Effect of high concentration of Co (II) on Enterobacter liquefaciens strain C-1: a bacterium highly resistant to heavy metals with an unknown genome

Heavy metals are required as nutrients for essential functions in microorganisms. However, higher concentrations of these cations are generally toxic and may produce contrasting effects on living organisms. Enterobacter liquefaciens strain C-1, a bacterium isolated from the Moa mine in Cuba, is able to survive in the presence of high concentrations of heavy metals. The proteomes of Enterobacter liquefaciens strain C-1, grown under aerobic conditions in the presence and absence of Co (II) were compared using two-dimensional gel electrophoresis analysis in the isoelectric point range of 4-7 and the mass range of 15-120 kDa. Significant changes in the expression level (> two-fold) were detected for 13 spots: seven and six were up- and down-regulated, respectively. Because the genome of this bacterium is unknown, identification by peptide mass fingerprinting only succeeded in four cases and most of the cross-species identifications were supported by de novo sequencing of tryptic peptides followed by sequence alignment using the MS BLAST program. Twelve different proteins were identified, ten are involved in cellular antioxidant defence probably induced by the presence of Co (II). This is the first step towards understanding the role of proteins participating in the mechanism of resistance to heavy metals in this bacterium.     

Local sequence information in cellular retinoic acid-binding protein I: specific residue roles in beta-turns

We have recently shown that two of the beta-turns (III and IV) in the ten-stranded, beta-clam protein, cellular retinoic acid-binding protein I (CRABP I), are favored in short peptide fragments, arguing that they are encoded by local interactions (K. S. Rotondi and L. M. Gierasch, Biochemistry, 2003, Vol. 42, pp. 7976-7985). In this paper we examine these turns in greater detail to dissect the specific local interactions responsible for their observed native conformational biases. Conformations of peptides corresponding to the turn III and IV fragments were examined under conditions designed to selectively disrupt stabilizing interactions, using pH variation, chaotrope addition, or mutagenesis to probe specific side-chain influences. We find that steric constraints imposed by excluded volume effects between near neighbor residues (i,i+2), favorable polar (i,i+2) interactions, and steric permissiveness of glycines are the principal factors accounting for the observed native bias in these turns. Longer-range stabilizing interactions across the beta-turns do not appear to play a significant role in turn stability in these short peptides, in contrast to their importance in hairpins. Additionally, our data add to a growing number of examples of the 3:5 type I turn with a beta-bulge as a class of turns with high propensity to form locally defined structure. Current work is directed at the interplay between the local sequence information in the turns and more long-range influences in the mechanism of folding of this predominantly beta-sheet protein.     

Relationships between the brain and the immune system

The concept that the brain can modulate activity the immune system stems from the theory of stress. Recent advances in the study of the inter-relationships between the central nervous system and the immune system have demonstrated a vast network of communication pathways between the two systems. Lymphoid organs are innervated by branches of the autonomic nervous system. Accessory immune cells and lymphocytes have membrane receptors for most neurotransmitters and neuropeptides. These receptors are functional, and their activation leads to changes in immune functions, including cell proliferation, chimiotactism and specific immune responses. Brain lesions and stressors can induce a number of changes in the functioning of the immune system. All these changes are not necessarily mediated by the neuroendocrine system. They can also be dependent on autonomic nerve function. The communication pathways that link the brain to the immune system are normally activated by signals from the immune system, and they serve to regulate immune responses. These signals originate from accessory immune cells such as monocytes and macrophages and they are represented mainly by proinflammatory cytokines. Proinflammatory cytokines produced at the periphery act on the brain via two major pathways: (1) a humoral pathway allowing pathogen specific molecular patterns to act on Toll-like receptors in those brain areas that are devoid of a functional blood-brain barrier, the so-called circumventricular areas; (2) a neural pathway, represented by the afferent nerves that innervate the bodily site of infection and injury. In both cases, peripherally produced cytokines induce the expression of brain cytokines that are produced by resident macrophages and microglial cells. These locally produced cytokines diffuse throughout the brain parenchyma to act on target brain areas so as to organise the central components of the host response to infection (fever, neuroendocrine activation, and sickness behavior).     

Length polymorphisms at two candidate genes explain variation of migratory behaviors in blackpoll warblers (Setophaga striata)

Migratory behaviors such as the timing and duration of migration are genetically inherited and can be under strong natural selection, yet we still know very little about the specific genes or molecular pathways that control these behaviors. Studies in candidate genes Clock and Adcyap1 have revealed that both of these loci can be significantly correlated with migratory behaviors in birds, though observed relationships appear to vary across species. We investigated geographic genetic structure of Clock and Adcyap1 in four populations of blackpoll warblers (Setophaga striata), a Neotropical–Nearctic migrant that exhibits geographic variation in migratory timing and duration across its boreal breeding distribution. Further, we used data on migratory timing and duration, obtained from light‐level geolocator trackers to investigate candidate genotype–phenotype relationships at the individual level. While we found no geographic structure in either candidate gene, we did find evidence that candidate gene lengths are correlated with five of the six migratory traits. Maximum Clock allele length was significantly and negatively associated with spring arrival date. Minimum Adcyap1 allele length was significantly and negatively associated with spring departure date and positively associated with fall arrival date at the wintering grounds. Additionally, we found a significant interaction between Clock and Adcyap1 allele lengths on both spring and fall migratory duration. Adcyap1 heterozygotes also had significantly shorter migration duration in both spring and fall compared to homozygotes. Our results support the growing body of evidence that Clock and Adcyap1 allele lengths are correlated with migratory behaviors in birds.     

Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER

Sec12p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER. Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain. Inhibition is attributable to titration of a limiting cytosolic protein. This limitation is overcome by addition of a highly enriched fraction of soluble Sar1p, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12. Furthermore, Sar1p binding to isolated membranes is enhanced at elevated levels of Sec12p. Sar1p-Sec12p interaction may regulate the initiation of vesicle budding from the ER.     

Biochemical and spectroscopic characterization of the covalent binding of heme to cytochrome b6

The three-dimensional structure of the cytochrome b(6)f complex disclosed the unexpected presence of a new heme c(i) [Stroebel, D., Choquet, Y., Popot, J.-L., and Picot, D. (2003) Nature 426, 413-418; Kurisu, G., Zhang, H., Smith, J. L., and Cramer, W. A. (2003) Science 302, 1009-1014]. Here we present a biochemical, spectroscopic, and mutagenesis study of this unusual heme binding in Chlamydomonas reinhardtii. As predicted by the structure data, we identify a Cys(35)-containing proteolytic fragment (Tyr(25)-Lys(111)) from cytochrome b(6) as a peptide that covalently binds a heme. Resonance Raman spectra of cyt b(6)f complexes show particular frequencies in nu(2), nu(3), nu(4), and nu(8) regions that identify this extra heme as a ferrous c'-like heme under a five-coordinated high-spin state. The set of frequencies is consistent with a coordination by either a water molecule or a hydroxide ion. Other changes in resonance Raman bands, observed in the mid- and low-frequency regions, point to a modification in conformation and/or environment of at least one b heme methyl and/or propionate group. Site-directed mutagenesis of apocytochrome b(6), leading to a Cys(35)Val substitution, generates Chlamydomonas strains that are unable to assemble cytochrome b(6)f complexes. On the basis of the mutant phenotype, we discuss the participation, in the covalent binding of heme c(i), of the nuclear CCB factors that we identified previously as controlling the apo to holo conversion of cytochrome b(6) [Kuras, R., de Vitry, C., Choquet, Y., Girard-Bascou, J., Culler, D., Büschlen, S., Merchant, S., and Wollman, F.-A. (1997) J. Biol. Chem. 272, 32427-32435].