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Architecture of the Bacteroides cellulosolvens Cellulosome: Description of a Cell Surface-Anchoring Scaffoldin and a Family 48 Cellulase. Qi Xu, 2004.A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced . The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an "X" domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module . In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected . Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed . Their interaction with ScaA and ScaB cohesins was examined by immunoassay . The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5 . Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module . Compared to previously described cellulosome systems, the apparent roles of the B . cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin . The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature . Cloning and Characterization of the Phosphatidylserine Synthase Gene of Agrobacterium sp . Strain ATCC 31749 and Effect of Its Inactivation on Production of High-Molecular-Mass (1  3)-ß-D-Glucan (Curdlan).Tara Karnezis, 2002.Genes involved in the production of the extracellular (1 3)-ß-glucan, curdlan, by Agrobacterium sp . strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999) . To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe . One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pssAG, encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted . The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased . In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the PssAG protein at its first step . Moreover, PC can be produced in a medium lacking choline . When the pssAG::TnphoA mutation was complemented by the intact pssAG gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics . The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport .
Repression of Glutamate Dehydrogenase Formation in Klebsiella aerogenes Requires Two Binding Sites for the Nitrogen Assimilation Control Protein, NAC. Thomas J. Goss, 2002.In Klebsiella aerogenes, the gdhA gene codes for glutamate dehydrogenase, one of the enzymes responsible for assimilating ammonia into glutamate . Expression of a gdhAp-lacZ transcriptional fusion was strongly repressed by the nitrogen assimilation control protein, NAC . This strong repression (>50-fold under conditions of severe nitrogen limitation) required the presence of two separate NAC binding sites centered at -89 and +57 relative to the start of gdhA transcription . Mutants lacking either or both of these sites lost the strong repression . The distance between the two sites was less important than the face of the helix on which they lay . Insertion or deletion of 10 bp between the sites had little effect on the strong repression, but insertion of 5 bp or deletion of either 5 or 15 bp decreased the repression significantly . We propose that the strong repression of gdhAp-lacZ expression requires an interaction between the NAC molecules bound at the two sites . A weaker repression of gdhAp-lacZ expression (about threefold) required only the NAC site centered at -89 . This weaker repression appears to result from NAC's ability to prevent the action of a positive effector the target of which overlaps the NAC binding site centered at -89 . Point mutations and deletions of this region result in the same threefold reduction in gdhAp-lacZ expression as the presence of NAC at this site . A Model To Estimate the Optimal Sample Size for Microbiological Surveys. S. F. Altekruse, 2003.Estimating optimal sample size for microbiological surveys is a challenge for laboratory managers . When insufficient sampling is conducted, biased inferences are likely; however, when excessive sampling is conducted valuable laboratory resources are wasted . This report presents a statistical model for the estimation of the sample size appropriate for the accurate identification of the bacterial subtypes of interest in a specimen . This applied model for microbiology laboratory use is based on a Bayesian mode of inference, which combines two inputs: (ii) a prespecified estimate, or prior distribution statement, based on available scientific knowledge and (ii) observed data . The specific inputs for the model are a prior distribution statement of the number of strains per specimen provided by an informed microbiologist and data from a microbiological survey indicating the number of strains per specimen . The model output is an updated probability distribution of strains per specimen, which can be used to estimate the probability of observing all strains present according to the number of colonies that are sampled . In this report two scenarios that illustrate the use of the model to estimate bacterial colony sample size requirements are presented . In the first scenario, bacterial colony sample size is estimated to correctly identify Campylobacter amplified restriction fragment length polymorphism types on broiler carcasses . The second scenario estimates bacterial colony sample size to correctly identify Salmonella enterica serotype Enteritidis phage types in fecal drag swabs from egg-laying poultry flocks . An advantage of the model is that as updated inputs from ongoing surveys are incorporated into the model, increasingly precise sample size estimates are likely to be made .
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