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PubMed2000

A peptide permease mutant of Mycobacterium bovis BCG resistant to the toxic peptides glutathione and S-nitrosoglutathione.

Oligopeptides play important roles in bacterial nutrition and signaling. Using sequences from the available genome database for Mycobacterium tuberculosis H37Rv, the oligopeptide permease operon (oppBCDA) of Mycobacterium bovis BCG was cloned from a cosmid library. An opp mutant strain was constructed by homologous recombination with an allele of oppD interrupted by kanamycin and streptomycin resistance markers. The deletion was complemented with a wild-type copy of the opp operon. Two approaches were taken to characterize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide variety of peptides as sole source of carbon and/or nitrogen. Among 25 peptides ranging from two to six amino acids in length, none was capable of supporting measurable growth as the sole carbon source in either wild-type or mutant strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (gamma-glutamyl-L-cyteinylglycine [GSH]) is toxic to wild-type BCG and was used successfully to characterize peptide uptake in the opp mutant. In 2 mM GSH, growth of the wild-type strain is inhibited, whereas the opp mutant is resistant to concentrations as high as 10 mM. Similar results were found with the tripeptide S-nitrosoglutathione (GSNO), thought to be a donor of NO in mammalian cells. Using incorporation of [(3)H]uracil to monitor the effects of GSH and GSNO on macromolecular synthesis in growing cells, it was demonstrated that the opp mutant is resistant, whereas the wild type and the mutant complemented with a wild-type copy of the operon are sensitive to both tripeptides. In uptake measurements, incorporation of [(3)H]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three strains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.

Cloning, Molecular↗

PubMed2001

Identification of proteins from Plasmodium falciparum that are homologous to reticulocyte binding proteins in Plasmodium vivax.

Plasmodium falciparum infections can be fatal, while P. vivax infections usually are not. A possible factor involved in the greater virulence of P. falciparum is that this parasite grows in red blood cells (RBCs) of all maturities whereas P. vivax is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from P. vivax, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the P. falciparum genome databases has identified genes that are homologous to the P. vivax rbp-1 and -2 genes. Two of these genes are virtually identical over a large region of the 5' end but are highly divergent at the 3' end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.

Amino Acid Sequence↗

PubMed2001

Disruption of Plasmodium falciparum chitinase markedly impairs parasite invasion of mosquito midgut.

To initiate invasion of the mosquito midgut, Plasmodium ookinetes secrete chitinolytic activity to penetrate the peritrophic matrix surrounding the blood meal. While ookinetes of the avian malaria parasite Plasmodium gallinaceum appear to secrete products of two chitinase genes, to date only one chitinase gene, PfCHT1, has been identified in the nearly completed Plasmodium falciparum strain 3D7 genome database. To test the hypothesis that the single identified chitinase of P. falciparum is necessary for ookinete invasion, the PfCHT1 gene was disrupted 39 bp upstream of the stop codon. PfCHT1-disrupted parasites had normal gametocytogenesis, exflagellation, and ookinete formation but were markedly impaired in their ability to form oocysts in Anopheles freeborni midguts. Confocal microscopy demonstrated that the truncated PfCHT1 protein was present in mutant ookinetes but that the concentration of mutant PfCHT1 within the apical end of the ookinetes was substantially reduced. These data suggest that full-length PfCHT1 is essential for intracellular trafficking and secretion and that the PfCHT1 gene product is necessary for ookinetes to invade the mosquito midgut.

Animals↗

PubMed2001

Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa.

The expression of many virulence factors in Pseudomonas aeruginosa is dependent upon environmental conditions, including iron levels, oxygen, temperature, and osmolarity. The virulence of P. aeruginosa PAO1 is influenced by the iron- and oxygen-regulated gene encoding the alternative sigma factor PvdS, which is regulated through the ferric uptake regulator (Fur). We observed that overexpression of PvdS in strain PAO1 and a DeltapvdS::Gm mutant resulted in increased pyoverdine production and proteolytic activity compared to when PvdS was not overexpressed. To identify additional PvdS-regulated genes, we compared extracellular protein profiles from PAO1 and the DeltapvdS::Gm mutant grown under iron-deficient conditions. A protein present in culture supernatants from PAO1 but not in supernatants from DeltapvdS::Gm was investigated. Amino acid sequence analysis and examination of the genomic database of PAO1 revealed that the N terminus of this 27-kDa protein is identical to that of protease IV of P. aeruginosa strain PA103-29 and is homologous to an endoprotease produced by Lysobacter enzymogenes. In this study, the gene encoding an endoprotease was cloned from PAO1 and designated prpL (PvdS-regulated endoprotease, lysyl class). All (n = 41) but one of the strains of P. aeruginosa, including clinical and environmental isolates, examined carry prpL. Moreover, PrpL production among these strains was highly variable. Analysis of RNase protection assays identified the transcription initiation site of prpL and confirmed that its transcription is iron dependent. In the DeltapvdS::Gm mutant, the level of prpL transcription was iron independent and decreased relative to the level in PAO1. Furthermore, transcription of prpL was independent of PtxR, a PvdS-regulated protein. Finally, PrpL cleaves casein, lactoferrin, transferrin, elastin, and decorin and contributes to PAO1's ability to persist in a rat chronic pulmonary infection model .

Amino Acid Sequence↗

PubMed2002

Purification and characterization of a second immunoreactive mannoprotein from Cryptococcus neoformans that stimulates T-Cell responses.

Although T-cell responses are known to be critical for effective host defenses against the fungal pathogen Cryptococcus neoformans, the antigens that stimulate protective responses are poorly characterized but are thought to be comprised, at least in part, of mannoproteins. Recently, we created a panel of murine CD4(+)-T-cell hybridomas that react with C. neoformans antigens. A mannoprotein antigen, MP98, that stimulated one of the hybridomas was purified, and the gene encoding MP98 was cloned. In the present study, the cryptococcal antigen, MP88, that stimulated a second T-cell hybridoma, X5A3, to secrete interleukin-2 was characterized. MP88 was purified from supernatants of glass bead-disrupted C. neoformans by anion-exchange and hydrophobic interaction chromatography. A single band with an apparent molecular mass of 88 kDa was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial internal amino acid sequencing. The gene encoding MP88 was cloned and sequenced. MP88 features a C-terminal serine/threonine-rich region, which presumably serves as a site for extensive O glycosylation, followed by a putative glycosylphosphatidylinositol anchor site. A search of C. neoformans genomic databases revealed that MP88 shares this feature with at least 11 other genes, including MP98. The mannoprotein nature of MP88 was established based upon the capacity of (i) the mannoprotein fraction of C. neoformans supernatants to stimulate X5A3 and (ii) mannosylated ligands to competitively inhibit this stimulation. Thus, a second cryptococcal mannoprotein has been identified which stimulates T-cell responses and is a vaccine candidate.

Amino Acid Sequence↗

PubMed2002

Novel 33-kilodalton lipoprotein from Mycobacterium leprae.

A novel Mycobacterium leprae lipoprotein LpK (accession no. ML0603) was identified from the genomic database. The 1,116-bp open reading frame encodes a 371-amino-acid precursor protein with an N-terminal signal sequence and a consensus motif for lipid conjugation. Expression of the protein, LpK, in Escherichia coli revealed a 33-kDa protein, and metabolic labeling experiments and globomycin treatment proved that the protein was lipidated. Fractionation of M. leprae demonstrated that this lipoprotein was a membrane protein of M. leprae. The purified lipoprotein was found to induce production of interleukin-12 in human peripheral blood monocytes. The studies imply that M. leprae LpK is involved in protective immunity against leprosy and may be a candidate for vaccine design.

Amino Acid Sequence↗

PubMed2003

Purification and characterization of a UDP-glucosyltransferase produced by Legionella pneumophila.

Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.

Amino Acid Sequence↗

PubMed2003

Two novel superantigens found in both group A and group C Streptococcus.

Two novel streptococcal superantigen genes (speL(Se) and speM(Se)) were identified from the Streptococcus equi genome database at the Sanger Center. Genotyping of 8 S. equi isolates and 40 Streptococcus pyogenes isolates resulted in the detection of the orthologous genes speL and speM in a restricted number of S. pyogenes isolates (15 and 5%, respectively). Surprisingly, the novel superantigen genes could not be found in any of the analyzed S. equi isolates. The results suggest that both genes are located on a mobile element that enables gene transfer between individual isolates and between streptococci from different Lancefield groups. S. equi pyrogenic exotoxin L (SPE-L(Se))/streptococcal pyrogenic exotoxin L (SPE-L) and SPE-M(Se)/SPE-M are most closely related to SMEZ, SPE-C, SPE-G, and SPE-J, but build a separate branch within this group. Recombinant SPE-L (rSPE-L) and rSPE-M were highly mitogenic for human peripheral blood lymphocytes, with half-maximum responses at 1 and 10 pg/ml, respectively. The results from competitive binding experiments suggest that both proteins bind major histocompatibility complex class II at the beta-chain, but not at the alpha-chain. The most common targets for both toxins were human Vbeta1.1 expressing T cells. Seroconversion against SPE-L and SPE-M was observed in healthy blood donors, suggesting that the toxins are expressed in vivo. Interestingly, the speL gene is highly associated with S. pyogenes M89, a serotype that is linked to acute rheumatic fever in New Zealand.

Amino Acid Sequence↗

PubMed2004

Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia.

Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.

Animals↗

PubMed1998

Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv.

An open reading frame in the genomic database of Mycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.

Amino Acid Sequence↗

PubMed1998

Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus.

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.

Amino Acid Sequence↗

PubMed1998

A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy.

A two-component signal transduction system encoded by the yycF and yycG genes is part of an operon containing three genes, yycH, yycI, and yycJ, with no known function and a gene, yycK, coding for an HtrA-like protease. This operon was transcribed during growth, and its transcription shut down as the cells approached stationary phase. This decreased transcription was not Spo0A dependent. The HtrA protease gene was separately controlled during sporulation from a sigmaG promoter. Studies using insertional inactivation plasmids revealed that neither yycF nor yycG could be inactivated, whereas the other genes were inactivated without loss of viability. A temperature-sensitive YycF response regulator mutant was isolated and shown to have an H215P mutation in a putative DNA-binding domain which is closely related to the OmpR family of response regulators. At the nonpermissive temperature, cultures of the mutant strain stopped growth within 30 min, and this was followed by a decrease in optical density. Microscopically, many of the cells appeared to retain their structure while being empty of their contents. The essential processes regulated by this two-component system remain unknown. A search of the genome databases revealed YycF, YycG, and YycJ homologues encoded by three linked genes in Streptococcus pyogenes. The high level of identity of these proteins (71% for YycF) suggests that this system may play a similar role in gram-positive pathogens.

Amino Acid Sequence↗

PubMed1999

Staphylococcus aureus cap5P encodes a UDP-N-acetylglucosamine 2-epimerase with functional redundancy.

The serotype 5 capsule gene cluster of Staphylococcus aureus comprises 16 genes (cap5A through cap5P), but little is known about how the putative gene products function in capsule biosynthesis. We propose that the N-acetylmannosaminuronic acid (ManNAcA) component of the S. aureus serotype 5 capsular polysaccharide (CP5) is synthesized from a UDP-N-acetylglucosamine (UDP-GlcNAc) precursor that is epimerized to UDP-N-acetylmannosamine (UDP-ManNAc) and then oxidized to UDP-ManNAcA. We report the purification and biochemical characterization of a recombinant UDP-GlcNAc 2-epimerase encoded by S. aureus cap5P. Purified Cap5P converted approximately 10% of UDP-GlcNAc to UDP-ManNAc as detected by gas chromatography-mass spectrometry. The epimerization of UDP-GlcNAc to UDP-ManNAc occurred over a wide pH range and was unaffected by divalent cations. Surprisingly, CP5 expression in S. aureus was unaffected by insertional inactivation of cap5P. Sequence homology searches of the public S. aureus genomic databases revealed the presence of another putative UDP-GlcNAc 2-epimerase on the S. aureus chromosome that showed 61% identity to Cap5P. Redundancy of UDP-GlcNAc 2-epimerase function in S. aureus was demonstrated by cloning the cap5P homologue from strain Newman and complementing an Escherichia coli rffE mutant defective in UDP-GlcNAc 2-epimerase activity. Our results confirm the putative function of the S. aureus cap5P gene product and demonstrate the presence of a second gene on the staphylococcal chromosome with a similar function.

Bacterial Proteins↗

PubMed1999

Purification and molecular characterization of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus: the third of a putative five-member tungstoenzyme family.

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100 degreesC by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS-) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 microM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.

Aldehyde Oxidoreductases↗

PubMed1999

The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force.

Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of V. cholerae. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V. cholerae. The energy source of its flagellar motor was investigated. We examined the Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V. cholerae strains and present the evidence that the polar flagellar motor of V. cholerae is driven by an Na+ motive force.

Flagella↗

PubMed2000

Cloning and characterization of the CSF1 gene of Saccharomyces cerevisiae, which is required for nutrient uptake at low temperature.

We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial baker's yeast that have practically no fermentation capacity at 5 degrees C and return to their normal capacity at 25 to 40 degrees C. CSF1 was cloned by functional complementation of the Csf phenotype. CSF1 contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids. The nucleotide sequence was identical to that of the YLR087C gene in the Saccharomyces genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein. Gene disruption shows that CSF1 is required for growth and fermentation only at low temperatures. Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10 degrees C. The disruptant cells had the same glucose uptake rates as the parental cells at 30 degrees C, but three- to fivefold-lower rates than the parental cells at 10 degrees C. These findings suggest that CSF1 associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.

Cloning, Molecular↗

PubMed2000

Purification and characterization of a membrane-bound hydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

Highly washed membrane preparations from cells of the hyperthermophilic archaeon Pyrococcus furiosus contain high hydrogenase activity (9.4 micromol of H(2) evolved/mg at 80 degrees C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with n-dodecyl-beta-D-maltoside and purified by multistep chromatography in the presence of Triton X-100. The purified preparation contained two major proteins (alpha and beta) in an approximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained approximately 1 Ni and 4 Fe atoms/mol. The reduced enzyme gave rise to an electron paramagnetic resonance signal typical of the so-called Ni-C center of mesophilic NiFe-hydrogenases. Neither highly washed membranes nor the purified enzyme used NAD(P)(H) or P. furiosus ferredoxin as an electron carrier, nor did either catalyze the reduction of elemental sulfur with H(2) as the electron donor. Using N-terminal amino acid sequence information, the genes proposed to encode the alpha and beta subunits were located in the genome database within a putative 14-gene operon (termed mbh). The deduced sequences of the two subunits (Mbh 11 and 12) were distinctly different from those of the four subunits that comprise each of the two cytoplasmic NiFe-hydrogenases of P. furiosus and show that the alpha subunit contains the NiFe-catalytic site. Six of the open reading frames (ORFs) in the operon, including those encoding the alpha and beta subunits, show high sequence similarity (>30% identity) with proteins associated with the membrane-bound NiFe-hydrogenase complexes from Methanosarcina barkeri, Escherichia coli, and Rhodospirillum rubrum. The remaining eight ORFs encode small (<19-kDa) hypothetical proteins. These data suggest that P. furiosus, which was thought to be solely a fermentative organism, may contain a previously unrecognized respiratory system in which H(2) metabolism is coupled to energy conservation.

Catalysis↗

PubMed2000

fleN, a gene that regulates flagellar number in Pseudomonas aeruginosa.

The single polar flagellum of Pseudomonas aeruginosa plays an important role in the pathogenesis of infection by this organism. However, regulation of the assembly of this organelle has not been delineated. In analyzing the sequence available at the Pseudomonas genome database, an open reading frame (ORF), flanked by flagellar genes flhF and fliA, that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found. The ORF was inactivated by inserting a gentamicin cassette in P. aeruginosa PAK and PAO1. The resulting mutants were nonmotile on motility agar plates, but under a light microscope they exhibited random movement and tumbling behavior. Electron microscopic studies of the wild-type and mutant strains revealed that the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type, indicating that this ORF was involved in regulating the number of flagella and chemotactic motility in P. aeruginosa. The ORF was named fleN. An intact copy of fleN on a plasmid complemented the mutant by restoring motility and monoflagellate status. The beta-galactosidase activities of eight flagellar operon or gene promoters in the wild-type and fleN mutant strains revealed a direct correlation between six promoters that were upregulated in the fleN mutant (fliLMNOPQ, flgBCDE, fliEFG, fliDS orf126, fleSR, and fliC) and positive regulation by FleQ, an NtrC-like transcriptional regulator for flagellar genes. Based on these results, we propose a model where FleN influences FleQ activity (directly or indirectly) in regulating flagellar number in P. aeruginosa.

Amino Acid Sequence↗