Effects of the Escherichia coli sfsA gene on mal genes expression and a DNA binding activity of SfsA

The sfsA gene was identified as one of the sfs genes the over-expression of which stimulates maltose fermentation of the Mal- Escherichia coli strain MK2001 (crp*1, cya:Km(r)). Expression from the malPQ promoter, which was measured using a chromosomally integrated malPp-lacZ fusion, was induced by over-expressing the sfsA gene in the crp*1, cya:Km(r) strain. The level of the MalE protein was increased in crp*1, cya:Km(r) cells over-producing SfsA. The SfsA protein was purified to homogeneity and tested for DNA binding activity. The purified SfsA protein binds to DNA non-specifically. All these results may suggest that SfsA functions as a DNA binding protein to induce the mal genes in coordination with CRP*1.     

Primary structure of the maltose-permease-encoding gene of Saccharomyces carlsbergensis

The MAL6 locus of Saccharomyces consists of a cluster of at least three genes: MAL6R encodes a positively acting regulatory protein; MAL6S encodes maltase; and MAL6T encodes maltose permease. A MAL6 Eco RI fragment, E1, that encompasses most of the MAL6T gene except for the first 90 bp of the ORF at its 5' end (sequenced previously), was cloned into a pGEM-Blue vector. Sequential deletions were generated and then sequenced. The MAL6T gene has a putative ORF of 1845 bp. The amino acid composition and sequence of the deduced protein shows that it is highly hydrophobic and has a size of 68.2 kDa. Computer-generated hydropathy profiles suggest that the MAL6T protein may have up to nine membrane-spanning regions. Generation of functional fusions of the MAL6T promoter region to Escherichia coli lacZ-containing vectors indicates that sequences in the intergenic region are responsible for the induction of MAL6T by maltose and for its carbon catabolite repression. We also demonstrated the suitability of E. coli lacZ as a reporter gene for promoter activity studies in yeast.     

Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae.

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.     

Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.     

Mapping, cloning, expression, and sequencing of the rhaT gene, which encodes a novel L-rhamnose-H+ transport protein in Salmonella typhimurium and Escherichia coli.

A L-rhamnose transport-negative strain of Escherichia coli was generated by Mu d(ApR,lac)I mutagenesis. This strain was used to isolate a clone of Salmonella typhimurium DNA that encoded L-rhamnose-H+ transport activity, the gene for which, rhaT, was sequenced. The rhaT gene was mapped on the E. coli chromosome between rhaR and sodA at 87.9 min, initially by Southern blot analysis and then by the isolation, expression, and sequencing of the rhaT gene. Both rhaT genes encoded a hydrophobic protein of 344 amino acids (91% identical) that contained 10 putative transmembrane regions. The RhaT protein represents a novel class of sugar transport protein.     

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.     

Bacteriophage T4 gene 59 helicase assembly protein binds replication fork DNA. The 1.45 A resolution crystal structure reveals a novel alpha-helical two-domain fold

The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection. T4 gene 59 helicase assembly protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro. T4 gene 59 helicase assembly protein binds to both T4 gene 41 helicase and T4 gene 32 single-stranded DNA binding protein, and to single and double-stranded DNA. We show here that T4 gene 59 helicase assembly protein binds most tightly to fork DNA substrates, with either single or almost entirely double-stranded arms. Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than six, but not more than 12 nucleotides. The 1.45 A resolution crystal structure of the full-length 217-residue monomeric T4 gene 59 helicase assembly protein reveals a novel alpha-helical bundle fold with two domains of similar size. Surface residues are predominantly basic (pI 9.37) with clusters of acidic residues but exposed hydrophobic residues suggest sites for potential contact with DNA and with other protein molecules. The N-terminal domain has structural similarity to the double-stranded DNA binding domain of rat HMG1A. We propose a speculative model of how the T4 gene 59 helicase assembly protein might bind to fork DNA based on the similarity to HMG1, the location of the basic and hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding. The fork-binding model suggests putative binding sites for the T4 gene 32 single-stranded DNA binding protein and for the hexameric T4 gene 41 helicase assembly.     

A method for the construction of in frame substitutions in operons: deletion of the essential Escherichia coli holB gene coding for a subunit of the DNA polymerase III holoenzyme

To investigate the putative five-gene operon at 24.9 min on the Escherichia coli genome, which comprises the genes pabC, yceG, tmk, holB and ycfH, a method for the construction of an in frame deletion strain of the essential E. coli holB gene was developed. HolB, also referred to as delta prime or delta', is a subunit of the DNA polymerase III (Pol III) holoenzyme. The holB gene was replaced by the kanamycin resistance gene kka1, coding for amino glycoside 3'-phosphotransferase kanamycin kinase. The kanamycin resistance gene was expressed under the control of the promoter(s) of the putative five-gene operon. The holB gene is essential for bacterial growth and the deletion of holB exhibits no polar effects on the adjacent genes tmk or ycfH in terms of cell viability. The method of the holB null construction presented in this work allows for a simplified studying of interactions between the different subunits of DNA polymerase III.     

Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster.

The genetic determinants of enterobacterial common antigen (ECA) include the rfe and rff genes located between ilv and cya near min 85 on the Escherichia coli chromosome. The rfe-rff gene cluster of E. coli K-12 was cloned in the cosmid pHC79. The cosmid clone complemented mutants defective in the synthesis of ECA due to lesions in the rfe, rffE, rffD, rffA, rffC, rffT, and rffM genes. Restriction endonuclease mapping combined with complementation studies of the original cosmid clone and six subclones revealed the order of genes in this region to be rfe-rffD/rffE-rffA/rffC-rffT-rffM . The rfe gene was localized to a 2.54-kilobase ClaI fragment of DNA, and the complete nucleotide sequence of this fragment was determined. The nucleotide sequencing data revealed two open reading frames, ORF-1 and ORF-2, located on the same strand of DNA. The putative initiation codon of ORF-1 was found to be 570 nucleotides downstream from the termination codon of rho. ORF-1 and ORF-2 specify putative proteins of 257 and 348 amino acids with calculated Mr values of 29,010 and 39,771, respectively. ORF-1 was identified as the rfe gene since ORF-1 alone was able to complement defects in the synthesis of ECA and 08-side chain synthesis in rfe mutants of E. coli. Data are also presented which suggest the possibility that the rfe gene is the structural gene for the tunicamycin sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase that catalyzes the synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), the first lipid-linked intermediate involved in ECA synthesis.     

Heterologously expressed polypeptide from the yeast meiotic gene HOP1 binds preferentially to yeast DNA.

HOP1 protein, present in sporulating cells of Saccharomyces cerevisiae and believed to be a component of the synaptonemal complex, has been expressed in Escherichia coli fused to a biotinylated tag protein. Once solubilized from bacterial inclusion bodies, the HOP1 fusion protein was purified by using a combination of avidin-affinity chromatography and gel filtration FPLC and refolded. Sequence comparisons indicate that the HOP1 gene product contains a zinc finger motif, which may confer DNA binding properties, and the recombinant polypeptide was used to assess the putative DNA binding properties of the product of native HOP1 protein using a gel-shift assay. Protein and protein-DNA complexes were detected by exploiting the affinity of streptavidin-alkaline phosphatase for the biotinylated tag protein after Western blotting. The HOP1 fusion protein bound unambiguously to digested genomic yeast DNA. This binding possessed some degree of specificity, was maintained under a wide range of salt concentrations, and was unaffected by the presence of high concentrations of competitor DNA (synthetic poly[dI-dC].poly[dI-dC]). In contrast, no shift was detected when the fusion protein was incubated with digested genomic DNA from Arabidopsis, or with lambda/HindIII DNA. Incubation with digested genomic DNA from Lilium produced a small change in the mobility of the protein. The biotinylated tag protein failed to show any DNA binding activity. Scatchard analysis indicated an apparent yeast genomic DNA:HOP1 fusion protein dissociation constant of K(d) = 5 x 10(-7) M.