Cell fusion between temperature-sensitive mutants of a Drosophila melanogaster cell line.

Temperature-sensitive (ts) mutants were isolated in a cell line of Drosophila melanogaster, GM1, by ethyl methanesulfate treatment. Two of them, ts15 and ts58, formed colonies at 23 degrees C but not at 30 degrees when inoculated at densities of/or less than 10(5) cells per 60 X 15-mm dish. By using these ts mutants, cell fusion was attempted with polyethylene glycol (PEG) 6000. Several colonies per dish developed at 30 degrees C when different ts mutants were mixed, treated with PEG, and inoculated at a density of 10(4) cells per dish. Cells in some of the colonies thus developed were propagated and their temperature-sensitive character and karyotypes were studied. The results indicated that cell fusion could be induced with PEG and that the cells which formed colonies at 30 degrees C after PEG treatment were the hybrids in which the temperature-sensitive lesions in the mutants were complemented.     

Adenovirus type 12 gene 401 function in transforming infection

The temperature-sensitive DNA-minus mutant, H12ts401, transformed two to eight times more hamster embryo cells than wild-type 12 adenovirus at 38.5 degrees C, but was unable to establish transformation of cultures of hamster embryo brain and rat 3Y1 cells at 41.5 and 40 degrees C, respectively. Another H12ts406 DNA-minus mutant was not defective in cell transformation at these restrictive temperatures. Both mutants, however, induced T-antigen and cell DNA synthesis after infection of 3Y1 cells at 40 degrees C.     

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.     

A mutant v-rel with increased ability to transform B lymphocytes.

We observed that two strains of REV-T differ in the ability to transform bursal cells in vitro. REV-TW, with v-rel derived from a well-characterized clone and considered the prototype of the wild type, fails to generate colonies in soft agar. In contrast, REV-S2A3, derived from the S2A3 cell line, readily transforms bursal cells. With PCR, a 1,591-bp fragment containing v-rel from the REV-S2A3 provirus was cloned into plasmid pREV-0. Except for the absence of v-rel, pREV-0 is identical to pREV-TW. Five clones of pREV-PCR, each produced by an independent amplification, were obtained. The REV-PCR viruses displayed the strong transforming phenotype of REV-S2A3. Two mutations were identified in the 5' region of v-rel from REV-PCR1 to REV-PCR5: a silent mutation and a G-to-T transversion, changing the alanine at position 40 to serine. To confirm the relevance of this amino acid substitution, a 478-bp fragment containing the mutations was exchanged between REV-TW and REV-PCR1. Only the mutant viruses were able to form large colonies of bursal cells in liquid culture and to generate bursal cell colonies in soft agar. When tested on splenocytes, the wild-type viruses induced predominantly non-B-cell colonies while the mutant viruses gave origin mainly to B-cell colonies. The above results indicate that the substitution of serine for alanine at position 40 of v-Rel enhances the ability of REV-T to transform B lymphocytes in vitro. This mutation is close to the DNA-binding region, and the variant v-Rel oncoprotein shows increased kappa B-binding activity, thus confirming the relevance of this property for transformation.     

Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles.

We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43 degrees C but grow well at 35 degrees C. In both strains peroxisomes were completely absent in cells grown at 43 degrees C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (< or = 37 degrees C). However, at intermediate temperatures (38-42 degrees C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one--or infrequently a few--small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.     

Mutants of Arthroderma benhamiae

The object of these studies was to isolate conditionally lethal mutants of Arthroderma benhamiae whose vegetative growth was temperature sensitive (ts). Three sorts of mutants were derived from nitrosoguanidine treatment of the microconidia of A. benhamiae: (i) ts "pleomorphic", (ii) ts "non-pleomorphic", and (iii) slow growers (sg) which were not temperature sensitive. The growth of wild type A. benhamiae, as measured by colony diameters, was 6 mm/day at 37 degrees C (chosen as non-permissive for screening mutants) and 5 mm/day at 25 degrees C (chosen as permissive for screening mutants). Growth of mutants of the first sort was virtually stopped at 37 degrees C but their growth at 25 degrees C was more rapid than that of wild type. The colony texture of these mutants was downy and their mycelium sterile. They failed to mate with parents of opposite mating type. These mutants were considered to be ts "pleomorphic". The second sort of mutants were ts "non-pleomorphic". Two isolates of this kind were recovered. They grew normally at 25 degrees C but their growth at 37 degrees C was reduced 50--70% of that of wild type. The nature of the temperature sensitive defect has not been identified. The third sort of mutant occurred very frequently. These isolates were slow growers (sg) regardless of temperature of incubation and their response to the respiratory inhibitors antimycin and salicyl hydroxamic acid suggested a defect in mitochondrial ribosome assembly and deficiencies in cytochromes not unlike those observed in the poky mutants of Neurospora. Two of the sg-mutants (sg 2 and sg3) produced abundant cleistothecia with asci and ascospores when back crossed to parents of opposite mating type. The sg 5 mutant produced cleistothecia but no ascospores. The ergosterol content of the sg 2 mutant was nearly the same as that of wild type while the ergosterol content of sg 3 was somewhat reduced and that of sg 5 was markedly reduced as compared to that of wild type. Thus the ergosterol content seems to play some role in sexual reproduction of A. benhamiae. The pattern of sensitivity to amphotericin B also reflected differences in the sterol content of the mutants, i.e., the two mutants with some alteration in their ergosterol content, sg 3 and sg 5 were more resistant to the antibiotic. During the course of these studies a number of variants were observed that produced different sorts and degrees of pigmentation of the reverse of their colonies. One stable variant of this kind gave rise to colonies with a red reverse when incubated at 37 degrees C and a yellow reverse when incubated at 25 degrees C. When plates were shifted from one temperature to another the next wave of growth was the color specified by the temperature.     

Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic characterization of Escherichia coli type 1 pilus adhesin mutants and identification of a novel binding phenotype

Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.     

Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium

Intragenic mutations were isolated that suppressed the dominant-lethal phenotype of the YPT1ile121 mutant gene in a temperature-dependent fashion. Among different amino acid substitutions resulting from single point mutations, two, Ala161----Val (A161V) and Met165----Ile (M165I), restored the function of the YPT1ile121 mutant protein. Mutants expressing the YPT1ile121/val161 allele (ypt1ts) only, grew normally at temperatures up to 30 degrees C but were arrested at 37 degrees C. At the restrictive temperature, ypt1ts mutants accumulated ER membranes, small vesicles, and unprocessed invertase, and they exhibited cytoskeletal defects and an enhanced 45Ca2+ uptake. Similar alterations were seen in YPT1-depleted cells. The ypt1ts mutant cells could be rescued from growth arrest by increasing extracellular Ca2+, and, even at the permissive temperature, they displayed increased trifluoperazine sensitivity.     

Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis.

A new method for detecting invertase activity in Saccharomyces cerevisiae colonies was used to screen for mutants resistant to catabolite repression of invertase. Mutations causing the highest level of derepression were located in two previously identified genes, cyc8 and tup1. Several of the cyc8 mutations, notably cyc8-10 and cyc8-11, were temperature dependent, repressed at 23 degrees C, and derepressed at 37 degrees C. The kinetics of derepression of invertase mRNA in cyc8-10 cells shifted from 23 to 37 degrees C was determined by Northern blots. Invertase mRNA was detectable at 5 min after the shift, with kinetics of accumulation very similar to that of wild-type cells shifted from high-glucose to low-glucose medium. Assays of representative enzymes showed that many but not all glucose-repressible enzymes are derepressed in both cyc8 and tup1 mutants. cyc8 and tup1 appear to be the major negative regulatory genes controlling catabolite repression in yeasts.     

Isolation of heat- and cold-sensitive mutants of chinese hamster lung cells affected in their ability to express the transformed state.

A clone of spontaneously transformed Chinese hamster lung cells was exposed to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), and six heat-sensitive and three cold-sensitive mutants were isolated after selection for inability to form colonies in soft agar at 39.5 degrees C and 34.5 degrees C, respectively. The heat-sensitive mutants had growth characteristics of transformed cells at 34.5 degrees C, but exhibited a normal phenotype at 39.5 degrees C. By contrast, cold-sensitive mutants displayed the characteristics of the normal cells at 34.5 degrees C and converted to a transformed phenotype at 39.5 degrees C. Transformed parent cells exhibited no obvious temperature-dependent properties. Temperature shift experiments showed that the colony-forming ability of both types of mutants was fully reversible. All of the mutants were able to grow well at both permissive and nonpermissive temperatures when grown on the surface of plastic dishes. Such mutants will be useful in analysis of factors involved in the expression of the transformed state or the maintenance of the nontransformed state.     

Toxoplasma gondii: characterization of temperature-sensitive tachyzoite cell cycle mutants

We mutagenized RH delta hxgprt strain tachyzoites of Toxoplasma gondii using N-nitroso-N-ethylurea and analyzed 40 clonal isolates (of 3680 ENU mutants) that were unable to grow in cell culture at 40 degrees C. These isolates grew normally at 34 degrees C, but showed variable growth at temperatures between 34 and 39 degrees C. The inability to grow at 40 degrees C was also correlated with a loss of virulence in mice for those mutants examined. We further characterized the temperature-sensitive (ts) isolates using flow cytometry and propidium iodide staining and identified three types of cell cycle-related mutations. Regardless of temperature, in the isolates ts1C12, ts7B4, and ts7B10, the distribution of parasites with a haploid DNA content was substantially higher (congruent with 85%) than that observed for RH delta hxgprt (congruent with 60%). Four other isolates, ts4F6, ts6C11, ts8G10, and ts11F5, contained G1-related mutations, and in each case, the DNA distribution among parasites at the permissive temperature was similar to that of the parental strain, but at 40 degrees C only a single population containing a 1N nuclear DNA complement was evident. Furthermore, there was no evidence of nuclear division or cytokinesis at 40 degrees C, and these parasites demonstrated a distended cytoplasm typical of G1 arrest in other cell types. Finally, parasites of the ts11C9 mutant arrested in two near-equal populations with either 1N or 2N complements of nuclear DNA. All arrested ts11C9 parasites contained a single nucleus, and a major subfraction of the 2N population contained abnormal and incompletely formed daughters-indicating that the initiation of daughter formation can occur in the absence of nuclear division.     

Temperature-sensitive autolysis-defective mutants of Escherichia coli.

Two independently isolated temperature-sensitive autolysis-defective mutants of Escherichia coli LD5 (thi lysA dapD) were characterized. The mutants were isolated by screening the survivors of a three-step enrichment process involving sequential treatments with bactericidal concentrations of D-cycloserine, benzyl-penicillin, and D-cycloserine at 42 degrees C. Cultures of the mutants underwent autolysis during beta-lactam treatment, D-cycloserine treatment, or diaminopimelic acid deprivation at 30 degrees C. The same treatments at 42 degrees C inhibited growth but did not induce lysis of the mutants. The minimum inhibitory concentrations of selected beta-lactam antibiotics and D-cycloserine were identical for the parent and mutant strains at both 30 and 42 degrees C. Both mutants failed to form colonies at 42 degrees C, and both gave rise to spontaneous temperature-resistant revertants. The revertants exhibited the normal lytic response when treated with D-cycloserine and beta-lactams or when deprived of diaminopimelic acid at 42 degrees C. The basis for the autolysis-defective phenotype of these mutants could not be determined. However, a nonspecific in vitro assay for peptidoglycan hydrolase activity in cell-free extracts indicated that both mutants were deficient in a peptidoglycan hydrolase. Both mutations were localized to the 56- to 61-min region of the E. coli chromosome by F' complementation.     

Viability of rep recA mutants depends on their capacity to cope with spontaneous oxidative damage and on the DnaK chaperone protein

Replication arrests due to the lack or the inhibition of replicative helicases are processed by recombination proteins. Consequently, cells deficient in the Rep helicase, in which replication pauses are frequent, require the RecBCD recombination complex for growth. rep recA mutants are viable and display no growth defect at 37 or 42 degrees C. The putative role of chaperone proteins in rep and rep recA mutants was investigated by testing the effects of dnaK mutations. dnaK756 and dnaK306 mutations, which allow growth of otherwise wild-type Escherichia coli cells at 40 degrees C, are lethal in rep recA mutants at this temperature. Furthermore, they affect the growth of rep mutants, and to a lesser extent, that of recA mutants. We conclude that both rep and recA mutants require DnaK for optimal growth, leading to low viability of the triple (rep recA dnaK) mutant. rep recA mutant cells form colonies at low efficiency when grown to exponential phase at 30 degrees C. Although the plating defect is not observed at a high temperature, it is not suppressed by overexpression of heat shock proteins at 30 degrees C. The plating defect of rep recA mutant cells is suppressed by the presence of catalase in the plates. The cryosensitivity of rep recA mutants therefore results from an increased sensitivity to oxidative damage upon propagation at low temperatures.     

Functional rescue of temperature-sensitive defects in the cell-cycle mutants of rat 3Y1 fibroblasts by the small t-antigen deletion mutant of simian virus 40

We examined the effects of large T antigen of simian virus 40 (SV40) on the proliferation phenotypes of temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which cease proliferating in the G1 phase of the cell cycle at a restrictive temperature (39.8 degrees C). Four ts mutants, each representing independent complementation groups, were transformed with the dl-884 mutant of SV40 which lacks the unique coding region for small t antigen. In the case of two ts mutants, their transformed derivatives did not cease proliferation at 39.8 degrees C. In the other two mutants, the transformed cells continued to enter the S phase but the cells became detached from the dishes thereafter, at 39.8 degrees C. The proliferation phenotypes of the dl-884-transformed cells at 39.8 degrees C were quite similar with those of the same mutants transformed with the wild-type SV40. These results indicate that large T antigen alone is sufficient to overcome the inhibition of cellular entry into S phase caused by four different ts defects and determines the proliferation phenotypes of the cells after entering the S phase at a restrictive temperature, and that small t antigen does not alter the cellular phenotypes determined by large T antigen.     

fipB and fipC: two bacterial loci required for morphogenesis of the filamentous bacteriophage f1.

We describe the identification of two mutations in bacterial genes, designated as fipB and fipC, which resulted in temperature-sensitive morphogenesis of bacteriophage f1. These mutations mapped at separate loci but had to be present simultaneously to block f1 production at 41.5 degrees C. One mutation defined the locus fipB at 85.3 min on the Escherichia coli linkage map; the other defined the locus fipC, which mapped very close to rpsL at 73 min. Since these mutations did not appear to affect phage DNA replication, gene expression, or protein localization, they probably interfered with the its life cycle at the level of assembly. fipB mutants were partially deficient in adsorption of bacteriophage lambda, and fipB and fipC mutants leaked beta-lactamase into the medium, suggesting that the mutations affect outer-membrane structure or function.