Upstream DNA Sequences Research Articles

Functional Analysis of DNA Sequences Required for Conidium-Specific Expression of the SpoC1-C1C Gene of Aspergillus nidulans

The SpoC1-C1C gene is centrally located within the A. nidulans conidium-specific SpoC1 gene cluster. With one exception, the 14 genes within the cluster are coordinately regulated. C1C transcript is first detected late in conidiation, coincidental with the appearance of mature conidia, and accumulates ∼1000-fold in conidia. We show that C1C expression is restricted to conidia, with mRNA abundance decreasing immediately after induction of germination. C1C transcription and translation are not temporally separated and, similar to C1C RNA abundance, a C1C::β-galactosidase fusion protein is first detected with the appearance of mature conidia and decreases after induction of germination. Cell-specific C1C expression requires both a position-dependent mechanism of regulation, responsible for repression in hyphae, and a position-independent mechanism of regulation, responsible for developmental expression. We show by functional analysis of upstream DNA sequences that a 10-bp sequence and two adjacent 6-bp direct repeats are necessary for position-independent, condium-specific expression of both the intact C1C gene and the reporter gene. At least one repeat (CAACAT) is required for normal levels of expression. We find that the C1C gene is not a direct target of the BrlAp and AbaAp developmental regulators, but of a yet unidentified conidium-specific transcriptional activator.

A GC-box is required for expression of the human vimentin gene.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. The promoter of the human vimentin gene (-1416 to +73) was shown to contain two positive-acting regions, separated by a negative region, and at least eight GC-boxes as determined by sequence homology (Rittling, S.R., Baserga, R., 1987. Mol. Cell. Biol. 7, 3908-3915). We have analyzed the region -900 to +41 for protein binding by in vivo footprinting experiments using ligation-mediated PCR. For the various GC-boxes, we detect protein binding only to that GC-box (at position -64 and -55) closest to the transcriptional start site. Transient transfection assays of various vimentin 5'-end fragments and mutations thereof fused to the reporter gene cat indicate that this sequence is indispensable for promoter function regardless of the inclusion of upstream DNA sequences. In vitro binding studies confirm that this region binds protein specifically. We suggest that this GC-box and its binding factor are required for regulated expression of the human vimentin gene.

Thyroid hormone inhibits transcription of the human tissue-type plasminogen activator gene promoter

Objective: to determine if triiodothyronine (T3) regulates the human tissue-type plasminogen activator (t-PA) gene through its upstream DNA sequences. Design: transient transfection analysis of t-PA reporter constructs and expression vectors for thyroid hormone receptor (TR) and retinoid X receptor (RXR) in HT 1080 fibrosarcoma and SK-N-SH neuroblastoma cells. Results: 10 −6 to 10 −9 M T3 was found to inhibit the expression of human t-PA promoter reporter constructs fivefold in transfected HT1080 fibroblast and SK-N-SH neuronal cells. Unliganded TR and RXR were shown to induce the basal level of the t-PA promoter twofold. Repression by T3 required the TR DNA binding domain. The region of the t-PA promoter mediating this repression was located within bp - 110-- 7 and comprised a sequence specifically interacting with TR homodimers. However, deletion of this sequence did not relieve T3 repression. Conclusion: T3 represses the human t-PA promoter activity in HT1080 and SK-N-SH cells. However, binding of TR homodimers to the minimal T3 responsive region was shown not to be involved in the T3 repression, suggesting the existence of an alternative T3 inhibitory mechanism.

Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation.

Whole-genome mRNA quantitation can be used to identify the genes that are most responsive to environmental or genotypic change. By searching for mutually similar DNA elements among the upstream non-coding DNA sequences of these genes, we can identify candidate regulatory motifs and corresponding candidate sets of coregulated genes. We have tested this strategy by applying it to three extensively studied regulatory systems in the yeast Saccharomyces cerevisiae: galactose response, heat shock, and mating type. Galactose-response data yielded the known binding site of Gal4, and six of nine genes known to be induced by galactose. Heat shock data yielded the cell-cycle activation motif, which is known to mediate cell-cycle dependent activation, and a set of genes coding for all four nucleosomal proteins. Mating type alpha and a data yielded all of the four relevant DNA motifs and most of the known a- and alpha-specific genes.

Functional characterization of the human thiopurine S-methyltransferase (TPMT) gene promoter.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S-methylation of aromatic and heterocyclic sulfhydryl compounds, including anticancer and immunosuppressive thiopurines. We recently isolated the human TPMT promoter, which does not contain TATA box or CCAAT element consensus sequences, but is GC rich with multiple GC boxes and other putative cis-regulatory elements. Here, we report the functional characterization of the TPMT promoter, revealing several positive regulatory elements and identifying stimulating protein 1 (Sp1) as an important trans-activator essential for constitutive activity in cell culture. One major and two closely located minor transcription start points were identified in HepG2 cells. Deletion analysis revealed positive cis-regulatory elements located in the regions -85 to -75, -68 to -58, -58 to -51 and +34 to +60 relative to the transcription start site. DNaseI footprinting analysis and cotransfection in Drosophila Schneider SL2 cells documented that Sp1 binds to the TPMT promoter and is important for constitutive activity. We conclude that constitutive transcription of the TPMT gene involves a limited upstream GC-rich DNA sequence, containing multiple GC boxes, and that transcription factor Sp1 [or related protein(s)] is an important trans-activator of this TATA-less promoter.

Molecular characterization of Mycoplasma arthritidis variable surface protein MAA2.

Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M. arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [3H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The maa2 genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The maa2 gene was expressed in Escherichia coli from the lacZ promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E. coli. The maa2 gene and upstream DNA sequences were cloned from M. arthritidis clonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative -10 box. Full-sized recombinant MAA2 was expressed in E. coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.

Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene.

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of approximately 100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5-10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

Metalloregulation in vitro of the aerobactin promoter of Escherichia coli by the Fur (ferric uptake regulation) protein.

The mechanism of transcriptional repression of the aerobactin operon of Escherichia coli by the Fe2+-responsive Fur (ferric uptake regulation) protein has been investigated. In the presence of a divalent metal, such as Mn2+, the Fur protein sequentially occupies two defined sites at the aerobactin promoter region, followed by a looser occupation of upstream DNA sequences. However, binding to the primary target site suffices for the entire repression effect. Comparison of transcription patterns generated with run-off experiments in the presence and absence of heparin showed that access of the RNA polymerase to the principal -35/-10 hexamers of the promoter region was fully prevented by Fur-Mn2+ bound to its primary site. Similarly, promoter-bound RNA polymerase could not be competed out from the DNA even in the presence of a large Fur-Mn2+ excess, although the repressor could immediately bind its target sequence at the region as soon as RNA polymerase moved away from the promoter during transcription. The high affinities of either protein for the promoter produce, in practice, a first-come, first-served effect that helps the system to respond instantly to changes in the iron status of the cells.

The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli.

We used partially purified NhaR and a highly purified His-tagged NhaR derivative to identify the cis-regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction. Gel retardation assay with DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR-binding motif. Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA. The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G-92, G-60, G-29 and A-24 form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a DeltanhaR strain; accordingly, these bases were protected in vitro by the purified His-tagged NhaR. 100 mM Na+, but not K+, removed the protection of G-60 conferred by His-tagged NhaR in vitro. Exposure of intact cells to 100 mM Na+, but not K+, exposed G-60. The maximal effect of Na+ in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5. In contrast to G-60, G-92 was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction. We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR-nhaA contact points.

Transcription of the Human Folylpoly-γ-glutamate Synthetase Gene

In mammals, folylpoly-gamma-glutamate synthetase (FPGS) activity is found in any cell undergoing sustained proliferative phases, but this enzyme also displays a tissue-specific pattern of expression in differentiated tissues. It is now reported that the steady state levels of FPGS mRNA in normal and neoplastic cells reflect these patterns, supporting the concept that the control mechanisms underlying this distribution are transcriptional. To initiate an understanding of these interacting levels of control, we have determined the position and properties of the minimal FPGS promoter controlling transcription of the FPGS gene in human CEM leukemia cells, a line which expresses high levels of this enzyme and its mRNA. The TATA-less region immediately upstream of the major transcriptional start site previously mapped in human tumor cells, which includes several GC- and Y-boxes, functioned as a remarkably efficient promoter when used to drive expression of a luciferase reporter in transient expression studies in CEM cells. The minimal region of the FPGS promoter required for maximal transcriptional activation in CEM cells included the 80 base pairs over which the multiple transcriptional start sites were located, and the 43 base pairs immediately upstream. DNase I footprint analysis detected the binding of Sp1 at all seven of the consensus sites within the probe used, two of which are contained within the minimal promoter region. The several Sp1 sites immediately upstream of the first major transcriptional start activated transcription in Drosophila cells when cotransfected with an Sp1 construct, including those in the region which functioned as a minimal promoter in CEM cells. An additional region of the minimal promoter, situated between the two translational start codons of the FPGS gene, was bound by protein(s) from HeLa cell nuclear extracts. We conclude that transcription of the FPGS gene in CEM cells involves transactivation events over a limited upstream DNA sequence and that the FPGS promoter used in proliferating human leukemic cells has strong similarity to other TATA-less promoters that utilize tandem, closely spaced Sp1 sites to initiate transcription.

Molecular characterization of the Serratia marcescens OmpF porin, and analysis of S. marcescens OmpF and OmpC osmoregulation

Serratia marcescens is a nosocomial pathogen with a high incidence of beta-lactam resistance. Reduced amounts of outer-membrane porins have been correlated with increased resistance to beta-lactams but only one porin, OmpC, has been characterized at the molecular level. In this study we present the molecular characterization of a second porin, OmpF, and an analysis of the expression of S. marcescens porins in response to various environmental changes. Two porins were isolated from the outer membrane using urea-SDS-PAGE and the relative amounts were shown to be influenced by the osmolarity of the medium and the presence of salicylate. From a S. marcescens genomic DNA library an 8 kb EcoRI fragment was isolated that hybridized with an oligonucleotide encoding the published N-terminal amino acid sequence of the S. marcescens 41 kDa porin. A 41 kDa protein was detected in the outer membrane of Escherichia coli NM522 carrying the cloned S. marcescens DNA. The cloned gene was sequenced and shown to code for a protein that shared 60-70% identity with other known OmpF and OmpC sequences. The upstream DNA sequence of the S. marcescens gene was similar to the corresponding E. coli ompF sequence; however, a regulatory element important in repression of E. coli ompF at high osmolarity was absent. The cloned S. marcescens OmpF in E. coli increased in expression in conditions of high osmolarity. The potential involvement of micF in the observed osmoregulation of S. marcescens porins is discussed.

Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism.

Escherichia coli hlyCABD operons encode the polypeptide component (HlyA) of an extracellular cytolytic toxin as well as proteins required for its acylation (HlyC) and sec-independent secretion (HlyBD). The E. coli protein RfaH is required for wild-type hemolysin expression at the level of hlyCABD transcript elongation (J. A. Leeds and R. A. Welch, J. Bacteriol. 178:1850-1857, 1996). RfaH is also required for the transcription of wild-type levels of mRNA from promoter-distal genes in the rfaQ-K, traY-Z, and rplK-rpoC gene clusters, supporting the role for RfaH in transcriptional elongation. All or portions of a common 39-bp sequence termed JUMPStart are present in the untranslated regions of RfaH-enhanced operons. In this study, we tested the model that the JUMPStart sequence and RfaH are part of the same functional pathway. We examined the effect of JUMPStart deletion mutations within the untranslated leader of a chromosomally derived hlyCABD operon on hly RNA and HlyA protein levels in either wild-type or rfaH null mutant E. coli. We also provide in vivo physical evidence that is consistent with RNA polymerase pausing at the wild-type JUMPStart sequences.

Defining a Functional Androgen Responsive Element in the 5′ Far Upstream Flanking Region of the Prostate-Specific Antigen Gene

Previously, an androgen responsive element (ARE or promoter ARE) was identified in the proximal promoter region of the prostate-specific antigen (PSA) gene. The proximal promoter fragment could mediate androgen induction of expression of a heterologous reporter gene in androgen receptor (AR)-less cells with exogenous AR in co-transfection assays. However, it exerted little androgen inducibility in androgen sensitive human prostate cells, LNCaP, which produce PSA mRNA and protein. In this study, we have identified a second functional ARE (or upstream ARE) approximately 4 kb upstream of the cap site of the PSA transcript. Interestingly, although the AREs are necessary for androgen induction, the DNA sequences surrounding the upstream ARE are also required for androgen induction by the PSA promoter in LNCaP cells. The results indicated that the upstream DNA sequences can cooperate with either ARE to mediate androgen induced gene expression in LNCaP cells.

The lipopolysaccharide-binding protein is a secretory class 1 acute-phase protein whose gene is transcriptionally activated by APRF/STAT/3 and other cytokine-inducible nuclear proteins.

Acute-phase reactants (APRs) are proteins synthesized in the liver following induction by interleukin-1 (IL-1), IL-6, and glucocorticoids, involving transcriptional gene activation. Lipopolysaccharide-binding protein (LBP) is a recently identified hepatic secretory protein potentially involved in the pathogenesis of sepsis, capable of binding the bacterial cell wall product endotoxin and directing it to its cellular receptor, CD14. In order to examine the transcriptional induction mechanisms by which the LBP gene is activated, we have investigated the regulation of expression of its mRNA in vitro and in vivo as well as the organization of 5' upstream regulatory DNA sequences. We show that induction of LBP expression is transcriptionally regulated and is dependent on stimulation with IL-1beta, IL-6, and dexamethasone. By definition, LBP thus has to be viewed as a class 1 acute-phase protein and represents the first APR identified which is capable of detecting pathogenic bacteria. Furthermore, cloning of the LBP promoter revealed the presence of regulatory elements, including the common APR promoter motif APRE/STAT-3 (acute-phase response element/signal transducer and activator of transcription 3). Luciferase reporter gene assays utilizing LBP promoter truncation and point mutation variants indicated that transcriptional activation of the LBP gene required a functional APRE/STAT-3 binding site downstream of the transcription start site, as well as an AP-1 and a C/EBP (CCAAT enhancer-binding protein) binding site. Gel retardation and supershift assays confirmed that upon cytokine stimulation APRF/STAT-3 binds to its recognition site, leading to strong activation of the LBP gene. Unraveling of the mechanism of transcriptional activation of the LBP gene, involving three known transcription factors, may contribute to our understanding of the acute-phase response and the pathophysiology of sepsis and septic shock.

Archaeal transcription factors and their role in transcription initiation.

Archaeal RNA polymerases show a weak ability in vitro to bind to promoter DNA and/or to initiate transcription with low activity independent of upstream regulatory DNA sequences. Active transcription in vitro and in vivo, however, depends strictly on a TATA box resembling the TATA box of eucaryal polII promoters. This TATA box is recognized by a polypeptide related to eucaryal TATA-binding protein (TBP) that was formerly designated aTFB. Template competition studies showed that this archaeal TATA-binding protein (aTBP) is stably sequestered at the promoter by interaction with the second archaeal transcription factor, aTFA, which is related to eucaryal transcription factor IIB (TFIIB). The association of archaeal TFIIB (aTFIIB) with the aTBP-promoter complex leads to template commitment, indicating that aTFIIB recruits archaeal RNA polymerase to the preinitiation complex. These analyses suggest the following order for assembly of transcription factors on the archaeal promoter: aTBP, aTFIIB, RNA polymerase, and provide evidence for a common molecular mechanism of transcription initiation by eucaryal RNA polymerase II and archaeal RNA polymerases. The sequence of the genes encoding aTBP and aTFIIB (TFB) showed all the characteristics conserved in their eucaryal counterparts. The degree of sequence similarity between archaeal and eucaryal transcription factors is between 27 to 35% for TFIIB and between 36 to 41% for TBP. The findings discussed here indicate that TBP and TFIIB perform analogous functions in Archaea and Eucarya and show that four essential components of archaeal and eucaryal transcriptional machineries. RNA polymerase, TATA box, TBP and TFIIB are homologous.

Regulation of Tissue-specific Expression of the Skeletal Muscle Ryanodine Receptor Gene

The ryanodine receptors (RYR) are a family of calcium release channels that are expressed in a variety of tissues. Three genes, i. e. ryr1, ryr2, and ryr3, have been identified coding for a skeletal muscle, cardiac muscle, and brain isoform, respectively. Although, the skeletal muscle isoform (RYR1) was shown to be expressed predominantly in skeletal muscle, expression was also detected in the esophagus and brain. To analyze the transcriptional regulation of the RYR1 gene, we have constructed chimeric genes composed of the upstream region of the RYR1 gene and the bacterial chloramphenicol acetyltransferase (CAT) gene and transiently transfected them into primary cultured porcine myoblasts, myotubes, and fibroblasts. A 443-base pair region upstream from the transcription start site was sufficient to direct CAT activity without tissue specificity. Deletion of a 61-base pair fragment from the 5'-end of the promoter resulted in a marked reduction of CAT activity in all three tissue types. A similar reduction of expression was observed when using a construct with the first intron in antisense orientation upstream from the promoter. In contrast, the first intron in sense orientation enhanced expression only in myotubes, while expression was repressed in fibroblasts and myoblasts. Gel retardation analyses showed DNA binding activity in nuclear extracts for two upstream DNA sequence elements. Our data suggest that (i) RYR1 gene expression is regulated by at least two novel transcription factors (designated RYREF-1 and RYREF-2), and (ii) tissue specificity results from a transcriptional repression in nonmuscle cells mediated by the first intron.

Genes and gene regulation of extracellular matrix proteins: an introduction.

Knowledge about the genes that encode extracellular matrix (ECM) proteins is expanding every day. Genes encoding long-known matrix proteins are being isolated while new genes for ECM proteins are being discovered using techniques that directly target the mRNA population. The regulation of expression of most genes occurs through the promoter domain upstream of the coding sequence, although this information may be supplemented by far upstream DNA sequences or DNA in the introns of the gene. Since this important information is embedded in the DNA, the study of gene structure is crucial to the analysis of gene regulation. With all of this new information, it will be possible to characterize common elements in genes that are coordinately regulated, and eventually target individual genes for control by exogenous agents such as drugs.

Characterization of late gene promoters of Chlamydia trachomatis.

Chlamydiae possess an intracellular developmental cycle defined by the orderly interconversion of infectious, metabolically inactive elementary bodies and noninfectious, dividing reticulate bodies. Only a few stage-specific genes have been cloned and sequenced, including the late-stage cysteine-rich protein operon and two late-stage genes encoding histone-like proteins. The aims of this study were to identify additional late-stage genes of Chlamydia trachomatis, analyze the upstream DNA sequence of late genes, and determine the sigma factor requirement of late genes. Stage-specific RNA, made by chlamydiae isolated from host cells, was used to probe C. trachomatis genomic libraries. Two new late genes, designated ltuA and ltuB, were identified, cloned, and sequenced. The predicted peptides encoded by ltuA and ltuB do not bear strong homology to known proteins, and the function of the new late genes is not known. The 5' ends of the transcripts of ltuA, ltuB, the cysteine-rich protein operon, and the two histone-like genes (hctA and hctB) were mapped, and a consensus -10 promoter region of TATAAT was derived from their upstream DNA sequences. In vitro transcription from templates encoding the promoter regions of ltuA, ltuB, and hctA cloned into the transcription assay vector pUC19-spf was found to be strongly stimulated by the addition of recombinant chlamydial sigma 66, while transcription from the putative hctB promoter region cloned in pUC19-spf was not detected in either the presence or absence of added sigma 66. These results suggest that the transcription of at least some chlamydial late-stage genes is dependent on sigma 66, which is homologous to the major sigma factors of other eubacteria.

Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

A homolog of the Rhizobium meliloti nitrogen fixation gene fixN is involved in the production of a microaerobically induced oxidase activity in the phytopathogenic bacterium Agrobacterium tumefaciens.

Hybridization analysis using the Rhizobium meliloti nitrogen fixation gene fixN as a probe revealed the presence of a homologous DNA region in the phytopathogenic bacterium Agrobacterium tumefaciens. Hybridization signals were also detected with total DNAs of Rhizobium leguminosarum bv. phaseoli, Rhodobacter capsulatus and Escherichia coli, but not those of Xanthomonas campestris pv. campestris and Pseudomonas putida. The hybridizing fragment from A. tumefaciens was cloned and sequenced. The predicted gene product of one of the two open reading frames identified on the sequenced fragment shows homology to FixN of different Rhizobiaceae as well as a low but significant similarity to subunit I of heme copper oxidases from various bacteria. The presence of five strictly conserved histidine residues previously implicated in forming ligands to heme and CuB in oxidases and the predicted membrane topology provide evidence that the A. tumefaciens fixN-like gene product is a component of the heme copper oxidase superfamily. The incomplete open reading frame starting only 8 nucleotides downstream of the fixN-like gene exhibits homology to Rhizobium fixO. Using an uidA (GUS) gene fusion it could be shown that the A. tumefaciens fixN-like gene is preferentially expressed under microaerobic conditions. Expression of the uidA fusion is abolished in R. meliloti fixJ and fixK mutants, indicating that an Fnr-like protein is involved in transcriptional regulation of the fixN-like gene in A. tumefaciens. The presence of an upstream DNA sequence motif identical to the Fnr-consensus binding site (anaerobox) further supports this hypothesis. A. tumefaciens mutated in the fixN-like gene shows decreased TMPD-specific oxidase activity under microaerobic conditions, indicating that the fixN-like gene or operon codes for proteins involved in respiration under reduced oxygen availability.