Temperature-Sensitive Mutants Blocked in the Folding or Subunit Assmbly of the Bacteriophage P22 Tailspike Protein. I. Fine-Structure Mapping

As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su^- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.—None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of thes ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein.     

Suppressors of Mutations in the rII Gene of Bacteriophage T4 Affect Promoter Utilization

Homyk, Rodriguez and Weil (1976) have described T4 mutants, called sip, that partially suppress the inability of T4rII mutants to grow in λ lysogens. We have found that mutants sip1 and sip2 are resistant to folate analogs and overproduce FH₂ reductase. The results of recombination and complementation studies indicate that sip mutations are in the mot gene. Like other mot mutations (Mattson, Richardson and Goodin 1974; Chace and Hall 1975; Sauerbier, Hercules and Hall 1976), the sip2 mutation affects the expression of many genes and appears to affect promoter utilization. The mot gene function is not required for T4 growth on most hosts, but we have found that it is required for good growth on E. coli CTr5X. Homyk, Rodriguez and Weil (1976) also described L mutations that reverse the effects of sip mutations. L₂ decreases the folate analog resistance and the inability of sip2 to grow on CTr5X. L₂ itself is partially resistant to a folate analog, and appears to reverse the effects of sip2 on gene expression. These results suggest that L₂ affects another regulatory gene related to the mot gene.     

Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNA^Tyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNA^Tyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA^Tyr. The endogenous TyrRS and tRNA^Tyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNA^Tyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.     

The sedimentation behaviour of ribonuclease-active and -inactive ribosomes from bacteria

1. The `30s' and `50s' ribosomes from ribonuclease-active (Escherichia coli B) and -inactive (Pseudomonas fluorescens and Escherichia coli MRE600) bacteria have been studied in the ultracentrifuge. Charge anomalies were largely overcome by using sodium chloride–magnesium chloride solution, I 0·16, made 0–50mm with respect to Mg²⁺. 2. Differentiation of enzymic and physical breakdown at Mg²⁺ concentrations less than 5mm was made by comparing the properties of E. coli B and P. fluorescens ribosomes. 3. Ribonuclease-active ribosomes alone showed a transformation of `50s' into 40–43s components. This was combined with the release of a small amount of `5s' material which may be covalently bound soluble RNA. Other transformations of the `50s' into 34–37s components were observed in both ribonuclease-active and -inactive ribosomes at 1·0–2·5mm-Mg²⁺, and also with E. coli MRE600 when EDTA (0·2mm) was added to a solution in 0·16m-sodium chloride. 4. Degradation of ribonuclease-active E. coli B ribosomes at Mg²⁺ concentration 0·25mm or less was coincident with the formation of 16s and 21s ribonucleoprotein in P. fluorescens, and this suggested that complete dissociation of RNA from protein was not an essential prelude to breakdown of the RNA by the enzyme. 5. As high Cs⁺/Mg²⁺ ratios cause ribosomal degradation great care is necessary in the interpretation of equilibrium-density-gradient experiments in which high concentrations of caesium chloride or similar salts are used. 6. The importance of the RNA moiety in understanding the response of ribosomes to their ionic environment is discussed.     

One-Step Biosynthesis of α-Keto-γ-Methylthiobutyric Acid from L-Methionine by an Escherichia coli Whole-Cell Biocatalyst Expressing an Engineered L-Amino Acid Deaminase from Proteus vulgaris

α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl₂, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 10⁴ mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca²⁺ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.     

One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Differentiation between Amber and Ochre Mutants of Yeast by Reversion with 4-Nitroquinoline-1-Oxide

4-nitroquinoline-1-oxide (NQO) induces high frequencies of intragenic revertants of amber (UAG) but not ochre (UAA) mutants of yeast. Distinction of the amber and ochre codons was made with well-characterized nonsense mutants of the iso-1-cytochrome c gene (cyc1 mutants) as well as with nonsense mutants having nutritional requirements. Thus the NQO-induced reversion frequencies corroborated the assignments that were based on the pattern of amino acid replacements in intragenic revertants and on the speficity of suppression. It was concluded from these results and from the results of a previous investigation with other cyc1 mutants (Prakash, Stewart and Sherman 1974) that NQO induces transversions of G:C base pairs at many sites and that the specificity is not strongly influenced by neighboring base pairs in at least the strains examined in these studies. NQO was previously shown to induce G:C → A:T transitions at least at one site and this and the previous study established that it does not significantly mutate A:T base pairs at numerous sites. Thus NQO can be used to selectively mutate G:C base pairs and to determine if the pathways of reverse mutations involve G:C base pairs. Suppressors that act on either amber or ochre mutants were induced with NQO, indicating that they can arise by mutations of G:C base pairs.     

Reversible Repression of Early Enzyme Synthesis in Bacteriophage T4-infected Escherichia coli

A mutant of Escherichia coli B, defective in its accumulation of K⁺, was found to synthesize protein at a rate proportional to the level of this cation in the growth medium. When bacteriophage T4-infected cells were incubated in growth medium containing 1 mm K⁺, phage deoxyribonucleic acid (DNA) was synthesized at a rate 25% that of normal, and phage protein was synthesized at a rate of 50% of normal. Deoxycytidine pyrophosphatase, a phage-directed early enzyme, shut off at a level of 55% that of normal when infected cells were incubated in medium containing 1 mm K⁺. However, deoxycytidine pyrophosphatase synthesis resumed in these cells when they were shifted to medium containing the normal K⁺ concentration (33 mm). DNA synthesis also attained the rate characteristic of this K⁺ concentration. These results suggest that phage DNA synthesis is not sufficient to repress early protein formation and also indicate that the inhibitor of early protein formation is an early function whose synthesis is sensitive to the same repression as that of the early proteins.     

Construction of a Proline-Producing Mutant of the Extremely Thermophilic Eubacterium Thermus thermophilus HB27

Growth of Thermus thermophilus HB27 was inhibited by a proline analog, 3,4-dehydroproline (DHP). This result suggested that the γ-glutamyl kinase (the product of the proB gene) was inhibited by feedback inhibition in T. thermophilus. DHP-resistant mutants were reported previously for Escherichia coli (A. M. Dandekar and S. L. Uratsu, J. Bacteriol. 170:5943–5945, 1988) and Serratia marcescens (K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol. 138:693–699, 1992), and their mutated sites in the proB gene were identified. Comparison of the amino acid sequence of T. thermophilus γ-glutamyl kinase with those of E. coli and S. marcescens mutants revealed that the DHP resistance mutations occurred in the amino acids conserved among the three organisms. For eliminating the feedback inhibition, we first constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis at the proB gene as reported for the proline-producing mutant of S. marcescens. The mutant, TH401, excreted about 1 mg of l-proline per liter at 70°C after 12 h of incubation. It was also suggested that T. thermophilus had a proline degradation and transport pathway since it was able to grow in minimal medium containing l-proline as sole nitrogen source. In order to disrupt the proline degradation or transport genes, TH401 was mutated by UV irradiation. Seven mutants unable to utilize l-proline for their growth were isolated. One of the mutants, TH4017, excreted about 2 mg of l-proline per liter in minimal medium at 70°C after 12 h of incubation.Thermus thermophilus, a gram-negative aerobic eubacterium, is one of the most widely studied species of extremely thermophilic microorganisms. We have been working on the molecular genetics and molecular reproduction of T. thermophilus HB27. We have already cloned and sequenced three proline biosynthetic genes, proB, proA, and proC, and reported that the proB and proA genes exist in tandem (7, 9).We have also constructed physical maps of the HB27 chromosome and of a large plasmid, pTT27, and determined the locations of all proline biosynthetic genes on the chromosomal DNA (20, 21). We have already succeeded in overproducing carotenoids in T. thermophilus HB27 (6), but at present there is no report about extracellular production of amino acids in extreme thermophiles. We have elucidated the consensus sequences for strong promoters of T. thermophilus (11) and developed a thermostable antibiotic resistance gene (12). It is also easy to disrupt or mutate genes on chromosomal DNA in T. thermophilus HB27 (8). Among the extreme thermophiles, a host-vector system has been established only in T. thermophilus. Generally, the reaction rate of thermostable enzymes which are produced from T. thermophilus is higher than those of enzymes from mesophiles. In a fermentation process such as amino acid production, T. thermophilus may contribute to the improvement of amino acid productivity since fermentation at a high temperature eliminates the problems of contamination and cooling procedures. So, we decided to attempt excretion of proline at a high temperature with T. thermophilus mutants.l-Proline is synthesized from glutamate by the sequential reaction of γ-glutamyl kinase, γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase in bacteria (1). Genes proB and proA, which encode γ-glutamyl kinase and γ-glutamyl phosphate reductase, respectively, were found to comprise an operon in T. thermophilus (9), Escherichia coli (5), and Serratia marcescens (13). In E. coli and S. marcescens, γ-glutamyl kinase is subject to feedback control by l-proline (3, 13), but γ-glutamyl phosphate reductase and pyrroline-5-carboxylate reductase are not inhibited by proline (3, 15). Meanwhile, E. coli and S. marcescens rapidly degrade proline by proline dehydrogenase (proline oxidase), encoded by the putA gene (3, 14, 22). So far, it has been reported that E. coli mutants resistant to proline analogs, dl-3,4-dehydroproline and l-azetidine-2-carboxylic acid, excreted l-proline into the medium. But the amount of l-proline excreted was too small for practical use because of the existence of the proline degradation pathway (2). For S. marcescens, Sugiura et al. (18, 19) have constructed a proline-overproducing strain, SP126, as a double mutant resistant to 3,4-dehydroproline and thiazolidine-4-carboxylate and derived from a proline dehydrogenase-deficient mutant (18). Strain SP126 produced about 20 mg of l-proline per ml in the fermentation medium (18).In T. thermophilus, the control system in proline biosynthesis has not been elucidated. However, we thought that the feedback control of proline biosynthesis in T. thermophilus should be similar to that of E. coli and S. marcescens, since the amino acid sequences of proline biosynthetic enzymes in T. thermophilus show a high similarity to sequences of those of E. coli and S. marcescens (7, 9). E. coli and S. marcescens mutants resistant to 3,4-dehydroproline have already been determined to be proB mutants (4, 14). The comparison of the amino acid sequences of γ-glutamyl kinases in E. coli, S. marcescens, and T. thermophilus showed that these mutations occurred in the positions conserved among the three microorganisms (Fig. ​(Fig.1).1). We thought that it was possible to construct a 3,4-dehydroproline-resistant mutant of T. thermophilus by introducing the same mutations into the proB gene found in the mutants of E. coli and S. marcescens. We determined the strategy for construction of a proline-producing strain of T. thermophilus by following two steps: first, construction of a 3,4-dehydroproline-resistant mutant by introduction of mutations into the proB gene, and second, isolation of a mutant which cannot utilize proline for its growth by mutagenizing the dehydroproline-resistant mutant. Open in a separate windowFIG. 1Comparison of the amino acid sequences of γ-glutamyl kinases in E. coli, S. marcescens, and T. thermophilus. The amino acid substitutions found in E. coli (4) and S. marcescens (14) are shown by arrows. Asterisks show the amino acid residues conserved in the three microorganisms.     

Overproduction of l-Cysteine and l-Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase

Organisms that overproduced l-cysteine and l-cystine from glucose were constructed by using Escherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg of l-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production of l-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production of l-cysteine plus l-cystine was markedly increased compared to production in JM39.l-Cysteine, one of the important amino acids used in the pharmaceutical, food, and cosmetics industries, has been obtained by extracting it from acid hydrolysates of the keratinous proteins in human hair and feathers. The first successful microbial process used for industrial production of l-cysteine involved the asymmetric conversion of dl-2-aminothiazoline-4-carboxylic acid, an intermediate compound in the chemical synthesis of dl-cysteine, to l-cysteine by enzymes from a newly isolated bacterium, Pseudomonas thiazoliniphilum (11). Yamada and Kumagai (13) also described enzymatic synthesis of l-cysteine from beta-chloroalanine and sodium sulfide in which Enterobacter cloacae cysteine desulfhydrase (CD) was used. However, high level production of l-cysteine from glucose with microorganisms has not been studied.Biosynthesis of l-cysteine in wild-type strains of Escherichia coli and Salmonella typhimurium is regulated through feedback inhibition by l-cysteine of serine acetyltransferase (SAT), a key enzyme in l-cysteine biosynthesis, and repression of expression of a series of enzymes used for sulfide reduction from sulfate by l-cysteine (4), as shown in Fig. ​Fig.1.1. Denk and Böck reported that a small amount of l-cysteine was excreted by a revertant of a cysteine auxotroph of E. coli. In this revertant, SAT encoded by the cysE gene was desensitized to feedback inhibition by l-cysteine, and the methionine residue at position 256 in SAT was replaced by isoleucine (2). These results indicate that it may be possible to construct organisms that produce high levels of l-cysteine by amplifying an altered cysE gene. Although the residue at position 256 is supposedly part of the allosteric site for cysteine binding, no attention has been given to the effect of an amino acid substitution at position 256 in SAT on feedback inhibition by l-cysteine and production of l-cysteine. It is also not known whether isoleucine is the best residue for desensitization to feedback inhibition. Open in a separate windowFIG. 1Biosynthesis and regulation of l-cysteine in E. coli. Abbreviations: APS, adenosine 5′-phosphosulfate; PAPS, phosphoadenosine 5′-phosphosulfate; Acetyl CoA, acetyl coenzyme A. The open arrow indicates feedback inhibition, and the dotted arrows indicate repression.On the other hand, l-cysteine appears to be degraded by E. coli cells. Therefore, in order to obtain l-cysteine producers, a host strain with a lower level of l-cysteine degradation activity must be isolated. In this paper we describe high-level production of l-cysteine plus l-cystine from glucose by E. coli resulting from construction of altered cysE genes. The methionine residue at position 256 in SAT was replaced by other amino acids or the termination codon in order to truncate the carboxy terminus from amino acid residues 256 to 273 by site-directed mutagenesis. A newly derived cysteine-nondegrading E. coli strain with plasmids having the altered cysE genes was used to investigate production of l-cysteine plus l-cystine.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

Whole-Genome Profiling of Mutagenesis in Caenorhabditis elegans

Deep sequencing offers an unprecedented view of an organism''s genome. We describe the spectrum of mutations induced by three commonly used mutagens: ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), and ultraviolet trimethylpsoralen (UV/TMP) in the nematode Caenorhabditis elegans. Our analysis confirms the strong GC to AT transition bias of EMS. We found that ENU mainly produces A to T and T to A transversions, but also all possible transitions. We found no bias for any specific transition or transversion in the spectrum of UV/TMP-induced mutations. In 10 mutagenized strains we identified 2723 variants, of which 508 are expected to alter or disrupt gene function, including 21 nonsense mutations and 10 mutations predicted to affect mRNA splicing. This translates to an average of 50 informative mutations per strain. We also present evidence of genetic drift among laboratory wild-type strains derived from the Bristol N2 strain. We make several suggestions for best practice using massively parallel short read sequencing to ensure mutation detection.MUTAGENESIS and the screening for mutants have long been a key tool of the practicing geneticist. The early work of T. H. Morgan and his colleagues relied on recovery of spontaneous mutations, which was limiting for the study of inheritance due to their infrequent occurrence (Morganet al. 1922; also see Sturtevant 1965). The discovery by H. J. Muller and others that X rays cause mutations ushered in the era of inducing mutations (Muller 1927). There is a long history of studies on mutagen specificity, both in prokaryotes and in eukaryotes, and today many mutagens are utilized in a variety of model organisms. In this article we use whole-genome deep sequencing in the model organism Caenorhabditis elegans to explore the types and frequencies of mutations induced by various mutagens and to document the feasibility of global identification of mutations.The mutagenic properties of ethyl methanesulfonate (EMS) were first demonstrated using the T4 viral system (Loveless 1959). Soon after, Lewis and Bacher (1968) demonstrated how to administer EMS to Drosophila melanogaster to generate mutations, and later Sydney Brenner did the same for the nematode C. elegans (Brenner 1974). The now classic article by Coulondre and Miller (1977) demonstrated the types of nucleotide substitutions generated by EMS and confirmed earlier observations (Bautz and Freese 1960) concerning the strong bias for GC to AT transitions. Today, EMS is still the most powerful and popular mutagen used by researchers studying D. melanogaster and C. elegans. Purely on the basis of genetic inference, when used at a concentration of 50 mm, EMS is calculated to induce ∼20 function-affecting variant alleles in C. elegans strains derived using this mutagen (Greenwald and Horvitz 1982; Anderson 1995).The chemical N-ethyl-N-nitrosourea (ENU) has been used as a mutagen since the 1970s but came to prominence when it was demonstrated to be the most effective chemical mutagen in mice (Russell et al. 1979). Today it is still the chemical mutagen of choice for this organism (Anderson 2000; Acevedo-Arozena et al. 2008). ENU has also been used for C. elegans mutagenesis (De Stasio et al. 1997). Although it appears to have different biases with regard to gene targets and base changes relative to EMS, the background mutational load after ENU mutagenesis has not been fully characterized (De Stasio and Dorman 2001).The chemical 4,5′,8-trimethylpsoralen is a crosslinking agent that is activated by near ultraviolet light. Studies in Escherichia coli have shown that it causes both single-base changes and deletions (Piette et al. 1985; Sladek et al. 1989). C. elegans researchers became interested in the potential of ultraviolet trimethylpsoralen (UV/TMP) to generate deletions in worms after the first deletions in this organism were isolated using this mutagen (Yandell et al. 1994). UV/TMP is now a major reagent in the arsenal of the C. elegans knockout consortium laboratories (Barstead and Moerman 2006). As a tool for generating deletions in eukaryotes it is quite useful but, outside of studies on prokaryotes, little else is known about the spectrum of mutagenic effects caused by UV/TMP.Massively parallel short read sequencing technologies offer unprecedented opportunities to study the complete genetic complement of an individual organism (Hillier et al. 2008). For genetic model systems the impact of this technology extends to the identification and correlation of induced mutations with selected phenotypes (Sarin et al. 2008). Several of the technological and bioinformatic issues that arise with next generation sequencing have already been addressed for the nematode C. elegans (Hillier et al. 2008; Sarin et al. 2008; Shen et al. 2008; Rose et al. 2010). Still, it is not clear how deeply one must sequence to confidently identify a relevant variant allele in a target mutant strain. Also of importance are questions concerning mutagen choice and dosage as they relate to the rate of induction of new mutations and background mutational load. We have undertaken the following study on mutagenesis and mutation detection to establish the parameters necessary to exploit next generation sequencing technologies for C. elegans genetics. For the first time we offer a whole-genome direct measure of mutation spectrum and background load for EMS, ENU, and UV/TMP. Readers interested in whole-genome sequencing of EMS mutagenized strains in C. elegans should also see the accompanying article in this issue by Sarin et al. (2010). In our study we also measured the single-nucleotide variation among currently used wild-type strains. In addition, we measured sequence read depth of all sequence and coding sequence and from this we make a recommendation of average genome coverage to ensure the correct identification of the causative mutation. We also examined the issue of false positive and false negative calls and make recommendations to eliminate most false positives without losing bona fide mutations.     

Adaptive Evolution of Escherichia coli K-12 MG1655 during Growth on a Nonnative Carbon Source,l-1,2-Propanediol

Laboratory adaptive evolution studies can provide key information to address a wide range of issues in evolutionary biology. Such studies have been limited thus far by the inability of workers to readily detect mutations in evolved microbial strains on a genome scale. This limitation has now been overcome by recently developed genome sequencing technology that allows workers to identify all accumulated mutations that appear during laboratory adaptive evolution. In this study, we evolved Escherichia coli K-12 MG1655 with a nonnative carbon source, l-1,2-propanediol (l-1,2-PDO), for ∼700 generations. We found that (i) experimental evolution of E. coli for ∼700 generations in 1,2-PDO-supplemented minimal medium resulted in acquisition of the ability to use l-1,2-PDO as a sole carbon and energy source so that the organism changed from an organism that did not grow at all initially to an organism that had a growth rate of 0.35 h⁻¹; (ii) six mutations detected by whole-genome resequencing accumulated in the evolved E. coli mutant over the course of adaptive evolution on l-1,2-PDO; (iii) five of the six mutations were within coding regions, and IS5 was inserted between two fuc regulons; (iv) two major mutations (mutations in fucO and its promoter) involved in l-1,2-PDO catabolism appeared early during adaptive evolution; and (v) multiple defined knock-in mutant strains with all of the mutations had growth rates essentially matching that of the evolved strain. These results provide insight into the genetic basis underlying microbial evolution for growth on a nonnative substrate.Evolution of microorganisms in the laboratory offers the possibility of relating acquired mutations to increased fitness of the organism under the conditions used. Complete identification of mutations over defined evolutionary periods is necessary to fully understand the evolutionary change because spontaneous mutation is the foundational biological source of phenotypic variation (52). Since microbes grow rapidly and have large population sizes and since ancestors can be preserved by freezing them for later direct comparison of evolved types, laboratory evolution using microorganisms provides a powerful context for studying the genetics of evolutionary adaptation (5, 12, 14, 19, 43) due to the advent of new technologies for genome-wide detection of mutations (30, 33). A large number of studies of experimental evolution with various microbes have been carried out using natural carbon sources, especially glucose (12, 19, 47, 55), since glucose is the preferred carbon and energy source for most bacteria and eukaryotic cells (4, 50). Recently, a few studies have investigated the adaptive evolution of Escherichia coli at the genetic and metabolic levels with gluconeogenic carbon sources, including lactate (34) and glycerol (20). Compared to experimental evolution with native carbon sources, microorganisms might be more capable of adapting to various nonnative carbon compounds because microorganisms are able to adapt to environmental changes by using a number of strategies to meet their growth requirements and to achieve optimal overall performance in the new conditions (20, 21, 34). However, a comprehensive analysis of the genetic basis of adaptation to nonnative carbon sources has not been performed.The K-12 MG1655 strain of E. coli is not able to utilize l-1,2-propanediol (l-1,2-PDO) as a sole carbon and energy source. However, E. coli has an enzyme, l-1,2-PDO oxidoreductase (POR), which is involved in fermentative l-fucose metabolism and catalyzes the oxidation of l-1,2-PDO to l-lactaldehyde (Fig. ​(Fig.11 A). The E. coli POR is encoded by the fucO gene of the fucose regulon (11, 23), which consists of two divergent operons (fucAO and fucPIKUR) under positive control of FucR (Fig. ​(Fig.1B)1B) (9). FucR is activated by fuculose-1-phosphate, which is the inducer of the fuc regulon (3). In E. coli, fucose metabolism is initiated by the sequential actions of a permease (encoded by fucP), an isomerase (encoded by fucI), a kinase (encoded by fucK), and an aldolase (encoded by fucA). The aldolase catalyzes the cleavage of fuculose-1-phosphate to dihydroxyacetone phosphate and l-lactaldehyde. Under aerobic respiratory conditions, l-lactaldehyde is oxidized to l-lactate by an NAD-linked aldehyde dehydrogenase with broad functions (encoded by aldA). l-Lactate is then oxidized to pyruvate by a flavin adenine dinucleotide (FAD)-dependent l-lactate dehydrogenase (encoded by the lldD gene of the lldPRD operon [formerly the lctPRD operon]). Under anaerobic fermentative conditions, however, redox balance requires sacrifice of the l-lactaldehyde as a hydrogen acceptor at the expense of NADH (Fig. ​(Fig.1A).1A). This reaction is catalyzed by the POR. The terminal fermentation product, l-1,2-PDO, is then released by a permease (57). Although the POR catalyzes the oxidation of l-1,2-PDO to l-lactaldehyde, l-1,2-PDO cannot be utilized by wild-type (WT) E. coli as a sole carbon source under aerobic conditions because this compound cannot induce expression of the fuc regulon (11). Indeed, the fuc regulon was not expressed under any conditions when a database of 213 expression profiles produced in our laboratory was examined (38). Furthermore, even if the POR is expressed, it is oxidatively inactivated by a metal-catalyzed oxidation (MCO) mechanism (7).Open in a separate windowFIG. 1.Metabolic pathway and fuc regulon for l-fucose and l-1,2-PDO. (A) Metabolic pathway for l-fucose and l-1,2-PDO. In E. coli, fucose metabolism is initiated by the sequential actions of a permease (encoded by fucP), an isomerase (encoded by fucI), a kinase (encoded by fucK), and an aldolase (encoded by fucA). The aldolase catalyzes cleavage of fuculose-1-phosphate to dihydroxyacetone phosphate and l-lactaldehyde. Under aerobic respiratory conditions, the l-lactaldehyde is further oxidized by a series of enzymes to pyruvate, which subsequently enters central metabolism. Under anaerobic fermentative conditions, the l-lactaldehyde is reduced to l-1,2-PDO by oxidoreductase (encoded by fucO). (B) Genetic organization of the fuc regulon. The fuc regulon for l-fucose uptake and metabolism consists of two divergent operons, fucAO and fucPIKUR.Sridhara et al. (48) previously described E. coli mutants that were isolated from an E. coli K-12 derivative treated with the mutagen ethyl methanesulfonate and were able to grow aerobically on l-1,2-PDO as a sole carbon source. Previous studies showed that an IS5 insertion between the fucAO and fucPIKUR operons caused constitutive expression of the fucAO operon (9, 41) at a level that enabled the E. coli mutant to grow on l-1,2-PDO. In addition, mutations resulting in increased resistance to MCO under aerobic conditions were found in the N-terminal domain of POR (39). However, at present, little is known about the accumulated genome-wide mutations and their effects on the fitness in E. coli that has acquired the ability to use l-1,2-PDO because previous studies have focused on mutations in POR and its regulatory region.In an attempt to investigate the genetic basis of adaptive evolution of E. coli during growth on l-1,2-PDO, we first isolated an E. coli mutant able to use l-1,2-PDO using experimental evolution without a mutagen, and we then characterized this evolved E. coli mutant. Using whole-genome sequencing, we identified all accumulated mutations of the evolved E. coli mutant related to the known ancestor and also determined the fitness benefits and phenotypic behaviors of the mutations discovered. Our results offer a systematic view of the genetic basis underlying microbial adaptation to a nonnative substrate.     

High-Level Heterologous Production of d-Cycloserine by Escherichia coli

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H₂S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM).     

An l-glucose Catabolic Pathway in Paracoccus Species 43P

An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD⁺-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism.