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The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.

Author(s): Per Uhlén [1, 2]; Ãsa Laestadius [1, 2]; Timo Jahnukainen [1]; Tomas Söderblom [3]; Fredrik Bäckhed [3]; Gianni Celsi [1]; Hjalmar Brismar [1]; Staffan Normark [3]; Anita Aperia [1]; [...]

The mppA genetic locus was isolated and characterized. The periplasmic binding protein was found to be essential for import of the bacterial cell wall peptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate. Two mutants, one with a mutation in groESL and the other with a mutation in oppB, were shown to be deficient in Opp function due to their rejection of triornithine toxicity. The oppB mutation suggested that murein tripeptide is transferred from MppA into the cytoplasm through membrane components of Opp.

An experiment designed to clone and characterize two operons for curli biogenesis from Salmonella typhimurium SR-11 was conducted. Results show similarities in the gene order and flanking genes of the curli fibers compared with their Escherichia coli counterparts. Biogenesis regulation was the same between S. typhimurium's Sr-11 and ATCC 14028-1 and E. coli's MC4100 and Y Mel.

The genetic characteristics of the novel TlpA protein was analyzed to characterize its thermosensing capacity in bacterial cells. Analysis of transcriptional fusion activity after the induction of tlpA in bacterial cells indicated the role of the protein as a novel gene regulator that exhibited a built-in thermosensing capacity. The sensor-regulator activity of TlpA was also based on a monomer-to-coiled-coil equilibrium which represented a unique adaptation of the coiled-structure for the regulation of gene function.

The potential interactions of the transcriptional regulator AmpR with intermediates of the degradative and/or biosynthetic pathways of murein metabolism in gram negative bacteria were investigated. Transcription assay results revealed that AmpR activates ampC beta-lactamase synthesis. The murein precursor, UDP-MurNAc-pentapeptide, on the other hand, inhibits AmpR-mediated transcriptional activation in vitro but such inhibition can be offset by the action of the muropeptide, anhMurNAc-tripeptide which accumulates in beta-lactamase-overproducing mutants. It is thus evident that cytosolic intermediates of murein biosynthesis and degradation operate as a cell-wall sensing device as they control beta-lactamase production.

The pilC1 gene, present in the MS11 strain of Neisseria gonorrhoeae, contains amino acid sequences similar to those present in the opa and pil gene locus. These amino acid sequences are repetitive in nature and are probably involved in recombination during the formation of pili, an antigen. The amino acid structure of pilC1 is 90% similar to that of pilC2 gene. The pilC1 gene is a part of the chromosome that encodes for pili and is formed by DNA duplication. Pili controls attachment of the bacterial cell to the host cell. The amino acid sequence of pilC1 is given.

The research of the role of asparagine 152 in the catalytic process of Escherichia coli AmpC beta-lactamase was done by site-directed mutagenesis. The substrate hydrolysis rate decreases by factors of 500-5000. The rate of acylation registers a 2-50-fold decrease and deacylation registers a 50-500-fold decrease. The wild-type AmpC beta-lactamase exists as an equilibrium mixture of the two forms.

An investigation of the requirement of the GII adhesin for the induction of acute pyelonephritis and/or cystitis by P-fimbriated Escherichia coli using an isogenic papG mutant DS17-8 reveals that the wild-type DS17 and DS17-8 induce bladder infection in cynomolgus monkeys while DS17 results in pyelonephritis. The absence of the Gal(alpha1-4)Gal-specific PapG adhesin in the mutant DS17-8 indicates that the incidence of acute pyelonephritis requires the ability of P-fimbriae to bind carbohydrates.

In beta lactam hydrolysis, serine hydroxyl groups occupy the position of the hydroxyl group of Try 150 in class A enzymes and the two hydroxyl groups function as a general base in catalysis or at least protonate the nitrogen as it is exuded during the formation of acyl intermediate. Tyr 150 action is similar to the action of essential histidine of serine proteases to enhance protein bond hydrolysis. The effects of mutagenesis of the serine residue on the activity of class A enzyme are different from those with AmpC enzyme due to perturbation of structure by the residue.

Site-directed mutagenesis enables the investigation of the function of conserved Lys315 residue in the catalytic process of class C beta-lactamase. Histidine replaces Lys315 in Enterobacter cloacae. The replacement causes introduction of a titratable group in the probe to help determine the function of Lys315.

The only family of bacterial proteins identified to have an immunoglobulin fold is needed for the assembly of adhesive pili. These proteins employ their unique immunoglobulin features in recognition processes that are being studied. Investigations into the action of periplasmic chaperones also reveal a great deal about the basic biological processes of assembly functions. Not all bacterial adhesins are assembled into pilus rods, and the ones that cannot are called nonpilus adhesins.

A new mechanism for the phagocytic processing of bacterial antigens by class I major histocompatibility complex I molecules has been discovered. This mechanism, which works for antigens lacking cytosolic penetration, produces resistance to the drugs cycloheximide and Brefeldin A. In contrast, these drugs are able to inhibit the classical class I pathway. This pathway may allow CD8 T-cells to be used against intracellular pathogens.

The in vivo processing of lipase in Staphylococcus aureus was elucidated by analysis of lipase gene products and culture supernatants. The results indicated that S. aureus lipase is secreted as an 82 KDa prolipase having full lipolytic activity, which is subsequently processed outside the cell. The processing is hypothesized to occur in a stepwise manner with the intermediates having full lipolytic activity as the mature 45 to 46 KDa lipase. A metallocysteine protease seems to be involved in the initial step, while other different proteases contribute to successive processing.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.04.032 Byline: Robert Daniels (1)(2), Staffan Normark (1)(2) Abstract: The chaperone/usher pathway is responsible for the assembly of adhesive pili on the surface of gram-negative pathogenic bacteria. In this issue, present the crystal structure of the PapC usher translocation domain and images of the FimD usher bound to a pilus translocation intermediate. These new structures provide the first detailed view of a translocase in action. Author Affiliation: (1) Swedish Institute for Infectious Disease Control, 171 82 Solna, Sweden (2) Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden