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Nine Mutants of Chlorobium tepidum Each Unable To Synthesize a Different Chlorosome Protein Still Assemble Functional Chlorosomes. Niels-Ulrik Frigaard, 2004.Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope . These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes . Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known . Nine mutants in which a single csm gene was inactivated were created; these mutants included genes encoding all chlorosome proteins except CsmA . All mutants had BChl c contents similar to that of the wild-type strain and had growth rates indistinguishable from or within  90%
(CsmC- and CsmJ-) of those of the wild-type
strain . Chlorosomes isolated from the mutants lacked only the protein
whose gene had been inactivated and were generally similar to those
from the wild-type strain with respect to size, shape, and BChl c,
BChl a, and carotenoid contents . However, chlorosomes from the
csmC mutant were about 25% shorter than those from the
wild-type strain, and the BChl c absorbance maximum was
blue-shifted about 8 nm, indicating that the structure of the BChl
c aggregates in these chlorosomes is altered . The results of the
present study establish that, except with CsmA, when the known
chlorosome proteins are eliminated individually, none of them are
essential for the biogenesis, light harvesting, or structural
organization of BChl c and BChl a within the
chlorosome . These results demonstrate that chlorosomes are remarkably
robust structures that can tolerate considerable changes in protein
composition .
Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans. Sergio Alvarez, 2004.For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance . To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM) . Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux . Copper in the range of 1 to 2 µM greatly stimulated exopolyphosphatase activity in cell extracts from A . ferrooxidans . The same was seen to a lesser extent with cadmium and zinc . Bioinformatic analysis of the available A . ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84) . Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A . ferrooxidans . Site-Specific Recombination System Encoded by Toluene Catabolic Transposon Tn4651. Hiroyuki Genka, 2002.The 56-kb class II toluene catabolic transposon Tn4651 from Pseudomonas putida plasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kb tnpT-res-tnpS region is 48 kb apart from the tnpA gene (M . Tsuda, K.-I . Minegishi, and T . Iino, J . Bacteriol . 171:1386-1393, 1989) . Detailed analysis of the 2.4-kb region revealed that the tnpS and tnpT genes encoding the putative 323- and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bp res site . The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS . In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination . Analysis of site-specific recombination under the Escherichia coli recA cells indicated that (i) the site-specific resolution between the two copies of the res site on a single molecule was catalyzed by TnpS, (ii) the functional res site was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution . It was also found that TnpS catalyzed the site-specific recombination between the res sites located at two different molecules to form a cointegrate molecule . Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination . Membrane Localization of Motility, Signaling, and Polyketide Synthetase Proteins in Myxococcus xanthus. Vesna Simunovic, 2003.Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways . In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization . Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient . The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM . Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction . The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers . As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions . Immunoblotting was used to localize important motility and signaling proteins within the protein peaks . CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm-3) . Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm-3) and low buoyant density (1.169 to 1.171 g cm-3) . FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM . The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles . Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM . Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm-3) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A . Identification and Characterization of a GDSL Esterase Gene Located Proximal to the swr Quorum-Sensing System of Serratia liquefaciens MG1. Kathrin Riedel, 2003.Serratia liquefaciens MG1 employs the swr quorum-sensing system to control various functions, including production of extracellular enzymes and swarming motility . Here we report the sequencing of the swr flanking DNA regions . We identified a gene upstream of swrR and transcribed in the same direction, designated estA, which encodes an esterase that belongs to family II of lipolytic enzymes . EstA was heterologously expressed in Escherichia coli, and the substrate specificity of the enzyme was determined in crude extracts . With the aid of zymograms visualizing EstA on polyacrylamide gels and by the analysis of a transcriptional fusion of the estA promoter to the promoterless luxAB genes, we showed that expression of the esterase is not regulated by the swr quorum-sensing system . An estA mutant was generated and was found to exhibit growth defects on minimal medium containing Tween 20 or Tween 80 as the sole carbon source . Moreover, we show that the mutant produces greatly reduced amounts of N-acyl-homoserine lactone (AHL) signal molecules on Tween-containing medium compared with the wild type, suggesting that under certain growth conditions EstA may be important for providing the cell with precursors required for AHL biosynthesis .
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