We have determined that the DNA sequence downstream of the well-characterized gonococcal fbp gene contains two open reading frames: one designated fbpB, which encodes a protein proposed to function as a cytoplasmic permease, and one designated fbpC, which encodes a protein proposed to function as a nucleotidebinding protein. The fpbABC operon composes an iron transport system that is homologous to the sfu and hit operons previously reported for Serratia marcescens and Haemophilus influenzae, respectively, and displays elements characteristic of ATP binding cassette transporters. The fpbABC operon differs from these loci in that it is lethal when overexpressed in Escherichia coli.

The ferric iron-binding protein (Fbp) expressed by pathogenic Neisseria spp. has been proposed to play a central role in the high-affinity acquisition of iron from human transferrin. The results of this investigation provide evidence that Fbp participates in this process as a functional analogue of a Gram-negative periplasmic-binding protein component, which operates as a part of a general active transport process for the receptor-mediated, high-affinity transport of iron from human transferrin. Known properties of Fbp are correlated with those of other well-characterized periplasmic-binding proteins, including structural features and the reversible binding of ligand. Predictive of a periplasmic-binding protein, which functions in the high-affinity acquisition of iron, is that Fbp is a transient participant in the process of iron acquisition from human transferrin. Evidence for this is demonstrated by results of pulse-chase experiments. Taken together, the data described here and elsewhere suggest that pathogenic Neisseria spp. use a periplasmic-binding protein-mediated active transport mechanism for the acquisition of iron from human transferrin.

This report describes the cloning and sequencing of the major iron-regulated protein (termed Fbp) of Neisseria gonorrhoeae strain F62. Attempts to identify recombinants expressing the Fbp using specific antibody proved unsuccessful. Therefore, an alternative cloning strategy using oligonucleotide probes derived from NH2-terminal and tryptic fragments of this protein was used to identify short fragments of the gene. Using this methodology, the gene encoding the precursor of Fbp was cloned on three separate overlapping fragments and sequenced, and the amino acid sequence was deduced. These data were unambiguously confirmed by the known NH2-terminal amino acid sequence and were supported by the sequences from tryptic fragments that lie outside of this region. Using oligonucleotide probes, we were unable to obtain clones encoding the potential regulatory region of this protein. Therefore, the technique of inverse polymerase chain reaction was used to amplify a fragment containing an additional 200 bp. This fragment was cloned and sequenced and found to contain a consensus ribosome binding site and potential -10 and -35 sequences. Hybridization analysis of genomic DNA from gonococcal strain F62 indicated that only a single copy of the Fbp gene exists per genome. These results complement the biochemical characterization of the Fbp expressed by gonococci and further suggest that it has a role in iron-acquisition.

The promoter region of the major iron-regulated protein ofNeisseria gonorrhoeae, Fbp, has two regions that exhibit homology with the Escherichia coli consensus Fur-binding sequences. Gel retardation assays suggested that purified E. coli Fur bound to two sites within the Fbp promoter. The presence of a gonococcal Fur homolog was suggested by Southern hybridization under conditions of low stringency, which revealed a DNA locus that exhibited homology to the E. colifur gene. Oligonucleotides derived from the conserved regions offur genes of * Corresponding author. protein (Fur) (2). Genetically diverse microorganisms appear to utilize a similar mechanism to control the expression of iron-regulated genes. Molecular homologs of Fur have been shown to exist in Yersinia pestis (42), Pseudomonas aeruginosa (37), Vibrio cholerae (22), and Vibrio vulnificus (23). Indirect evidence suggests that a similar Fur homolog exists in N. gonorrhoeae. The 37,000-Da major iron-regulated protein (Fbp) is expressed by all strains of N. gonorrhoeae when grown under iron-restricted conditions (29, 30). Little if any of this protein is present when gonococci are grown in iron-replete medium. Fbp mRNA is not detectable in gonococci grown in the presence of excess iron (33), suggesting that Fbp synthesis is regulated at the level of transcription. The 5' flanking sequence of the Fbp gene contains two regions which share considerable homology with the E. coli consensus Fur-binding iron boxes (3, 33). One of the potential iron boxes overlaps the Fbp promoter. From these data, we postulated the existence of a gonococcal Fur homolog. This report describes the identification, cloning, and sequence of the gonococcal Fur homolog. Our results show that E. coli Fur recognizes putative gonococcal Fur-binding sequences and that cloned gonococcal Fur complements two E. coli Furmutants. Additional data on the regulation of Fur in N. gonorrhoeae are presented. MATERIALS AND METHODS Bacterial strains, plasmids, and medium. N. gonorrhoeae F62 was obtained from R. P. Williams (Baylor College of Medicine, Houston, Tex.). E. coli 71.18 and TB1 were provided by K. Birkness (Centers for Disease Control and Prevention, Atlanta, Ga.). E. coli XL1 Blue was purchased from Stratagene, La Jolla, Calif., and E. coli H1681 was kindly provided by K. Hantke (Universitat Tubingen, Tubingen, Germany) (19). E. coli W3110 fur' (lacU169 Tna2) and W3110 fur (lacU169 Tna2 fir::TnS) (13) were provided by J. B. Neilands (University of California, Berkeley). pUC9, pUC19, and pBR322 were purchased from Bethesda Research Laboratories, Gaithersburg, Md. Plasmid pLAFR3

The ability to acquire iron from a human host is a major determinant in the pathogenesis of Neisseria gonorrhoeae and Neisseria meningitidis. Pathogenic Neisseria spp. do not synthesize siderophores and instead express a receptor-mediated, high-affinity iron acquisition system in the iron-restricted environment of its host. A ferric-iron-binding protein (Fbp) of Neisseria spp. is also iron-regulated and may play a central role in this novel iron-uptake system. To define the physical properties of Fbp further, we used polymerase chain reaction to synthesize DNA fragments containing the fbp structural gene with and without the sequence encoding the Fbp leader peptide. These fragments were ligated into pUC13 to create in-frame fusions with the alpha peptide of lacZ. The expression of Fbp was under the control of the lacZ promoter. Both fusion clones produced Fbp in large amounts, facilitating the purification of quantities of Fbp sufficient for elucidating the biochemical, immunologic, and functional properties of this protein.

Several clones containing alpha tropomyosin sequences were isolated from cDNA libraries prepared fram quail skeletal or smooth muscle RNA. All of these clones contain identical sequences coding for amino acids 81-257 of alpha skeletal muscle tropomyosin where they overlap, strongly indicating they are derived from the same gene. However, there are differences among these clones in sequences coding for the final 27 amino acids, as well as 3' untranslated sequences. In addition, S1 nuclease and Northern analyses indicate that coding sequences at the 5' end of the tropomyosin mRNA are also differentially expressed in smooth and skeletal muscle. These data point to the likelihood that differential splicing at both the 5' and 3' ends of a single tropomyosin gene results in the appearance of multiple tissue-specific transcripts. INTRODUCTICNContractile proteins are the primary components of muscle cells. Most of these proteins are expressed as members of multigene families and the expression of specific isoforms of each contractile protein in different muscles plays a major role in determining muscle fiber structure and function. Furthermore, during myogenesis, different developmental and muscle-specific isoforms of these genes are expressed. Thus formation of specific muscle types such as smooth, skeletal, and cardiac muscle is associated with the differential accumulation of specific contractile protein isoforms.Although many of the contractile protein genes have been isolated and characterized, very little is known about the tropanyosin gene family in vertebrates. Tropcmyosins are a closely related family of proteins which are bound to actin and troponin and serve to mediate the effect of calcium on muscle contraction (1,2,3). Skeletal muscle contains two tropomyosin subunits, alpha and beta, which are present in different molar ratios in individual muscle types, and at different times during development (4,5). Manmalian cardiac muscle apparently contains only an alpha type subunit (5), © I R L Press Limited, Oxford, England.

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