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.

which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chi4 genes encoding various deletions of chitinase Al were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase Al specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase Al does not bind were not significantly afected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type HI modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.Various organisms, including bacteria, fungi, plants, and some vertebrates, produce chitinases, enzymes which hydrolyze the ,-1,4 linkage of chitin. Bacterial chitinases are thought to be important in the digestion of chitin for utilization as a carbon and energy source, and, from the ecological point of view, such chitinases serve an important role in recycling chitin in nature.Bacillus circulans WL-12 is one of the bacteria which excrete chitinases into culture media (28). The chitinase system of the bacterium includes at least six different chitinase molecules: chitinases Al, A2, Bi, B2, C, and D. Chitinase A2 is a derivative of Al and is generated by proteolytic modification; likewise, B2 is thought to be a derivative of Bi. Chitinase Al is assumed to be the key enzyme in the chitinase system, as it (and A2) is the most abundantly produced form as well as the enzyme with the highest colloidal chitin-hydrolyzing activity and very high affinity toward chitin. The gene encoding chitinase Al (chiA) and the one encoding chitinase D (chiD), which is located immediately upstream of the chiA gene, have been cloned and sequenced (27,29). Amino acid sequence analysis suggests that chitinase Al comprises at least three discrete functional domains; namely, a C-terminal domain, a region consisting of two type III modules (domains), and a large N-terminal domain. In previous reports, we showed that the type III modules of chitinase Al were related to those of fibronectin, a multifunctional extracellular and plasma protein of higher eukaryotes (29), and that glutamic acid 2...

The minimal set of genetic information necessary and sufficient to sustain a functioning cell contains not only trans-acting genes, but also cis-acting chromosomal regions that cannot be complemented by plasmids carrying these regions. In Escherichia coli (E. coli), only one chromosomal region, the origin of replication has been identified to be cis-acting. We constructed a series of mutants with long-range deletions, and the chromosomal regions containing trans-acting essential genes were deleted in the presence of plasmids complementing the deleted genes. The deleted regions cover all regions of the chromosome except for the origin and terminus of replication. The terminus affects cell growth, but is not essential. Our results indicate that the origin of DNA replication is the only vital, unique cis-acting DNA sequence in the E. coli chromosome necessary for survival.

Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillinbinding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two D,L-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the D,L-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these D,L-endopeptidases showed LytF and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of D,L-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed.

(ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD ChiA1 was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD ChiA1 specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD ChiA1 was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD ChiA1 to regenerated chitin. These observations suggest that ChBD ChiA1 recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD ChiA1 exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBD ChiA1 is mediated mainly by hydrophobic interactions. Hydrolysis of ␤-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of ␤-chitin microcrystals but greatly enhances the efficiency of degradation.Chitin, an insoluble linear ␤-1,4-linked homopolymer of Nacetylglucosamine, is a common constituent of fungal cell walls, exoskeletons of insects, and shells of crustaceans and is one of the most abundant polysaccharides in nature. Chitinase degrades chitin by hydrolyzing ␤-1,4-glycosidic-linkages, and the activity has been found in a variety of organisms. Bacteria, which do not contain chitin as a constituent, also produce chitinase, mainly for utilization of chitin as a carbon and energy source. From the ecological point of view, bacterial chitinases play an important role in recycling chitin in nature.

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.Carbamate insecticides such as carbaryl (1-naphthyl N-methylcarbamate) are broad-spectrum insecticides that comprise the major proportion of agricultural pesticides used in today's agricultural industry. These compounds are considered hazardous because they potently inhibit acetylcholine esterase (9) and the N-nitrosocarbamates formed are potent mutagens (8). On the other hand, these pesticides generally do not persist in soil for a long time, and the persistence of these compounds in agricultural soil is due to repeated applications (10, 38). From these perspectives, an understanding of the degradation mechanism is needed to control the persistence of these pesticides in soil.Soil microorganisms are thought to play a significant role in the reduction of pesticides in soil, and many soil bacteria capable of degrading carbamate pesticides have been isolated and characterized (4,6,16,17,21,28). The biochemical characteristics of carbamate pesticide hydrolases have also been reported (5,17,18,20,25). However, little is known about the genes for these enzymes. The mcd gene, which encodes a carbofuran hydrolase, is located on a 100-kb plasmid called pPDL11, and it has been cloned from Achromobacter sp. strain WM111 (41). This gene was shown to be present in many bacteria and to be encoded on a 100-, 105-, 115-, or 124-kb plasmid found in diverse bacteria isolated from geographically distant areas (6, 29). However, the structure of the carbamate insecticide degradative gene has not as yet been reported, although the nucleotide sequence of mcd gene is available (accession no. AF160188).The present study characterizes a carbaryl hydrolase purified from Rhizobium sp. strain AC100. From the result of a plasmid-curing experiment, it was suggested that the carbamate insecticide degradative gene is encoded on the plasmid. We then cloned the degradative gene from the plasmid DNA and determined the nucleotide sequence of the 10-kb region containing the degradative gene. The sequence analysis suggested that the g...

The three-dimensional structure of the chitin-binding domain (ChBD) of chitinase A1 (ChiA1) from a Grampositive bacterium, Bacillus circulans WL-12, was determined by means of multidimensional heteronuclear NMR methods. ChiA1 is a glycosidase that hydrolyzes chitin and is composed of an N-terminal catalytic domain, two fibronectin type III-like domains, and C-terminal ChBD ChiA1 (45 residues, Ala 655 -Gln 699 ), which binds specifically to insoluble chitin. ChBD ChiA1 has a compact and globular structure with the topology of a twisted ␤-sandwich. This domain contains two antiparallel ␤-sheets, one composed of three strands and the other of two strands. The core region formed by the hydrophobic and aromatic residues makes the overall structure rigid and compact. The overall topology of ChBD ChiA1 is similar to that of the cellulose-binding domain (CBD) of Erwinia chrysanthemi endoglucanase Z (CBD EGZ ). However, ChBD ChiA1 lacks the three aromatic residues aligned linearly and exposed to the solvent, which probably interact with cellulose in CBDs. Therefore, the binding mechanism of a group of ChBDs including Ch-BD ChiA1 may be different from that proposed for CBDs.

Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (beta/alpha)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline beta-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the K(m)-value when reduced (GlcNAc)5 (where GlcNAc is N -acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.