Studies on Inc-P plasmids in Erwinia carotovora subsp. carotovora

Abstract Inc-P plasmids, RP4, R751, pMO850, and pRK2013 were transferred to Erwinia carotovora . These plasmids were stably maintained in E. carotovora and the transconjugants were efficient donors of respective plasmids to other strains of E. carotovora and Escherichia coli . These plasmids were not able to mobilize chromosomal markers from one strain of E. carotovora to another strain of E. carotovora even in the presence of homologous DNA sequences on the plasmid and the bacterial chromosome. The presence of Inc-P plasmid does not affect the pathogenic phenotype of E. carotovora . A broad host range Inc-P cosmid, pLAFR1, was transferred to E. carotovora with the help of pRK2013, suggesting the potential use of a binary plasmid system for genetic complementation in E. carotovora .     

Application of Urine Analysis to Diagnosis and Treatment of Heroin Addiction

Experience with urine analysis for morphine using thin-layer chromatography in 310 cases of real or possible heroin abuse showed that it was valuable not only in detecting improper drug use but also in monitoring treatment. The results of this test can be available routinely in 24, and exceptionally in five hours. A negative result implies that the subject has taken less than 10 mg. of heroin in the past 24 hours.     

Chirality differences in amino acid retention and release from the acid-extractable pool of cultured mammalian cells

In previous work, no chiral differences were found between D and L enantiomers of Leu in their ability to displace one another from the acid-extractable pool in mammalian cells. Recent evidence suggested otherwise. Our aim is to examine whether, in physiological range, D-amino acids have an equivalent ability to displace L-amino acids from the acid-extractable pool of HeLa cells, and vice versa. In the millimolar range, D-Leu and L-Leu have similar uptake and displacement properties with regard to the acid-extractable pool in HeLa cells, despite only the latter isomer being incorporated into protein. Below millimolar concentrations however, a distinct difference was found in the displacement of tritium-labelled L-Leu from the pool by unlabelled D-Leu compared with unlabelled L-Leu. Thus, unlabelled L-Leu in the external medium at 10⁻⁴ or 10⁻⁵ M displaced an equivalent amount of label from the pool as D-Leu introduced at a concentration approx. one order of magnitude higher, respectively. Reciprocal experiments, in which the acid-extractable pool was preloaded with ³H-D-Leu, confirmed this finding. The chirality difference was noted whether pool prelabelling was carried out at 37 or 0°C; but in order to avoid the complications of active transport mechanisms, the competition work reported here was done at 0°C. Similar chirality differences were observed with other hydrophobic amino acids, including His, Ile and Phe, such as, preferential displacement by the L-Leu racemer compared with the D-Leu racemer below mM levels. This was also true for the D and L forms of the non-utilisable isomer of Leu, norleucine (nLeu). We conclude that D-forms of hydrophobic amino acids have lower affinity for similar or the same intracellular binding sites involved in the acid-extractable pool than their L-forms. The significance of these chirality findings to amino acid pools in cells, and to the predominance of L-forms of amino acids in the biosphere is considered.     

Biochemical changes during germination and seedling growth in Cuscuta campestris

Changes in the contents of starch, protein, DNA, RNA, total phosphorus, acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis.
Starch content in Cuscuta seeds showed a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination and then decreased. Total phosphorus in the seeds remained almost unchanged during the period of study. Both trichloroacetic acid soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed.     

Possible Links Between Stress Defense and the Tricarboxylic Acid (TCA) Cycle in Francisella Pathogenesis

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. In vivo, this facultative intracellular bacterium survives and replicates mainly in the cytoplasm of infected cells. We have recently identified a genetic locus, designated moxR that is important for stress resistance and intramacrophage survival of F. tularensis. In the present work, we used tandem affinity purification coupled to mass spectrometry to identify in vivo interacting partners of three proteins encoded by this locus: the MoxR-like ATPase (FTL_0200), and two proteins containing motifs predicted to be involved in protein–protein interactions, bearing von Willebrand A (FTL_0201) and tetratricopeptide (FTL_0205) motifs. The three proteins were designated here for simplification, MoxR, VWA1, and TPR1, respectively. MoxR interacted with 31 proteins, including various enzymes. VWA1 interacted with fewer proteins, but these included the E2 component of 2-oxoglutarate dehydrogenase and TPR1. The protein TPR1 interacted with one hundred proteins, including the E1 and E2 subunits of both oxoglutarate and pyruvate dehydrogenase enzyme complexes, and their common E3 subunit. Remarkably, chromosomal deletion of either moxR or tpr1 impaired pyruvate dehydrogenase and oxoglutarate dehydrogenase activities, supporting the hypothesis of a functional role for the interaction of MoxR and TPR1 with these complexes. Altogether, this work highlights possible links between stress resistance and metabolism in F. tularensis virulence.Francisella tularensis is responsible for the disease tularamia in a large number of animal species. This highly infectious bacterial pathogen can be transmitted to humans in numerous ways (1, 2, 3), including direct contact with sick animals, inhalation, ingestion of contaminated water or food, or by bites from ticks, mosquitoes, or flies. Four different subspecies (subsp.) of F. tularensis that differ in virulence and geographic distribution exist, designated subsp. tularensis (type A), subsp. holarctica (type B), subsp. Novicida, and subsp. mediasiatica, respectively. F. tularensis subsp. tularensis is the most virulent subspecies causing a severe disease in humans, whereas F. tularensis subsp. holarctica causes a similar disease but of less severity (4). Because of its high infectivity and lethality, F. tularensis is considered a potential bioterrorism agent (5).F. tularensis is able to survive and to replicate in the cytoplasm of a variety of infected cells, including macrophages. To resist this stressful environment, the bacterium must have developed stress resistance mechanisms, most of which are not yet well characterized. We recently reported the identification of a novel genetic locus that is important for stress resistance and intracellular survival of F. tularensis (6). This locus was designated moxR because the first gene FTL_0200, encodes a protein belonging to the AAA+ ATPase of the MoxR family ((7) and references therein). The data obtained in that first study had led us to suggest that the F. tularensis MoxR-like protein might constitute, in combination with other proteins of the locus, a chaperone complex contributing to F. tularensis pathogenesis.To further validate this hypothesis and expand our initial observations, we here decided to perform tandem affinity purification (TAP),¹ using a dual affinity tag approach coupled to mass spectroscopy analyses (8), to identify proteins interacting in vivo with three proteins encoded by the proximal portion of the moxR locus. For this, we chose as baits: the MoxR-like protein (FTL_0200) and two proteins bearing distinct motifs possibly involved in protein–protein interactions, FTL_0201 (Von Willebrand Factor Type A domain, or VWA) and FTL_0205 (tetratrichopeptide repeat or TPR). The three proteins were designated here for simplification, MoxR, VWA1, and TPR1; and the corresponding genes moxR, vwa1, and tpr1, respectively.VWA domains are present in all three kingdoms of life. They consist of a β-sheet sandwiched by multiple α helices. Frequently, VWA domain-containing proteins function in multiprotein complexes (9). TPR typically contain 34 amino acids. Many three-dimensional structures of TPR domains have been solved, revealing amphipathic helical structures (10). TPR-containing proteins are also found in all kingdoms of life. They can be involved in a variety of functions, and generally mediate protein–protein interactions. In the past few years, several TPR-related proteins have been shown to be involved in virulence mechanisms in pathogenic bacteria ((11) and references therein).Our proteomic approach allowed us to identify a series of protein interactants for each of the three moxR-encoded proteins. Remarkably, the protein TPR1 interacted with all the subunits of the pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (OGDH) complexes. Furthermore, inactivation of tpr1 also severely impaired the activities of these two enzymes. Inactivation of tpr1 affected bacterial resistance to several stresses (and in particular oxidative stress), intramacrophagic bacterial multiplication and bacterial virulence in the mouse model. Functional implications and possible relationship between bacterial metabolism, stress defense, and bacterial virulence are discussed.     

Mitochondrial and Nuclear Genes-Based Phylogeography of Arvicanthis niloticus (Murinae) and Sub-Saharan Open Habitats Pleistocene History

A phylogeographic study was conducted on the Nile grass rat, Arvicanthis niloticus, a rodent species that is tightly associated with open grasslands from the Sudano-Sahelian regions. Using one mitochondrial (cytochrome b) and one nuclear (intron 7 of Beta Fibrinogen) gene, robust patterns were retrieved that clearly show that (i) the species originated in East Africa concomitantly with expanding grasslands some 2 Ma, and (ii) four parapatric and genetically well-defined lineages differentiated essentially from East to West following Pleistocene bioclimatic cycles. This strongly points towards allopatric genetic divergence within savannah refuges during humid episodes, then dispersal during arid ones; secondary contact zones would have then stabilized around geographic barriers, namely, Niger River and Lake Chad basins. Our results pertinently add to those obtained for several other African rodent as well as non-rodent species that inhabit forests, humid zones, savannahs and deserts, all studies that now allow one to depict a more comprehensive picture of the Pleistocene history of the continent south of the Sahara. In particular, although their precise location remains to be determined, at least three Pleistocene refuges are identified within the West and Central African savannah biome.