Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis

Abstract The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis . Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD₅₀ of 10³ colony forming units (CFU) or greater (low virulence) from those having an LD₅₀ of approximately 10¹ or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica , a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD₅₀ of 10² were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Characterization of the dual start motif of a class II holin gene

Holins are small membrane proteins that, at a genetically programmed time in a bacteriophage infective cycle, allow bacteriolytic enzymes, or endolysins, to escape to the periplasm and to attack the cell wall. Most holins fall into two sequence classes, I and II, based on the number of potential transmembrane domains (three for class I and two for class II). The prototype class I holin gene, S  ^λ, has a dual start motif and encodes not only the effector holin, S^λ105, but also an inhibitor, S^λ107, with a Met–Lys … extension at the terminus. The prototype class II holin gene of phage 21, S  ²¹, begins with the motif Met–Lys–Ser–Met … , and a potential RNA secondary structure overlaps the Shine–Dalgarno sequence. Here, we demonstrate that (i) two protein products are elaborated from S  ²¹, S²¹71 and S²¹68; (ii) the shorter product is required for lysis; (iii) the longer product, S²¹71, inhibits S  ²¹ function; and (iv) the Lys-2 residue is important for the inhibitor function. Moreover, the RNA stem–loop structure is involved in the downregulation of S²¹71 synthesis. However, our results suggest that, in S  ²¹, different segments of the single consensus Shine–Dalgarno sequence serve the two translational starts. These results show that the dual start motifs of class II holin genes are functionally homologous to those of class I holin genes.     

Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Aims:  The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae .
Methods and Results:  A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65°C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non- cholerae Vibrio isolates and 37 non- Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2·2 × 10³ CFU ml⁻¹ or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2·2 × 10⁴ CFU g⁻¹ or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction.
Conclusion:  The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection.
Significant and Impact of the study:  The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae . This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.     

Known bacterial virulence factors do not explain the variation in urinary cytokine levels in patients with urosepsis

Abstract We measured urinary endotoxin, IL-6 and IL-8 levels in 23 patients with gram-negative urosepsis. The endotoxin and cytokine levels showed a 100–1000 fold range. No correlation was found between levels of urinary endotoxin, and IL-6 or IL-8 levels. In all cases bacterial numbers were ≥ 10⁵ CFU ml⁻¹ urine. The endotoxin content of the isolated microorganisms neither correlated with the urinary cytokine levels, nor with IL-6 and IL-8 levels obtained in vitro when 10³ log-phase CFU of each of the bacteria were incubated with heparinized whole blood of three healthy donors. Neither the haemolysin phenotype of the bacteria, nor the presence of the P-pili gene was correlated with the cytokine response in vivo or in vitro. Other factors than known bacterial virulence factors apparently contribute to the wide variation in urinary cytokine levels in urinary tract infection.     

Effect of Lactobacillus gasseri BNR17 on blood glucose levels and body weight in a mouse model of type 2 diabetes

Aims:  To investigate the effect of Lactobacillus gasseri BNR17 isolated from human breast milk on blood glucose and body weight in type 2 diabetic animals.
Methods and results:  db/db mice were divided into one control group and five sample groups; the sample groups received BNR17 (10⁷, 10⁸, 10⁹ and 10¹⁰ CFU) or rosiglitazone (8 mg kg⁻¹) orally twice a day for 12 weeks. BNR17 groups had a dose-dependent reduction in food, water intake and amount of excrement. Body weight loss was not seen in the BNR17 groups. Fasting and postprandial 2 h blood glucose levels were significantly lower in the BNR17 (10¹⁰ CFU) group compared with the control group. HbA1c decreased in the BNR17 group, although it was not statistically significant. During the oral glucose tolerance test, the BNR17 groups exhibited dose-dependent improvement in glucose sensitivity.
Conclusions:  Lactobacillus gasseri BNR17 has a suppressing effect on blood glucose levels and improved diabetic symptoms in db/db mice.
Significance and Impact of the Study:  Blood glucose-lowering lactic acid bacteria are expected to be useful as a therapeutic for treating type 2 diabetes in humans.     

Virulence of luminescent and non-luminescent isogenic vibrios towards gnotobiotic Artemia franciscana larvae and specific pathogen-free Litopenaeus vannamei shrimp

Aims:  This study was conducted to test the virulence of luminescent (L) and non-luminescent (NL) isogenic strains of Vibrio campbellii LMG21363, Vibrio harveyi BB120 (wild type) and quorum-sensing mutant strains derived from the wild type such as Vibrio harveyi BB152, BB170, MM30 and BB886.
Methods and Results:  The NL strains could be obtained by culturing rifampicin-resistant luminescent strains in the dark under static condition. The virulence of the L and NL strains was tested in gnotobiotic Artemia franciscana larvae challenged with 10⁴ CFU ml⁻¹ of bacteria. All luminescent isogenic tested strains showed higher virulence compared to the NL strains. The virulence of L and NL V. campbellii and V. harveyi BB120 was also tested in specific pathogen-free juvenile shrimp upon intramuscular injection with 10⁶ CFU of bacteria. In contrast with Artemia , there was no significant difference in mortality between the groups challenged with L and NL strains ( P  > 0·05). The non-luminescent strains were not able to revert back to the luminescent state and quorum sensing did not influence this phenotypic shift.
Conclusions:  Luminescent Vibrio strains can switch to a non-luminescent state by culturing them in static conditions. The NL strains become less virulent as verified in Artemia .
Significance and Impact of the Study:  The luminescent state of Vibrio cells in a culture needs to be verified in order to assure maintenance of virulence.     

A CHEMILUMINESCENCE BIOSENSOR COUPLED WITH IMMUNOMAGNETIC SEPARATION FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM

A chemiluminescence biosensor, using a fiber-optic-linked photometer and a data acquisition unit connected to a PC, was developed in conjunction with immunomagnetic separation for rapid detection of Salmonella Typhimurium. Magnetic microbeads coated with Anti-Salmonella antibodies and anti-Salmonella antibodies conjugated with horseradish peroxidase (HRP) were added to artificially-inoculated samples, and the immuno-reaction was completed in 60 min resulting in a sandwich complex. A magnetic field was applied to collect magnetic beads and the addition of luminol to HRP-conjugated antibodies resulted in a chemiluminescence reaction. The signal was collected through a fiber optic light guide, measured with a photometer, and recorded in the data acquisition unit. The minimum detection limit of the chemiluminescence biosensor for S. Typhimurium was 1.97 × 10³ CFU/mL and the range of the detectable signal was from 8.6 to 350 mV for cell numbers from 1.97 × 10³ to 1.97 × 10⁶ CFU/mL. Signal values for 10⁶ CFU/mL of S. Typhimurium were at least 97 and 394% higher than the corresponding values for S. enteritidis and four times the signal values for others including S. montevideo, S. california, S. heidlberg, and S. seftenberg, respectively. The biosensor response showed a significant difference (P < 0.05) between 10³ CFU/mL S. Typhimurium and 10⁶ CFU/mL of commonly-occurring bacteria in foods including Listeria monocytogenes, Pseudomonas aeruginosa, Citrobacter freundii, Campylobacter jejuni, Escherichia coli O157, and generic Escherichia coli. A regression equation, V = 0.0262 N ^5.7713, with R²= 0.9713 was obtained for the calibration curve over the detection range for S. Typhimurium. The whole procedure could be completed within 90 min.     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).     

Improvement in the growth performance of white shrimp, Litopenaeus vannamei, by a protease-producing probiotic, Bacillus subtilis E20, from natto

Aims:  To isolate and identify a benefic bacterium, Bacillus subtilis E20, from natto (fermented soybeans), and incorporate it into shrimp feed to promote shrimp growth performance.
Methods and Results:  A protease-producing bacterium, E20, isolated from natto was identified as B. subtilis by an API 50 CHB kit and the 16S rDNA sequence. B. subtilis E20 was able to grow at a broad range of temperatures (10–50°C), pH values (5–10), and NaCl levels (0–9%). The best culture conditions for B. subtilis E20 to produce the protease were 40°C, a pH of 6–8 and 0% NaCl. No shrimp died after being injected with B. subtilis E20 [up to 10⁹ colony-forming units (CFU) per shrimp]. Bacillus subtilis E20 was incorporated in diets at the levels of 0 (control), 10⁶, 10⁷, and 10⁸ CFU kg⁻¹ for shrimp grow-out culture, and results showed that after feeding on B. subtilis E20-containing diets (10⁸ CFU kg⁻¹ of diet), shrimp had excellent growth performance and production compared to the control because protease activities in the digestive tract were improved by B. subtilis E20.
Conclusions:  Bacillus subtilis E20 isolated from natto is a great protease producer and is able to improve shrimp growth performance through increasing the digestibility of food.
Significance and Impact of the Study:  Results suggest that B. subtilis E20 is a potential candidate for use as a probiotic to improve shrimp growth performance, and consequently reduce feed costs.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .     

Microbial colonization of an in vitro model of a tissue engineered human skin equivalent – a novel approach

This was a preliminary investigation to define the conditions of colonization of a human skin equivalent (SE) model with cutaneous microorganisms. SEs of 24 mm diameter were constructed with a dermal matrix of fibrin containing fibroblasts and a stratified epidermis. Microbial colonization of the SEs was carried out in a dry environment, comparable to ' in vivo ' skin, using a blotting technique to remove inoculation fluid. The microbial communities were sampled by scrub washing and viable cells enumerated on selective growth medium. Staphylococcus epidermidis , Propionibacterium acnes and Malassezia furfur (human skin commensals) and Staphylococcus aureus (transient pathogen) were colonized at inoculum densities of 10²–10⁶ CFU SE⁻¹ on the surface of replicate SEs. Growth of all species was supported for upto 72–120 h, with recovery densities of between 10⁴–10⁹ CFU SE⁻¹. A novel, real-time growth monitoring method was also developed, using S. aureus containing a lux cassette. Light output increased from 20 to 95 h, and colonization increased from 10² to 10⁸ CFU SE⁻¹, as confirmed by conventional recovery. Thus, the SE model has potential to investigate interactions between resident and transient microbial communities with themselves and their habitat, and for testing treatments to control pathogen colonization of human skin.     

Isolation and characterization of a novel simazine-degrading bacterium from agricultural soil of central Chile, Pseudomonas sp. MHP41

s -Triazine herbicides are used extensively in South America in agriculture and forestry. In this study, a bacterium designated as strain MHP41, capable of degrading simazine and atrazine, was isolated from agricultural soil in the Quillota valley, central Chile. Strain MHP41 is able to grow in minimal medium, using simazine as the sole nitrogen source. In this medium, the bacterium exhibited a growth rate of μ=0.10 h⁻¹, yielding a high biomass of 4.2 × 10⁸ CFU mL⁻¹. Resting cells of strain MHP41 degrade more than 80% of simazine within 60 min. The atzA, atzB, atzC, atzD, atzE and atzF genes encoding the enzymes of the simazine upper and lower pathways were detected in strain MHP41. The motile Gram-negative bacterium was identified as a Pseudomonas sp., based on the Biolog microplate system and comparative sequence analyses of the 16S rRNA gene. Amplified ribosomal DNA restriction analysis allowed the differentiation of strain MHP41 from Pseudomonas sp. ADP. The comparative 16S rRNA gene sequence analyses suggested that strain MHP41 is closely related to Pseudomonas nitroreducens and Pseudomonas multiresinovorans . This is the first s -triazine-degrading bacterium isolated in South America. Strain MHP41 is a potential biocatalyst for the remediation of s -triazine-contaminated environments.     

Mutation of rpoS gene decreased resistance to environmental stresses, synthesis of extracellular products and virulence of Vibrio anguillarum

Vibrio anguillarum is a gram-negative halophilic bacterium that causes vibriosis in marine fish, freshwater fish and other aquatic animals. Bacteria have developed strategies to survive in harsh environments. The alternative σ factor, RpoS (σ^S), plays a key role in surviving under stress conditions in some gram-negative bacteria. An rpoS mutant of pathogenic V. anguillarum W-1 was constructed by homologous recombination. The sensitivity of the rpoS mutant to osmotic stress [2.4 M NaCl in artificial seawater (ASW)] did not change obviously, but the sensitivity of the rpoS mutant to high temperature (45 °C in ASW), UV-irradiation and oxidative stress (5 mM H₂O₂ in ASW) increased 33-fold, sixfold and 10-fold, respectively. The production of extracellular phospholipase, diastase, lipase, caseinase, hemolysin, catalase and protease of the rpoS mutant decreased markedly compared with those of the wild-type strain. Virulence of the rpoS mutant strain was also decreased when it was inoculated intraperitoneally into zebra fish; the lethal dose 50% of the wild type and the mutant was 8.66 × 10⁴ and 2.55 × 10⁶ CFU per fish, respectively. These results indicated that the RpoS of V. anguillarum plays important roles in bacterial adaptation to environmental stresses and its pathogenicity.     

Fine mapping of the FecL locus influencing prolificacy in Lacaune sheep

In the Lacaune sheep population, two major loci influencing ovulation rate are segregating: FecX and FecL . The FecX ^L mutation is a non-conservative substitution (p.Cys53Tyr) in BMP15 that prevents the processing of the protein. Using a statistical approach, FecL has been shown to be an autosomal major gene. A full genome scan localized the FecL locus on sheep chromosome 11. Fine mapping reduced the interval containing FecL to markers BM17132 and FAM117A , corresponding to a synteny block of 1.1 megabases on human chromosome 17, which encompasses 20 genes. The expression of 16 genes from this interval was observed in tissues of the reproductive axis, but expression was not affected in homozygous FecL ^L females. In this interval, a unique haplotype was associated with the FecL ^L mutation. This particular haplotype could be predicted by the DLX3 :c.*803A>G SNP in the 3' UTR sequence of the DLX3 gene. This SNP provided accurate classification of animals (99.5%) as carriers or non-carriers of the mutation and therefore maybe useful in marker assisted selection. A synergistic action of FecL ^L and FecX ^L mutations on both ovulation rate and litter size was demonstrated. Until now, all the Fec genes identified in sheep belong to the bone morphogenetic protein (BMP) system. Based on the human orthologous region, none of the 20 genes in the FecL region corresponds to known molecules in the BMP system. The identification of the FecL ^L mutation could lead to the discovery of a new pathway involved in the regulation of ovulation rate.     

Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler

Aims:  To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus.
Methods and Results:  Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates ( n  = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10⁷–10¹ CFU ml⁻¹ and that for tdh2 was 10⁷–10⁴ CFU ml⁻¹.
Conclusions:  The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min.
Significance and Impact of the Study:  The assay developed allows accurate detection of pathogenic V. parahaemolyticus , which is valuable for rapid tracing of infection source during outbreaks.     

Aboveground biomass estimation of the shrubs, Echiochilon fruticosum (Desf.) and Helianthemum kahiricum (Del.) in the arid zone rangelands of Tunisia

The knowledge of the plant biomass is very important for the assessment of the rangeland productivity. It could help to select the appropriate species for the improvement of natural ecosystems (rehabilitation, restoration and seedling). By examining different correlations between the biomass production and the volume parameters of two North African shrub species of high range value ( Echiochilon fruticosum Desf. and Helianthemum kahiricum Del.), we aimed to establish the appropriate regression models, which could be useful for the prediction of the productivity of these species. The data showed a significant relationship between the total biomass (TB) production and the mean diameter (MD) of the studied species ( R ² = 0.65 for Echiochilon and R ² = 0.75 for Helianthemum ). Likewise, annual fresh production (leaves and current-year shoots) was well correlated with MD of Helianthemum ( R ² = 0.82). However, the correlation between these two parameters was relatively low for Echiochilon ( R ² = 0.42).     

Modulation of 1O2-mediated retrograde signaling by the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein, a central integrator of stress and energy signaling

Shortly after the release of singlet oxygen (¹O₂) in chloroplasts, changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Extensive genetic screens aimed at identifying constituents involved in ¹O₂-mediated plastid-to-nucleus signaling have failed to identify extraplastidic signaling components. This finding suggests that ¹O₂-mediated signals are not translocated to the nucleus via a single linear pathway, but rather through a signaling network that is difficult to block by single mutations. The complexity of this signaling network has been tackled by mutagenizing a transgenic flu line expressing the luciferase reporter gene under the control of the promoter of a ¹O₂-responsive AAA-ATPase gene (At3g28580) and isolating second site mutants that constitutively express the reporter gene at a high level. One of the mutants was shown by map-based cloning and sequencing to contain a single amino acid change in the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein. PRL1 suppresses the expression of AAA-ATPase and other ¹O₂-responsive genes. PRL1 seems to play a major role in modulating responses of plants to environmental changes by interconnecting ¹O₂-mediated retrograde signaling with other signaling pathways.     

The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli

In Escherichia coli , lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn²⁺. In the presence of 0.5 mM EGTA, only the parental strain was able to take up ⁶⁵Zn²⁺. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI ) and localized at 42 min on the genetic map of E. coli . At high Zn²⁺ concentrations, the znu mutants took up more ⁶⁵Zn²⁺ than the parental strain. The high-affinity ⁶⁵Zn²⁺ uptake was repressed by growth in the presence of 10 μM Zn²⁺. A znuA–lacZ operon fusion was repressed by 5 μM Zn²⁺ and showed a more than 20-fold increase in β-galactosidase activity when Zn²⁺ was bound to 1.5 μM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn²⁺-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli . A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity ⁶⁵Zn²⁺ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB .