Na(+)/H(+) antiporters are membrane proteins that play a major role in pH and Na(+) homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants. The emerging genomic sequence projects already have started to reveal that the Na(+)/H(+) antiporters cluster in several families. Structure and function studies of a purified antiporter protein have as yet been conducted mainly with NhaA, the key Na(+)/H(+) antiporter of Escherichia coli. This antiporter has been overexpressed, purified and reconstituted in a functional form in proteoliposomes. It has recently been crystallized in both 3D as well as 2D crystals. The NhaA 2D crystals were analyzed by cryoelectron microscopy and a density map at 4 A resolution was obtained and a 3D map was reconstructed. NhaA is shown to exist in the 2D crystals as a dimer of monomers each composed of 12 transmembrane segments with an asymmetric helix packing. This is the first insight into the structure of a polytopic membrane protein. Many Na(+)/H(+) antiporters are characterized by very dramatic sensitivity to pH, a property that corroborates their role in pH homeostasis. The molecular mechanism underlying this pH sensitivity has been studied in NhaA. Amino acid residues involved in the pH response have been identified. Conformational changes transducing the pH change into a change in activity were found in loop VIII-IX and at the N-terminus by probing trypsin digestion or binding of a specific monoclonal antibody respectively. Regulation by pH of the eukaryotic Na(+)/H(+) antiporters involves an intricate signal transduction pathway (recently reviewed by Yun et al., Am. J. Physiol. 269 (1995) G1-G11). The transcription of NhaA has been shown to be regulated by a novel Na(+)-specific regulatory network. It is envisaged that interdisciplinary approaches combining structure, molecular and cell biology as well as genomics should be applied in the future to the study of this important group of transporters.

We sequenced the 2 botulinum toxin gene clusters of Clostridium botulinum strain IBCA10-7060 type Bh. The sequence of bont/H differed substantially from the sequences of the 7 known bont genes for toxin types A-G. The 5' one-third terminus of bont/H that codes for the botulinum toxin light chain differed markedly from the light chain coding sequences of toxin types A-G. The 3' two-thirds terminus of bont/H that codes for the botulinum toxin heavy chain contained a novel Hn translocation domain coding sequence and a nonneutralizing type A-like Hc binding domain coding sequence. bont/H was part of an orfX toxin gene cluster that was located at a unique chromosomal site distant from those used by other botulinum toxin gene clusters. The bont/B sequence was similar to that of subtype bont/B2 and was located within its ha toxin gene cluster at the oppA/brnQ site. Our findings further establish that C. botulinum IBCA10-7060 produces novel BoNT/H.

Escherichia coli responses to four inhibitors that interfere with translation were monitored at the transcriptional level. A DNA microarray method provided a comprehensive view of changes in mRNA levels after exposure to these agents. Real-time reverse transcriptase PCR analysis served to verify observations made with microarrays, and a chromosomal grpE::lux operon fusion was employed to specifically monitor the heat shock response. 4-Azaleucine, a competitive inhibitor of leucyl-tRNA synthetase, surprisingly triggered the heat shock response. Administration of mupirocin, an inhibitor of isoleucyl-tRNA synthetase activity, resulted in changes reminiscent of the stringent response. Treatment with kasugamycin and puromycin (targeting ribosomal subunit association as well as its peptidyl-transferase activity) caused accumulation of mRNAs from ribosomal protein operons. Abundant biosynthetic transcripts were often significantly diminished after treatment with any of these agents. Exposure of a relA strain to mupirocin resulted in accumulation of ribosomal protein operon transcripts. However, the relA strain's response to the other inhibitors was quite similar to that of the wild-type strain.

H-NS is a major component of bacterial chromatin and influences the expression of many genes. H-NS has beenshown to exhibit a binding preference for certain ATrich curved DNA elements in vitro. In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo. In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding. In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo. A K d of 1-2 M was estimated for binding of H-NS to each of these sequences. In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation. None of the synthetic curved sequences tested could functionally replace the DRE in vivo. These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo. Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter. Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription.

ᰔBotulinum neurotoxin, the most poisonous substance known and a potential biothreat agent (1), causes human and animal botulism worldwide (4,9). This toxin is encoded by the bont gene as part of a cluster that includes nontoxic accessory genes (10). Two main gene clusters are known: the hemagglutinincontaining (haϩ) cluster in toxin serotypes A1, B, C, D, and G and the orfX cluster in toxin serotypes A, E, and F (7, 10). In addition, some Clostridium botulinum type A strains (e.g., NCTC2916) contain a silent type B gene in their chromosome, designated A(B). The silent B toxin genes described to date contain the full length of the active type B gene cluster and one or more mutations that result in an unexpressed type B neurotoxin (3,8).Here we report the first infant botulism case resulting from a rare bont/A5 subtype, which we unexpectedly found to be part of a novel neurotoxin type A/B gene arrangement. Our bont/A5 gene sequence differs by 2 bp from the bont/A5 sequence previously identified in four wound botulism cases (2). The California infant strain, designated IBCA94-0216, was characterized by sequencing the toxin complex genes and their flanking regions (GenBank accession number FJ959094). Sequence analysis confirmed that the bont/A5 is located within a hemagglutinin complex (haϩ) that contains the genes ha70, ha17, ha33, botR, ntnh, and bont/A5 (Fig. 1). This type of toxin cluster is commonly associated with strains bont/A1 and bont/B but is not found in strains bont/A2, bont/A3, and bont/A4 (7, 10). The IBCA94-0216 neurotoxin gene cluster has an unexpected 76-bp deletion between the genes ha33 and botR. This is the first example of such a deletion.Downstream from bont/A5 are two copies of transposases belonging to the is3 family, a structure identical to the downstream flanking sequence of the type A1 Hall neurotoxin gene cluster ( Fig. 1) (10). Interestingly, 2,041 bp downstream from the bont/A5 gene and immediately following the second is3 is a partial bont/B gene that is only 65% of a full-length bont/B (6). The partial bont/B gene lacks the nucleotide sequence coding for the light chain of the toxin and begins at nucleotide 1362 of the 3,876-nucleotide full-length toxin gene. Comparisons of this region (1,362 to 3,876 nucleotides) revealed it is most similar to the rare bont/B3 subtype (5). The entire IBCA94-0216 bont/A/B cluster is flanked by a 5Ј flagellin gene and a 3Ј transposase, as is found in the silent type B gene cluster of strain NCTC2916 (7).Nucleic acid pairwise identities (using the Kimura two-parameter method in MEGA4 software) of the toxin cluster genes show that the IBCA94-0216 A5 neurotoxin and ntnh genes are most similar to the type A1 Hall neurotoxin and ntnh genes, respectively, while the hemagglutinin genes are most similar to the hemagglutinin genes of type B strains. This mosaic gene arrangement and the presence of the partial type B neurotoxin gene lead us to speculate that some form of genetic rearrangement occurred between type A and type B neurotoxin gene clusters in the c...

Sanger and shotgun sequencing of Clostridium botulinum strain Af84 type Af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. The three toxin gene clusters consisted of bont subtypes A2, F4 and F5. The bont/A2 and bont/F4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/F5 toxin gene cluster was located within a large 246 kb plasmid. These findings are the first identification of a C. botulinum strain that contains three botulinum neurotoxin gene clusters.

nhaA encodes an Na+/H+ antiporter in Escherichia coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na+. We used Northern (RNA) analysis to measure directly the cellular levels of nhaA mRNA. NhaR belongs to the LysR family of regulatory proteins. Consistent with our previous data with an nhaA'-'lacZ fusion, NhaR was found to be a positive regulator and Na+ was found to be a specific inducer of nhaA transcription. In the nhaA'-'lacZ fusion, maximal induction was observed at alkaline pH. In contrast, in the nhaA+ strain both the level of nhaA expression and the induction ratio were lower at alkaline pH. This difference may be due to the activity of NhaA in the wild-type strain as NhaA efficiently excreted Na+ at alkaline pH and reduced the intracellular concentration of Na+, the signal for induction. We also showed that although the global regulator rpoS was not involved in nhaA regulation, the global regulator hns played a role. Thus, the expression of nhaA'-'lacZ was derepressed in strains bearing hns mutations and transformation with a low-copy-number plasmid carrying hns repressed expression and restored Na+ induction. The derepression in hns strains was nhaR independent. Most interestingly, multicopy nhaR, which in an hns+ background acted only as an Na+-dependent positive regulator, acted as a repressor in an hns strain in the absence of Na+ but was activated in the presence of the ion. Hence, an interplay between nhaR and hns in the regulation of nhaA was suggested.

The Na ⍣ -specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na ⍣ /H ⍣ antiporter of Escherichia coli et al., 1994) Northern analysis of nhaA mRNA (Dover et al., 1996)