The mechanisms of HAMP-mediated signaling in transmembrane receptors

HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.     

Vanillate Metabolism in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.     

Comparison of traditional and molecular typing methods of Escherichia coli O157

An account is given using typing methods and detection of virulence genes of different serotypes of Escherichia coli isolated in Hungary. By hybridization using SLT-I and SLT-II probes and PCR method using stx1-2, eae and ehx primers we could differentiate O157 strains of different serotypes into eight (stx, eae, ehxA positive; stx, eae positive; stx, ehxA positive; stx positive; eae, ehxA positive; eae positive; ehxA positive; stx, eae, ehxA negative) types. The discriminatory power of phage typing proves to be much higher than that of the plasmid profile. RAPD typing with different primers could confirm or exclude the subtypes identity of the isolated E. coli O157 serotypes. Escherichia coli O157:HNM isolates could be sorted in six different phage types and six different RAPD types with ERIC-1, in five RAPD types with ERIC-2 and in seven types with M13 primers. Escherichia coli O157:H7 showed six different phage types and three RAPD types with ERIC-1 and ERIC-2 and five types with M13 primers. According to our results the standard PFGE protocol [32] gives the opportunity to differentiate epidemiologically independent but evolutionary related or unrelated isolates, but the practical value of PFGE method for epidemiological purposes must be confirmed by other or more restriction enzymes or using an other protocol. Summarizing our results we suggest the use of phage and RAPD typing and in doubtful cases the PFGE method.     

Mechanism of regulation of receptor histidine kinases

Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity.     

Flipping the switch: bringing order to flagellar assembly

The bacterial flagellum is a complex self-assembling nanomachine that contains its own type III protein export apparatus. Upon completion of early flagellar structure, this apparatus switches substrate specificity to export late structural subunits, thereby coupling sequential flagellar gene expression with flagellar assembly. The switch is achieved by a conformational change of the export apparatus component FlhB driven by the flagellar hook-length control protein FliK. Two basic models of FliK- and FlhB-based switching are currently being pursued, together with the investigation of another factor, Flk, which prevents premature export of late substrates. Here, we review in detail each of these three export switch components and present the current understanding of how they work in concert in the making of a flagellum.     

Feasibility of using prokaryote biosensors to assess acute toxicity of polycyclic aromatic hydrocarbons

The aim of this study was to assess the acute toxicity of polycyclic aromatic hydrocarbons using lux-marked bacterial biosensors. Standard solutions of phenanthrene, pyrene and benzo[a]pyrene were produced using 50 mM hydroxpropyl-β-cyclodextrin solution which contained each respective polycyclic aromatic hydrocarbon at 6.25 times the aqueous solubility limit of the compound. The polycyclic aromatic hydrocarbon solutions were incubated with each of the biosensors for 280 min and the bioluminescence monitored every 20 min. Over the incubation time period, there was no significant decrease in bioluminescence in any of the biosensors tested with the exception of Rhizobium leguminosarum biovar trifolii TA1 luxAB. In this series of incubations, there was a dramatic increase in bioluminescence in the presence of phenanthrene (2.5 times) and benzo[a]pyrene (3 times) above that of the background control (biosensor without polycyclic aromatic hydrocarbon) after 20 min. Over the next 3 h, bioluminescence decreased to that of the control. An ATP assay was carried out on the biosensors to assess if uncoupling of the oxidative phosphorylation mechanisms in the respiratory chain of the cells had occurred. However, it was found that the polycyclic aromatic hydrocarbons had no effect on the organisms indicating that there was no uncoupling. Additionally, mineralisation studies using ¹⁴C-labelled polycyclic aromatic hydrocarbons showed that the biosensors could not mineralise the compounds. This study has shown that the three polycyclic aromatic hydrocarbons tested are not acutely toxic to the prokaryotic biosensors tested, although acute toxicity has been shown in other bioassays. These results question the rationale for using prokaryote biosensors to assess the toxicity of hydrophobic chemicals, such as polycyclic aromatic hydrocarbons.     

Lack of phosphate incorporation into TN-I in live frog muscle

The phosphorylation of TN-I was investigated in muscles of live frogs injected with [³²P]orthophosphate. Isolation of TN-I was carried out by the rapid and specific technique of affinity chromatography developed by Syska, Perry, and Trayer [FEBS Lett.40, 253–257 (1974)] followed by gel electrophoresis in the presence of sodium dodecyl sulfate. No significant labeling of TN-I was found even in frogs which were exposed to the ³²P-treatment for several days. A comparison of the specific radioactivity of TN-I from resting and contracting frog muscle showed no change in the ³²P content of TN-I during muscle contraction.