Combining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120

The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.     

How essential are nonessential genes?

Gene essentiality in bacteria has been identified in silico, focusing on gene persistence, or experimentally, focusing on the growth of knockouts in rich media. Comparing 55 genomes of Firmicutes and Gamma-proteobacteria to identify the genes which, while persistent among genomes, do not lead to a lethal phenotype when inactivated, we show that the characteristics of persistence, conservation, expression, and location are shared between persistent nonessential (PNE) genes and experimentally essential genes. PNE genes show an overrepresentation of genes related to maintenance and stress response. This outlines the limits of current experimental techniques to define gene essentiality and highlights the essential role of genes implicated in maintenance which, although dispensable for growth, are not dispensable from an evolutionary point of view. Firmicutes and Gamma-proteobacteria are mostly differing in the construction of the cell envelope, DNA replication and proofreading, and RNA degradation. In addition to suggesting functions for persistent genes that had until now resisted identification, we show that these genes have many characters in common with experimentally identified essential genes. They should then be regarded as truly essential genes.     

Malaria “epidemic” in Quebec: diagnosis and response to imported malaria

Background

Imported malaria is an increasing problem. The arrival of 224 African refugees presented the opportunity to investigate the diagnosis and management of imported malaria within the Quebec health care system.

Methods

The refugees were visited at home 3–4 months after arrival in Quebec. For 221, a questionnaire was completed and permission obtained for access to health records; a blood sample for malaria testing was obtained from 210.

Results

Most of the 221 refugees (161 [73%]) had had at least 1 episode of malaria while in the refugee camps. Since arrival in Canada, 87 (39%) had had symptoms compatible with malaria for which medical care was sought. Complete or partial records were obtained for 66 of these refugees and for 2 asymptomatic adults whose children were found to have malaria: malaria had been appropriately investigated in 55 (81%); no malaria smear was requested for the other 13. Smears were reported as positive for 20 but confirmed for only 15 of the 55; appropriate therapy was verified for 10 of the 15. Of the 5 patients with a false-positive diagnosis of malaria, at least 3 received unnecessary therapy. Polymerase chain reaction testing of the blood sample obtained at the home visit revealed malaria parasites in 48 of the 210 refugees (23%; 95% confidence interval [CI] 17%– 29%). The rate of parasite detection was more than twice as high among the 19 refugees whose smears were reported as negative but not sent for confirmation (47%; 95% CI 25%– 71%).

Interpretation

This study has demonstrated errors of both omission and commission in the response to refugees presenting with possible malaria. Smears were not consistently requested for patients whose presenting complaints were not “typical” of malaria, and a large proportion of smears read locally as “negative” were not sent for confirmation. Further effort is required to ensure optimal malaria diagnosis and care in such high-risk populations.In many industrialized countries, the incidence of imported malaria is rising because of changing immigration patterns and refugee policies as well as increased travel to malaria-endemic regions.^{1,2,3,4,5,6,7,8,9,10} Imported malaria is not rare in Canada (300–1000 cases per year),³ the United States^2,3,4 or other industrialized countries.^5,6,7,8,9,10 Malaria can be a serious challenge in these countries because of its potentially rapid and lethal course.^11,12,13,14 The task of front-line health care providers is made particularly difficult by the protean clinical presentations of malaria. Classic periodic fevers (tertian or quartan) are seen infrequently.^{9,15,16,17,18,19} Atypical and subtle presentations are especially common in individuals who have partial immunity (e.g., immigrants and refugees from disease-endemic areas) or are taking malaria prophylaxis (e.g., travellers).^9,16,17 Even when malaria is considered, an accurate diagnosis can remain elusive or can be delayed as a result of inadequate or distant specialized laboratory support.^19,20In Quebec, the McGill University Centre for Tropical Diseases collaborates with the Laboratoire de santé publique du Québec to raise awareness of imported malaria, to offer training and quality-assurance testing, and to provide reference diagnostic services. A preliminary diagnosis is typically made by the local laboratory, and smears (with or without staining) are sent to the McGill centre, where they are reviewed within 2–48 hours, depending on the urgency of the request. Initial medical decisions are usually based on local findings and interpretations. Although malaria is a reportable disease, there is no requirement to use the reference service.On Aug. 9, 2000, 224 refugees from Tanzanian camps landed in Montréal aboard an airplane chartered by Canadian immigration authorities. Over the ensuing 5 weeks, the McGill University Centre for Tropical Diseases noted an increase in demand for malaria reference services and an apparent small “epidemic” of imported malaria. This “epidemic” prompted us to investigate the performance of the health care system in the diagnosis and management of imported malaria.     

Universal biases in protein composition of model prokaryotes

The levels of cellular organization in living organisms are the results of a variety of selection pressures. We have investigated here the final outcome of this integrated selective process in proteins of the best known microbial models Escherichia coli, Bacillus subtilis, and Methanococcus jannaschii, supposed to have undergone separate evolution for more than 1 billion years. Using multivariate analysis methods, including correspondence analysis, we studied the overall amino acid composition of all proteins making a proteome. Starting from and further developing previous results that had pointed out some general forces driving the amino acid composition of the proteomes of these model bacteria, we explored the correlations existing between the structure and functions of the proteins forming a proteome and their amino acid composition. The electric charge of amino acids measured against hydrophobicity creates a highly homogeneous cluster, made exclusively of proteins that are core components of the cytoplasmic membrane of the cell (integral inner membrane proteins). A second bias is imposed by the G+C content of the genome, indicating that protein functions are so robust with respect to amino acid changes that they can accommodate a large shift in the nucleotide content of the genome. A remarkable role of aromatic amino acids was uncovered. Expressed orphan proteins are enriched in these residues, suggesting that they might participate in a process of gain of function during evolution.     

Canine herpesvirus-1 (CHV-1): clinical, serological and virological patterns in breeding colonies

Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with reproductive disorders and neonatal mortality. To advise dog breeders towards an effective management of CHV-1 infected colonies, 27 breeding bitches were studied during one reproductive cycle in field conditions: the effect of cycle stage, kennel size, initial antibody titre, mating and gestation on serologic and viral excretion patterns was evaluated, while the association between reproductive disorders and CHV-1 antibody titres and viral excretion was also analysed. All initially seronegative bitches seroconverted, while 40% of the initially seropositive bitches became seronegative at one or two occasions. No difference in antibody patterns was observed between mated and unmated bitches. Of the mated bitches, 46% experienced infertility, foetal resorption or mummification. No difference in antibody patterns was observed depending on the occurrence of reproductive disorders even if a decrease in antibody titres during early or late-di-oestrus was often present. Significantly higher titres were observed at all cycle stages in large kennels. None of the vaginal and nasal samples or buffy coats tested positive for CHV-1 DNA. The mixed image of clinical and sub-clinical carriage in this study demonstrated CHV-1 has a complex and difficult to predict clinical behavior. Preventive management with vaccination of reproducing bitches in kennels with reproductive disorders should therefore be advised.     

Clinical and environmental isolates of Legionella pneumophila serogroup 1 cannot be distinguished by sequence analysis of two surface protein genes and three housekeeping genes

We used gene sequencing to determine whether clinical (sporadic, epidemic, and endemic) and environmental isolates of Legionella pneumophila serogroup (sg) 1 belong to specific lineages. A total of 178 clinical and environmental L. pneumophila sg 1 isolates, defined by pulsed-field gel electrophoresis and epidemiological data as sporadic, epidemic, or endemic, were analyzed for polymorphisms in five gene fragments. The fragments belonged to three housekeeping genes (coding for aconitase [acn], aspartate-beta-semialdehyde dehydrogenase [asd], and RNA polymerase beta subunit [rpoB]) and two surface protein genes (coding for the macrophage infectivity potentiator [mip] and the major outer membrane protein [mompS]). The phylogenetic tree inferred from sequence polymorphisms of the five genes identified two large clusters, one consisting of 133 poorly differentiated strains and containing two smaller clusters (10 and 2 strains) unrelated to each other and the other consisting of 42 strains. Clinical and environmental isolates could not be distinguished on this basis, and no link between genetic background and epidemiological type was found, suggesting that other factors are responsible for differences in pathogenicity.     

Surface structure and nanomechanical properties of Shewanella putrefaciens bacteria at two pH values (4 and 10) determined by atomic force microscopy

The nanomechanical properties of gram-negative bacteria (Shewanella putrefaciens) were investigated in situ in aqueous solutions at two pH values, specifically, 4 and 10, by atomic force microscopy (AFM). For both pH values, the approach force curves exhibited subsequent nonlinear and linear regimens that were related to the progressive indentation of the AFM tip in the bacterial cell wall, including a priori polymeric fringe (nonlinear part), while the linear part was ascribed to compression of the plasma membrane. These results indicate the dynamic of surface ultrastructure in response to changes in pH, leading to variations in nanomechanical properties, such as the Young's modulus and the bacterial spring constant.