Members of the ferritin-like Dps protein family are found in a number of bacterial species, where they demonstrate the potential to bind iron, and have been implicated in tolerance to oxidative stress. In this study of the food-borne pathogen Listeria monocytogenes, the fri gene encoding a Dps homologue was deleted, and, compared to wild-type cells, it was found that the resulting mutant was less resistant to hydrogen peroxide, and demonstrated reduced survival following long-term (7–11 days) incubation in laboratory media. In view of this, it is shown that fri gene expression is controlled by the hydrogen peroxide regulator PerR, as well as the general stress sigma factor σ B. When fri mutant cells were transferred to iron-limiting conditions, growth was retarded relative to wild-type cells, indicating that Fri may be required for iron storage. This notion is supported by the observation that the L. monocytogenes genome appears not to encode other ferritin-like proteins. Given the role of Fri in resistance to oxidative stress, and growth under iron-limiting conditions, the ability of the fri mutant to infect mice was examined. When injected by the intraperitoneal route, the fri mutant demonstrated a reduced capacity to proliferate in the organs of infected mice relative to the wild-type, whereas when the bacteria were supplied intravenously this effect was mitigated. In addition, the mutant was impaired in its ability to survive and grow in J774.A1 mouse macrophage cells. Thus, the data suggest that Fri contributes to the ability of L. monocytogenes to survive in environments where oxidative stress and low iron availability may impede bacterial proliferation.

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pH i ) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pH i , allowing fast and noninvasive determination of pH i that is ideally suited for direct analysis of individual bacterial cells present in complex environments.Bacteria are often subjected to various forms of environmental stress, and in recent years, there has been increasing focus on the different mechanisms they employ to protect themselves against these environmental changes. One of the most extensively studied stress responses is the ability of bacteria to survive low pH by adjusting their intracellular pH (pH i ) in response to changes in extracellular pH (pH ex ) (1, 4). Several methods have been developed for measuring bacterial pH i (2,14,17,19). However, as these techniques require radioactive labeling and time-consuming staining procedures, noninvasive methods for continuous measurement of pH i in bacteria are in demand. Since the discovery of green fluorescent protein (GFP), a wide range of mutant variants has been created. In eukaryotic cells, several GFP variants with altered excitation and emission spectra have been examined for their use as pH i probes (8,11,12,13,16). One of these GFP variants, ratiometric GFP, was obtained by introducing specific amino acid substitutions to the chromophore, causing the resulting protein to alter its excitation spectrum according to the pH of the surrounding environment (13).In order to express the ratiometric GFP protein in both gram-positive and gram-negative bacterial cells, we inserted the corresponding gene downstream of the P32 promoter in the chloramphenicol-resistant expression vector pMG36c, which replicates in both cell types (20). The ratiometric gfp gene (GenBank accession no. AF058694) was amplified by PCR by using the oligonucleotides C-GFP (5Ј-TAT CCC AAG CTT TTA TTT GTA TAG TTC ATC CAT GCC ATG TG-3Ј) and N-GFP (5Ј-TGC TCT AGA GTA ATA AGG AGG AAA AAA TAT GAG TAA AGG AGA AGA ACT TTT CAC TGG AGT TGT CCC-3Ј) (DNA Technology, Å rhus, Denmark) that additionally introduced an initiating ATG codon as well as a ribosomal binding site (5). The resulting 750-bp DNA fragment was inserted into the XbaI-and EcoRI-digested pMG36c, and the correct DNA sequence of the ratiometric gfp gene present in the resulting plasmid (pGFPratiometric) was confirmed by DNA sequence analysis (data not shown).When we introduced pGFPratiometric in the gram-positive bacterium Lactococcus lactis subsp. lactis CNRZ 157 as previously described (9) and grew cells at 30°C in M17 broth containing 0.5% (wt/vol) glucose and 5 g of chloramphenicol per ml, we found that excitation at 410 nm gave a strong pHdependent fluorescent signal. Furthermore, a pH-independent isosbestic point was seen at 430 nm, which is in accordance with previous results obtained in mammali...

A biosecurity survey was performed to gather information on the biosecurity level and laboratory capacity in Kenya for the purpose of providing information outlining relevant components for biosecurity legislation, biosecurity implementation, and enforcement of biosecurity measures in Kenya. This survey is, to the authors' knowledge, the first to be published from an African country. A total of 86 facilities with laboratories covering relevant categories, such as training laboratories, human diagnostic laboratories, veterinary diagnostic laboratories, and research laboratories, were selected to participate in the survey. Each facility was visited by a survey team and staff were asked to answer 29 groups of questions from a questionnaire. The survey showed that Kenyan laboratory facilities contain biological agents of biosecurity concern. The restrictions for these agents were found to be limited for several of the facilities, in that many laboratory facilities and storage units were open for access by either students or staff who had no need of access to the laboratory. The survey showed a great deal of confusion in the terms biosecurity and biosafety and a generally limited biosecurity awareness among laboratory personnel. The survey showed that the security of biological agents of biosecurity concern in many facilities does not meet the international requirements. The authors recommend developing a legal framework in Kenya for effective controls, including national biosecurity regulations, guidelines, and procedures, thereby reducing the risk that a Kenyan laboratory would be the source of a future biological attack.