RESUMO
AIMS: To develop an effective gene transfer system for Streptomyces ipomoeae, the causative agent of soil rot disease of sweet potatoes. METHODS AND RESULTS: Of the several methods investigated, introduction of genes into S. ipomoeae could only be achieved using intergeneric conjugation from Escherichia coli. However, even results for that method varied greatly and were dependent on using particular media for S. ipomoeae spore preparation and conjugation. Transconjugant to recipient ratios as great as 4.1 x 10(-5) were achieved when International Streptomyces Project Medium 4 was used for both sporulation and conjugation protocols. Both site-specifically integrating and autonomously replicating plasmids could be introduced and maintained in S. ipomoeae, and plasmids could be introduced with approximate equivalent frequencies from either methyl-proficient or methyl- deficient E. coli donors; the latter result indicates a likely absence of relevant methyl-specific restriction in S. ipomoeae. CONCLUSIONS: Efficient transfer of genes into S. ipomoeae was achieved here by using an optimized intergeneric mating procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: The described protocol will facilitate further genetic manipulation of this agriculturally important pathogen.
Assuntos
Conjugação Genética , Escherichia coli/genética , Transferência Genética Horizontal , Biologia Molecular/métodos , Streptomyces/genética , Meios de Cultura/química , Escherichia coli/fisiologia , Genes Bacterianos , Ipomoea batatas/microbiologia , Plasmídeos , Streptomyces/fisiologiaRESUMO
Intermycelial transfer of Streptomyces plasmid pIJ101 occurs prior to cellular differentiation and is mediated by plasmid functions that are also required for production of zones of growth-inhibited recipient cells (i.e., pocks) that develop around individual donors during mating on agar medium. Several other pIJ101 functions, including that of the kilB gene, whose unregulated expression on pIJ101 is lethal, are required for normal pock size and so have been postulated to mediate intramycelial spread of the plasmid throughout recipient cells. Using antibodies raised against a KilB fusion protein expressed in Escherichia coli, native KilB protein was detected throughout development of pIJ101-containing Streptomyces lividans cells, with the concentration of KilB increasing dramatically and reaching a maximum during the final stages (i.e., sporulation and secondary metabolism) of cellular differentiation. Insertion of the kilB gene of pIJ101 into the S. lividans chromosome in cells lacking the pIJ101 KorB protein, which normally represses kilB gene transcription, resulted in elevated but still temporally increasing amounts of KilB. The increased expression or accumulation of the KilB spread protein throughout cellular differentiation of S. lividans, which leads to maximum KilB concentrations during developmental stages that occur far later than when intermycelial transfer of pIJ101 is mediated, supports the existence of a subsequent intramycelial component to the pIJ101 spread function. The results also suggest that intramycelial spread of pIJ101 molecules within the recipient extends beyond intercompartmental movements within the substrate mycelia and includes undetermined steps within the spore-yielding aerial hyphae as well.
Assuntos
Proteínas de Bactérias/genética , Plasmídeos/genética , Streptomyces/genética , Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Streptomyces/citologiaRESUMO
Plasmid pIJ101 from Streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. The tra gene product (Tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces.
Assuntos
Proteínas de Bactérias/genética , Conjugação Genética , Plasmídeos/genética , Streptomyces/genética , Cromossomos Bacterianos , Regulação Bacteriana da Expressão Gênica , Mutagênese Insercional , Streptomyces/crescimento & desenvolvimentoRESUMO
Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.
Assuntos
Proteínas de Bactérias/genética , Conjugação Genética , DNA Bacteriano , Plasmídeos , Proteínas Repressoras/genética , Streptomyces/genética , Sequência de Bases , Genes Bacterianos , Dados de Sequência MolecularRESUMO
The tra gene of Streptomyces lividans plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid transfer and the interbacterial transfer of chromosomal genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutagenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the tra gene affected plasmid and chromosomal gene transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211-6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA transfer.
Assuntos
Proteínas de Bactérias/genética , Plasmídeos/genética , Streptomyces/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , Análise Mutacional de DNA , Técnicas de Transferência de Genes , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24. 2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.
Assuntos
Conjugação Genética , Plasmídeos , Streptomyces/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Replicação do DNA , Bases de Dados Factuais , Escherichia coli , Teste de Complementação Genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Streptomyces/classificaçãoRESUMO
ABSTRACT Streptomyces soil rot is a destructive disease of sweetpotato (Ipomoea batatas) that causes yield loss resulting from decay of the feeder root system and reduced quality due to the presence of necrotic lesions on the storage roots. It is managed by the use of resistant cultivars, but variability of the pathogen has not been previously assessed. This study compared 36 strains of the pathogen Streptomyces ipomoeae from different locations in the United States and Japan. The strains could be separated into three groups on the basis of their ability to inhibit the growth of one another in in vitro assays. Although some strains contained plasmids of approximately 18, 42, or 270 kb in size, plasmid profiles did not correspond to inhibition grouping. Fingerprinting by repetitive element-based polymerase chain reaction (rep-PCR) using outwardly facing primers for the BOX, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) sequences indicated relatively high genomic homogeneity within S. ipomoeae. However, cluster analysis of similarity coefficients among strains using rep-PCR data revealed clusters that correlated with the inhibition grouping. The neotype strain of S. ipomoeae had lower similarity values by rep-PCR than any of the other strains and could not be grouped by inhibitory interactions.
RESUMO
We previously have shown that a chromosomally integrated copy of the tra gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates transcription of tra as well as its own synthesis, can promote the conjugal transfer of both chromosomal and plasmid genes in Streptomyces lividans. Using an antibody generated against a fusion protein containing the C-terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface-grown S. lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found. Expression of Tra during the S. lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites. Comparison of the membrane concentration of Tra protein with tra mRNA concentration during the S. lividans life cycle indicated that the disappearance of Tra is post-transcriptionally controlled and thus is not mediated by KorA. The results of 'interrupted mating' experiments, together with the time of appearance of Tra in S. lividans membranes, indicate that the intermycelial transfer of pIJ101 in S. lividans is complete by the onset of cellular differentiation.
Assuntos
Proteínas de Bactérias/genética , Conjugação Genética , Proteínas de Escherichia coli , Proteínas de Membrana/genética , Plasmídeos , Streptomyces/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Processamento Pós-Transcricional do RNA , Streptomyces/metabolismoRESUMO
The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra+ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.
Assuntos
Proteínas de Bactérias/fisiologia , Cromossomos Bacterianos , Conjugação Genética , DNA Bacteriano/genética , Proteínas de Escherichia coli , Plasmídeos/genética , Streptococcus/genética , Proteínas de Bactérias/genética , Mutagênese Insercional , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Deleção de SequênciaRESUMO
CD68 antigen is expressed by tissue macrophages and cells of myeloid/mononuclear lineage. CD68 is recognized by many monoclonal antibodies (mAbs), including KP1, EMB11, Y1/82A, Y2/131, Ki-M6, and Ki-M7. Using the labeled strept-avidin-biotin (LSAB) immunoperoxidase technique, we examined CD68 antigen expression in human peripheral blood T cells. The anti-CD68 mAbs KP1 and EMB11 stained virtually all fresh isolated gamma/delta T cells and CD3-CD56+ natural killer (NK) cells in a granular cytoplasmic pattern. In contrast, fresh isolated CD4+ and CD8+ T lymphocytes showed no detectable reactivity with these two anti-CD68 mAbs. However, in vitro stimulation with T-cell mitogen or recombinant interleukin-2 (rIL-2) induced expression of CD68 antigen in activated CD4+ and CD8+ T lymphocytes. Similarly, the lymphokine-activated killer (LAK) cells generated after long-term (14 to 21 days) culture of peripheral blood mononuclear cells (PBMC) in rIL-2 also showed strong granular cytoplasmic staining by the anti-CD68 antibodies. This study shows that the CD68 antigen is constitutively expressed in NK cells and gamma/delta T cells and that its expression is strongly induced in activated CD4+ and CD8+ T lymphocytes as well as LAK cells.
Assuntos
Antígenos CD/sangue , Antígenos de Diferenciação Mielomonocítica/sangue , Linfócitos T/imunologia , Anticorpos Monoclonais , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
The fepA-entD and fes-entF operons in the enterobactin synthesis and transport system are divergently transcribed from overlapping promoters, and both are inhibited by the Fur repressor protein under iron-replete conditions. A plasmid harboring divergent fepA'-phoA and fes-entF'-'lacZ fusions, both under the control of this bidirectional regulatory region, was constructed for the purpose of monitoring changes in expression of the two operons simultaneously. Deletion analysis, site-directed mutagenesis, and primer extension were employed to define both a single promoter governing the expression of fes-entF and two tandemly arranged promoters giving rise to the opposing fepA-entD transcript. A single Fur-binding site that coordinately regulates the expression of all transcripts emanating from this control region was identified by in vitro protection from DNase I nicking. The substitution of one base pair in the Fur recognition sequence relieved Fur repression but did not change the in vitro affinity of Fur for its binding site. Additional mutations in a limited region outside of the promoter determinants for either transcript inhibited expression of both fes and fepA. These observations suggest a mechanism of Fur-mediated regulation in this compact control region that may involve other regulatory components.
Assuntos
Proteínas da Membrana Bacteriana Externa , Enterobactina/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ferro/farmacologia , Receptores de Superfície Celular , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Transporte Biológico , Hidrolases de Éster Carboxílico/genética , Proteínas de Transporte/genética , Análise Mutacional de DNA , Expressão Gênica , Genes Bacterianos/genética , Vetores Genéticos , Dados de Sequência Molecular , Regiões Operadoras Genéticas/genética , Óperon/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão , Proteínas Repressoras/metabolismo , Deleção de SequênciaRESUMO
SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage lambda integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf61) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.
Assuntos
DNA Nucleotidiltransferases/genética , Elementos de DNA Transponíveis/genética , Genes Bacterianos/genética , Plasmídeos/genética , Recombinação Genética/genética , Streptomyces/genética , Proteínas Virais , Sequência de Aminoácidos , Sequência de Bases , Integrases , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Recombinases , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
This study evaluated the tissue reactions to two materials: ceramic hydroxyapatite (CHA), and a composite material of demineralized bone powder (DBP) and CHA (ratio of 4:1) in a collagen vehicle. The materials were tested in a subcutaneous pocket, a mandibular onlay, and in a calvarial onlay model. Specimens were evaluated histologically at 7, 10, 14, and 21 days postimplantation. Ceramic hydroxyapatite, implanted subcutaneously, elicited a fibrous response with minimal inflammation, but did not induce bone formation. In specimens of subcutaneously implanted composite material, induced bone was evident in association with the DBP. In CHA onlay specimens, there was a small amount of reactive bone extending from the host bone into the implant. In composite onlays, bone filled the entire body of the implant. The results of this study indicate that CHA particles were not osteoinductive in heterotopic sites and that osteoconductive ingrowth was minimal in onlays. Bone was induced by DBP even when mixed with CHA particles and implanted in subcutaneous and intraosseous sites. It was concluded that composite implants may provide a means of combining the osteoinductive properties of DBP with the bulk and structural support of osteoconductive CHA particles.
Assuntos
Transplante Ósseo , Osso e Ossos/patologia , Cerâmica , Hidroxiapatitas , Próteses e Implantes , Pele/patologia , Processo Alveolar/patologia , Animais , Materiais Biocompatíveis , Reabsorção Óssea/patologia , Osso e Ossos/cirurgia , Cerâmica/química , Colágeno , Tecido Conjuntivo/patologia , Procedimentos Cirúrgicos Dermatológicos , Durapatita , Hidroxiapatitas/química , Masculino , Mandíbula , Osteoblastos/patologia , Osteogênese , Ratos , Crânio , CicatrizaçãoRESUMO
To investigate the etiologic role of Chlamydia trachomatis in cystitis, the authors used the immunoperoxidase technique with a monoclonal antibody against Chlamydia and examined paraffin sections from 36 cases of histologically proven cystitis. The average patients' age was 60 (range, 2-85) years. Biopsies were taken for follow-up of treated bladder carcinoma (19), hematuria (8), and other nonneoplastic conditions (9). Chlamydial antigens were detected by immunohistochemistry in 12 (33%) of these 36 cases. Staining for Chlamydia occurred in the upper layers of the transitional epithelium and involved long stretches of epithelium. Underlying inflammation was usually chronic but did not have specific distinguishing features. Eight of the Chlamydia-positive biopsies were taken for follow-up of treated carcinoma, two were for hematuria, one for neurogenic bladder, and one for evaluation of sterile pyuria. Eleven (92%) of these 12 positive cases had a history of recent urologic instrumentation, in contrast to only 11 (46%) of 24 negative cases (P less than 0.02). There was no significant difference in the age or sex distribution between the two groups. The authors conclude that Chlamydia trachomatis can ascend the urethra and infect the bladder urothelium. Urologic instrumentation enhances the ability of Chlamydia to reach the bladder. Chlamydia trachomatis may play an etiologic role in cystitis.
Assuntos
Antígenos de Bactérias/análise , Chlamydia trachomatis/imunologia , Cistite/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Criança , Pré-Escolar , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Coloração e RotulagemRESUMO
Immunofluorescence (IF) of skin biopsies for detection of Rickettsia rickettsii (RR) has proven useful as a rapid test for confirmation of Rocky Mountain spotted fever (RMSF). However, IF lacks sensitivity, requires special equipment and training, and is difficult to interpret. The authors have developed an indirect avidin-biotin immunoperoxidase (IP) technique to detect RR in fixed and frozen tissue sections. The technique was evaluated on fixed cutaneous specimens from patients dying of RMSF and compared to specimens from control patients dying of an acute febrile illness with skin manifestations and vasculitis. IP correctly identified RR in 9 of 12 cases with probable identification in 2 additional cases. Of 11 controls, 10 were negative and one was uninterpretable. RR was easily visualized within cytoplasm and nuclei of endothelial cells in association with perivascular lymphocytic infiltrates and less frequently with vasculitis or non-inflamed vasculature. IP is rapid, amplifies small quantities of antigen, gives excellent histologic detail as compared with IF, and is easily adapted for use in hospitals with immunoperoxidase capabilities.
Assuntos
Técnicas Imunoenzimáticas/instrumentação , Rickettsia rickettsii/isolamento & purificação , Febre Maculosa das Montanhas Rochosas/diagnóstico , Dermatopatias Infecciosas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos/análise , Criança , Pré-Escolar , Estudos de Avaliação como Assunto , Feminino , Técnicas Histológicas , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos , Rickettsia rickettsii/imunologia , Febre Maculosa das Montanhas Rochosas/microbiologia , Febre Maculosa das Montanhas Rochosas/patologia , Dermatopatias Infecciosas/microbiologia , Dermatopatias Infecciosas/patologia , Fatores de TempoRESUMO
The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a transmembrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA-entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5' to entD, along with RNA hybridization data, suggest that an iron-regulated transcript contains both fepA and entD.
Assuntos
Enterobactina/genética , Escherichia coli/genética , Genes Bacterianos , Serina/análogos & derivados , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Eletroforese em Gel de Poliacrilamida , Endopeptidase K , Enterobactina/análise , Immunoblotting , Proteínas de Membrana/análise , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Serina Endopeptidases/metabolismo , Esferoplastos/metabolismo , Transcrição GênicaRESUMO
The iron-controlled fepA and fes-entF transcripts from the Escherichia coli enterobactin gene complex are expressed divergently from a limited genetic region, thereby suggesting the existence of a single, possibly overlapping promoter junction for these genes. The nucleotide sequence of a 1,997-base pair HpaI fragment specific for this genetic region allowed for the identification of an 1,122-base pair open reading frame as the previously uncharacterized fes gene. Its product, Fes (approximately Mr 42,573) plays an essential but as yet ambiguous role in the release of ferric iron from the ligand. An additional small open reading frame of 216 nucleotides (encoding a potential product of calculated Mr 8,271) was also identified between fes and entF. A portion of the remaining nucleotide sequence defined a 320-base pair control region for both the fepA and fes-entF messages. Primer extension analyses placed the major in vivo transcription initiation sites to within 18 nucleotides of one another, thereby revealing a novel, extensively overlapping bidirectional promoter as well as long dual leader transcripts. This promoter region contains multiple overlapping nucleotide stretches which show strong homology to the consensus Fur repressor-binding sequence, forms of which are found in all E. coli iron-regulated promoters characterized to date.
Assuntos
Enterobactina/genética , Escherichia coli/genética , Regiões Promotoras Genéticas , Serina/análogos & derivados , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Dados de Sequência Molecular , Peso MolecularRESUMO
The Escherichia coli entF gene, which encodes the serine-activating enzyme involved in enterobactin synthesis, has been localized to a 4.7-kilobase-pair DNA fragment inserted in the vector pBR328. This recombinant molecule, pITS32, restored the ability of an entF mutant to grow on low-iron medium and to produce enterobactin. Examination of its translation products by minicell and electrophoretic analyses revealed a protein of approximately 160,000 daltons, which we identified as the EntF protein. A small DNA segment from pITS32 containing the translational start site for entF allowed the low constitutive expression of beta-galactosidase when cloned (pITS301) upstream of the lacZ structural gene in the vector pMC1403. In contrast, a clone (pITS312) containing the identical entF-lacZ fusion and a larger region upstream of entF including the entire fes gene and extending into the fepA gene (whose transcription is in the opposite direction relative to entF) expressed beta-galactosidase in high yet inducible amounts in response to fluctuations in the metabolic iron concentration. Transposon insertion mutations in the fes gene but not an insertion near the 5' region of fepA in pITS312 reduced this high inducible expression to the low constitutive level seen for pITS301. These observations are most readily explained by the presence of a regulatory region located upstream of fes which mediates the iron-regulated expression of a transcript that includes the fes and entF genes.