Is the lactose-utilization ability ofRhizobium ofSesbania procumbens a vestigeal function?

Rhizobium SBS-R100, isolated from the stem nodules ofSesbania procumbens, synthesized -galactosidase constitutively. Transposon mutagenesis by Tn9 induced mutants defective in lactose utilization; the mutations did not interfere with growth, nodulation or N₂ fixation. Mouse monoclonal antibody raised against -galactosidase ofEscherichia coli reacted with soluble proteins of wild typeRhizobium SBS-R100. Anin vivo constructed recombinant plasmid pSBS-4 complemented aRhizobium mutant defective in lactose utilization.     

An improved selection method for λlacZ − phages based on galactose sensitivity

The determination of thelacZ mutant frequency in gt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on -galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl--d-galactopyranoside, a substrate for -galactosidase, this leads to the suppression of lacZ ⁺ phage propagation without affecting the ability of lacZ ^– phages to form plaques. With this method it is possible to screen 1.5×10⁶ phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.     

Identification of σs-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri

Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor σ^s. Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS ^? derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.     

Investigation of the Relationship between the Lectin Activity of Azospirilla and Their α-Glucosidase, β-Glucosidase,and β-Galactosidase

The activities of -glucosidase, -glucosidase, and -galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilenseSp7 and Azospirillum lipoferum59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol–acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied for the azospirilla studied. The cofunction of the A. brasilenseSp7 lectin and -galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum59b lectin and glucosidases was revealed. The lectin from A. lipoferum59b may possess saccharolytic activity.     

Comparison of wild-type and UV-mutant β-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications

A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal -glucans. Activity against -(1, 3)(1, 4)-d-glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less -glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley -glucan (13.0–16.9%), but were more active against crude -glucan from barley (16.0–24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash -glucan. Finally, TC2 and TC5 produce more efficient -glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications.     

Gene Delivery into Rat Glomerulus Using a Mesangial Cell Vector

To develop an effective protocol of gene transfer into glomeruli, an ex vivo gene delivery system using rat mesangial cells (RMC) as a vector was examined. RMC genetically engineered with a retrovirus harboring the Escherichia coli -galactosidase gene was used to estimate the efficacy of gene delivery and the location of the cells within the kidney. The RMC expressing -galactosidase, RMCLZ1, was cultured in vitro and the cells were injected into the left kidney through the renal artery of a normal Sprague Dawley rat. At least 1 x 10⁶ RMCLZ1 was required for effective gene delivery into glomeruli. One hour and 1, 4, and 14 d after injection, glomeruli were isolated from the left kidneys injected with the cells and the expression of -galactosidase in each glomeruli was evaluated. One hour and 1 d after injection, more than 90 and 80%, respectively, of glomeruli from the left kidney showed strong -galactosidase activity, while no activity of -galactosidase was found in the glomeruli from the right kidneys. The number of glomeruli stained by X-gal and the intensity decreased with time. Fourteen days after injection, about 35% of the glomeruli retained the RMCLZ1. X-gal and periodic acid-Schiff staining of frozen sections obtained 14 d after injection allowed the estimation of the site where the mesangial cells injected were located. The mesangial cells were found mainly in two different locations, the glomerular capillary and the mesangium. The majority (about 90%) of the mesangial cells were located in the glomerular capillary and about 9% of the cells were in the mesangial area. Occasionally, the positive staining was found in proximal tubules and the interlobular artery. Although additional methods are required for the site-specific targeting of the mesangial area, the ex vivo gene transfer to glomerli uis feasible and may be a useful tool for future investigations in the pathological mechanisms of glomerular injury.     

Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: Use of AUA as a restart codon

Summary An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the -galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active -galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the -galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.     

lacZ fusions to genes that specify exported proteins: A general technique

Summary We have devised a general, one-step technique for isolation of strains in which the gene coding for an exported protein is fused to the gene for -galactosidase (lacZ). These fusions specify a hybrid protein comprised of an NH₂-terminal portion of the exported protein and a large functional COOH-terminal portion of -galactosidase. The fusions are constructed with a derivative of the MudII(lac, Ap) phage. To overcome the lethality that is often associated with the expression of such a hybrid gene, we have recombined an early lacZ nonsense mutation onto this phage. With the use of strains that carry a temperaturesensitive nonsense suppressor, expression of the full-length hybrid protein can be controlled by varying the growth temperature. We demonstrated the utility of this technique by isolating a series of fusions to a gene, ompA, coding for a major outer membrane protein. As expected, strains containing these fusions are not viable under conditions that permit synthesis of a functional nonsense suppressor. Accordingly, this method should also be useful for direct selection of export-defective mutants.     

Lytic activity localized to membrane-spanning region of ?X174 E protein

Summary Lytic activity of the X174E (lysis) protein had previously been localized to the amino terminal 51 amino acids (a.a.) of the molecule (Blasi and Lubitz 1985). This E gene lytic activity has here been further localized to the amino terminal 29 a.a., a region of the protein which is thought to just span the cell membrane (Young and Young 1982). X174 E gene fusions to both the lacZ gene and the chloramphenicol acetyl transferase (CAT) gene resulted in fusion proteins with lytic activity. Fusion to a third protein, trpE, did not result in lytic activity. These results support a model of oligomerization of the X174 E protein for lytic activity since both -galactosidase and CAT exist as tetramers in their native state. A difference in the composition of the charged amino acids at the cytoplasmic boundary between the various fusion proteins could also account for these results, since these amino acids may play a role in proper anchoring of the E protein in the cell membrane. In a spontaneous E gene mutant, which introduces a proline residue at position 9 of the E protein, lytic activity of the E protein was decreased, but not abolished. The presence of the helix-breaking proline at this position may interfere with insertion of the lysis protein into the cell membrane, leading to the decreased functional activity of the protein.     

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmic-galactosidase (-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that all-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions.     

Analysis of the reduction in expression of tetracycline resistance determined by transposon Tn10 in the multicopy state

The tet genes of transposon Tn10 have been mapped in a 2,200 bp DNA sequence by analysing deletion and Tn5 insertion mutations. When the tet genes were present on multi-copy plasmids the level of resistance expressed was about ten-fold lower than that determined by a single copy of Tn10 in the E. coli chromosome. The 36K tet protein known to be encoded by R100 in E. coli minicells was not detected when they harboured a multicopy tet plasmid. However, normal high levels of resistance were expressed when the tet genes were recombined into the host chromosome as part of a lambda lysogen, showing that the multicopy effect was phenotypic. Most of the Tn5 insertions and deletions in tet which caused Tc^s mutations also prevented expression of high level Tc^r from a chromosomal Tn10 element present in the same cell. Only those insertions in the promoter-proximal 90–130 bp of a 1,275 bp HindII fragment known to carry the gene encoding the 36K tet protein did not reduce the single copy Tn10 resistance level.A gene fusion system that results in the constitutive synthesis of -galactosidase from a tet promoter has been used to assay tet repressor activity. The basal (uninduced) -galactosidase level in cells carrying multicopy tet plasmids was 10–20 fold lower than those carrying a single copy. The tet:: Tn5 mutants defective in the trans-dominant multicopy effect still made normal amounts of tet repressor showing that repressor overproduction was not responsible for this effect. In addition a repressor-defective constitutive mutant did not exhibit a higher resistance level when located on a multicopy plasmid vector. We postulate that a regulatory mechanism recognises the amino-terminus of the tet structural gene product when attempts are being made to overproduce the protein and prevents further translation.     

Several phenotypic changes in the cell envelope of Agrobacterium tumefaciens chvB mutants are prevented by calcium limitation

The chvB gene of Agrobacterium tumefaciens encodes a 235 kDa proteinaceous intermediate involved in the synthesis of -1,2-glucan. chvB mutants show a pleiotropic phenotype. Besides not to produce cyclic -1,2-glucan, chvB mutants have been reported to be avirulent, attachment-deficient, and nonmotile. In this study we report additional differences from the parent strain, probably all linked to changes in the cell envelope. This pleiotropic phenotype — except for attachment and virulence — could largely be prevented by growing chvB cells with low levels of calcium. Although a role for -1,2-glucan in osmoadaptation has been proposed, the mode of action of -1,2-glucan is not known. We speculate that in A. tumefaciens -1,2-glucan stabilizes membranes, which would be important especially in hypotonic media containing calcium.Abbreviations Cb carbenicillin - Km kanamycin - TCA trichloroacetic acid - K_av fraction of the stationary gel volume available for diffusion - LPS lipopolysaccharide - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Heterologous Kluyveromyces lactis β-galactosidase secretion by Saccharomyces cerevisiae super-secreting mutants

Several Saccharomyces cerevisiae strains with a super-secreting phenotype have been transformed using a secretion plasmid containing the LAC4 gene and have proven to be effective in the secretion of Kluyveromyces lactis -galactosidase. The strain CGY1585 (ssc1-1) showed the highest secretion (1.7 EU ml^–1) in the culture medium. As far as we know, Kluyveromyces lactis -galactosidase is the largest sized protein and the only intracellular one among those secreted by these mutants hitherto. The recombinant strains all grew in lactose media.     

Reversion of a truncated gene for ampicillin resistance by genetic rearrangements in Escherichia coli K12

Summary The composite transposon Tn2672 is a derivative of the Tn3-related transposon Tn902 whose bla gene providing ampicillin resistance had been inactivated by the insertion of the IS1-flanked multiple drug resistance transposon Tn2671. Most ampicillin resistant revertants of Tn2672 are due to precise excision of Tn2671. However, a rare Bla⁺ revertant which still retains all the previously acquired drug resistance markers was isolated. On this revertant, the 5 part of the split bla gene on Tn2672 has converted to an intact, active bla gene, and the entire Tn902 is structurally restored. In contrast, the adjacent IS1b element belonging to Tn2671 has its terminal 142 base pairs deleted. Despite of this rearragement, the split 3 part of bla and its adjacent sequences have remained unchanged. Models are presented to explain the observed DNA rearrangements, and their similarity with gene conversion events is discussed.     

Germ line transmission and expression of alacZ containing transgene in tilapia (Oreochromis niloticus)

Transgenic tilapia were produced using a carp -actinlacZ gene construct and the transmission of the transgene to F₁ progeny was followed. Of 36 founder fish analysed, 9 (25%) were found to be transgenic in at least one tissue and two of these were found to transmit the transgene to progeny. One male produced only three out of 28 transgene DNA positive progeny (10.7%) but none of these were found to express the transgene. The other male produced 243 out of 1601 (15.2%)lacZ expressing embryos; 40 of these were analysed by Southern blotting and all were found to be positive for the presence of the transgene but revealed two different patterns of transgene arrangement. Three different expression patterns were also observed and presumed to be a result of multiple integration events and resulting position effects.     

Round-cell mutants of Salmonella typhimurium produced by transposition mutagenesis: Lethality of rodA and mre mutations

Summary Thirty-three insertions of transposon Tn10l6l7 into genes involved in the control of rod cell shape were isolated in Salmonella typhimurium by the characteristic glossy appearance of colonies composed of spherical cells. Genetic tests demonstrated that 25 (76%) were insertions in the rodA gene, 7 (21 %) were mre mutants, and 1 (3%) was a divD mutant. No insertion in the pbpA gene were found. Insertions in cell shape genes only appeared when strains displaying resistance to mecillinam (not caused by -lactamase production) were employed. Neither rodA nor mre insertions could be transduced to wild-type strains but they were normally accepted by mecillinam-resistant derivatives and by cya and crp mutants, which, unlike the corresponding Escherichia coli strains, did not display resistance to mecillinam. On the other hand, the divD insertion could be efficiently transduced to any strain. It is concluded that the rodA, mre, and divD genes are involved in the control of rod cell shape but, in addition, the RodA and Mre products perform some function(s) that is essential for wild-type cells but dispensable for some mecillinam-resistant strains, and for cya and crp mutants.     

Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives

Summary Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli -galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac⁺ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.Abbreviations bp base pairs - Nm neomycin - Km kanamycin - Sm streptomycin - Sp spectinomycin - Gm gentamycin - Tc tetracycline - Tp trimethoprim - Ot oxytetracycline - Rf rifampicin - Xgal 5-bromo-4-chloro-3-indolyl--d-galactoside