Environmentally constrained mutation and adaptive evolution in Salmonella

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3], [4] and [5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7], [8], [9] and [10], or as a consequence of evolutionary processes [11], [12], [13], [14], [15] and [16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that chnages in the mutability of specific loci can be influenced by alterations in DNA topology [10] and [17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not ‘directed’ and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.     

Detection of Chlamydia trachomatis in Papanicolaou-stained cervical smears by an indirect immunoperoxidase method

A two-step (indirect) immunoperoxidase method directed against Chlamydia trachomatis was developed. The method was then used to evaluate the specificity of cytologic changes suggestive of C. trachomatis in Papanicolaou smears of cervical specimens from women who were culture-negative for the organism. Positive immunoperoxidase staining was detected in 9 of 21 cases (43%) tested. Technical problems, especially background staining, precluded interpretation in the remainder of the cases. Cervical cytology, as demonstrated by immunoperoxidase staining, may, in some instances, be more sensitive than the culture. However, because the etiology of cytologic changes not specifically identified by immunoperoxidase staining may be due to other organisms or factors, immunoperoxidase procedures, as described, should not replace culture for confirmation of cytologic findings suggestive of C. trachomatis.     

Separation and characterization of pyrimidine deoxyribonucleoside 2'-hydroxylase and thymine 7-hydroxylase from Aspergillus nidulans

Partially purified preparations from Aspergillus nidulans were shown to catalyze two alpha-ketoglutarate dependent dioxygenase reactions: the pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3) and the thymine 7-hydroxylase (EC 1.14.11.6) reactions. These reactions showed an absolute requirement for alpha-ketoglutarate and molecular oxygen and were stimulated by Fe(II), ascorbate and catalase. Both reactions demonstrated a stoichiometry such that for each mole of substrate (deoxyribonucleoside or pyrimidine) hydroxylated one mole of CO2 was produced from alpha-ketoglutarate. These two activities were separated using DEAE-Sephacel chromatography.     

Optimal Triage Test Characteristics to Improve the Cost-Effectiveness of the Xpert MTB/RIF Assay for TB Diagnosis: A Decision Analysis

Background

High costs are a limitation to scaling up the Xpert MTB/RIF assay (Xpert) for the diagnosis of tuberculosis in resource-constrained settings. A triaging strategy in which a sensitive but not necessarily highly specific rapid test is used to select patients for Xpert may result in a more affordable diagnostic algorithm. To inform the selection and development of particular diagnostics as a triage test we explored combinations of sensitivity, specificity and cost at which a hypothetical triage test will improve affordability of the Xpert assay.

Methods

In a decision analytical model parameterized for Uganda, India and South Africa, we compared a diagnostic algorithm in which a cohort of patients with presumptive TB received Xpert to a triage algorithm whereby only those with a positive triage test were tested by Xpert.

Findings

A triage test with sensitivity equal to Xpert, 75% specificity, and costs of US$5 per patient tested reduced total diagnostic costs by 42% in the Uganda setting, and by 34% and 39% respectively in the India and South Africa settings. When exploring triage algorithms with lower sensitivity, the use of an example triage test with 95% sensitivity relative to Xpert, 75% specificity and test costs $5 resulted in similar cost reduction, and was cost-effective by the WHO willingness-to-pay threshold compared to Xpert for all in Uganda, but not in India and South Africa. The gain in affordability of the examined triage algorithms increased with decreasing prevalence of tuberculosis among the cohort.

Conclusions

A triage test strategy could potentially improve the affordability of Xpert for TB diagnosis, particularly in low-income countries and with enhanced case-finding. Tests and markers with lower accuracy than desired of a diagnostic test may fall within the ranges of sensitivity, specificity and cost required for triage tests and be developed as such.     

Genetic footprinting in bacteria

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.