The morphogenesis of rotaviruses follows a unique pathway in which immature double-layered particles (DLPs) assembled in the cytoplasm bud across the membrane of the endoplasmic reticulum (ER), acquiring during this process a transient lipid membrane which is modified with the ER resident viral glycoproteins NSP4 and VP7; these enveloped particles also contain VP4. As the particles move towards the interior of the ER cisternae, the transient lipid membrane and the nonstructural protein NSP4 are lost, while the virus surface proteins VP4 and VP7 rearrange to form the outermost virus protein layer, yielding mature infectious triple-layered particles (TLPs). In this work, we have characterized the role of NSP4 and VP7 in rotavirus morphogenesis by silencing the expression of both glycoproteins through RNA interference. Silencing the expression of either NSP4 or VP7 reduced the yield of viral progeny by 75 to 80%, although the underlying mechanism of this reduction was different in each case. Blocking the synthesis of NSP4 affected the intracellular accumulation and the cellular distribution of several viral proteins, and little or no virus particles (neither DLPs nor TLPs) were assembled. VP7 silencing, in contrast, did not affect the expression or distribution of other viral proteins, but in its absence, enveloped particles accumulated within the lumen of the ER, and no mature infectious virus was produced. Altogether, these results indicate that during a viral infection, NSP4 serves as a receptor for DLPs on the ER membrane and drives the budding of these particles into the ER lumen, while VP7 is required for removing the lipid envelope during the final step of virus morphogenesis.Rotaviruses are nonenveloped icosahedral viruses whose capsid is formed by three concentric layers of protein. The innermost layer is formed by 60 dimers of VP2 that surrounds the viral genome composed of 11 segments of double-stranded RNA and 12 copies of each VP1, the virus polymerase, and VP3, the virus capping enzyme. The second layer of protein is formed by 280 trimers of VP6, which sits on top of VP2 to form double-layered particles (DLPs). Finally, the addition of 280 trimers of glycoprotein VP7 which constitute the outermost layer of the virus and 60 dimeric spikes of the VP4 protein to DLPs form triple-layered particles (TLPs) that represent the mature infectious virus (13).Rotavirus morphogenesis occurs by an unusual process where DLPs, which are thought to assemble in cytoplasmic inclusions termed viroplasms, bud across the membrane of the endoplasmic reticulum (ER). During this process, the DLPs acquire a transient lipid envelope which is subsequently lost to yield the mature infectious TLPs (33). The ER membrane through which DLPs bud is modified by two viral proteins, the virion surface protein VP7 and the nonstructural polypeptide NSP4 (5, 7, 21). NSP4 has a large cytosolic domain that interacts with DLPs, and it has been proposed that this interaction drives the translocation of the double-layered particles into the lume...
The study of the biosynthesis of alginate, the exopolysaccharide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, has biotechnological and medical significance. We report here the identification of the A. vinelandii genes coding for the putative sigma factor AlgU and its negative regulators MucA and MucB through the suppression of the highly mucoid phenotype of an A. vinelandii strain by a plasmid encoding MucA and MucB. The sequences of the A. vinelandii algU, mucA, and mucB genes are highly homologous to those of the corresponding P. aeruginosa genes, AlgU shows 93% identity, and MucA and MucB are 64.4 and 63.9% identical, respectively. Forming part of the same operon as algU, mucA, and mucB, two additional genes (mucC and mucD) were identified and sequenced; the product of the former gene is homologous to ORF4 of Photobacterium sp. strain SS9, and that of the latter gene belongs to the HtrA serine protease family. Interestingly, the nonmucoid A. vinelandii UW136 had a 0.9-kb insertion within the algU gene. A strong correlation between AlgU activity and alginate production by A. vinelandii was also found, as reflected in the level of algD transcription.
Different structural changes of the Sym plasmid were found in a Rhizobium phaseoli strain that loses its symbiotic phenotype at a high frequency. These rearrangements affected both nif genes and TnS mob insertions in the plasmid, and in some cases they modified the expression of the bacterium's nodulation ability. One of the rearrangements was more frequent in heat-treated cells, but was also found under standard culture conditions; other structural changes appeared to be related to the conjugal transfer of the plasmid.The genus Rhizobium comprises the gram-negative bacteria that form nodules on legumes. In this association, the bacteria fix atmospheric nitrogen that is then assimilated by the plant.The genetic information controlling symbiotic activity in the fast-growing rhizobia is encoded in plasmids (10,11,13,19). A symbiotic (Sym) plasmid has been defined as one that determines the plant species specificity for nodulation and contains the nitrogenase enzyme structural genes (nifgenes) (12).Plasmids participate very frequently in recombination events (14,20). It has been proposed (27) that this plasticity enables the bacteria that harbor plasmids to adapt to different environmental changes and permits the rapid spread of newly created functions among very diverse bacteria. There is one report (24) of a change in the structure of a plasmid that resulted in modified metabolic activity in the recipient bacteria. This strongly suggests that plasmid plasticity is important in the generation of new functions in bacteria.In Rhizobium phaseoli, the nitrogen fixation gene sequences are reiterated (25). In strain CFN42, there are three regions of the Sym plasmid that contain nitrogenase structural genes (nifregions) (22). These three regions contain the nitrogenase reductase gene (nifH); the nucleotide sequence of the three copies is identical (26). In addition, two of the regions contain also nifD and nifK genes. The identity of the nifH genes suggests that a recombination event could be involved in the generation or maintenance of their reiteration. Reiterated sequences are not common in bacteria, but they have been found in some strains of R. phaseoli, Rhizobium trifolii, and Rhizobium japonicum (2) and also Streptomyces sp. (21), Halobacterium halobium (29), and Pseudomonas syringae p.v. "phaseolicola" (32). The presence of these reiterations may be related to the instability and genetic rearrangements of these organisms (1, 6, 23).We have found that symbiotically unstable isolates are very common among the R. phaseoli strains isolated in different regions of Mexico, and all have reiterated nifH genes (L. Castrejon and G. Soberon, manuscript in preparation). We suppose that the symbiotic instability of these R. phaseoli strains is due to genetic rearrangements caused by the presence of reiterated sequences. We report here that the loss of the symbiotic phenotype of an unstable R. phaseoli strain is due to changes in the structure of its Sym plasmid, * Corresponding author. and that this Sym plasmid can be involve...
A Pseudomonas aeruginosa gene homologous to thefabG gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of thisfabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-β-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by therhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent β-ketoacyl reductase absolutely required for the synthesis of the β-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.
Bacteria resembling Rhizobium leguminosarum, but lacking symbiotic information, were isolated from soil of two different geographical origins. One of these bacteria belongs to a previously described Rhizobium leguminosarum bv. phaseoli somatic serogroup, is fully complemented for nodulation and nitrogen fixation by an R. leguminosarum bv. phaseoli symbiotic plasmid, and is able to compete for bean nodulation with indigenous R. leguminosarum bv. phaseoli strains. This is the first report giving evidence for persistence in soil of Rhizobium lacking symbiotic information.Key words: Rhizobium ecology, symbiotic plasmid, nodulation, plasmid transfer.
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