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In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription. [ABSTRACT FROM AUTHOR]

Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, the Shigella autotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library of Escherichia coli proteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitive ftsZ allele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm of E. coli. DnaK was also required for IcsA secretion at the pole in Shigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A second Shigella autotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm. doi: 10.1128/JB.00833-09

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci coiocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in [DELTA]datA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.

Dynamic organization of chromosomal DNA in Escherichia coli, organization critical in cell division, chromosome replication, and partitioning in the bacterial cell cycle, is discussed. The subcellular localization of 22 DNA segments of the E. coli chromosome has been studied by the FISH method. The bacterial chromosome is 1000 times the length of the bacterium and folded by some unknown means into a nucleoid, a compact form. The circular chromosomal DNA has been shown to be organized to form a compact ring structure in the cell.

Three migration patterns are discernible in the replication origin (oriC), the chromosome terminus and other DNA regions during the partitioning of daughter chromosomes in Escherichia coli. In cell division cycle by FISH, oriC is at the old-pole-proximal and the terminus is at the new-pole-proximal nucleoid borders. OriC, which travel to opposite nucleoid borders, are retained and stay a constant distance from each cell pole.

F plasmid, the fertility factor of E. coli, is partitioned with fidelity to daughter cells during cell division, due to sopA and sopB, trans-acting genes, and sopC, a cis-acting site. The subcellular distribution of mini-F-plasmid molecules was visualized through fluorescence in situ hyrbidization. Molecules lacking the sopABC segment showed random distribution in spaces not occupied by nucleoids.

The production of RNase E polypeptides lacking the carboxyl-terminal due to rne (smbB) mutations inhibits the formation of temperature sensitive colonies and anucleated cells in Escherichia coli mukB mutants. The polypeptides also lack the hydrophilic arginine-rich sequence. Unlike the wild-type polypeptides, the mutant polypeptides do not bind to polynucleotide phosphorylase. The suppressor mutations increase the formation of MukB mutant protein that removes the defects caused by the mukB mutation. The smbB mutants in rne(super +) strains show normal growth.

Western blot analysis and immunohistochemical techniques were utilized in the investigation of the subcellular distribution of the FtsH protein in Escherichia coli. Results show that the adenosine triphosphate-binding protein is putative in the cytoplasmic phase of the cell membrane. Analysis of the amino acid sequence and the predicted structure reveal that FtsH is an integralmembrane protein spanning the lipid bilayer twice. The carboxy terminal is cytoplasmic and contains the nucleotide binding region.

Cloning and sequencing analysis were carried out to study the wild type and a temperature-sensitive mutant Escherichia coli ftsH gene. The protein product ofthese genes (FtsH) was identified to be an adenosine triphosphate-binding protein consisting of 644 amino acids with a molecular weight of 70.7 kiloDaltons. The mutant gene had two base substitutions corresponding to two amino acid residue alteration. FtsH was demonstrated to be homologous with a class of putative adenosine triphosphatases that have important role in cell cycle regulation, genetic expression and membrane functions in eukaryotes.

The isolation and characterization of the smbA gene, whose mutations suppress temperature-sensitive growth of the mukB null mutant of Escherichia coli, were reported. The smbA suppressor gene is located at 4 min on the E. coli chromosome. The smbA gene product is essential for cell growth, and cells show a novel morphological phenotype in the absence of the normal function of the SmbA protein.