Tools for characterization of Escherichia coli genes of unknown function

Despite the power of sequencing and of emerging high-throughput technologies to collect data rapidly, the definitive functional characterization of unknown genes still requires biochemical and genetic analysis in case-by-case studies. This often involves the deletion of target genes and phenotypic characterization of the deletants. We describe here modifications of an existing deletion method which facilitates the deletion process and enables convenient analysis of the expression properties of the target gene by replacing it with an FRT-lacZ-aph-P(lac)-FRT cassette. The lacZ gene specifically reports the activity of the deleted gene and therefore allows the determination of the conditions under which it is actively expressed. The aph gene, encoding resistance to kanamycin, provides a selectable means of transducing a deleted locus between strains so that the deletion can be combined with other relevant mutations. The lac promoter helps to overcome possible polar effects on downstream genes within an operon. Because the cassette is flanked by two directly repeated FRT sites, the cassette can be excised by the Flp recombinase provided in trans. Removing the cassette leaves an in-frame deletion with a short scar which should not interfere with downstream expression. Replacements of yacF, yacG, yacH, yacK (cueO), yacL, ruvA, ruvB, yabB, and yabC made with the cassette were used to verify its properties.     

Reduction of marker discrimination in transductional recombination

Summary The recovery of phage P1 mediated transductants varies with the marker selected in a manner which cannot be fully accounted for by dosage differences in the donor gene population. This variation in transduction frequency is due primarily to recombinational discrimination in the recipient cell. We show here that increasing the intracellular level of recA protein, which might be expected to increase the contribution of recF mediated events to recombinant formation, decreases this discrimination slightly, and that replacing recBC mediated recombination by a recF dependent process, augmented by an additional, as yet uncharacterized mutation, dramatically reduces recombinational discrimination. We conclude that although recBC mediated transductional recombination is selective, recombination which relies on recF need not be so. We also show that UV-damaged DNA can be successfully recombined in the absence of the recB product (even in sbcB ^* cells) and that eliminating exonuclease I (the sbcB product) facilitates the recombination of heavily irradiated DNA.     

Defining the need for public health control of scabies in Solomon Islands

Pacific Island countries have a high burden of scabies and impetigo. Understanding of the epidemiology of these diseases is needed to target public health interventions such as mass drug administration (MDA). The aim of this study is to determine the prevalence of scabies and impetigo in Solomon Islands as well as the relationship between them and their distribution. We conducted a prevalence study in 20 villages in Western Province in Solomon Islands. All residents of the village were eligible to participate. Nurses conducted clinical assessments including history features and skin examination. Diagnosis of scabies was made using the 2020 International Alliance for the Control of Scabies diagnostic criteria. Assessments were completed on 5239 participants across 20 villages. Overall scabies prevalence was 15.0% (95%CI 11.8–19.1). There was considerable variation by village with a range of 3.3% to 42.6%. There was a higher prevalence of scabies in males (16.7%) than females (13.5%, adjusted relative risk 1.2, 95%CI 1.1–1.4). Children aged under two years had the highest prevalence (27%). Overall impetigo prevalence was 5.6% (95%CI 4.2–7.3), ranging from 1.4% to 19% by village. The population attributable risk of impetigo associated with scabies was 16.1% (95% CI 9.8–22.4). The prevalence of scabies in our study is comparable to previous studies in Solomon Islands, highlighting a persistent high burden of disease in the country, and the need for public health strategies for disease control.     

The tail of a chaperonin: the C-terminal region of Esctierichia coli GroEL protein

The active form of the KSP60 molecular chaperone of Escherichia coli, GroEL, is a pair of seven-membered rings. We have used site-directed mutagenesis to construct forms of the 547-amino-acid monomer truncated at the C-terminus. We show here that forms that are 520 amino acids long or longer are close to being fully functional. Removing one further amino acid, however, results in a protein, GroEL519, which retains little function. This truncated form is metabolically stable but is not recovered from the cell in particle form. When synthesized at high levels, it prevents the normal assembly of GroEL547 present in the same cell. When synthesized at low levels, it can be included, probably at low molar ratios, in particles formed by assembly-competent forms of GroEL. This can be seen as partial complementation of the temperature-sensitive mutant groEL44. We conclude that amino acid 520 is cruical for particle assembly. GroEL516 has in vivo properties similar to those of GroEL519, but the still shorter form, GroEL504, appears to be inactive.     

Constraints on the Structure of Combinatorial “Chick-a-dee” Calls

“Chick-a-dee” calls of the black-capped chickadee (Parus atricapillus) constitute a combinatorial system of animal communication, apparently the only such system yet described. Four note-types, which may be omitted or repeated a variable number of times, occur in a fixed sequence (A–B–C–D) to constitute calls. Quantitative analyses determining the nature of departures from first-order transitional probabilities between successive notes revealed a variety of results that point consistently to two underlying features of calls. (1) Some constraint operates to limit the length of calls, as manifest in shortening of repetition-strings and omitting of note-types expected to follow. (2) There is an opposing tendency to include at least one or two D-notes at the end of a call, regardless of the overall length. The second feature suggests important semantic properties of D-notes, especially the ratio of D s to other note-types and the flock-specific acoustical structure of D-notes. The constraint on length of call does not appear intimately related to semantic causes, but may instead be determined by the maximum duration of continuous phonation, in parallel with the fundamental breath-group of human speech. In this respect, a “chick-a-dee” call resembles an entire “natural sentence” of spoken human language.