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An Evolutionary Hot Spot: the pNGR234b Replicon of Rhizobium sp . Strain NGR234. W. R. Streit, 2004.Rhizobium sp . strain NGR234 has an exceptionally broad host range and is able to nodulate more than 112 genera of legumes . Since the overall organization of the NGR234 genome is strikingly similar to that of the narrow-host-range symbiont Rhizobium meliloti strain 1021 (also known as Sinorhizobium meliloti), the obvious question is why are the spectra of hosts so different? Study of the early symbiotic genes of both bacteria (carried by the SymA plasmids) did not provide obvious answers . Yet, both rhizobia also possess second megaplasmids that bear, among many other genes, those that are involved in the synthesis of extracellular polysaccharides (EPSs) . EPSs are involved in fine-tuning symbiotic interactions and thus may help answer the broad- versus narrow-host-range question . Accordingly, we sequenced two fragments (total, 594 kb) that encode 575 open reading frames (ORFs) . Comparisons revealed 19 conserved gene clusters with high similarity to R . meliloti, suggesting that a minimum of 28% (158 ORFs) of the genetic information may have been acquired from a common ancestor . The largest conserved cluster carried the exo and exs genes and contained 31 ORFs . In addition, nine highly conserved regions with high similarity to Agrobacterium tumefaciens C58, Bradyrhizobium japonicum USDA110, and Mesorhizobium loti strain MAFF303099, as well as two conserved clusters that are highly homologous to similar regions in the plant pathogen Erwinia carotovora, were identified . Altogether, these findings suggest that  40%
of the pNGR234b genes are not strain specific and were
probably acquired from a wide variety of other microbes . The presence
of 26 ORFs coding for transposases and site-specific integrases
supports this contention . Surprisingly, several genes involved in the
degradation of aromatic carbon sources and genes coding for a type IV
pilus were also found .
Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule. Tomoko Yoshino, 2004.G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery . GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation . In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1 . A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies . Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region . D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule . D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants . After washing, the complexes were ready to use for analysis . This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption . Bacillus anthracis pXO1 Plasmid Sequence Conservation among Closely Related Bacterial Species. James Pannucci, 2002.The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45 . Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera . Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs . Three ORFs were conserved in most of the bacteria tested . Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B . anthracis isolates . Three isolates, Bacillus cereus D-17, B . cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined . The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1 . Pulsed-field gel electrophoresis revealed large potential plasmids present in both B . cereus 43881 (341 kb) and B . thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs . Envelope Disorder of Escherichia coli Cells Lacking Phosphatidylglycerol. Motoo Suzuki, 2002.Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, is considered to play specific roles in various cellular processes that are essential for cell viability . A null mutation of pgsA, which encodes phosphatidylglycerophosphate synthase, does indeed confer lethality . However, pgsA null mutants are viable if they lack the major outer membrane lipoprotein (Lpp) (lpp mutant) (S . Kikuchi, I . Shibuya, and K . Matsumoto, J . Bacteriol . 182:371-376, 2000) . Here we show that Lpp expressed from a plasmid causes cell lysis in a pgsA lpp double mutant . The envelopes of cells harvested just before lysis could not be separated into outer and inner membrane fractions by sucrose density gradient centrifugation . In contrast, expression of a mutant Lpp (Lpp  K) lacking the COOH-terminal lysine residue (required for covalent linking to peptidoglycan) did not cause lysis and allowed for the clear separation of the outer and inner membranes . We propose that in pgsA mutants LppK could not be modified by the addition of a diacylglyceryl moiety normally provided by phosphatidylglycerol and that this defect caused unmodified LppK to accumulate in the inner membrane . Although LppK accumulation did not lead to lysis, the accumulation of unmodified wild-type Lpp apparently led to the covalent linking to peptidoglycan, causing the inner membrane to be anomalously anchored to peptidoglycan and eventually leading to lysis . We suggest that this anomalous anchoring largely explains a major portion of the nonviable phenotypes of pgsA null mutants .
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