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19000.9970The Two TpsB-Like Proteins in Anabaena sp. Strain PCC 7120 Are Involved in Secretion of Selected Substrates. The outer membrane of Gram-negative bacteria acts as an initial diffusion barrier that shields the cell from the environment. It contains many membrane-embedded proteins required for functionality of this system. These proteins serve as solute and lipid transporters or as machines for membrane insertion or secretion of proteins. The genome of Anabaena sp. strain PCC 7120 codes for two outer membrane transporters termed TpsB1 and TpsB2. They belong to the family of the two-partner secretion system proteins which are characteristic of pathogenic bacteria. Because pathogenicity of Anabaena sp. strain PCC 7120 has not been reported, the function of these two cyanobacterial TpsB proteins was analyzed. TpsB1 is encoded by alr1659, while TpsB2 is encoded by all5116 The latter is part of a genomic region containing 11 genes encoding TpsA-like proteins. However, tpsB2 is transcribed independently of a tpsA gene cluster. Bioinformatics analysis revealed the presence of at least 22 genes in Anabaena sp. strain PCC 7120 putatively coding for substrates of the TpsB system, suggesting a rather global function of the two TpsB proteins. Insertion of a plasmid into each of the two genes resulted in altered outer membrane integrity and antibiotic resistance. In addition, the expression of genes coding for the Clp and Deg proteases is dysregulated in these mutants. Moreover, for two of the putative substrates, a dependence of the secretion on functional TpsB proteins could be confirmed. We confirm the existence of a two-partner secretion system in Anabaena sp. strain PCC 7120 and predict a large pool of putative substrates.IMPORTANCE Cyanobacteria are important organisms for the ecosystem, considering their contribution to carbon fixation and oxygen production, while at the same time some species produce compounds that are toxic to their environment. As a consequence, cyanobacterial overpopulation might negatively impact the diversity of natural communities. Thus, a detailed understanding of cyanobacterial interaction with the environment, including other organisms, is required to define their impact on ecosystems. While two-partner secretion systems in pathogenic bacteria are well known, we provide a first description of the cyanobacterial two-partner secretion system.202133257527
43910.9970Sequence and organization of pMAC, an Acinetobacter baumannii plasmid harboring genes involved in organic peroxide resistance. Acinetobacter baumannii 19606 harbors pMAC, a 9540-bp plasmid that contains 11 predicted open-reading frames (ORFs). Cloning and transformation experiments using Acinetobacter calcoaceticus BD413 mapped replication functions within a region containing four 21-bp direct repeats (ori) and ORF 1, which codes for a predicted replication protein. Subcloning and tri-parental mating experiments mapped mobilization functions to the product of ORF 11 and an adjacent predicted oriT. Three ORFs code for proteins that share similarity to hypothetical proteins encoded by plasmid genes found in other bacteria, while the predicted products of three others do not match any known sequence. The product of ORF 8 is similar to Ohr, a hydroperoxide reductase responsible for organic peroxide detoxification and resistance in bacteria. This ORF is immediately upstream of a coding region whose product is related to the MarR family of transcriptional regulators. Disk diffusion assays showed that A. baumannii 19606 is resistant to the organic peroxide-generating compounds cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (t-BHP), although to levels lower than those detected in Pseudomonas aeruginosa PAO1. Cloning and introduction of the ohr and marR ORFs into Escherichia coli was associated with an increase in resistance to CHP and t-BHP. This appears to be the first case in which the genetic determinants involved in organic peroxide resistance are located in an extrachromosomal element, a situation that can facilitate the horizontal transfer of genetic elements coding for a function that protects bacterial cells from oxidative damage.200616530832
998720.9969Four genes essential for recombination define GInts, a new type of mobile genomic island widespread in bacteria. Integrases are a family of tyrosine recombinases that are highly abundant in bacterial genomes, actively disseminating adaptive characters such as pathogenicity determinants and antibiotics resistance. Using comparative genomics and functional assays, we identified a novel type of mobile genetic element, the GInt, in many diverse bacterial groups but not in archaea. Integrated as genomic islands, GInts show a tripartite structure consisting of the ginABCD operon, a cargo DNA region from 2.5 to at least 70 kb, and a short AT-rich 3' end. The gin operon is characteristic of GInts and codes for three putative integrases and a small putative helix-loop-helix protein, all of which are essential for integration and excision of the element. Genes in the cargo DNA are acquired mostly from phylogenetically related bacteria and often code for traits that might increase fitness, such as resistance to antimicrobials or virulence. GInts also tend to capture clusters of genes involved in complex processes, such as the biosynthesis of phaseolotoxin by Pseudomonas syringae. GInts integrate site-specifically, generating two flanking direct imperfect repeats, and excise forming circular molecules. The excision process generates sequence variants at the element attachment site, which can increase frequency of integration and drive target specificity.201728393892
907330.9968EpitoCore: Mining Conserved Epitope Vaccine Candidates in the Core Proteome of Multiple Bacteria Strains. In reverse vaccinology approaches, complete proteomes of bacteria are submitted to multiple computational prediction steps in order to filter proteins that are possible vaccine candidates. Most available tools perform such analysis only in a single strain, or a very limited number of strains. But the vast amount of genomic data had shown that most bacteria contain pangenomes, i.e., their genomic information contains core, conserved genes, and random accessory genes specific to each strain. Therefore, in reverse vaccinology methods it is of the utmost importance to define core proteins and core epitopes. EpitoCore is a decision-tree pipeline developed to fulfill that need. It provides surfaceome prediction of proteins from related strains, defines core proteins within those, calculate their immunogenicity, predicts epitopes for a given set of MHC alleles defined by the user, and then reports if epitopes are located extracellularly and if they are conserved among the core homologs. Pipeline performance is illustrated by mining peptide vaccine candidates in Mycobacterium avium hominissuis strains. From a total proteome of ~4,800 proteins per strain, EpitoCore predicted 103 highly immunogenic core homologs located at cell surface, many of those related to virulence and drug resistance. Conserved epitopes identified among these homologs allows the users to define sets of peptides with potential to immunize the largest coverage of tested HLA alleles using peptide-based vaccines. Therefore, EpitoCore is able to provide automated identification of conserved epitopes in bacterial pangenomic datasets.202032431712
836740.9968A hybrid NRPS-PKS gene cluster related to the bleomycin family of antitumor antibiotics in Alteromonas macleodii strains. Although numerous marine bacteria are known to produce antibiotics via hybrid NRPS-PKS gene clusters, none have been previously described in an Alteromonas species. In this study, we describe in detail a novel hybrid NRPS-PKS cluster identified in the plasmid of the Alteromonasmacleodii strain AltDE1 and analyze its relatedness to other similar gene clusters in a sequence-based characterization. This is a mobile cluster, flanked by transposase-like genes, that has even been found inserted into the chromosome of some Alteromonasmacleodii strains. The cluster contains separate genes for NRPS and PKS activity. The sole PKS gene appears to carry a novel acyltransferase domain, quite divergent from those currently characterized. The predicted specificities of the adenylation domains of the NRPS genes suggest that the final compound has a backbone very similar to bleomycin related compounds. However, the lack of genes involved in sugar biosynthesis indicates that the final product is not a glycopeptide. Even in the absence of these genes, the presence of the cluster appears to confer complete or partial resistance to phleomycin, which may be attributed to a bleomycin-resistance-like protein identified within the cluster. This also suggests that the compound still shares significant structural similarity to bleomycin. Moreover, transcriptomic evidence indicates that the NRPS-PKS cluster is expressed. Such sequence-based approaches will be crucial to fully explore and analyze the diversity and potential of secondary metabolite production, especially from increasingly important sources like marine microbes.201324069455
28950.9966A genetic system that reports transient activation of genes in Bacillus. Site-specific recombination is a powerful tool for precise excision of DNA fragments. We used this characteristic to construct a genetic system to report the transient activation of a promoter by promoting the stable acquisition of an antibiotic resistance marker by the bacterium. The system is composed of two compatible plasmid derivatives from Gram-positive bacteria. One of the plasmids allows the insertion of promoters upstream from tnpI, which encodes the site-specific recombinase of Tn4430. The second plasmid carries two selectable resistance genes: one is flanked by two site-specific recombination sequences and is lost following recombination; in contrast, the other resistance gene becomes functional after the site-specific recombination event. By inserting conditionally controlled promoters (the xylose-inducible xylA promoter or the plcA promoter whose expression is dependent on the growth medium) upstream of tnpI, we demonstrated that our genetic system responds to signals inducing transcription by conferring a new resistance phenotype to the host bacteria. Thus, this system can be used to identify genes which are transiently or conditionally expressed.19979427554
24360.9966Phylogenetic distribution of translational GTPases in bacteria. BACKGROUND: Translational GTPases are a family of proteins in which GTPase activity is stimulated by the large ribosomal subunit. Conserved sequence features allow members of this family to be identified. RESULTS: To achieve accurate protein identification and grouping we have developed a method combining searches with Hidden Markov Model profiles and tree based grouping. We found all the genes for translational GTPases in 191 fully sequenced bacterial genomes. The protein sequences were grouped into nine subfamilies. Analysis of the results shows that three translational GTPases, the translation factors EF-Tu, EF-G and IF2, are present in all organisms examined. In addition, several copies of the genes encoding EF-Tu and EF-G are present in some genomes. In the case of multiple genes for EF-Tu, the gene copies are nearly identical; in the case of multiple EF-G genes, the gene copies have been considerably diverged. The fourth translational GTPase, LepA, the function of which is currently unknown, is also nearly universally conserved in bacteria, being absent from only one organism out of the 191 analyzed. The translation regulator, TypA, is also present in most of the organisms examined, being absent only from bacteria with small genomes.Surprisingly, some of the well studied translational GTPases are present only in a very small number of bacteria. The translation termination factor RF3 is absent from many groups of bacteria with both small and large genomes. The specialized translation factor for selenocysteine incorporation--SelB--was found in only 39 organisms. Similarly, the tetracycline resistance proteins (Tet) are present only in a small number of species. Proteins of the CysN/NodQ subfamily have acquired functions in sulfur metabolism and production of signaling molecules. The genes coding for CysN/NodQ proteins were found in 74 genomes. This protein subfamily is not confined to Proteobacteria, as suggested previously but present also in many other groups of bacteria. CONCLUSION: Four of the translational GTPase subfamilies (IF2, EF-Tu, EF-G and LepA) are represented by at least one member in each bacterium studied, with one exception in LepA. This defines the set of translational GTPases essential for basic cell functions.200717214893
37270.9966A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens. A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.19968692990
26280.9966Genome scanning in Haemophilus influenzae for identification of essential genes. We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.199910438768
837290.9966A Plasmid-Encoded Putative Glycosyltransferase Is Involved in Hop Tolerance and Beer Spoilage in Lactobacillus brevis. Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species.IMPORTANCELactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.202031757821
8375100.9966Genome-scale identification method applied to find cryptic aminoglycoside resistance genes in Pseudomonas aeruginosa. BACKGROUND: The ability of bacteria to rapidly evolve resistance to antibiotics is a critical public health problem. Resistance leads to increased disease severity and death rates, as well as imposes pressure towards the discovery and development of new antibiotic therapies. Improving understanding of the evolution and genetic basis of resistance is a fundamental goal in the field of microbiology. RESULTS: We have applied a new genomic method, Scalar Analysis of Library Enrichments (SCALEs), to identify genomic regions that, given increased copy number, may lead to aminoglycoside resistance in Pseudomonas aeruginosa at the genome scale. We report the result of selections on highly representative genomic libraries for three different aminoglycoside antibiotics (amikacin, gentamicin, and tobramycin). At the genome-scale, we show significant (p<0.05) overlap in genes identified for each aminoglycoside evaluated. Among the genomic segments identified, we confirmed increased resistance associated with an increased copy number of several genomic regions, including the ORF of PA5471, recently implicated in MexXY efflux pump related aminoglycoside resistance, PA4943-PA4946 (encoding a probable GTP-binding protein, a predicted host factor I protein, a delta 2-isopentenylpyrophosphate transferase, and DNA mismatch repair protein mutL), PA0960-PA0963 (encoding hypothetical proteins, a probable cold shock protein, a probable DNA-binding stress protein, and aspartyl-tRNA synthetase), a segment of PA4967 (encoding a topoisomerase IV subunit B), as well as a chimeric clone containing two inserts including the ORFs PA0547 and PA2326 (encoding a probable transcriptional regulator and a probable hypothetical protein, respectively). CONCLUSIONS: The studies reported here demonstrate the application of new a genomic method, SCALEs, which can be used to improve understanding of the evolution of antibiotic resistance in P. aeruginosa. In our demonstration studies, we identified a significant number of genomic regions that increased resistance to multiple aminoglycosides. We identified genetic regions that include open reading frames that encode for products from many functional categories, including genes related to O-antigen synthesis, DNA repair, and transcriptional and translational processes.200919907650
9876110.9965The Facts and Family Secrets of Plasmids That Replicate via the Rolling-Circle Mechanism. Plasmids are self-replicative DNA elements that are transferred between bacteria. Plasmids encode not only antibiotic resistance genes but also adaptive genes that allow their hosts to colonize new niches. Plasmid transfer is achieved by conjugation (or mobilization), phage-mediated transduction, and natural transformation. Thousands of plasmids use the rolling-circle mechanism for their propagation (RCR plasmids). They are ubiquitous, have a high copy number, exhibit a broad host range, and often can be mobilized among bacterial species. Based upon the replicon, RCR plasmids have been grouped into several families, the best known of them being pC194 and pUB110 (Rep_1 family), pMV158 and pE194 (Rep_2 family), and pT181 and pC221 (Rep_trans family). Genetic traits of RCR plasmids are analyzed concerning (i) replication mediated by a DNA-relaxing initiator protein and its interactions with the cognate DNA origin, (ii) lagging-strand origins of replication, (iii) antibiotic resistance genes, (iv) mobilization functions, (v) replication control, performed by proteins and/or antisense RNAs, and (vi) the participating host-encoded functions. The mobilization functions include a relaxase initiator of transfer (Mob), an origin of transfer, and one or two small auxiliary proteins. There is a family of relaxases, the MOB(V) family represented by plasmid pMV158, which has been revisited and updated. Family secrets, like a putative open reading frame of unknown function, are reported. We conclude that basic research on RCR plasmids is of importance, and our perspectives contemplate the concept of One Earth because we should incorporate bacteria into our daily life by diminishing their virulence and, at the same time, respecting their genetic diversity.202234878299
351120.9965Rapid and efficient cloning of proviral flanking fragments by kanamycin resistance gene complementation. We have developed a technique for the rapid cloning of unknown flanking regions of transgenic DNA. We complemented a truncated kanamycin resistance gene of a bacterial plasmid with a neomycin resistance gene fragment from a gene transfer vector. Optimized transformation conditions allowed us to directly select for kanamycin-resistant bacteria. We cloned numerous proviral flanking fragments from growth factor-independent cell mutants that were obtained after infection with a replication incompetent retroviral vector and identified integrations into the cyclin D2 and several unknown genomic sequences. We anticipate that our method could be adapted to various vector systems that are used to tag and identify genes and to map genomes.19999863001
6349130.9965High-level chromate resistance in Arthrobacter sp. strain FB24 requires previously uncharacterized accessory genes. BACKGROUND: The genome of Arthrobacter sp. strain FB24 contains a chromate resistance determinant (CRD), consisting of a cluster of 8 genes located on a 10.6 kb fragment of a 96 kb plasmid. The CRD includes chrA, which encodes a putative chromate efflux protein, and three genes with amino acid similarities to the amino and carboxy termini of ChrB, a putative regulatory protein. There are also three novel genes that have not been previously associated with chromate resistance in other bacteria; they encode an oxidoreductase (most similar to malate:quinone oxidoreductase), a functionally unknown protein with a WD40 repeat domain and a lipoprotein. To delineate the contribution of the CRD genes to the FB24 chromate [Cr(VI)] response, we evaluated the growth of mutant strains bearing regions of the CRD and transcript expression levels in response to Cr(VI) challenge. RESULTS: A chromate-sensitive mutant (strain D11) was generated by curing FB24 of its 96-kb plasmid. Elemental analysis indicated that chromate-exposed cells of strain D11 accumulated three times more chromium than strain FB24. Introduction of the CRD into strain D11 conferred chromate resistance comparable to wild-type levels, whereas deletion of specific regions of the CRD led to decreased resistance. Using real-time reverse transcriptase PCR, we show that expression of each gene within the CRD is specifically induced in response to chromate but not by lead, hydrogen peroxide or arsenate. Higher levels of chrA expression were achieved when the chrB orthologs and the WD40 repeat domain genes were present, suggesting their possible regulatory roles. CONCLUSION: Our findings indicate that chromate resistance in Arthrobacter sp. strain FB24 is due to chromate efflux through the ChrA transport protein. More importantly, new genes have been identified as having significant roles in chromate resistance. Collectively, the functional predictions of these additional genes suggest the involvement of a signal transduction system in the regulation of chromate efflux and warrants further study.200919758450
9350140.9965Genome DNA Sequence Variation, Evolution, and Function in Bacteria and Archaea. Comparative genomics has revealed that variations in bacterial and archaeal genome DNA sequences cannot be explained by only neutral mutations. Virus resistance and plasmid distribution systems have resulted in changes in bacterial and archaeal genome sequences during evolution. The restriction-modification system, a virus resistance system, leads to avoidance of palindromic DNA sequences in genomes. Clustered, regularly interspaced, short palindromic repeats (CRISPRs) found in genomes represent yet another virus resistance system. Comparative genomics has shown that bacteria and archaea have failed to gain any DNA with GC content higher than the GC content of their chromosomes. Thus, horizontally transferred DNA regions have lower GC content than the host chromosomal DNA does. Some nucleoid-associated proteins bind DNA regions with low GC content and inhibit the expression of genes contained in those regions. This form of gene repression is another type of virus resistance system. On the other hand, bacteria and archaea have used plasmids to gain additional genes. Virus resistance systems influence plasmid distribution. Interestingly, the restriction-modification system and nucleoid-associated protein genes have been distributed via plasmids. Thus, GC content and genomic signatures do not reflect bacterial and archaeal evolutionary relationships.201322772895
176150.9965The mercury resistance (mer) operon in a marine gliding flavobacterium, Tenacibaculum discolor 9A5. Genes conferring mercury resistance have been investigated in a variety of bacteria and archaea but not in bacteria of the phylum Bacteroidetes, despite their importance in many environments. We found, however, that a marine gliding Bacteroidetes species, Tenacibaculum discolor, was the predominant mercury-resistant bacterial taxon cultured from a salt marsh fertilized with mercury-contaminated sewage sludge. Here we report characterization of the mercuric reductase and the narrow-spectrum mercury resistance (mer) operon from one of these strains - T. discolor 9A5. This mer operon, which confers mercury resistance when cloned into Flavobacterium johnsoniae, encodes a novel mercury-responsive ArsR/SmtB family transcriptional regulator that appears to have evolved independently from other mercury-responsive regulators, a novel putative transport protein consisting of a fusion between the integral membrane Hg(II) transporter MerT and the periplasmic Hg(II)-binding protein MerP, an additional MerP protein, and a mercuric reductase that is phylogenetically distinct from other known mercuric reductases.201322816663
764160.9964Fungal ATP-binding cassette (ABC) transporters in drug resistance & detoxification. Pleiotropic drug resistance (PDR) is a well-described phenomenon occurring in fungi. PDR shares several similarities with processes in bacteria and higher eukaryotes. In mammalian cells, multidrug resistance (MDR) develops from an initial single drug resistance, eventually leading to a broad cross-resistance to many structurally and functionally unrelated compounds. Notably, a number of membrane-embedded energy-consuming ATP-binding cassette (ABC) transporters have been implicated in the development of PDR/MDR phenotypes. The yeast Saccharomyces cerevisiae genome harbors some 30 genes encoding ABC proteins, several of which mediate PDR. Therefore, yeast served as an important model organism to study the functions of evolutionary conserved ABC genes, including those mediating clinical antifungal resistance in fungal pathogens. Moreover, yeast cells lacking endogenous ABC pumps are hypersensitive to many antifungal drugs, making them suitable for functional studies and cloning of ABC transporters from fungal pathogens such as Candida albicans. This review discusses drug resistance phenomena mediated by ABC transporters in the model system S. cerevisiae and certain fungal pathogens.200616611035
290170.9964Utility of the clostridial site-specific recombinase TnpX to clone toxic-product-encoding genes and selectively remove genomic DNA fragments. TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.201424682304
3768180.9964The Concerted Action of Two B3-Like Prophage Genes Excludes Superinfecting Bacteriophages by Blocking DNA Entry into Pseudomonas aeruginosa. In this study, we describe seven vegetative phage genomes homologous to the historic phage B3 that infect Pseudomonas aeruginosa Like other phage groups, the B3-like group contains conserved (core) and variable (accessory) open reading frames (ORFs) grouped at fixed regions in their genomes; however, in either case, many ORFs remain without assigned functions. We constructed lysogens of the seven B3-like phages in strain Ps33 of P. aeruginosa, a novel clinical isolate, and assayed the exclusion phenotype against a variety of temperate and virulent superinfecting phages. In addition to the classic exclusion conferred by the phage immunity repressor, the phenotype observed in B3-like lysogens suggested the presence of other exclusion genes. We set out to identify the genes responsible for this exclusion phenotype. Phage Ps56 was chosen as the study subject since it excluded numerous temperate and virulent phages. Restriction of the Ps56 genome, cloning of several fragments, and resection of the fragments that retained the exclusion phenotype allowed us to identify two core ORFs, so far without any assigned function, as responsible for a type of exclusion. Neither gene expressed separately from plasmids showed activity, but the concurrent expression of both ORFs is needed for exclusion. Our data suggest that phage adsorption occurs but that phage genome translocation to the host's cytoplasm is defective. To our knowledge, this is the first report on this type of exclusion mediated by a prophage in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium frequently isolated from infected immunocompromised patients, and the strains are resistant to a broad spectrum of antibiotics. Recently, the use of phages has been proposed as an alternative therapy against multidrug-resistant bacteria. However, this approach may present various hurdles. This work addresses the problem that pathogenic bacteria may be lysogenized by phages carrying genes encoding resistance against secondary infections, such as those used in phage therapy. Discovering phage genes that exclude superinfecting phages not only assigns novel functions to orphan genes in databases but also provides insight into selection of the proper phages for use in phage therapy.202032461312
179190.9964The genetics and biochemistry of mercury resistance. The ability of bacteria to detoxify mercurial compounds by reduction and volatilization is conferred by mer genes, which are usually plasmid located. The narrow spectrum (Hg2+ detoxifying) Tn501 and R100 determinants have been subjected to molecular genetic and DNA sequence analysis. Biochemical studies on the flavoprotein mercuric reductase have elucidated the mechanism of reduction of Hg2+ to Hg0. The mer genes have been mapped and sequenced and their protein products studied in minicells. Based on the deduced amino acid sequences, these proteins have been assigned a role in a mechanistic scheme for mercury flux in resistant bacteria. The mer genes are inducible, with regulatory control being exerted at the transcriptional level both positively and negatively. Attention is now focusing on broad-spectrum resistance involving detoxification of organomercurials by an additional enzyme, organomercurial lyase. Lyase genes have recently been cloned and sequencing studies are in progress.19872827958