Chromosomal tet(O)-harboring regions in Campylobacter coli isolates from turkeys and swine.

In turkey-derived Campylobacter coli isolates of a unique lineage (cluster II), the tetracycline resistance determinant tet(O) was chromosomal and was part of a gene cassette (transposon) interrupting a Campylobacter jejuni-associated putative citrate transporter gene. In contrast, the swine-derived C. coli strain 6461 harbored a chromosomal tet(O) in a different genomic location.

The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation.

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1-A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

Superoxide dismutase-deficient mutants of Helicobacter pylori are hypersensitive to oxidative stress and defective in host colonization.

Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among organisms that are exposed to oxic environments. The single SOD of Helicobacter pylori, encoded by the sodB gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously. We have isolated mutants with interruptions in the sodB gene and have characterized them with respect to their response to oxidative stress and ability to colonize the mouse stomach. The sodB mutants are devoid of SOD activity, based on activity staining in nondenaturing gels and quantitative assays of cell extracts. Though wild-type H. pylori is microaerophilic, the mutants are even more sensitive to O(2) for both growth and viability. While the wild-type strain is routinely grown at 12% O(2), growth of the mutant strains is severely inhibited at above 5 to 6% O(2). The effect of O(2) on viability was determined by subjecting nongrowing cells to atmospheric levels of O(2) and plating for survivors at 2-h time intervals. Wild-type cell viability dropped by about 1 order of magnitude after 6 h, while viability of the sodB mutant decreased by more than 6 orders of magnitude at the same time point. The mutants are also more sensitive to H(2)O(2), and this sensitivity is exacerbated by increased O(2) concentrations. Since oxidative stress has been correlated with DNA damage, the frequency of spontaneous mutation to rifampin resistance was studied. The frequency of mutagenesis of an sodB mutant strain is about 15-fold greater than that of the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became H. pylori positive, while 15 out of 17 mice inoculated with the wild-type strain were shown to harbor the organism. Therefore, SOD is a virulence factor which affects the ability of this organism to colonize the mouse stomach and is important for the growth and survival of H. pylori under conditions of oxidative stress.

Regulation of ornithine decarboxylase activity and polyamine transport by agmatine in rat pulmonary artery endothelial cells.

Agmatine, a product of arginine decarboxylation in mammalian cells, is believed to govern cell polyamines by inducing antizyme, which in turn suppresses ornithine decarboxylase (ODC) activity and polyamine uptake. However, since agmatine is structurally similar to the polyamines, it is possible that it exerts antizyme-independent actions on polyamine regulatory pathways. The present study determined whether agmatine inhibited ODC activity and polyamine transport in rat pulmonary artery endothelial cells (PAECs) by an antizyme-dependent mechanism. Agmatine caused time-dependent reductions in ODC activity, which occurred before increases in antizyme. Interventions that suppressed proteasome function caused large increases in ODC activity but failed to attenuate inhibitory effects of agmatine. When agmatine was present in the culture medium, 14C-polyamine uptake was competitively inhibited as evidenced by substantial elevations in K(m) values. If PAECs were incubated with agmatine for periods sufficient to increase antizyme, there were modest decreases in V(max) for putrescine and spermidine but not for spermine. These effects of agmatine on polyamine transport were insensitive to protein synthesis inhibition. Collectively, our findings show that agmatine decreases ODC activity and polyamine transport in PAECs, but a causal role for antizyme in these actions of agmatine is difficult to establish. Nevertheless, these observations are consistent with a model in which PAECs express both antizyme-1 and -2, but only the latter contributes to agmatine-mediated suppression of ODC activity.

Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori.

The nickel-containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)- and 200 (HypB:kan)-fold lower than for the wild-type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H(2)-oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease-containing fractions of the mutant strains contained about four (HypA:kan)- and five (HypB:kan)-fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.

Characterization of the NifU and NifS Fe-S cluster formation proteins essential for viability in Helicobacter pylori.

The Fe-S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori. Obtaining viable H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O(2) atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency. This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H. pylori viability. H. pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized. Purified NifU is a red protein that contains approximately 1.5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S](2+,+) cluster by characteristic UV-visible, EPR, and MCD spectra. The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein. Purified NifS has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters. The results here indicate that the NifU type of Fe-S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H. pylori lacks other Fe-S cluster assembly proteins, that the H. pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clusters.

Odontogenic carcinoma occurring in a dentigerous cyst: case report and clinical management.

This case report describes an unusual odontogenic carcinoma, which was detected during routine periodontal examination. The lesion occurred in a dentigerous cyst associated with an impacted third molar in an otherwise asymptomatic 66-year-old male patient. The impacted tooth and lesion were excised based on evidence of radiographic change and clinical findings. An unusual histopathologic presentation is reported. The treatment provided for this tumor and the management of impacted teeth is reviewed.

Cellular disposition of transported polyamines in hypoxic rat lung and pulmonary arteries.

The polyamines putrescine, spermidine (SPD), and spermine are a family of low-molecular-weight organic cations essential for cell growth and differentiation and other aspects of signal transduction. Hypoxic pulmonary vascular remodeling is accompanied by depressed lung polyamine synthesis and markedly augmented polyamine uptake. Cell types in which hypoxia induces polyamine transport in intact lung have not been delineated. Accordingly, rat lung and rat main pulmonary arterial explants were incubated with [(14)C]SPD in either normoxic (21% O(2)) or hypoxic (2% O(2)) environments for 24 h. Autoradiographic evaluation confirmed previous studies showing that, in normoxia, alveolar epithelial cells are dominant sites of polyamine uptake. In contrast, hypoxia was accompanied by prominent localization of [(14)C]SPD in conduit, muscularized, and partially muscularized pulmonary arteries, which was not evident in normoxic lung tissue. Hypoxic main pulmonary arterial explants also exhibited substantial increases in [(14)C]SPD uptake relative to control explants, and autoradiography revealed that enhanced uptake was most evident in the medial layer. Main pulmonary arterial explants denuded of endothelium failed to increase polyamine transport in hypoxia. Conversely, medium conditioned by endothelial cells cultured in hypoxic, but not in normoxic, environments enabled hypoxic transport induction in denuded arterial explants. These findings in arterial explants were recapitulated in rat cultured main pulmonary artery cells, including the enhancing effect of a soluble endothelium-derived factor(s) on hypoxic induction of [(14)C]SPD uptake in smooth muscle cells. Viewed collectively, these results show in intact lung tissue that hypoxia enhances polyamine transport in pulmonary artery smooth muscle by a mechanism requiring elaboration of an unknown factor(s) from endothelial cells.

Dual roles of Bradyrhizobium japonicum nickelin protein in nickel storage and GTP-dependent Ni mobilization.

The hydrogenase accessory protein HypB, or nickelin, has two functions in the N(2)-fixing, H(2)-oxidizing bacterium Bradyrhizobium japonicum. One function of HypB involves the mobilization of nickel into hydrogenase. HypB also carries out a nickel storage/sequestering function in B. japonicum, binding nine nickel ions per monomer. Here we report that the two roles (nickel mobilization and storage) of HypB can be separated in vitro and in vivo using molecular and biochemical approaches. The role of HypB in hydrogenase maturation is completely dependent on its intrinsic GTPase activity; strains which produce a HypB protein that is severely deficient in GTPase activity but that fully retains nickel-sequestering ability cannot produce active hydrogenase even upon prolonged nickel supplementation. A HypB protein that lacks the nickel-binding polyhistidine region near the N terminus lacks only the nickel storage capacity function; it is still able to bind a single nickel ion and also retains complete GTPase activity.

Dental endosseous implant assessments in a type 2 diabetic population: a prospective study.

Diabetes mellitus, a prevalent disorder worldwide, is associated with systemic adverse sequelae, such as wound healing alterations, which may affect osseointegration of dental implants. This prospective multicenter study assessed the success of 2-stage endosseous root-form implants (3 different implant systems) placed in the mandibular symphysis of 89 male type 2 diabetic subjects. The implants were uncovered approximately 4 months after placement, restored with an implant-supported, Hader bar clip-retained overdenture, and maintained at scheduled follow-up data collection examinations for 60 months after loading. Sixteen (9.0%) of the 178 implants failed. Life table methods calculated implant survival at approximately 88%, from prosthesis placement through the 60-month follow-up, and at approximately 90% from implant placement through the observation period. No implants failed between surgical placement and uncovering, 5 failed at uncovering, 7 failed after uncovering before prosthesis placement, and 4 failed after prosthesis placement. Fasting plasma glucose (FPG) and glycosylated hemoglobin (HbA1c) values were determined before implant placement (baseline) and approximately 4 months later at surgical uncovering (follow-up). The 5-year implant outcomes (successes versus failures) were analyzed against the following predictor variables: (1) baseline and follow-up FPG values, (2) baseline and follow-up HbA1c values, (3) subject age, (4) duration of diabetes (years), (5) baseline diabetic therapy, (6) smoking history, and (7) implant length. Regression analysis found only duration of diabetes (P < .025) and implant length (P < .001) to be statistically significant predictors of implant failure. There was no statistically significant difference in failure rates between the 3 different implant systems used. This study supports the use of dental implants in type 2 diabetic patients.

Interactions between agmatine and polyamine uptake pathways in rat pulmonary artery endothelial cells.

Agmatine, a product of arginine metabolism in vascular endothelial cells, is structurally similar to the natural polyamines, putrescine, spermidine and spermine. To test the hypothesis that agmatine and polyamines interacted at the level of the polyamine transporter, we determined if polyamines competed with agmatine for import and whether interventions modulating polyamine import exerted coordinate effects on agmatine uptake. Multiple lines of evidence were obtained to suggest that agmatine enters pulmonary artery endothelial cells (PAECs) via the polyamine transporter, though its intracellular disposition after uptake appears different from the natural polyamines.

Regulation of gadd153 mRNA expression by hypoxia in pulmonary artery smooth muscle cells.

Hypoxia causes pulmonary hypertension and induces oxygen radicals in pulmonary artery smooth muscle cells (PASMCs). Since oxidative stress regulates gaddl53 expression, we examined gaddl53 mRNA in PASMCs cultured in a hypoxic environment. Gadd153 mRNA content was increased in PASMCs cultured for 24 hours in 1% oxygen. This increase was not abrogated by inhibition of protein synthesis. To explore the signaling pathways mediating hypoxic regulation of gaddl53 mRNA, the impact of calcium channel blockade by verapamil, G protein inhibition by pertussis toxin, and protein kinase C (PKC) down-regulation, was examined. Although none of these interventions reduced basal expression of gaddl53 mRNA in PASMCs, all of them suppressed the induction by hypoxia. In contrast, antioxidants had no effect. These observations indicate hypoxia induces gaddl53 expression in PASMCs through common signaling pathways.

Long-term assessment (5 to 71 months) of endosseous dental implants placed in the augmented maxillary sinus.

BACKGROUND: It is not uncommon for the placement of endosseous dental implants in the maxillary posterior jaw region to be complicated by the pneumatization of the maxillary sinus. When this occurs, the residual bone between the floor of the sinus and the crestal ridge is inadequate for the placement of implants. The sinus lift procedure provides a way to increase the amount of available bone and the placement of longer implants. METHODS: One hundred twenty (120) implants were placed in 45 augmented maxillary sinuses. Patients ranged in age from 34 to 78 years. The implant design included a limited number of non-hydroxyapatite (HA)-coated titanium screws, with the majority of the implants being HA-coated cylinders, grooved cylinders, and screws. The augmentation materials were autogenous bone, allogenic bone (demineralized freeze-dried bone allograft, DFDBA), alloplastic bone (HA), combination grafts of HA and DFDBA, and combination grafts of autogenous bone and DFDBA. All the cases were successfully restored with implant-supported, bar-retained overdentures or fixed partial dentures. The follow-up began at Stage 2 uncovering and ranged from 5 to 71 months, with a mean of 38.2 and standard deviation of 14.6 months. RESULTS: Three (2.5%) of the 120 implants failed between the period of implant placement and 36 months. Failures appeared to be associated with a history of smoking. Other complications encountered during the study are presented. Implant survival was higher in those placed in grafted sinuses (97.5%) than in those placed in the posterior maxilla without sinus grafting (90.3%). CONCLUSION: These findings support the use of implants placed in augmented sinuses to support dental prostheses.

The influence of preoperative antibiotics on success of endosseous implants at 36 months.

The benefits of prophylactic antibiotics are well recognized in dentistry. However, their routine use in the placement of endosseous dental implants remains controversial. As part of the comprehensive Dental Implant Clinical Research Group (DICRG) clinical implant study, the preoperative or postoperative use of antibiotics, the type used, and the duration of coverage were left to the discretion of the surgeon. These data for 2,973 implants were recorded and correlated with failure of osseointegration during healing (Stage 1), at surgical uncovering (Stage 2), before loading the prosthesis (Stage 3), and from prosthesis loading to 36 months (Stage 4). The results showed a significantly higher survival rate at each stage of treatment in patients who had received preoperative antibiotics.

Periodontal-type measurements associated with hydroxyapatite-coated and non-HA-coated implants: uncovering to 36 months.

BACKGROUND: While the use of hydroxyapatite (HA)-coated endosseous dental implants has gained in popularity over the past 10 years, the short-term and long-term predictability and indications for their use remain highly controversial. Some reports suggest that the HA coating may separate from the substructure, undergo dissolution in tissue fluids, and/or contribute to rapid osseous breakdown around the implant. Other reports, however, relate favorable responses to HA-coated implants, which include rapid bone adaptation to the HA, greater stability at uncovering, and increased coronal bone growth. These contradictions may be related to differences in chemical composition of the HA on the implant surface. Most clinicians and researchers may agree that long-term, independent, scientific clinical studies are needed to compare HA-coated and non-HA-coated (titanium-alloy and CP-titanium) implants under the same conditions. Concerns appear in the literature that HA-coated implants experience greater breakdown because they are more susceptible to bacterial colonization due to their roughness and hydrophilicity. Some studies suggest that specific putative periodontal pathogens may adhere to the HA, thereby predisposing the implant to greater peri-implantitis than that experienced by non-HA implants. METHODS: A total of 32 clinical research centers, located in various geographic regions of the United States, were selected to participate in a comprehensive clinical study. More than 2,900 HA-coated and non-HA implants were randomized as to location within one of three jaw regions--maxillary anterior, mandibular anterior, and mandibular posterior--and followed for 36 months. It can be assumed that in each of these jaw regions, the conditions associated with both implant surface types would be similar enough to permit meaningful comparisons of periodontal-type measurements that have not previously been reported. Periodontal-type measurements (gingiva, plaque, suppuration, and calculus indices; probing depth; attachment levels; recession; and keratinized tissue width) for each aspect of each implant (mesial, facial, distal, and lingual) were recorded at 3, 6, 9, 12, 18, 24, and 36 months following implant uncovering. The implant was considered the experimental unit for analysis using generalized estimating equation and repeated measure methods. Data for the four aspects of each implant, as well as measurements over time, were all clustered in the unit of analysis. RESULTS: The percentages of implants with zeros recorded for the indices was remarkably similar for both HA-coated and non-HA implants. While statistically significant differences were found for some of the measurements associated with HA-coated and non-HA implants under certain conditions, these differences were too small to be considered clinically significant. CONCLUSIONS: Overall, there was no clinically significant difference between the periodontal-type measurements for HA-coated and non-HA-coated implants followed for a period from 3 through 36 months. The concerns about HA-coated implants being associated with adverse periodontal responses for the HA chemical composition included in this study appear to be unfounded for a period of clinical performance up to 36 months.

The influence of preoperative antibiotics on success of endosseous implants up to and including stage II surgery: a study of 2,641 implants.

According to the American College of Surgeons, complex oral surgical procedures, including the transoral placement of endosseous implants, are of the type that may require prophylactic antibiotics. However, the routine use of prophylactic antibiotics in the field of dental implantology continues to be controversial, and their utilization varies widely. No data from a randomized prospective clinical study of the prophylactic use of antibiotics in implant surgery have been previously published. As part of the comprehensive Dental Implant Clinical Research Group clinical implant study, the preoperative or postoperative use of antibiotics, the type used, and the duration of coverage was left to the discretion of the surgeon. These data were recorded and correlated with failure of osseointegration during healing (stage I) and at stage II surgery (uncovering). The results showed that significantly fewer failures occurred when preoperative antibiotics were used.

Translation of ODC mRNA and polyamine transport are suppressed in ras-transformed CREF cells by depleting translation initiation factor 4E.

Rapid tumor growth and metastasis require increased polyamine metabolism, which is coordinately regulated by ornithine decarboxylase (ODC) and the polyamine transporter. Both activities are stimulated by ras signalling and are dependent upon protein biosynthesis. T24ras oncogene expression in rat embryo fibroblasts (CREFT24) induces cellular transformation and malignancy, in part, by stimulating the rate-limiting translation initiation factor, eIF-4E. CREFT24 expressing antisense RNA to eIF-4E (AS4E) have markedly decreased tumor growth rates and metastatic capacity, without altered monolayer growth rates. Herein, we demonstrate that in AS4E, ODC is translationally suppressed resulting in decreased ODC activity. Additionally, exogenous polyamine uptake is suppressed in AS4E cells indicating that AS4E can neither generate nor import the polyamines necessary to support rapid tumor growth. These data provide evidence that eIF-4E is the link between ras-induced malignancy and increased polyamine metabolism and support the hypothesis that eIF-4E plays a pivotal role in mediating ras-induced malignancy.

The sequences of hypF, hypC and hypD complete the hyp gene cluster required for hydrogenase activity in Bradyrhizobium japonicum.

A region of DNA 6 kb downstream of the hydrogenase (H2ase) structural genes and directly downstream of the hypB gene of Bradyrhizobium japonicum was shown by mutational analysis to be necessary for H2ase synthesis. Sequencing of this region revealed two complete open reading frames, and the 5' fragment of a third ORF. They encode proteins with homologies to the HypF, HypC and the N-terminus of HypD from other H2ase-containing organisms. The hypF of B. japonicum encodes a 753-aa protein with a predicted molecular mass of 80.3 kDa that contains the two zinc-finger motifs characteristic of other HypF proteins. The hypC encodes a 85-aa protein with a predicted molecular mass of 8.4 kDa. The 5' portion of hypD, which encodes the first 35 aa, upon combining with the previously reported C-terminus of HypD, designated HypD' (Van Soom et al. (1993) Mol. Gen. Genet. 239, 235-240) encodes a protein with a predicted molecular mass of 42.4 kDa. Complementation studies on a H2 uptake defective strain of B. japonicum containing a polar mutation in the hyp operon revealed that the products of the hyp F, C, D, E genes are required for H2ase production. Evidence is also presented that the hyp genes are co-transcribed from a large operon together with the downstream genes hupGHIJK, making a polycistronic message of 11 genes.

Multiple polyamine regulatory pathways control compensatory cardiovascular hypertrophy in coarctation hypertension.

While a number of factors may initiate structural alterations within the cardiovascular system in response to hypertension, there are obligate cellular signaling mechanisms, such as the polyamines, through which they must operate. This study examined the effects of polyamine synthesis inhibition using eflornithine, a suicide inhibitor of ornithine decarboxylase on blood pressure, compensatory remodeling of the cardiovascular system, and cardiac and aortic polyamine contents using an aortic coarctation model in rats. Eflornithine treatment failed to reduce carotid arterial blood pressure and actually significantly elevated vascular pressure above and below the coarctation site by 14 days of hypertension. Eflornithine only transiently reduced aortic polyamine content of hypertensive rats while this agent reduced coarctation-induced aortic medial wall thickening and the synthesis/deposition of fibronectin and laminin in the hypertensive aorta. Increases in left ventricular mass and polyamine content were concomitantly reduced in hypertensive rats administered eflornithine. These results suggest that multiple polyamine regulatory pathways may maintain vascular polyamine content in response to aortic coarctation; however de novo polyamine synthesis is essential for select aspects of vascular remodeling, including matrix synthesis. Cardiac tissue, in contrast, may rely principally on de novo polyamine synthesis.

The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase.

The HypB protein from Bradyrhizobium japonicum is a metal-binding GTPase required for hydrogenase expression. In-frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene. Complete deletion of the gene yielded a strain (JH delta Eg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JH delta 23H lacking only the N-terminal histidine-rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JH delta 23H was low in comparison to the wild type in 10-50 nM nickel levels, but could be cured to nearly wild-type levels by including 50 microM nickel during the derepression incubation. Studies on strains harbouring the hup promoter-lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild-type promoter activity in 50 microM NiCl2. Further evidence that HypB is required for hup promoter-binding activity was obtained from gel-shift assays. HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O2, 10% partial pressure H2) HypB was detected, and its expression preceded hydrogenase synthesis by 3-6 h. 63Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild-type strain at all time points tested during the derepression incubation. Wild-type cultures that received nickel during the HypB expression-specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period. In contrast to the wild type, strain JH delta 23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period. These results indicate that the loss of the metal-binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps.