Las21 participates in extracellular/cell surface phenomena in Saccharomyces cerevisiae

Las21 (Yj1062W) is a member of the major facilitator super family, possessing multimembrane spanning domains. The LAS21 gene was identified as a responsible gene for a Saccharomyces cerevisiae mutan which shows sensitivity to a local anestheticum, tetracaine. The null las21 mutant (las21 delta) is viable but shows temperature sensitive growth. We found, in addition to this phenotype, that the las21 delta strain shows a number of defects; mating deficiency, calcofluor resistance, and formation of Zymolyase sensitive spores. Temperature sensitive growth of the las21 delta mutant was found to be suppressed by 0.1 M MgSO4. Two multicopy suppressors were obtained. They are ECM33 (YBR078W) and PIR2/HSP150 (YJR159W) both have some roles in an extracellular function. The common features of the suppressors, genetic and physiological, of the las21 delta mutation suggest that Las21 participates in a global activity of extracellular phenomena. The las 21 phenotypes are consistent with the idea that Las21/Gpi7 acts in metabolism of glycosylphosphatidylinositol.     

LAS24/KOG1, a component of the TOR complex 1 (TORC1), is needed for resistance to local anesthetic tetracaine and normal distribution of actin cytoskeleton in yeast

It is known that some local anesthetics inhibit the growth of budding yeast cells. To investigate the pathway of local anesthetics' action, we isolated and characterized mutants that were hyper-sensitive to tetracaine, and at the same time, temperature-sensitive for growth. They were collectively called las (local anesthetic sensitive) mutants. One of the LAS genes, LAS24, was found to be identical to KOG1, which had been independently discovered as a member of the TOR complex 1 (TORC1). Las24p/Kog1p is a widely conserved TOR binding protein containing the NRC domain, HEAT repeats and WD-40 repeats, but its function remains unknown. Like the tor mutants, the las24 mutants were found to have a defect in cell wall integrity and to show sensitivity to rapamycin. Furthermore, Las24p is required not only in TORC1-mediated (rapamycin-sensitive) pathways such as translation initiation control and phosphorylation of Npr1p and Gln3p, but also for the normal distribution of the actin cytoskeleton, which has been regarded as a TORC2-mediated event. Intriguingly, the temperature-sensitivity of the las24 mutant was suppressed by either activation of Tap42/PPase or by down-regulation of the RAS/cAMP pathway. Suppressors of the temperature-sensitivity of the las24-1 mutant were found not to be effective for suppression of the tetracaine-sensitivity of the same mutant. These observations along with the facts that tetracaine and high temperature differentially affected the las24-1 mutant suggest that Las24p/Kog1p is not a target of tetracaine and that the tetracaine-sensitive step may be one of downstream branches of the TORC1 pathway. Consistent with the broad cellular functions exerted by the TOR pathway, we found that Las24p was associated with membranes and was localized at vacuoles, the plasma membrane and small vesicles.     

An improved integration replacement/disruption method for mutagenesis of yeast essential genes

We improved the integration replacement/disruption method (Shortle, D., Novic, P., and Botstein, D. Proc. Natl. Acad. Sci. USA 81: 4889-4893, 1984) for isolating mutants in any of essential genes of the yeast Saccharomyces cerevisiae by integrating mutagenized DNA into the wild type gene of interest. We adopted this method to isolate temperature-sensitive mutants of the MPC1 gene encoding the YLL031C ORF. To facilitate integration of the mutagenic plasmid at a site near the 5' end of the ORF, a BamHI site was created at 300 bp downstream of the 5' end of the truncated ORF to be mutagenized. The MPC1 gene was disrupted in the wild type haploid strain by integrating a 5'-truncated derivative of the gene with mutations induced by in vitro mutagenesis. Transformants thus obtained were subjected for diagnosis of conditional lethality by replica-plating onto an appropriate selection medium to detect mutants. A primary mutant isolated by this method reverted in a high frequency due to a tandem repeat created by mutagenic integration. We deviced a method to obtain a stable temperature-sensitive strain by disrupting the tandem duplication. Two stable temperature-sensitive mutants thus obtained were found to be remedial either with 1 M sorbitol or with 0.1 M Mg2+ and to be sensitive to local anestheticum, tetracaine, at 25 degrees C.     

Plasma membrane expansion terminates in Saccharomyces cerevisiae secretion-defective mutants while phospholipid synthesis continues.

Phospholipid synthesis activity and plasma membrane growth have been studied in the Saccharomyces cerevisiae temperature-sensitive, secretion-defective mutants isolated by Novick and Schekman (Proc. Natl. Acad. Sci. U.S.A. 76:1858-1862, 1979; Novick et al., Cell 21:205-215, 1980). The mutants, sec1 through sec23, do not grow at 37 degrees C and exhibit lower rates of phospholipid synthesis than does the wild-type strain X2180. None of the mutants exhibits a decline in lipid synthesis rapid enough to explain secretion failure. Plasma membrane growth was assessed indirectly by examining the osmotic sensitivity of spheroplasts derived from cultures transferred from 24 to 37 degrees C. Spheroplasts from the normal-growing strain X2180 exhibited a small rapid increase in osmotic sensitivity and stabilized at a more sensitive state. Spheroplasts from the sec mutants exposed to the same temperature shift exhibited progressively increasing osmotic sensitivity. Cycloheximide treatment prevented progressive increases in osmotic fragility. These data are compatible with the hypothesis that plasma membrane expansion is restricted in the sec mutants. During incubation at 37 degrees C, the accumulation of intracellular materials within the no-longer expanding plasma membrane exerts osmotic stress on the membrane, increasing with time. The gene products defective in Novick and Schekman's sec mutants appear to be required for both extracellular protein secretion and plasma membrane growth in yeast cells.     

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae

Las21/Gpi7 contains a heavy-metal-associated motif at its N-terminus. When this motif was disrupted by amino acid substitution, the cells acquired weak copper-resistance. We found that the previously isolated las21 mutants were strongly resistant to copper. Metallothionein is necessary for the expression of the copper-resistance of the las21 mutants. However, hyper-production of metallothionein is unlikely to be the cause of copper-resistance of the las21 mutants. Copper-sensitive mutants (collectively called Cus mutants) were isolated from the las21delta and characterized. One of the Cus genes was found to be PBS2, which encodes Hog1 MAP kinase kinase, indicating that the Hog1 MAP kinase pathway is needed for the expression of copper-resistance of the las21 mutants. As expected, the las21delta hog1delta strain was no longer copper-resistant. We found that Hog1 was constitutively activated in las21delta cells and in ssk1delta las21delta cells but not in sho1delta las21delta cells. Inactivation of either FSR2/MCD4 or MPC1/GPI13, both of which are involved in GPI anchor synthesis, like LAS21, caused a similar level of constitutive activation of Hog1 kinase and copper-resistance as found in the las21delta strain. The constitutive activation was canceled by introducing the sskl mutation, but not the sho1 mutation, in each GPI anchor mutant tested, suggesting that the defect in GPI anchor synthesis specifically affects the Slnl branch of the MAP kinase pathway. Since the wild-type cells grown in YPD containing 0.5 M NaCl do not show copper-resistance, mere activation of Hog1 is not sufficient for expression of copper-resistance. We propose that a defect in GPI anchor synthesis has multiple consequences, including activation of the Hog1 MAP kinase cascade and conferring copper-resistance.     

Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation.

The Schizosaccharomyces pombe sts1+ gene, identified by supersensitive mutations to a protein kinase inhibitor, staurosporine, was isolated by complementation by the use of a fission yeast genomic library. Nucleotide sequencing shows that the sts1+ gene encodes a 453 amino acid putative membrane-associated protein that is significantly similar (26% identity) to the chicken lamin B receptor. It is also highly related (53% identity) to a budding yeast ORF, YGL022. These three proteins contain a similar hydrophobicity pattern consisting of eight or nine putative transmembrane domains. By gene disruption we demonstrate that the sts1+ gene is not essential for viability. These disruptants exhibit pleiotropic defects, such as cold-sensitivity for growth and at the permissive temperature, a supersensitivity to divalent cations and several unrelated drugs including staurosporine, caffeine, chloramphenicol, sorbitol, and SDS. Disruption of the sts1+ gene does not lead to a sensitivity to thiabendazole or hydroxyurea.     

Cloning and characterization of the rad4 gene of Schizosaccharomyces pombe; a gene showing short regions of sequence similarity to the human XRCC1 gene.

The rad4.116 mutant of the fission yeast Schizosaccharomyces pombe is temperature-sensitive for growth, as well as being sensitive to the killing actions of both ultraviolet light and ionizing radiation. We have cloned the rad4 gene by complementation of the temperature sensitive phenotype of the rad4.116 mutant with a S. pombe gene bank. The rad4 gene fully complemented the UV sensitivity of the rad4.116 mutant. The gene is predicted to encode a protein of 579 amino acids with a basic tail, a possible zinc finger and a nuclear location signal. The amino terminal part of the predicted rad4 ORF contains two short regions of similarity to the C-terminal part of the human XRCC1 gene. Codon usage suggests that the gene is very poorly expressed, and this was confirmed by RNA studies. Gene disruption showed that the rad4 gene was essential for the mitotic growth of S. pombe.     

Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.     

TIP 1, a cold shock-inducible gene of Saccharomyces cerevisiae

Using differential hybridization, genes whose expression is induced at low temperatures were identified in yeast Saccharomyces cerevisiae. One of these genes that corresponds to an mRNA that is induced 6-8-fold within 2 h after shifting the culture temperature from 30 to 10 degrees C was further characterized. Surprisingly, its expression was also induced by heat shock, and thus the gene was designated TIP 1 (temperature shock-inducible protein gene). Southern hybridization analysis demonstrated that there are several genes homologous to the TIP 1 gene on the yeast genome. A TIP 1 disruption mutation exerted an observable effect neither on growth nor on viability after being exposed to freezing temperatures. The TIP 1 gene encodes a protein of 210 amino acid residues with a molecular weight of 20,727, containing 20.0% alanine and 23.3% serine. The TIP 1 protein has a typical signal peptide at the amino-terminal end and an extremely hydrophobic sequence at the carboxyl-terminal end. The TIP 1 protein is thus likely to be secreted across the membrane and anchored on the outside surface of the plasma membrane. These results indicate that the TIP 1 protein is a new type of stress inducible protein in yeast.     

Isolation of a Xanthomonas oryzae pv. oryzae flagellar operon region and molecular characterization of flhF

An 8.1-kb DNA fragment from Xanthomonas oryzae pv. oryzae that contains six open reading frames (ORF) was cloned. The ORF encodes proteins similar to flagellar proteins FlhB, FlhA, FlhF, and FliA, plus two proteins of unknown function, ORF234 and ORF319, from Bacillus subtilis and other organisms. These ORF have a similar genomic organization to those of their homologs in other bacteria. TheflhF gene product, FlhF, has a GTP-binding motif conserved in its homologs. Unlike its homologs, however, X. oryzae pv. oryzae FlhF carries two transmembrane-like domains. Insertional mutations of theflhF gene with the omega cassette or the kanamycin resistance gene significantly retard but do not abolish the motility of the bacteria. Complementation of the mutants with the wild-type flhF gene restored the motility. The X. oryzae pv. oryzae FlhF interacts with itself; the disease resistance gene product XA21; and a protein homologous to the Pill protein of Pseudomonas aeruginosa, XooPilL, in the yeast two-hybrid system. The biological relevance of these interactions remains to be determined.     

Characterization of Null Mutants of the RAD55 Gene of Saccharomyces cerevisiae: Effects of Temperature, Osmotic Strength and Mating Type

RAD55 belongs to a group of genes required for resistance to ionizing radiation, RAD50-RAD57, which are thought to define a pathway of recombinational repair. Since all four alleles of RAD55 are temperature conditional (cold sensitive) for their radiation phenotype, we investigated the phenotype produced by null mutations in the RAD55 gene, constructed in vitro and transplaced to the yeast chromosome. The X-ray sensitivity of these null mutant strains was surprisingly suppressed by increased temperature, osmotic strength of the growth medium and heterozygosity at the mating-type locus. These first two properties, temperature conditionality and osmotic remediability, are commonly associated with missense mutations; these rad55 null mutants are unique in that they exhibit these properties although the mutant gene cannot be expressed. X-ray-induced mitotic recombination was also cold sensitive in rad55 mutant diploids. Although mitotic growth was unaffected in these strains, meiosis was a lethal event at both high and low temperatures. Whereas the phenotype of rad55 null mutants is consistent with a role of RAD55 in recombination and recombinational repair, there is evidence for considerable RAD55-independent recombination, at least in mitotic cells, which is influenced by temperature and MAT. We discuss models for the role of RAD55 in recombination to explain the unusual properties of rad55 mutants.     

Characterisation of Schizosaccharomyces pombe rad31, a UBA-related gene required for DNA damage tolerance.

The fission yeast rad31-1 mutant is sensitive to both UV and ionising radiation and exhibits a growth defect at 35 degrees C. In addition, the mutant displays defects in cell morphology and nuclear division at 26 degrees C which are exaggerated at 35 degrees C. We have cloned the rad31 gene and have shown that it is not essential for viability, although cells containing a disrupted rad31 gene grow slowly. The null allele has similar cell and nuclear morphologies to the original allele and displays an extremely high frequency of loss of minichromosomes. rad31 is not required for either the S/M or G2/M checkpoint, however double mutant analysis indicates that rad31 acts in a process which is defective in the checkpoint rad mutants and which involves hus5 . Sequence analysis indicates that rad31 encodes a protein which is related to ubiquitin activating proteins and more particularly to an ORF in Saccharomyces cerevisiae and to the Arabidopsis thaliana AXR1 and human APP-BP1 genes. We have isolated the S.cerevisiae sequence, which we have named RHC31 ( ad31homologue in S. erevisiae), since we show that it can complement the slow growth phenotype and radiation sensitivity of S.pombe rad31.     

Genetic characterization of genes encoding enzymes catalyzing addition of phospho-ethanolamine to the glycosylphosphatidylinositol anchor in Saccharomyces cerevisiae

MPC1/GPI13/YLL031C, one of the genes involved in the addition of phospho-ethanolamine to the glycosylphosphatidylinositol (GPI) anchor core, is an essential gene. Three available temperature-sensitive mutant alleles, mpc1-3, mpc1-4, and mpc1-5, displayed different phenotypes to each other and, correspondingly, these mutants were found to have different mutations in the MPC1 ORF. Temperature-sensitivity of mpc1-5 mutants was suppressed by 5 mM ZnSO(4) and by 5 mM MnCl(2). Multicopy suppressors were isolated from mpc1-5 mutant. Suppressors commonly effective to mpc1-4 and mpc1-5 mutations are PSD1, encoding phosphatidylserine decarboxylase, and ECM33, which were found to suppress the temperature-sensitive phenotype shown by the fsr2-1 and las21delta mutants, those of which have defects in the GPI anchor synthesis. PSD2, encoding another phosphatidylserine decarboxylase that is localized in Golgi/vacuole, was found to be able to serve as a multicopy suppressor of mpc1 and fsr2-1 mutants but not of the las21 delta mutant. In contrast to psd1delta, psd2delta showed a synthetic growth defect with mpc1 mutants but not with fsr2-1 or las21delta. Furthermore, psd1delta psd2delta mpc1 triple mutants did not form colonies on nutrient medium unless ethanolamine was supplied to the medium, whereas psd1delta psd2 delta fsr2-1 or psd1delta psd2 delta las21delta triple mutants grew on nutrient medium without supplementation of ethanolamine. These observations suggest that Mpc1 preferentially utilizes phosphatidylethanolamine produced by Psd2 that is localized in Golgi/vacuole. fsr2-1 dpl1 Delta psd1delta strains showed slower growth than fsr2-1 dpl1delta psd2 delta, suggesting that Fsr2 enzyme depends more on Dpl1 and Psd1 for production of phosphatidylethanolamine. Las21 did not show preference for the metabolic pathway to produce phosphatidylethanolamine.     

A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae.

The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP-binding protein, which is implicated in various actin cytoskeleton-dependent cell functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature-sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two-hybrid analysis demonstrated that GTP-Rho1p, but not GDP-Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain. MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1-6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T). These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.     

Characterization of an outer membrane protein gene, pgmA, and its gene product from Porphyromonas gingivalis

A gene upstream from fimA, the gene encoding fimbrilin, on the chromosome of Porphyromonas gingivalis was sequenced and shown to be the gene encoding an outer membrane protein in this organism based on homology and biochemical analyses. Therefore, the gene (formerly ORF5) was designated pgmA, the P. gingivalis outer membrane protein A gene. The gene product, PgmA, was sensitive to protease, and was detected as a 60-kDa protein from wild-type strains with trichloroacetic acid treatment, which was carried out to destroy intrinsic proteases, and from protease-deficient mutants without this treatment prior to electrophoresis. PgmA was indeed present in the membrane fraction. Its nature was determined to be that of outer membrane proteins in gram-negative bacteria based on attempts at differential extraction of inner membrane proteins with detergents. No evidence has been found thus far from functional analyses that this protein is related to fimbrial morphogenesis and functions or to serum resistance of this organism.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers.

Calcineurin, or PP2B, plays a critical role in mediating Ca2+-dependent signaling in many cell types. In yeast cells, this highly conserved protein phosphatase regulates aspects of ion homeostasis and cell wall synthesis. We show that calcineurin mutants are sensitive to high concentrations of Mn2+ and identify two genes, CCC1 and HUM1, that, at high dosages, increase the Mn2+ tolerance of calcineurin mutants. CCC1 was previously identified by complementation of a Ca2+-sensitive (csg1) mutant. HUM1 (for "high copy number undoes manganese") is a novel gene whose predicted protein product shows similarity to mammalian Na+/Ca2+ exchangers. hum1 mutations confer Mn2+ sensitivity in some genetic backgrounds and exacerbate the Mn2+ sensitivity of calcineurin mutants. Furthermore, disruption of HUM1 in a calcineurin mutant strain results in a Ca2+-sensitive phenotype. We investigated the effect of disrupting HUM1 in other strains with defects in Ca2+ homeostasis. The Ca2+ sensitivity of pmc1 mutants, which lack a P-type ATPase presumed to transport Ca2+ into the vacuole, is exacerbated in a hum1 mutant strain background. Also, the Ca2+ content of hum1 pmc1 cells is less than that of pmc1 cells. In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function. These genetic interactions suggest that Hum1p may participate in vacuolar Ca2+/H+ exchange. Therefore, we prepared vacuolar membrane vesicles from wild-type and hum1 cells and compared their Ca2+ transport properties. Vacuolar membrane vesicles from hum1 mutants lack all Ca2+/H+ antiport activity, demonstrating that Hum1p catalyzes the exchange of Ca2+ for H+ across the yeast vacuolar membrane.     

Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells

Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution.     

Use of polymyxin B, levallorphan, and tetracaine to isolate novel envelope mutants of Escherichia coli.

Mutants of Escherichia coli were isolated by their resistance to the bacteriocidal effects of the membrane-active drugs polymyxin B, levallorphan, and tetracaine. The mutants were examined for additional changes in cellular physiology evoked by the lesions; many polymyxin-resistant strains had a concomitant increased sensitivity to anionic detergents, and several strains of each type had concomitant alterations in generation time and morphology. Mutants of each class (polymyxin resistant, tetracaine resistant, and levallorphan resistant) were transduced into recipient strains. The levallorphan resistance site (lev) was located at approximately 9 min on the E. coli chromosome. Polymyxin (pmx) and tetracaine (tec) resistance loci were also transduced. The lev and tec strains had a slight prolongation of generation time, in contrast with their isogenic wild-type strains. The tec transductant produced long filaments in the absence of tetracaine and had an altered colonial morphology, it reverted at high frequency, with the morphological abnormalities reverting along with the tetracaine resistance. The pmx transductant had an increased sensitivity to levallorphan and to anionic detergents. In contrast, both lev and tec mutants were more resistant to acriflavine than was the wild type or the pmx transductant. The pmx, lev, and tec loci differed in sensitivity to mitomycin C; the lev strain was more resistant, the tec strain was more sensitive, and the pmx strain was much more sensitive than the wild type. There was no difference in sensitivity to several other dyes and detergents, colicins, or T bacteriophage between the transductant and isogenic wild-type strains. Thus, lev, tec, and pmx loci confer more subtle alterations in the permeability barrier than do lipopolysaccharide-deficient mutants previously studied.