This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mitani, Y.
Right arrow Articles by Tamura, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mitani, Y.
Right arrow Articles by Tamura, T.

 Previous Article  |  Next Article 

Journal of Bacteriology, April 2005, p. 2582-2591, Vol. 187, No. 8
0021-9193/05/$08.00+0     doi:10.1128/JB.187.8.2582-2591.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of LtsA from Rhodococcus erythropolis, an Enzyme with Glutamine Amidotransferase Activity

Yasuo Mitani,1 XianYing Meng,2 Yoichi Kamagata,2 and Tomohiro Tamura1,3*

Proteolysis and Protein Turnover Research Group, Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku,1 Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo,3 Microbial and Genetic Resources Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba-Higashi, Tsukuba, Japan2

Received 10 October 2004/ Accepted 4 January 2005


arrow
ABSTRACT
 
The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ltsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ltsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ltsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH4Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH4Cl, indicating that NH3 can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.


arrow
INTRODUCTION
 
The genera Rhodococcus, Corynebacterium, and Mycobacterium are members of the mycolic acid-containing actinomycetes (4, 7). One of their characteristic features is a cell wall structure that consists of mycolic acid, arabinogalactan, and peptidoglycan (2). The genus Mycobacterium is one of the most studied genera because it includes several clinically important pathogens, such as Mycobacterium tuberculosis and Mycobacterium leprae. Mycobacteria are resistant to not only hydrophobic molecules but also hydrophilic molecules, mainly because of the presence of mycolic acid in their cell walls (2, 6). Although the carbon chain length of mycolic acid in rhodococci is rather short compared with that of mycobacteria, the cell wall components of rhodococci are similar to those of mycobacteria (2, 4).

Lysozyme (muramidase) is an enzyme that cleaves at the ß-1,4-linkage between N-acetylglucosamine and N-acetylmuramic acid in the peptidoglycan, which forms the huge polymeric scaffold of the cell wall of gram-positive bacteria. Gram-positive bacteria are generally sensitive to lysozyme because the peptidoglycan directly faces the outer environment. On the other hand, in the case of gram-negative bacteria, the outer membrane of the cell wall, mainly composed of lipopolysaccharide, acts as a barrier to cell wall-lytic enzymes (14). Notably, M. tuberculosis is resistant to lysozyme, most likely because of steric hindrance caused by the massive attachment of mycolyl-arabinogalactan complexes (1, 11).

Hirasawa and coworkers (10) demonstrated that a mutation in the ltsA gene of Corynebacterium glutamicum (ltsACg) causes a lysozyme-sensitive phenotype. Although the amino acid sequences of LtsACg (the product of ltsACg) and Escherichia coli asparagine synthetase (AS-BEc, the product of asnBEc) are similar, LtsA does not display asparagine synthetase activity (10), and its function remains to be elucidated.

We developed a recombinant protein expression system using Rhodococcus erythropolis as the host. This system can express proteins over a wide temperature range (4 to 35°C) and can successfully produce proteins that cannot be expressed in E. coli cells (26, 27). We isolated lysozyme-sensitive mutants in order to simplify the method of extracting proteins because the cell wall is too rigid to be disrupted easily. The lysozyme susceptibility was caused by mutations in the R. erythropolis homologue of ltsA.

AS-B functions as an asparagine synthetase and produces asparagine from aspartic acid by using glutamine as an amide donor (31). AS-B belongs to the huge glutamine amidotransferase family, whose members catalyze amide nitrogen transfer from glutamine to acceptor substrate molecules in different biosynthetic pathways (36, 37). An X-ray crystallographic analysis of AS-B showed that it comprises two domains, each harboring an active site (17, 23). The N-terminal domain, referred to as the glutaminase domain, hydrolyzes glutamine to glutamic acid and ammonia, and the C-terminal domain, the synthetase domain, catalyzes the adenylation of aspartic acid and the subsequent amidation of this reaction intermediate to produce asparagine (36, 37). These two active sites are connected by a molecular tunnel through which the ammonia molecules pass from the N-terminal active site to the C-terminal active site (17). In the glutamine amidotransferase family, molecular tunnels between two or more active sites are commonly observed (12, 22), as described previously for GMP synthetase (33), glutamine phosphoribosylpyrophosphate amidotransferase (15, 16), and carbamyl phosphate synthetase (34). Most glutamine amidotransferases can utilize NH3 (supplied as NH4Cl) as an amide donor in place of glutamine both in vivo and in vitro (36).

Here, we report the isolation of R. erythropolis lysozyme-sensitive mutants and the identification of the gene responsible for the phenotype, a homologue of ltsACg. We also show that the ltsA homologues from M. tuberculosis (ltsAMt) and Bacillus subtilis (ltsABs) could complement the lysozyme-sensitive phenotype, but ltsA from Streptomyces coelicolor (ltsASc) and asnB from E. coli (asnBEc) could not. Furthermore, we characterized the enzymatic function of LtsA as a glutamine amidotransferase and detected glutaminase activity in vitro and synthetase activity with the substrate NH4Cl in vivo.


arrow
MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. R. erythropolis JCM3201 and E. coli S17-1 were purchased from the Japan Collection of Microorganisms and the American Type Culture Collection, respectively. The vector pHN144 was used as the E. coli-R. erythropolis shuttle vector (26). In this work, recombinant proteins were produced by using R. erythropolis as the host and pTip as the expression vector (26). Plasmid pK18mobsacB was purchased from the American Type Culture Collection. R. erythropolis JCM3201, L-65, and L-88 were cultured at 30°C in Luria-Bertani (LB) medium (1% Bacto tryptone [Difco], 0.5% yeast extract, 1% NaCl). The medium was solidified with 1.5% agar. Antibiotics were used at the following concentrations: kanamycin, 200 µg ml–1; and tetracycline, 8 and 20 µg ml–1 in liquid and solid media, respectively.

UV mutagenesis of R. erythropolis. R. erythropolis cells were grown to the late-exponential phase, and serial dilutions of cells were spread on LB medium plates and irradiated for 20 s with UV light (254 nm). The UV irradiation killed 90% of the cells. After 2 days of incubation at 30°C, individual colonies were inoculated into two 96-well plates containing LB medium with or without lysozyme (50 µg ml–1; from chicken egg white; Sigma). Cells which could not grow in the presence of lysozyme were analyzed further. Lysozyme sensitivity was confirmed by spotting organisms on LB medium plates containing 50 µg of lysozyme ml–1, as shown in Fig. 1.



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 1. R. erythropolis mutants L-65 and L-88 are susceptible to lysozyme. Growth curves for wild-type (WT) and mutant strains were plotted after the OD600 was determined. Lysozyme (12.5 µg ml–1) was added when the OD600 was approximately 0.2 (indicated by arrows). (A) Cells grown at 30°C in the presence ({circ}) or absence ({blacksquare}) of lysozyme. (B) ltsA disruptant (KO) could not grow on LB medium plates containing 12.5 µg of lysozyme ml–1. Serial dilutions of the cultures were spotted on LB medium plates, and the approximate numbers of cells are indicated below the plates.

Isolation of the ltsA gene and complementation tests. ltsA and homologous genes from C. glutamicum and members of related genera were aligned, and degenerate primers matching highly conserved regions were designed. Degenerate PCR was performed by using primers Re-34 (5'-TTCAATGG[G/T]GAGATCTA[C/T]AA[C/T]TA-3') as the sense primer and Re-37 (5'-TTGTC[G/T]GCTTT[G/T]AC[G/T]AGGATGTC-3') as the antisense primer. The amplified fragment was isolated and subjected to sequence analysis. On the basis of the sequence obtained by degenerate PCR, inverse PCR was carried out to obtain a full-length open reading frame (ORF) by using templates of circularized chromosomal DNA after they were digested with either BamHI or BssHII. After the amplified DNA fragments were sequenced, an approximately 2.5-kbp DNA fragment containing the ltsA ORF was amplified from the R. erythropolis chromosome with primers Re-44 (5'-GGATCCGACCTTGCGCCACGTTCACGCCTTC-3') and Re-45 (5'-GCGCGCCGCACAGCTGGGATCTTTGGTCGC-3'). Next, this DNA fragment was cloned into pHN144 to obtain pHN144-ltsA. Complementation of the phenotype of the R. erythropolis L-65 and L-88 mutants was achieved by transformation with pHN144-ltsA.

Cloning of ltsA homologues. The sequences of homologues of the ltsA gene were obtained from the DDBJ/EMBL/GenBank database. The accession numbers for the C. glutamicum, M. tuberculosis, B. subtilis, S. coelicolor, and E. coli sequences were AB029550, BX842579, AF008220, AL939105, and J05554, respectively. Each ORF was PCR amplified by using chromosomal DNA as the template and was cloned into the pTip expression vector (26). Chromosomal DNAs from M. tuberculosis and B. subtilis were kindly provided by N. Ohara (Nagasaki University) and K. Kobayashi (Nara Institute of Science and Technology), respectively. The vectors were designated pTip-ltsACg, pTip-ltsAMt, pTip-ltsABs, pTip-ltsASc, and pTip-asnBEc, respectively.

Site-directed mutagenesis. To construct the pTip-ltsAC2A plasmid, the ltsA ORF was amplified with primers Re-90 (5'-AAGGCATATGGCCGGACTGCTCGGGCTACTGACATCTG-3') and Re-54 and subcloned into the pTip vector as described below for pTip-ltsAWT. pTip-ltsAC2S was constructed with primers Re-91 (5'-AAGGCATATGTCCGGACTGCTCGGGCTACTGACATCTG-3') and Re-54. To construct pTip-ltsAD278N, overlap PCRs were performed. The first PCR was carried out by using pTip-ltsAWT as the template and primers Re-52 and Re-93 (5'-ATCGCCGTGGAGTTGATACCACCGG-3') and primers Re-92 (5'-CCGGTGGTATCAACTCCACGGCGAT-3') and Re-54 (mutated nucleotides are underlined). Then the second PCR was performed by using a mixture of the first PCR product as the template and primers Re-52 and Re-54, and the product was cloned into the pTip vector. pTip-ltsAD376N, pTip-ltsAK494A, and pTip-ltsAK494R were constructed by using the following primers for the first PCR. The mutation primers were primers Re-94 (5'-GCGAGGGTGCCAACGAGCTGTTCGG-3') and Re-95 (5'-CCGAACAGCTCAACGGCACCCTCGC-3') for pTip-ltsAD376N, primers Re-98 (5'-GACATCCTCGTCGCCGCCGACAAGATG-3') and Re-99 (5'-CATCTTGTCGGCGGCGACGAGGATGTC-3') for pTip-ltsAK494A, and primers Re-96 (5'-GACATCCTCGTCCGCGCCGACAAGATG-3') and Re-97 (5'-CATCTTGTCGGCGCGGACGAGGATGTC-3') for pTip-ltsAK494R (mutated nucleotides are underlined). Every mutation was confirmed by sequencing of the plasmids.

Conjugative plasmid transfer from E. coli S17-1 to R. erythropolis. Conjugation was performed as previously described (32), with some modifications. The cells of E. coli S17-1 and R. erythropolis were grown separately in liquid cultures to the log phase, and 100 µl of the E. coli culture was mixed with 500 µl of the R. erthropolis culture. Two hundred microliters of the mating mixture was spread onto a nitrocellulose filter on an LB agar plate. After 20 h of incubation at 30°C, the cells were washed from the filter with 2 ml of LB medium, and 200-µl aliquots were spread onto LB agar plates supplemented with kanamycin (200 µg ml–1) and nalidixic acid (25 µg ml–1). R. erythropolis transconjugants appeared after 4 days of incubation at 30°C.

Production of recombinant protein and antibody. To construct the LtsA expression vectors pTip-ltsAWT, pTip-ltsAL65, and pTip-ltsAL88, ltsA ORFs were PCR amplified by using chromosomal DNA of R. erythropolis JCM3201, the L-65 mutant, or the L-88 mutant as the template along with primers Re-52 (5'-GGAATTCCATATGTGTGGACTGCTCGGGCTACTGACAT-3') and Re-54 (5'-CCCAAGCTTGATGTTGACCGGGTACACCGGTTCCTG-3'). The amplified DNA fragments were digested with NdeI and HindIII and introduced into the pTip vector, which had been digested with the same enzymes. The resulting plasmids coded for full-length LtsA plus six additional residues (KLRSRG) and a C-terminal six-His tag. The expression vectors were transformed into R. erythropolis by electroporation as previously described (26). To produce LtsA proteins, a 10-ml preculture of cells carrying an expression vector was inoculated into 100 ml of LB medium supplemented with 1 µg of thiostrepton ml–1. After 14 h of incubation at 30°C, cells were harvested and resuspended in buffer (50 mM sodium phosphate [pH 8.0], 300 mM NaCl). Lysozyme was added to a final concentration of 2 mg ml–1, and the cells were incubated on ice for 1 h. Subsequently, they were disrupted by sonication for 15 min, followed by centrifugation (4°C, 20,000 x g, 15 min). The recombinant proteins were isolated by using Ni-nitrilotriacetic acid Superflow (QIAGEN) according to the manufacturer's instructions. His-tagged LtsA was >95% pure as judged by using Coomassie blue-stained gels. The eluate was dialyzed against buffer (50 mM Tris-HCl [pH 7.5], 10% glycerol). The dialyzed recombinant protein was immediately used for the assay because this protein was extremely labile, similar to AS-BEc (13). To generate anti-LtsA antibody, isolated His-tagged LtsA was used to immunize a rabbit.

Preparation of cell wall extracts. The L-65 mutant of R. erythropolis was grown to the mid-log to late log phase in LB medium supplemented with 1 mg of isoniazid ml–1. The culture was cooled in an ice bath, and cells were harvested by centrifugation at 4°C. The cells were washed with cold distilled water, resuspended in buffer S (50 mM Tris-HCl, pH 7.5), sonicated in an ice bath for 15 min, and centrifuged at 20,000 x g for 20 min. The pellet was treated with 2% sodium dodecyl sulfate (SDS) in buffer S at 60°C for 2 h. After centrifugation (20,000 x g, 20 min), the pellets were washed three times with buffer S, and this was followed by treatment with 10 mg of lysozyme ml–1 in buffer S to solubilize muropeptides and then heat denaturation at 60°C for 1 h. After centrifugation (20,000 x g, 20 min), the supernatant was used as the cell wall extract. One hundred microliters of soluble extract was obtained from a 100-ml culture.

Glutaminase activity assays. LtsA assays for glutaminase activity were performed at 30°C by using a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 25 mM KCl, 1 mM glutamine, 0.2 mM dithiothreitol, and 2 mM ATP. Four microliters of cell wall extract was used per 50-µl reaction mixture. Aliquots were quenched as previously described (9). After appropriate dilution of reaction mixtures, the concentration of glutamic acid was determined with an Amplex red glutamic acid/glutamate oxidase assay kit (Molecular Probes).

Transmission electron microscopy. Bacterial pellets were fixed overnight with 2.5% glutaraldehyde and 0.05% ruthenium red in 0.1 M cacodylate buffer (pH 7.4) at 4°C. Cells were washed in the same buffer and postfixed for 3 h in 1% osmium tetroxide containing 0.05% ruthenium red at 4°C. Subsequently, the cells were rinsed with the buffer. Bacteria were suspended for 1 h at room temperature in 2% aqueous uranyl acetate and then washed in distilled water. Suspended cells were embedded in 1.5% agarose before dehydration with a graded ethanol series. The dehydrated blocks were embedded in Spurr resin. Ultrathin sections were cut with an ultramicrotome (Ultracut-N; Leichert-Nissei), mounted on copper grids, and stained with uranyl acetate and lead citrate. Images of sections were obtained by using a transmission electron microscope (75 kV; H-7000; Hitachi, Tokyo, Japan).

Nucleotide sequence accession number. The DNA sequence of R. erythropolis ltsA has been deposited in the DDBJ/EMBL/GenBank database under accession number AB183824.


arrow
RESULTS
 
Screening of lysozyme-sensitive mutants of R. erythropolis and isolation of ltsA. R. erythropolis provides an alternative system for expressing recombinant proteins that are difficult to express in E. coli cells because of their toxicity to cells, because of the production of inclusion bodies, and because of low productivity (26). On the other hand, it is difficult to disrupt cells not only by physical treatments, such as sonication or beating with glass beads, but also by enzymatic treatments, such as treatment with the cell wall-lytic enzyme lysozyme. In order to purify recombinant proteins from Rhodococcus cells by simple methods, we isolated lysozyme-sensitive mutants.

We performed UV irradiation of R. erythropolis cells as described in Materials and Methods. Two lysozyme-sensitive mutants were isolated from approximately 9,000 irradiated growing colonies, and these mutants were designated L-65 and L-88. Both mutant strains were sensitive to lysozyme and could not grow on LB medium plates containing 12.5 µg of lysozyme ml–1, while the wild-type strain could grow on plates containing more than 800 µg of lysozyme ml–1 (data not shown). To confirm the susceptibility to lysozyme, we directly added lysozyme (final concentration, 12.5 µg ml–1) to the culture media of mutants L-65 and L-88 and measured the absorbance. The cell density (optical density at 600 nm [OD600]) of both cultures drastically decreased after the addition of lysozyme, but no significant change was observed for the wild-type strain culture (Fig. 1A). The susceptibility of both mutants to antibiotics, such as ampicillin, kanamycin, tetracycline, chloramphenicol, and thiostrepton, was not different from that of wild-type cells (data not shown), suggesting that the cell wall permeability of the mutants was not changed. We produced and isolated several recombinant proteins in the lysozyme-sensitive mutants and found that the production level was as high as that in wild-type cells. Since the mutant cells were sensitive to lysozyme, cell disruption could be done under mild conditions, avoiding extensive physical treatments, such as sonication. The transformation efficiencies and growth rates of the mutant and wild-type cells were the same (data not shown). These results demonstrate that using the lysozyme-sensitive mutants has no disadvantages but has the unique advantage of mild lysis conditions compared to the conditions for wild-type host cells.

The phenotypic similarities of the R. erythropolis mutants and the C. glutamicum ltsA mutant (10) suggested that the R. erythropolis ltsA gene could complement the L-65 and L-88 mutants. Therefore, we cloned the ltsA locus from the wild-type R. erythropolis chromosome and constructed the self-replicating vector pHN144-ltsA, which contained the cognate promoter. The vector was introduced into L-65 and L-88 by electroporation, and the resultant transformants were found to be lysozyme resistant (data not shown). Sequence analysis of the ltsA genes of both mutants revealed the same transition (TT to TG) that resulted in replacement of Phe-557 with Cys and replacement of Phe-48 with Cys in the L-65 and L-88 mutants, respectively (the first methionine was designated Met-1). To confirm that the disruption of ltsA causes lysozyme sensitivity, an R. erythropolis ltsA disruptant was constructed by using the conjugation method described in Materials and Methods. Chromosomal disruption of the ltsA gene was verified by PCR (data not shown). The ltsA disruptant could not grow on plates containing 12.5 µg of lysozyme ml–1 (Fig. 1B). These results indicated that ltsA was responsible for the observed lysozyme resistance of R. erythropolis.

Sequence analysis and functional implications of LtsA. To deduce the function of LtsA, we compared the amino acid sequences of various AS-B and LtsA homologues from both gram-positive and gram-negative bacteria (Fig. 2); all of the proteins except LtsA of C. glutamicum (LtsACg) and R. erythropolis (LtsARe) were annotated as AS-B in the database. The alignment was constructed by using the DIALIGN program (25). The sequence of the N-terminal region (amino acids 1 to 232, corresponding to amino acids 1 to 195 of AS-BEc), which formed the N-terminal domain (17), was well conserved; Cys-2, which was essential for glutaminase activity (21), and Arg-50, Asn-79, Glu-81, and Asp-105, which were shown to stabilize glutamine by hydrogen bonding interactions with AS-BEc (23), were absolutely conserved (Fig. 2A). The presence of these highly conserved functional residues strongly suggested that the LtsARe N-terminal domain exhibited glutaminase activity. In contrast, the C-terminal region appeared to be rather diverse. The amino acid residues Ser-274, Asp-278, Ser-279, Gly-372, Asp-376, Lys-534, and Lys-554, which were shown to interact with the ATP/Mg2+ complex in the C-terminal active site in order to produce a catalytic intermediate for AS-BEc (23), were also conserved (Fig. 2). Besides these residues, additional conserved regions with unknown functions were identified (amino acids 507 to 512, corresponding to amino acids 401 to 406 of AS-BEc). Other regions, including the aspartic acid recognition residues of AS-BEc (Asp-385 and Arg-388), were not conserved.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 2. Schematic diagram and partial sequence alignment of E. coli AS-B, R. erythropolis LtsA, and homologues. (A) Protein sequence alignment was performed by using the DIALIGN program. Highly conserved regions are indicated by solid boxes. The regions containing residues important for the enzymatic activities are shown in detail (identical residues are indicated by boldface type). The position of Phe-557 of LtsA is also indicated because it was the residue mutated in L-65 and was conserved in AS-B (Phe-451). The mutated residue of L-88 (Phe-48) is conserved only in C. glutamicum, M. tuberculosis, and B. subtilis and is not shown. In this study, the first methionine was consistently included in the numbering and was designated Met-1. (B) Alignment of selected, highly conserved regions of R. erythropolis, C. glutamicum, M. tuberculosis, B. subtilis, and S. coelicolor LtsA homologues and E. coli AS-B. Residues identical in all species are indicated by a black background, and residues identical only in R. erythropolis, C. glutamicum, M. tuberculosis, and B. subtilis are indicated by a grey background. Residues that were mutated to characterize the synthetase activity are indicated by asterisks (the N terminus, including Cys-2, is not shown in the alignment).

To examine whether the genes described above complemented the function of LtsARe, the homologous genes of ltsA were PCR amplified, and expression vectors were constructed as described in Materials and Methods. The mutant cells transformed with each expression vector showed that LtsARe homologues from C. glutamicum, M. tuberculosis, and B. subtilis restored cells to lysozyme resistance, but LtsA homologues from S. coelicolor and E. coli did not change the susceptibility to lysozyme (Fig. 3A). The colonies obtained after transformation were cultured, and protein expression was confirmed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis with anti-LtsA antibodies (Fig. 3B). We could not clearly demonstrate expression of AS-BEc due to a low expression level and low cross-reactivity with anti-LtsA antibodies, but production of AS-BEc was confirmed by SDS-PAGE after isolation by Ni-nitrilotriacetic acid chromatography (data not shown). These results demonstrated that certain homologues of LtsA are functionally conserved and may have functions similar to those of LtsARe in vivo.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 3. Complementation of lysozyme sensitivity by LtsA homologues. (A) Cells were grown in LB media containing 200 µg of kanamycin ml–1, 8 µg of tetracycline ml–1, and 0.1 µg of thiostrepton ml–1. Growth curves were plotted as described in the legend to Fig. 1. Symbols: {circ}, growth with lysozyme (12.5 µg ml–1) added at the time indicated by arrows; {blacksquare}, growth in the absence of lysozyme. Panels a to g indicate ltsA disruptant R. erythropolis cells carrying the following plasmids: a, empty pTip vector; b, pTip-ltsARe; c, pTip-ltsACg; d, pTip-ltsAMt; e, pTip-ltsABs; f, pTip-ltsASc; g, pTip-asnBEc. (B) Analysis of the amounts of recombinant proteins produced by the clones used for the complementation tests by SDS-PAGE (top gel) and immunoblotting with anti-LtsA antibodies (bottom gel). Lanes a to g correspond to the description for panel A.

Cell wall structure of mutant cells. The unique cell wall of Rhodococcus functions as a barrier not only to hydrophobic molecules but also to hydrophilic molecules (2). Although the target substrate of lysozyme is peptidoglycan, which is the innermost layer of the cell wall, mutant cells may have defects in the outer layer of the cell wall, as shown for a Mycobacterium smegmatis mutant which is more susceptible to several antibiotics than wild-type cells (35). To determine whether the cell wall structure of the R. erythropolis lysozyme-sensitive mutants was different from that of the wild-type strain, both types of cells were analyzed by transmission electron microscopy (TEM). As shown in Fig. 4, the observed cell wall structures were similar to those of bacteria containing mycolic acids, such as mycobacteria (6, 19, 35). The cell walls of both the wild-type and mutant strains contained an inner electron-dense layer, followed by an electron-transparent layer that is believed to consist mainly of mycolic acids (6) and an outer layer. Although the outermost layer of the cell wall of the mutants appeared to be comparatively broader and rougher, no severe damage was observed. On the other hand, if the outer layer of the cell wall functions as a barrier to lysozyme, cells with a damaged cell wall should be susceptible to lysozyme. To examine this possibility, we treated wild-type cells with two different inhibitors of cell wall synthesis, isoniazid and ethambutol, at sublethal concentrations (1 mg ml–1 for isoniazid and 6.25 µg ml–1 for ethambutol) (3, 20), but this treatment did not change the susceptibility of the cells to lysozyme (data not shown).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 4. TEM analysis of thin sections of R. erythropolis wild-type cells (A), L-65 cells (B), and L-88 cells (C). The micrographs are typical of the appearance of the cells in several different fields. Digitally magnified (x3) views of typical cell surface areas are shown in the insets. Scale bars = 0.2 µm. OL, outer layer; ETL, electron-transparent layer; EDL, electron-dense layer.

Glutaminase activity of LtsA. To determine whether LtsA exhibits glutaminase activity, glutamic acid production was monitored by using recombinant LtsARe, as described in Materials and Methods. Recombinant wild-type and mutant LtsAs were produced by using the Rhodococcus expression system (26, 27), because LtsARe can hardly be expressed in E. coli cells (approximately 0.8 mg liter of culture–1). The glutaminase activity of wild-type LtsA was 0.74 µM h–1 (Fig. 5A), whereas the glutaminase activity of the mutant proteins could hardly be detected (Fig. 5B and C).



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 5. Glutaminase activity of LtsA proteins. (A to C) Recombinant LtsAWT (A), mutant LtsAL65 (B), and mutant LtsAL88 (C) (0.1 µg) were subjected to the glutaminase activity test as described in Materials and Methods. Five microliters of a reaction mixture (initial volume, 50 µl) was quenched at 0, 2, and 4 h to estimate the concentration of glutamic acid. Glutaminase activity was monitored in the presence ({circ}) or absence ({square}) of cell wall extract. In the case of the wild-type protein, AMP-PNP was tested in the presence of cell wall extract (x). The error bars indicate the standard errors of the means for triplicate determinations. (D) ltsA disruptant cells carrying pTip-ltsAWT, pTip-ltsAL65, or pTip-ltsAL88 were spotted on an LB medium plate containing 50 µg of lysozyme ml–1 (left plate). Identical amounts of cells were spotted on an LB medium plate containing 50 µg of lysozyme ml–1 and 10 mg of NH4Cl ml–1 (right plate). Serial dilutions of the cultures were spotted on the LB medium plates as described in the legend to Fig. 1.

In several cases, glutaminase activity is coupled to synthetase activity, meaning that the synthetase activity of the C-terminal domain recognizes an amide acceptor and forms an intermediate before the glutaminase activity of the N-terminal domain hydrolyzes a glutamine (36). The glutaminase activity of AS-BEc is dramatically enhanced when the amide acceptor aspartic acid is present in the reaction mixture (5). Therefore, we assumed that the glutaminase activity of LtsA is also enhanced when an amide acceptor is present in the reaction mixture. To test this hypothesis, we treated a cell wall extract of the R. erythropolis L-65 mutant with lysozyme in order to generate amide acceptors. Interestingly, the rate of release of glutamic acid from LtsARe was dramatically enhanced (2.05 µM h–1) (Fig. 5A) when the lysozyme-treated cell wall extract was added to the reaction mixture. The glutaminase activity of the wild type of LtsARe was not enhanced when aspartic acid was included instead of the cell wall extract in the reaction mixture (data not shown). These results strongly suggested that components of the lysozyme-treated cell wall extract serve as amide acceptors and that the glutaminase activity of LtsA is coupled to the synthetase activity. The glutaminase activity of LtsA from the L-65 mutant was slightly enhanced (0 to 0.55 µM h–1) when the lysozyme-treated cell wall extract was added (Fig. 5B). However, no enhancement was observed for LtsA from the L-88 mutant (Fig. 5C). These data indicated that the reduced activity of the L-65 and L-88 mutant LtsA enzymes resulted in high levels of susceptibility to lysozyme.

In the case of AS-BEc, ATP and aspartic acid form a catalytic intermediate, and then the NH3 is transferred to the amide acceptor (17). To confirm that the activity of LtsARe is enhanced by ATP hydrolysis, a nonhydrolyzable ATP analog, AMP-PNP [adenosine 5'-(ß{gamma}-imido)triphosphate], was used in the reaction instead of ATP. In the presence of AMP-PNP and in the absence of lysozyme-treated cell wall extract, the basal glutaminase activity of LtsAWT was reduced (0.29 µM h–1) (data not shown), and no significant enhancement of the glutaminase activity was observed in the presence of cell wall extract (0.32 µM h–1) (Fig. 5A).

The NH3-dependent synthetase activities of some glutamine amidotransferases have been shown to function in vivo by using NH4Cl (18, 28, 29, 38). To investigate whether LtsARe could utilize free NH3 as an amide donor, the lysozyme sensitivity of R. erythropolis ltsA disruptant cells carrying the inducible expression vector pTip-ltsAL65 or pTip-ltsAL88 was examined in the presence and in the absence of NH4Cl. Both types of cells expressing the mutant proteins could efficiently grow on a plate containing lysozyme supplemented with NH4Cl (Fig. 5D), although the cells could hardly grow on a plate without NH4Cl (Fig. 5D). Since both mutant proteins were deficient for glutaminase activity, these data suggest that NH3 is transferred to an amide acceptor, as shown previously for other glutamine amidotransferases. It should be noted that cells carrying the inducible expression vector pTip-ltsAL65 or pTip-ltsAL88 showed weak lysozyme resistance, as shown in Fig. 5D. This was caused by weak expression of the mutant proteins even without induction, resulting in a level of expression of these proteins that was slightly higher than that in the mutant L-65 and L-88 strains. The leaked LtsA expression made cells rather resistant to lysozyme, and the MICs were 50 µg ml–1 for cells containing pTip-ltsAL65 and 200 µg ml–1 for cells containing pTip-ltsAL88. We also tested the sensitivity of the cells to lysozyme by a method described above (Fig. 1A) using 12.5 µg of lysozyme ml–1. The growth of cells containing the vector pTip-ltsAL65 was affected, and a small reduction in the OD600 was observed after the addition of lysozyme (data not shown). In cells containing pTip-ltsAL88, the growth was slightly affected, and the growth rate was reduced after the addition of lysozyme, but no reduction in the OD600 was observed (data not shown). These data suggest that an elevated level of expression of mutant LtsA can result in a functional level similar to that of wild-type LtsA.

Mutational analysis of LtsA. To characterize LtsARe further, conserved residues of LtsARe were mutated by site-directed mutagenesis as described in Materials and Methods. Similar levels of each mutant protein were observed in ltsA disruptant cells by Western blot analysis (Fig. 6B). The primary structure of LtsARe indicates that it belongs to the Ntn-glutamine amidotransferases (37), meaning that it has an N-terminal glutaminase domain followed by a synthetase domain (Fig. 2A). Cys-2 is essential for catalysis of glutaminase activity by Ntn-glutamine amidotransferases, and replacement of this residue with alanine or serine inactivates the enzyme (5).



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 6. (A) Growth curves for ltsA disruptants complemented with various mutant alleles of ltsA were plotted based on the OD600. Cells were grown at 30°C in LB medium in the presence ({circ}) or absence ({blacktriangleup}) of 100 mM NH4Cl. Lysozyme (12.5 µg ml–1) was added during the early log phase (indicated by arrows). Panels a to i indicate ltsA-disruptant R. erythropolis cells carrying the following plasmids producing mutant LtsA proteins: a, empty pTip vector; b, pTip-ltsAWT; c, pTip-ltsAC2A; d, pTip-ltsAC2S; e, pTip-ltsAD278N; f, pTip-ltsAD376N; g, pTip-ltsAD278N,D376N; h, pTip-ltsAK494A; i, pTip-ltsAK494R. (B) The amounts of proteins produced by the cells were determined by using anti-LtsA antibodies. Total protein was analyzed by SDS-PAGE (top gel), and the same preparations were probed with LtsA antibodies after blotting onto nylon membranes (bottom gel). Lanes b to i correspond to the description for panel A. vector; lane b, pTip-ltsAWT; lane c, pTip-ltsAC2A; lane d, pTip-ltsAC2S; lane e, pTip-ltsAD278N; lane f, pTip-ltsAD376N; lane g, pTip-ltsAD278N, D376N; lane h, pTip-ltsAK494A; lane I, pTip-ltsAK494R.

In order to determine the extent of lysozyme sensitivity more precisely, we cultured the cells in liquid media and monitored the OD600 in the presence and absence of NH4Cl (100 mM). First, we confirmed that the cell density of ltsA disruptant cells carrying the empty pTip vector was drastically decreased after the addition of lysozyme, both in the presence and in the absence of NH4Cl (Fig. 6A). Next, we assayed the same mutant strain containing pTip-ltsAWT as a positive control. In this case, the addition of lysozyme did not affect the growth either in the presence or in the absence of NH4Cl (Fig. 6A, panel b).

Next, we replaced Cys-2 of LtsAWT with either alanine or serine. In both cases the growth was drastically inhibited by the addition of lysozyme in the absence of NH4Cl, while the growth was only slightly inhibited and no decrease in the absorbance was observed in the presence of NH4Cl (Fig. 6A, panels c and d). These results indicated that the NH3 molecule can be used in vivo as an amide donor in place of glutamine.

Residues Asp-278 and Asp-376 are located in the putative ATP-binding pocket of the synthetase domain. With ltsA disruptant cells expressing LtsA proteins with a mutation(s) in these residues, a decrease in the absorbance was observed both in the presence and in the absence of NH4Cl (Fig. 6A, panels e, f, and g). These results suggested that in the absence of the catalytic intermediate, resulting from activation of the amide acceptor from the cell wall with ATP, NH3 cannot be transferred.

Next, we tried to examine the residues important for the recognition of substrate on the basis of the in vivo synthetase function. Based on a sequence comparison, the data suggested that Arg-387 of AS-BEc, which is the residue involved in the recognition of aspartic acid, corresponds to Lys-494 of LtsARe (Fig. 2B). To examine the importance of this residue, we constructed two mutants, in which the lysine was replaced by alanine or arginine. The arginine mutant was not affected by the addition of lysozyme even in the absence of NH4Cl (Fig. 6A, panel i), while the alanine mutant was dramatically affected in the absence of NH4Cl and was slightly affected in the presence of NH4Cl (Fig. 6A, panel h). These results suggested that Lys-494 is an amino acid residue that is critically involved in substrate recognition and that the positive charge of this residue is important for the synthetase activity of LtsA.


arrow
DISCUSSION
 
In this report, we describe the isolation of lysozyme-sensitive mutants of R. erythropolis, the identification of ltsA as the gene responsible for the phenotype, and characterization of the gene product, LtsA, as a member of the glutamine amidotransferase family, which amidates an unidentified component of the R. erythropolis cell wall. Although ltsA is homologous to asnB, it was shown not to function as an asparagine synthetase (10). We cloned several homologues of ltsA from members of closely related genera and analyzed complementation of the lysozyme-sensitive phenotype of the R. erythropolis ltsA disruptant. We found that ltsA from C. glutamicum, M. tuberculosis, and B. subtilis could complement the phenotype, whereas ltsA from S. coelicolor and asnB from E. coli did not complement the phenotype. These observations were in accordance with the phylogenetic tree described by Min and coworkers (24), in which they compared LtsA/AS-B homologues from various species. C. glutamicum, M. tuberculosis, and B. subtilis form a subgroup; in contrast, S. coelicolor does not belong to this subgroup. Our results suggest that the function of LtsA is conserved, at least among rhodococci, corynebacteria, and mycobacteria, and that the function of LtsA is not connected to the presence of mycolic acids, because the cell wall structure of B. subtilis is apparently different from that of R. erythropolis and the cell wall does not contain mycolic acids. An investigation of the phenotype of a B. subtilis ltsA/asnB disruptant may contribute further to our understanding of the functional conservation among these species.

We detected the glutaminase activity in vitro and assayed the synthetase activity using NH4Cl in vivo. Functional analysis of LtsA indicated that it may be composed of three distinct but linked functions, namely, glutaminase, synthetase, and a connecting tunnel between these two functional domains, as found in the structure of AS-B (17). Although it is not clear whether wild-type LtsA can directly utilize NH3 in vivo, our data obtained with site-directed mutants suggest that this is the case. An experimental system may be required to assay the synthetase activity of LtsA in vitro and to investigate whether LtsA has an NH3-dependent synthetase activity.

The mutated residue of L-88 was in the glutaminase domain and was conserved among homologous proteins, but the importance of this residue has not been reported. On the other hand, the mutation of L-65 was in the synthetase domain, but it resulted in reduced glutaminase activity, which was compensated for by the addition of NH4Cl. The molecular reasons for the importance of this residue may be revealed by a crystallographic analysis of LtsA. Alternatively, this mutant may be defective in the connection between the glutaminase and synthetase domains, resulting in inefficient coupling.

In this study, we characterized the synthetase activity of LtsARe by mutational analysis and examined important residues for substrate recognition. We showed that the LtsAK494A mutant was sensitive to lysozyme but was rather moderately affected compared with the null mutant. Since the LtsAK494A mutant turned out to be lysozyme resistant in the presence of NH4Cl, we suggest that the LtsAK494A mutant is defective in the synthetase activity; i.e., the positive charge of LtsAK494A is important for substrate recognition.

TEM analysis suggested that there is no apparent change in the appearance of the outer layers of the cell wall of the lysozyme-sensitive mutants, which could explain the lysozyme sensitivity. Moreover, our studies with isoniazid and ethambutol suggested that the mycolate-containing cell wall structure does not cause the lysozyme sensitivity of R. erythropolis LtsA mutants. However, the addition of cell wall components solubilized by lysozyme enhanced the glutaminase activity. This strongly suggests that LtsA is involved in the biosynthesis of the cell wall. The cell wall components solubilized by our procedure are mainly peptidoglycan fragments containing disaccharide polypeptides. Since peptidoglycan precursors have carboxyl groups in meso-diaminopimelic acid and D-glutamic acid, which are amidated to some extent in R. erythropolis peptidoglycan (8, 30), these molecules might be a substrate of LtsA. The peptidoglycan of E. coli is the same type as that of R. erythropolis; it contains meso-diaminopimelic acid and D-glutamic acid (8, 30), and cell wall extracts of E. coli prepared by the procedure described here for R. erythropolis also enhanced the gultaminase activity of LtsARe (unpublished data). This might be one reason for the morphological defects and aggregation observed for E. coli cells producing recombinant LtsARe. In order to characterize the LtsA enzyme further, the natural substrate present in the cell wall lysate must be identified.


arrow
ACKNOWLEDGMENTS
 
We are grateful to P. Zwickl for critical reading of the manuscript. We also thank all the members of our group for their helpful discussions and Y. Miura for the construction and sequencing of plasmids.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan. Phone: 81-11-857-8938. Fax: 81-11-857-8990. E-mail: t-tamura{at}aist.go.jp. Back


arrow
REFERENCES
 
    1
  1. Azuma, I., D. W. Thomas, A. Adam, J.-M. Ghuysen, R. Bonaly, J.-F. Peti, and E. Lederer. 1970. Occurrence of N-glycolylmuramic acid in bacterial cell walls. A preliminary survey. Biochim. Biophys. Acta 208:444-451.[Medline]
    2. 2
  2. Barry, C. E., III, R. E. Lee, K. Mdluli, A. E. Sampson, B. G. Schroeder, R. A. Slayden, and Y. Yuan. 1998. Mycolic acids: structure, biosynthesis and physiological functions. Prog. Lipid Res. 37:143-179.[CrossRef][Medline]
    3. 3
  3. Belanger, A. E., G. S. Besra, M. E. Ford, K. Mikosova, J. T. Belisle, P. J. Brennan, and J. M. Inamine. 1996. The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc. Natl. Acad. Sci. USA 93:11919-11924.[Abstract/Free Full Text]
    4. 4
  4. Bell, K. S., J. C. Philp, Aw, D. W. J., and N. Christofi. 1998. The genus Rhodococcus. J. Appl. Microbiol. 85:195-210.[CrossRef][Medline]
    5. 5
  5. Boehlein, S. K., N. G. J. Richards, E. S. Walworth, and S. M. Schuster. 1994. Arginine 30 and asparagine 74 have functional roles in the glutamine dependent activities of Escherichia coli asparagine synthetase B. J. Biol. Chem. 269:26789-26795.[Abstract/Free Full Text]
    6. 6
  6. Brennan, P. J., and H. Nikaido. 1995. The envelope of mycobacteria. Annu. Rev. Biochem. 64:29-63.[CrossRef][Medline]
    7. 7
  7. Finnerty, W. R.. 1992. The biology and genetics of the genus Rhodococcus. Annu. Rev. Microbiol. 46:193-218.[CrossRef][Medline]
    8. 8
  8. Ghuysen, J. M.. 1968. Use of bacteriologic enzymes in determination of wall structure and their role in cell metabolism. Bacteriol. Rev. 32:425-464.[Free Full Text]
    9. 9
  9. Harpel, M. R., K. Y. Horiuchi, Y. Luo, L. Shen, W. Jiang, D. J. Nelson, K. C. Rogers, C. P. Decicco, and R. A. Copeland. 2002. Mutagenesis and mechanism-based inhibition of Streptococcus pyogenes Glu-tRNAGln amidotransferase implicate a serine-based glutaminase site. Biochemistry 41:6398-6407.[CrossRef][Medline]
    10. 10
  10. Hirasawa, T., M. Wachi, and K. Nagai. 2000. A mutation in the Corynebacterium glutamicum ltsA gene causes susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production. J. Bacteriol. 182:2696-2701.[Abstract/Free Full Text]
    11. 11
  11. Hirschfield, G. R., M. McNeil, and P. J. Brennan. 1990. Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis. J. Bacteriol. 172:1005-1013.[Abstract/Free Full Text]
    12. 12
  12. Huang, X., H. M. Holden, and F. M. Raushel. 2001. Channeling of substrates and intermediates in enzyme-catalyzed reactions. Annu. Rev. Biochem. 70:149-180.[CrossRef][Medline]
    13. 13
  13. Humbert, R., and R. D. Simoni. 1980. Genetic and biological studies demonstrating a second gene coding for asparagine synthetase in Escherichia coli. J. Bacteriol. 142:212-220.[Abstract/Free Full Text]
    14. 14
  14. Ibrahim, H. R., M. Yamada, K. Matsushita, K. Kobayashi, and A. Kato. 1994. Enhanced bactericidal action of lysozyme to Escherichia coli by inserting hydrophobic pentapeptide into its C terminus. J. Biol. Chem. 269:5059-5063.[Abstract/Free Full Text]
    15. 15
  15. Kim, J. H., J. M. Krahn, D. R. Tomchick, J. L. Smith, and H. Zalkin. 1994. Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site. J. Biol. Chem. 271:15549-15557.
    16. 16
  16. Krahn, J. M., J. H. Kim, M. R. Burns, R. J. Parry, H. Zalkin, and J. L. Smith. 1997. Coupled formation of an amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site. Biochemistry 36:11061-11068.[CrossRef][Medline]
    17. 17
  17. Larsen, T. M., S. K. Boehlein, S. M. Schuster, N. G. J. Richards, J. B. Thoden, H. M. Holden, and I. Rayment. 1999. Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product. Biochemistry 38:16146-16157.[CrossRef][Medline]
    18. 18
  18. Mäntsälä, P, and H. Zalkin. 1984. Glutamine amidotransferase function. J. Biol. Chem. 259:14230-14236.[Abstract/Free Full Text]
    19. 19
  19. Mdluli, K., J. Swanson, E. Fischer, R. E. Lee, and C. E. Barry III. 1998. Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium. Mol. Microbiol. 27:1223-1233.[CrossRef][Medline]
    20. 20
  20. Mdluli, K., R. A. Slayden, Y. Zhu, S. Ramaswamy, X. Pan, D. Mead, D. D. Crane, J. M. Musser, and C. E. Barry III. 1998. Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid. Science 280:1607-1610.[Abstract/Free Full Text]
    21. 21
  21. Mei, B., and H. Zalkin. 1989. A cysteine-histidine-aspartate catalytic triad is involved in glutamine amide transfer function in purF-type glutamine amidotransferase. J. Biol. Chem. 264:16613-16619.[Abstract/Free Full Text]
    22. 22
  22. Miles, E. W., S. Rhee, and D. R. Davies. 1999. The molecular basis of substrate channeling. J. Biol. Chem. 274:12193-12196.[Free Full Text]
    23. 23
  23. Miller, M. T., B. O. Bachman, C. A. Townsend, and A. C. Rosenzweig. 2001. Structure of ß-lactam synthetase reveals how to synthesize antibiotics instead of asparagine. Nat. Struct. Biol. 8:684-689.[CrossRef][Medline]
    24. 24
  24. Min, B., J. T. Pelaschier, D. E. Graham, D. T. Hansen, and D. Söll. 2002. Transfer RNA-dependent amino acid biosynthesis: an essential route to asparagine formation. Proc. Natl. Acad. Sci. USA 99:2678-2683.[Abstract/Free Full Text]
    25. 25
  25. Morgenstern, B., A. Dress, and T. Werner. 1996. Multiple DNA and protein sequence alignment based on segment-to-segment composition. Proc. Natl. Acad. Sci. USA 93:12098-12103.[Abstract/Free Full Text]
    26. 26
  26. Nakashima, N., and T. Tamura. 2004. A novel system for expressing recombinant proteins over a wide temperature from 4 to 35°C. Biotechnol. Bioeng. 86:136-148.[CrossRef][Medline]
    27. 27
  27. Nakashima, N., and T. Tamura. 2004. Isolation and characterization of a rolling circle type plasmid from Rhodococcus erythropolis: application in multiple recombinant protein expression. Appl. Environ. Microbiol. 70:5557-5568.[Abstract/Free Full Text]
    28. 28
  28. Paluh, J. L., H. Zalkin, D. Betsch, and H. L. Weith. 1985. Study of anthranilate synthase function by replacement of cysteine 84 using site-directed mutagenesis. J. Biol. Chem. 260:1889-1894.[Abstract/Free Full Text]
    29. 29
  29. Rubino, S. D., H. Nyunoya, and C. J. Lusty. 1986. Catalytic domains of carbamyl phosphate synthetase. J. Biol. Chem. 261:11320-11327.[Abstract/Free Full Text]
    30. 30
  30. Schleifer, K. H., and O. Kandler. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36:407-477.[Free Full Text]
    31. 31
  31. Scofield, M. A., W. S. Lewis, and S. M. Schuster. 1990. Nucleotide sequence of Escherichia coli asnB and deduced amino acid sequence of asparagine synthetase B. J. Biol. Chem. 265:12895-12902.[Abstract/Free Full Text]
    32. 32
  32. Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784-789.[CrossRef]
    33. 33
  33. Tesmer, J. J. G., T. J. Klem, M. L. Deras, V. J. Davisson, and J. L. Smith. 1996. The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families. Nat. Struct. Biol. 3:74-86.[CrossRef][Medline]
    34. 34
  34. Thoden, J. B., H. M. Holden, G. Wesenberg, F. M. Raushel, and I. Rayment. 1997. Structure of carbamyl phosphate synthetase: a journey of 96 Å from substrate to product. Biochemistry 36:6305-6316.[CrossRef][Medline]
    35. 35
  35. Wang, L., R. A. Slayden, C. E. Barry III, and J. Liu. 2000. Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids. J. Biol. Chem. 275:7224-7229.[Abstract/Free Full Text]
    36. 36
  36. Zalkin, H.. 1993. The amidotransferases. Adv. Enzymol. Relat. Areas Mol. Biol. 66:203-309.[Medline]
    37. 37
  37. Zalkin, H., and J. L. Smith. 1998. Enzymes utilizing glutamine as an amide donor. Adv. Enzymol. Relat. Areas Mol. Biol. 72:87-144.[Medline]
    38. 38
  38. Zalkin, H., and T. Murphy. 1975. Utilization of ammonia for tryptophan synthesis. Biochem. Biophys. Res. Commun. 67:1370-1377.[CrossRef][Medline]

Journal of Bacteriology, April 2005, p. 2582-2591, Vol. 187, No. 8
0021-9193/05/$08.00+0     doi:10.1128/JB.187.8.2582-2591.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mitani, Y.
Right arrow Articles by Tamura, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mitani, Y.
Right arrow Articles by Tamura, T.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»