pyrE Gene Research Articles

Intrinsic Natural Resistance of Various Plant Pathogens to Ipflufenoquin, a New DHODH (Dihydroorotate Dehydrogenase)-inhibiting Fungicide, in Relation to Unaltered Amino Acid Sequence of the Target Site.

In recent years, increasingly stringent pesticide regulations have made the development of new chemistries challenging. Under these regulations, the new fungicide ipflufenoquin (FRAC Code 52) was first released in Japan. Its mode of action is new; it inhibits dihydroorotate dehydrogenase (DHODH), a key enzyme in the biosynthesis of pyrimidine-based nucleotides. However, because it is a single-site inhibitor, the risk of resistance developing in pathogens must be carefully considered. The risk for dual use of DHODH inhibitors in agriculture and medicine has also become a great concern because a new antifungal olorofim is under development against human pathogens now and cross-resistance has recently been reported between ipflufenoquin and olorofim in Aspergillus fumigatus. In this study, the sensitivity to ipflufenoquin was examined in culture and in plants using "baseline" isolates, which had never been exposed to DHODH inhibitors. Isolates of Alternaria alternata, Botrytis cinerea, B. elliptica, Colletotrichum fioriniae, C. fructicola, C. nymphaeae, C. orbiculare, C. siamense, C. tropicale, C. truncatum, and Sclerotinia sclerotiorum were highly sensitive to ipflufenoquin in culture, but isolates of Coniella vitis, Corynespora cassiicola, Pseudocercospora fuligena, and Rhizoctonia solani were inherently resistant. Ipflufenoquin had low efficacy against C. cassiicola and C. vitis after inoculation of cucumber and grapevine leaves, respectively. To understand the mechanism of natural resistance, we analyzed the partial sequence of pyrE genes, which encode the DHODH enzyme, but did not find any differences in the deduced amino acids that were thought to be associated with resistance. Thus, mechanisms other than target-site mutations might be involved in the intrinsic resistance.

Regulation of the pyrimidine biosynthetic pathway by lysine acetylation of E. coli OPRTase.

The de novo pyrimidine biosynthesis pathway is an important route due to the relevance of its products, its implications in health and its conservation among organisms. Here, we investigated the regulation by lysine acetylation of this pathway. To this aim, intracellular and extracellular metabolites of the route were quantified, revealing a possible blockage of the pathway by acetylation of the OPRTase enzyme (orotate phosphoribosyltransferase). Chemical acetylation of OPRTase by acetyl-P involved a decrease in enzymatic activity. To test the effect of acetylation in this enzyme, K26 and K103 residues were selected to generate site-specific acetylated proteins. Several differences were observed in kinetic parameters, emphasizing that the kcat of these mutants showed a strong decrease of 300 and 150-fold for OPRTase-103AcK and 19 and 6.3-fold for OPRTase-26AcK, for forward and reverse reactions. In vivo studies suggested acetylation of this enzyme by a nonenzymatic acetyl-P-dependent mechanism and a reversion of this process by the CobB deacetylase. A complementation assay of a deficient strain in the pyrE gene with OPRTase-26AcK and OPRTase-103AcK was performed, and curli formation, stoichiometric parameters and orotate excretion were measured. Complementation with acetylated enzymes entailed a profile very similar to that of the ∆pyrE strain, especially in the case of complementation with OPRTase-103AcK. These results suggest regulation of the de novo pyrimidine biosynthesis pathway by lysine acetylation of OPRTase in Escherichia coli. This finding is of great relevance due to the essential role of this route and the OPRTase enzyme as a target for antimicrobial, antiviral and cancer treatments.

NIH4215: A mutation-prone thiamine auxotrophic clinical Aspergillus fumigatus isolate.

Aspergillus fumigatus is the main cause of life-threatening invasive aspergillosis. Despite the availability of various antifungals, therapy remains challenging and requires further studies. Accordingly, the clinical A. fumigatus isolate NIH4215 deriving from a fatal case of human pulmonary aspergillosis has frequently been used in drug efficacy studies. Unexpectedly, our initial attempts to generate a bioluminescent reporter of strain NIH4215 for in vivo drug efficacy studies failed, as NIH4215 was unable to grow on defined minimal medium. Subsequent analyses discovered a previously undescribed thiamine auxotrophy of strain NIH4215 and transformation with thiamine biosynthesis genes from A. fumigatus strain Af293 identified the nmt1 gene as cause of the thiamine auxotrophy. Sequencing of the defective nmt1 gene revealed the loss of a cysteine codon within an essential iron-binding motif. Subsequently, the wild-type nmt1 gene was successfully used to generate a bioluminescent reporter strain in NIH4215 by simultaneously deleting the akuB locus. The resulting bioluminescent ΔakuB strains showed a high frequency of homologous integration as confirmed by generation of pyrG and niaD deletion mutants. When tested in a Galleria mellonella infection model, neither thiamine auxotrophy nor the deletion of the akuB locus had a significant effect on virulence. However, besides thiamine auxotrophy, sectors with altered morphology and albino mutants frequently arose on colony edges of strain NIH4215 and its derivatives, and stable albino mutants were successfully isolated. A proposed increased mutation rate of NIH4215 was confirmed by screening for spontaneous occurrence of fluoorotic acid resistant mutants. Independent mutations in the pyrG and pyrE gene were identified in the fluoroorotic acid resistant NIH4215 isolates and the frequency of mutation was by at least one order of magnitude higher than that observed for the clinical A. fumigatus isolate CBS144.89. In summary, despite its virulence in animal models, strain NIH4215 is a thiamine auxotroph and prone to accumulate mutations. Our results suggest that thiamine biosynthesis is dispensable for host infection and mutation-prone strains such as NIH4215 could potentially facilitate the evolution of azole resistant strains as increasingly observed in the environment.

A clean in-frame knockout system for gene deletion in Acetobacterium woodii

Acetogenic bacteria produce acetate following the fixation of CO2 via the Wood-Ljungdahl pathway. As such, they represent excellent process organisms for the production of novel chemicals and fuels from this waste greenhouse gas. Acetobacterium woodii is the model acetogen and numerous studies have been conducted investigating its biochemistry, gas consumption and use as a production chassis. However, there are a dearth of available tools for A. woodii gene modification which limits the research options available for genetic studies. Here, the previously proposed Clostridia Roadmap is implemented in A. woodii leading to the derivation of a knockout system for the generation of clean, in-frame deletions. The replicon of the Gram-positive plasmid pCD6 that originated in Clostridioides difficile was identified as being replication-defective in A. woodii, a property that was exploited to construct a pseudo-suicide knockout plasmid which was used to generate an auxotrophic, pyrE mutant. This allowed the subsequent use of a heterologous pyrE gene (from Clostridium acetobutylicum) as a counter selection marker and the deletion of a number of genes by allelic exchange. Specific mutants generated were affected in growth on glucose, fructose and ethanol as a consequence of deletion of fruA, pstG and adhE, respectively.

De novo pyrimidine synthesis pathway inhibition reduces motility virulence of Pseudomonas aeruginosa despite complementation

ObjectivesFinding alternative methods to overcome antibiotic resistance in the medically important bacterium Pseudomonas aeruginosa has become increasingly vital as antibiotic resistance becomes more difficult to overcome. DNA synthesis inhibition is a strategy that has been used to inhibit cancer cells from proliferating by using pyrimidine analogues. Gateway site-directed mutagenesis is an effective way to create precise knockouts which creates good models to understand the relationship between biochemical pathways and the physiology of the organism. This article demonstrates the effect of pyrimidine synthesis inhibition on the motility virulence and physiology of P. aeruginosa. MethodsWild type Pseudomonas aeruginosa (PA01) was genetically modified using gateway cloning technology to inhibit the pyrimidine synthesis pathway through the complete knockout of the pyrE gene. The mutant strain was compared to the wild type PA01 in terms of its twitching, swarming and swimming motility forms and biofilm formation abilities. In addition to that, the production of the bacterial surfactant, rhamnolipids, was also measured. Results and conclusionsThe inhibition of the pyrimidine synthesis pathway resulted in significant reduction in motility and biofilm capabilities, despite complementing the mutation with uracil to bypass the mutation. Inhibition of the pyrimidine synthesis pathway is thus an effective way to reduce the virulence of Pseudomonas aeruginosa. Even though the bacterium is able to acquire pyrimidines from its surrounding, it was not sufficient enough to overcome the effects of the mutation.

Resistance profiling of Aspergillus fumigatus to olorofim indicates absence of intrinsic resistance and unveils the molecular mechanisms of acquired olorofim resistance

ABSTRACT Olorofim (F901318) is a new antifungal currently under clinical development that shows both in vitro and in vivo activity against a number of filamentous fungi including Aspergillus fumigatus. In this study, we screened A. fumigatus isolates for intrinsic olorofim-resistant A. fumigatus and evaluated the ability of A. fumigatus to acquire an olorofim-resistant phenotype. No intrinsic resistance was found in 975 clinical A. fumigatus isolates. However, we found that isolates with increased olorofim MICs (> 8 mg/L) could be selected using a high number of conidia and olorofim exposure under laboratory conditions. Assessment of the frequency of acquired olorofim resistance development of A. fumigatus was shown to be higher than for voriconazole but lower than for itraconazole. Sequencing the PyrE gene of isogenic isolates with olorofim MICs of >8 mg/L identified various amino acid substitutions with a hotspot at locus G119. Olorofim was shown to have reduced affinity to mutated target protein dihydroorotate dehydrogenase (DHODH) and the effect of these mutations was proven by introducing the mutations directly in A. fumigatus. We then investigated whether G119 mutations were associated with a fitness cost in A. fumigatus. These experiments showed a small but significant reduction in growth rate for strains with a G119V substitution, while strains with a G119C substitution did not exhibit a reduction in growth rate. These in vitro findings were confirmed in an in vivo pathogenicity model.

Improving riboflavin production by modifying related metabolic pathways in Bacillus subtilis.

Riboflavin is a feed additive, food additive and clinical drug, with a significant annual demand of nearly 8000 t. Fermentation using recombinant Bacillus subtilis is currently one of the most important industrial production method for riboflavin. First, a suitable medium was selected and the expression of the ureABC operon was modified. The ykgB gene was overexpressed in B. subtilis RX10, the production of the derivative strain RX20 was increased to 4·61 g l-1 riboflavin, and the yield was increased to 52 mg riboflavin g-1 glucose. The relative transcription level of pyr operon in RX20 was reduced to 71%, the production of the derivative strain RX21 was increased to 5·82 g l-1 riboflavin, and the yield was 76 mg riboflavin g-1 glucose. The start codon of the pyrE gene in RX21 was modified to 'TTG', the production of the derivative strain RX22 was increased to 7·01 g l-1 riboflavin, and the yield was 89 mg riboflavin g-1 glucose. These results indicated that overexpression of the ykgB gene and reduction of the metabolic flux of de novo synthesis of pyrimidine nucleotides were beneficial to the synthesis of riboflavin.

Generation of Trichoderma harzianum with pyr4 auxotrophic marker by using the CRISPR/Cas9 system

Trichoderma harzianum is a filamentous fungus used as a biological control agent for agricultural pests. Genes of this microorganism have been studied, and their applications are patented for use in biofungicides and plant breeding strategies. Gene editing technologies would be of great importance for genetic characterization of this species, but have not yet been reported. This work describes mutants obtained with an auxotrophic marker in this species using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas (CRISPR-associated) system. For this, sequences for a guide RNA and Cas9 overexpression were inserted via biolistics, and the sequencing approach confirmed deletions and insertions at the pyr4 gene. Phenotypic characterization demonstrated a reduction in the growth of mutants in the absence of uridine, as well as resistance to 5-fluorotic acid. In addition, the gene disruption did not reduce mycoparasitc activity against phytopathogens. Thus, target disruption of the pyr4 gene in T. harzianum using the CRISPR/Cas9 system was demonstrated, and it was also shown that endogenous expression of the system did not interfere with the biological control activity of pathogens. This work is the first report of CRISPR Cas9-based editing in this biocontrol species, and the mutants expressing Cas9 have potential for the generation of useful technologies in agricultural biotechnology.

An efficient transformation system for Trichoderma atroviride using the pyr4 gene as a selectable marker.

The development of an efficient transformation system is essential to enrich the genetic understanding of Trichoderma atroviride. To acquire an additional homologous selectable marker, uracil auxotrophic mutants were generated. First, the pyr4 gene encoding OMP decarboxylase was replaced by the hph marker gene, encoding a hygromycin phosphotransferase. Then, uracil auxotrophs were employed to determine that 5mM uracil restores their growth and conidia production, and 1mgml-1 is the lethal dose of 5-fluoroorotic acid in T. atroviride. Subsequently, uracil auxotrophic strains, free of a drug-selectable marker, were selected by 5-fluoroorotic acid resistance. Two different deletions in pyr4 were mapped in four auxotrophs, encoding a protein with frameshifts at the 310 and 335 amino acids in their COOH-terminal. Six auxotrophs did not have changes in the pyr4 ORF even though a specific cassette to delete the pyr4 was used, suggesting that 5-FOA could have mutagenic activity. The Ura-1 strain was selected as a genetic background to knock out the MAPKK Pbs2, MAPK Tmk3, and the blue light receptors Blr1/Blr2, using a short version of pyr4 as a homologous marker. The ∆tmk3 and ∆pbs2 mutants selected with pyr4 or hph marker were phenotypically identical, highly sensitive to different stressors, and affected in photoconidiation. The ∆blr1 and ∆blr2 mutants were not responsive to light, and complementation of uracil biosynthesis did not interfere in the expression of blu1, grg2, phr1, and env1 genes upregulated by blue light. Overall, uracil metabolism can be used as a tool for genetic manipulation in T. atroviride.

A copper-controlled RNA interference system for reversible silencing of target genes in Trichoderma reesei

BackgroundTrichoderma reesei is a primary lignocellulosic enzyme producer in industry. However, the mechanisms underlying cellulase synthesis as well as other physiological processes are insufficiently understood partly due to the sophisticated process for its genetic manipulation. Target gene knockdown by RNA interference (RNAi) is a powerful tool for genetic research and biotechnology in eukaryotes including filamentous fungi. Previously reported RNAi system in T. reesei was either uncontrollable or only applicable in certain nutrition state.ResultsIn the present study, we incorporated the copper-responsive tcu1 promoter into an RNAi-mediated silencing system to develop a controllable RNAi-mediated silencing system in T. reesei. As the proof-of-concept, a prototrophic pyr4 gene, highly expressed cel7a and xyr1 genes induced by Avicel and a fab1 gene, whose knockout has proved to be intractable, were successfully knocked down in the absence of copper when the respective RNAi fragment was expressed. Importantly, the phenotype of RNAi strains was shown to be reversed easily to mimic the complementation for excluding any unwanted effects resulted from the random integration of the hpRNA cassette by adding copper in the media. Thus, this controllable RNAi-mediated silencing system can be turned on and turned off only depending on the absence and presence of copper ions in the media, respectively, and not on the nutritional states.ConclusionsThe copper-controlled RNA interference system represents an effective tool for reversible silencing of target genes in T. reesei. This reported strategy to conditionally knock down or turn off genes will contribute to our understanding of T. reesei gene functions, especially those that are difficult to be knocked out due to various reasons. In addition, this simple and cost-effective method holds great potential for the application in synthetic biology and genetic engineering of T. reesei.

Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955

BackgroundThe thermophile Geobacillus thermoglucosidasius has considerable attraction as a chassis for the production of chemicals and fuels. It utilises a wide range of sugars and oligosaccharides typical of those derived from lignocellulose and grows at elevated temperatures. The latter improves the rate of feed conversion, reduces fermentation cooling costs and minimises the risks of contamination. Full exploitation of its potential has been hindered by a dearth of effective gene tools.ResultsHere we designed and tested a collection of vectors (pMTL60000 series) in G. thermoglucosidasius NCIMB 11955 equivalent to the widely used clostridial pMTL80000 modular plasmid series. By combining a temperature-sensitive replicon and a heterologous pyrE gene from Geobacillus kaustophilus as a counter-selection marker, a highly effective and rapid gene knock-out/knock-in system was established. Its use required the initial creation of uracil auxotroph through deletion of pyrE using allele-coupled exchange (ACE) and selection for resistance to 5-fluoroorotic acid. The turnaround time for the construction of further mutants in this pyrE minus strain was typically 5 days. Following the creation of the desired mutant, the pyrE allele was restored to wild type, within 3 days, using ACE and selection for uracil prototrophy. Concomitant with this process, cargo DNA (pheB) could be readily integrated at the pyrE locus. The system’s utility was demonstrated through the generation in just 30 days of three independently engineered strains equivalent to a previously constructed ethanol production strain, TM242. This involved the creation of two in-frame deletions (ldh and pfl) and the replacement of a promoter region of a third gene (pdh) with an up-regulated variant. In no case did the production of ethanol match that of TM242. Genome sequencing of the parental strain, TM242, and constructed mutant derivatives suggested that NCIMB 11955 is prone to the emergence of random mutations which can dramatically affect phenotype.ConclusionsThe procedures and principles developed for clostridia, based on the use of pyrE alleles and ACE, may be readily deployed in G. thermoglucosidasius. Marker-less, in-frame deletion mutants can be rapidly generated in 5 days. However, ancillary mutations frequently arise, which can influence phenotype. This observation emphasises the need for improved screening and selection procedures at each step of the engineering processes, based on the generation of multiple, independent strains and whole-genome sequencing.

Development of a pyrE-based selective system for Thermotoga sp. strain RQ7.

To fully unlock the biotechnological potentials of Thermotoga species, this study aimed to expand the genetic toolbox of Thermotoga by developing a new selective system. The developed system was composed of two components: a recipient strain bearing a deletion in its orotate phosphoribosyltransferase gene pyrE and a shuttle vector expressing a heterologous pyrE as the selectable marker. A spontaneous uracil auxotroph, T. sp. strain RQ7-15, was isolated at 70°C with 2mg/ml 5-fluoroorotic acid. The mutant carried a 112bp deletion in pyrE and was a suitable recipient strain. To avoid homologous recombination, the pyrE gene from another thermophilic bacterium Caldicellulosiruptor saccharolyticus was used as the selectable marker. The gene was cloned into two Thermotoga-E. coli shuttle vectors, controlled by different promoters: the promoter of Thermus S-layer protein (P slpA ) in pDH25 and the promoter of the pyrimidine synthesis operon of T. sp. strain RQ7 (P RQ7.pyr ) in pDH28. After being introduced into the mutant strain RQ7-15 through natural transformation, both vectors allowed the host to thrive in a minimal medium. Single colonies of transformants were isolated and confirmed by polymerase chain reactions and restriction digestions. In summary, a pyrE-based selective system has been established in T. sp. strain RQ7.

A novel integrative expression vector for Sulfolobus species.

With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 (pyrE(sso)) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli. For stable expression of the target gene, an α-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene (saci_1160) encoding an α-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase (gdhA(saci)) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the "pop-in" and "pop-out" method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The α-glucosidase activity was confirmed by the hydrolysis of pNPαG. The pINEX vector should be applicable in delineating gene functions in this organism.

Genetic variations of live attenuated plague vaccine strains (Yersinia pestis EV76 lineage) during laboratory passages in different countries

Plague, one of the most devastating infectious diseases in human history, is caused by the bacterial species Yersinia pestis. A live attenuated Y. pestis strain (EV76) has been widely used as a plague vaccine in various countries around the world. Here we compared the whole genome sequence of an EV76 strain used in China (EV76-CN) with the genomes of Y. pestis wild isolates to identify genetic variations specific to the EV76 lineage. We identified 6 SNPs and 6 Indels (insertions and deletions) differentiating EV76-CN from its counterparts. Then, we screened these polymorphic sites in 28 other strains of EV76 lineage that were stored in different countries. Based on the profiles of SNPs and Indels, we reconstructed the parsimonious dissemination history of EV76 lineage. This analysis revealed that there have been at least three independent imports of EV76 strains into China. Additionally, we observed that the pyrE gene is a mutation hotspot in EV76 lineages. The fine comparison results based on whole genome sequence in this study provide better understanding of the effects of laboratory passages on the accumulation of genetic polymorphisms in plague vaccine strains. These variations identified here will also be helpful in discriminating different EV76 derivatives.

The pyrE Gene as a Bidirectional Selection Marker in Bifidobacterium Longum 105-A.

We constructed a deletion mutant of the pyrE gene in Bifidobacterium longum 105-A. A pyrE knockout cassette was cloned into pKKT427, a Bifidobacterium-Escherichia coli shuttle vector, and then introduced into B. longum 105-A by electroporation. The transformants were propagated and spread onto MRS plates containing 5-fluoroorotic acid (5-FOA) and uracil. 5-FOA-resistant mutants were obtained at a frequency of 4.7 × 10−5 integrations per cell. To perform pyrE gene complementation, the pyrE gene was amplified by PCR and used to construct a complementation plasmid (pKKT427-pyrE+). B. longum 105-A ∆pyrE harboring this plasmid could not grow on MRS plates containing 5-FOA, uracil and spectinomycin. We also developed a chemically defined medium (bifidobacterial minimal medium; BMM) containing inorganic salts, glucose, vitamins, isoleucine and tyrosine for positive selection of pyrE transformants. B. longum 105-A ∆pyrE could not grow on BMM agar, but the same strain harboring pKKT427-pyrE+ could. Thus, pyrE can be used as a counterselection marker in B. longum 105-A and potentially other Bifidobacterium species as well. We demonstrated the effectiveness of this system by constructing a knockout mutant of the xynF gene in B. longum 105-A by using the pyrE gene as a counterselection marker. This pyrE-based selection system will contribute to genetic studies of bifidobacteria.

Low Species Barriers in Halophilic Archaea and the Formation of Recombinant Hybrids

Speciation of sexually reproducing organisms requires reproductive barriers. Prokaryotes reproduce asexually butoften exchange DNA by lateral gene transfer mechanisms and recombination [1], yet distinct lineages are still observed. Thus, barriers to gene flow such as geographic isolation, genetic incompatibility or a physiological inability to transfer DNA represent potential underlying mechanisms behind preferred exchange groups observed in prokaryotes [2-6]. In Bacteria, experimental evidence showed that sequence divergence impedes homologous recombination between bacterial species [7-11]. Here we study interspecies gene exchange in halophilic archaea that possess a parasexual mechanism of genetic exchange that is functional between species [12, 13]. In this process, cells fuse forming a diploid state containing the full genetic repertoire of both parental cells, which facilitates genetic exchange and recombination. Later, cells separate, occasionally resulting in hybrids of the parental strains [14]. We show high recombination frequencies between Haloferax volcanii and Haloferax mediterranei, two species that have an average nucleotide sequence identity of 86.6%. Whole genome sequencing of Haloferax interspecies hybrids revealed the exchange of chromosomal fragments ranging from 310Kb to 530Kb. These results show that recombination barriers may be more permissive in halophilic archaea than they are in bacteria.

Heteroduplex Formation, Mismatch Resolution, and Genetic Sectoring During Homologous Recombination in the Hyperthermophilic Archaeon Sulfolobus Acidocaldarius

Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR) remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldarius pyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr+ clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells, or to conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which regions of heteroduplex DNA formed and then segregated after partial resolution of inter-strand differences. Surprisingly, sectoring was also frequent in cells transformed with single-stranded DNAs. Oligonucleotides produced more sectored transformants when electroporated as single strands than as a duplex, although all forms of donor DNA (positive-strand, negative-strand, and duplex) produced a diversity of genotypes, despite the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells. Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.

Sulfolobus mutants, generated via PCR products, which lack putative enzymes of UV photoproduct repair.

In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5′ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

Homologous recombination in Sulfolobus acidocaldarius: genetic assays and functional properties

HR (homologous recombination) is expected to play important roles in the molecular biology and genetics of archaea, but, so far, few functional properties of archaeal HR have been measured in vivo. In the extreme thermoacidophile Sulfolobus acidocaldarius, a conjugational mechanism of DNA transfer enables quantitative analysis of HR between chromosomal markers. Early studies of this system indicated that HR occurred frequently between closely spaced mutations within the pyrE gene, and this result was later supported by various analyses involving defined point mutations and deletions. These properties of intragenic HR suggested a non-reciprocal mechanism in which donor sequences become incorporated into the recipient genome as short segments. Because fragmentation of donor DNA during cell-to-cell transfer could not be excluded from contributing to this result, subsequent analyses have focused on electroporation of selectable donor DNA directly into recipient strains. For example, S. acidocaldarius was found to incorporate synthetic ssDNA (single-stranded DNA) of more than approximately 20 nt readily into its genome. With respect to various molecular properties of the ssDNA substrates, the process resembled bacteriophage lambdaRed-mediated 'recombineering' in Escherichia coli. Another approach used electroporation of a multiply marked pyrE gene to measure donor sequence tracts transferred to the recipient genome in individual recombination events. Initial results indicate multiple discontinuous tracts in the majority of recombinants, representing a relatively broad distribution of tract lengths. This pattern suggests that properties of the HR process could, in principle, account for many of the apparent peculiarities of intragenic recombination initiated by S. acidocaldarius conjugation.

Lactobacillus plantarum response to inorganic carbon concentrations: PyrR2-dependent and -independent transcription regulation of genes involved in arginine and nucleotide metabolism

Lactobacillus plantarum susbp. plantarum is a capnophilic Gram-positive heterotroph with optimal growth in 4 % CO(2)-enriched air. At low inorganic carbon (C(i)) concentrations, the pyr genes encoding the enzymes of the pyrimidine biosynthetic pathway were overexpressed, in agreement with a previous study showing that these genes are regulated at the transcription level in response to C(i) via a PyrR(2)-mediated mechanism. A previous study of high-CO(2)-requiring (HCR) mutants revealed an unknown genetic link between arginine regulation and C(i)-dependent nutritional needs. To better understand L. plantarum's adaptation to C(i) availability, additional C(i)-responsive genes were sought in the arginine biosynthetic pathway (arg and car genes) using slot-blot hybridization and a proteomic differential 2D gel electrophoresis (DIGE) global approach. Besides the nine pyr-encoded proteins, 16 new Icr (inorganic-carbon-regulated) proteins accumulated differentially in response to C(i) availability, suggesting that the C(i) response involves several metabolic pathways and adaptation processes. Among these Icr proteins only argininosuccinate lyase, encoded by argH, was involved in arginine biosynthesis. Three proteins involved in the purine biosynthetic pathway and nucleotide conversion, adenylate kinase (Adk), GMP synthase (GuaA), and IMP dehydrogenase (GuaB), accumulated differentially in response to changes in C(i) levels. Expression of the Icr protein-encoding genes argH and guaB was regulated at the transcription level or by RNA stability in response to C(i) availability, as previously demonstrated for the pyr genes. However, PyrR(2) was not essential for the C(i)-regulated transcription of argH and guaB, demonstrating that PyrR(2) modulates only a subset of C(i)-regulated genes. These results suggest that the C(i) response may involve at least two regulatory mechanisms in L. plantarum.