To investigate whether mice genetically unaltered by many generations of laboratory selection exhibit similar hormonal and demographic responses to caloric restriction (CR) as laboratory rodents, we performed CR on cohorts of genetically heterogeneous male mice which were grandoffspring of wild-caught ancestors. Although hormonal changes, specifically an increase in corticosterone and decrease in testosterone, mimicked those seen in laboratory-adapted rodents, we found no difference in mean longevity between ad libitum (AL) and CR dietary groups, although a maximum likelihood fitted Gompertz mortality model indicated a significantly shallower slope and higher intercept for the CR group. This result was due to higher mortality in CR animals early in life, but lower mortality late in life. A subset of animals may have exhibited the standard demographic response to CR in that the longest-lived 8.1% of our animals were all from the CR group. Despite the lack of a robust mean longevity difference between groups, we did note a strong anticancer effect of CR as seen in laboratory rodents. Three plausible interpretations of our results are the following: (1) animals not selected under laboratory conditions do not show the typical CR effect; (2) because wild-derived animals eat less when fed AL, our restriction regime was too severe to see the CR effect; or (3) there is genetic variation for the CR effect in wild populations; variants that respond to CR with extended life are inadvertently selected for under conditions of laboratory domestication.
Ageratum conyzoides L., a weed species widely distributed throughout southeast Asia, frequently exhibits striking yellow vein symptoms associated with infection by Ageratum yellow vein virus (AYVV), a member of the Geminiviridae (genus Begomovirus). Most begomoviruses have bipartite genomes (DNAs A and B), but only a DNA A has been identified for AYVV. We demonstrate that yellow vein disease of A. conyzoides results from co-infection by AYVV DNA A (2,741 nt) and a circular DNA that is approximately half its size (1,347 nt) that we designate DNA beta. Apart from the sequence TAATATTAC, common to all geminiviruses and containing the initiation site of rolling circle replication, DNA beta shows negligible sequence homology either to AYVV DNA A or to DNA B associated with bipartite begomoviruses. DNA beta depends on DNA A for replication and is encapsidated by DNA A-encoded coat protein and so has characteristics of a DNA satellite. However, systemic infection of A. conyzoides by DNA A alone is sporadic and asymptomatic, and DNA A accumulation is reduced to 5% or less of its accumulation in the presence of DNA beta. Therefore, DNA A and DNA beta together form a previously unrecognized disease-inducing complex. Our data also demonstrate that the nanovirus-like DNA 1 component associated with infected A. conyzoides plays no essential role in the disease and represents a satellite-like DNA. Furthermore, the satellite DNA previously found associated with tomato leaf curl virus is probably a defective DNA beta homologue.
Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2. Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure. We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18. SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD. SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes. We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein. Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex. The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30. The N-terminal domain of p33 is present in several uncharacterized human proteins. We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain.
The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin-independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization signal (NLS)-deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53(-/-) cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19(arf), and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1-binding-defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half-life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine-free mutant. Consequently, MDM2 suppressed p21waf1/cip1-induced cell growth arrest of human p53(-/-) and p53(-/-)/Rb(-/-)cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin-independent fashion.
Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.
We study a situation that arises in the somatic evolution of cancer. Consider a finite population of replicating cells and a sequence of mutations: type 0 can mutate to type 1, which can mutate to type 2. There is no back mutation. We start with a homogeneous population of type 0. Mutants of type 1 emerge and either become extinct or reach fixation. In both cases, they can generate type 2, which also can become extinct or reach fixation. If mutation rates are small compared to the inverse of the population size, then the stochastic dynamics can be described by transitions between homogeneous populations. A "stochastic tunnel" arises, when the population moves from all 0 to all 2 without ever being all 1. We calculate the exact rate of stochastic tunneling for the case when type 1 is as fit as type 0 or less fit. Type 2 has the highest fitness. We discuss implications for the elimination of tumor suppressor genes and the activation of genetic instability. Although our theory is developed for cancer genetics, stochastic tunnels are general phenomena that could arise in many circumstances.
A coagglutinating reagent has recently been developed and utilized in capsule typing of pneumococci. The reagent consists of stabilized staphylococci coated with specific antibody via the gamma globulin Fc-protein A reaction thereby orienting the antibody molecules with the antigen-reactive Fab parts outwards. Reagent staphylococci coated with antibodies directed against streptococcal group-specific antigens have been used in the present investigations. Overnight cultures of streptococci were treated with trypsin and then directly tested for coagglutination of selected reagent staphylococci on glass slides. In all, 179 strains of streptococci were analyzed by the method described. The results were compared with grouping by using Lancefield extracts in counter-immunoelectrophoresis and were shown to agree completely. The coagglutination method described proved to be accurate, rapid, and simple.
Enzymes that use the cofactor pyridoxal phosphate (PLP) constitute a ubiquitous class of biocatalysts. Here, we analyse their variety and genomic distribution as an example of the current opportunities and challenges for the study of protein families. In many free-living prokaryotes, almost 1.5% of all genes code for PLP-dependent enzymes, but in higher eukaryotes the percentage is substantially lower, consistent with these catalysts being involved mainly in basic metabolism. Assigning the function of PLP-dependent enzymes simply on the basis of sequence criteria is not straightforward because, as a consequence of their common mechanistic features, these enzymes have intricate evolutionary relationships. Thus, many genes for PLP-dependent enzymes remain functionally unclassified, and several of them might encode undescribed catalytic activities. In addition, PLP-dependent enzymes often show catalytic promiscuity (that is, a single enzyme catalyses different reactions), implying that an organism can have more PLP-dependent activities than it has genes for PLP-dependent enzymes. This observation presumably applies to many other classes of protein-encoding genes.
The Src homology 2-containing phosphotyrosine phosphatase (SHP2) is primarily a positive effector of receptor tyrosine kinase signaling. However, the molecular mechanism by which SHP2 effects its biological function is unknown. In this report, we provide evidence that defines the molecular mechanism and site of action of SHP2 in the epidermal growth factor-induced mitogenic pathway. We demonstrate that SHP2 acts upstream of Ras and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell by interfering with the process of Ras inactivation catalyzed by Ras GTPase-activating protein (RasGAP). It does so by inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane, to its substrate (GTP-Ras) microdomain. Inhibition is achieved through the dephosphorylation of RasGAP binding sites at the level of the plasma membrane. We have identified Tyr992 of the epidermal growth factor receptor (EGFR) to be one such site, since its mutation to Phe renders the EGFR refractory to the effect of dominant-negative SHP2. To our knowledge, this is the first report to outline the site and molecular mechanism of action of SHP2 in EGFR signaling, which may also serve as a model to describe its role in other receptor tyrosine kinase signaling pathways.
Sodalis glossinidius is a maternally transmitted secondary endosymbiont residing intracellularly in tissues of the tsetse flies, Glossina spp. In this study, we have used Tn5 mutagenesis and a negative selection procedure to derive a S. glossinidius mutant that is incapable of invading insect cells in vitro and is aposymbiotic when microinjected into tsetse. This mutant strain harbors Tn5 integrated into a chromosomal gene sharing high sequence identity with a type III secretion system invasion gene (invC) previously identified in Salmonella enterica. With the use of degenerate PCR, we have amplified a further six Sodalis inv/spa genes sharing high sequence identity with type III secretion system genes encoded by Salmonella pathogenicity island 1. Phylogenetic reconstructions based on the inv/spa genes of Sodalis and other members of the family Enterobacteriaceae have consistently identified a well-supported clade containing Sodalis and the enteric pathogens Shigella and Salmonella. These results suggest that Sodalis may have evolved from an ancestor with a parasitic intracellular lifestyle, possibly a latter-day entomopathogen. These observations lend credence to a hypothesis suggesting that vertically transmitted mutualistic endosymbionts evolve from horizontally transmitted parasites through a parasitism-mutualism continuum.
The pattern of Epstein-Barr virus (EBV) RNAs expressed in Raji cells superinfected with P3HR1 EBV was examined. RNAs whose expression was of an immediate-early type (resistant to treatment of the cells with anisomycin) were identified. These RNAs, encoding the EBV reading frames BZLF1 and BRLF1, were probably expressed from defective virus within the P3HR1 preparation, and some of them were responsible for the induction of the EBV productive cycle in the Raji cells. The structures of the B95-8 RNAs equivalent to the anisomycin-resistant RNAs were determined. The RNA encoding the BZLF1 reading frame contained two splices which extended and modified the reading frame from that previously described.
In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.
We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed "immunoRCA. " In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag-Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide. ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Abs, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.
Circadian rhythms in mammals are generated by a feedback loop in which the three PERIOD (PER) proteins, acting in a large complex, inhibit the transcriptional activity of the CLOCK-BMAL1 dimer, which represses their own expression. Although fundamental, the mechanism of negative feedback in the mammalian clock, or any eukaryotic clock, is unknown. We analyzed protein constituents of PER complexes purified from mouse tissues and identified PSF (polypyrimidine tract-binding protein-associated splicing factor). Our analysis indicates that PSF within the PER complex recruits SIN3A, a scaffold for assembly of transcriptional inhibitory complexes and that the PER complex thereby rhythmically delivers histone deacetylases to the Per1 promoter, which repress Per1 transcription. These findings provide a function for the PER complex and a molecular mechanism for circadian clock negative feedback.
The Self-care of Heart Failure Index (SCHFI) is a measure of self-care defined as a naturalistic decision-making process involving the choice of behaviors that maintain physiological stability (maintenance) and the response to symptoms when they occur (management). In the 5 years since the SCHFI was published, we have added items, refined the response format of the maintenance scale and the SCHFI scoring procedure, and modified our advice about how to use the scores.
The objective of this article was to update users on these changes.
In this article, we address 8 specific questions about reliability, item difficulty, frequency of administration, learning effects, social desirability, validity, judgments of self-care adequacy, clinically relevant change, and comparability of the various versions.
The addition of items to the self-care maintenance scale did not significantly change the coefficient alpha, providing evidence that the structure of the instrument is more powerful than the individual items. No learning effect is associated with repeated administration. Social desirability is minimal. More evidence is provided of the validity of the SCHFI. A score of 70 or greater can be used as the cut-point to judge self-care adequacy, although evidence is provided that benefit occurs at even lower levels of self-care. A change in a scale score more than one-half of an SD is considered clinically relevant. Because of the standardized scores, results obtained with prior versions can be compared with those from later versions.
The SCHFI v.6 is ready to be used by investigators. By publication in this format, we are putting the instrument in the public domain; permission is not required to use the SCHFI.
Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.
Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of the common serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiae wild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.
At what age can children attribute false beliefs to others? Traditionally, investigations into this question have used elicited-response tasks in which children are asked a direct question about an agent's false belief. Results from these tasks indicate that the ability to attribute false beliefs does not emerge until about age 4. However, recent investigations using spontaneous-response tasks suggest that this ability is present much earlier. Here we review results from various spontaneous-response tasks that suggest that infants in the second year of life can already attribute false beliefs about location and identity as well as false perceptions. We also consider alternative interpretations that have been offered for these results, and discuss why elicited-response tasks are particularly difficult for young children.
Many cell division-related proteins are located at specific positions in the bacterial cell, and this organized distribution of proteins requires energy. Here, we report that the proton motive force, or more specifically the (trans)membrane potential, is directly involved in protein localization. It emerged that the membrane potential modulates the distribution of several conserved cell division proteins such as MinD, FtsA, and the bacterial cytoskeletal protein MreB. We show for MinD that this is based on the membrane potential stimulated binding of its C-terminal amphipathic helix. This function of the membrane potential has implications for how these morphogenetic proteins work and provide an explanation for the effects observed with certain antimicrobial compounds.
Mechanical ventilation is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged mechanical ventilation is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during mechanical ventilation, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of mechanical ventilation- induced reactive oxygen species production in the diaphragm is important.
These experiments tested the hypothesis that elevated mitochondrial reactive oxygen species emission is required for mechanical ventilation-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm.
Cause and effect was determined by preventing mechanical ventilation-induced mitochondrial reactive oxygen species emission in the diaphragm of rats using a novel mitochondria-targeted antioxidant (SS-31).
Compared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against mechanical ventilation-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against mechanical ventilation-induced myofiber atrophy and contractile dysfunction.
These results reveal that prevention of mechanical ventilation-induced increases in diaphragmatic mitochondrial reactive oxygen species emission protects the diaphragm from mechanical ventilation-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of reactive oxygen species production in the diaphragm during prolonged mechanical ventilation. These results could lead to the development of a therapeutic intervention to impede mechanical ventilation-induced diaphragmatic weakness.
This study examined racial/ethnic disparities in mental health service access and use at different poverty levels.
We compared demographic and clinical characteristics and service use patterns of Whites, Blacks, Hispanics, and Asians living in low-poverty and high-poverty areas. Logistic regression models were used to assess service use patterns of minority racial/ethnic groups compared with Whites in different poverty areas.
Residence in a poverty neighborhood moderates the relationship between race/ethnicity and mental health service access and use. Disparities in using emergency and inpatient services and having coercive referrals were more evident in low-poverty than in high-poverty areas.
Neighborhood poverty is a key to understanding racial/ethnic disparities in the use of mental health services.
We examined associations of race/ethnicity, gender, and sexual orientation with mental disorders among lesbian, gay, bisexual, and transgender (LGBT) youths.
We assessed mental disorders by administering a structured diagnostic interview to a community sample of 246 LGBT youths aged 16 to 20 years. Participants also completed the Brief Symptom Inventory 18 (BSI 18).
One third of participants met criteria for any mental disorder, 17% for conduct disorder, 15% for major depression, and 9% for posttraumatic stress disorder. Anorexia and bulimia were rare. Lifetime suicide attempts were frequent (31%) but less so in the prior 12 months (7%). Few racial/ethnic and gender differences were statistically significant. Bisexually identified youths had lower prevalences of every diagnosis. The BSI 18 had high negative predictive power (90%) and low positive predictive power (25%) for major depression.
LGBT youths had higher prevalences of mental disorder diagnoses than youths in national samples, but were similar to representative samples of urban, racial/ethnic minority youths. Suicide behaviors were similar to those among representative youth samples in the same geographic area. Questionnaires measuring psychological distress may overestimate depression prevalence among this population.
Pleiomorphic mouse sarcoma S180 cells were transfected with cDNAs for the liver cell adhesion molecule (L-CAM), the neural cell adhesion molecule (N-CAM), or both CAMs. Transfected cells expressed the appropriate CAMs at their surface and those expressing L-CAM (S180L cells) changed from adjoining spindle or round shapes to a closely linked "epithelioid" sheet when grown to confluence. Cells transfected with cDNA for N-CAM (S180N cells) also expressed this CAM on the cell surfaces and bound brain vesicles containing N-CAM but showed no phenotypic change to an epithelioid state. In S180L cells and doubly transfected (S180L/N) cells, L-CAM was concentrated at regions of cell contact and was codistributed with cortical actin. In S180N cells, N-CAM was uniformly distributed on the cell surface. When S180L cells were cocultured with S180L/N cells, N-CAM was not concentrated at boundaries between the S180L and S180L/N cells but was concentrated at boundaries between pairs of S180L/N cells. Fab' fragments of anti-L-CAM dissociated the epithelioid sheets of S180L or S180L/N cells into cells with shapes resembling those of untransfected cells. Cells in epithelioid sheets were polygonal in shape but, unlike cells in true epithelia, had no basement membrane or polar structure; they also lacked tight junctions and desmosomes. Ultrastructural examination showed that, in contrast to the untransfected phenotype, cells in epithelioid sheets had large increases in adherens junctions and gap junctions. Dye coupling experiments indicated that the gap junctions were functional. The frequency of expression of both kinds of junctions was sharply decreased by treatment with anti-L-CAM Fab' fragments. These experiments provide support for the precedence hypothesis, which proposes that the linkage of cells by means of CAMs is a necessary event for the extensive expression of junctional structures.
Resistance to cobalt, zinc, and cadmium specified by the czc determinant on plasmid pMOL30 in Alcaligenes eutrophus results from a cation efflux system. Five membrane-bound polypeptides that were expressed in Escherichia coli from this determinant under the control of a phage T7 promoter were assigned to four open reading frames identified in the nucleotide sequence of the 6881-base-pair fragment containing the czc putative operon. The contributions of the polypeptides to the cation efflux system were analyzed with deletion derivatives of the 6.9-kilobase fragment, constructed, and expressed in E. coli under the control of the phage T7 promoter and in A. eutrophus under the control of the lac promoter.