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Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this study, the mitochondrial DNA polymerase γ (Mip1) of the entomopathogenic fungus Metarhizium brunneum is characterized and analyzed with disruption experiments and its in silico interactions with key proteins implicated in mt gene transcription, i.e., mt RNA polymerase Rpo41 and mt transcription factor Mtf1. Disruption of mip 1 gene and its partial expression influences cell growth, morphology, germination and stress tolerance. A putative in silico model of Mip1-Rpo41-Mtf1, which is known to be needed for the initiation of replication, was proposed and helped to identify potential amino acid residues of Mip1 that interact with the Rpo41-Mtf1 complex. Moreover, the reduced expression of mip 1 indicates that Mip1 is not required for efficient transcription but only for replication. Functional differences between the M. brunneum Mip1 and its counterparts from Saccharomyces cerevisiae and higher eukaryotes are discussed.

The enzymatic arsenal of several soil microorganisms renders them particularly suitable for the degradation of lignocellulose, a process of distinct ecological significance with promising biotechnological implications. In this study, we investigated the spatiotemporal diversity and distribution of bacteria and fungi with 16S and Internally Trascribed Spacer (ITS) ribosomal RNA next-generation-sequencing (NGS), focusing on forest mainland Abies cephalonica and insular Quercus ilex habitats of Greece. We analyzed samples during winter and summer periods, from different soil depths, and we applied optimized and combined targeted meta-omics approaches aiming at the peroxidase-catalase family enzymes to gain insights into the lignocellulose degradation process at the soil microbial community level. The microbial communities recorded showed distinct patterns of response to season, soil depth and vegetation type. Overall, in both forests Proteobacteria , Actinobacteria , Acidobacteria were the most abundant bacteria phyla, while the other phyla and the super-kingdom of Archaea were detected in very low numbers. Members of the orders Agaricales , Russulales , Sebacinales , Gomphales , Geastrales , Hysterangiales , Thelephorales , and Trechisporales ( Basidiomycota ), and Pezizales , Sordariales , Eurotiales , Pleosporales , Helotiales , and Diaporthales ( Ascomycota ) were the most abundant for Fungi . By using optimized "universal" PCR primers that targeted the peroxidase-catalase enzyme family, we identified several known and novel sequences from various Basidiomycota , even from taxa appearing at low abundance. The majority of the sequences recovered were manganese peroxidases from several genera of Agaricales , Hysterangiales , Gomphales , Geastrales , Russulales , Hymenochaetales , and Trechisporales , while lignin -and versatile-peroxidases were limited to two to eight species, respectively. Comparisons of the obtained sequences with publicly available data allowed a detailed structural analysis of polymorphisms and functionally relevant amino-acid residues at phylogenetic level. The targeted metagenomics applied here revealed an important role in lignocellulose degradation of hitherto understudied orders of Basidiomycota , such as the Hysterangiales and Gomphales , while it also suggested the auxiliary activity of particular members of Proteobacteria , Actinobacteria , Acidobacteria, Verrucomicrobia , and Gemmatimonadetes . The application of NGS-based metagenomics approaches allows a better understanding of the complex process of lignocellulolysis at the microbial community level as well as the identification of candidate taxa and genes for targeted functional investigations and genetic modifications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kalntremtziou, Papaioannou, Vangalis, Polemis, Pappas, Zervakis and Typas.)

In the present work the entomopathogenic fungus B. bassiana lipids were studied against the potent pro-inflammatory and thrombotic mediators implicated in several disorders, platelet-activating factor (PAF) and thrombin. Bioactivities of lipid extracts from B. bassiana cells and culture supernatants and of their lipid fractions separated by a one-step HPLC-analysis ere assessed against the PAF/Thrombin-induced aggregation of washed rabbit platelets. Lipid extracts from both cell-biomass and supernatant inhibited strongly PAF/Thrombin-activities and platelet-aggregation, exhibiting higher specificity against PAF. Similarly, HPLC-derived lipid-fractions of phenolics/glycolipids, Sphingomyelins and Phosphatidylcholines (PC) showed strong anti-PAF potency. PC PAF-like molecules exhibited the strongest antagonistic anti-PAF effects, while in higher amounts they agonistically inhibited PAF-activities. Some bioactive lipids with strong anti-PAF effects are exo-cellularly secreted in the culture media during fungal growth, while others are not. The presence of such lipid bioactives in B. bassiana with strong anti-inflammatory and anti-thrombotic properties, provide new perspectives and putative future applications for this entomopathogenic fungus., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lipids are a very heterogeneous class of biomolecules with distinct structures and functions. Total lipids (TLs) obtained from natural sources are regularly further separated into lipid subclasses, with the two major ones being the polar lipids (PLs) and neutral lipids (NLs). Traditional analytical methods for fractionating TLs into NLs, PLs, and their subclasses, usually comprise difficult, costly and time-consuming steps. Instead, several benefits and applications are derived by implementing a novel one-step semi-preparative and reversed-phase HPLC-analysis for separating TLs into all kinds of lipid subclasses. This method allows a one-step separation/fractionation of several subclasses of bio-functional PLs (i.e. phospholipids, glycolipids, phenolic compounds, N-acyl-homoserine-lactones, etc.) and NLs (i.e. triacylglycerols, fatty acids, esters, etc.) from TL-extracts of a natural source, prior to further testing them for their bio-functionality (i.e. in bioassays/cell models) and structure-activity relationships (i.e. LC-MS/GC-MS).•This method can be applied in several natural sources, such as animal and marine sources, plants, microorganisms of biotechnological and agricultural interest, foods, beverages and related products, and by-products.•This method can also be applied for separating specific bio-functional lipids from complex medical and pharmaceutical samples (i.e. cells, tissues, blood, plasma, liposomes, etc.), either for evaluating their role in diseases (i.e. PAF/PAF-like molecules) or by elucidating their protective roles (i.e. PLs rich in ω3 PUFA) for supplements and nutraceuticals' applications., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s). Published by Elsevier B.V.)

Zymomonas mobilis is an α-proteobacterium that interests the biofuel industry due to its perfect ethanol fermentation yields. From its first description as a bacterial isolate in fermented alcoholic beverages to date, Z. mobilis has been rigorously studied in directions basic and applied. The Z. mobilis powerful Entner-Doudoroff glycolytic pathway has been the center of rigorous biochemical studies and, aside from ethanol, it has attracted interest in terms of high-added-value chemical manufacturing. Energetic balances and the effects of respiration have been explored in fundamental directions as also in applications pursuing strain enhancement and the utilization of alternative carbon sources. Metabolic modeling has addressed the optimization of the biochemical circuitry at various conditions of growth and/or substrate utilization; it has been also critical in predicting desirable end-product yields via flux redirection. Lastly, stress tolerance has received particular attention, since it directly determines biocatalytical performance at challenging bioreactor conditions. At a genetic level, advances in the genetic engineering of the organism have brought forth beneficial manipulations in the Z. mobilis gene pool, e.g., knock-outs, knock-ins and gene stacking, aiming to broaden the metabolic repertoire and increase robustness. Recent omic and expressional studies shed light on the genomic content of the most applied strains and reveal landscapes of activity manifested at ambient or reactor-based conditions. Studies such as those reviewed in this work, contribute to the understanding of the biology of Z. mobilis, enable insightful strain development, and pave the way for the transformation of Z. mobilis into a consummate organism for biomass conversion., (© 2020 Elsevier Ltd. All rights reserved.)

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis , this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis , it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD + under conditions when acetaldehyde, but not ethanol, is the desired catabolic product. In the present work, we sought to improve the capacity Z. mobilis to synthesize acetaldehyde, based on predictions of a stoichiometric model of its central metabolism developed herein. According to the model analysis, the main objectives in the course of engineering acetaldehyde producer strains were determined to be: (i) reducing ethanol synthesis via reducing the activity of alcohol dehydrogenase (ADH), and (ii) enhancing the respiratory capacity, either by overexpression of the respiratory NADH dehydrogenase (NDH), or by mutation of other components of respiratory metabolism. Several mutants with elevated respiration rate, decreased alcohol dehydrogenase activity, or a combination of both, were obtained. They were extensively characterized by determining their growth rates, product yields, oxygen consumption rates, ADH, and NDH activities, transcription levels of key catabolic genes, as well as concentrations of central metabolites under aerobic culture conditions. Two mutant strains were selected, with acetaldehyde yield close to 70% of the theoretical maximum value, almost twice the previously published yield for Z. mobilis . These strains can serve as a basis for further development of industrial acetaldehyde producers., (Copyright © 2019 Kalnenieks, Balodite, Strähler, Strazdina, Rex, Pentjuss, Fuchino, Bruheim, Rutkis, Pappas, Poole, Sawodny and Bettenbrock.)

Methicillin-resistant Staphylococcus aureus (MRSA) strains are important human pathogens and a significant health hazard for hospitals and the food industry. They are resistant to β-lactam antibiotics including methicillin and extremely difficult to treat. In this study, we show that the Staphylococcus aureus COL (MRSA) strain, with a known complete genome, can easily survive and grow under acidic and alkaline conditions (pH5 and pH9, respectively), both planktonically and as a biofilm. A microarray-based analysis of both planktonic and biofilm cells was performed under acidic and alkaline conditions showing that several genes are up- or down-regulated under different environmental conditions and growth modes. These genes were coding for transcription regulators, ion transporters, cell wall biosynthetic enzymes, autolytic enzymes, adhesion proteins and antibiotic resistance factors, most of which are associated with biofilm formation. These results will facilitate a better understanding of the physiological adjustments occurring in biofilm-associated S. aureus COL cells growing in acidic or alkaline environments, which will enable the development of new efficient treatment or disinfection strategies., (Copyright © 2019 Efthimiou, Tsiamis, Typas and Pappas.)

Background: Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain., Results: In this study, we determined the complete chromosome and plasmid sequences of ZM4 and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1-2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion., Conclusions: The complete genome of ZM4 and two xylose-utilizing derivatives is reported in this study, with an emphasis on identifying and characterizing plasmid genes. Plasmid gene annotation, validation, expression levels at growth conditions of interest, and contribution to host fitness are reported for the first time.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in food has provoked a great concern about the presence of MRSA in associated foodstuff. Although MRSA is often detected in various retailed meat products, it seems that food handlers are more strongly associated with this type of food contamination. Thus, it can be easily postulated that any food could be contaminated with this pathogen in an industrial environment or in household and cause food poisoning. To this direction, the effect of rocket (Eruca sativa ) extract on MRSA growth and proteome was examined in the present study. This goal was achieved with the comparative study of the MRSA strain COL proteome, cultivated in rocket extract versus the standard Luria-Bertani growth medium. The obtained results showed that MRSA was able to grow in rocket extract. In addition, proteome analysis using 2-DE method showed that MRSA strain COL is taking advantage of the sugar-, lipid-, and vitamin-rich substrate in the liquid rocket extract, although its growth was delayed in rocket extract compared to Luria-Bertani medium. This work could initiate further research about bacterial metabolism in plant-based media and defense mechanisms against plant-derived antibacterials.

Zymomonas mobilis subsp. mobilis is one of the most rigorous ethanol-producing organisms known to date, considered by many to be the prokaryotic alternative to yeast. The two most applied Z. mobilis subsp. mobilis strains, ZM4 and CP4, derive from Recife, Brazil, and have been isolated from sugarcane fermentations. Of these, ZM4 was the first Z. mobilis representative strain to be sequenced and analyzed. Here, we report the finishing of the genome sequence of strain CP4, which is highly similar but not identical to that of ZM4.

Zymomonas mobilis is an ethanologenic bacterium that has been studied for use in biofuel production. Of the sequenced Zymomonas strains, ATCC 29191 has been described as the phenotypic centrotype of Zymomonas mobilis subsp. mobilis, the taxon that harbors the highest ethanol-producing Z. mobilis strains. ATCC 29191 was isolated in Kinshasa, Congo, from palm wine fermentations. This strain is reported to be a robust levan producer, while in recent years it has been employed in studies addressing Z. mobilis respiration. Here we announce the finishing and annotation of the ATCC 29191 genome, which comprises one chromosome and three plasmids.

To ensure faithful transmission of low-copy plasmids to daughter cells, these plasmids must replicate once per cell cycle and distribute the replicated DNA to the nascent daughter cells. RepABC family plasmids are found exclusively in alphaproteobacteria and carry a combined replication and partitioning locus, the repABC cassette, which is also found on secondary chromosomes in this group. RepC and a replication origin are essential for plasmid replication, and RepA, RepB and the partitioning sites distribute the replicons to predivisional cells. Here, we review our current understanding of the transcriptional and post-transcriptional regulation of the Rep proteins and of their functions in plasmid replication and partitioning.

Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

Zymomonas mobilis is an alphaproteobacterium studied for bioethanol production. Different strains of this organism have been hitherto sequenced; they all belong to the Z. mobilis subsp. mobilis taxon. Here we report the finished and annotated genome sequence of strain ATCC 29192, a cider-spoiling agent isolated in the United Kingdom. ATCC 29192 is the lectotype of the second-best-characterized subspecies of Z. mobilis, Z. mobilis subsp. pomaceae. The nucleotide sequence of ATCC 29192 deviates from that of Z. mobilis subsp. mobilis representatives, which justifies its distinct taxonomic positioning and proves particularly useful for comparative and functional genomic analyses., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

When animals are under stress, glucocorticoids commonly inhibit adult neurogenesis by acting through glucocorticoid receptors (GRs). However, in some cases, conditions that elevate glucocorticoids promote adult neurogenesis, and the role of glucocorticoid receptors in these circumstances is not well understood. We examined the involvement of GRs in social enhancement of brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus. In this species, long-term social interaction simultaneously elevates plasma cortisol, enhances brain cell addition and increases production of aggressive electrocommunication signals ("chirps"). We implanted isolated and paired fish with capsules containing nothing (controls) or the GR antagonist, RU486, recorded chirp production and locomotion for 7d, and measured the density of newborn cells in the periventricular zone. Compared to isolated controls, paired controls showed elevated chirping in two phases: much higher chirp rates in the first 5h and moderately higher nocturnal rates thereafter. Treating paired fish with RU486 reduced chirp rates in both phases to those of isolated fish, demonstrating that GR activation is crucial for socially induced chirping. Neither RU486 nor social interaction affected locomotion. RU486 treatment to paired fish had a partial effect on cell addition: paired RU486 fish had less cell addition than paired control fish but more than isolated fish. This suggests that cortisol activation of GRs contributes to social enhancement of cell addition but works in parallel with another GR-independent mechanism. RU486 also reduced cell addition in isolated fish, indicating that GRs participate in the regulation of cell addition even when cortisol levels are low., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The potential genotoxic effects of several pure secondary metabolites produced by fungi used as biological control agents (BCAs) were studied with the Ames Salmonella/microsome mutagenicity assay and the Vitotox test, with and without metabolic activation. A complete set of Salmonella tester strains was used to avoid false negative results. To detect possible mutagenic and/or cytotoxic effects of fungal secondary metabolites due to synergistic action, crude extracts and fungal cell extracts of the BCAs were also examined. Although the sensitivity of the methods varied depending on the metabolite used, clearly no genotoxicity was observed in all cases. The results of the two assays are discussed in the light of being used in a complementary fashion for a convincing risk-assessment evaluation of fungal BCAs and their secondary metabolites., (2011 Elsevier B.V. All rights reserved.)

Zymomonas mobilis is a facultatively anaerobic α-proteobacterium with a considerable potential for industrial ethanol production. An important tool in the generation of stable mutants for this organism is described in this chapter; it entails insertional mutagenesis with the help of the transposable element mini-Mu. The latter is delivered into Z. mobilis with the use of plasmid pULB113 (RP4::mini-Mu) that self-transfers in the organism at notable frequencies and remains highly stable even under nonselective conditions. Transposition of mini-Mu and subsequent mutagenesis occur readily in Z. mobilis pULB113 transconjugants and result in the generation of large numbers of random mutants. This can be demonstrated by the isolation of various auxotrophs with single or multiple nutritional requirements, the vast majority of which bears insertions at different chromosomal locations, even when exhibiting the same requirement. Therefore, transposon mutagenesis with the use of mini-Mu serves as a simple and effective tool for indiscriminate mutant production in Z. mobilis.

Zymomonas mobilis is an ethanol-producing alphaproteobacterium currently considered a major candidate organism for bioethanol production. Here we report the finished and annotated genome sequence of Z. mobilis subsp. mobilis strain NCIMB 11163, a British ale-infecting isolate. This is the first Z. mobilis strain whose genome, chromosomal and plasmid, is presented in its entirety.

The Agrobacterium tumefaciens oncogenic Ti plasmids replicate and segregate to daughter cells via repABC cassettes, in which repA and repB are plasmid partitioning genes and repC encodes the replication initiator protein. repABC cassettes are encountered in a growing number of plasmids and chromosomes of the alpha-proteobacteria, and findings from particular representatives of agrobacteria, rhizobia and Paracoccus have began to shed light on their structure and functions. Amongst repABC replicons, Ti plasmids and particularly the octopine-type Ti have recently stood as model in regulation of repABC basal expression, which acts in plasmid copy number control, but also appear to undergo pronounced up-regulation of repABC, upon interbacterial and host-bacterial signaling. The last results in considerable Ti copy number increase and collective elevation of Ti gene expression. Inhibition of the Ti repABC is in turn conferred by a plant defense compound, which primarily affects Agrobacterium virulence and interferes with cell-density perception. Altogether, the above suggest that the entire Ti gene pool is subjected to the bacterium-eukaryote signaling network, a phenomenon quite unprecedented for replicons thought of as stringently controlled. It remains to be seen whether similar copy number variations characterize related replicons or if they are of even broader significance in plasmid biology.

Cell-cell communication via the production and detection of chemical signal molecules has been the focus of a great deal of research over the past decade. One class of chemical signals widely used by proteobacteria consists of N-acyl-homoserine lactones, which are synthesized by proteins related to LuxI of Vibrio fischeri and are detected by proteins related to the V. fischeri LuxR protein. A related marine bacterium, Vibrio harveyi, communicates using two chemical signals, one of which, autoinducer-2 (AI-2), is a furanone borate diester that is synthesized by the LuxS protein and detected by a periplasmic protein called LuxP. Evidence from a number of laboratories suggests that AI-2 may be used as a signal by diverse groups of bacteria, and might permit intergeneric signalling. These two families of signalling systems have been studied from the perspectives of physiology, ecology, biochemistry, and more recently, structural biology. Here, we review the biochemistry and structural biology of both acyl-homoserine-lactone-dependent and AI-2-dependent signalling systems., (Copyright 2004 Blackwell Publishing Ltd)

Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.

The replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine-type Ti plasmid repABC operon, three promoters were recently shown to be activated by the LuxR-type regulator TraR. Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number. Here we describe a fourth promoter, designated P4. This promoter lies directly upstream of repA and is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the major cis-acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA-mediated repression in trans. Purified RepA bound to an approximately 70-nucleotide operator site overlapping the P4 promoter and extending well downstream. Binding affinity was increased by adenosine di- and tri-phosphates and also by purified RepB. Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters. These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid.

TraR, a LuxR-type quorum-sensing transcription factor in Agrobacterium tumefaciens, activates genes required for conjugal transfer of the Ti plasmid and also enhances the copy number of a nopaline-type Ti plasmid. Here, we show that TraR increases the copy number of an octopine-type Ti plasmid up to eightfold and that TraR activates transcription of the repABC operon up to 25-fold. The ability of TraR to increase copy number was strictly dependent on several TraR-activated promoters of this operon, indicating that TraR affects copy number solely at the level of transcription. Promoter resections and mRNA transcript analysis revealed the presence of three TraR-dependent promoters. Two TraR-dependent transcription start sites are located 45.5 and 65.5 nucleotides downstream of a site called tra box II, whereas the third start site lies 42.5 nucleotides downstream of a site called tra box III. Purified TraR bound to both tra boxes with comparable affinities, causing moderate DNA bending. TraR bound and bent these two sites independently rather than synergistically. Alteration of tra box III to match the consensus sequence dramatically increased TraR-dependent expression of repABC and plasmid copy number. TraR-dependent elevation of Ti plasmid copy number caused a three- to fourfold increase in plant tumorigenesis.

Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 A as a complex with the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an alpha/beta/alpha sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix turn helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90 degree angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.

A new insertion sequence, designated ISZm1068, was isolated from Zymomonas mobilis strain CP4. This element consists of 1,068 bp and contains one major ORF which shows similarities both at the nucleotide and at the amino acid sequence level with the corresponding ORFs encoding the transposases of many IS5 family elements, in particular the IS1031 group. Moreover, the Z. mobilis ORF shares the conserved N2, N3 and C1 signature motifs of the IS4 and IS5 families. Six out of seven Z. mobilis wild-type strains were shown by hybridisation to contain a single copy of the ISZm1068 element. Nucleotide sequences of the insertion elements from these strains exhibited extremely high levels of identity, varying from 94.25 to 99.25%. ISZm1068 was shown to be active in Escherichia coli cells and led to plasmid replicon fusions within the host cell. Sequence analysis of rare cointegration and resolution derivatives suggests that ISZm1068 has putative imperfect inverted repeats at its extremities of 18 bp (IR-right) and 14 bp (IR-left), and that a 3-bp (5'-TCA-3') target sequence is duplicated upon insertion.

The complete nucleotide sequences of two small cryptic Zymomonas mobilis ATCC 10988 plasmids (pZMO1 and pZMO2) were determined. The plasmids showed 67% homology to each other at their nucleotide level. Plasmid pZMO1 was 1651 bp long with 38% G + C content and contained an open reading frame (ORFZMO1) of 1044 nucleotides. ORFZMO1 is predicted to encode a polypeptide of 348 amino acids and shows a high degree of homology with gram-negative replication proteins of rolling circle replicating plasmids, which belong to the pC194/pUB110 family. Plasmid pZMO2 was found to be 1669 bp long, with a 38.5% G + C content, and it contained an ORF of 552 nucleotides (ORFZMO2) encoding a putative polypeptide of 184 amino acids. This polypeptide also shows a high degree of homology with the replication proteins of RCR plasmids of gram-negative bacteria, but only at their N-termini. The region necessary for replication of both plasmids was determined by stability tests under nonselective conditions, following cloning in pBR325 and introduction in Z. mobilis ATCC 10988 by pRK2013 assisted conjugation. Double- and single-strand origin regions were predicted by sequence analysis. Detection of single-stranded DNA in the extract of exponentially growing cells confirmed experimentally the rolling circle replication mode of at least pZMO2., (Copyright 2000 Academic Press.)

Conjugative or mobilizable plasmids carrying the transposable elements Tn5, Tn501 or mini Mu were readily transferred from Escherichia coli donors into Zymomonas mobilis recipients with frequencies depending both on donor and recipient strain used. With the exception of pULB113 (RP4::mini Mu), all foreign plasmids exhibited high instability in Z. mobilis transconjugants under both selective and non-selective conditions. Transposition events and consequent mutagenesis occurred readily in Z. mobilis transconjugant strains, with Tn5 and Tn501 being far less successful than mini Mu. Transposon mutagenesis with the help of mini Mu resulted in the isolation of a large number of independent auxotrophs with polyauxotrophs, cysteine, methionine and isoleucine requiring-isolates being the most frequent. When chromosomal DNA from all these mutants was digested with various restriction enzymes and the resulting restriction patterns were hybridized with a mini Mu probe, the majority of these mutants appeared to have insertions at different sites of the chromosome. Thus, transposon mutagenesis by mini Mu is proven to be a simple and efficient tool for mutant production and the genetic analysis of Z. mobilis.