resistance was cloned from one of the mutants (FSC2.4). Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes α-isopropyl malate synthase I (α-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3′ end of the LEU4 ORF. Six different point mutations – four transitions and two transversions – were found in the remaining mutants. α-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains. In the other two the enzyme was resistant to Zn²⁺-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism. The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn²⁺-mediated inactivation by CoA. Deletion experiments involving the R-region showed that it is also necessary for enzyme activity.     

An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.     

Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation

Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein's pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.     

Protein A Mutants of Staphylococcus aureus

Staphylococcus aureus Cowan I was exposed to nitrosoguanidine or ethyl-methanesulfonate, and survivors were screened on nutrient agar plates containing rabbit anti-protein A serum for loss of protein A production. More than half of all protein A-deficient mutants also lacked nuclease, coagulase, alpha hemolysin, fibrinolysin, mannitol utilization, and the phage-type pattern. Mutants with a spectrum of these properties were also isolated. Induced or spontaneous reversions of the mutants were observed. The properties of the protein A-deficient mutants suggest that synthesis or release (or both) of a number of extracellular products of S. aureus is controlled by a common regulatory mechanism.     

Loop B is a major structural component of the 5-HT3 receptor

The 5-HT₃ receptor belongs to a family of therapeutically important neurotransmitter-gated receptors whose ligand binding sites are formed by the convergence of six peptide loops (A-F). Here we have mutated 15 amino acid residues in and around loop B of the 5-HT₃ receptor (Ser-177 to Asn-191) to Ala or a residue with similar chemical properties. Changes in [³H]granisetron binding affinity (K_d) and 5-HT EC₅₀ were determined using receptors expressed in human embryonic kidney 293 cells. Substitutions at all but one residue (Thr-181) altered or eliminated binding for one or both mutants. Receptors were nonfunctional or EC₅₀ values were altered for all but two mutants (S182T, I190L). Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the β-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor. The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein. Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.