Expression and purification of the SsbB protein from Streptococcus pneumoniae

The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein.     

Control of developmental gene expression by cell-to-cell interactions in Myxococcus xanthus.

The ssbA mutants of Myxococcus xanthus behave as if they are unable to produce a cell-to-cell signal required for normal development. They are unable to form fruiting bodies or spores on developmental medium. They do sporulate, however, if allowed to develop in mixtures with wild-type cells. Fusions of developmentally induced promoters of M. xanthus to the Escherichia coli lacZ gene were used to characterize the effect of the ssbA mutations on developmental gene expression. Each of the five independent fusions tested was found to be dependent upon the ssbA+ allele for full expression. The ssbA mutants were able to express each of these fusions if the mutants were allowed to develop in mixtures with wild-type (Lac-) cells. These results cannot be explained on the basis of genetic exchange. The data are consistent with regulation of gene expression mediated by cell-to-cell interactions.     

Obligatory coupling of colicin release and lysis in mitomycin-treated Col+ Escherichia coli

Previous work has shown that Escherichia coli K12 strains carrying the small, high copy number ColE2-P9 plasmid produce large amounts of colicin and then lyse and release colicin when grown in broth culture containing mitomycin C. Strains carrying the larger, low copy number ColIa-CA53 plasmid produced much less colicin and did not lyse or discharge more than 15% of their colicin when grown under the same conditions. Naturally-occurring Col+ strains and E. coli K12 derivatives carrying different Col plasmids could be classified either as ColE2+-like or ColIa+-like according to whether or not they produced large amounts of colicin and lysed and discharged colicin when grown in the presence of mitomycin, and also by the size and presumed copy number of the Col plasmid they carried. Strains carrying multiple copies of the cloned colicin Ia structural gene produced large amounts of colicin but did not lyse or release colicin when grown in the presence of mitomycin. This result rules out the possibility that high level accumulation of colicin is sufficient to cause lysis. Conditions were sought under which colicin Ia could be released from the producing cells. It was found that mitomycin-treated cultures of strains carrying both ColE2 and ColIa plasmids released both colicins when they lysed, although colicin Ia release occurred later than colicin E2 release. It was also noted that colicin Ia-laden cells released their colicin when diluted into fresh culture medium.     

Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4.

A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.     

Colicin E2 production and release by Escherichia coli K12 and other Enterobacteriaceae

Previous work has shown that Escherichia coli K12 ColE2+ cells undergo a form of partial lysis and exhibit increases in lysophosphatidylethanolamine (lysoPE) and free fatty acid content due to activation of phospholipase A when induced to produce and release colicin E2. The increase in lysoPE content was assumed to be essential for efficient colicin release. These same characteristics are also presented by some natural ColE2+ isolates, and by other representatives of the Enterobacteriaceae after transformation with derivatives of a ColE2 plasmid. However, Salmonella typhimurium strains carrying ColE2 plasmids released colicin without partial lysis and without increasing their lysoPE content. A previously undetected minor phospholipid, which appeared in these and other strains only when they were induced to produce colicin, may be an important factor in colicin release. In ColE2+ E. coli K12, production of this new lipid was dependent on phospholipase A activation following expression of the ColE2 lysis gene. Some other ColE2+ strains did not respond to induction of colicin production in the same way as ColE2+ E. coli K12. These strains were less sensitive to inducer (mitomycin C) or unable to produce increased amounts of colicin in response to induction, or unable to degrade colicin once it was released. In general, the results suggest that colicin release occurs by the same or similar processes in the various strains tested, and support the continued use of E. coli K12 as the model strain for studying the mechanisms of colicin release.     

The dnaK protein modulates the heat-shock response of Escherichia coli

E. coli bacteria respond to a sudden upward shift in temperature by transiently overproducing a small subset of their proteins, one of which is the product of the dnaK gene. Mutations in dnaK have been previously shown to affect both DNA and RNA synthesis in E. coli. Bacteria carrying the dnaK756 mutation fail to turn off the heat-shock response at 43 degrees C. Instead, they continue to synthesize the heat-shock proteins in large amounts and underproduce other proteins. Both reversion and P1 transduction analyses have shown that the failure to turn off the heat-shock response is the result of the dnaK756 mutation. In addition, bacteria that overproduce the dnaK protein at all temperatures undergo a drastically reduced heat-shock response at high temperature. We conclude that the dnaK protein is an inhibitor of the heat-shock response in E. coli.     

The ntr genes of Escherichia coli activate the hut and nif operons of Klebsiella pneumoniae

Regulation of the synthesis of glutamine synthetase and of the arginine and glutamine transport systems (Ntr phenotype) in Salmonella have been shown to require two regulatory genes on the C-terminal side of the glnA gene (McFarland et al., 1981). We have cloned a HindIII-EcoRI DNA fragment from Escherichia coli coding for analogous properties with respect to the Ntr phenotype in E. coli. A plasmid containing this E. coli DNA fragment joined to another fragment carrying a cyanobacterial glnA gene (but no functional regulatory genes) was introduced into a Klebsiella pneumoniae mutant with a Gln-Ntr- phenotype, i.e., which could not derepress nitrogenase. The cyanobacterial gene made the Klebsiella strain Gln+ and the E. coli DNA fragment made the strain Ntr+, including the ability to derepress nitrogenase fully. Thus the products of the glnA-linked ntr genes of E. coli can regulate expression of the Ntr-dependent genes of Klebsiella.     

Phosphatidylinositol, a phospholipid of ice-nucleating bacteria.

The nature of the phospholipids of the various bacteria that have ice nucleation activity in supercooled water has been determined. The seven bacteria studied included Pseudomonas syringae, Erwinia herbicola, three Escherichia coli K-12 strains that are phenotypically Ice+ because they contain plasmids with different amounts of either P. syringae or E. herbicola cloned DNA, and two E. coli K-12 strains without cloned ice gene DNA. All five Ice+ bacterial strains contained small amounts (0.1 to 1.0% of the total phospholipids) of phosphatidylinositol (PI), a phospholipid not previously detected in E. coli, Pseudomonas, or Erwinia species. The Ice- E. coli strains also contained trace level of PI that amounted to 2 to 30% of the level found in the Ice+ E. coli strains. Extracts of Ice+ strains contained low but measurable activities of PI synthase, while the activities in Ice- strains amounted to only 8 to 12% or less of that found in extracts of Ice+ bacteria. The functioning of the ice gene apparently increased both the PI synthase activity and the PI content of Ice+ strains from low endogenous levels. The relative ice nucleation activity at -4 degrees C or above (class A nucleation activity) of all Ice+ strains was found to be proportional to their PI content. The addition of myo-inositol (5 x 10(-4) M) to synthetic culture media increased the class A nucleation activity of both Ice+ E. coli strains and P. syringae up to sevenfold but had no stimulating effect on ice nucleation at lower temperatures (class B and class C nucleation activities). If these cells after fusion with PI vesicles were incubated with an energy source, the class A nucleation activity increased 70-fold over that present before fusion. These results indicate that PI plays an important role in ice nucleation at warm temperatures and is a likely precursor or component of the class A structure.     

Isolation and characterization of a lambda transducing bacteriophage carrying the cysE gene of Escherichia coli K-12

A specialized lambda transducing phage carrying the cysE and gpsA genes of E. coli K-12 has been isolated. The transducing phage has been separated from the helper phage on equilibrium gradients and has been shown to be defective. Evidence is presented that the phage kil gene is not expressed.     

Involvement of the AtoS-AtoC signal transduction system in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli

The AtoS-AtoC signal transduction system in E. coli, which induces the atoDAEB operon for the growth of E. coli in short-chain fatty acids, can positively modulate the levels of poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles in E. coli. Increased amounts of cPHB were synthesized in E. coli upon exposure of the cells to acetoacetate, the inducer of the AtoS-AtoC two-component system. While E. coli that overproduce both components of the signal transduction system synthesize higher quantities of cPHB (1.5-4.5 fold), those that overproduce either AtoS or AtoC alone do not display such a phenotype. Lack of enhanced cPHB production was also observed in cells overexpressing AtoS and phosphorylation-impaired AtoC mutants. The results were not affected by the nature of the carbon source used, i.e., glucose, acetate or acetoacetate. An E. coli strain with a deletion in the atoS-atoC locus (delta atoSC) synthesized lower amounts of cPHB compared to wild-type cells. When the delta atoSC strain was transformed with a plasmid carrying a 6.4-kb fragment encoding the AtoS-AtoC system, cPHB biosynthesis was restored to the level of the atoSC+ cells. Introduction of a multicopy plasmid carrying a functional atoDAEB operon, but not one with a promoterless operon, resulted in increased cPHB synthesis only in atoSC+ cells in the presence of acetoacetate. These results indicate that the presence of both a functional AtoS-AtoC two-component signal transduction system and a functional atoDAEB operon is critical for the enhanced cPHB biosynthesis in E. coli.     

Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli

A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene.     

Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by MNNG both in E. coli and in S. typhimurium. The AdaST protein encoded by the plasmid increased expression of the ada'-lacZ chromosome fusion about 5-fold when an E. coli strain carrying both the fusion operon and the plasmid was exposed to a low concentration of MNNG, whereas the E. coli Ada protein encoded by a low-copy-number plasmid increased it about 40-fold under the same conditions. The low ability of AdaST to function as a positive regulator could account for the apparent lack of an adaptive response to alkylation damage in S. typhimurium.     

The cloning and expression of the RNAse structural gene of Bacillus intermedius]

The structure gene of extracellular alkaline ribonuclease Bacillus intermedius (binase) has been cloned in E. coli cells in composition of pMT 316 plasmid carrying the inhibitor gene (barstar of barnase--binase structure homologue. The possibility to use such vector has been proved during the barstar action on binase catalytic activity. Using biochemical immunochemical analysis the expression of binase gene in E. coli cells has been confirmed. The recombinant clone E. coli which contains both plasmids simultaneously--carrying gene for barster and for benase has been produced. The given vector is suggested to be used for cloning of inhibitor gene to obtain a viable producer of alkaline intracellular ribonuclease.     

The heat-shock-regulated grpE gene of Escherichia coli is required for bacterial growth at all temperatures but is dispensable in certain mutant backgrounds.

Previous work has established that the grpE+ gene product is a heat shock protein that is essential for bacteriophage lambda growth at all temperatures and for Escherichia coli growth at temperatures above 43 degrees C. Here it is shown that the grpE+ gene product is essential for bacterial viability at all temperatures. The strategy required constructing a grpE deletion derivative carrying a selectable chloramphenicol drug resistance marker provided by an omega insertion and showing that this deletion construct can be crossed into the bacterial chromosome if and only if a functional grpE+ gene is present elsewhere in the same cell. As a control, the same omega insertion could be placed immediately downstream of the grpE+ coding sequence without any observable effects on host growth. This result demonstrates that the inability to construct a grpE-deleted E. coli strain is not simply due to a lethal polar effect on neighboring gene expression. Unexpectedly, it was found that the grpE deletion derivative could be crossed into the bacterial chromosome in a strain that was defective in DnaK function. Further analysis showed that it was not the lack of DnaK function per se that allowed E. coli to tolerate a deletion in the grpE+ gene. Rather, it was the presence of unknown extragenic suppressors of a dnaK mutation that somehow compensated for the deficiency in both DnaK and GrpE function.     

Expression of a Saccharomyces cerevisiae photolyase gene in Escherichia coli.

A 3.3-kilobase PvuII fragment carrying the PHR1 gene of Saccharomyces cerevisiae has been cloned into an Escherichia coli expression vector and introduced into E. coli strains deficient in DNA photolyase. Complementation of the E. coli phr-1 mutation was observed, strongly suggesting that the yeast PHR1 gene encodes a DNA photolyase.     

Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli.

We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. Malonyl transacylase has been overexpressed 155-fold compared to a wild-type strain. Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of cis-vaccenate are incorporated into the membrane phospholipids.     

The tsr gene-coding plasmid pIJ702 prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans

Streptomyces lividans normally accumulated high levels of ppGpp during nutritional shift-down. Its accumulation was, however, severely inhibited when a small amount of thiopeptin (an analogue of thiostrepton) was included in the transfer medium. In contrast, a S. lividans strain, which harbours the plasmid pIJ702 carrying the tsr gene resist to thiopeptin through methylation of the 23S rRNA, was still capable of accumulating ppGpp in the presence of large amounts of thiopeptin. These results indicate that the rRNA methylation resulting from the action of tsr gene prevents thiopeptin not only from inhibiting cell-growth but also from inhibiting ppGpp synthesis. The results also indicate that the observed accumulation of ppGpp during nutritional shift-down was associated with ribosomal function, as has been shown in E. coli and B. subtilis.     

Cloning and expression of the gene for thermostable pullulanase from Clostridium thermohydrosulfuricum in Escherichia coli

Using a pUC19-based genomic library of the anaerobic thermophilic bacterium C. thermohydrosulfuricum a DNA fragment that confers pullulanase activity to E. coli cells has been identified. Subcloning and restriction mapping procedures was carried out and the primary structure of the 5'-region of the pullulanase gene (pul) was determined. The pul enzyme was shown to be a protein with molecular weight of approximately 60,000. It was found that both pullulanase and glucoamylase activities resides in pullulanase. The intracellular distribution of pullulanase was studied. An E. coli strain that produces large amounts of thermostable pullulanase has been constructed.