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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The virD operon of the resident Ti plasmid of Agrobacterium tumefaciens contains loci involved in T-DNA processing and undefined virulence functions. Nucleotide sequence of the entire virD operon of pTiC58 revealed similarities to the virD operon of the root-inducing plasmid pRiA4b and to that of the octopine-type plasmid pTiA6NC. However, comparative sequence data show that virD of pTiC58 is more akin to that of the pRiA4b than to that of the pTiA6NC. T7f10::virD gene fusions were used to generate polypeptides that confirm the presence of four open reading frames virD1, virD2, virD3, and virD4 within virD which have a coding capacity for proteins of 16.1, 49.5, 72.6, and 73.5 kDa, respectively. virD3 therefore encodes a polypeptide 3.4 times larger (72.6 versus 21.3 kDa) than that encoded by virD3 of octopine Ti plasmids. Non-polar virD4 mutants could not be complemented by a distant homologue, TraG protein of plasmid RP4. An independently regulated fifth ORF (orf5) is located immediately downstream of 3′ end of virD4 and encodes a polypeptide of 97.4 kDa. The expression of orf5 is dependent on its own promoter and is independent of acetosyringone induction in A. tumefaciens. Recently, it has been shown that virD3 of octopine Ri or Ti plasmids is not required for virulence. In this report, we confirm and extend these findings on a nopaline Ti plasmid by using several virD non-polar mutants that were tested for virulence. virD3 and orf5 non-polar mutants showed no effect on tumorigenicity on 14 different plant species, while virD4 mutants lost their tumorigenicity completely on all these test plants. These data suggest that virD3 and orfS are not essential for virulence whereas virD4 is absolutely required on a wide range of host plants.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: SCG1/GPA1, STE4, and STE18 encode the α, β and λ components of the G protein involved in mating pheromone signal transduction in Saccharomyces cerevisiae. Responses, including G1 arrest and expression of genes such as FUS1, are activated by βλ, which is negatively controlled by α(GDP), We previously demonstrated that overexpression of Scg1 suppresses responses to α factor and that expression of certain hybrids between Scg1 and mammalian Gα proteins has the same effect and also suppresses growth arrest in an scg1-null mutant. Effects were attributed to sequestration of βλ. We now show that effects on growth rate, morphology and FUS1 expression are consistent with this model. The STE4HPL allele causes dominant activation of the response pathway, and is presumed to encode a β subunit insensitive to control by α(GDP). Scg1 overexpression suppresses the growth arrest due to STE4HPL; normal α-factor responses and fertility are restored. A model based on sequestration of βγ reconciles this result with the apparent paradox that the same level of Scg1 overexpression inhibits responses and mating in wild-type cells. A Gαi hybrid also restores growth and allows inefficient mating in the STEHPL strain.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The DNA cytosine methylase gene of Escherichia coli, dem, overlaps an open reading frame (ORF) that continues in +1 register past the end of dcm. This ORF codes for a gene, vsr, that is required for a T:G to C:G base mismatch correction process. In this study, mutants that affect the level of expression of the two genes were constructed and characterized. Further, a previously isolated mutant, dcm-6, was cloned and mutations within it were identified. Northern blots were used to identify dcm-specific RNA species in wild type and dcm-6 cells. Based on these studies we conclude that there is a six-codon overlap between vsr and dcm. The two proteins appear to be made from a single RNA transcript and translation of dcm is required for the efficient synthesis of Vsr. Further, Vsr is active by itself and may not be produced as a fusion with Dem. This is the first example of chromosomal genes that overlap in their coding regions and produce proteins with distinct functions.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Two genomic sequences from the halophilic archaeon Haloferax mediterranei, where we had found PstI restriction-pattern modifications depending on the salinity of the growth medium, have been studied. A markedly salt-dependent differential expression has been detected in the nearby regions. Two of the open reading frames characterized correspond to two of the differentially expressed transcripts. In both cases the PstI sites were included in purine–pyrimidine alternancies suggestive of Z-DNA structures and located in non–coding regions with frequent repetitive motifs. A long alternating adenine-thymine tract also appears in the upstream regions of one of these open reading frames. A possible role of local DNA configuration in osmoregulation in this organism is discussed.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The LipB protein of Pseudomonas glumae is essential for the production of active extracellular lipase encoded by the lipA gene. When lipase is overproduced in P. glumae in the absence of a functional lipB gene, the enzyme accumulates intracellularly in an inactive conformation. Heterologous expression of the lipase in Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli indicated that LipB is not directly involved in the trans location of the lipase across the inner or outer membrane. However, the presence of LipB was essential for obtaining active lipase and had a profound influence on the stability of the protein to proteolytic degradation. Inactive iipase, produced in the absence of LipB could be activated in vitro by unfolding and refolding, which demonstrates that LipB activity is not responsible for an essential covalent modification of the enzyme. We propose that LipB is a lipase-specific foldase. Furthermore, proper folding of the lipase in the periplasm appears to be essential for Xcp-mediated translocation across the outer membrane.
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Myxococcus xanthus is a Gram-negative bacterium that glides on a solid surface and displays a wide range of social behaviour including microbial development. The frz genes are homologues to the chemotaxis genes of Escherichia coli and Salmonella typhimurium and have been shown to be involved in microbial development. However, chemotaxis has never been clearly demonstrated in Myxococcus. In this study, we showed that M. xanthus exhibited tactic movements to many chemicals when they were subjected to steep and stable chemical gradients. M. xanthus was observed to spread into areas with abundant nutrients like yeast extract or Casitone and avoid areas with no nutrients or repellents (short-chain alcohols or DMSO. Responses to attractants and repellents were additive. Movement towards attractants or away from repellents required the frz genes and was correlated with methylation or demethylation of FrzCD, a methyl-accepting taxis protein. Furthermore, the frz genes were found to be required for both fruiting body formation during starvation and swarming in nutrient-rich medium. In wild-type strains, cells near the colony edge were observed to swarm towards the surrounding growth medium and to contain highly methylated FrzCD; cells near the colony centre contained mainly demethylated FrzCD and showed directed movement towards the colony edge. FrzCD was also found to be methylated during the aggregation stage of fruiting body formation on agar but largely demethylated in cells shaken in liquid starvation media. An frzf mutant failed to exhibit directed cell movements and no longer showed modification of FrzCD under these conditions. These observations suggest that M. xanthus does show chemotactic movements, that these movements require the frz genes, and that chemotaxis plays a very important role in the social behaviour of this organism.
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We report the purification of a minor Bordetella pertussis fimbrial subunit, designated FimD, and the identification of its gene (fimD.) FimD could be purified from the bulk of major fimbrial subunits by exploiting the fact that major subunit-subunit interactions are more stable in the presence of SDS than minor-major subunit interactions. To locate the gene for FimD, internal peptides of FimD were generated, purified and sequenced. Subsequently, an oligonucleotide probe, based on the primary sequence of one peptide, was used to clone fimD. The primary structure of FimD, derived from the DNA sequence of its gene, showed homology with a number of fimbrial adhesins. Most pronounced homology was observed with MrkD, a fimbrial adhesin derived from Klebsieila pneumoniae. These observations suggest that FimD may represent a B. pertussis fimbrial adhesin. With a fimD-specific probe we detected the presence of a fimD homologue in Bordetella parapertussis and Bordetella bron-chiseptica but not in Bordetella avium. Cloning and sequencing revealed that the B. parapertussis and B. bronchiseptica fimD product differed from the B. pertussis fimD product in 20 and 1 amino acid residues, respectively. Since B. bronchiseptica is normally not a human pathogen, but causes respiratory disease in a wide range of non-human mammalian species, this may suggest that FimD recognizes a receptor that is well conserved in mammalian species. An in-frame deletion in fimD completely abolished FimD expression and also affected the expression of the major subunits Fim2 and Fim3 suggesting that, in contrast to other adhesins that are minor components of fimbriae, FimD is required for formation of the fimbrial structure.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Helicobacter pylori is a human pathogen that has been associated with gastritis, peptic ulcer and gastric carcinoma. The role of the direct action of H. pylori virulence factors and of the induction of autoreactive immunity in the development of chronic gastritis has not been clarified yet. Here we report the cloning and molecular characterization of a gene of H. pylori coding for a protein of 58kDa, recognized by sera of patients affected by H. pylori-induced gastroduodenal diseases. This antigen is present in all the H. pylori strains tested and it belongs to the Hsp60 family of heat-shock proteins, with high homology with other bacterial and eukaryotic proteins of the same family. This class of homologous proteins has been implicated in the induction of autoimmune disorders in different systems. The presence in infected patients of anti-H. pylori Hsp60 antibodies, potentially cross-reacting with the human homologue, and cross-reactivity between human Hsp60 and a rabbit antiserum against H. pylori Hsp60 suggest that a role of this protein in gastroduodenal diseases is possible.
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Aerolysin is a channel-forming protein secreted by Aeromonas hydrophila. To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E. coli, Aeromonas salmonicida, and A. hydrophila. We were surprised to find that secretion of the enzyme by both Aeromonas spp. was independent of the aerolysin segments fused to it. The smallest fusion product contained only the signal sequence and two amino acids of aerolysin. The largest had more than 90% of the aerolysin molecule. The fusion proteins were found in the periplasms of E. coli and A. salmonicida grown in LB medium containing glucose, as well as in the shocked cells. Aerolysin itself was secreted by A. salmonicida under these conditions. In contrast, when A. salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas β-lactamase remained in its normal periplasmic location. Similar results were obtained with A. hydrophila. The change in location of the enzyme in A. salmonicida appeared to be related to the pH of the growth medium. A. salmonicida and A. hydrophila also secreted native E. coli alkaline phosphatase, but A. hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme. We conclude that the Aeromonas secretion system can recognize the E. coli enzyme as an extracellular protein and direct it outside the cell.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The two reaction-centre proteins of the photosystem I (PSI) complex are encoded by two adjacent genes named psaA and psaB. We have performed targeted mutagenesis to insertionally inactivate each of these genes in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. The resulting mutant strains, termed psaA:: NmR and psaB:: NmR, were blue because of a high ratio of phycobilin to chlorophyll and were unable to grow in light. These mutant cells also lacked chemically reducible P700 (the reaction-centre chlorophylls of PSI) and as a consequence did not exhibit any PSI-mediated photochemical activity. However, their photosystem II (PSII) complexes were fully active. The loss of the PsaA and PsaB proteins and their associated chlorophyll molecules resulted in a five- to sevenfold decrease in the chlorophyll/PSII ratio in the mutant cells relative to the wild-type cells. Interestingly, the psaS:: NmR and not the psaA:: NmR mutant strain retained a small fluorescence peak (77K) at 721 nm originating from chlorophyll molecule(s) presumably bound to a small amount of the PsaA protein present in the psaB mutant. These results demonstrate that this organism is suitable for the manipulation of PSI reaction-centre proteins.
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