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The bacteriumHelicobacter pylorihas a large flagellar motor that generates significantly higher torque than the archetypicalEscherichia colimotor. To understand howH. pylorinavigates the viscous environment of the stomach, it is essential to establish how specific motor components contribute to efficient motility. We show here that the protein FlgV, required for motility inCampylobacter jejuni, forms a novel ring associated with the MS and C rings inH. pylori. Deletion offlgVfromH. pyloriB128 or a highly motile variant ofH. pyloriG27 (G27M) resulted in reduced motility in soft agar medium. Based on comparative analyses ofin-situflagellar motor structures ofH. pyloriwild-type and ΔflgVmutants, the reduced motility of the ΔflgVmutants and the location of the FlgV ring suggest it stabilizes interactions between the MS and C rings and/or plays a role in switching the direction of flagellar rotation. Overall, these results identify a novel motor accessory likely adapted to promote flagellar function for bacterial colonization of high-load environments such as the gastric mucosa.

Helicobacter pylori uses a cluster of polar, sheathed flagella for swimming motility. A search for homologs of H. pylori proteins that were conserved in Helicobacter species that possess flagellar sheaths but were underrepresented in Helicobacter species with unsheathed flagella identified several candidate proteins. Four of the identified proteins are predicted to form part of a tripartite efflux system that includes two transmembrane domains of an ABC transporter (HP1487 and HP1486), a periplasmic membrane fusion protein (HP1488), and a TolC-like outer membrane efflux protein (HP1489). Deleting hp1486/hp1487 and hp1489 homologs in H. pylori B128 resulted in reductions in motility and the number of flagella per cell. Cryo-electron tomography studies of intact motors of the Δhp1489 and Δhp1486/hp1487 mutants revealed many of the cells contained a potential flagellum disassembly product consisting of decorated L and P rings, which has been reported in other bacteria. Aberrant motors lacking specific components, including a cage-like structure that surrounds the motor, were also observed in the Δhp1489 mutant. These findings suggest a role for the H. pylori HP1486-HP1489 tripartite efflux system in flagellum stability. Three independent variants of the Δhp1486/hp1487 mutant with enhanced motility were isolated. All three motile variants had the same frameshift mutation in fliL, suggesting a role for FliL in flagellum disassembly.

The regulated uptake and consumption of d-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as Escherichia coli, which utilizes only l-Asp, Acinetobacter baylyi ADP1 can consume both d-Asp and l-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control d- and l-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use d-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of aspT, aspY, racD, and aspA (which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N(7)-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed l- and d-Asn and l-Glu, but not d-Glu, as the sole carbon or nitrogen source using interrelated pathways. IMPORTANCE A regulatory scheme was revealed in which AalR responds to l-Asp and DarR responds to d-Asp, a molecule with critical signaling functions in many organisms. The RacD-mediated interconversion of these isomers causes overlap in transcriptional control in A. baylyi. Our studies improve understanding of transport and regulation and lay the foundation for determining how regulators distinguish l- and d-enantiomers. These studies are relevant for biotechnology applications, and they highlight the importance of d-amino acids as natural bacterial growth substrates.

Helicobacter pylori uses a cluster of polar, sheathed flagella for motility, which it requires for colonization of the gastric epithelium in humans. As part of a study to identify factors that contribute to localization of the flagella to the cell pole, we disrupted a gene encoding a cardiolipin synthase (clsC) in H. pylori strains G27 and B128. Flagellum biosynthesis was abolished in the H. pylori G27 clsC mutant but not in the B128 clsC mutant. Transcriptome sequencing analysis showed that flagellar genes encoding proteins needed early in flagellum assembly were expressed at wild-type levels in the G27 clsC mutant. Examination of the G27 clsC mutant by cryo-electron tomography indicated the mutant assembled nascent flagella that contained the MS ring, C ring, flagellar protein export apparatus, and proximal rod. Motile variants of the G27 clsC mutant were isolated after allelic exchange mutagenesis using genomic DNA from the B128 clsC mutant as the donor. Genome resequencing of seven motile G27 clsC recipients revealed that each isolate contained the flgI (encodes the P-ring protein) allele from B128. Replacing the flgI allele in the G27 clsC mutant with the B128 flgI allele rescued flagellum biosynthesis. We postulate that H. pylori G27 FlgI fails to form the P ring when cardiolipin levels in the cell envelope are low, which blocks flagellum assembly at this point. In contrast, H. pylori B128 FlgI can form the P ring when cardiolipin levels are low and allows for the biosynthesis of mature flagella. IMPORTANCEH. pylori colonizes the epithelial layer of the human stomach, where it can cause a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. To colonize the stomach, H. pylori must penetrate the viscous mucous layer lining the stomach, which it accomplishes using its flagella. The significance of our research is identifying factors that affect the biosynthesis and assembly of the H. pylori flagellum, which will contribute to our understanding of motility in H. pylori, as well as other bacterial pathogens that use their flagella for host colonization.

Helicobacter pylori uses a cluster of polar, sheathed flagella for motility, which it requires for colonization of the gastric epithelium in humans. As part of a study to identify factors that contribute to localization of the flagella to the cell pole, we disrupted a gene encoding a cardiolipin synthase (clsC) in H. pylori strains G27 and B128. Flagellum biosynthesis was abolished in the H. pylori G27 clsC mutant but not in the B128 clsC mutant. Transcriptome sequencing analysis showed that flagellar genes encoding proteins needed early in flagellum assembly were expressed at wild-type levels in the G27 clsC mutant. Examination of the G27 clsC mutant by cryo-electron tomography indicated the mutant assembled nascent flagella that contained the MS ring, C ring, flagellar protein export apparatus, and proximal rod. Motile variants of the G27 clsC mutant were isolated after allelic exchange mutagenesis using genomic DNA from the B128 clsC mutant as the donor. Genome resequencing of seven motile G27 clsC recipients revealed that each isolate contained the flgI (encodes the P-ring protein) allele from B128. Replacing the flgI allele in the G27 clsC mutant with the B128 flgI allele rescued flagellum biosynthesis. We postulate that H. pylori G27 FlgI fails to form the P ring when cardiolipin levels in the cell envelope are low, which blocks flagellum assembly at this point. In contrast, H. pylori B128 FlgI can form the P ring when cardiolipin levels are low and allows for the biosynthesis of mature flagella. IMPORTANCE H. pylori colonizes the epithelial layer of the human stomach, where it can cause a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. To colonize the stomach, H. pylori must penetrate the viscous mucous layer lining the stomach, which it accomplishes using its flagella. The significance of our research is identifying factors that affect the biosynthesis and assembly of the H. pylori flagellum, which will contribute to our understanding of motility in H. pylori, as well as other bacterial pathogens that use their flagella for host colonization.

The helical shape of the human stomach pathogen Helicobacter pylori has been suggested to provide mechanical advantage for penetrating the viscous stomach mucus layer. Using single-cell tracking and quantitative morphology analysis, we document marked variation in cell body helical parameters and flagellum number among H. pylori strains leading to distinct and broad speed distributions in broth and viscous gastric mucin media. These distributions reflect both temporal variation in swimming speed and morphologic variation within the population. Isogenic mutants with straight-rod morphology showed 7-21% reduction in speed and a lower fraction of motile bacteria. Mutational perturbation of flagellum number revealed a 19% increase in speed with 4 versus 3 median flagellum number. Resistive force theory modeling incorporating variation of both cell shape and flagellum number predicts qualitative speed differences of 10-30% among strains. However, quantitative comparisons suggest resistive force theory underestimates the influence of cell body shape on speed for helical shaped bacteria.

Salmonella enterica serovar Typhimurium utilizes a wide range of growth substrates, some of which are relatively novel. One of these unusual substrates is d-glucosaminate, which is metabolized by the enzymes encoded in the dga operon. d-Glucosaminate is transported and converted to d-glucosaminate-6-phosphate (G6P) by a phosphotransferase system, composed of DgaABCD. The protein product of dgaE, d-glucosaminate-6-phosphate ammonia lyase (DGL), converts G6P to 2-keto-3-deoxygluconate-6-phosphate, which undergoes a retroaldol reaction catalyzed by the DgaF protein to give d-glyceraldehyde-3-phosphate and pyruvate. We have now developed an improved synthesis of G6P which gives a higher yield. The DGL reaction is of mechanistic interest because it is one of only a few enzymes in the pyridoxal-5'-phosphate (PLP) dependent aminotransferase superfamily known to catalyze reaction of a d-amino acid substrate. The pH dependence of DGL shows an optimum at 7.5-8.5, suggesting a requirement for a catalytic base. α-Glycerophosphate and inorganic phosphate are weak competitive inhibitors, with Ki values near 30mM, and d-serine is neither a substrate nor an inhibitor. We have found in rapid-scanning stopped-flow experiments that DGL reacts rapidly with its substrate to form a quinonoid intermediate with λmax=480nm, within the dead time (ca. 2msec), which then rapidly decays (k=279s-1) to an intermediate with absorption between 330 and 350nm, probably an aminoacrylate complex. We suggest a mechanism for DGL and propose that the unusual stereochemistry of the DGL reaction requires a catalytic base poised on the opposite face of the PLP-substrate complex from the other members of the aminotransferase superfamily.

STM2360 is a gene located in a small operon of undetermined function in Salmonella enterica serovar Typhimurium LT2. The amino acid sequence of STM2360 shows significant similarity (∼30% identity) to diaminopimelate decarboxylase (DapDC), a Fold III pyridoxal-5′-phosphate (PLP) dependent enzyme involved in l -lysine biosynthesis. We have found that the protein coded by STM2360 has a previously undocumented catalytic activity, d -ornithine/ d -lysine decarboxylase (DOKDC). The reaction products, cadaverine and putrescine, respectively, were identified by NMR and mass spectrometry. The substrate specificity of DOKDC is d -Lysine > d -Ornithine. This is the first pyridoxal-5′-phosphate dependent decarboxylase identified to act on d -amino acids. STM2358, located in the same operon, has ornithine racemase activity. This suggests that the physiological substrate of the decarboxylase and the operon is ornithine. Homologs of STM2360 with high sequence identity (>80%) are found in other common enterobacteria, including species of Klebsiella, Citrobacter, Vibrio and Hafnia, as well as Clostridium in the Firmicutes, and Pseudomonas.

The serine protease HtrA of C. jejuni has been identified as a novel secreted virulence factor which opens cell-to-cell junctions by cleaving E-cadherin. Efficient C. jejuni transmigration across polarized human epithelial cells requires the intact flagellum and HtrA; however, the mechanism of HtrA secretion into the supernatant is unknown. Here we show that HtrA secretion is highly efficient and does not require its proteolytic activity because the protease-inactive S197A mutant is secreted like wild-type HtrA. In addition, the flagellar mutants ΔflaA/B, ΔfliI, ΔflgH, ΔflhA, ΔflhB, and ΔflgS were also able to secrete HtrA in high amounts, while they were strongly attenuated in secreting the well-known invasion antigen CiaB. We also tested several culture media and cell lines of different origin such as human, mouse, hamster, dog, and chicken for their ability to influence HtrA secretion. Interestingly, HtrA was effectively secreted in the presence of most but not all cell lines and media, albeit at different levels, but secretion was significantly higher when fetal calf serum (FCS) was added. These results demonstrate that HtrA secretion by Campylobacter proceeds independent of HtrA’s protease activity, the flagellum and origin of cell lines, but can be strongly enhanced by molecular compound(s) present in FCS.

Capsule depolymerase (CapD) is aγ-glutamyl transpeptidase and a product of theBacillus anthraciscapsule biosynthesis operon. In this study, we examined the effect of modulatingcapDexpression onB. anthraciscapsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions ofcapDwere made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and tocapA,aB. anthraciscapsule biosynthesis gene. Translation signals were altered to improve expression ofcapD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-typeB. anthracisAmes but was detected in strains engineered with a consensus ribosome-binding site forcapD. Strains overexpressingcapDat amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression ofcapDincreased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest thatB. anthracismay have evolved weakcapDexpression resulting in optimized capsule-mediated virulence.

The flagellum, a rotary engine required for motility in many bacteria, plays key roles in gene expression. It has been known for some time that flagellar substructures serve as checkpoints that coordinate flagellar gene expression with assembly. Less well understood, however, are other more global effects on gene expression. For instance, the flagellum acts as a 'wetness' sensor in Salmonella typhimurium, and as a mechanosensor in other bacteria. Additionally, it has been implicated in a variety of bacterial processes, including biofilm formation, pathogenesis and symbiosis. Although for many of these processes it might be simply that motility is required, in other cases it seems that the flagellum plays an underappreciated role in regulating gene expression.

The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes ) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the “motor” and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae .

Regulation of theHelicobacter pyloriflagellar gene cascade involves the transcription factorsσ54(RpoN), employed for expression of genes required midway through flagellar assembly, andσ28(FliA), required for expression of late genes. Previous studies revealed that mutations in genes encoding components of the flagellar protein export apparatus block expression of theH. pyloriRpoN and FliA regulons. FlhB is a membrane-bound component of the export apparatus that possesses a large cytoplasmic domain (FlhBC). The hook length control protein FliK interacts with FlhBCto modulate the substrate specificity of the export apparatus. FlhBCundergoes autocleavage as part of the switch in substrate specificity. Consistent with previous reports, deletion offlhBinH. pyloriinterfered with expression of RpoN-dependent reporter genes, while deletion offliKstimulated expression of these reporter genes. In the ΔflhBmutant, disruptingfliKdid not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the ΔflhBmutation is not due to the inability to export FliK. Amino acid substitutions (N265A and P266G) at the putative autocleavage site ofH. pyloriFlhB prevented processing of FlhB and export of filament-type substrates. The FlhB variants supported wild-type expression of RpoN- and FliA-dependent reporter genes. In the strain producing FlhBN265A, expression of RpoN- and FliA-dependent reporter genes was inhibited whenfliKwas disrupted. In contrast, expression of these reporter genes was unaffected or slightly stimulated whenfliKwas disrupted in the strain producing FlhBP266G.H. pyloriHP1575 (FlhX) shares homology with the C-terminal portion of FlhBC(FlhBCC) and can substitute for FlhBCCin flagellar assembly. DisruptingflhXinhibited expression of aflaBreporter gene in the wild-type but not in the ΔfliKmutant or strains producing FlhB variants, suggesting a role for FlhX or FlhBCCin normal expression of the RpoN regulon. Taken together, these data indicate that the mechanism by which the flagellar protein export apparatus exerts control over theH. pyloriRpoN regulon is complex and involves more than simply switching substrate specificity of the flagellar protein export apparatus.

Co-ordinated regulation of gene expression is required for the transmission and survival of Borrelia burgdorferi in different hosts. The sigma factor RpoS (sigma(S)), as regulated by RpoN (sigma(54)), has been shown to regulate key virulence factors (e.g. OspC) required for these processes. As important, multiple signals (e.g. temperature, pH, cell density, oxygen) have been shown to increase the expression of sigma(S)-dependent genes; however, little is known about the signal transduction mechanisms that modulate the expression of rpoS. In this report we show that: (i) rpoS has a sigma(54)-dependent promoter that requires Rrp2 to activate transcription; (ii) Rrp2Delta123, a constitutively active form of Rrp2, activated sigma(54)-dependent transcription of rpoS/P-lacZ reporter constructs in Escherichia coli; (iii) quantitative reverse transcription polymerase chain reaction (QRT-PCR) experiments with reporter cat constructs in B. burgdorferi indicated that Rrp2 activated transcription of rpoS in an enhancer-independent fashion; and finally, (iv) rpoN is required for cell density- and temperature-dependent expression of rpoS in B. burgdorferi, but histidine kinase Hk2, encoded by the gene immediately upstream of rrp2, is not essential. Based on these findings, a model for regulation of rpoS has been proposed which provides mechanisms for multiple signalling pathways to modulate the expression of the sigma(S) regulon in B. burgdorferi.

Helicobacter pylori extracellular proteins are of interest because of possible roles in pathogenesis, host recognition, and vaccine development. We utilized a unique approach by growing two strains (including one nonsequenced strain) in a defined serum-free medium and directly analyzing the proteins present in the culture supernatants by LC-MS/MS. Over 125 proteins were identified in the extracellular proteomes of two H. pylori strains. Forty-five of these proteins were enriched in the extracellular fraction when compared to soluble cell-associated protein samples. Our analysis confirmed and expanded on the previously reported H. pylori extracellular proteome. Extracellular proteins of interest identified here included cag pathogenicity island protein Cag24 (CagD); proteases HP0657 and HP1012; a polysaccharide deacetylase, HP0310, possibly involved in the hydrolysis of acetyl groups from host N-acetylglucosamine residues or from residues on the cell surface; and HP0953, an uncharacterized protein that appears to be restricted to Helicobacter species that colonize the gastric mucosa. In addition, our analysis found eight previously unidentified outer membrane proteins and two lipoproteins that could be important cell surface proteins.

Protective antigen is essential for the pathology of Bacillus anthracis and is the proposed immunogen for an improved human anthrax vaccine. Known since discovery to comprise differentially charged isoforms, the cause of heterogeneity has eluded specific structural definition until now. Recombinant protective antigen (rPA) contains similar isoforms that appear early in fermentation and are mostly removed through purification. By liquid chromatography-tandem mass spectrometry sequencing of the entire protein and inspection of spectral data for amino acid modifications, pharmaceutical rPA contained measurable deamidation at seven of its 68 asparagine residues. A direct association between isoform complexity and percent deamidation was observed such that each decreased with purity and increased with protein aging. Position N537 consistently showed the highest level of modification, although its predicted rate of deamidation ranked 10th by theoretical calculation, and other asparagines of higher predicted rates were observed to be unmodified. rPA with more isoforms and greater deamidation displayed lower activities for furin cleavage, heptamerization, and holotoxin formation. Lethal factor-mediated macrophage toxicity correlated inversely with deamidation at residues N466 and N408. The described method measures deamidation without employing theoretical isotopic distributions, comparison between differentially treated samples or computational predictions of reactivity rates, and is broadly applicable to the characterization of other deamidated proteins. Proteins 2007. © 2007 Wiley-Liss, Inc.

Background Francisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms. Results We initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response. Conclusions Direct interactions with specific host proteins and the ability to influence interactions among host proteins are important components for F. tularensis to avoid host-cell defense mechanisms and successfully establish an infection. Although direct host-pathogen protein-protein binding is only one aspect of Francisella virulence, it is a critical component in directly manipulating and interfering with cellular processes in the host cell. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2351-1) contains supplementary material, which is available to authorized users.

The helical shape of the human stomach pathogen Helicobacter pylori has been suggested to provide mechanical advantage for penetrating the viscous stomach mucus layer. Using single-cell tracking and quantitative morphology analysis, we document marked variation in cell body helical parameters and flagellum number among H. pylori strains leading to distinct and broad speed distributions in broth and viscous gastric mucin media. These distributions reflect both temporal variation in swimming speed and morphologic variation within the population. Isogenic mutants with straight-rod morphology showed 7-21% reduction in speed and a lower fraction of motile bacteria. Mutational perturbation of flagellum number revealed a 19% increase in speed with 4 versus 3 median flagellum number. Resistive force theory modeling incorporating variation of both cell shape and flagellum number predicts qualitative speed differences of 10-30% among strains. However, quantitative comparisons suggest resistive force theory underestimates the influence of cell body shape on speed for helical shaped bacteria.

Salmonella enteric serovar Typhimurium, a major cause of food-borne illness, is capable of using a variety of carbon and nitrogen sources. Fructoselysine and glucoselysine are Maillard reaction products formed by the reaction of glucose or fructose, respectively, with the ε-amine group of lysine. We report here that S . Typhimurium utilizes fructoselysine and glucoselysine as carbon and nitrogen sources via a mannose family phosphotransferase (PTS) encoded by gfrABCD (glucoselysine/fructoselysine PTS components EIIA, EIIB, EIIC, and EIID; locus numbers STM14_5449 to STM14_5454 in S . Typhimurium 14028s). Genes coding for two predicted deglycases within the gfr operon, gfrE and gfrF , were required for growth with glucoselysine and fructoselysine, respectively. GfrF demonstrated fructoselysine-6-phosphate deglycase activity in a coupled enzyme assay. The biochemical and genetic analyses were consistent with a pathway in which fructoselysine and glucoselysine are phosphorylated at the C-6 position of the sugar by the GfrABCD PTS as they are transported across the membrane. The resulting fructoselysine-6-phosphate and glucoselysine-6-phosphate subsequently are cleaved by GfrF and GfrE to form lysine and glucose-6-phosphate or fructose-6-phosphate. Interestingly, although S . Typhimurium can use lysine derived from fructoselysine or glucoselysine as a sole nitrogen source, it cannot use exogenous lysine as a nitrogen source to support growth. Expression of gfrABCDEF was dependent on the alternative sigma factor RpoN (σ 54 ) and an RpoN-dependent LevR-like activator, which we designated GfrR. IMPORTANCE Salmonella physiology has been studied intensively, but there is much we do not know regarding the repertoire of nutrients these bacteria are able to use for growth. This study shows that a previously uncharacterized PTS and associated enzymes function together to transport and catabolize fructoselysine and glucoselysine. Knowledge of the range of nutrients that Salmonella utilizes is important, as it could lead to the development of new strategies for reducing the load of Salmonella in food animals, thereby mitigating its entry into the human food supply.

Flagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. In Helicobacter pylori , transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes, flaB and flgE . Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhA NT ). The K D (equilibrium dissociation constant) was 21 nM and was characterized by fast-on ( k on = 2.9 × 10 4 M −1 s −1 ) and slow-off ( k off = 6.2 × 10 −4 s −1 ) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhA NT sequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhA NT . FlhA NT binding did not stimulate FlgS autophosphorylation in vitro , suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation. IMPORTANCE The high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.

Several Helicobacter pylori flagellar genes require sigma(54) for their transcription. Predicted H. pylori sigma(54)-dependent promoters display a preference for A at position -23 instead of C or T as occurs in promoters from most other bacteria. Substitution of the A at position -23 of the H. pylori flaB promoter with a C did not effect expression of a flaB'-'xylE reporter gene in H. pylori, whereas T or G substitutions at this position drastically reduced expression. Results of gel mobility shift assays that used DNA probes corresponding to core promoter sequences and a H. pylori sigma(54) protein fused to the Escherichia coli maltose-binding protein suggested that H. pylori sigma(54) has a higher affinity for promoters with an A at the -23 position. The failure to observe an effect on expression for the flaB mutant promoter with the A to C substitution at the -23 position indicates that sequences flanking the core promoter region may assist binding of H. pylori sigma(54) to the mutant flaB promoter. Alternatively, H. pylori RNA polymerase or the sigma(54)-dependent activator FlgR may compensate for the reduced affinity of sigma(54) for the mutant flaB promoter.

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyltransferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90–100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine. Published by Elsevier Ltd.

Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhalational anthrax because they initiate germination and dissemination of spores. Found in high frequency throughout the respiratory track, dendritic cells (DCs) routinely take up foreign particles and migrate to lymph nodes. However, the participation of DCs in phagocytosis and dissemination of spores has not been investigated previously. We found that human DCs readily engulfed fully pathogenic Ames and attenuated B. anthracis spores predominately by coiling phagocytosis. Spores provoked a loss of tissue-retaining chemokine receptors (CCR2, CCR5) with a concurrent increase in lymph node homing receptors (CCR7, CD11c) on the membrane of DCs. After spore infection, immature DCs displayed a mature phenotype (CD83bright, HLA-DRbright, CD80bright, CD86bright, CD40bright) and enhanced costimulatory activity. Surprisingly, spores activated the MAPK cascade (ERK, p38) within 30 min and stimulated expression of several inflammatory response genes by 2 h. MAPK signaling was extinguished by 6 h infection, and there was a dramatic reduction of secreted TNF-α, IL-6, and IL-8 in the absence of DC death. This corresponded temporally with enzymatic cleavage of proximal MAPK signaling proteins (MEK-1, MEK-3, and MAP kinase kinase-4) and may indicate activity of anthrax lethal toxin. Taken together, these results suggest that B. anthracis may exploit DCs to facilitate infection.

Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA(63)), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA(63) pore (approximately 12 angstroms) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA(63). In this study, we found that purified (PA(63))(7)-LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA(63) heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogen-activated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA(63) pore. Complexes of (PA(63))(7)-LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.

Sinorhizobium meliloti DctD is an activator of sigma(54)-RNA polymerase holoenzyme and member of the AAA+ superfamily of ATPases. DctD uses energy released from ATP hydrolysis to stimulate the isomerization of a closed promoter complex to an open complex. DctD binds to upstream activation sequences (UAS) and contacts the closed complex through DNA looping to activate transcription, but the UAS is not essential for activation if DctD is expressed at higher than normal levels. Introduction of specific substitutions within or near the conserved ESELFG motif in the C3 region of a truncated, constitutively active form of DctD produced several mutant forms of the protein that had increased dependence on the UAS for activation. Removing the DNA-binding domain from one UAS-dependent mutant and from one activation-deficient mutant significantly increased transcriptional activation, indicating that the DNA-binding domain interfered with the activities of these mutant proteins. A UAS-dependent mutant with a P315L substitution in the C6 region was identified from a genetic screen. Alanine scanning mutagenesis of conserved amino acid residues around Pro-315 produced two additional UAS-dependent mutants as well as several mutants that failed to activate transcription but retained ATPase activity. In contrast to the two mutant proteins with substitutions in the C3 region, removal of the DNA-binding domain from the mutant proteins with substitutions in the C6 region did not stimulate their activity. The residues in the C6 region that were altered are in a probable hinge region between the alpha/beta and alpha-helical subdomains of the AAA+ domain. The alpha-helical subdomain contains the sensor II helix that has been implicated in other AAA+ proteins as sensing changes in the nucleotide during the hydrolysis cycle. Substitutions in the hinge region may have abolished nucleotide sensing by interfering with subdomain interactions, altering the relative orientation of the sensor II helix or interfering with oligomerization of the protein.

Helicobacter pyloriFlgR activates transcription with σ54-RNA polymerase holoenzyme (σ54-holoenzyme) from at least five flagellar operons. Activators of σ54-holoenzyme generally bind enhancer sequences located >70 bp upstream of the promoter and contact σ54-holoenzyme bound at the promoter through DNA looping to activate transcription.H. pyloriFlgR lacks the carboxy-terminal DNA-binding domain present in most σ54-dependent activators. As little as 42 bp of DNA upstream of theflaBpromoter and 26 bp of DNA sequence downstream of the transcriptional start site were sufficient for efficient FlgR-mediated expression from aflaB′-′xylEreporter gene inH. pylori, indicating that FlgR does not use an enhancer to activate transcription. Other examples of σ54-dependent activators that lack a DNA-binding domain includeChlamydia trachomatisCtcC and activators from the otherChlamydiaspp. whose genomes have been sequenced. FlgR fromHelicobacter hepaticusandCampylobacter jejuni, which are closely related toH. pylori, appear to have carboxy-terminal DNA-binding domains, suggesting that the loss of the DNA-binding domain fromH. pyloriFlgR occurred after the divergence of these bacterial species. Removal of the amino-terminal regulatory domain of FlgR resulted in a constitutively active form of the protein that activated transcription from σ54-dependent genes inEscherichia coli. The truncated FlgR protein also activated transcription withE. coliσ54-holoenzyme in an in vitro transcription assay.

After repeated passages through embyronated eggs, the Nine Mile strain ofCoxiella burnetiiexhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol.132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G+C contents were found within the deletions.

BenM is a LysR-type bacterial transcriptional regulator that controls aromatic compound degradation in Acinetobacter sp. ADP1. Here, in vitro transcription assays demonstrated that two metabolites of aromatic compound catabolism, benzoate and cis,cis -muconate, act synergistically to activate gene expression. The level of BenM-regulated benA transcription was significantly higher in response to both compounds than the combined levels due to each alone. These compounds also were more effective together than they were individually in altering the DNase I footprint patterns of BenM-DNA complexes. This type of dual-inducer synergy provides great potential for rapid and large modulations of gene expression and may represent an important, and possibly widespread, feature of transcriptional control.

Flagellar biogenesis in Helicobacter pylori is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ 80 ), RpoN (σ 54 ), and FliA (σ 28 ), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent ( flaB and flgE ) and FliA-dependent ( flaA ) genes. The MS ring (encoded by fliF ) is one of the earliest flagellar structures assembled. Deletion of fliF resulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease in flaA transcript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions of fliH and genes encoding C ring components ( fliM and fliY ) decreased transcript levels of flaB and flgE but had little or no effect on transcript levels of flaA . Transcript levels of flaB and flgE were elevated in mutants where genes encoding rod proteins ( fliE and flgBC ) were deleted, while transcript levels of flaA was reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure. IMPORTANCE The mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of the H. pylori flagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of the H. pylori RpoN regulon, a hypothesis that can be tested by examining protein-protein interactions in vitro .

Flagellar biogenesis in Helicobacter pylori involves the coordinated expression of flagellar genes with assembly of the flagellum. The H. pylori flagellar genes are organized into three regulons based on the sigma factor needed for their transcription (RpoD [σ 80 ], RpoN [σ 54 ], or FliA [σ 28 ]). Transcription of RpoN-dependent genes is activated by a two-component system consisting of the sensor kinase FlgS and the response regulator FlgR. While the cellular cues sensed by the FlgS/FlgR two-component system remain to be elucidated, previous studies revealed that disrupting certain components of the flagellar export apparatus inhibited transcription of the RpoN regulon. FliO is the least conserved of the membrane-bound components of the export apparatus and has not been annotated for any of the H. pylori genomes sequenced to date. A PSI-BLAST analysis identified a potential H. pylori FliO protein which membrane topology algorithms predict to possess a large N-terminal periplasmic domain that is absent from FliO of Escherichia coli and Salmonella , the paradigms for flagellar structure/function studies. FliO was necessary for flagellar biogenesis as well as wild-type levels of motility and transcription of RpoN-dependent and FliA-dependent flagellar genes in H. pylori strain B128. FliO also appears to be required for wild-type levels of the export apparatus protein FlhA in the membrane. Interestingly, the periplasmic and cytoplasmic domains were somewhat dispensable for flagellar gene regulation and assembly, suggesting that these domains have relatively minor roles in flagellar synthesis.

Transcription by ς 54 -RNA polymerase holoenzyme requires an activator that catalyzes isomerization of the closed promoter complex to an open complex. We examined mutant forms of Salmonella enterica serovar Typhimurium ς 54 that were defective in transcription initiation but retained core RNA polymerase- and promoter-binding activities. Four of the mutant proteins allowed activator-independent transcription from a heteroduplex DNA template. One of these mutant proteins, L124P V148A, had substitutions in a sequence that had not been shown previously to participate in the prevention of activator-independent transcription. The remaining mutants did not allow efficient activator-independent transcription from the heteroduplex DNA template and had substitutions within a conserved 20-amino-acid segment (Leu-179 to Leu-199), suggesting a role for this sequence in transcription initiation.

Transcription initiation with ς 54 -RNA polymerase holoenzyme (ς 54 -holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. ς 54 ’s binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation. Following random mutagenesis, we isolated eight mutant forms of Salmonella typhimurium ς 54 that were deficient in transcription initiation but still directed ς 54 -holoenzyme to the promoter to form a closed complex. Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for ς 54 -holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of ς 54 at some point after closed-complex formation. These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.

The phosphate-binding loop of ς 54 -dependent activators is thought to participate in ATP binding and/or hydrolysis. Alanine substitutions at positions 3, 4, 6, 7, and 8 of this motif in Rhizobium meliloti DctD disrupted transcriptional activation and ATP hydrolysis. Interestingly, substitution of alanine at position 7 also affected DNA binding.

Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between sigma54-RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with sigma54-holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for sigma54-holoenzyme. Mutagenesis was confined to the well-conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in sigma54-dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C-terminal half of the C3 region were efficiently cross-linked to sigma54 and the beta-subunit of RNA polymerase, suggesting that they bound normally to sigma54-holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N-terminal half of the C3 region had reduced affinities for sigma54 and the beta-subunit in the cross-linking assay. These data suggest that the N-terminal half of the C3 region of DctD contains a site that may contact sigma54-holoenzyme during open complex formation.

We cloned the era gene of Francisella tularensis from a plasmid library by heterologous genetic complementation of an Escherichia coli mutant conditionally defective for the production of Era, an essential protein for cell growth. Nucleotide sequence analysis indicated that, in F. tularensis , era constitutes a single gene operon. ORFs aspC and mdh encoding aspartate aminotransferase and malate dehydrogenase, respectively, flank era in F. tularensis . Although classified as Gram − , the flanking regions and the relative location of era in F. tularensis are distinctly different from those of typical Gram − and Gram + bacteria. Computer analysis of bacterial Era protein sequences identified conserved domains in addition to the common G domains of most GTP-binding proteins. © 1997 Elsevier Science B.V. All rights reserved.

Background Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ54 regulon in a bacterium is that the σ54 RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ54-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP—the AAA+ ATPase domain of DctD from Sinorhizobium meliloti—to activate transcription from all σ54-dependent promoters for the characterization of the σ54 regulon of Salmonella Typhimurium LT2. Results The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ54-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ54-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ54 binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ54 binding sites suggest that many of these sites are capable of functioning as σ54-dependent promoters. Conclusion Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse σ54-dependent promoters of the S. Typhimurium LT2 σ54 regulon under a single growth condition, this approach is likely to be valuable for examining σ54 regulons in other bacterial species. The S. Typhimurium σ54 regulon included a high number of intragenic σ54 binding sites/promoters, suggesting that σ54 may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.

Background Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ54 regulon in a bacterium is that the σ54 RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ54-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP—the AAA+ ATPase domain of DctD from Sinorhizobium meliloti—to activate transcription from all σ54-dependent promoters for the characterization of the σ54 regulon of Salmonella Typhimurium LT2. Results The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ54-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ54-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ54 binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ54 binding sites suggest that many of these sites are capable of functioning as σ54-dependent promoters. Conclusion Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse σ54-dependent promoters of the S. Typhimurium LT2 σ54 regulon under a single growth condition, this approach is likely to be valuable for examining σ54 regulons in other bacterial species. The S. Typhimurium σ54 regulon included a high number of intragenic σ54 binding sites/promoters, suggesting that σ54 may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.

A 1.2-kb EcoRI genomic DNA fragment of Coxiella burnetti, when cloned onto a multicopy plasmid, was found to induce capsule synthesis (mucoidy) in Escherichia coli. Nucleotide sequence analysis revealed the presence of an open reading frame that could encode a protein of 270 amino acids. Insertion of a tet cassette into a unique NruI restriction site resulted in the loss of induction of mucoidy. Because of its ability to induce mucoidy, we designated this gene mucZ. Computer search for homologies to mucZ revealed 42% identity to an open reading frame located at 1 min of the E. coli chromosome. Interestingly, the C-terminal amino acid residues of MucZ share significant homology with the J domain of the DnaJ protein and its homologs, suggesting potential interactions between MucZ and components of the DnaK-chaperone machinery. Results presented in this paper suggest that E. coli requires DnaK-chaperone machinery for Lon-RcsA-mediated induction of capsule synthesis, as noticed first by S. Gottesman (personal communication). The induction caused by MucZ is independent of Lon-RcsA and is mediated through the two-component regulators RcsC and RcsB. DnaK and GrpE but not DnaJ are also required for the RcsB-mediated MucZ induction, and we propose that MucZ is a DnaJ-like chaperone protein that might be required for the formation of an active RcsA-RcsB complex and for the RcsC-dependent phosphorylation of RcsB. Discussions are presented that suggest three different roles for alternative forms of the DnaK-chaperone machinery in capsule production.

The dctD gene product (DCTD) activates transcription from dctA by the sigma 54-holoenzyme form of RNA polymerase in Rhizobium meliloti. We have purified a constitutively active form of R. meliloti DCTD that lacks 142 amino acid residues from the N terminus (designated DCTDL143). Purified DCTDL143 recognized the DCTD-binding sites at the dctA promoter region and catalyzed the isomerization of closed complexes between sigma 54-holoenzyme and the dctA promoter to open complexes. Like the related sigma 54-dependent activators NTRC and NIFA, a purine nucleoside triphosphate with a hydrolyzable beta-gamma bond was required prior to transcription initiation for this isomerization. DCTDL143 hydrolyzed purine nucleoside triphosphates but not pyrimidine nucleoside triphosphates. As observed with NTRC-phosphate, the specific activity for the ATPase of DCTDL143 was strongly dependent on the enzyme concentration and was stimulated by DNA fragments bearing the binding sites for the protein. These DNA fragments increased the Vmax for MgATP hydrolysis but did not significantly lower the apparent Km for MgATP. These data are consistent with the idea proposed for related activators that DCTDL143 must assemble into an active, oligomeric form before it can hydrolyze MgATP and presumably activate transcription.

Summary Rhizobium meliloti DctD is believed to have three functional domains: an N-terminal, two-component receiver domain; and like other σ54-dependent activators, C-terminal and central domains for DNA binding and transcription activation. We have characterized a progressive series of M-terminal deletions of R meliloti DctD. The N-terminal domain was not needed for binding the dctA upstream activation sequence. Only 25% of the C-terminal end of the receiver domain was needed to significantly inhibit the central domain, and proteins lacking up to 60% of the N-terminal end of the receiver domain were‘inducible’in R. meliloti cells. We hypothesize that the W-terminal two-thirds of the DctD receiver domain augments and controls an adjacent subdomain for inhibiting the central domain.

A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.