Identification and characterization of the Myxococcus xanthus bsgA gene product.

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.     

Mutations in the Sup-38 Gene of Caenorhabditis Elegans Suppress Muscle-Attachment Defects in Unc-52 Mutants

Mutations in the unc-52 locus of Caenorhabditis elegans have been classified into three different groups based on their complex pattern of complementation. These mutations result in progressive paralysis (class 1 mutations) or in lethality (class 2 and 3 mutations). The paralysis exhibited by animals carrying class 1 mutations is caused by disruption of the myofilaments at their points of attachment to the cell membrane in the body wall muscle cells. We have determined that mutations of this class also have an effect on the somatic gonad, and this may be due to a similar disruption in the myoepithelial sheath cells of the uterus, or in the uterine muscle cells. Mutations that suppress the body wall muscle defects of the class 1 unc-52 mutations have been isolated, and they define a new locus, sup-38. Only the muscle disorganization of the Unc-52 mutants is suppressed; the gonad abnormalities are not, and the suppressors do not rescue the lethal phenotype of the class 2 and class 3 mutations. The suppressor mutations on their own exhibit a variable degree of gonad and muscle disorganization. Putative null sup-38 mutations cause maternal-effect lethality which is rescued by a wild-type copy of the locus in the zygote. These loss-of-function mutations have no effect on the body wall muscle structure.     

Identification of esg, a genetic locus involved in cell-cell signaling during Myxococcus xanthus development.

JD258, a Tn5 insertion mutant of Myxococcus xanthus, was shown to have major defects in three development-associated properties: expression of the developmentally regulated tps gene, spore formation, and production of multicellular fruiting bodies. The defects in tps gene expression and sporulation could be substantially corrected, at the phenotypic level, by mixing JD258 with wild-type cells (extracellular complementation). By this criterion, JD258 appeared to be a new member of a group of conditional developmental mutants that were previously characterized and placed in four extracellular complementation groups (A to D) based on the ability of mutants in one group to stimulate development in mutants belonging to a different group (D. C. Hagen, A. P. Bretscher, and D. Kaiser, Dev. Biol. 64:284-296, 1978). Mutants from groups A, B, C, and D all displayed extracellular complementation activity when mixed with JD258. These results, and other aspects of the phenotype of JD258, indicate that this mutant defines a fifth extracellular complementation group, group E. The M. xanthus esg locus identified by the Tn5 insertion in JD258 was cloned in Escherichia coli and used for further genetic analysis of the locus. These studies indicated that the esg locus resides within a 2.5-kb region of the M. xanthus chromosome and that the locus contains at least two genetic complementation groups. Our results are consistent with a model in which the esg locus controls the production of a previously unrecognized extracellular signal that must be transmitted between cells for the completion of M. xanthus development.     

Second Site Mutations Specifically Suppress the Fix(-) Phenotype of Rhizobium Meliloti Ndvf Mutations on Alfalfa: Identification of a Conditional Ndvf-Dependent Mucoid Colony Phenotype

Rhizobium meliloti mutants carrying ndvF insertion or deletion mutations induce nodules on alfalfa which contain very few infected cells and fail to fix N(2) (Fix(-)). We have characterized five independent second site mutations (designated sfx) which completely suppress the Fix(-) phenotype of ndvF mutants on Medicago sativa but not on another R. meliloti host Melilotus alba. Genetic mapping and phenotypic analysis revealed that the suppressor mutations sfx-1, sfx-4 and sfx-5 mapped to a single locus which was distinct from another locus defined by the sfx-2 and sfx-3 mutations. Tn5-mob-mediated conjugal mapping experiments showed that the sfx-1 locus was located clockwise from trp-33 on the R. meliloti chromosome and a detailed cotransduction map of this region was generated. To clone the sfx-1 locus, we prepared a cosmid library from total DNA obtained from an sfx-1, ndvF deletion strain. From this library, a cosmid pTH56, which converted Fix(-) ndvF mutants to Fix(+), was isolated. Southern blot analysis provided direct physical evidence that the insert DNA in plasmid pTH56 was contiguous with the sfx-1 region. On low osmolarity glutamate-yeast extract-mannitol-salts medium (GYM) agar medium, ndvF insertion and deletion mutants were found to have a mucoid colony phenotype, as opposed to the dry colony phenotype of the wild-type strain. This phenotype was shown to be dependent on the exoB and expE genes required for synthesis of exopolysaccharide II in R. meliloti but not to be dependent on genes required exclusively for the synthesis of the succinoglycan or exopolysaccharide I. Transduction of either sfx-1 or sfx-2 or transfer of the cosmid pTH56 into the ndvF mutants restored them to a wild-type dry colony phenotype. The mucoid phenotype is not responsible for the Fix(-) phenotype of ndvF mutants as the Fix(-), ndvF exp double mutants can be complemented to Fix(+) by introducing plasmids which carry only the wild-type ndvF genes.     

AsgD, a new two-component regulator required for A-signalling and nutrient sensing during early development of Myxococcus xanthus

Myxococcus xanthus has a complex life cycle that includes fruiting body formation. One of the first stages in development has been called A-signalling. The asg (A-signalling) mutants have been proposed to be deficient in producing A-signal, resulting in development arresting at an early stage. In this paper, we report the identification of a new asg locus asgD. This locus appears to be involved in both environmental sensing and intercellular signalling. Expression of asgD was undetected during vegetative growth, but increased dramatically within 1 h of starvation. The AsgD protein is predicted to contain 773 amino acids and to be part of a two-component regulatory system because it has a receiver domain located at the N-terminus and a histidine protein kinase at the C-terminus. An asgD null mutant was defective in fruiting body formation and sporulation on CF medium. However, the defects of the mutant were complemented extracellularly when cells were mixed with wild-type strains or with bsgA, csgA, dsgA or esgA mutants, but were not complemented extracellularly by asgA, asgB or asgC mutants. In addition, the mutant was rescued by a subset of A-factor amino acids. Surprisingly, when the mutant was plated on stringent starvation medium rather than CF, cells were able to form fruiting bodies. Thus, it appears that AsgD is directly or indirectly involved in sensing nutritionally limiting conditions. The discovery of the asgD locus provides an important sensory transduction component of early development in M. xanthus.     

β-Glucuronidase Mutants of the Nematode CAENORHABDITIS ELEGANS

Using a screening procedure that is based on a histochemical stain for the enzyme beta-glucuronidase, we have isolated several mutants of the nematode Caenorhabditis elegans affected in beta-glucuronidase activity. All of the mutations fall into one complementation group and identify a new gene, gus-1, which has been mapped on the right arm of linkage group I (LG I), 1.1 map units to the left of unc-54. The mutants have no visible phenotype, and their viabilities and fertilities are unaffected. Linked revertants of two of the mutations have been isolated. They restore enzyme activity to almost wild-type levels; the beta-glucuronidase that one of the revertants produces differs from that of the wild type. We propose that gus-1 is the structural locus for beta-glucuronidase.     

Identification of a Bdellovibrio bacteriovorus genetic locus, hit, associated with the host-independent phenotype.

Bdellovibrios invade and grow within the periplasmic space of suitable gram-negative bacteria. Wild-type bdellovibrios are obligately dependent on host cells for growth, but spontaneous host-independent (H-I) mutants that grow axenically on standard rich culture media can be isolated. Such mutants generally retain the ability to grow intraperiplasmically, although the plaques that they produce on lawns of host cells are smaller and more turbid than those produced by wild-type bdellovibrios. Here, we identify the first genetic locus associated with the H-I phenotype: hit (host interaction). We show that three individual H-I mutants suffered mutations at the hit locus and that recombination of wild-type hit sequences into the genomes of the H-I mutants greatly enhanced their plaquing ability. DNA sequence analysis localized the hit mutation in each of the H-I mutants to a 135-bp region of the genome. Mutations at hit may not fully account for the H-I phenotype, however, as recombination of wild-type hit sequences into the genomes of the H-I mutants had little effect on the axenic-growth phenotype of the mutants. Possible explanations for this result and potential roles for the hit locus are discussed.     

Intercellular signaling is required for developmental gene expression in Myxococcus xanthus

Certain developmental mutants of Myxococcus xanthus can be complemented (extracellularly) by wild-type cells. Insertions of Tn5 lac (a transposon which couples beta-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A- mutations reduced developmental beta-galactosidase expression from 18 of 21 Tn5 lac insertions tested and that expression was restored to A- Tn5 lac cells by adding wild-type cells. The earliest A-dependent Tn5 lac normally expresses beta-galactosidase at 1.5 hr of development indicating a developmental block at 1-2 hr in A- mutants. A substance which can rescue the expression of this early Tn5 lac is released by wild-type (A+) but not by A- cells. This substance appears in a cell-free wash of wild-type cells or in starvation buffer conditioned by wild-type cells 1-2 hr after development is initiated. The conditioned starvation buffer also restores normal morphological development to an A- mutant.     

Homo-oligomer formation by basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domain.

Basigin (Bsg) is a highly glycosylated transmembrane protein with two immunoglobulin (Ig)-like domains. A number of studies, including gene targeting, have demonstrated that Bsg plays pivotal roles in spermatogenesis, implantation, neural network formation and tumor progression. In the present study, to understand the mechanism of action of Bsg, we determined its expression status on the plasma membrane. Cotransfection of Bsg expression vectors with two different tags clarified that Bsg forms homo-oligomers in a cis-dependent manner on the plasma membrane. If the disulfide bond of the more N-terminally located Ig-like domain was destroyed by mutations, Bsg could not form oligomers. In contrast, the mutations of the C-terminal Ig-like domain or N-glycosylation sites did not affect the association. The association of mouse and human Bsgs, which exhibit high homology in the transmembrane and intracellular domains but low homology in the extracellular domain, was very weak as compared with that within the same species, suggesting the importance of the extracellular domain in the association. If the extracellular domain of the human Ret protein was replaced with the N-terminal Ig-like domain of Bsg, the resulting chimera protein was associated with intact wild-type Bsg, but not if the C-terminal Ig-like domain, instead of the N-terminal one, of Bsg was used. No oligomer formation took place between the intact wild-type Ret and Bsg proteins. In conclusion, these data indicate that the N-terminal Ig-like domain is necessary and sufficient for oligomer formation by Bsg on the plasma membrane.     

A Genetic Analysis of Suppressors of the Pf10 Mutation in Chlamydomonas Reinhardtii

A mutation at the PF10 locus of the unicellular green alga Chlamydomonas reinhardtii leads to abnormal cell motility. The asymmetric form of the ciliary beat stroke characteristic of wild-type flagella is modified by this mutation to a nearly symmetric beat. We report here that this abnormal motility is a conditional phenotype that depends on light intensity. In the absence of light or under low light intensities, the motility is more severely impaired than at higher light intensities. By UV mutagenesis we obtained 11 intragenic and 70 extragenic strains that show reversion of the pf10 motility phenotype observed in low light. The intragenic events reverted the motility phenotype of the pf10 mutation completely. The extragenic events define at least seven suppressor loci; these map to linkage groups IV, VII, IX, XI, XII and XVII. Suppressor mutations at two of the seven loci (LIS1 and LIS2) require light for their suppressor activity. Forty-eight of the 70 extragenic suppressors were examined in heterozygous diploid cells; 47 of these mutants were recessive to the wild-type allele and one mutant (bop5-1) was dominant to the wild-type allele. Complementation analysis of the 47 recessive mutants showed unusual patterns. Most mutants within a recombinationally defined group failed to complement one another, although there were pairs that showed intra-allelic complementation. Additionally, some of the mutants at each recombinationally defined locus failed to complement mutants at other loci. They define dominant enhancers of one another.     

Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii.

We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region.     

A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro

We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B. avium. The mutants also acquired resistance to a newly identified B. avium-specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O-antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild-type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.     

Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion

Bacillus subtilis succinate dehydrogenase (SDH) is composed of two unequal subunits designated Fp (Mr, 65,000) and Ip (Mr. 28,000). The enzyme is structurally and functionally complexed to cytochrome b 558 (Mr, 19,000) in the membrane. A total of 21 B. subtilis SDH-negative mutants were isolated. The mutants fall into five phenotypic classes with respect to the presence and localization of the subunits of the SDH-cytochrome b558 complex. One class contains mutants with an inactive membrane-bound complex. Membrane-bound enzymatically active SDH could be reconstituted in fused protoplasts of selected pairs of SDH-negative mutants. Most likely reconstitution is due to the assembly of preformed subunits in the fused cells. On the basis of the reconstitution data, the mutants tested could be divided into three complementation groups. The combined data of the present and previous work indicate that the complementation groups correspond to the structural genes for the three subunits of the membrane-bound SDH-cytochrome b558 complex. A total of 31 SDH-negative mutants of B. subtilis have now been characterized. The respective mutations all map in the citF locus at 255 degrees on the B. subtilis chromosomal map. In the present paper, we have revised the nomenclature for the genetics of SDH in B. subtilis. All mutations which give an SDH-negative phenotype will be called sdh followed by an isolation number. The designation citF will be omitted, and the citF locus will be divided into three genes: sdhA, sdhB, and sdhC. Mutations in sdhA affect cytochrome b558, mutations in sdhB affect Fp, and mutations in sdhC affect Ip.     

Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors.

The establishment of bovine papillomavirus type 1 in somatic mammalian cells is mediated by extrachromosomal replication and stable maintenance of the viral genome as a multicopy nuclear plasmid. Previous studies indicated the requirement of viral gene expression for bovine papillomavirus type 1 replication and plasmid maintenance (M. Lusky and M. R. Botchan, Cell 36:391-401, 1984; Turek et al., Proc. Natl. Acad. Sci. U.S.A. 79:7914-7918, 1982). To define the viral genes which are necessary for this process, we constructed a series of specific mutations within the viral genome and assayed the resulting mutants for their ability to replicate extrachromosomally in mouse C127 cells. We report here that the bovine papillomavirus type 1 trans-acting replication factors were encoded by at least two distinct viral genes since the mutants fell into two complementation groups, rep and cop. Mutants (rep-) affecting the E1 open reading frame (ORF) failed to replicate bovine papillomavirus type 1 DNA extrachromosomally and would integrate into chromosomal DNA. We suggest that this gene product is one of the factors required to specifically preclude the integration event. Mutants (cop-) affecting the E7 ORF were maintained in the extrachromosomal state; however, the copy number of the mutant genomes was reduced 100-fold compared with that of wild-type DNA. Analysis of single-cell subclones showed that each cell contained the mutant genomes at a copy number of one to two, indicating that the cop- phenotype did not reflect a simple segregation defect. We propose that the gene defined by mutations in the E7 ORF played a crucial role in stably maintaining the copy number of the viral plasmid at high levels. Genomes with mutations in the cop and rep complementation groups, when cotransfected, rescued the wild-type phenotype, extrachromosomal replication with a high, stable copy number for both types of plasmids. Therefore, the gene products acted in trans, and the mutations were recessive to the wild-type functions. One specific rep- mutant showed a 30-fold-increased transformation efficiency when compared with that of the wild-type genome. In addition, morphological transformation mediated by the cop- mutants appeared to be unstable. These results imply that either or both of the replication functions played some role in regulating the expression of the viral transforming functions.     

Molecular organization of the cut locus of Drosophila melanogaster

Mutations of the cut locus (ct) of Drosophila can be divided into four groups based on their phenotypes and complementation patterns. Each group alters the phenotype of a different set of tissues. Two hundred kilobases of ct DNA, located in 7B1-2, have been cloned by chromosomal walking, and the cloned sequences have been used to analyze more than 40 mutants. Based on the location of transposable element mutations and the extent of deficiencies and an inversion, four cut locus regions can be defined. Mutations in each region affect the phenotype of a different set of tissues. The most centromere proximal region contains mutations that are null for cut locus function. Within individual regions, a higher level of organization can be detected.     

Drosophila basement membrane collagen col4a1 mutations cause severe myopathy

Recent data from clinical and mammalian genetic studies indicate that COL4A1 mutations manifest with basement membrane defects that result in muscle weakness, cramps, contractures, dystrophy and atrophy. In-depth studies of mutant COL4A1-associated muscle phenotype, however, are lacking and significant details of the muscle-specific pathomechanisms remain unknown. In this study, we have used a comprehensive set of Drosophila col4a1 and col4a2 mutants and a series of genetic and mutational analyses, gene, protein expression, and immunohistochemistry experiments in order to establish a Drosophila model and address some of these questions. The Drosophila genome contains two type IV collagen genes, col4a1 and col4a2. Mutant heterozygotes of either gene are viable and fertile, whereas homozygotes are lethal. In complementation analysis of all known mutants of the locus and a complementation matrix derived from these data we have identified the dominant lesions within the col4a1, but not within the col4a2 gene. Expression of a col4a1 transgene partially rescued the dominant and recessive mutant col4a1 alleles but not the col4a2 mutations that were all recessive. Partial complementation suggested that col4a1 gene mutations have strong antimorph effect likely due to the incorporation of the mutant protein into the triple helix. In col4a1 mutants, morphological changes of the oviduct muscle included severe myopathy with centronuclear myofibers leading to gradual development of female sterility. In larval body wall muscles ultrastructural changes included disturbance of A and I bands between persisting Z bands. In the most severely affected DTS-L3 mutant, we have identified four missense mutations within the coding region of the col4a1 gene two of which affected the Y within the Gly-X-Y unit and a 3' UTR point mutation. In conclusion, our Drosophila mutant series may serve as an effective model to uncover the mechanisms by which COL4A1 mutations result in compromised myofiber-basement membrane interactions and aberrant muscle function.     

A Conditional Mutant Having Paralyzed Cilia and a Block in Cytokinesis Is Rescued by Cytoplasmic Exchange in Tetrahymena Thermophila

Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.