Development of a novel continuous culture device for experimental evolution of bacterial populations.

The availability of a robust and reliable continuous culture apparatus that eliminates wall growth problems would lead to many applications in the microbial field, including allowing genetically engineered strains to recover high fitness, improving biodegradation strains, and predicting likely antibiotic resistance mechanisms. We describe the design and implementation of a novel automated continuous culture machine that can be used both in time-dependent mode (similar to a chemostat) and turbidostat modes, in which wall growth is circumvented through the use of a long, variably divisible tube of growth medium. This tube can be restricted with clamps to create a mobile growth chamber region in which static portions of the tube and the associated medium are replaced together at equal rates. To functionally test the device as a tool for re-adaptation of engineered strains, we evolved a strain carrying a highly deleterious deletion of Elongation Factor P, a gene involved in translation. In 200 generations over 2 weeks of dilution cycles, the evolved strain improved in generation time by a factor of three, with no contaminations and easy manipulation.

Artificially ambiguous genetic code confers growth yield advantage.

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli.

BACKGROUND: To maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate. RESULTS: To overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate. CONCLUSIONS: This approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected.

An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes.

Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs. There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each. These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs. Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret. Here we describe a different cysteinyl-tRNA synthetase from M. jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo. This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin. The existence of this protein contrasts with proposals that aminoacylation with cysteine in M. jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase.

Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway.

Aminoacyl transfer RNA (tRNA) synthetases establish the rules of the genetic code by catalyzing the aminoacylation of tRNAs. For some synthetases, accuracy depends critically on an editing function at a site distinct from the aminoacylation site. Mutants of Escherichia coli that incorrectly charge tRNA(Val) with cysteine were selected after random mutagenesis of the whole chromosome. All mutations obtained were located in the editing site of valyl-tRNA synthetase. More than 20% of the valine in cellular proteins from such an editing mutant organism could be replaced with the noncanonical aminobutyrate, sterically similar to cysteine. Thus, the editing function may have played a central role in restricting the genetic code to 20 amino acids. Disabling this editing function offers a powerful approach for diversifying the chemical composition of proteins and for emulating evolutionary stages of ambiguous translation.

Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces.

The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP-dependent protease family Clp were cloned, mapped and characterized. S. coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes. Disruption of the clpP1 gene in S. lividans and S. coelicolor blocks differentiation at the substrate mycelium step. Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S. lividans, S. albus and S. coelicolor. Overproduction of ClpX accelerates actinorhodin production in S. coelicolor and activates its production in S. lividans.

The ClpB ATPase of Streptomyces albus G belongs to the HspR heat shock regulon.

The clpB gene of Streptomyces albus was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides. Transcriptional analysis showed that the clpB gene was heat induced. Primer extension identified a transcription start site preceded by typical vegetative -10 and -35 hexamer sequences. The Streptomyces HspR repressor is known to bind to three inverted repeat motifs (IR1, IR2, IR3) upstream from the S. coelicolor dnaK operon. We identified an inverted repeat motif identical to IR3 upstream from the S. albus clpB gene. DNA-binding experiments showed that HspR regulates clpB transcription by interacting directly with this motif. Streptomyces albus is the first Gram-positive organism for which the co-regulation of DnaK and ClpB has been described. Such co-regulation suggests that there is a physiological relationship between these two proteins in this bacterium. Genes similar to hspR were also identified in Mycobacterium leprae, M. tuberculosis and in bacteria unrelated to the actinomycetales order, such as Helicobacter pylori and Aquifex aeolicus. HspR binding sites were found in these bacteria upstream from various heat shock genes, suggesting that these genes are regulated by HspR. The HspR binding site, here called HAIR (HspR associated inverted repeat), has the consensus sequence CTTGAGT N7 ACTCAAG.

Streptogramin B biosynthesis in Streptomyces pristinaespiralis and Streptomyces virginiae: molecular characterization of the last structural peptide synthetase gene.

Streptomyces pristinaespiralis and S. virginiae both produce closely related hexadepsipeptide antibiotics of the streptogramin B family. Pristinamycins I and virginiamycins S differ only in the fifth incorporated precursor, di(mono)methylated amine and phenylalanine, respectively. By using degenerate oligonucleotide probes derived from internal sequences of the purified S. pristinaespiralis SnbD and SnbE proteins, the genes from two streptogramin B producers, S. pristinaespiralis and S. virginiae, encoding the peptide synthetase involved in the activation and incorporation of the last four precursors (proline, 4-dimethylparaaminophenylalanine [for pristinamycin I(A)] or phenylalanine [for virginiamycin S], pipecolic acid, and phenylglycine) were cloned. Analysis of the sequence revealed that SnbD and SnbE are encoded by a unique snbDE gene. SnbDE (4,849 amino acids [aa]) contains four amino acid activation domains, four condensation domains, an N-methylation domain, and a C-terminal thioesterase domain. Comparison of the sequences of 55 amino acid-activating modules from different origins confirmed that these sequences contain enough information for the performance of legitimate predictions of their substrate specificity. Partial sequencing (1,993 aa) of the SnbDE protein of S. virginiae allowed comparison of the proline and aromatic acid activation domains of the two species and the identification of coupled frameshift mutations.

Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes.

Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed.

Multienzymatic non ribosomal peptide biosynthesis: identification of the functional domains catalysing peptide elongation and epimerisation.

Peptide synthetases are multienzymatic complexes that synthesize bioactive peptides molecules by the thiotemplate mechanism. Comparison of the known sequences of peptide synthetases led us to the identification of a 350 amino acids domain catalysing elongation and containing the motif HHxxxDG. This motif is present as many times as acyltransfer or epimerisation reactions occur during biosynthesis of the peptide. The distance between this motif and the phosphopantetheinyl attachment site is nearly invariant. An identical motif is found in other enzymes effecting acyl transfer such as chloramphenicol acetyltransferase from Tn9 and dihydrolipoamide acyltransferase. Altogether, the HHxxxDG motif may constitute the signature of a superfamily sharing a common catalytic mechanism based on the acid-base properties of the second histidine for effecting acyl transfer or peptide epimerisation.

Fructose phosphotransferase system of Xanthomonas campestris pv. campestris: characterization of the fruB gene.

In the plant pathogen Xanthomonas campestris pv. campestris, fructose is transported by a specific phosphotransferase system (PTS). This PTS involves a multiphosphoryl transfer protein (MTP) encoded by the fruB gene, which was cloned and sequenced. fruB is part of a transcriptional unit together with the fruK gene, coding for 1-phosphofructokinase, which is located upstream from the fruA gene, coding for the fructose-specific permease (EIIB'BCFru). The amino acid sequence of the X. campestris MTP deduced from the fruB sequence shared 46% identical residues with an MTP identified in Rhodobacter capsulatus. The X. campestris MTP (837 amino acid residues) consists of three moieties: a fructose-specific enzyme-IIA-like N-terminal moiety (residues 1-148), followed by an HPr-like moiety (161-251) and an enzyme-I-like C-terminal moiety (274-837). The three domains are separated by two flexible hinge regions rich in proline and alanine residues. The construction of a fruB mutant confirmed the role of the MTP in fructose transport and phosphorylation in X. campestris.

ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. Sequence and in vivo activities.

The ATP-dependent Clp protease of Escherichia coli consists of two subunits, the ClpP subunit, which has the proteolytic active site, and ClpA, which possesses ATPase activity and activates the proteolytic activity of ClpP in vitro. Recently, Zylicz and co-workers (Wojtkowiak, D., Georgopoulos, C., and Zylicz, M. (1993) J. Biol. Chem. 268, 22609-22617) identified another E. coli protein that activated ATP-dependent degradation of lambda O protein in the presence of ClpP. The amino-terminal sequence of this protein corresponds to the translated amino-terminal sequence of a gene that we have named clpX. clpX encodes a protein with M(r) 46,300, similar to that observed for the protein purified by Wojtkowiak et al. clpX is an operon with clpP; both genes are cotranscribed in a single heat-inducible 2200-base mRNA, with clpP the promoter proximal gene. The sequence of ClpX includes a single consensus ATP-binding site motif and has limited homology to regions of ClpA and other members of the ClpA/B/C family. A third group of proteins, ClpY, closely related to ClpX, has been identified by sequence homology. Mutations in either clpX or clpP abolish degradation of the highly unstable lambda O protein in vivo. clpX mutants are not defective in degradation of previously identified ClpA/ClpP substrates such as a ClpA-beta-galactosidase fusion protein. It appears that selectivity of degradation by ClpP in vivo is determined by interaction of ClpP with different regulatory ATPase subunits.

Fructose catabolism in Xanthomonas campestris pv. campestris. Sequence of the PTS operon, characterization of the fructose-specific enzymes.

In Xanthomonas campestris pv. campestris, fructose is transported and phosphorylated into fructose 1-phosphate through a phosphoenolpyruvate-dependent phosphotransferase system. The nucleotide sequence of the fruA gene encoding the phosphotransferase system permease specific of fructose (EIIFru) was determined. The fructose 1-phosphate produced by the phosphotransferase system is phosphorylated into fructose 1,6-bisphosphate by a 1-phosphofructokinase. This enzyme was characterized and the corresponding gene (fruK) was sequenced. Sequence comparisons revealed that FruK is a member of a new family of ATP-binding proteins composed of sugar (or sugar-phosphate) kinases. In phosphotransferase system-deficient strains, fructose can still be transported by an unidentified permease. The intracellular fructose is then phosphorylated by a multimeric fructokinase of 135 kDa specific for fructose and inhibited by fructose, fructose 1,6-bisphosphate, and mannose. Several other enzymes of fructose metabolism were assayed and a potential pathway for fructose catabolism is presented.

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Fructose was shown to be phosphorylated by a specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose EII. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.

A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity.

A DNA fragment from Xanthomonas campestris pv. campestris that partially restored the carbohydrate fermentation pattern of a cya crp Escherichia coli strain was cloned and expressed in E. coli. The nucleotide sequence of this fragment revealed the presence of a 700-base-pair open reading frame that coded for a protein highly similar to the catabolite activation factor (CAP) of E. coli (accordingly named CLP for CAP-like protein). An X. campestris pv. campestris clp mutant was constructed by reverse genetics. This strain was not affected in the utilization of various carbon sources but had strongly reduced pathogenicity. Production of xanthan gum, pigment, and extracellular enzymes was either increased or decreased, suggesting that CLP plays a role in the regulation of phytopathogenicity.