Genetic characterization of Escherichia coli type 1 pilus adhesin mutants and identification of a novel binding phenotype.

Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.

Rapid, synchronous, and stable induction of type 1 piliation in Escherichia coli by using a chromosomal lacUV5 promoter.

Type 1 pili are filamentous proteinaceous appendages produced by certain members of the family Enterobacteriaceae. In Escherichia coli, the adhesive properties of these pili are due to the binding of at least one minor pilus component to mannose, a sugar common to cell surface molecules of many eukaryotic cells. The study of pilus assembly may be benefited by a rapid way of inducing pilus synthesis de novo. We describe herein the construction and characterization of a strain in which piliation can be rapidly induced by the addition of lactose or its analog isopropyl-beta-D-thiogalactopyranoside. This was accomplished by placing the chromosomal fimA gene (encoding the major structural subunit of pili) under lacUV5 promoter control. Further experiments suggested that transcription of genes downstream of fimA, whose products are required for normal pilus assembly and function, may also be controlled by the lacUV5 promoter. The construction described herein may have a variety of applications apart from aiding the study of pilus assembly since its adhesive properties can be rapidly and easily turned on and off.

Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding.

We describe the characterization of two genes, fimF and fimG (also called pilD), that encode two minor components of type 1 pili in Escherichia coli. Defined, in-frame deletion mutations were generated in vitro in each of these two genes. A double mutation that had deletions identical to both single lesions was also constructed. Examination of minicell transcription and translation products of parental and mutant plasmids revealed that, as predicted from the nucleotide sequence and previous reports, the fimF gene product was a protein of ca. 16 kDa and that the fimG gene product was a protein of ca. 14 kDa. Each of the constructions was introduced, via homologous recombination, into the E. coli chromosome. All three of the resulting mutants produced type 1 pili and exhibited hemagglutination of guinea pig erythrocytes. The latter property was also exhibited by partially purified pili isolated from each of the mutants. Electron microscopic examination revealed that the fimF mutant had markedly reduced numbers of pili per cell, whereas the fimG mutant had very long pili. The double mutant displayed the characteristics of both single mutants. However, pili in the double mutant were even longer than those seen in the fimG mutant, and the numbers of pili were even fewer than those displayed by the fimF mutant. All three mutants could be complemented in trans with a single-copy-number plasmid bearing the appropriate parental gene or genes to give near-normal parental piliation. On the basis of the phenotypes exhibited by the single and double mutants, we believe that the fimF gene product may aid in initiating pilus assembly and that the fimG product may act as an inhibitor of pilus polymerization. In contrast to previous studies, we found that neither gene product was required for type 1 pilus receptor binding.

Concomitants of asymptomatic retinal cholesterol emboli.

BACKGROUND AND PURPOSE: Asymptomatic retinal cholesterol emboli are sometimes encountered on ophthalmoscopic examination. They are associated with decreased survival, but their clinical significance is not fully known. We sought to determine which vascular risk factors are associated with such emboli. METHODS: We studied 70 consecutive men (55-84 years old) with asymptomatic retinal cholesterol emboli diagnosed in an eye clinic. Twenty-one men (57-78 years old) from the same eye clinic without retinal emboli or retinal ischemic events were randomly selected as control subjects. We determined vascular risk factors, presence of ischemic heart disease, and extracranial carotid artery disease. RESULTS: Patients had a higher prevalence of hypertension, smoked more, and had a higher prevalence of heterogeneous or echolucent carotid plaques on either side than did control subjects (p less than 0.001 for all three factors). Patients also had a higher prevalence of carotid artery stenosis greater than or equal to 50% on either side and a higher prevalence of ischemic heart disease than did control subjects, but these did not reach statistical significance (p = 0.06 and p = 0.08, respectively). CONCLUSIONS: Our findings suggest that hypertension and cigarette smoking may be important in the pathogenesis of asymptomatic retinal cholesterol emboli and that these emboli indicate systemic atherosclerosis rather than ipsilateral carotid artery stenosis.

CooB is required for assembly but not transport of CS1 pilin.

CS1 pili are filamentous proteinaceous appendages found on many enterotoxigenic Escherichia coli (ETEC) strains isolated from human diarrhoeal disease. They are thought to effect colonization of the upper intestine by facilitating binding to human ileal epithelial cells. We have identified a gene, cooB, which lies directly upstream of cooA, the gene that encodes the major structural CS1 protein. When translated in vitro, the protein product of cooB migrates in sodium dodecyl sulphate/polyacrylamide gel with an apparent molecular mass of 26 kDa, which is consistent with that predicted from its DNA sequence. We constructed a mutant allele (cooB-1) by insertion of the omega fragment, which inhibits transcription and translation, into the cooB gene in vitro. In a derivative of an ETEC strain with the cooB-1 mutation (JEF100) and a plasmid that encodes Rns (pEU2030), the positive regulator required for CS1 expression, no cooB and a greatly reduced level of cooA product was detectable in total cell extracts. The reduction of cooA in this strain appears to result from polarity of the cooB mutation because introduction of the wild-type cooA gene in trans causes production of CooA protein, which is found in cell pellet extracts, in extracts containing only surface proteins and in the culture supernatant. Therefore, in the absence of CooB, CooA is stable and it is transported through both inner and outer membranes. However, the cooB-1 strain with cooA in trans does not cause haemagglutination of bovine erythrocytes (the model system used to assay adherence mediated by coli surface antigen 1 (CS1) pili).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of type 1 piliation on in vitro killing of Escherichia coli by mouse peritoneal macrophages.

Escherichia coli K-12 mutants possessing defined lesions affecting type 1 pilus production, receptor binding, or length were examined for their ability to resist killing by mouse peritoneal macrophages in vitro. Mutants were mixed pairwise at known ratios in wells containing macrophages, and after incubation, the ratio of the survivors was assayed. The difference in phagocytic killing between type 1 piliated cells and isogenic nonpiliated cells was significant, the piliated cells being approximately threefold more resistant. Pilus length had little effect upon survival, as the long-piliated mutants were no more resistant to killing than the normal-length parents. Interestingly, the receptor-binding function of type 1 pili was most important in effecting resistance, as mutants lacking the ability to bind receptor were killed as effectively as nonpiliated mutants. These data are consistent with the notion that pili actually impede killing by macrophages rather than serve as passive physical barriers to uptake.

Visual function after pan-retinal photocoagulation: a survey.

Thirty-five diabetic patients who had undergone pan-retinal photocoagulation were surveyed to determine the frequency and severity of visual difficulties they experience. Among the most frequent problems were difficulty adjusting to dim lighting, difficulty adjusting to bright lighting, and trouble in sorting dark colors. Judging distances, negotiating stairways, and avoiding obstacles were identified as having become more difficult since the laser treatment. According to a correlation analysis, the difficulties encountered in some important tasks, such as driving in the daytime, were highly related to visual acuity. However, many of the problems reported most frequently, and many of the problems whose frequency had increased the most since the laser therapy, were not related to acuity. Despite their many visual complaints, the patients expressed very positive attitudes toward the photocoagulation treatment.

Construction of bacteriophage luminal diameterX174 mutants with maximum genome sizes.

The bacteriophage phi X174 strain ins6 constructed previously was used to investigate the maximum genome size that could be packaged into the icosahedral phage without concomitant loss of phage viability. The J-F intercistronic region of ins6, which already contains an insert of 117 base pairs with a unique PvuII site, was enlarged further by insertion of HaeIII restriction fragments of the plasmid pBR322 into that PvuII site. By using a biochemical approach for the site-specific mutagenesis as well as selection of mutant genomes, a series of mutants was isolated with genomes of up to 5,730 nucleotides, 6.4% larger than that of the wild-type DNA. Phages with genomes larger than 5,550 nucleotides were highly unstable and were rapidly outgrown by spontaneously occurring deletion mutants. The data predict that genomes of at least 6,090 nucleotides could be constructed and, most likely, packaged, but the resulting phages would not grow well. We speculate that the volume of the phage capsid is not the limiting factor of genome size or is not the only limiting factor.

Scotopic sensitivity to ON and OFF stimulus transients.

The luminance of a large diffuse field, viewed peripherally, was temporally modulated near absolute detection threshold. The field luminance either increased abruptly and decreased gradually (ON stimulus) or increased gradually and decreased abruptly (OFF stimulus). For all wavelengths shorter than 620 nm, sensitivity to the ON stimulus was greater than to the OFF. The spectral sensitivity curves obtained indicate that rods and/or their associated neural pathways are more sensitive to ON stimulus transients than to OFF.

Red - green opponent spectral sensitivity: disparity between cancellation and direct matching methods.

The spectral sensitivity at the opponent stage of the visual system is traditionally measured by a hue-cancellation procedure. Comparison with a direct hue-matching method shows that cancellation overestimates short-wavelength sensitivity by as much as a factor of 30. The observation implies that different mechanisms control the perception of short-wavelength and long-wavelength redness.