Mutants of Dictyostelium discoideum with altered carbohydrate moieties of contact site A.

Mutants of Dictyostelium discoideum were isolated and found to be defective in the epitope recognized by the monoclonal antibody 120 against the carbohydrate moieties of an integral membrane glycoprotein, contact site A, with the apparent molecular mass of 80 x 10(3). One mutant, HG764, did not express any contact site A and had lost cell contact resistant to EDTA. The others, including HG794, expressed a 68-kDa form of contact site A. In comparison with the parental strain HG592, HG794 showed weaker EDTA-resistant cell contact and the same degree of EDTA-sensitive cell contact. This suggested that the moieties which HG794 lacked were involved in EDTA-resistant cell contact. The 68-kDa contact site A in HG794 could be labeled with wheat germ agglutinin and incorporated [35S] sulfate. The modB mutant HL220 also expresses 68-kDa contact site A, although it cannot be labeled with wheat germ agglutinin. Therefore, the mutants HG794 and HL220 were compared by a complementation test. The diploid strain DG701 expressed 80-kDa contact site A and showed the same degree of EDTA-resistant cell contact as strain HG592. In its EDTA-resistant cell contact, HG794 was stronger than HL220. These results suggest that HG794 is a new mutant, and that there might be at least two processes in the glycosylation of 68-kDa contact site A to the 80-kDa form. The carbohydrate moieties recognized by monoclonal antibody 120 and by wheat germ agglutinin might be involved in EDTA-resistant cell contact.     

Attenuated virulence of min operon mutants of Neisseria gonorrhoeae and their interactions with human urethral epithelial cells

Neisseria gonorrhoeae, a sexually-transmitted gram-negative bacterium, causes gonorrhoea in humans. The min genes of N. gonorrhoeae are involved in cell division site selection with oxyR co-transcribed with these genes. The mutation in min genes and oxy R cause aberrant cell morphology and aggregation patterns, respectively. Our objective was to assess the contribution of neisserial min operon cell division genes i.e. minC, minD and oxyR in virulence. Compared to the N. gonorrhoeae parental strain (Ng CH811Str(R)), its isogenic mutants with insertionally inactivated minC (Ng CSRC1), minD (Ng CJSD1) or oxyR (Ng KB1) showed reduced adherence to and invasion of urethral epithelial cells. This may be explained by defective microcolony formation in the mutant strains, possibly owing to abnormal morphology and aggregation. The expression levels of surface virulence factors like Opa, pilin and lipooligosaccharide in the mutants were unchanged relative to Ng CH811Str(R). Furthermore, in urethral epithelial cells, the min and oxyR mutants induced the release of proinflammatory cytokines like IL6 and IL8 to levels similar to that induced by the parental strain. Taken together, our studies indicate that inactivation of minC, minD or oxyR in N. gonorrhoeae attenuates its ability to bind to and invade urethral epithelial cells without altering its potential to induce IL6 and IL8 release.     

Selection of Dictyostelium mutants defective in cytoskeletal proteins: use of an antibody that binds to the ends of alpha-actinin rods.

A monoclonal antibody, mAb 47-19-2, was used to study the subunit topology of the rod-shaped alpha-actinin molecules of Dictyostelium discoideum and to screen for mutants defective in the production of alpha-actinin. Electron microscopy of rotary-shadowed alpha-actinin-antibody complexes showed binding of mAb 47-19-2 to both ends of the alpha-actinin rods and cleavage of the rods into its subunits, indicating that the two subunits of alpha-actinin extend in an anti-parallel mode through the whole length of the rod. The antibody binding sites were located in close proximity to the sites responsible for actin cross-linking, which is consistent with the blocking activity of the antibody. In a mutant, HG1130, no antibody label was detected in colony blots, and by immunoblotting of mutant proteins separated by SDS-PAGE, only trace amounts of alpha-actinin were found. The mutant showed normal binding of antibodies directed against the actin-binding proteins severin and capping protein. The mutation responsible for the alpha-actinin defect was recessive and located on linkage group I of the genetic map of D. discoideum. HG1130 cells grew on bacteria at a normal rate and also axenically like cells of the parent strain AX2. After starvation the mutant cells expressed the contact site A glycoprotein, a marker of the aggregation-competent stage, and reacted chemotactically to cyclic AMP. The aggregation patterns and fruiting bodies of the mutant appeared to be normal. Patching and capping on the surface of HG1130 cells was induced by antibodies against the contact site A glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of Pseudomonas aeruginosa PAO mutant that produces altered elastase.

Pseudomonas aeruginosa PAO mutants defective in elastase were isolated by plate assays of nitrosoguanidine-mutagenized clones. A total of 75 elastase mutants were isolated from 43,000 mutagenized clones. One mutant (PAO-E64) was apparently identical to the parental strain except for its deficiency in elastase activity. This mutant produced an enzyme which was antigenically indistinguishable from parental elastase. Furthermore, equal levels of elastase antigen were produced by this mutant and its parental strain. The mutant elastase, however, had greatly reduced enzymatic activity. Mutant PAO-E64 is presumed to have a mutation in the structural gene for elastase. We have designated the genotype of the mutation in PAO-E64 as lasA1.     

Siderophore-controlled iron assimilation in the enterobacterium Erwinia chrysanthemi: evidence for the involvement of bacterioferritin and the Suf iron-sulfur cluster assembly machinery

The intracellular fate of iron acquired by bacteria during siderophore-mediated assimilation is poorly understood. We investigated this question in the pathogenic enterobacterium Erwinia chrysanthemi. This bacterium produces two siderophores, chrysobactin and achromobactin, during plant infection. We analyzed the distribution of iron into cytosolic proteins in bacterial cells supplied with 59Fe-chrysobactin using native gel electrophoresis. A parental strain and mutants deficient in bacterioferritin (bfr), miniferritin (dps), ferritin (ftnA), bacterioferredoxin (bfd), or iron-sulfur cluster assembly machinery (sufABCDSE) were studied. In the parental strain, we observed two rapidly 59Fe-labeled protein signals identified as bacterioferritin and an iron pool associated to the protein chain-elongation process. In the presence of increased 59Fe-chrysobactin concentrations, we detected mini-ferritin-bound iron. Iron incorporation into bacterioferritin was severely reduced in nonpolar sufA, sufB, sufD, sufS, and sufE mutants but not in a sufC background. Iron recycling from bacterioferritin did not occur in bfd and sufC mutants. Iron depletion caused a loss of aconitase activity, whereas ferric chrysobactin supplementation stimulated the production of active aconitase in parental cells and in bfr and bfd mutants. Aconitase activity in sufA, sufB, sufD, sufS, and sufE mutant strains was 10 times lower than that in parental cells. In the sufC mutant, it was twice as low as that in the parental strain. Defects observed in the mutants were not caused by altered ferric chrysobactin transport. Our data demonstrate a functional link between bacterioferritin, bacterioferredoxin, and the Suf protein machinery resulting in optimal bacterial growth and a balanced distribution of iron between essential metalloproteins.     

Effects of Mutational Loss of Specific Intracellular Proteases on the Sporulation of Bacillus subtilis

Two protease-deficient mutants of Bacillus subtilis 168 (Trp(-)) were isolated and compared with the parental strain with respect to production of intracellular proteases and sporulation. A mutant lacking the metal-requiring "neutral" protease intracellularly sporulated as well as the parental strain. A second mutant, deficient in an as yet uncharacterized intracellular protease, failed to sporulate normally. It is proposed that this new protease is also involved in intracellular protein turnover.     

Analysis of Different Types of Competitive Capacity in the Alfalfa Rhizobia (Sinorhizobium meliloti) Tn5 Mutants

Nodulation, rhizospheral, and saprophytic types of competitiveness (NC, RC, and SC, respectively) were studied in the highly active strains CXM1-105 and CXM1-188 of the alfalfa rhizobium Sinorhizobium meliloti.The competitiveness was estimated with the use of markers of antibiotic resistance. It was found that the mutant strain T37, which was characterized by a drastically decreased NC, had higher SC and RC than the parental strain. The mutant T107 (with a moderately decreased NC) did not differ from the parental strain with respect to RC but had a higher SC. The mutant T27 (with the lowest NC) did not differ from the parental strain with respect to SC or RC. In the mutant Tb1, the NC and RC were decreased and the SC was the same as in the parental strain. In Tb7, the SC was decreased and RC was increased. In the mutant T795, all of the three types of competitiveness were decreased. The difference between the mutants studied and the parental strain with respect to NC and RC was confirmed using an indirect method (the ability to form effective symbiosis after mixed inoculation together with the an ineffective tester strain CXM1-48) and the X-Gluc staining method (using the S. melilotiRmM4gustester strain carrying the gene of -glucuronidase). However, the decreased SC that the mutants exhibited when they were cultivated together with parental strains in a plant-growth substrate (vermiculite) was not observed in the case of their cocultivation in liquid media. The independent variation of different types of competitiveness indicate that rhizobia have several separate gene systems determining their survival in in plantaandex plantaecological niches.     

Post-translational glycosylation of the contact site A protein of Dictyostelium discoideum is important for stability but not for its function in cell adhesion

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.     

Mediation of cell-cell adhesion by the altered contact site A glycoprotein expressed in modB mutants of Dictyostelium discoideum

In Dictyostelium discoideum, a surface glycoprotein with Mr 80,000 (gp80) has been found to mediate the EDTA-resistant contact sites A at the aggregation stage of development. To evaluate the role of the carbohydrate moiety in cell-cell adhesion, we have examined the accumulation and activity of an altered gp80 molecule in two glycosylation (modB) mutants. Both mutants synthesize an altered gp80 of lower molecular size. This modB-gp80 can be detected by the monoclonal antibody 80L5C4, which is capable of blocking cell-cell adhesion (C. -H. Siu, T. Y. Lam, and A. Choi, (1985) J. Biol. Chem. 260, 16,030-16,036). The mutant cells exhibit both EDTA-sensitive and EDTA-resistant types of cell-cell binding, though to a lesser extent than that of the parental strain, and the EDTA-resistant binding sites are blocked in the presence of 80L5C4 Fab. Mutant cells can also bind Covaspheres conjugated with gp80. These results suggest that the modB-gp80 protein still retains the domain essential for its cell binding activity and the carbohydrate moiety affected by the modB mutation is not directly involved in cell-cell adhesion.     

Isolation and characterization of Escherichia coli tolC mutants defective in secreting enzymatically active alpha-hemolysin.

This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed alpha-helical domain, while the remaining two mapped within the outer membrane-embedded beta-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.     

Selection and properties of Escherichia coli mutants defective in the synthesis of cyclopropane fatty acids.

Mutants of Escherichia coli K-12 defective in the synthesis of cyclopropane fatty acids (CFA) have been selected and isolated by a L-[methyl-3H]methionine suicide procedure. Two mutants were isolated. Stationary-phase cultures of both mutants contain less than 0.7% of the CFA content found in the parental strain. The CFA deficiency is attributed to a deficiency of CFA synthetase activity. Extracts of both mutants contain less than 10% of the CFA synthetase activity found in extracts of the parental strain. Experiments in which parental and mutant extracts were mixed indicate that the lack of activity in the mutant strains is not due to an inhibitor of CFA synthetase present in the mutant extracts. We have not yet detected a physiological phenotype for these mutants. These strains grow normally at various temperatures in a variety of media. We have tested survival (colony-forming ability) in response to (i) prolonged incubation in stationary phase, (ii) exposure to drying, and (iii) exposure to detergents, heavy metals, low pH, high salt concentration, and a variety of other environmental conditions. The survival of both mutants is identical to that of the parental strain under all conditions tested. The compositions (excepting the CFA deficiency) and metabolic turnover rates of the phospholipids of both mutant strains are indistinguishable from those of the wild-type strain. The transport of several amino acids also seems normal in these mutants.     

Isolation of Cadmium- and Mercury-sensitive Mutants of Escherichia coli and Some Factors Influencing Their Sensitivities

Several Cd²⁺ hypersensitive and Hg²⁺ sensitive mutants of Escherichia coli K-12 were isolated after repeated mutagenesis with nitrosoguanidine. The Cd-hypersensitive mutant, CD17P, could not grow in a chemically defined liquid medium containing 0.5 μm Cd²⁺, and the growth of Hg-sensitive mutant, HG17P-52, was inhibited by 0.9 μm Hg²⁺. Thus, CD 17P and HG17P-52 were respectively about 1000 fold and 4 fold more sensitive than the parental strain.

Both mutants were also considerably more sensitive to Zn²⁺ than their parent, but they did not show any appreciable change in sensitivity to other metals tested. Among the various metabolites and chemicals tested, reducing agents such as cysteine, dithiothreitol and vitamin C showed a protective effect against metal toxicity. Reduced glutathione was effective only against Hg²⁺. EDTA specifically and efficiently reversed only Cd²⁺ toxicity.     

Defects in contact-stimulated gliding during aggregation by Myxococcus xanthus.

During development, Myxococcus xanthus cells glide toward foci of aggregation and produce compact multicellular mounds. We studied development in strains with defects in contact-stimulated gliding. Contact stimulation involves a mechanism influenced by contacts between neighboring cells which stimulates the gliding motility of single cells (Hodgkin and Kaiser, Proc. Natl. Acad. Sci. USA 74:2938-2942, 1977; Hodgkin and Kaiser, Mol. Gen. Genet. 171:167-176, 1979). Most mutants containing a mutation in a single gene affecting contact stimulation (cgl gene) were able to form foci of aggregation during development. However, the aggregates were diffuse, suggesting that contact stimulation is important for morphogenetic movements during aggregation. A mutant containing a mutation in the cglF3 gene showed a striking delay in aggregation, suggesting that the cglF3 gene affects a mechanism stimulating cells moving to foci or affects a mechanism for coordinating early cell behavior. Mutants containing the cglF3 mutation in combination with a cglB, cglC, cglE, or cglF1 mutation had severe defects in aggregation and failed to recover from the early delay. The severity of the defects in mutants containing two cgl mutations suggests that cgl genes are critical for development. We propose that cgl genes stimulate cell movement or control specific contacts between cells during aggregation.     

A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2)

In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.     

Characterization of a Schizosaccharomyces pombe morphological mutant altered in the galactomannan content

In a search for Schizosaccharomyces pombe mutants resistant to the antifungal agent papulacandin B, a morphological mutant was isolated. The mutant is round shaped in contrast to the rod shaped parental strain. This morphological defect segregated as a recessive Mendelian character and was not observed in other papulacandin B resistant mutants belonging to the same complementation group. The mutation mapped in the right arm of S. pombe chromosome III very close to pap1 marker. Mutant cell walls were more susceptible to alkali extraction and Novozyme degradation than those from the wild-type. A specific reduction in the cell wall galactomannan fraction was the only significant difference detected as compared to the wild-type strain. Levels of beta (1,3)-glucan and mannan synthases as well as other enzymic periplasmic mannoproteins were very similar in wild type and mutant strains.     

Evidence for the involvement of vacuolar activity in metal(loid) tolerance: vacuolar-lacking and -defective mutants of Saccharomyces cerevisiae display higher sensitivity to chromate, tellurite and selenite

The responses of Saccharomyces cerevisiae towards the oxyanions tellurite, selenite and chromate were investigated in order to establish the involvement of the yeast vacuole in their detoxification. Three mutants of S. cerevisiae with defective vacuolar morphology and function were used; mutant JSR180D1 is devoid of any vacuolar-like structure while ScVatB and ScVatC are deficient in specific protein subunits of the vacuolar (V)-H -ATPase. All the mutant strains showed increased sensitivity to tellurite and chromate compared to their parental strains. Such sensitivity of the mutants was associated with increased accumu-lation of tellurium and chromium. These results indicate that accumulation of both tellurium and chromium occurred mainly in the cytosolic compartment of the cell, with detoxification influenced by the presence of a functionally-active vacuole which may play a role in compartmentation as well as regulation of the cytostolic compartment for optimal expression of a detoxification mechanism, e.g. reduction. In contrast, the vacuolar-lacking mutant, JSR180D1, and the defective V-H ATPase mutant ScVatB displayed lower selenium accu-mulation than their parental strains. Additionally, the mutant strain ScVatB displayed a higher tolerance to selenite than the parental strain. This result suggests that accumulation of selenium occurs mainly in the vacuolar compartment of the cell with tolerance depending on the ability of the cytosolic component to reduce selenite to elemental selenium, which might, in turn, be related to activity of the V-H -ATPase. These results are discussed in relation to vacuolar compartmentation and the significance of the vacuolar H -ATPase in cytosolic homeostasis of H both of which may affect the accumulation, reduction, and toler-ance to the tested metal(loids). © Rapid Science 1998     

Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategy

During growth of Pseudomonas aeruginosa strain PAO1 with the toxic detergent SDS, a part of the population actively formed macroscopic cell aggregates while the other part grew as freely suspended cells. The physiological function of aggregation for growth with SDS was investigated. Three mutants growing with SDS without aggregation were isolated: the spontaneous mutant strain N and two mutants with transposon insertions in the psl operon for exopolysaccharide synthesis. SDS-induced aggregation in strain N but not in a pslJ mutant was restored by complementation with two genes encoding diguanylate cyclases responsible for synthesis of cyclic-di-guanosine monophosphate (c-di-GMP). By expressing a c-di-GMP-specific phosphodiesterase SDS-induced aggregation of strain PAO1 was reduced. Upon exposure to SDS in the presence of the uncoupler carbonyl cyanide chlorophenylhydrazone, the aggregating strains had ca. 500-fold higher survival rates than the non-aggregating strains. Co-incubation experiments revealed that strain N could integrate into aggregates of strain PAO1 and thereby increase its survival rate more than 1000-fold. These results showed that SDS-induced aggregation involved c-di-GMP signalling with the psl operon as a possible target. Cell aggregation could serve as a pre-adaptive strategy ensuring survival and growth of P. aeruginosa populations in environments with multiple toxic chemicals.     

Production of l-DOPA by Mutants of Pseudomonas melanogenum

Several kinds of mutants of Pseudomonas melanogenum were derived by mutational treatment with N-methyl-N’-nitro-N-nitrosoguanidine, and selected for 3,4-dihydroxyphenyl-l-alanine (l-DOPA) production by newly devised screening method which was carried out on agar plates based on violet-black colour formation by the reaction of l-DOPA with iron ion. Mutants tested were; glucose-insensitive mutant, cysteine-insensitive mutant, 3-amino-tyrosine-resistant mutant and p-fluorophenylalanine-resistant mutant. Some colonies isolated by monocolony procedure without mutagenic treatment were also tested. Among the 3-aminotyrosine-resistant mutants many good l-DOPA producers were found.

An 3-aminotyrosine-resistant mutant, strain ATN–36, produced 14 to 15 mg/ml of l-DOPA from 26 mg/ml of l-tyrosine (68 % in molar conversion ratio). When the cell concentration in reaction mixture was increased to 4-times the concentration of culture broth, l-DOPA production reached to 21 mg/ml from 52 mg/ml of tyrosine. An enzymatic basis of the high l-DOPA productivity of the improved mutants was found to be due to the increased tyrosinase activity (150 to 160% of the parental strain) of the mutants.