Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12.

Assembly of the OmpF and LamB proteins was kinetically retarded in deep rough lipopolysaccharide mutants of Escherichia coli K-12. OmpF assembly was affected at the step of conversion of metastable trimers to stable trimers, whereas LamB assembly was influenced both at the monomer-to-metastable trimer and metastable-to-stable trimer steps. These assembly defects were reversed in the presence of the sfaA1 and sfaB3 suppressor alleles, which were isolated by using ompF assembly mutants.     

Site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide

The site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide was assessed by collecting high-resolution phosphorus nuclear magnetic resonance spectra in the presence of manganese, a paramagnetic divalent cation. This technique revealed high-affinity interactions between the cation and all of the lipopolysaccharide phosphoryl groups. To ascertain whether the carboxyl groups of 2-keto-3-deoxyoctonate contributed to the metal cation binding, lipopolysaccharide was chemically modified using a glycine ethyl ester - carbodiimide reaction. Of the three available carboxyl groups, only one was neutralized by the exogenously added ligand; the others appeared to be cross-linked within the molecule. By analogy, only one carboxyl group should be freely available for binding metallic ions, while the others are probably neutralized by the close proximity of endogenous amino substituents. Although high-resolution phosphorus nuclear magnetic resonance showed that an intermolecular conformational change had occurred after the carboxyl groups were neutralized, titration with manganese revealed no differences in the apparent strength of the interactions between the cation and the phosphoryl groups. Together, these data suggest that the high affinity of lipopolysaccharide for divalent metallic ions can be attributed primarily to the phosphoryl substituents and not free carboxyl groups.     

Associations of Escherichia coli K-12 OmpF trimers with rough and smooth lipopolysaccharides.

The associations of both rough and smooth lipopolysaccharides (LPS) with the OmpF porin of Escherichia coli K-12 were examined in galE strains deleted for ompC. Transformation with pSS37 and growth with galactose conferred the ability to assemble a Shigella dysenteriae O antigen onto the core oligosaccharide of E. coli K-12 LPS. The association of LPS with OmpF trimers was assessed by staining, autoradiography of LPS specifically labeled with [1-14C]galactose, and Western immunoblotting with a monoclonal antibody specific for OmpF trimers. These techniques revealed that the migration distances and multiple banding patterns of OmpF porin trimers in sodium dodecyl sulfate-polyacrylamide gels were dictated by the chemotype of associated LPS. Expression of smooth LPS caused almost all of the trimeric OmpF to run in gels with a slower mobility than trimers from rough strains. The LPS associated with trimers from a smooth strain differed from the bulk-phase LPS by consisting almost exclusively of molecules with O antigen.     

Electron microscopic observation of crystals of Escherichia coli K-12 lipopolysaccharide

Previously we showed that Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates or solid columns, when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2, and stored at 4 C for 10 days. Later, Escherichia coli K-12 LPS thus treated was found to form discoid crystals as well as hexagonal plate crystals and solid column crystals. Analysis by electron diffraction of the discoid crystals from the direction perpendicular to the basal plane showed that they consisted of hexagonal lattices with the a axis of 4.62 A. This result was quite the same as that of the hexagonal plate crystals. Electron micrographs of the edges of the discoid crystals revealed stacked sheets of the hexagonal plate crystals. From these results it was concluded that formation of the discoid crystals results from irregular overlapping of the hexagonal plate crystals.     

In vitro hexagonal assembly of lipopolysaccharide of Escherichia coli K-12

We examined Escherichia coli K-12 lipopolysaccharide (LPS), which is known to be an R-form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K-12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2 +/- 0.3 nm and that of the calcium salt form 18.5 +/- 0.3 nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.     

Identification of the purI locus in Escherichia coli K-12

A genetic locus has been identified in Escherichia coli that is analogous to the purI locus in Salmonella.     

Role of lipopolysaccharide and outer membrane protein of Escherichia coli K-12 in the receptor activity for bacteriophage T4.

Lipopolysaccharide isolated from Escherichia coli K-12 did not inactivate phage T4, although the cell envelopes with 1% sodium deoxycholate resulted in the release of cytoplasmic membrane proteins, 70% of the lipopolysaccharide, and almost all of the phospholipid. The reconstitution of phage receptor activity was achieved from deoxycholate-soluble and -insoluble fractions by dialysis against a solution of magnesium chloride. Lipopolysaccharide was the only essential component in the deoxycholate-soluble fraction. PhageT4-resistant mutants YA21-6 and YA21-82, having defects in the deoxycholate-soluble and -insoluble fractions, respectively, were isolated. The deoxycholate-soluble fraction of YA21-6 possessed heptoseless lipopolysaccharide, and this defect was responsible for the phage resistance. The deoxycholate-insoluble fraction of YA21-82 lacked outer membrane protein O-8. The addition of O-8 to this fraction together with the wild-type lipopolysaccharide resulted in the appearance of the receptor activity. Furthermore, the reconstitution was successfully achieved with only O-8 and the wild-type lipopolysaccharide, indicating that O-8 was an essential component in the deoxycholate-insoluble fraction.     

Roles of lipopolysaccharide and outer membrane protein OmpC of Escherichia coli K-12 in the receptor function for bacteriophage T4

The roles of lipopolysaccharide and OmpC, a major outer membrane protein, in the receptor function for bacteriophage T4 were studied by using Escherichia coli K-12 strains having mutations in the ompC gene or in genes controlling different stages of lipopolysaccharide synthesis. The receptor activity for T4 was monitored by (i) T4 sensitivity of intact cells, (ii) phage inactivation activity of cell envelopes, and (iii) phage inactivation activity of specimens reconstituted from purified OmpC and lipopolysaccharide. It was found that (i) in the presence of the OmpC protein, the essential region of the lipopolysaccharide for the receptor activity was the core-lipid A region that includes the heptose region, whereas the glucose region was not necessarily required for the receptor function; (ii) the OmpC protein was not required at all when the distal end of the lipopolysaccharide was removed to expose a glucose residue at the distal end; and (iii) when cells lacked both the OmpC protein and the glucose region, they became extremely resistant to T4. Based on these findings, the roles of the OmpC protein and lipopolysaccharide in T4 infection are discussed.     

Peptidoglycan association of bacteriophage T5 receptor in Escherichia coli K-12.

A total of 50% of the FhuA proteins (also called TonA proteins) present in Escherichia coli cells were associated with the peptidoglycan and 50% were free, whether or not this protein was overproduced. This FhuA-peptidoglycan association was made via the lipoprotein.     

Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12

The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the lambda rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.     

Expression and regulation of protein K, an Escherichia coli K1 porin, in Escherichia coli K-12

Using a modified lambda phage as a vector and a procedure developed in Dr. C. Schnaitman's laboratory, we have cloned the structural gene for protein K from an Escherichia coli K1 strain to an E coli K-12 strain. The cloned inserts consist of two HindIII fragments, 4 kb and 6.5 kb in size. The protein produced by the insert is nearly identical to "authentic" protein K when chymotryptic peptides of 125I-labeled proteins are compared. Protein K was found to respond to changes in the osmolarity of the medium, being favored in trypticase soy broth (high osmolarity). This fluctuation was not dependent on a functional ompR gene. However, protein K was not expressed in strains carrying the envZ-473 mutation. Thus, protein K appears to be within a class of exported proteins whose expression is regulated by the envZ gene independent of the ompR gene.     

OmpF assembly mutants of Escherichia coli K-12: isolation, characterization, and suppressor analysis.

This paper describes a novel genetic method used to isolate mutations that alter proper assembly of OmpF in the outer membrane. The thermolabile nature of assembly intermediates allowed selection of temperature-sensitive mutations within the ompF gene. A variant allele of ompF (ompF-Dex) was used because it provided a convenient selectable phenotype (Dex+). Assembly mutants were isolated in two steps. First, amber mutations were obtained that mapped in ompF-Dex. This resulted in a Dex- phenotype. Starting with these Dex- strains, Dex+ revertants were isolated. Mutants that displayed a temperature-sensitive Dex+ phenotype were further characterized. Three such mutants possessed a single substitution within ompF that reverted the nonsense codon to a sense codon which replaced W214 with either an E or Q and Y231 with a Q residue in the mature OmpF protein. All three mutant OmpF proteins showed an assembly defect. This defect led to a substantial reduction in the amount of stable OmpF trimers with the concomitant increase of a high-molecular-weight form of OmpF which migrated at the top of the gel. Suppressor mutations were sought that corrected the assembly defect of OmpF. These extragenic suppressor mutations were mapped at 45 min on the Escherichia coli chromosome. The suppressor mutations displayed no allele specificity and were recessive to the wild-type allele. In the presence of a suppressor, mutant stable trimers appeared in an almost normal manner. The appearance of stable trimers concurred with a substantial loss of the high-molecular-weight OmpF species. At this stage, it is not clear whether the high-molecular-weight species of OmpF is a normal assembly intermediate or a dead-end assembly product. The results presented in this study raise the intriguing possibility of a chaperone-like activity for the wild-type suppressor gene product.     

Two-component nature of bacteriophage T4 receptor activity in Escherichia coli K-12.

Escherichia coli bacteriophage T4 uses the lipopolysaccharide of the outer cell envelope membrane as a receptor. Lipopolysaccharide from E. coli K-12 required a major outer membrane protein, polypeptide Ib, for phage inactivation.     

大肠杆菌E.coli K-12转录因子结合位点的理论预测

基于实验验证的22种大肠杆菌K12的转录因子结合位点序列,分析了转录因子结合位点每一位置的碱基保守性,提出了预测转录因子结合位点的位置权重矩阵打分函数算法(PWMSA)。利用self-consistency和cross-validation两种检验方法对此算法进行检验,self-consistency检验总的预测成功率达到87.59%,cross-validation检验成功率达到85.48%。对基因间序列进行搜索,获得了多个可能的转录因子结合位点。     

Cloning of bacteriophage PM2 DNA in Escherichia coli K12.

Summary DNA fragments of phage PM2 restricted with Hin dIII endonuclease was cloned in the vector pBR 322 in an Escherichia coli K 12 host. The attempt to clone full length PM2 DNA restricted with PstI endonuclease has been unsuccesful. From six randomly chosen recombinant clones DNA was purified and analysed with EcoRI, PstI and HindIII endonucleases. The physical map of three chimeric plasmids was unequivocally established. It was shown, that the whole PM2 genome was cloned, although in separate fragments. However, most of the recombinant clones were instable in the absence of selective pressure.     

Identification of a novel genetic element in Escherichia coli K-12.

Induction of the SOS repair processes of Escherichia coli K-12 caused a 14.4-kilobase species of circular deoxyribonucleic acid, called element e14, to be excised from the chromosome. To aid further characterization of this species, an 11.6-kilobase segment of e14 was inserted into the HindIII site of plasmid pBR313. To map e14 on the E. coli K-12 chromosome, the recombinant plasmid, pAG2, was used to transform a polA recipient, an event which required integration of pAG2 into the recipient chromosome. This recombinational event was dependent upon the region of homology between the incoming plasmid and the chromosome, as no transformants were scored when either a strain cured of the element was the recipient or pBR313 was the transforming deoxyribonucleic acid. Using these transformants, we have shown that e14 maps between the purB and pyrC loci near min 25. Several strains of E. coli K-12 were found to contain e14; however, one strain, Ymel trpA36, did not. In addition, e14 was found to be absent in both E. coli B/5 and E. coli C. The approach to mapping developed for this work could be used to map other fragments of E. coli deoxyribonucleic acid which have no known phenotype.