Cryo-EM structure of the bifunctional secretin complex of Thermus thermophilus.

Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.

Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus.

Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural transformation of DNA in the thermophilic bacterium Thermus thermophilus requires a unique secretin complex comprising six stacked rings, a membrane-embedded cone structure, and two gates that open and close a central channel. To investigate the role of distinct domains in ring and gate formation, we examined a set of deletion derivatives by cryomicroscopy techniques. Here we report that maintaining the N0 ring in the deletion derivatives led to stable PilQ complexes. Analyses of the variants unraveled that an N-terminal domain comprising a unique ßßßαß fold is essential for the formation of gate 2. Furthermore, we identified four ßαßßα domains essential for the formation of the N2 to N5 rings. Mutant studies revealed that deletion of individual ring domains significantly reduces piliation. The N1, N2, N4, and N5 deletion mutants were significantly impaired in T4P-mediated twitching motility, whereas the motility of the N3 mutant was comparable with that of wild-type cells. This indicates that the deletion of the N3 ring leads to increased pilus dynamics, thereby compensating for the reduced number of pili of the N3 mutant. All mutants exhibit a wild-type natural transformation phenotype, leading to the conclusion that DNA uptake is independent of functional T4P.

Identification and characterization of a unique, zinc-containing transport ATPase essential for natural transformation in Thermus thermophilus HB27.

Thermus thermophilus is a model strain to unravel the molecular basis of horizontal gene transfer in hot environments. Previous genetic studies led to the identification of a macromolecular transport machinery mediating DNA uptake in an energy-dependent manner. Here, we have addressed how the transporter is energized. Inspection of the genome sequence revealed four putative transport (AAA) ATPases but only the deletion of one, PilF, led to a transformation defect. PilF is similar to transport ATPases of type IV and type II secretions systems but has a unique N-terminal sequence that carries a triplicated GSPII domain. To characterize PilF biochemically it was produced in Escherichia coli and purified. The recombinant protein displayed NTPase activity with a preference for ATP. Gel filtration analyses combined with dynamic light scattering demonstrated that PilF is monodispersed in solution and forms a complex of 590 ± 30 kDa, indicating a homooligomer of six subunits. It contains a tetracysteine motif, previously shown to bind Zn(2+) in related NTPases. Using atomic absorption spectroscopy, indeed Zn(2+) was detected in the enzyme, but in contrast to all known zinc-binding traffic NTPases only one zinc atom was bound to the hexamer. Deletion of the four cysteine residues led to a loss of Zn(2+). Nevertheless, the mutant protein retained ATPase activity and hexameric complex formation.

Nocardichelins A and B, siderophores from Nocardia strain acta 3026.

An actinomycete, strain Acta 3026, isolated from mangrove soil was characterized and found to belong to the genus Nocardia. The strain produces two new cytotoxic metabolites, nocardichelins A (1) and B (2). Each of the compounds strongly inhibited human cell lines from gastric adenocarcinoma, breast carcinoma, and hepatocellular carcinoma with GI50 values in a low micromolar to nanomolar range. The structural characterization of the compounds was performed by mass spectrometry and NMR spectroscopy. The nocardichelins represent a new group of siderophores that combine the structural elements of mycobactin-type siderophores from mycobacteria and hydroxamate-type siderophores (desferrioxamine B) produced by streptomycetes. The chromazurol S assay, characteristic for iron(III) complexation, was positive, confirming the role as a siderophore.

The effect of prenatal hypoxia and malnutrition on memory consolidation in the chick.

The contribution of hypoxia and malnutrition to cognitive impairments was investigated in chicks incubated in conditions of reduced gas exchange. Previous research has shown that reducing gas exchange during incubation by wrapping half the eggshell with an impermeable membrane results in impaired cognitive ability in young chicks. The results were interpreted within a three stage sequential model of memory using discriminated bead avoidance learning. Reducing gas exchange for 4 days from day 10 or 14, of the 21-day incubation, inhibits memory formation and consolidation into permanent storage. The nature of the cognitive deficit depended on the timing of the insult. Environmental hypoxia (14% oxygen), induced from days 10 to 14 and from days 14 to 18, replicated the memory deficits found previously when eggs were partially wrapped with a membrane. Oxygen is necessary to break down food and to provide energy to build tissue proteins, and therefore hypoxia (partial wrapping or environmental incubation) may indirectly cause malnutrition. Malnutrition, induced by removing 5%, 7.5% or 10% albumin from the egg prior to incubation, had no significant effect on memory consolidation. Raised corticosterone levels occurred in chicks malnourished by 5% and 7.5%, but brain sparing was only evident in chicks with 7.5% albumin removal. Hatch rates were very low in 10% malnourished chicks. Using the chick as a model of prenatal stress, we have been able to isolate the effects of hypoxia from contributing maternal factors.