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).     

The yiaE Gene,Located at 80.1 Minutes on the Escherichia coli Chromosome,Encodes a 2-Ketoaldonate Reductase

An open reading frame located in the bisC-cspA intergenic region, or at 80.1 min on the Escherichia coli chromosome, encodes a hypothetical 2-hydroxyacid dehydrogenase, which was identified as a result of the E. coli Genome Sequencing Project. We report here that the product of the gene (yiaE) is a 2-ketoaldonate reductase (2KR). The gene was cloned and expressed with a C-terminal His tag in E. coli, and the protein was purified by metal-chelate affinity chromatography. The determination of the NH₂-terminal amino acid sequence of the protein defined the translational start site of this gene. The enzyme was found to be a 2KR catalyzing the reduction of 2,5-diketo-d-gluconate to 5-keto-d-gluconate, 2-keto-d-gluconate (2KDG) to d-gluconate, 2-keto-l-gulonate to l-idonate. The reductase was optimally active at pH 7.5, with NADPH as a preferred electron donor. The deduced amino acid sequence showed 69.4% identity with that of 2KR from Erwinia herbicola. Disruption of this gene on the chromosome resulted in the loss of 2KR activity in E. coli. E. coli W3110 was found to grow on 2KDG, whereas the mutant deficient in 2KR activity was unable to grow on 2KDG as the carbon source, suggesting that 2KR is responsible for the catabolism of 2KDG in E. coli and the diminishment of produced 2KDG from d-gluconate in the cultivation of E. coli harboring a cloned gluconate dehydrogenase gene.

We previously reported the cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase (GADH) from Erwinia cypripedii in Escherichia coli (26). In the course of further study on the conversion of d-gluconate to 2-keto-d-gluconate (2KDG) with a recombinant E. coli strain, we observed that the level of 2KDG produced in the medium gradually decreased after the exhaustion of d-gluconate in the medium (see Fig. ​Fig.1).1). In an effort to find the reason, the NADPH-dependent reductase activity catalyzing the conversion of 2KDG to d-gluconate was detected in extracts of E. coli cells. This result suggested the existence of enzymes involved in ketogluconate metabolism in E. coli, as reported for several species of the genera Corynebacterium, Brevibacterium, Erwinia, Acetobacter, Gluconobacter, Serratia, and Pseudomonas (20, 23, 25). In Erwinia, Acetobacter, Gluconobacter, Serratia, and Pseudomonas, oxidation of glucose to ketogluconates such as 2KDG, 5-keto-d-gluconate (5KDG), and 2,5-diketo-d-gluconate (25DKG) has been shown to proceed via membrane-bound dehydrogenases, which are linked to the electron transport chain (2, 21). The ketogluconates or their phosphorylated forms are unique substrates in that they enter into central metabolism only after they are reduced by NADPH-dependent reductases (20, 23). NADPH-dependent 2-ketoaldonate reductase (2KR), which catalyzes the reduction of 2KDG to d-gluconate, 25DKG to 5KDG, and 2-keto-l-gulonate (2KLG) to l-idonate (IA), has been purified and characterized from Brevibacterium ketosoreductum (25) and Erwinia herbicola (23). Even if the substrate specificity has not been examined with 25DKG as a substrate, 2KDG reductases from acetic acid bacteria also catalyze the reduction of 2KLG to IA as well as of 2KDG to d-gluconate (1).Open in a separate windowFIG. 1Time course of bioconversion of d-gluconate to 2KDG by E. coli harboring the cloned GADH gene. E. coli W3110(pGA313) was grown in a 2-liter fermentor at 37°C with aeration at 1 vvm and agitation at 500 rpm.Until now, no ketoaldonate reductase has been reported for E. coli. We report here that the product of the yiaE gene, located in the bisC-cspA intergenic region at 80.1 min on the E. coli chromosome, is a 2KR; in addition, the diminishment of produced 2KDG from d-gluconate in the cultivation of recombinant E. coli harboring a cloned membrane-bound GADH gene is due to 2KR as the cytosolic enzyme responsible for conversion of 2KDG to d-gluconate. We found also that E. coli W3110 grows on 2KDG as the sole carbon source.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Using RNA Interference to Identify Specific Modifiers of a Temperature-Sensitive,Embryonic-Lethal Mutation in the Caenorhabditis elegans Ubiquitin-Like Nedd8 Protein Modification Pathway E1-Activating Gene rfl-1

The essential Caenorhabditis elegans gene rfl-1 encodes one subunit of a heterodimeric E1-activating enzyme in the Nedd8 ubiquitin-like protein conjugation pathway. This pathway modifies the Cullin scaffolds of E3 ubiquitin ligases with a single Nedd8 moiety to promote ligase function. To identify genes that influence neddylation, we used a synthetic screen to identify genes that, when depleted with RNAi, enhance or suppress the embryonic lethality caused by or198ts, a temperature-sensitive (ts) mutation in rfl-1. We identified reproducible suppressor and enhancer genes and employed a systematic specificity analysis for each modifier using four unrelated ts embryonic lethal mutants. Results of this analysis highlight the importance of specificity controls in identifying genetic interactions relevant to a particular biological process because 8/14 enhancers and 7/21 suppressors modified lethality in other mutants. Depletion of the strongest specific suppressors rescued the early embryonic cell division defects in rfl-1(or198ts) mutants. RNAi knockdown of some specific suppressors partially restored Cullin neddylation in rfl-1(or198ts) mutants, consistent with their gene products normally opposing neddylation, and GFP fusions to several suppressors were detected in the cytoplasm or the nucleus, similar in pattern to Nedd8 conjugation pathway components in early embryonic cells. In contrast, depletion of the two strongest specific enhancers did not affect the early embryonic cell division defects observed in rfl-1(or198ts) mutants, suggesting that they may act at later times in other essential processes. Many of the specific modifiers are conserved in other organisms, and most are nonessential. Thus, when controlled properly for specificity, modifier screens using conditionally lethal C. elegans mutants can identify roles for nonessential but conserved genes in essential processes.UBIQUITIN-mediated proteolysis regulates many biological processes (Nandi et al. 2006). In the early Caenorhabditis elegans embryo, these include oocyte maturation, cell cycle progression, cell polarization, and cell fate patterning, all of which require the timely destruction of maternally expressed proteins (Bowerman and Kurz 2006; Greenstein and Lee 2006). One C. elegans protein targeted for proteolysis early in embryogenesis is MEI-1, the AAA-ATPase subunit of the microtubule-severing complex called katanin (Mains et al. 1990; Dow and Mains 1998; Srayko et al. 2000; Kurz et al. 2002; Pintard et al. 2003a; Xu et al. 2003). Katanin is a heterodimer of two subunits called p60 and p80 in vertebrates and MEI-1 and MEI-2 in C. elegans. Katanin in C. elegans is required for proper assembly and function of the small, barrel-shaped meiotic spindles (Albertson and Thomson 1993; McNally et al. 2006) and must be degraded after meiotic divisions to permit assembly of the much larger first mitotic spindle in the one-cell zygote. In mutants that fail to degrade katanin after the completion of meiosis, the first mitotic spindle is fragmented and mis-oriented, cytokinesis is defective, and the embryos die without hatching (Dow and Mains 1998; Srayko et al. 2000; Kurz et al. 2002).The katanin subunit MEI-1 is targeted for poly-ubiquitylation and proteolytic destruction by a Cullin-based E3 ligase (Kurz et al. 2002). This complex includes the Cullin scaffolding protein CUL-3 and a substrate-specific adaptor called MEL-26 that binds to CUL-3 through a BTB domain and to MEI-1 through a MATH domain (Pintard et al. 2003b). Cullin 3-based E3 ligases in mammals also utilize substrate-specific adaptor proteins that, like MEL-26, have both a Cullin-binding BTB/POZ domain and another protein–protein interaction domain that binds to the substrate (Geyer et al. 2003; Cullinan et al. 2004; Angers et al. 2006). While MEI-1/Katanin downregulation by the CUL-3/MEL-26 E3 ligase is essential at most growth temperatures, a mel-26 null mutation is viable at the low growth temperature of 15° (Lu and Mains 2007). This bypass of mel-26 at 15° depends at least in part on the anaphase-promoting complex and its targeting of MEI-1 for proteolytic degradation (Lu and Mains 2007). Phosphorylation by the kinase MBK-2 primes MEI-1 for proteolysis (Quintin et al. 2003; Stitzel et al. 2007) and also promotes the downregulation of MEI-1 by the anaphase-promoting complex (Lu and Mains 2007).CUL-3 is the only C. elegans Cullin thus far identified that requires modification by the ubiquitin-like protein Nedd8 (Bowerman and Kurz 2006). In contrast, C. elegans CUL-2 is required for progression through meiosis and for the localized degradation of cell fate determinants in one-cell-stage embryos (Liu et al. 2004; Sonneville and Gonczy 2004), but neddylation-defective mutants do not exhibit these early defects (Bowerman and Kurz 2006). Cullin neddylation is mediated by the Nedd8 protein conjugation pathway, which begins with a heterodimeric E1-activating enzyme consisting of ULA-1 and RFL-1 (Uba3p in budding yeast) and also includes the E2-conjugating enzyme UBC-12 (Jones and Candido 2000; Srayko et al. 2000; Kurz et al. 2002) and the E3 ligase DCN-1 (Kurz et al. 2005).The downregulation of MEI-1/katanin by the CUL-3/MEL-26 E3 ligase requires a balance of both CUL-3 neddylation, which is mediated by the Nedd8 conjugation pathway, and deneddylation, which is mediated by the conserved COP-9 Signalosome (Pintard et al. 2003a). Other Cullin-based E3 ubiquitin ligases also require a balance of neddylation and deneddylation (Lyapina et al. 2001; Schwechheimer et al. 2001; Bornstein et al. 2006; Hetfeld et al. 2008). Deneddylation may modulate activation of the E3 ligase and thereby prevent the premature degradation of substrate adaptor proteins that also can become poly-ubiquitylated and degraded as a result of E3 ligase function.To identify additional factors that influence neddylation, and the downregulation of MEI-1/katanin after the completion of meiosis in C. elegans, we report here our use of RNA interference (RNAi) to reduce gene functions in a temperature-sensitive (ts) neddylation-defective mutant, rfl-1(or198ts). The discovery of RNAi and its systemic properties in C. elegans have made it possible to systematically target C. elegans genes for depletion by feeding worms bacterial strains that express double-strand RNAs corresponding to C. elegans gene sequences (Fire et al. 1998; Timmons et al. 2001; Feinberg and Hunter 2003; Baugh et al. 2005; Lehner et al. 2006; van Haaften et al. 2006). Furthermore, chemical mutagenesis screens have identified temperature-sensitive mutations in many essential C. elegans genes, which can be used for synthetic screens by choosing intermediate-growth temperatures that sensitize the genetic background and also optimize visual scoring of embryonic viability. Recently, genomewide RNAi screens have been used to identify C. elegans genes that, when reduced in function, restore viability to temperature-sensitive, embryonic-lethal mutants (Labbe et al. 2006; O''Rourke et al. 2007). Because a loss of suppressor function restores mutant viability, the suppressors may negatively regulate either the wild-type gene product or the process that requires the wild-type gene product.Here we report our identification of C. elegans genes that, when reduced in function by feeding RNAi, reproducibly suppressed or enhanced rfl-1(or198ts) embryonic lethality. Most suppressors were specific for rfl-1(or198ts), while specific enhancement was less common. Many of the rfl-1-specific suppressors and enhancers are conserved but appear nonessential. GFP fusions to several specific suppressors exhibit localization patterns that resemble those known for neddylation pathway components, and depletion of some of these partially restored CUL-3 neddylation in rfl-1(or198ts) mutants. In addition to identifying possible roles for conserved genes in cullin neddylation, we report the first quantitative analysis of specificity for both the enhancement and the suppression of a conditionally lethal mutant in C. elegans. Our results highlight the importance of testing genetic modifiers of conditionally lethal mutants for locus specificity.     

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.     

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.     

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.     

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.     

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N⁶-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA^Pyl_CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host''s ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.     

Genetic Analysis of Regulatory Mutants of Alkaline Phosphatase of E. COLI

A fine structure map of the phoR region of E. coli , mutations of which affect the rate of alkaline phosphatase synthesis, was constructed by Hfr x F^- crosses. Mutations causing three different phenotypes (previously reported as phoRa, phoRb, phoRc (Garen and Echols 1962a, b)) are clustered in three closely linked genetic loci. PhoR mutants of all three types, including the phoRb type not previously tested, are recessive to wild-type phoR⁺. In addition, phoRa and phoRc complement each other, while phoRa and phoRb do not. Our results support the hypothesis of Morris et al. (1974) that phoRc mutants represent a cistron (phoB) different from phoR.     

Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms and Escherichia coli

Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.Indicators of pollution (e.g., coliforms, fecal coliforms, and Escherichia coli) are traditionally used for monitoring the microbiological safety of water supplies and recreational water. Several techniques for detection of coliforms and E. coli are based on enzymatic hydrolysis of fluorogenic or chromogenic substrates for β-d-galactosidase and β-d-glucuronidase (9, 20). Current methods of recovery are usually culture based, and the analysis time is 18 to 24 h. In addition to enzymatic activity, these techniques use growth at appropriate temperatures in the presence of inhibitors, combined with demonstration of enzymatic activity, to selectively detect target bacteria.Rapid methods which require less than 6 h and are based on chromogenic, fluorogenic, or chemiluminogenic substrates for detection of coliforms, fecal coliforms, or E. coli have been described (1–3, 10, 27, 28). These rapid assays are based on the assumption that β-d-galactosidase and β-d-glucuronidase are markers for coliforms and E. coli, respectively. However, when the incubation time is 1 h or less, growth is not a selective step, and all β-d-galactosidase-positive or β-d-glucuronidase-positive microorganisms in a water sample contribute to the activity measured. At low initial concentrations of target bacteria (i.e., E. coli and total coliforms), increasing the preincubation time to 5 to 6 h did not result in a predominance of target bacteria compared to nontarget bacteria (28).The β-d-galactosidase or β-d-glucuronidase activity calculated per cultivable coliform or fecal coliform bacterium in environmental samples can be 1 to 2 log units higher than the activity per induced E. coli cell in pure culture (11, 26). The presence of active, noncultivable bacteria can be one reason for this. Studies of survival (7, 24, 25) and disinfection (26) of E. coli have shown that loss of cultivability does not necessarily result in a loss of β-d-galactosidase activity. The presence of false-positive bacteria can be another reason.β-d-Galactosidase has been found in numerous microorganisms, including gram-negative bacteria (e.g., strains belonging to the Enterobacteriaceae, Vibrionaceae, Pseudomonadaceae, and Neisseriaceae), several gram-positive bacteria, yeasts, protozoa, and fungi (17, 29). β-d-Glucuronidase is produced by most E. coli strains and also by other members of the Enterobacteriaceae, including some Shigella and Salmonella strains and a few Yersinia, Citrobacter, Edwardia, and Hafnia strains. Production of β-d-glucuronidase by Flavobacterium spp., Bacteroides spp., Staphylococcus spp., Streptococcus spp., anaerobic corynebacteria, and Clostridium has also been reported (12).High numbers of false-positive bacteria in sewage and contaminated water have been revealed by enumeration of β-d-galactosidase- and β-d-glucuronidase-positive CFU on nonselective agar supplemented with fluorogenic or chromogenic substrates (11, 28). Whether the activity from nontarget organisms can be neglected in a rapid assay depends on the number of nontarget organisms compared with the number of target bacteria and also on the level of their enzyme activity. Plant and algal biomass must be present at high concentrations to interfere in rapid bacterial β-d-galactosidase and β-d-glucuronidase assays (8).The main objective of this study was to investigate the enzyme characteristics of β-d-galactosidase- and β-d-glucuronidase-positive bacteria isolated from environmental water samples and to evaluate the potential influence of false-positive bacteria in rapid assays for coliform bacteria or E. coli in water. The effect of temperature on enzyme activity and on the interference of nontarget bacteria in the rapid assays was investigated as an important factor.(Some of the results were presented at the 97th General Meeting of the American Society for Microbiology 1997, Miami Beach, Fla., 4 to 8 May 1997.)     

Molecular evidence that the genes for dioecism and monoecism in Spinacia oleracea L. are located at different loci in a chromosomal region

Spinach (Spinacia oleracea L.) is widely known to be dioecious. However, monoecious plants can also occur in this species. Sex expression in dioecious spinach plants is controlled by a single gene pair termed X and Y. Our previous study showed that a single, incompletely dominant gene, which controls the monoecious condition in spinach line 03–336, should be allelic or linked to X/Y. Here, we developed 19 AFLP markers closely linked to the monoecious gene. The AFLP markers were mapped to a 38.2-cM chromosomal region that included the monoecious gene, which is bracketed between flanking markers with a distance of 7.1 cM. The four AFLP markers developed in our studies were converted into sequence-characterized amplified region (SCAR) markers, which are linked to both the monoecious gene and Y and are common to both populations segregating for the genes. Linkage analysis using the SCAR markers suggested that the monoecious gene (M) and Y are located in different intervals, between different marker pairs. Analysis of populations segregating for both M and Y also directly demonstrates linkage of the genes at a distance of ∼12 cM. The data presented in this study may be useful for breeding dioecious and highly male monoecious lines utilized as the pollen parents for hybrid seed production, as well as for studies of the evolutionary history of sexual systems in this species, and can provide a molecular basis for positional cloning of the sex-determining genes.     

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.     

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.