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• Integrated bioinformatics system for identifying operons in bacterial genomes. • Combining computational and experimental data improves accuracy of operon prediction. • Provides insight into co-expressed genes, functional relationships, and regulatory networks. An operon is a set of neighboring genes in a genome that is transcribed as a single polycistronic message. Genes that are part of the same operon often have related functional roles or participate in the same metabolic pathways. The majority of all bacterial genes are co-transcribed with one or more other genes as part of a multi-gene operon. Thus, accurate identification of operons is important in understanding co-regulation of genes and their functional relationships. Here, we present a computational system that uses RNA-seq data to determine operons throughout a genome. The system takes the name of a genome and one or more files of RNA-seq data as input. Our method combines primary genomic sequence information with expression data from the RNA-seq files in a unified probabilistic model in order to identify operons. We assess our method's ability to accurately identify operons in a range of species through comparison to external databases of operons, both experimentally confirmed and computationally predicted, and through focused experiments that confirm new operons identified by our method. Our system is freely available at https://cs.wellesley.edu/~btjaden/Rockhopper/. [ABSTRACT FROM AUTHOR]

Abstract We have identified an IncX1 plasmid named pQJDSal1 from Salmonella enterica subsp. enterica serovar Pullorum (S. Pullorum). The plasmid is 67,685 bp in size and has 72 putative genes. pQJDSal1 harbors a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin plasmid addiction system. Two regions (A and B) that have not been previously reported in IncX1 plasmids are inserted into the backbone. Region A (10.7 kb), inserted between parA and taxD , consists of a new Tn 6168- like transposon containing an arsenic resistant operon arsB2CHR and sulfonamide resistance gene sul2. Region B contains another arsenic resistant operon arsADHR , resistance gene bla TEM-1B and three transposable elements. Conjugation experiments showed that pQJDSal1 could transfer from S. Pullorum to Escherichia coli (E. coli) J53. Statistical analysis of 70 sequenced IncX1 plasmids revealed that IncX1 plasmids harbored various antibiotic resistance genes. The results highlight the importance of IncX1 plasmids in disseminating antibiotic resistance genes. Highlights • The first time to identify an IncX1 plasmid in Salmonella Pullorum • A new Tn 6168 _like transposon is identified. • Tn 1772 is inserted into Tn 2. • Madding statistics on the antibiotic resistance genes in IncX1 plasmids [ABSTRACT FROM AUTHOR]

Abstract Bacillus megaterium belongs to the group of pigmented bacilli producing carotenoids that ensure self-protection from UV radiation-induced and collateral oxidative damage. Metabolite profiling of strain MS941 revealed the presence of the C30 carotenoids 4,4′-diapophytofluene and 4,4′-diaponeurosporenic acid. A gene function analysis demonstrated the presence of a corresponding C30 carotenoid biosynthetic pathway with pharmaceutical importance. We identified a gene cluster comprising putative genes for a farnesyl diphosphate synthase (IspA), a diapophytoene synthase (CrtM) and three distinct diapophytoene desaturases (CrtN1–3). Intriguingly, crtM was organized in an operon together with two of the identified crtN genes. The individual activities of the encoded enzymes were determined by heterologous expression and product analysis in the non-carotenogenic model organism Escherichia coli. Our experimental data show that the first catalytic steps of C30 carotenoid biosynthesis in B. megaterium share significant similarity to the corresponding biosynthetic pathway of Staphylococcus aureus. The biosynthesis of farnesyl diphosphates and their subsequent condensation to form 4,4′-diapophytoene are catalyzed by the identified IspA and CrtM, respectively. The following desaturation reactions to form 4,4′-diaponeurosporene, however, require the activities of multiple diapophytoene desaturases. A biosynthetic operon was engineered and successfully expressed in an E. coli whole-cell system creating a cell factory for a high-yield production of the C30 carotenoid 4,4′-diaponeurosporene which has promising potential in the treatment of various inflammatory diseases. Highlights • Identification and characterization of a carotenoid biosynthesis operon from Bacillus megaterium. • The identified operon is involved in C30 carotenoid biosynthesis. • Engineering of an E. coli based microbial cell factory for the high-yield production of 4,4'-diaponeurosporene. • 4,4′-diaponeurosporene has promising potential in the treatment of various inflammatory diseases. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin–Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET- pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans , indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence. [ABSTRACT FROM AUTHOR]

Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, which, as the main systemic disease in the Chilean salmon industry, causes significant economic losses. This bacterium can produce biofilm as a persistence and survival strategy in adverse conditions. In other bacteria, cheA is a key gene for modulating the onset of bacterial chemotaxis, as well as having a secondary role in biofilm production. Notwithstanding this association, the potential relationships between biofilm formation and genes involved in P. salmonis chemotaxis are poorly understood. This study aimed to determine P. salmonis cheA gene expression when grown in different culture media known to induce biofilm production. Piscirickettsia salmonis AUSTRAL-005 produced moderate/high biofilm levels after 144 h of incubation in the AUSTRAL-SRS and marine broths. In contrast, LF-89 biofilm production was weak/nonexistent in the aforementioned broths. Both assessed P. salmonis strains contained the cheYZA operon. Additionally, AUSTRAL-005 cheA transcripts increased in both culture media. In conclusion, these results suggest potential relationships between biofilm formation and genes related to chemotaxis in the fish pathogen P. salmonis . [ABSTRACT FROM AUTHOR]

Ti/Ri plasmids in pathogenic Agrobacterium species are repABC replicons that are stably maintained by the function of repABC genes. Two Ti plasmids, pTiBo542 and pTiS4, belonging to incRh2 and incRh4 incompatibility groups, respectively, were reported to carry two repABC loci. In the present study, to reveal the roles of the two repABC loci in the two plasmids, we constructed mini-replicons carrying any one or both of the repABC loci (referred to as repABC1 and repABC2 here) and examined their replication and incompatibility properties. The introduction of mini-replicons into A . tumefaciens C58C1 strains suggested that repABC1 functions as replicator genes but repABC2 does not in both the Ti plasmids. Because the components of repABC2 of pTiBo542 have highly similar amino acid and nucleotide sequences to those of the incRh1-type repABC replicon, we introduced repABC2 -containing replicons into cells harboring an incRh1 plasmid in order to check their incompatibility traits. As a result, the repABC2 -containing replicon expelled the resident incRh1 plasmid, indicating that the extra repABC locus is dispensable for replication and could work as an incompatibility determinant against incRh1 group plasmids. We suggest that the locus contributes to plasmid retention by eliminating the burden of co-existing competitive plasmids in host cells through its incompatibility. [ABSTRACT FROM AUTHOR]

Lux-operon of psychrophilic bacteria Aliivibrio logei contains two copies of luxR and is regulated by Type I quorum sensing (QS). Activation of lux -operon of psychrophilic bacteria A. logei by LuxR1 requires about 100 times higher concentrations of autoinducer (AI) than the activation by LuxR2. On the other hand, LuxR1 does not require GroEL/ES chaperonin for its folding and cannot be degraded by protease Lon, while LuxR2 sensitive to Lon and requires GroEL/ES. Here we show that at 10 −5 – 10 −4 М concentrations of AI a combination of luxR1 and luxR2 products is capable of activating the Pr-promoters of A. logei lux -operon in Escherichia coli independently of GroEL/ES and protease Lon. The presence of LuxR1 assists LuxR2 in gro − cells when AI was added at high concentration, while at low concentration of AI in a cell LuxR1 decreases the LuxR2 activity. These observations may be explained by the formation of LuxR1/LuxR2 heterodimers that act in complex with AI independently from GroEL/ES and protease Lon. Importance This study expands current understanding of QS regulation in A. logei as it implies cooperative regulation of lux -operon by LuxR1 and LuxR2 proteins. [ABSTRACT FROM AUTHOR]

Infections of pathogenic bacteria are very common in acquired immunodeficiency syndrome (AIDS) patients. However, the biological effects of HIV-1 Tat on bacteria are incompletely understood. In this study, HIV-1 Tat was expressed in Escherichia coli and Pseudomonas aeruginosa (PA01) to investigate its biological effects on bacteria. Bacterial cells expressing either HIV-1 Tat 1-86 (Tat 1-86 ) or HIV-1 Tat 1-72 (Tat 1-72 ) grow significantly faster than those with either only an empty vector or an unrelated control (GFP or Rluc). Supplementation of purified HIV-1 Tat 1-86 or Tat 1-101 protein into bacterial culture medium stimulated the growth of both E . coli and PA01. The expression profile of certain cell division-associated genes, such as those in the division cell wall ( dcw ) operon ( ftsA , ftsQ , ftsW and ftsZ ), yafO and zipA , was altered in HIV-1 Tat 1-86 expressing E . coli BL21(DE3). Furthermore, the expression of firefly luciferase (Fluc) reporter gene, when engineered for control by the dcw promoter and terminator, was enhanced by HIV-1 Tat in E . coli , confirming that HIV-1 Tat transcriptionally regulates the expression of the dcw operon. The finding that HIV-1 Tat stimulates bacterial growth whether it is produced intracellularly or applied extracellularly may have relevance for HIV patients who are highly susceptible to opportunistic bacterial infections. Contents category: Viruses –Retroviruses. The GenBank accession number for the sequence of HIV-1 Tat1-86 is AF324439.1 . [ABSTRACT FROM AUTHOR]

Clostridium butyricum is a Gram-positive bacterium involved in the development of necrotizing enterocolitis (NEC) in preterm infants. To colonize the digestive tract, components of the cell wall of C. butyricum must interact with the intestinal mucosa. The d -alanylation of cell wall components such as teichoic acids results in a net positive charge on the cell wall, which is important for many functions of Gram-positive bacteria. Notably, d -alanylation mediates resistance to antimicrobial peptides and antibiotics. Here, we show that the dlt operon of C. butyricum encodes the enzymes responsible for the d -alanylation of cell wall components and influences the surface properties of the cell wall. We show that the d -alanylation of cell wall components controls the septation of C. butyricum , which is an essential mechanism during vegetative growth. Furthermore, we find that d -alanylation is involved in the resistance of C. butyricum to some cationic antimicrobial peptides (CAMPs) and lysozyme. Finally, we show that the d -alanylation of cell wall components influences vancomycin-induced lysis. [ABSTRACT FROM AUTHOR]

Analysis of the Bifidobacterium longum subsp. infantis ATCC 15697 genome sequence for the presence of toxin-antitoxin genes revealed two relBE -like operons, three relB-mazF -like operons, one relB-vapC -like operon, one solitary gene coding for the MazF toxin and one gene coding for the RelB antitoxin. An attempt to clone the selected relE and mazF toxin genes from B. longum subsp. infantis ATCC 15697 revealed their toxic effects on Escherichia coli, which could be neutralized by coexpression of these toxins with their cognate antitoxins. The only two toxin proteins, RelE and VapC, that were found to be non-toxic to E. coli, were overproduced and purified. Electrophoretic assays showed that both RelE and VapC possessed direct endoribonuclease activity. The expression levels of toxin genes in B. longum subsp. infantis ATCC 15697 increased during the nutrient starvation and entry into the late stationary phase. The two relBE bicistronic operons r elE2-relB1 and relE1-relB4 from B. longum subsp. infantis ATCC 15697 were cloned and overexpressed in B. longum subsp. longum NCC2705 strain. The strain B. longum NCC2705 [pCESH80::relE1-relB4] showed a significantly decreased growth rate with later onset of the log phase and decreased cells density in the stationary phase. [ABSTRACT FROM AUTHOR]

Mercury resistance is the most widespread of all anti-microbial resistance occurring in a wide variety of Gram-negative and Gram-positive bacterial genera. The systems that are most studied and best understood are those encoded in mercury resistance (Mer) operons in Gram-negative bacteria. The mercury detoxification functions by the importation of highly toxic Hg 2+ into cytoplasm and enzymic reduction to volatile Hg 0 . MerT is a small (13 kDa) inner membrane protein involved in mercuric ion transport system. We have overexpressed recombinant 6His-tagged MerT from Escherichia coli in a native folded form and purified it to homogeneity in n-dodecyl-β- d -maltopyranoside (DDM) by immobilized metal affinity chromatography (IMAC). Circular dichroism showed that the protein is largely α-helical. Size-exclusion chromatography (SEC) in a variety of detergents showed that the protein exists in a multiple of oligomeric states as also confirmed by SEC coupled with multiple-angle light scattering. [ABSTRACT FROM AUTHOR]

The bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. We isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5 Å resolution. The structure reveals a β-sandwich fold, with no close structural homologs. Possible roles of FlhE, including that of a chaperone, are discussed. [ABSTRACT FROM AUTHOR]

We report the construction of a tetracycline inducible expression vector that allows regulated gene expression in the enteric pathogen Vibrio cholerae . The expression vector, named pXB300, contains the tetracycline regulatory elements from Tn10, a multiple cloning site downstream of the tetA promoter and operator sequences, a ColE1 origin of replication, a β-lactamase resistance gene for positive selection, and the hok/sok addiction system for selection in the absence of antibiotic. The function of the tetracycline expression system was demonstrated by cloning lacZ under control of the tetA promoter and quantifying β-galactosidase expression in Escherichia coli and V. cholerae . The utility for pXB300 was documented by complementation of V. cholerae virulence mutants during growth under virulence inducing conditions. The results showed that pXB300 allowed high-level expression of recombinant genes with linear induction in response to the exogenous concentration of the inducer anhydrotetracycline. We further show that pXB300 was reliably maintained in V. cholerae during growth in the absence of antibiotic selection. [ABSTRACT FROM AUTHOR]

Salmonella enterica Serovar Typhimurium U288 is an emerging pathogen of pigs. The strain contains three plasmids of diverse origin that encode traits that are of concern for food security and safety, these include antibiotic resistant determinants, an array of functions that can modify cell physiology and permit genetic mobility. At 148,711 bp, pSTU288-1 appears to be a hybrid plasmid containing a conglomerate of genes found in pSLT of S . Typhimurium LT2, coupled with a mosaic of horizontally-acquired elements. Class I integron containing gene cassettes conferring resistance against clinically important antibiotics and compounds are present in pSTU288-1. A curious feature of the plasmid involves the deletion of two genes encoded in the Salmonella plasmid virulence operon ( spvR and spvA ) following the insertion of a tnpA IS26-like element coupled to a bla TEM gene. The spv operon is considered to be a major plasmid-encoded Salmonella virulence factor that is essential for the intracellular lifecycle. The loss of the positive regulator SpvR may impact on the pathogenesis of S . Typhimurium U288. A second 11,067 bp plasmid designated pSTU288-2 contains further antibiotic resistance determinants, as well as replication and mobilization genes. Finally, a small 4675 bp plasmid pSTU288-3 was identified containing mobilization genes and a pleD -like G-G-D/E-E-F conserved domain protein that modulate intracellular levels of cyclic di-GMP, and are associated with motile to sessile transitions in growth. [ABSTRACT FROM AUTHOR]

The ica operon and aap gene are important factors for Staphylococcus epidermidis biofilm formation. However, we found 15 out of 101 S. epidermidis strains isolated from patients had both the ica operon and the aap gene in the genome but could not form biofilms (ica + aap + /BF − isolates). Compared with standard strain RP62A, the 15 ica + aap + /BF − isolates had similar growth curves and initial attachment abilities, but had much lower apparent transcription levels of the icaA gene and significantly less production of polysaccharide intercellular adhesion (PIA). Furthermore, the expression of accumulation-associated protein in ica + aap + /BF − isolates was much weaker than in RP62A. The mRNA levels of icaADBC transcription-related regulatory genes, including icaR , sarA , rsbU , srrA , arlRS and luxS , were measured in the 15 ica + aap + /BF − clinical isolates. The mRNA levels of arlR and rsbU in all of the ica + aap + /BF − isolates were lower than in RP62A at 4 h. At 10 h, 14/15 of the isolates showed lower mRNA levels of arlR and rsbU than shown by RP62A. However, expression of sarA , luxS , srrA and icaR varied in different ica + aap + /BF − isolates. To further investigate the role of arlRS in biofilm formation, we analyzed icaA , sarA and rsbU transcription, PIA synthesis, Aap expression and biofilm formation in an arlRS deletion mutant of S. epidermidis strain 1457 and all were much less than in the wild type strain. This is consistent with the hypothesis that ArlRS may play an important role in regulating biofilm formation by the ica + aap + /BF − S. epidermidis clinical isolates and operate via both ica-dependent and Aap-dependent pathways. [ABSTRACT FROM AUTHOR]

Abstract: Using a computational pipeline based on similarity networks reconstruction we analysed the 1133 genes of the Burkholderia vietnamiensis (Bv) G4 five plasmids, showing that gene and operon duplication played an important role in shaping the plasmid architecture. Several single/multiple duplications occurring at intra- and/or interplasmids level involving 253 paralogous genes (stand-alone, clustered or operons) were detected. An extensive gene/operon exchange between plasmids and chromosomes was also disclosed. The larger the plasmid, the higher the number and size of paralogous fragments. Many paralogs encoded mobile genetic elements and duplicated very recently, suggesting that the rearrangement of the Bv plastic genome is ongoing. Concerning the “molecular habitat” and the “taxonomical status” (the Preferential Organismal Sharing) of Bv plasmid genes, most of them have been exchanged with other plasmids of bacteria belonging (or phylogenetically very close) to Burkholderia, suggesting that taxonomical proximity of bacterial strains is a crucial issue in plasmid-mediated gene exchange. [Copyright &y& Elsevier]

Abstract: Toxin–antitoxin systems are widely distributed among many bacterial species, including human pathogens. Typically, these systems consist of two genes in an operon which encodes a stable toxin disrupting essential cellular processes and a labile antitoxin preventing toxicity. Regulation of type II TA system in which both components are proteins, relies on proteolysis. In this paper, we outline the significant features of antitoxin proteins important for proteolysis. We present examples of best known processes of antitoxin degradation by specific proteases mainly in Escherichia coli, but are also included intensively studied systems from other bacteria. The effect of environmental conditions on regulation and activity of TA systems and on consequences of proteolytic activity are discussed. [Copyright &y& Elsevier]

Abstract: The key components of the major secretion pathway in bacteria, the Sec pathway, are the proteins SecA, an ATPase that generates the energy required for protein translocation, and the heterotrimeric protein complex SecYEG, which functions as the preprotein-conducting channel through the cytoplasmic membrane, named translocon. Overexpression of exoproteins can cause jamming of the membrane, e.g., due to a shortage of translocons. Therefore, we decided to increase the number of translocons by first creating an artificial secYEG operon and then fusing it to an inducible promoter. By Western- and Northern-blot analysis, we could first show that the amount of the SecY protein and the secYEG transcript can be increased after addition of the inducer. Next, we proved by immunoblot experiments that the amount of α-amylase secreted in the presence of increased amounts of SecYEG proteins is enhanced. Therefore, increasing the number of translocons is accompanied by a concomitant increase in the amount exoenzymes. This finding will be of importance for high-level secretion of recombinant proteins. [Copyright &y& Elsevier]

Abstract: Studies on some purple photosynthetic bacteria have shown that the genes required for photosynthesis form a large cluster. This is often called a photosynthesis gene cluster (PGC) and has been described as a unique feature of purple photosynthetic bacteria. Here, photosynthesis genes were retrieved from the rapidly increasing genomic data of purple bacteria. Arrangements of these genes on the genomes were compared among 24 species and strains were selected from the wide distribution of alpha, beta, and gamma classes of purple bacteria. The presence of PGC was confirmed in the all of these, although it is divided into two or several islands in some species. Gene arrangements in several subclusters possibly forming an operon or a superoperon are well conserved while the directions and locations of these subclusters in the PGC vary according to the strains. Phylogenetic trees based on the sequences of the suspected products of the genes in the PGCs consistently place the species of the beta and gamma classes in a cluster within the branch of the alpha class, showing that a horizontal transfer of PGC had occurred in the process of evolution. Information obtained from the increasing genome projects on bacteria is very useful for the study on evolution of photosynthesis and even for its application in metabolic engineering. [Copyright &y& Elsevier]

Abstract: The Bacteroides conjugative transposon, CTnDOT, is an integrated conjugative element (ICE), found in many human colonic Bacteroides spp. strains. It has a complex regulatory system for both excision from the chromosome and transfer and mobilization into a new host. It was previously shown that a cloned DNA segment encoding the xis2c, xis2d, orf3, and exc genes was required for tetracycline dependent activation of the Ptra promoter. The Xis2c and Xis2d proteins are required for excision while the Exc protein stimulates excision. We report here that neither the Orf3 nor the Exc proteins are involved in activation of the Ptra promoter. Deletion analysis and electromobility shift assays showed that the Xis2c and Xis2d proteins bind to the Ptra promoter to activate the tra operon. Thus, the recombination directionality factors of CTnDOT excision also function as activator proteins of the Ptra promoter. [Copyright &y& Elsevier]