malB region in Escherichia coli K-12: specialized transducing bacteriophages and first restriction map.

By starting from an Escherichia coli K-12 strain with a lambda phage integrated in the malB region, series of transducing phages carrying part or all of the malB region have been isolated. Genetic mapping of the transduced malB fragments was accomplished by complementation and recombination with known mutations in the region. By using the DNA of these phages, it was found that the malB region is cleaved by the restriction enzymes BglII, EcoRI, HaeII, HincII, SalI, and SstI, but not BamHI, HindIII, KpnI, PstI, XbaI, or XhoI. A physical map was constructed and tentatively correlated with the genetic map.     

malB Region in Escherichia coli K-12: Characterization of New Mutations

Phenotypic characterization and mapping of more than 50 Mal(-) mutations located in the malB region lead one to divide the site for Mal(-)lambdas mutations (formerly called gene malB) in that region, into two adjacent genetic segments malJ and malK. malJ and malK are both involved in maltose permeation. It is suggested that (i) malK and lamB, the only known gene specifically involved in phage lambda adsorption (20), constitute an operon of polarity malK lamB. (ii) malJ and malK correspond to two different genes, and (iii) a promoter for the malK lamB operon is located between malJ and malK. Since lambda receptors and maltose permease are inducible by maltose and absent in malT mutants, it is likely that the expression of the malK lamB operon is controlled by the product of gene malT, the positive regulatory gene of the maltose system.     

On Some Genetic Aspects of Phage λ Resistance in E. COLI K12

Most mutations rendering E. coli K12 resistant to phage lambda, map in two genetic regions malA and malB.-The malB region contains a gene lamB specifically involved in the lambda receptor synthesis. Twenty-one independent lamB mutations studied by complementation belonged to a single cistron. This makes it very likely that lamB is monocistronic. Among the lamB mutants some are still sensitive to a host range mutant of phage lambda. Mutations mapping in a proximal gene essential for maltose metabolism inactivate gene lamB by polarity confirming that both genes are part of the same operon. Because cases of intracistronic complementation have been found, the active lamB product may be an oligomeric protein.-Previously all lambda resistant mutations in the malA region have been shown to map in the malT cistron. malT is believed to be a positive regulatory gene necessary for the induction of the "maltose operons" in the malA region and in the malB region of the E. coli K12 genetic map. No trans dominant malT mutation have been found. Therefore if they exist, they occur at a frequency of less than 10(-8), or strongly reduce the growth rate of the mutants.     

Sustained hydrostatic pressure tolerance of the shallow water shrimp Palaemonetes varians at different temperatures: insights into the colonisation of the deep sea

We investigated the tolerance of adult specimens of the shallow-water shrimp Palaemonetes varians to sustained high hydrostatic pressure (10 MPa) across its thermal tolerance window (from 5 to 27 °C) using both behavioural (survival and activity) and molecular (hsp70 gene expression) approaches. To our knowledge, this paper reports the longest elevated hydrostatic pressure exposures ever performed on a shallow-water marine organism. Behavioural analysis showed a 100% survival rate of P. varians after 7 days at 10 MPa and 5 or 10 °C, whilst cannibalism was observed at elevated temperature (27 °C), suggesting no impairment of specific dynamic action. A significant interaction of pressure and temperature was observed for both behavioural and molecular responses. Elevated pressure was found to exacerbate the effect of temperature on the behaviour of the animals by reducing activity at low temperature and by increasing activity at high temperature. In contrast, only high pressure combined with low temperature increased the expression of hsp70 genes. We suggest that the impressive tolerance of P. varians to sustained elevated pressure may reflect the physiological capability of an ancestral species to colonise the deep sea. Our results also support the hypothesis that deep-sea colonisation may have occurred during geological periods of time when the oceanic water column was warm and vertically homogenous.     

Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein

Mal+ lacZ operon fusions, inducible by maltose, were isolated in Escherichia coli, strain MC4100. One fusion strain, SF1707, was analyzed in detail. This fusion did not map in any of the known genes of the malA or malB region, but its expression was under control of malT, the positive regulator gene of the maltose regulon. The gene in which the fusion occurred mapped between xyl and mtl at 80 min on the linkage map and was transcribed clockwise. We define this gene as malS. The malS-lacZ fusion was transferred onto a phage lambda vector and the 5' portion of malS was subcloned into pBR322. The resulting plasmid was used as a probe to identify the intact malS gene in a lambda library of E. coli chromosomal HindIII fragments. The phage that hybridized with the probe contained a 12-kilobase insert. The malS containing portion was subcloned into pBR322 as a 4-kilobase ClaI-HindIII fragment. This plasmid directed the malT and maltose-dependent synthesis of a periplasmic protein of 66,000 apparent molecular weight. The purified enzyme hydrolyzed maltodextrins longer than maltose including cyclic dextrins. The primary products of hydrolysis were glucose, maltose, and maltotriose, even when maltotetraose was used as a substrate. These properties differentiate this periplasmic enzyme from the cytoplasmic amylomaltase and define it as an alpha-amylase.     

Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme.

Model-free approaches (random mutagenesis, DNA shuffling) in combination with more "rational," three-dimensional information-guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage lambda, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque-forming ability. Compensating mutations in W138P T4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid lambda phage with sufficient lysozyme activity to produce normal-size plaques, even at elevated temperature (42 degrees C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target-randomized variants.     

Interference with phage lambda development by the small subunit of the phage 21 terminase, gp1.

Bacteriophage lambda development is blocked in cells carrying a plasmid that expresses the terminase genes of phage 21. The interference is caused by the small subunit of phage 21 terminase, gp1. Mutants of lambda able to form plaques in the presence of gp1 include sti mutants. One such mutation, sti30, is an A. T-to-G.C transition mutation at base pair 184 on the lambda chromosome. The sti30 mutation extends the length of the ribosome-binding sequence of the Nul gene that is complementary to the 3' end of the 16S rRNA from GGA to GGAG. The sti30 mutation causes a approximately 50-fold increase in the level of expression of a Nul-lacZ reporter gene, indicating that the sti30 mutation overcomes the gp1 inhibition by increasing the level of expression of gpNul. Although the Nul and A genes of lambda overlap, the sti30 mutation has little effect on the level of gpA expression, indicating that translational coupling does not occur.     

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.     

Characterization of the SOS response of Pseudomonas fluorescens strain DC206 using whole-genome transcript analysis

DNA microarray technology was used to survey changes in gene expression in Pseudomonas fluorescens after mitomycin C treatment. As expected, genes associated with the SOS response were upregulated, such as those encoding the recombination protein RecA, DNA repair protein RecN, excinuclease ABC subunit A UvrA, and the LexA repressor protein. Interestingly, expression of 33 clustered bacteriophage-like genes was upregulated, suggesting that mitomycin C (MMC) may induce a prophage resident in the P. fluorescens genome. However, no phage particles were detected in P. fluorescens strain DC206 that had been treated with MMC using transmission electron microscopy. The same preparation failed to produce phage plaques on lawns of any of 10 different Pseudomonas strains tested, indicating that the 33 bacteriophage-like gene cluster represents a defective prophage.     

Oligopeptidase A is required for normal phage P22 development.

The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing.     

Synchronous effects of temperature, hydrostatic pressure, and salinity on growth, phospholipid profiles, and protein patterns of four Halomonas species isolated from deep-sea hydrothermal-vent and sea surface environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30 degrees C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30 degrees C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30 degrees C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30 degrees C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

Control of lysogeny and immunity of Bacillus subtilis temperate bacteriophage SP beta by its d gene.

The d gene from the Bacillus subtilis temperate bacteriophage SP beta was isolated. When introduced into an SP beta-sensitive strain of B. subtilis, the cloned d gene directed the synthesis of a 22-kilodalton protein and conferred on the host immunity to SP beta phage. A frameshift mutation, designated d2, was introduced into the cloned d gene, and it was subsequently crossed back into the SP beta phage genome. The resulting SP beta phage grew lytically and formed clear plaques on sensitive bacteria. Although the cloned d gene confers immunity to the host, we could not detect complementation of the d gene by mixed infection with SP beta d2 and various SP beta c mutants. The nucleotide sequence of the 1,033-base-pair PstI-to-EcoRI fragment containing the d gene was determined; it includes an open reading frame that could potentially encode a protein of 227 amino acids. The gene was mapped within the PstI H fragment on the phage genome, which positions the d gene about 25 kilobases from the right end of the phage genome. It is transcribed from right to left.     

Analysis of intracellular pH in the yeast Saccharomyces cerevisiae under elevated hydrostatic pressure: a study in baro- (piezo-) physiology

Hydrostatic pressure is a distinctive feature of deep-sea environments, and this thermodynamic parameter has potentially inhibitory effects on organisms adapted to living at atmospheric pressure. In the yeast Saccharomyces cerevisiae, hydrostatic pressure causes a delay in or cessation of growth. The vacuole is a large acidic organelle involved in degradation of cellular proteins or storage of ions and various metabolites. Vacuolar pH, as determined using the pH-sensitive fluorescent dye 6-carboxyfluorescein, was analyzed in a hydrostatic chamber with transparent windows under elevated hydrostatic pressure conditions. A pressure of 40–60 MPa transiently reduced the vacuolar pH by approximately 0.33. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH after application of hydrostatic pressure, indicating that the transient acidification is mediated through the function of vacuolar H⁺-ATPase. The vacuolar acidification was observed only in the presence of fermentable sugars, and never observed in the presence of ethanol, glycerol, or 3-o-methyl-glucose as the carbon source. Analysis of a glycolysis-defective mutant suggested that glycolysis or CO₂ production is involved in the pressure-induced acidification. Hydration and ionization of CO₂ is facilitated by elevated hydrostatic pressure because a negative volume change (ΔV < 0) accompanies the chemical reaction. Moreover the glucose-induced cytoplasmic alkalization is inhibited by elevated hydrostatic pressure, probably because of inhibition of the plasma membrane H⁺-ATPase. Therefore, the cytoplasm tends to become acidic under elevated hydrostatic pressure conditions, and this could be crucial for cell survival. To maintain a favorable cytoplasmic pH, the yeast vacuoles may serve as proton sequestrants under hydrostatic pressure. We are investigating the physiological effects of hydrostatic pressure in the course of research in a new experimental field, baro- (piezo-) physiology. Received: January 22, 1998 / Accepted: February 16, 1998     

A PCR-based method for high stringency screening of DNA libraries.

A rapid method for cloning genomic DNA utilizing a PCR-based screening protocol is described. A murine genomic library in lambda phage was subdivided into 64 wells, each containing 1000 clones, and propagated in bacteria. Amplified phage from each of 8 wells across columns, and each of 8 wells down rows, were pooled. The pooled phage were screened for the presence of murine M-CSF DNA by PCR using specific oligonucleotide primers. A single well that contained an M-CSF genomic clone was identified by the synthesis of a PCR product of the correct size that hybridized to an internal M-CSF oligonucleotide probe. This well was subdivided into 64 wells, each containing approximately 30 individual phage, reamplified, and rescreened utilizing the same protocol. A positive well was then subdivided and amplified a third time starting with an average of 2 phage per well, and rescreened for M-CSF DNA by PCR. Phage from a PCR-positive well, now highly enriched for M-CSF DNA, were grown as individual plaques. PCR-screening of randomly picked plaques demonstrated that the majority contained an M-CSF genomic insert. This method obviates the more labor and time intensive method of plaque hybridization screening of DNA libraries, and is more stringent since three oligonucleotides (the two PCR primers, and the hybridization probe) are required to give a true positive signal. Similar methodology has also been used to clone a cDNA gene contained within a plasmid library.