Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.     

Knock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H₂O₂ production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H₂O₂ production in the dark but a two to threefold increase of H₂O₂ in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.     

Determination of the Diversity of Rhodopirellula Isolates from European Seas by Multilocus Sequence Analysis

In the biogeography of microorganisms, the habitat size of an attached-living bacterium has never been investigated. We approached this theme with a multilocus sequence analysis (MLSA) study of new strains of Rhodopirellula sp., an attached-living planctomycete. The development of an MLSA for Rhodopirellula baltica enabled the characterization of the genetic diversity at the species level, beyond the resolution of the 16S rRNA gene. The alleles of the nine housekeeping genes acsA, guaA, trpE, purH, glpF, fumC, icd, glyA, and mdh indicated the presence of 13 genetically defined operational taxonomic units (OTUs) in our culture collection. The MLSA-based OTUs coincided with the taxonomic units defined by DNA-DNA hybridization experiments. BOX-PCR supported the MLSA-based differentiation of two closely related OTUs. This study established a taxon-area relationship of cultivable Rhodopirellula species. In European seas, three closely related species covered the Baltic Sea and the eastern North Sea, the North Atlantic region, and the southern North Sea to the Mediterranean. The last had regional genotypes, as revealed by BOX-PCR. This suggests a limited habitat size of attached-living Rhodopirellula species.The biogeography of microorganisms describes the habitat size of the species and the distribution of microorganisms on Earth. The experimental approaches depend on the focus of the studies. Habitats are often analyzed by environmental microbiologists with genetic-fingerprinting techniques, with up to 200 bands or fragments representing the whole community. Although the taxonomic resolution of these operational taxonomic units (OTUs) is limited, the studies revealed a community biogeography (22). Medical microbiologists analyze the alleles of housekeeping genes of microorganisms to gain insight into the epidemiology of pathogens, the population biogeography (2). This strain-specific, fine-scale taxonomic resolution within a species is well suited to observance of recent dispersal events. At the species level, multilocus sequence typing (MLST) and analysis (MLSA), which were developed for intraspecies and intragenus specific studies, respectively, consist of the sequences of several (at least seven) housekeeping gene fragments concatenated to an approximately 5-kilobase alignment (17). Recent MLSA studies revealed its applicability to marine isolates and the analysis of biogeographic patterns: Alteromonas macleodii isolates could be grouped in an epipelagic and an abyssal clade (6), and strains of Pseudomonas aeruginosa were genetically well separated into groups of coastal and oceanic origin (8). However, for Salinibacter ruber strains, biogeographical distinctness was not resolved in an MLSA study but showed allopatry in a metabolic analysis (31). Several studies used MLSA together with DNA-DNA hybridization (DDH) for the delineation of new species, e.g., for Vibrio and Ensifer spp. (20, 36).In the biogeography of microorganisms, the experimental proof of a local genetic evolution was first revealed at sample sites that were physically separated by over 18,000 km (39). Large populations and the small size of microbes have been considered as facilitators for dispersal over long distances, eventually establishing cosmopolitan microbial populations. On the other hand, the smallest spatial scale of a microbial species in an open system has not been investigated. Attached-living bacteria disperse only during a distinct, short time span in their lives. This limitation of the dispersal time stimulated this study of the biogeography of Rhodopirellula baltica in European seas.R. baltica is a planctomycete with typical morphological features. The peptidoglycanless bacteria have an intracellular compartmentation: the riboplasm with the nucleoid is separated by a membrane from the surrounding paryphoplasm. Cells attach with a holdfast substance to surfaces or, in culture, to themselves, forming typical rosettes. Proliferation occurs by budding, and offspring cells live free in the water column: they are motile with a flagellum until they settle on the sediment (4).Seventy recently isolated strains affiliated according to the 16S rRNA gene analysis with R. baltica SH1^T as the closest validly described species (40). The 16S rRNA gene sequences do not offer sufficient information at the species level. A dissimilarity of the 16S rRNA genes of more than 3%, recently reduced to 1.3% (34, 35), indicates that the strains under consideration belong to two species. These thresholds yielded in our strain collection, according to an ARB-based calculation, five or eight operational taxonomic units besides the species R. baltica (40). For strains with highly identical sequences, whole-genome DDH experiments have to be performed to identify the affiliation to established species. Recently, multilocus sequence analyses have emerged as a possible alternative method. Our strain collection comprised many strains with a 16S rRNA gene sequence very closely related to that of R. baltica SH1^T. To gain insight into the genetic identity of the isolates on the species level and the habitat sizes of the species, we developed a multilocus sequence analysis and applied it to the strain collection. The MLSA results were calibrated with a DDH study. The closely related strains were additionally characterized by BOX-PCR, a fingerprinting method (15). Transmission electron microscopy (EM) was performed on some isolates to support the identification as Planctomycetes and to visualize morphological differences between strains.     

Homologous expression of the nrdF gene of Corynebacterium ammoniagenes strain ATCC 6872 generates a manganese-metallocofactor (R2F) and a stable tyrosyl radical (Y˙) involved in ribonucleotide reduction

Ribonucleotide reduction, the unique step in the pathway to DNA synthesis, is catalyzed by enzymes via radical-dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene (nrdF) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac-promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron (30:1), high activity (69 μmol 2'-deoxyribonucleotide·mg?1 ·min?1) and distinct absorption at 408 nm, characteristic of a tyrosyl radical (Y˙), which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of Y˙ in ribonucleotide reduction because 0.2 nmol 2'-deoxyribonucleotide was formed, driven by 0.4 nmol Y˙ located on R2F. X-band electron paramagnetic resonance spectroscopy demonstrated a tyrosyl radical at an effective g-value of 2.004. Temperature dependent X/Q-band EPR studies revealed that this radical is coupled to a metallocofactor. Similarities of the native C. ammoniagenes ribonucleotide reductase to the in vitro activated Escherichia coli class Ib enzyme containing a dimanganese(III)-tyrosyl metallocofactor are discussed.     

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.     

Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I–F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I–F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans- homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2- and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM-mediated signaling.     

Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4

N-terminal truncation of NPY has important physiological consequences, because the truncated peptides lose their capability to activate the Y1-receptor. The sources of N-terminally truncated NPY and related peptides are unknown and several proline specific peptidases may be involved. First, we therefore provide an overview on the peptidases, belonging to structural and functional homologues of dipeptidyl peptidase 4 (DP4) as well as aminopeptidase P (APP) and thus, represent potential candidates of NPY cleavage in vivo. Second, applying selective inhibitors against DP4, DP8/9 and DP2, respectively, the enzymatic distribution was analyzed in brain extracts from wild type and DP4 deficient F344 rat substrains and human plasma samples in activity studies as well as by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry. Third, co-transfection of Cos-1 cells with Dpp4 and Npy followed by confocal lasermicroscopy illustrated that hNPY-dsRed1-N1 was transported in large dense core vesicles towards the membrane while rDP4-GFP-C1 was transported primarily in different vesicles thereby providing no clear evidence for co-localization of NPY and DP4. Nevertheless, the review and experimental results of activity and mass spectrometry studies support the notion that at least five peptidases (DP4, DP8, DP9, XPNPEP1, XPNPEP2) are potentially involved in NPY cleavage while the serine protease DP4 (CD26) could be the principal peptidase involved in the N-terminal truncation of NPY. However, DP8 and DP9 are also capable of cleaving NPY, whereas no cleavage could be demonstrated for DP2.     

Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.