New Findings on Evolution of Metal Homeostasis Genes: Evidence from Comparative Genome Analysis of Bacteria and Archaea

In order to examine the natural history of metal homeostasis genes in prokaryotes, open reading frames with homology to characterized P_IB-type ATPases from the genomes of 188 bacteria and 22 archaea were investigated. Major findings were as follows. First, a high diversity in N-terminal metal binding motifs was observed. These motifs were distributed throughout bacterial and archaeal lineages, suggesting multiple loss and acquisition events. Second, the CopA locus separated into two distinct phylogenetic clusters, CopA1, which contained ATPases with documented Cu(I) influx activity, and CopA2, which contained both efflux and influx transporters and spanned the entire diversity of the bacterial domain, suggesting that CopA2 is the ancestral locus. Finally, phylogentic incongruences between 16S rRNA and P_IB-type ATPase gene trees identified at least 14 instances of lateral gene transfer (LGT) that had occurred among diverse microbes. Results from bootstrapped supported nodes indicated that (i) a majority of the transfers occurred among proteobacteria, most likely due to the phylogenetic relatedness of these organisms, and (ii) gram-positive bacteria with low moles percent G+C were often involved in instances of LGT. These results, together with our earlier work on the occurrence of LGT in subsurface bacteria (J. M. Coombs and T. Barkay, Appl. Environ. Microbiol. 70:1698-1707, 2004), indicate that LGT has had a minor role in the evolution of P_IB-type ATPases, unlike other genes that specify survival in metal-stressed environments. This study demonstrates how examination of a specific locus across microbial genomes can contribute to the understanding of phenotypes that are critical to the interactions of microbes with their environment.     

Isolation, Sequence Analysis, and Comparison of Two Plasmids (28 and 29 Kilobases) from the Biomining Bacterium Leptospirillum ferrooxidans ATCC 49879

Two plasmids, of 28,878 bp and 28,012 bp, were isolated from Leptospirillum ferrooxidans ATCC 49879. Altogether, a total of 67 open reading frames (ORFs) were identified on both plasmids, of which 32 had predicted products with high homology to proteins of known function, while 11 ORFs had predicted products with homology to previously identified proteins of unknown function. Twenty-four ORFs had products with no homologues in the GenBank/NCBI database. An analysis of the ORFs and other features of the two plasmids, the first to be isolated from a bacterium of the genus Leptospirillum, is presented.     

Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c₅₅₄; and cycB, cytochrome c_m552. The deduced protein sequences of HAO, c₅₅₄, and c_m552 were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c_m552, NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c₅₅₄ gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c₅₅₄ gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.     

Molecular Analysis of Human, Porcine, and Poultry Enterococcus faecium Isolates and Their erm(B) Genes

Fifty-nine erm(B)-positive Enterococcus faecium strains isolated from pigs, broilers, and humans were typed using multilocus sequence typing (MLST), and the coding sequence of the erm(B) gene was determined. Identical erm(B) gene sequences were detected in genetically unrelated isolates. Furthermore, genetically indistinguishable strains were found to contain different erm(B) alleles. This may suggest that horizontal exchange of the erm(B) gene between animal and human E. faecium strains or the existence of a common reservoir of erm(B) genes might be more important than direct transmission of resistant strains.     

Porous membranes for reconstitution of ion channels

Functional biological synthetic composite (BSC) membranes were made using phospholipids, biological membrane proteins and permeable synthetic supports or membranes. Lipid bilayers were formed on porous polycarbonate (PC), polyethylene terephthalate (PETE) and poly (l-lactic acid) (PLLA) membranes and in 10-100 μm laser-drilled pores in a 96-well plastic plate as measured by increased resistance or decreased currents. Bilayers in 50 μm and smaller pores were stable for up to 4 h as measured by resistance changes or a current after gramicidin D reconstitution. Biological membrane transport reconstitution was then carried out. Using vesicles containing Kv1.5 K⁺ channels, K⁺ currents and decreased resistance were measured across bilayers in 50 μm pores in the plastic plate and PLLA membranes, respectively, which were inhibited by compound B, a Kv1.5 K⁺ channel inhibitor. Functional reconstitution of Kv1.5 K⁺ channels was successful. Incorporation of membrane proteins in functional form in stable permeable membrane-supported lipid bilayers is a simple technology to create BSC membranes that mimic biological function which is readily adaptable for high throughput screening.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in <Emphasis Type="Italic">Taxus chinensis</Emphasis> cultures

The roles of glucose-6-phosphate dehydrogenase (G6PDH) in paclitaxel production were investigated in cell suspension cultures of Taxus chinensis. In the normal cultures, the trend of G6PDH activity was similar to that of cell growth. Addition of glutamate increased G6PDH activity, while dehydroepiandrosterone (DHEA) decreased G6PDH activity. In elicitor-treated cultures, cell growth was depressed, while G6PDH activity and taxol production were enhanced compared with the control. Glutamate recovered the depression of cell growth, and resulted in further increase in G6PDH activity and taxol production. Contrarily, DHEA exacerbated the depression of cell growth, and decreased G6PDH activity and taxol production induced by fungal elicítor. The results indicated that G6PDH played a critic role of taxol production by affecting cell viability.     

Phylogenetics of New World Astragalus: Screening of novel nuclear loci for the reconstruction of phylogenies at low taxonomic levels

This study explores methods to use information gathered from genomics technology to understand evolutionary relationships in the hyperdiverse legume group Neo-Astragalus. These species inhabit deserts and mountains of North and South America, and even though the monophyly of the group is well established, relationships within it are still poorly understood. Plastid genes, commonly used to infer phylogenies in plants, are usually not useful for closely related taxa because of low levels of genetic variation. TheMedicago truncatula genome project provided a suite of candidate nuclear loci with high levels of variation that might prove suitable for low-level phylogenetics. This paper reports the development of methods for screening a large number of these nuclear loci, and detailed analysis of four of them. Four different patterns of phylogenetic diversification occur in the loci sampled from these genomes ofAstragalus species. One locus (CNGC4) was single copy and could be directly used in phylogenetic analyses. Two loci (ARG10 and FENR) showed patterns strongly suggestive of duplication events in some taxa, and one locus (tRALS) has apparently undergone a cryptic duplication, making it very difficult to diagnose. Potential methods for using the information provided by these loci are discussed.