Isolation of a novel protein involved in the transport of fructose by an inducible phosphoenolpyruvate fructose phosphotransferase system in Streptococcus mutans.

Fructose transport in Streptococcus mutans LG-1 is mediated by at least two distinct phosphoenolpyruvate fructose phosphotransferase systems. One system is constitutive and consists of membrane components enzyme II as well as enzyme I and heat-stable protein. The other system is inducible and requires, in addition to enzyme I and heat-stable protein, a soluble substrate-specific protein for catalytic activity. This protein factor, designated IIIfru, was purified by DEAE-cellulose chromatography, hydroxylapatite chromatography, molecular sieving on Sephadex G-75, and preparative electrophoresis. The purified preparation showed only one protein band, with a molecular weight of 12,600, on sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, on gel electrophoresis with the discontinuous buffer Tris-glycine, and after electrofocusing in gel (pI congruent to 3.7). The molecular weight of the native protein determined by gel filtration at 4 degrees C was 51,000. Immunodiffusion experiments performed with immunoglobulins prepared against the purified IIIfru from S. mutans LG-1 suggested that other S. mutans strains possessed a IIIfru. No precipitin bands, however, were detected with extracts from S. salivarius, S. sanguis, S. lactis, S. faecalis, Staphylococcus aureus, Bacillus subtilis, Lactobacillus casei, and Escherichia coli.     

The multifunctional fructose-specific component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli K12--fruA gene product

Mutational damage of the ptsH gene leads to pleiotropic disturbance of sugar utilization in Escherichia coli K12. A fruS mutation suppresses the defect because of a constitutional expression of the fruB and fruA genes. FruB protein possessing a pseudo-HPr activity replaces the HPr. It was shown that wild type allele fruS+ dominates over the fruS1156 mutation in heterozygous merodiploid. The existence of thermosensitive mutations (fruS4 and fruS12) which repair the ptsH damage was also demonstrated. The fruS mutations were located in the fru operon. Fructose utilization was not disturbed in fruS1156 mutant, but fruS2 and fruS12 mutants were unable to utilize fructose. Spontaneous mutations (fruS6 and fruS13) possessing the same phenotype at any temperature similar to the thermosensitive ones under nonpermissive conditions were isolated. They were mapped using the P1vir transduction. The fruS mutations were found in the structural gene of the fructose operon. Presumably it is the fruA gene that cods for the fructose-specific multidomain protein IIB'Bc of the phosphoenolpyruvate-dependent phosphotransferase system.     

A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity.

A DNA fragment from Xanthomonas campestris pv. campestris that partially restored the carbohydrate fermentation pattern of a cya crp Escherichia coli strain was cloned and expressed in E. coli. The nucleotide sequence of this fragment revealed the presence of a 700-base-pair open reading frame that coded for a protein highly similar to the catabolite activation factor (CAP) of E. coli (accordingly named CLP for CAP-like protein). An X. campestris pv. campestris clp mutant was constructed by reverse genetics. This strain was not affected in the utilization of various carbon sources but had strongly reduced pathogenicity. Production of xanthan gum, pigment, and extracellular enzymes was either increased or decreased, suggesting that CLP plays a role in the regulation of phytopathogenicity.     

Molecular cloning and sequencing of an operon, carRS of Azospirillum brasilense, that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism.

A pleiotropic carbohydrate mutant, CR17, of Azospirillum brasilense RG (wild type) that assimilates C4 dicarboxylates (succinate and malate) but not carbohydrate (fructose, arabinose, galactose, glycerol, and gluconate) as C sources for growth was used to identify the car (carbohydrate regulation) locus by complementation analysis. The 2.8-kb genomic fragment that complemented the Car- defect of CR17 and overlapped the fru operon (S. Chattopadhyay, A. Mukherjee, and S. Ghosh, J. Bacteriol. 175:3240-3243, 1993) has now been completely sequenced. The sequence contains an operon, carRS, coding for two proteins, CARR and CARS, having 236 and 352 amino acid residues, respectively. The 3'-flanking region of the carRS operon showed sequence homology with the 5' terminus of the fruB gene of a related bacterium, Rhodobacter capsulatus. A complementation study with carRS deletion clones showed that only the carR+ gene was required to complement the Car- defect of CR17, signifying that the carbohydrate pleiotropy was due to a lesion within this gene. Although the 2.8-kb DNA containing the carRS operon when introduced by conjugation into CR17 also complemented the Car- defect, the complemented transconjugant was unable to utilize succinate as a C source. The reason for this is not clear. A sequence analysis of the two protein products strongly suggests that the protein pair may constitute a novel two-component regulatory system for global expression of carbohydrate catabolic pathways in A. brasilense.     

A new gene responsible for an energy-transducing system in Escherichia coli

A mutant strain (ttr-3) of Escherichia coli was originally isolated as a strain resistant to tributyltin exhibiting temperature-sensitive depressions of growth and ATP synthesis on succinate plates at 42 degrees C. The ttr gene was mapped between the pyrE and dnaA genes (in the 82-83 min region) on the chromosome by P1-transduction experiments. Comparison of proline transport and oxygen uptake by membrane vesicles of the wild-type transductant and the mutant (ttr-3) transductant showed that membrane vesicles of the mutant exhibited temperature-sensitive decrease of proline transport and increase of oxygen uptake at the restrictive temperature (42 degrees C), compatible with depression of growth of the mutant at this temperature. Therefore, the ttr gene seems to code for some factor involved in the respiratory chain that is present in the inner membrane of Escherichia coli.     

The role of cytotoxic necrotizing factor-1 in colonization and tissue injury in a murine model of urinary tract infection

Cytotoxic necrotizing factor-1 (CNF1) is commonly found in Escherichia coli isolates from patients with urinary tract infection (UTI). To determine whether CNF1 is an important UTI virulence factor we compared the ability of a clinical E. coli UTI isolate and a CNF1-negative mutant of that isolate to colonize and induce histological changes in the urinary tract in a murine model of ascending UTI. We found no evidence that the mutant strain was attenuated.     

Differentiation between mutants of Escherichia coli K defective in oxidative phosphorylation.

Hybrid membrane particles from two mutants of Escherichia coli K12, Bv4 and K11, defective in oxidative phosphorylation, have been prepared, in which ATP-driven membrane energization is restored. A soluble factor of mutant K11 was found to have properties similar to parental crude coupling factor, ATPase (EC 3.6.1.3). Membrane particles of this mutant could not be reconstituted by parental coupling factor. Either parental coupling factor, or the soluble factor of mutant K11 could reconstitute both respiration-driven and ATP-driven energization to membrane particles of mutant Bv14 or to parental particles depleted of ATPase. Mutant Bv4 was found to be devoid of coupoing factor activity, while retaining the ability to hydrolyze ATP. Both mutants possess an ATPase with an altered binding to the membrane. Mutant K11 is impaired in respiration-driven amino acid transport, in contrast to mutant Bv4. The three major subunits of parental Escherichia coli ATPase have been isolated and antibodies have been prepared against these subunits. Antibodies against the largest subunit (alpha component) or against the intact catalytic subunits (alpha + beta components) inhibit both ATP-Pi exchange in the parent organism as well as ATP hydrolytic activity in parent and mutants. Antibodies against the two other subunits (beta or gamma components) also inhibit these two reactions, but were found to be less effective. Mutant N144, which lacks ATPase activity, shows no precipitin lines with anti-alpha, anti-beta, anti-gamma, or anti (alpha + beta) preparations. In contrast, mutants Bv4 and K11, exhibit cross-reactivity with all of the antisera.     

The use of bacteria as probes for lectins in preparations of solubilized human tonsil cell membranes

In earlier work we have shown that some bacteria bind naturally to lymphocyte subpopulations and that this binding may be due to lectin-carbohydrate interactions. Here we determined the possibility of using bacteria to probe for these lectins in solubilized tonsil cell membrane preparations. Since lectins are capable of agglutination, we determined the ability of human tonsil cell membrane extract (TCME) to agglutinate bacteria. We used Escherichia coli strain YS57 which does not bind to human lymphocytes and a mutant strain derived from it, E. coli UI 2023, which binds to about 50 percent of human lymphocytes. The UI 2023 was agglutinated while the YS57 was not; this agglutination was not due to antibodies or DNA. When E. coli UI 2023 was treated with periodate, it lost its ability to be agglutinated. The agglutination of E. coli UI 2023 was not blocked by any of the monosaccharides and disaccharides used but was blocked by the E. coli LPS, more specifically, by its carbohydrate moiety. Also, the E. coli UI 2023 absorbed the agglutinating factor while its parental strain, YS57, did not. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of TCME after absorption with bacteria showed that a band around 67kD was absent in the TCME absorbed by E. coli prevented the absorption by E. coli UI 2023 whereas Na2IO4-treated LPS did not. In addition, tonsil cell membrane was radioiodinated before obtaining the TCME; sodium dodecylsulfate-polyacrylamide gel electrophoresis of the radioiodinated TCME recovered after elution from E. coli UI 2023, but not from E. coli YS57, showed again a band around 67 kD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.     

Purification and characterization of the fructose-inducible HPr-like protein, FPr, and the fructose-specific enzyme III of the phosphoenolpyruvate: sugar phosphotransferase system of Salmonella typhimurium

The proteins comprising the fructose-specific phosphoenolpyruvate:sugar phosphotransferase system were investigated using a strain of Salmonella typhimurium which lacks the general phosphotransferase system proteins, HPr and Enzyme I, synthesizes the fructose phosphotransferase system proteins, FPr, Enzyme IIfru, Enzyme IIIfru, and fructose-1-phosphate kinase, constitutively, and expresses the Enzyme I-like protein Enzyme I. Enzyme I activity was found in the cytoplasmic fraction, Enzyme IIfru in the membrane fraction, and FPr and Enzyme IIIfru activities were distributed between the two fractions. Extraction of membranes with butanol and urea led to quantitative release of the membrane-associated Enzyme IIIfru and FPr activities, while Enzyme IIfru remained with the membranes. FPr was purified to homogeneity using ion exchange chromatography, gel filtration, and reversed phase high pressure liquid chromatography (HPLC), and its amino acid composition and N-terminal sequence were determined. A complex of FPr and Enzyme IIIfru (Mr 50,000) was also purified to near homogeneity using ion exchange chromatography, gel filtration, and chromatography on hydroxylapatite. When the purified complex was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was visualized as two protein bands with mobilities corresponding to molecular weights of about 40,000 (Enzyme IIIfru) and 9,000 (FPr). Neither the FPr and Enzyme IIIfru activities nor the proteins represented by these two bands separated during the above chromatography steps or using any of several other techniques, including reversed phase HPLC, indicating a very tight association. Active Enzyme IIIfru free of FPr was never isolated or observed. The proteins could be separated in denatured form by gel filtration in the presence of guanidine HCl or urea. Free FPr and the FPr-Enzyme IIIfru complex were characterized, and the properties of free and complexed FPr were compared to those of HPr.     

High-frequency transformation of Rhizobium meliloti.

Transformation of R factor RP4 and its derivative pRK290 from Escherichia coli to Rhizobium meliloti is reported. The efficiency of transformation was in the range of 10(-5) per viable cell. In addition, chromosomal DNA prepared from one R. meliloti strain resistant to streptomycin was transferred to the isoleucine-valine-requiring mutant susceptible to streptomycin.     

SOS system induction in Escherichia coli cells with distinct levels of ribonucleotide reductase activity.

The UV-mediated induction of recA and sfiA genes in Escherichia coli cells with distinct levels of dATP has been studied. Low levels of dATP were obtained by using either a temperature-sensitive ribonucleotide (RDP) reductase-deficient (nrdA) mutant or a wild-type strain treated with hydroxyurea. High pools of dATP were achieved by using a plasmid overproducing RDP reductase. The results obtained show that expression of the recA and sfiA genes was inhibited neither in the UV-irradiated nrdA mutant at 42 degrees C nor in the wild-type strain in the presence of hydroxyurea. Likewise, the increase of the dATP pool did not enhance recA and sfiA gene expression after UV irradiation. All these data suggest that the basal level of dATP is not a limiting factor in the process of induction of the SOS system in Escherichia coli.     

The core lipopolysaccharide of Escherichia coli is a ligand for the dendritic-cell-specific intercellular adhesion molecule nonintegrin CD209 receptor

The dendritic-cell-specific intercellular adhesion molecule nonintegrin (DC-SIGN) CD209 is a receptor for Escherichia coli K-12 that promotes bacterial adherence and phagocytosis. However, the ligand of E. coli for DC-SIGN has not yet been identified. In this study, we found that DC-SIGN did not mediate the phagocytosis of several pathogenic strains of E. coli, including enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, and uropathogenic E. coli, in dendritic cells or HeLa cells expressing human DC-SIGN antigen. However, we showed that an outer core lipopolysaccharide (LPS) (rough) mutant, unlike an inner core LPS (deep rough) mutant or O-antigen-expressing recombinant of E. coli K-12 was phagocytosed. These results demonstrate that the host cells expressing DC-SIGN can phagocytose E. coli in part by interacting with the complete core region of the LPS molecule. These results provide a mechanism for how O antigen acts as an antiphagocytic factor.     

Functional effects of deleting the coiled-coil motif in Escherichia coli elongation factor Ts.

Elongation factor Ts (EF-Ts) is the guanine nucleotide-exchange factor for elongation factor Tu (EF-Tu) that is responsible for promoting the binding of aminoacyl-tRNA to the mRNA-programmed ribosome. The structure of the Escherichia coli EF-Tu-EF-Ts complex reveals a protruding antiparallel coiled-coil motif in EF-Ts, which is responsible for the dimerization of EF-Ts in the crystal. In this study, the sequence encoding the coiled-coil motif in EF-Ts was deleted from the genome in Escherichia coli by gene replacement. The growth rate of the resulting mutant strain was 70-95% of that of the wild-type strain, depending on the growth conditions used. The mutant strain sensed amino acid starvation and synthesized the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate at a lower cell density than the wild-type strain. Deletion of the coiled-coil motif only partially reduced the ability of EF-Ts to stimulate the guanine nucleotide exchange in EF-Tu. However, the concentration of guanine nucleotides (GDP and GTP) required to dissociate the mutant EF-Tu-EF-Ts complex was at least two orders of magnitude lower than that for the wild-type complex. The results show that the coiled-coil motif plays a significant role in the ability of EF-Ts to compete with guanine nucleotides for the binding to EF-Tu. The present results also indicate that the deletion alters the competition between EF-Ts and kirromycin for the binding to EF-Tu.     

Novel mutants of elongation factor G

A novel mutant form of elongation factor G (EF-G) in Escherichia coli is described. This variant EF-G restricts reading frame errors by a factor of 2 to 3 in vivo at two different positions in a lacIZ fusion. In addition, a conventional fusidic acid resistant (fusR) mutant of EF-G was compared with the restrictive mutant. Both mutants were characterized in vitro in a steady-state poly(U) translating system. The data indicate that the restrictive EF-G variant has an altered interaction with the ribosome both in vivo and in vitro. In contrast, the conventional fusR variant is altered in its interaction with GTP, which is evident in vitro.     

ompC突变造成大肠杆菌在碱性条件下对渗透压敏感

大肠杆菌DH42突变株碱性条件下对高渗透压敏感。采用mini-Tn5转座突变质粒,同源重组构建突变菌株和DNA片段亚克隆等技术确定了造成大肠杆菌DH42在碱性条件下,对高渗透压敏感的原因是ompC基因突变。通过P1转导,构建了大肠杆菌D9（W3110 ompC：：kan）菌株。比较D9菌株和DH42菌株在不同pH和不同盐浓度条件下的生长,发现大肠杆菌ompC基因是大肠杆菌在碱性条件下应对高渗透压环境胁迫的必须基因。