SummaryThe minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.

No abstract

Escherichia coli YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family. A yibP disrupted mutant grows in rich medium at 37°C but not at 42°C. In the yibP null mutant, cell division and FtsZ ring formation are inhibited at 42°C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis. Five percent betaine suppresses the temperature sensitivity of the yibP disrupted mutation. The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain. YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction. Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space. Purified YibP has a protease activity that split the substrate ␤-casein.The complete genome sequence of Escherichia coli has been determined (1). There are a lot of open reading frames whose biological functions are still unknown. The physiological function of the yibP gene is unknown so far. Computer analysis of the deduced amino acid sequences of YibP showed that YibP protein has a membrane-spanning region, two long coiled-coil regions, and a C-terminal globular domain. The C-terminal domain of YibP has a region homologous to members of the M23/M37 family (http://www.sanger.ac.uk/cgi-bin/Pfam /getacc?PF01551).Members of the peptidase M23/37 family are zinc metallopeptidases with a range of specificities. Members of the M37 family are Gly-Gly endopeptidases (19). Members of the M23 family are also endopeptidases. The M37 family includes some bacterial lipoproteins, such as E. coli NlpD (9, 12), for which no proteolytic activity has been demonstrated. B-lytic endopeptidases are bacterial metallopeptidases that belong to the M23 protease family (Medline entry 95405261). Cleavage is specific for glycine bounds, especially in Gly-Gly-Xaa sequences, where Xaa is any aliphatic hydrophobic residue. Blytic endopeptidases exist in the cell wall of gram-positive bacteria in which the peptidoglycan cross-links contain glycine residues. These endopeptidases contain zinc, but the exact position of the metal-binding ligands is uncertain.In this work, we found that yibP disrupted mutant cells were unable to form colonies at 42°C. We report here various properties of yibP disrupted mutant cells and the subcellular localization of the YibP protein. We found that the purified YibP protein had a proteolytic activity for the substrate ␤-casein. MATERIALS AND METHODSBacterial strains, plasmids, and media. Bacterial strains and plasmids are listed in Tables 1 and 2. Bacterial cells were grown in L medium (1% Bactotryptone, 0.5% Bacto-yeast extract, and 0.5% sodium chloride, pH 7.2) and synthetic medium ...

Iron sulfide (FeS) particles produced by sulfate-reducing bacteria are an excellent adsorbent for heavy metal ions. To select the bacteria which produce a magnetic FeS as an adsorbent for the high gradient magnetic separation, an open gradient magnetic separator operating at a 10 tesla magnetic field was used. Magnetization study showed that the magnetic FeS particles consist of paramagnetic like and ferro-magnetic component.Index Terms-Heavy metal, high gradient magnetic separation, magnetic iron sulfide, sulfate reducing bacteria.

The genes for acid-stable a-atnylase and glucoamylase were cloned from white shochu-koji, Aspergillus kawachii. Both genes were used to transform the parent strain of white shochu-koji which carried a dominant selective marker gene, amdS, that originated from A. oryzae. Three lines of transformants were identified that secreted about 6-fold more acid-stable a-amylase activity and about 7-fold more glucoamylase activity than the parent strain in liquid culture. In solid culture, all three transformants had 2-fold higher acid-stable a-amylase activity and 2.4-fold higher glucoamylase activity than the parent strain. When koji was prepared on a laboratory scale, acid-stable a-amylase activity was 5.7-fold higher and glucoamylase activity was 3.8-fold higher than when the parent strain was used. Shochu was produced with a koji ratio of 33% or 10% using one line of transformants on a laboratory scale. Even with a koji ratio of 10%, the weight of the mash obtained with the transformant decreased to almost the same extent as with a koji ratio of 33% and the parent strain. Levels offlavour compounds in shochu produced with koji of the transformant were higher than in the shochu prepared with koji of the parent strain.In particular, levels ofisoamyl acetate and $-phenethyl acetate were as high as 12.9 mgllitre and 3.8 mgllitre, respectively.