Gavin H. Thomas

Escherichia coli K-12: a cooperatively developed annotation snapshot—2005

The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.

Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB

The Amt proteins are ammonium transporters that are conserved throughout all domains of life, being found in bacteria, archaea and eukarya. In bacteria and archaea, the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the PII signal transduction protein family, proteins that regulate enzyme activity and gene expression in response to the intracellular nitrogen status. We have now shown that in Escherichia coli and Azotobacter vinelandii, GlnK binds to the membrane in an AmtB‐dependent manner and that GlnK acts as a negative regulator of the transport activity of AmtB. Membrane binding is dependent on the uridylylation state of GlnK and is modulated according to the cellular nitrogen status such that it is maximal in nitrogen‐sufficient situations. The membrane sequestration of GlnK by AmtB represents a novel form of signal transduction in which an integral membrane transport protein functions to link the extracellular ammonium concentration to the intracellular responses to nitrogen status. The results also offer new insights into the evolution of PII proteins and a rationale for their trigonal symmetry.

Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm

The ‘aeg46.5 ’ operon was originally detected as an ‘anaerobically expressed gene’ located at minute 46.5 on the Escherichia coli linkage map. Subsequent results from the E. coli Genome Sequencing Project revealed that the ‘aeg46.5 ’ promoter was located in the centisome 49 (minute 47) region. Downstream from this promoter are 15 genes, seven of which are predicted to encode a periplasmic nitrate reductase and eight encode proteins homologous to proteins essential for cytochrome c assembly in other bacteria. All of these genes, together with the ‘aeg46.5 ’ promoter, have been subcloned on a 20 kb EcoRI fragment from Kohara phage 19D1. Evidence is presented that, as predicted, the region includes structural genes for two c‐type cytochromes of mass 16 kDa and 24 kDa, which are transcribed from the previously described ‘aeg46.5 ’ promoter, and that the first seven genes encode a functional nitrate reductase. We, therefore, propose that they should be designated nap (nitrate reductase in the periplasm) genes. Plasmids encoding the entire 20 kb region, or only the downstream eight genes, complemented five mutations resulting in total absence of all five known c‐type cytochromes in E. coli, providing biochemical evidence that these are ccm (for cytochrome c maturation) genes. The ccm region was transcribed both from the FNR‐dependent, NarL‐ and NarP‐regulated nap promoter (formerly the ‘aeg46.5 ’ promoter) and from constitutive or weakly regulated promoters apparently located within the downstream nap and ccm genes.

Genomic insight into the amino acid relations of the pea aphid, Acyrthosiphon pisum, with its symbiotic bacterium Buchnera aphidicola.

The pea aphid genome includes 66 genes contributing to amino acid biosynthesis and 93 genes to amino acid degradation. In several respects, the pea aphid gene inventory complements that of its symbiotic bacterium, Buchnera aphidicola (Buchnera APS). Unlike other insects with completely sequenced genomes, the pea aphid lacks the capacity to synthesize arginine, which is produced by Buchnera APS. However, consistent with other insects, it has genes coding for individual reactions in essential amino acid biosynthesis, including threonine dehydratase and branched‐chain amino acid aminotransferase, which are not coded in the Buchnera APS genome. Overall the genome data suggest that the biosynthesis of certain essential amino acids is shared between the pea aphid and Buchnera APS, providing the opportunity for precise aphid control over Buchnera metabolism.

The glnKamtB operon a conserved gene pair in prokaryotes

G.T. and M.M. were supported by EU Contract No. BIO4 CT 972310 and G.C. acknowledges a BBSRC studentship. We thank S. Austin, R. Dixon and G. Sawers for constructive comments on the manuscript.

Membrane topology of the Mep/Amt family of ammonium transporters

The Mep/Amt proteins constitute a new family of transport proteins that are ubiquitous in nature. Members from bacteria, yeast and plants have been identified experimentally as high‐affinity ammonium transporters. We have determined the topology of AmtB, a Mep/Amt protein from Escherichia coli, as a representative protein for the complete family. This was established using a minimal set of AmtB–PhoA fusion proteins with a complementary set of AmtB–LacZ fusions. These data, accompanied by an in silico analysis, indicate that the majority of the Mep/Amt proteins contain 11 membrane‐spanning helices, with the N‐terminus on the exterior face of the membrane and the C‐terminus on the interior. A small subset, including E. coli AmtB, probably have an additional twelfth membrane‐spanning region at the N‐terminus. Addition of PhoA or LacZ α‐peptide to the C‐terminus of E. coli AmtB resulted in complete loss of transport activity, as judged by measurements of [14C]‐methylammonium uptake. This C‐terminal region, along with four membrane‐spanning helices, contains multiple residues that are conserved within the Mep/Amt protein family. Structural modelling of the E. coli AmtB protein suggests a number of secondary structural features that might contribute to function, including a putative ammonium binding site on the periplasmic face of the membrane at residue Asp‐182. The implications of these results are discussed in relation to the structure and function of the related human Rhesus proteins.

Sialic acid transport in Haemophilus influenzae is essential for lipopolysaccharide sialylation and serum resistance and is dependent on a novel tripartite ATP‐independent periplasmic transporter

Sialylation of the lipopolysaccharide (LPS) is an important mechanism used by the human pathogen Haemophilus influenzae to evade the innate immune response of the host. We have demonstrated that N‐acetylneuraminic acid (Neu5Ac or sialic acid) uptake in H. influenzae is essential for the subsequent modification of the LPS and that this uptake is mediated through a single transport system which is a member of the tripartite ATP‐independent periplasmic (TRAP) transporter family. Disruption of either the siaP (HI0146) or siaQM (HI0147) genes, that encode the two subunits of this transporter, results in a complete loss of uptake of [14C]‐Neu5Ac. Mutant strains lack sialylated glycoforms in their LPS and are more sensitive to killing by human serum than the parent strain. The SiaP protein has been purified and demonstrated to bind a stoichiometric amount of Neu5Ac by electrospray mass spectrometry. This binding was of high affinity with a Kd of ∼0.1 µM as determined by protein fluorescence. The inactivation of the SiaPQM TRAP transporter also results in decreased growth of H. influenzae in a chemically defined medium containing Neu5Ac, supporting an additional nutritional role of sialic acid in H. influenzae physiology.

Tripartite ATP‐independent periplasmic (TRAP) transporters in bacteria and archaea

The tripartite ATP-independent periplasmic (TRAP) transporters are the best-studied family of substrate-binding protein (SBP)-dependent secondary transporters and are ubiquitous in prokaryotes, but absent from eukaryotes. They are comprised of an SBP of the DctP or TAXI families and two integral membrane proteins of unequal sizes that form the DctQ and DctM protein families, respectively. The SBP component has a structure comprised of two domains connected by a hinge that closes upon substrate binding. In DctP-TRAP transporters, substrate binding is mediated through a conserved and specific arginine/carboxylate interaction in the SBP. While the SBP component has now been relatively well characterized, the membrane components of TRAP transporters are still poorly understood both in terms of their structure and function. We review the expanding repertoire of substrates and physiological roles for experimentally characterized TRAP transporters in bacteria and discuss mechanistic aspects of these transporters using data primarily from the sialic acid-specific TRAP transporter SiaPQM from Haemophilus influenzae, which suggest that TRAP transporters are high-affinity, Na(+)-dependent unidirectional secondary transporters.

In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase

The Escherichia coli AmtB protein is member of the ubiquitous Amt family of ammonium transporters. Using a variety of [14C]methylammonium-uptake assays in wild-type E. coli, together with amtB and glutamine synthetase (glnA) mutants, we have shown that the filtration method traditionally used to measure [14C]methylammonium uptake actually measures intracellular accumulation of methylglutamine and that the kinetic data deduced from such experiments refer to the activity of glutamine synthetase and not to AmtB. Furthermore, the marked difference between the K(m) values of glutamine synthetase calculated in vitro and those calculated in vivo from our data suggest that ammonium assimilation by glutamine synthetase is coupled to the function of AmtB. The use of a modified assay technique allows us to measure AmtB activity in vivo. In this way, we have examined the role that AmtB plays in ammonium/methylammonium transport, in the light of conflicting proposals with regard to both the mode of action of Amt proteins and their substrate, i.e. ammonia or ammonium. Our in vivo data suggest that AmtB acts as a slowly conducting channel for NH3 that is neither dependent on the membrane potential nor on ATP. Furthermore, studies on competition between ammonium and methylammonium suggest that AmtB has a binding site for NH4+ on the periplasmic face.

A fragile metabolic network adapted for cooperation in the symbiotic bacterium Buchnera aphidicola

BackgroundIn silico analyses provide valuable insight into the biology of obligately intracellular pathogens and symbionts with small genomes. There is a particular opportunity to apply systems-level tools developed for the model bacterium Escherichia coli to study the evolution and function of symbiotic bacteria which are metabolically specialised to overproduce specific nutrients for their host and, remarkably, have a gene complement that is a subset of the E. coli genome.ResultsWe have reconstructed and analysed the metabolic network of the γ-proteobacterium Buchnera aphidicola (symbiont of the pea aphid) as a model for using systems-level approaches to discover key traits of symbionts with small genomes. The metabolic network is extremely fragile with > 90% of the reactions essential for viability in silico; and it is structured so that the bacterium cannot grow without producing the essential amino acid, histidine, which is released to the insect host. Further, the amount of essential amino acid produced by the bacterium in silico can be controlled by host supply of carbon and nitrogen substrates.ConclusionThis systems-level analysis predicts that the fragility of the bacterial metabolic network renders the symbiotic bacterium intolerant of drastic environmental fluctuations, whilst the coupling of histidine production to growth prevents the bacterium from exploiting host nutrients without reciprocating. These metabolic traits underpin the sustained nutritional contribution of B. aphidicola to the host and, together with the impact of host-derived substrates on the profile of nutrients released from the bacteria, point to a dominant role of the host in controlling the symbiosis.