The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe²⁺‐ and Fe³⁺‐citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was ≈ 10‐fold lower than in the wild type (with 0.5 μM Fe), irrespective of whether Fe was supplied in the Fe²⁺ or Fe³⁺ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe²⁺ and Fe³⁺ transport. The growth inhibition exhibited by the feoB mutant in Fe‐deficient media was relieved by human holo‐transferrin, holo‐lactoferrin and Fe³⁺‐dicitrate, but not by FeSO₄. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe²⁺ transport in the wild type suggested the presence of high‐ and low‐affinity uptake systems. The high‐affinity system (apparent K_s = 0.54 μM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe²⁺ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe²⁺ and Fe³⁺ uptake implied the concerted involvement of both an Fe³⁺ reductase and FeoB in the uptake of Fe supplied as Fe³⁺. Taken together, the results are consistent with FeoB‐mediated Fe²⁺ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low‐pH, low‐O₂ environment of the stomach.

The dct locus of Rhodobacter capsulatus encodes a high-affinity transport system for the C4-dicarboxylates malate, succinate, and fumarate. The nucleotide sequence of the region downstream of the previously sequenced dctP gene (encoding a periplasmic C4-dicarboxylate-binding protein) was determined. Two open reading frames (ORFs) of 681 bp (dctQ) and 1,320 bp (dctM) were identified as additional dct genes by insertional mutagenesis and complementation studies. DctQ (24,763 Da) and DctM (46,827 Da) had hydropathic profiles consistent with the presence of 4 and 12 potential transmembrane segments, respectively, and were localized in the cytoplasmic membrane fraction after heterologous expression of the dctQM ORFs in Escherichia coli. DctP, DctQ, and DctM were found to be unrelated to known transport proteins in the ABC (ATP-binding cassette) superfamily but were shown to be homologous with the products of previously unidentified ORFs in a number of gram-negative bacteria, including Bordetella pertussis, E. coli, Salmonella typhimurium, Haemophilus influenzae, and Synechocystis sp. strain PCC6803. An additional ORF (rypA) downstream of dctM encodes a protein with sequence similarity to eukaryotic protein-tyrosine phosphatases, but interposon mutagenesis of this ORF did not result in a Dct- phenotype. Complementation of a Rhizobium meliloti dctABD deletion mutant by heterologous expression of the dctPQM genes from R. capsulatus demonstrated that no additional structural genes were required to form a functional transport system. Transport via the Dct system was vanadate insensitive, and in uncoupler titrations with intact cells, the decrease in the rate of succinate transport correlated closely with the fall in membrane potential but not with the cellular ATP concentration, implying that the proton motive force, rather than ATP hydrolysis, drives uptake. It is concluded that the R. capsulatus Dct system is a new type of periplasmic secondary transporter and that similar, hitherto-unrecognized systems are widespread in gram-negative bacteria. The name TRAP (for tripartite ATP-independent periplasmic) transporters is proposed for this new group.

The human gastrointestinal pathogenCampylobacter jejuniis a microaerophilic bacterium with a respiratory metabolism. The genome sequence ofC. jejunistrain 11168 reveals the presence of genes that encode terminal reductases that are predicted to allow the use of a wide range of alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, andN- orS-oxides. All of these reductase activities were present in cells of strain 11168, and the molybdoenzyme encoded byCj0264cwas shown by mutagenesis to be responsible for both trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) reduction. Nevertheless, growth ofC. jejuniunder strictly anaerobic conditions (with hydrogen or formate as electron donor) in the presence of any of the electron acceptors tested was insignificant. However, when fumarate, nitrate, nitrite, TMAO, or DMSO was added to microaerobic cultures in which the rate of oxygen transfer was severely restricted, clear increases in both the growth rate and final cell density compared to what was seen with the control were obtained, indicative of electron acceptor-dependent energy conservation. TheC. jejunigenome encodes a single class I-type ribonucleotide reductase (RNR) which requires oxygen to generate a tyrosyl radical for catalysis. Electron microscopy of cells that had been incubated under strictly anaerobic conditions with an electron acceptor showed filamentation due to an inhibition of cell division similar to that induced by the RNR inhibitor hydroxyurea. An oxygen requirement for DNA synthesis can thus explain the lack of anaerobic growth ofC. jejuni. The results indicate that strict anaerobiosis is a stress condition forC. jejunibut that alternative respiratory pathways can contribute significantly to energy conservation under oxygen-limited conditions, as might be found in vivo.

Campylobacter jejuni is a microaerophilic, asaccharolytic bacterium. The identity of the carbon and energy sources used by C. jejuni in vivo is unknown, but the genome sequence of strain NCTC11168 indicates the presence of genes for catabolism of a limited range of amino acids, including serine. Specific omission of l -serine from a defined medium containing a mixture of amino acids led to a dramatic decrease in cell yields. As C. jejuni does not have a biosynthetic serine requirement, this supports earlier suggestions that l -serine is a preferentially catabolized amino acid. Serine transport was found to be mediated by at least two systems in strain 11168; a high-capacity, low-affinity l -serine-specific system encoded by Cj1625c ( sdaC ) and a higher-affinity l -serine/ l -threonine system responsible for residual l -serine transport in an sdaC mutant. Catabolism of l -serine to pyruvate and ammonia is carried out by SdaA (encoded by Cj1624c ), which was overexpressed, purified, and shown to be an oxygen-labile iron-sulfur enzyme. l -Serine dehydratase activity in an sdaA mutant was reduced 10-fold compared to that in the wild type, but the residual activity (due to the anabolic l -threonine dehydratase) could not support either growth on or utilization of l -serine in defined media. However, although sdaA mutants showed no obvious growth defect in complex media, they completely failed to colonize 3-week-old chickens as assayed both by cloacal swabs taken over a 6-week period and by cecal colony counts postmortem. In contrast, the isogenic parent strain colonized chickens to high levels within 1 week of inoculation. The results show that an active SdaA is essential for colonization of the avian gut by C. jejuni and imply that catabolism of l -serine is crucially important for the growth of this bacterium in vivo.

The ability to efficiently scavenge nutrients in the host is essential for the viability of any pathogen. All catabolic pathways must begin with the transport of substrate from the environment through the cytoplasmic membrane, a role executed by membrane transporters. Although several classes of cytoplasmic membrane transporters are described, high-affinity uptake of substrates occurs through Solute Binding-Protein (SBP) dependent systems. Three families of SBP dependant transporters are known; the primary ATP-binding cassette (ABC) transporters, and the secondary Tripartite ATP-independent periplasmic (TRAP) transporters and Tripartite Tricarboxylate Transporters (TTT). Far less well understood than the ABC family, the TRAP transporters are found to be abundant among bacteria from marine environments, and the TTT transporters are the most abundant family of proteins in many species of β-proteobacteria. In this review, recent knowledge about these families is covered, with emphasis on their physiological and structural mechanisms, relating to several examples of relevant uptake systems in pathogenicity and colonization, using the SiaPQM sialic acid uptake system from Haemophilus influenzae and the TctCBA citrate uptake system of Salmonella typhimurium as the prototypes for the TRAP and TTT transporters, respectively. High-throughput analysis of SBPs has recently expanded considerably the range of putative substrates known for TRAP transporters, while the repertoire for the TTT family has yet to be fully explored but both types of systems most commonly transport carboxylates. Specialized spectroscopic techniques and site-directed mutagenesis have enriched our knowledge of the way TRAP binding proteins capture their substrate, while structural comparisons show conserved regions for substrate coordination in both families. Genomic and protein sequence analyses show TTT SBP genes are strikingly overrepresented in some bacteria, especially in the β-proteobacteria and some α-proteobacteria. The reasons for this are not clear but might be related to a role for these proteins in signaling rather than transport.