Temperature-sensitive mutants of Escherichia coli affecting beta-galactoside transport

Six different temperature-sensitive (ts) mutants have been isolated which have parental beta-galactoside permease levels at low temperatures but have decreased permease levels when grown at high temperatures. These mutants were derived from Escherichia coli ML308 (lacI(-)Y(+)Z(+)A(+)). After N-methyl-N'-nitro-N'-nitro-soguanidine mutagenesis, ampicillin was used to select for cells unable to grow on low lactose concentrations at 42 C. Temperature-sensitive mutants were assayed for galactoside permease activity after growth in casein hydrolysate medium at 25 or 42 C by measuring both radioactive methylthio-beta-d-galactoside uptake and in vivo o-nitrophenyl-beta-d-galactoside hydrolysis. The six conditional isolates have decreased levels of galactoside permease which are correlated with decreased growth rates at elevated temperatures. The low permease levels are not due to a temperature labile lacY gene product but rather to a temperature labile synthesis rate of functional permease. Some of the mutants exhibit a ts increase in permeability as shown by the increased leakage of intracellular beta-galactosidase and by the increased rate of in vivo o-nitrophenyl-beta-d-galactoside hydrolysis via the nonpermease mediated entry mechanism. Preliminary evidence indicates that transport in general is decreased in these mutants, yet there is some specificity in the mutational lesion since glucoside transport is unaffected. All these observations suggest that these mutants have ts alterations in membrane synthesis which results in pleiotropic effects on various membrane functions.     

Mutants of Escherichia coli Unable to Make Protein at 42 C

Members of a collection of mutants of Escherichia coli unable to form colonies on nutrient agar at 42 C have been characterized on the basis of their growth response to a shift from 32 to 42 C in liquid medium. Forty-four mutants, which show an abrupt, nonlethal cessation of growth when moved to the restrictive temperature, have been characterized with respect to the effect of the mutation responsible for temperature sensitivity on deoxyribonucleic acid, ribonucleic acid, and protein synthesis. In 12 mutants, the mutation causing temperature sensitivity of growth primarily affects protein synthesis, in each case through an altered aminoacyl-transfer ribonucleic acid synthetase. Mutants with temperature-sensitive glutamyl-, phenylalanyl-, and valyl-transfer ribonucleic acid synthetases have been obtained, and the genes specifying these enzymes have been mapped by conjugation and transduction. Another mutant has been shown to possess a temperature-sensitive tryptophanyl-transfer ribonucleic acid synthetase, but this is not responsible for inability to grow at 42 C on media containing tryptophan.     

Supersensitivity of Escherichia coli Cells to Several β-Lactam Antibiotics Caused by Rod− Morphology Mutations at the mrd Gene Cluster

Two kinds of spherical mutants, mrdA and mrdB mutants, have been isolated from Escherichia coli strain K12. The mrdA mutants have thermosensitive penicillin-binding protein 2, while the mrdB mutants have normal penicillin-binding proteins. Both kinds of mutants form spherical cells at 42°C and are resistant to the amidinopenicillin, mecillinam, at the same temperature. The two mutations have been mapped very close to lip at 14.2 min (revised chromosome linkage map, 1980) on the E. coli chromosome. Both mutations cause supersensitivities of cell growth to various β-lactam antibiotics, such as ampicillin, cephalexin, cefoxitin and nocardicin A at 42°C.     

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.     

Cell shape and division in Escherichia coli: experiments with shape and division mutants.

Double mutants which carry mutations in genes (rodA, pbpA) required for cell elongation (i.e., maintenance of rod shape) in combination with mutations in genes (ftsA, ftsI, ftsQ, or ftsZ) required for septation were constructed. Such mutants were able to grow for about two mass doublings at a normal rate at the restrictive temperature (42 degrees C). The morphology of the cells formed under these conditions was interpreted by assuming the existence of a generalized system for peptidoglycan growth together with two additional systems which modify the shape of the growing peptidoglycan layer. The results also showed that different fts genes probably control different stages in septation. ftsZ (sulB or sfiB) appears to be required for the earliest step in septation, ftsQ and ftsI (pbpB or sep) are required for a later step or steps, and ftsA is required only for the latest stages in septation.     

Conditional mutants of Staphylococcus aureus defective in cell wall precursor synthesis

Temperature-sensitive (ts) mutants of Staphylococcus aureus with defective cell wall biosynthesis have been differentiated from other ts mutants by their ability to grow at the restrictive temperature (43 C) in the presence of 1 m NaCl. Under all conditions they possess normal colonial and cellular morphology at the level of resolution of the light microscope and are, therefore, not protoplasts. However, differences between mutant and wild-type cells can be seen by scanning electron microscopy. Many of the mutants contained concentrations of nucleotide precursors of peptidoglycan synthesis in excess of those present in wild-type cells, at both 30 and 43 C. The types of peptidoglycan precursors accumulated by six of the mutants have been determined, and specific enzymatic defects in three of these have been identified.     

Autolysins and shape change in rodA mutants of Bacillus subtilis.

The biochemical phenotype of rodA mutants was not affected by the simultaneous presence in double mutants of the lyt gene which makes them 90 to 95% deficient in autolysin action. The only morphological effect of this deficiency on the expression of the rod gene was that both the rod and the coccal forms of the mutant failed to separate and grew as long chains of cells. Inhibition of protein synthesis stopped the increase in peptidoglycan that occurred when the growth temperature for the mutants was raised to 45 degrees C. These observations support the idea that a derepression of peptidoglycan synthesis occurs at this temperature. The increased amount of cellular peptidoglycan at the higher growth temperature is not likely to be the result of the concomitant switching off of autolytic enzyme action.     

Small heat shock proteins in the development of thermotolerance in Pisolithus sp.

Small heat shock proteins (HSPs) have been shown to confer thermotolerance in many organisms. Here, we demonstrate that small HSPs (sHSPs) can also be involved in development of thermotolerance in Pisolithus sp. In heat shock response, Pisolithus isolate RV82 synthesized proteins of molecular mass 28, 26 and 15–18 kDa. These group of proteins are synthesized when mycelial mass are exposed to heat shock temperature (42 °C) for short period (30 min) and incubated back at 28 °C, the optimal temperature for growth. Our results show sHSPs are an important biochemical alteration in ectomycorrhizal fungi under thermal stress.     

Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics

Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli chi2452, which showed a tolerant response against beta-lactam antibiotics when grown at 42 degrees C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli chi1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of chi1776 or in chi2452 grown at 32 degrees C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of chi2452 grown at 42 degrees C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecillinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of chi2452 cells grown at 42 degrees C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of chi2452 grown either at 42 or 32 degrees C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings.     

An unusual mutation results in the replacement of diaminopimelate with lanthionine in the peptidoglycan of a mutant strain of Mycobacterium smegmatis

Mycobacterial peptidoglycan contains L-alanyl-D-iso-glutaminyl-meso-diaminopimelyl-D-alanyl-D-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the L-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate D-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to beta-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine beta-synthase, an enzyme that is a part of the cysteine biosynthetic pathway.     

Thermosensitive Bacillus subtilis mutants which lyse at the non-permissive temperature

A collection of 655 thermosensitive mutants of Bacillus subtilis 168, obtained by indirect selection, was screened for those lysing at the non-permissive temperature. Thirty-three mutations thus identified were distributed by transformation into eight linkage groups designated lssA to lssH. The distribution was non-random. With the exception of group A, all groups were small, suggesting that mutations identified in each of them may map in one gene only. Linkage groups identified here were mapped in four different regions of the B. subtilis chromosome and their positions relative to reference markers were the following: (i) aroI-lssA-dal-purB; (ii) metC-lssB-lssC-furA-pyrE-cysC-lssD; (iii) lssF-gtaA-lssG-hisA-lssH-cysB; and (iv) cysA-lssE-dnaC-purA. Kinetics of N-acetyl-D-[1-14C]glucosamine incorporation revealed that groups A, B, C, D and F are deficient in peptidoglycan synthesis at the restrictive temperature. In group G, anomalies at the cell wall level were suggested by incorporation and growth curves. It appears that in almost all known cases, thermosensitive lysis mutations in B. subtilis either affect genes involved in peptidoglycan synthesis or lead, more or less directly, to induction of prophages.     

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)     

Temperature-Sensitive Mutants of Bacteriophage Mu

Temperature-sensitive mutants of bacteriophage Mu, which grow at 32 C but not at 42 C, have been isolated. These mutants fall into two groups. Group 1 mutants fail to lyse host cells at nonpermissive temperatures, whereas lysis occurs normally with the group 2 mutants. All of the group 1 mutants apparently belong to the cistrons mapping to the left of gene C, whereas the group 2 mutants have lesions in various genes between D and S.     

Temperature-sensitive beta-lactam-tolerant mutants of Escherichia coli

Seven temperature-sensitive penicillin-tolerant mutants of Escherichia coli strain LD5 (thi lysA dapD) were isolated and characterized. Treatment with beta-lactams caused lysis of the mutants at 30 degrees C. Although growth of the mutants at 42 degrees C was inhibited by beta-lactams, no lysis occurred. The mutants were also slightly tolerant to D-cycloserine at 42 degrees C but lysed readily when deprived of diaminopimelate or when treated with moenomycin. The minimum inhibitory concentrations of various antibiotics were the same for the mutants and their parent. The mutations conferring penicillin tolerance were phenotypically suppressed in the presence of a variety of compounds which may act as chaotropic or antichaotropic agents. No defects in penicillin-binding proteins and peptidoglycan hydrolases were detected. Temperature-resistant revertants of the mutants were no longer tolerant to penicillin-induced autolysis at 42 degrees C. The mutations in five isolates were localized to the 56 to 61 min region of the E. coli linkage map and to the 44 to 51 min region in the case of two other isolates.     

A mutant of Streptococcus pneumoniae that exhibits thermosensitive penicillin tolerance and the paradoxical effect

Mutants of Streptococcus pneumoniae that contain active autolysin and yet cannot be induced to lyse during treatment with penicillin (Lyt+Tol+ mutants) have been described. We have now shown that these mutants are temperature dependent (32 degrees C); at 37 degrees C these bacteria underwent penicillin-induced lysis. In addition, mutants at the lysis-permissive temperature showed the so-called 'paradoxical response' to penicillin. Temperature shift experiments indicated that the change from tolerant to lytic response or vice versa is a fast process. No differences were detected in autolysin specific activity or in the kinetics of inhibition of protein, peptidoglycan and teichoic acid syntheses in cells treated with penicillin at 32 and 37 degrees C. The results of genetic crosses indicated that the thermosensitivity of penicillin-induced autolysis in the Lyt+Tol+ mutants is not a property of the autolytic enzyme itself. The observations suggest that the thermosensitive process in the mutants represents either a step(s) in autolysin regulation or involves some difference in the structure of the cell walls produced at 32 degrees C versus 37 degrees C.     

Regulation of Bacterial Cell Division: Genetic and Phenotypic Analysis of Temperature-Sensitive, Multinucleate, Filament-Forming Mutants of Escherichia coli

Three mutants of Escherichia coli K-12 which form filaments during 42 C incubation have been characterized. The mutant strains AX621, AX629, and AX655 continued to grow and to synthesize deoxyribonucleic acid at 42 C for 150 to 180 min, after which time growth ceased. When cultures of the mutants were transferred from 42 to 28 C, septation of the filaments began after a 25- to 30-min period and continued at a greater than normal rate until no filaments remained. Addition of chloramphenicol at the time of transfer from 42 to 28 C prevented cell division in strain AX655 and caused lysis of strains AX621 and AX629. The temperature sensitivity mutation in each strain mapped near leu. For strain AX621, the mutation was specifically located between leu and nadC by P1 transduction. Properties of these strains are compared with those of other cell division mutants.     

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.     

Non-histone proteins and long-range organization of HeLa interphase DNA

We have studied the association of the Sindbis virus glycoproteins in mature virions and infected cells. The glycoproteins were cross-linked with bifunctional amino-reactive reagents (11 Å cross-linking distance), some of which could be subsequently cleaved by reduction. Using monospecific rabbit antisera against each viral envelope glycoprotein it was found that >90% of the cross-linked glycoprotein dimers from intact virions or virions solubilized with Triton X100 prior to cross-linking were heterodimers of E1 and E2. The pattern of cross-linked glycoproteins from intact virions as well as infected cells suggested that three E1-E2 dimers may be associated to form a hexameric subunit. Cross-linking of pulselabeled infected monolayers showed that PE2 was cross-linked to E1 less efficiently than was E2, suggesting that if PE2 and E1 are associated as a complex in infected cells then their conformation with respect to reactive amino groups is distinct from that of the mature virion glycoproteins. ts mutants of Sindbis virus in the complementation groups corresponding to E1 and PE2 fail to cleave PE2 at the non-permissive temperature (40 °C). In monolayers infected with these mutants or a heat-resistant variant of Sindbis virus, the glycoprotein precursors synthesized at the elevated temperature were readily cross-linked into large aggregates, indicating a temperature-sensitive tendency for the proteins to aggregate.