Survival of Escherichia coli and Vibrio harveyi in Dead Sea water

Abstract When suspensions of Escherichia coli or the marine luminescent bacterium Vibrio harveyi were mixed with Dead Sea water, the number of viable bacteria decreased by 90% in a time varying from less than 1 h to several hours, depending on the bacterial strain tested. Survival was better at low temperatures, and diluting the Dead Sea water permitted prolonged survival of both coliform bacteria and V. harveyi . The death rate of E. coli in Dead Sea water was comparable to that in water from the Great Salt Lake (Utah). The high concentrations of calcium and magnesium in Dead Sea water, rather than the high total salinity, was identified as the main factor responsible for the rapid die-off. Exposure to direct solar irradiation significantly increased the die-off rate of E. coli in Dead Sea water.
Large numbers of coliform bacteria were recovered from the lake at distances of at least 20 m from a sewage discharge site on the western shore of the Dead Sea.     

Expression of bioluminescence by Escherichia coli containing recombinant Vibrio harveyi DNA.

When isogenic strains of Escherichia coli, RR1 (rec+) and HB101 (recA), were transformed with mapped recombinant plasmids known to contain Vibrio harveyi luciferase genes and large regions of DNA flanking on both sides, a small percentage (0.005%) of the colonies expressed high levels of luminescence (up to 10(12) quanta s-1 ml-1) in the absence of added aldehyde. The altered ability to express light was found to be due to a mutation in the host and not to an alteration in the recombinant DNA. When these bright colonies were cured of plasmid, they could be retransformed with cloned V. harveyi gene fragments in cis and in trans to yield luminescent colonies at 100% frequency. The maximum length of V. harveyi DNA required to produce light-emitting E. coli was shorter (6.3 kilobase pairs) than that required for expression of the V. fischeri system in E. coli. Cell extracts from bright clones contained wild-type levels of activity for the heteropolymeric (alpha beta) luciferase; fatty acid labeling revealed the presence of the three acylated polypeptides of the fatty acid reductase system which is involved in aldehyde biosynthesis for the luminescence reaction. The increased light emission in the mutant bacteria appeared to arise in part from production of higher levels of polycistronic mRNAs coding for luciferase.     

Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli.

Binding of the DnaA protein to its binding sites, the DnaA-boxes (TTATCCACA), was measured by a simple physiological approach. The presence of extra DnaA-boxes in growing cells leads to a derepression of dnaA gene expression, measured as beta-galactosidase activity of a dnaA-lacZ fusion polypeptide. Different DnaA-boxes caused different degrees of derepression indicating that the DnaA protein requires sequences in addition to the DnaA-box for efficient binding. The DnaA-boxes in oriC might act cooperatively in binding of the DnaA protein. The derepressed levels of DnaA protein obtained in a strain carrying an oriC+-pBR322 chimera were very high and sufficient to activate oriC on the chimeric plasmid, which was maintained at a copy number more than three times that of pBR322.     

Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region.

Initiation of chromosomal replication in Escherichia coli is dependent on availability of the initiator protein DnaA. We have introduced into E. coli cells plasmids carrying the chromosomal locus datA, which has a high affinity for DnaA. To be able to monitor oriC initiation as a function of datA copy number, we introduced a minichromosome which only replicates from oriC, using a host cell which replicates its chromosome independently of oriC. Our data show that a moderate increase in datA copy number is accompanied by increased DnaA protein synthesis that allows oriC initiation to occur normally, as measured by minichromosome copy number. As datA gene dosage is increased dnaA expression cannot be further derepressed, and the minichromosome copy number is dramatically reduced. Under these conditions the minichromosome was maintained by integration into the chromosome. These findings suggest that the datA locus plays a significant role in regulating oriC initiation, by its capacity to bind DnaA. They also suggest that auto regulation of the dnaA gene is of minor importance in regulation of chromosome initiation.     

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

Genetic analysis of the Escherichia coli K-12 srl region.

Specialized transducing lambda derivatives, deletion mapping, and Plkc transductional crosses have been used to analyze the genetic organization and regulation of the srl genes. Transducing phages obtained from a secondary site lambda insertion in srlA are of two types: lambdapsrlC1 and lambdaprecA are substituted in the b2 region of the lambda chromosome (galtype) and carry the srlC gene but not srlD; lambdapsrlD is substituted in the early region of the phage deoxyribonucleic acid (biotype) and carries the srlD gene but not srlC. The lambdapsrlC1 phage, which lysogenizes at attlambda, complements srlC mutants in trans, indicating that this gene codes for a diffusable positive regulatory element. The srl genes have been ordered relative to the cysC, recA, and alaS genes by two- and three-factor P1kc crosses. The order, cysC...srlD-srlA-srlC-recA-alaS, has been obtained. The srlA and srlD genes comprise an operon with srlD operator distal. From the secondary site lysogen, it has been possible to obtain deletion mutants of this region that are sensitive to ultraviolet light and are recombination deficient. Genetic evidence suggests that these deletions extend from srl into the recA gene.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

Analysis of the DNA-binding domain of Escherichia coli DnaA protein

The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374-467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC:lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC:lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.     

Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene

One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds. Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen. In the present work we have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase. We show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes. The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures. The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production. The maximum rate of DBT removal was 8 mg/h. g dry cell weight. Experiments were also conducted using resting cells with the addition of various carbon sources. It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate (51 mg/h. g dry cell weight). The culture with acetate and no oxidoreductase expression had the highest level of HBP production. For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase. Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting.     

Translocation of Vibrio harveyi N,N''-diacetylchitobiase to the outer membrane of Escherichia coli.

The gene encoding N,N'-diacetylchitobiase (chitobiase) of the chitinolytic marine bacterium Vibrio harveyi has been isolated. While expression of the chitobiase gene (chb) was inducible by N,N'-diacetylchitobiose in V. harveyi, it was expressed constitutively when cloned in Escherichia coli, suggesting that controlling elements are not closely linked to chb. Chitobiase was found in the membrane fraction of E. coli cells containing plasmids with the cloned V. harveyi chb gene. When membranes of such cells were separated on Osborn gradients, chitobiase activity was found mainly in the outer membrane band. Translocation of the enzyme to the outer membrane was accompanied by cleavage of a signal peptide. A fusion protein, in which 22 amino acids from the amino terminus of prechitobiase were replaced with 21 amino acids from the pUC19 lacZ amino terminus, was not processed, and 99% of the activity was located in the cytoplasmic fraction. A homology to six amino acids surrounding the lipoprotein processing and modification site was found near the amino terminus of prechitobiase.     

The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E. coli replication origin

Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763-4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB.     

Genetic analysis of transpositions in the lac region of Escherichia coli

The lac region of Escherichia coli, carried on an F′ lacproB episome, was used as a target for the transposition of several transposable elements. Tn9 shows a preferential integration (by a factor of 50) into a region extending from the end of the Z gene through the Y gene. Throughout the remainder of the lacI, Z and Y genes one other short region, located in the middle of the I gene, is favored for integration. Within these favored regions many different integration points are evident. Inspection of the DNA sequence for the I and Y genes, and parts of the Z gene, shows a strong correlation between A + T richness and regions of preferential integration. Tn5 insertions follow a similar pattern, although with less preference; whereas Tn10 insertions (provided by T. J. Foster), also favor the Y gene and the end of Z, but are distributed among fewer integration points. Most of the Tn3 insertions into the episome are accompanied by a nearby or adjacent deletion.     

Topology analysis of the colicin V export protein CvaA in Escherichia coli.

The antibacterial protein toxin colicin V is secreted from Escherichia coli cells by a dedicated export system that is a member of the multicomponent ATP-binding cassette (ABC) transporter family. At least three proteins, CvaA, CvaB, and TolC, are required for secretion via this signal sequence-independent pathway. In this study, the subcellular location and transmembrane organization of membrane fusion protein CvaA were investigated. First, a series of CvaA-alkaline phosphatase (AP) protein fusions was constructed. Inner and outer membrane fractionations of cells bearing these fusions indicated that CvaA is inner membrane associated. To localize the fusion junctions, the relative activities of the fusion proteins, i.e., the amounts of phosphatase activity normalized to the rate of synthesis of each protein, as well as the stability of each fusion, were determined. These results indicated that all of the fusion junctions occur on the same side of the inner membrane. In addition, the relative activities were compared with that of native AP, and the protease accessibility of the AP moieties in spheroplasts and whole cells was analyzed. The results of these experiments suggested that the fusion junctions occur within periplasmic regions of CvA. We conclude that CvaA is an inner membrane protein with a single transmembrane domain near its N terminus; the large C-terminal region extends into the periplasm. This study demonstrates the application of AP fusion analysis to elucidate the topology of a membrane-associated protein having only a single transmembrane domain.     

The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA

Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo-oligomerization of DnaA via its N-terminus (amino acid residues 1-86) is also essential for initiation. Results from solid-phase protein-binding assays indicate that residues 1-86 (or 1-77) of DnaA are necessary and sufficient for self interaction. Using a 'one-hybrid-system' we found that the DnaA N-terminus can functionally replace the dimerization domain of coliphage lambda cl repressor: a lambdacl-DnaA chimeric protein inhibits lambda plasmid replication as efficiently as lambdacI repressor. DnaA derivatives with deletions in the N-terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N-terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N-termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross-talk with the nucleotide-binding domain III or the DNA-binding domain IV. We propose that E. coli DnaA is composed of largely independent domains - or modules - each contributing a partial, though essential, function to the proper functioning of the 'holoprotein'.     

Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium.

The DnaA protein concentration was determined in five different Escherichia coli strains and in Salmonella typhimurium LT2 growing at different growth rates. The DnaA protein concentration was found to be invariant over a wide range of growth rates in the four E. coli K-12 strains and in S. typhimurium. In E. coli B/r the DnaA protein concentration was generally higher than in the K-12 strains, and it increased with decreasing growth rates. For all the strains, there appears to be a correlation between the DnaA protein concentration and the initiation mass. This supports the concept of the concentration of DnaA protein setting the initiation mass and, thus, that the DnaA protein is a key molecule in the regulation of initiation of chromosome replication in members of the family Enterobacteriaceae.     

New non-detrimental DNA-binding mutants of the Escherichia coli initiator protein DnaA

The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain. Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure. By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.