Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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A locus involved in kanamycin, chloramphenicol and L-serine resistance is located in the bglY-galU region of the Escherichia coli K12 chromosome

Summary Spontaneous mutants of Escherichia coli K12 displaying an increased level of the kanamycin resistance conferred by plasmid pGR71 were selected. Several mutants obtained in this way apparently carry large chromosomal deletions extending into galU and/or bglY (27 min). This positive selection of deletions allowed detection of a new locus located between galU and bglY. Deletions of this locus are responsible for increased resistance to kanamycin (Irk), decreased resistance to l-serine in minimal medium (Drs) and decreased resistance to chloramphenicol (Drc) when a cat gene is present in the bacteria.     

Environmental vibrio phage–bacteria interaction networks reflect the genetic structure of host populations

Phages depend on their bacterial hosts to replicate. The habitat, density and genetic diversity of host populations are therefore key factors in phage ecology, but our ability to explore their biology depends on the isolation of a diverse and representative collection of phages from different sources. Here, we compared two populations of marine bacterial hosts and their phages collected during a time series sampling program in an oyster farm. The population of Vibrio crassostreae, a species associated specifically to oysters, was genetically structured into clades of near clonal strains, leading to the isolation of closely related phages forming large modules in phage–bacterial infection networks. For Vibrio chagasii, which blooms in the water column, a lower number of closely related hosts and a higher diversity of isolated phages resulted in small modules in the phage–bacterial infection network. Over time, phage load was correlated with V. chagasii abundance, indicating a role of host blooms in driving phage abundance. Genetic experiments further demonstrated that these phage blooms can generate epigenetic and genetic variability that can counteract host defence systems. These results highlight the importance of considering both the environmental dynamics and the genetic structure of the host when interpreting phage–bacteria networks.     

Escherichia coli Heat Shock Protein DnaK: Production and Consequences in Terms of Monitoring Cooking

Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F₇₀¹⁰ of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55°C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50°C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55°C, F₇₀¹⁰ = 3 min) accompanied by a 12-h recovery (containing 76,786 ± 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 ± 6,056 molecules/cell) when later heated to 60°C for 50 min (F₇₀¹⁰ = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.     

Single-Step Purification of Two Functional Human Apolipoprotein E Variants Hyperexpressed inEscherichia coli

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH₂-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into theEscherichia colistrain BL21(DE3). Recombinant strains were grown at 37°C in a Luria and Bertani medium and the addition of isopropyl β-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.     

Division of Listeria monocytogenes serovar 4b strains into two groups by PCR and restriction enzyme analysis.

Altogether, 133 strains of Listeria monocytogenes serovar 4b were investigated. A segment of 2,916 bp containing parts of the two genes inlA and inlB in L. monocytogenes was amplified by the PCR technique. The PCR product obtained was cleaved with the restriction enzyme AluI, and the fragments generated were separated by gel electrophoresis, leading to two distinct groups: PCR-restriction enzyme analysis groups I and II, containing 37 and 96 strains, respectively. The PCR-restriction enzyme analysis method described in this paper could be a useful tool for the subtyping of L. monocytogenes serovar 4b strains.     

Evaluation of the Organon-Teknika MICRO-ID LISTERIA system.

The MICRO-ID LISTERIA system, designed to identify Listeria isolates to species level within 24 h, was compared with conventional biochemical identification. MICRO-ID LISTERIA used in combination with the CAMP test correctly identified 409 (98.8%) of 414 strains isolated from human, animal, food, and environmental sources belonging to the seven species currently defined within the genus Listeria. The kit was easy to use and simple to interpret. However, 8 of the 15 tests (i.e., phenylalanine deaminase, hydrogen sulfide, indole, ornithine decarboxylase, lysine decarboxylase, malonate, urease, and o-nitrophenyl-beta-D-galactopyranoside) were considered superfluous for the differentiation of Listeria spp. The CAMP test was indispensable when using the MICRO-ID LISTERIA system, in particular to differentiate CAMP test-positive L. monocytogenes from the nonhemolytic, rhamnose-positive L. innocua. The hemolytic L. seeligeri and L. ivanovii strains and the nonhemolytic, non-rhamnose-acidifying L. welshimeri strains could also be differentiated from one another only on the basis of their CAMP test results. The very few strains of L. grayi and L. murrayi were easily differentiated from the other nonhemolytic species. Catalase-negative cocci should not be tested, because 12 out of 19 catalase-negative strains (all enterococci) in our test were misidentified as Listeria spp. The MICRO-ID LISTERIA system identified strains within 18 to 24 h and is thus less time-consuming than conventional tests. The system could, therefore, be used together with correctly done CAMP tests for the rapid identification of Listeria isolates, especially food and environmental isolates, for which rapid species differentiation is important.