Construction of scrA::lacZ gene fusions to investigate regulation of the sucrose PTS of Streptococcus mutans

The scrA gene coding for sucrose EnzymeII of the phosphoenolpyruvate dependent phosphotransferase system previously isolated from Streptococcus mutans was fused in vitro to the promoterless lacZ' gene to monitor the expression of the scrA gene. The scrA::lacZ gene fusion was introduced back into S. mutans GS-5IS3 by two independent transformation procedures involving either linear or plasmid DNA to produce both scrA and scrA+ mutants. These mutants should prove useful for analyzing the regulation of sucrose transport in S. mutans.     

Amino-sugar transport systems of Escherichia coli K12

Glucosamine, mannose and 2-deoxyglucose enter Escherichia coli by the phosphotransferase system coded for by the gene ptsM. The glucosamine- and mannose-negative, deoxyglucose-resistant phenotype of ptsM mutants can be suppressed by a mutation mapping near ptsG that allows constitutive expression of the glucose phosphotransferase coded for by the gene ptsG. N-Acetylglucosamine enters E. coli by two distinct phosphotransferase systems (White, 1970). One of these is the PtsM system, the other is coded for by a gene which maps near the nagA,B genes at about min 15 on the E. coli chromosome. We propose that this gene be designated ptsN. Strains with either of these components of the phosphotransferase system will utilize N-acetylglucosamine as sole carbon source.     

Suppression of IIIGlc-defects by Enzymes IINag and IIBgl of the PEP:carbohydrate phosphotransferase system

The Enzymes II of the PEP:carbohydrate phosphotransferase system (PTS) specific for N-acetylglucosamine (IINag) and beta-glucosides (IIBgl) contain C-terminal domains that show homology with Enzyme IIIGlc of the PTS. We investigated whether one or both of the Enzymes II could substitute functionally for IIIGlc. The following results were obtained: (i) Enzyme IINag, synthesized from either a chromosomal or a plasmid-encoded nagE+ gene could replace IIIGlc in glucose, methyl alpha-glucoside and sucrose transport via the corresponding Enzymes II. An Enzyme IINag with a large deletion in the N-terminal domain but with an intact C-terminal domain could also replace IIIGlc in IIGlc-dependent glucose transport. (ii) After decryptification of the Escherichia coli bgl operon, Enzyme IIBgl could substitute for IIIGlc. (iii) Phospho-HPr-dependent phosphorylation of methyl alpha-glucoside via IINag/IIGlc is inhibited by antiserum against IIIGlc as is N-acetylglucosamine phosphorylation via IINag. (iv) In strains that contained the plasmid which coded for IINag, a protein band with a molecular weight of 62,000 D could be detected with antiserum against IIIGlc. We conclude from these results that the IIIGlc-like domain of Enzyme IINag and IIBgl can replace IIIGlc in IIIGlc-dependent carbohydrate transport and phosphorylation.     

Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 has four enzyme II (EII) genes of the phosphotransferase system in its genome encoding transporters for sucrose, glucose, fructose, and an unidentified EII. To analyze the function of these EII genes, they were inactivated via homologous recombination and the resulting mutants characterized for sugar utilization. Whereas the sucrose EII was the only transport system for sucrose in C. glutamicum, fructose and glucose were each transported by a second transporter in addition to their corresponding EII. In addition, the ptsF ptsG double mutant carrying deletions in the EII genes for fructose and glucose accumulated fructose in the culture broth when growing on sucrose. As no fructokinase gene exists in the C. glutamicum genome, the fructokinase gene from Clostridium acetobutylicum was expressed in C. glutamicum and resulted in the direct phosphorylation of fructose without any fructose efflux. Accordingly, since fructokinase could direct fructose flux to the pentose phosphate pathway for the supply of NADPH, fructokinase expression may be a potential strategy for enhancing amino acid production.     

Plasmid instabilities of single and three-plasmid systems in Escherichia coli during continuous cultivation.

Plasmid instabilities in E. coli JM103 carrying three plasmids (pRK248cI, pMTC48, pEcoR4) and a single plasmid system (pTG206) for the production of fusion EcoRI (SPA::EcoRI) and catechol 2,3-dioxygenase, respectively, were investigated in continuous cultures under selective and non-selective conditions. In a three-plasmid system, pRK248cI was lost gradually together with pMTC48 from the host under non-selective conditions. The selective pressure against pRK248cI stabilized the pMTC48. This indicates that the loss of pMTC48 under non-selective conditions was caused by the loss of cI857 gene (coded by pRK248cI) which resulted in the overproduction of the toxic gene product (coded by pMTC48). In the case of single plasmid (pTG206) system, the plasmid lost from the host under non-selective conditions. This plasmid was stabilized in the host growing under selective conditions. During this period we obtained some ampicillin resistant colonies which gave low levels of enzyme activities compared to the normal plasmid bearing cells. Plasmid analysis from the above cells showed that the plasmid has undergone structural instability. Further, restriction analysis of this plasmid exhibited an additional PvuII site in a 0.9 kbp fragment that was integrated near the tet promoter which controls the expression of the xyl E gene, thereby resulting low levels of enzyme activities. Our results indicate that some of the IS elements which are present in the host chromosome were responsible for such instabilities to turn off the synthesis by inserting into the tet promoter region to lower the protein formation during the bioprocess.     

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.     

Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12.

The conjugative plasmid pUR400 determines tetracycline resistance and enables cells of Escherichia coli K-12 to utilize sucrose as the sole carbon source. Three types of mutants affecting sucrose metabolism were derived from pUR400. One type lacked a specific transport system (srcA); another lacked sucrose-6-phosphate hydrolase (scrB); and the third, a regulatory mutant, expressed both of these functions constitutively (scrR). In a strain harboring pUR400, both transport and sucrose-6-phosphate hydrolase were inducible by fructose, sucrose, and raffinose; if a scrB mutant was used, fructose was the only inducer. These data suggested that fructose or a derivative acted as an endogenous inducer. Sucrose transport and sucrose-6-phosphate hydrolase were subject to catabolite repression; these two functions were not expressed in an E. coli host (of pUR400) deficient in the adenosine 3-,5'-phosphate receptor protein. Sucrose uptake (apparent Km = 10 microM) was dependent on the scrA gene product and on the phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) of the host. The product of sucrose uptake (via group translocation) was identified as sucrose-6-phosphate, phosphorylated at C6 of the glucose moiety. Intracellular sucrose-6-phosphate hydrolase catalyzed the hydrolysis of sucrose-6-phosphate (Km = 0.17 mM), sucrose (Km = 60 mM), and raffinose (Km = 150 mM). The active enzyme was shown to be a dimer of Mr 110,000.     

Stable transformation of moth bean Vigna aconitifolia via direct gene transfer

Direct gene transfer proved to be an efficient transformation method for Vigna aconitifolia, a member of the legume family. Kanamycin resistant calli and plants were regenerated from heat shocked protoplasts treated with PEG and plasmid DNA containing the coding region for aminoglycoside phosphotransferase gene (NPT II). The plant cultivar used was an important factor in attaining higher transformation frequencies. Transformation was confirmed by Southern blot analysis using a non-radioactive detection system. Attempts to transform mesophyll and suspension cultured cells by this method were unsuccessful. Protoplasts electroporated with the plasmid pCAP212, which codes for chloramphenicol acetyltransferase, exhibited transient expression of this gene two days after treatment while electroporated cells did not show this enzyme activity. It is therefore assumed that the DNA uptake is prevented by the cell wall.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

Modified binary plant transformation vectors with the wild-type gene encoding NPTII.

The defective gene encoding neomycin phosphotransferase (NPTII) present in the binary plasmid vector, pBin19, was replaced with the wild-type (wt) gene. Plasmid vectors analogous to pBin19, pBI121 and pBI101 were constructed carrying the gene encoding the wt NPTII enzyme activity.     

Molecular analysis of thescrA andscrB genes fromKlebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme IIScr of the phosphotransferase system and a sucrose-6-phosphate invertase

TheKlebsiella pneumoniae genesscrA andscrB are indispensable for sucrose (Scr) utilisation. GenescrA codes for an Enzyme II^Scr (II^Scr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), whilescrB encodes a sucrose 6-phosphate specific invertase. A 3.7 kbscrAB DNA fragment has been cloned fromK. pneumoniae and expressed inEscherichia coli. Its nucleotide sequence was determined and the coding regions forscrA (1371 bp) andscrB (1401 bp) were identified by genetic complementation, enzyme activity tests and radiolabelling of the gene products. In addition, the nucleotide sequence of thescrB gene from the conjugative plasmid pUR400 isolated fromSalmonella typhimurium was also determined and errors in the previously published sequence of thescrA gene of pUR400 were corrected. Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes. Based on the analysis of seven II^Scr proteins, a hypothetical model of the secondary structure of II^Scr is proposed.     

Molecular analysis of thescrA andscrB genes fromKlebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme IIScr of the phosphotransferase system and a sucrose-6-phosphate invertase

TheKlebsiella pneumoniae genesscrA andscrB are indispensable for sucrose (Scr) utilisation. GenescrA codes for an Enzyme II^Scr (II^Scr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), whilescrB encodes a sucrose 6-phosphate specific invertase. A 3.7 kbscrAB DNA fragment has been cloned fromK. pneumoniae and expressed inEscherichia coli. Its nucleotide sequence was determined and the coding regions forscrA (1371 bp) andscrB (1401 bp) were identified by genetic complementation, enzyme activity tests and radiolabelling of the gene products. In addition, the nucleotide sequence of thescrB gene from the conjugative plasmid pUR400 isolated fromSalmonella typhimurium was also determined and errors in the previously published sequence of thescrA gene of pUR400 were corrected. Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes. Based on the analysis of seven II^Scr proteins, a hypothetical model of the secondary structure of II^Scr is proposed.     

Nature and properties of hexitol transport systems in Escherichia coli.

In Escherichia coli K-12 the naturally occurring hexitols D-mannitol, D-glucitol, and galactitol are taken up and phosphorylated via three distinct transport systems by a mechanism called either group translocation or vectorial phosphorylation. For every system, a membrane-bound enzyme II-complex of the phosphoenolpyruvate-dependent phosphotransferase system has been found, each requiring phosphoenolpyruvate, enzyme I, and HPr or alternatively P-HPr as the phosphate donor. Cells with a constitutive synthesis of all hexitol transport systems but with low P-HPr levels have very low transport and phosphorylating activities in vivo, although 40 to 90% of the enzyme II-complex activities are detected in cell extracts of such mutants. No indications for additional hexitol transport systems, especially for systems able to transport and accumulate free hexitols as in Klebsiella aerogenes, have been found. Substrate Km, and Vmax of the three transport systems for several hexitols and hexitol analogues have been determined by growth rates, transport activities, and in vitro phosphorylating activities. Each system was found to take up several hexitols, but only one hexitol serves as the inducer. This inducer invariably is the substrate with the highest affinity. Since bacterial transport systems, as a general rule, seem to have a relatively broad substrate specificity, in contrast to a more restricted inducer specificity, we propose to name the system inducible by D-mannitol and coded by the gene mtlA the D-mannitol transport system, the system inducible by D-glucitol and coded by gutA the D-glucitol transport system, and the system inducible by galactitol and coded by gatA the galactitol transport system.     

Food-grade host/vector expression system for <Emphasis Type="Italic">Lactobacillus casei</Emphasis> based on complementation of plasmid-associated phospho-β-galactosidase gene <Emphasis Type="Italic">lacG</Emphasis>

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.     

Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli.

We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system. We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations. Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response. Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system. Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars. Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars.     

Nucleotide sequence analysis of the gene specifying the bifunctional 6''-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.