Early antitermination in the atypical coliphage mEp021 mediated by the Gp17 protein.

The coliphage mEp021 belongs to a phage group with a unique immunity repressor, and its life cycle requires the host factor Nus. mEp021 has been classified as non-lambdoid based on its specific characteristics. The mEp021 genome carries a gene encoding an Nλ-like antiterminator protein, termed Gp17, and three nut sites (nutL, nutR1, and nutR2). Analysis of plasmid constructs containing these nut sites, a transcription terminator, and a GFP reporter gene showed high levels of fluorescence when Gp17 was expressed, but not in its absence. Like lambdoid N proteins, Gp17 has an arginine-rich motif (ARM), and mutations in its arginine codons inhibit its function. In infection assays using the mutant phage mEp021ΔGp17::Kan (where gp17 has been deleted), gene transcripts located downstream of transcription terminators were obtained only when Gp17 was expressed. In contrast to phage lambda, mEp021 virus particle production was partially restored (>1/3 relative to wild type) when nus mutants (nusA1, nusB5, nusC60, and nusE71) were infected with mEp021 and Gp17 was overexpressed. Our results suggest that RNA polymerase reads through the third nut site (nutR2), which is more than 7.9 kbp downstream of nutR1.

Transcriptional analysis in bacteriophage Fc02 of Pseudomonas aeruginosa revealed two overlapping genes with exclusion activity.

Little is known about the gene expression program during the transition from lysogenic to lytic cycles of temperate bacteriophages in Pseudomonas aeruginosa. To investigate this issue, we developed a thermo-sensitive repressor mutant in a lysogen and analyzed the phage transcriptional program by strand-specific RNA-Seq before and after thermo-induction. As expected, the repressor gene located on the phage DNA forward strand is transcribed in the lysogen at the permissive temperature of 30°C. Upstream the repressor gene, we noticed the presence of two overlapped ORFs apparently in the same transcript. One ORF is a gene that encodes a protein of 7.9 kDa mediating the exclusion of various super-infecting phages. The other ORF, placed in an alternate reading frame with a possible AUG initiation codon at 25 nucleotide downstream of the AUG of the first gene, is expected to encode a 20.7 kDa polypeptide of yet an unknown function. Upon lifting repression at 40°C, the transcription of an operon which is involved in the lytic cycle is started from a promoter on the reverse phage DNA strand. The first gene in the operon is a homolog of the antirepresor ner, a common gene in the lysis-lysogeny regulation region of other phages. Interestingly, the next gene after ner is gene 10 that on the reverse strand overlaps the overlapped gene olg1 on the forward strand. Curiously, gene 10 expression also shows superinfection exclusion. Strand-specific RNA-Seq also has uncovered the transcription succession of gene modules expressed during the phage lytic stage. The conservation of overlapped genes with similar functions may be evolutionarily selected.

The 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.

Selection of Functional Quorum Sensing Systems by Lysogenic Bacteriophages in Pseudomonas aeruginosa.

Quorum sensing (QS) in Pseudomonas aeruginosa coordinates the expression of virulence factors, some of which are used as public goods. Since their production is a cooperative behavior, it is susceptible to social cheating in which non-cooperative QS deficient mutants use the resources without investing in their production. Nevertheless, functional QS systems are abundant; hence, mechanisms regulating the amount of cheating should exist. Evidence that demonstrates a tight relationship between QS and the susceptibility of bacteria against the attack of lytic phages is increasing; nevertheless, the relationship between temperate phages and QS has been much less explored. Therefore, in this work, we studied the effects of having a functional QS system on the susceptibility to temperate bacteriophages and how this affects the bacterial and phage dynamics. We find that both experimentally and using mathematical models, that the lysogenic bacteriophages D3112 and JBD30 select QS-proficient P. aeruginosa phenotypes as compared to the QS-deficient mutants during competition experiments with mixed strain populations in vitro and in vivo in Galleria mellonella, in spite of the fact that both phages replicate better in the wild-type background. We show that this phenomenon restricts social cheating, and we propose that temperate phages may constitute an important selective pressure toward the conservation of bacterial QS.

Comparative genomic analysis of Pseudomonas aeruginosa phage PaMx25 reveals a novel siphovirus group related to phages infecting hosts of different taxonomic classes.

Bacteriophages (phages) are estimated to be the most abundant and diverse entities in the biosphere harboring vast amounts of novel genetic information. Despite the genetic diversity observed, many phages share common features, such as virion morphology, genome size and organization, and can readily be associated with clearly defined phage groups. However, other phages display unique genomes or, alternatively, mosaic genomes composed of regions that share homology with those of phages of diverse origins; thus, their relationships cannot be easily assessed. In this work, we present a functional and comparative genomic analysis of Pseudomonas aeruginosa phage PaMx25, a virulent member of the Siphoviridae family. The genomes of PaMx25 and a highly homologous phage NP1, bore sequence homology and synteny with the genomes of phages that infect hosts different than Pseudomonas. In order to understand the relationship of the PaMx25 genome with that of other phages, we employed several computational approaches. We found that PaMx25 and NP1 effectively bridged several phage groups. It is expected that as more phage genomes become available, more gaps will be filled, blurring the boundaries that currently separate phage groups.

Genomic and Transcriptional Mapping of PaMx41, Archetype of a New Lineage of Bacteriophages Infecting Pseudomonas aeruginosa.

Previously, a collection of virulent phages infecting Pseudomonas aeruginosa was isolated from open water reservoirs and residual waters. Here, we described the comparative genomics of a set of five related phages from the collection, the physical structure of the genome, the structural proteomics of the virion, and the transcriptional program of archetypal phage PaMx41. The phage genomes were closely associated with each other and with those of two other P. aeruginosa phages, 119X and PaP2, which were previously filed in the databases. Overall, the genomes were approximately 43 kb, harboring 53 conserved open reading frames (ORFs) and three short ORFs in indel regions and containing 45% GC content. The genome of PaMx41 was further characterized as a linear, terminally redundant DNA molecule. A total of 16 ORFs were associated with putative functions, including nucleic acid metabolism, morphogenesis, and lysis, and eight virion proteins were identified through mass spectrometry. However, the coding sequences without assigned functions represent 70% of the ORFs. The PaMx41 transcription program was organized in early, middle, and late expressed genomic modules, which correlated with regions containing functionally related genes. The high genomic conservation among these distantly isolated phages suggests that these viruses undergo selective pressure to remain unchanged. The 119X lineage represents a unique set of phages that corresponds to a novel phage group. The features recognized in the genomes and the broad host range of clinical strains suggest that these phages are candidates for therapy applications. IMPORTANCE: Pseudomonas aeruginosa is an opportunistic pathogen that causes stubborn nosocomial infections that are frequently resistant to multiple antibiotics. Bacterial viruses (bacteriophages or phages) represent a natural mechanism for pathogenic bacterial control. Here, a group of virulent phages, previously shown to infect a broad range of clinical P. aeruginosa strains, was characterized at the genomic and molecular levels. These phages belong to a unique and tightly related group. In addition, we conducted a transcriptional study of an archetypal phage of this group to characterize the role of many unknown coding sequences based on expression temporalities. These results contribute to our knowledge of 119X-like phages and, in general, provide information concerning P. aeruginosa podophage diversity and lytic cycles.

New insights into the interaction between the quorum-sensing receptor NprR and its DNA target, or the response regulator Spo0F.

The NprR protein and NprRB signaling peptide comprise a bifunctional quorum-sensing system from the Bacillus cereus group that is involved in transcriptional activation through DNA-binding and in sporulation initiation by binding to Spo0F. We characterized in vitro the direct interactions established by NprR that may be relevant for performing its two functions. Apo-NprR interacted with Spo0F, but not with the target DNA. The NprRB signaling peptide SSKPDIVG that binds strongly to Apo-NprR, failed to bind and disrupt the NprR-Spo0F complex. Finally, the NprR-NprRB complex bound both to Spo0F and the target DNA with similar affinity. Based on our findings, we propose that rather than a switch triggered by NprRB, the NprR/NprRB ratio and the availability of Spo0F binding sites define the function of NprR.

Regulation of sporulation initiation by NprR and its signaling peptide NprRB: molecular recognition and conformational changes.

NprR belongs to the RNPP family of quorum-sensing receptors, a group of intracellular regulators activated directly by signaling oligopeptides in Gram-positive bacteria. In Bacillus thuringiensis (Bt), nprR is located in a transcriptional cassette with nprRB that codes for the precursor of the signaling peptide NprRB. NprR is a transcriptional regulator activated by binding of reimported NprRB; however, several reports suggest that NprR also participates in sporulation but the mechanism is unknown. Our in silico results, based on the structural similarity between NprR from Bt and Spo0F-binding Rap proteins from Bacillus subtilis, suggested that NprR could bind Spo0F to modulate the sporulation phosphorelay in Bt. Deletion of nprR-nprRB cassette from Bt caused a delay in sporulation and defective trigger of the Spo0A∼P-activated genes spoIIA and spoIIIG. The DNA-binding domain of NprR was not necessary for this second function, since truncated NprRΔHTH together with nprRB gene was able to restore the sporulation wild type phenotype in the ΔnprR-nprRB mutant. Fluorescence assays showed direct binding between NprR and Spo0F, supporting that NprR is a bifunctional protein. To understand how the NprR activation by NprRB could result in two different functions, we studied the molecular recognition mechanism between the signaling peptide and the receptor. Using synthetic variants of NprRB, we found that SSKPDIVG displayed the highest affinity (Kd = 7.19 nM) toward the recombinant NprR and demonstrated that recognition involves conformational selection. We propose that the peptide concentration in the cell controls the oligomerization state of the NprR-NprRB complex for switching between its two functions.

The transcription factors Sox5 and Sox9 regulate Catsper1 gene expression.

Catsper is a Ca(2+)permeable channel required for sperm hyperactivation. In spite of its central role in male fertility, the transcriptional mechanisms that regulate Catsper1 expression are ill defined. In this work, we describe the identification and characterization of important regulatory elements in the murine Catsper1 gene proximal promoter. Four transcription start sites and three functional Sox-binding sites were identified in the Catsper1 promoter. Interestingly, transcription factors Sox5 and Sox9 caused a significant increase in transactivation of the Catsper1 promoter in heterologous systems, and chromatin immunoprecipitation assays showed that both transcription factors interact with the Catsper1 promoter in vivo. These results provide new insights into the molecular mechanisms that control Catsper channel expression.

Narrow-host-range bacteriophages that infect Rhizobium etli associate with distinct genomic types.

In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ≈ 45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species.

High diversity and novel species of Pseudomonas aeruginosa bacteriophages.

The diversity of Pseudomonas aeruginosa bacteriophages was investigated using a collection of 68 phages isolated from Central Mexico. Most of the phages carried double-stranded DNA (dsDNA) genomes and were classified into 12 species. Comparison of the genomes of selected archetypal phages with extant sequences in GenBank resulted in the identification of six novel species. This finding increased the group diversity by ~30%. The great diversity of phage species could be related to the ubiquitous nature of P. aeruginosa.

Comparative genomic analysis of two brucellaphages of distant origins.

Here, we present the first complete genome sequence of brucellaphage Tbilisi (Tb) and compared it with that of Pr, a broad host-range brucellaphage recently isolated in Mexico. The genomes consist of 41,148 bp (Tb) and 38,253 bp (Pr), they differ mainly in the region encoding structural proteins, in which the genome of Tb shows two major insertions. Both genomes share 99.87% nucleotide identity, a high percentage of identity among phages isolated at so globally distant locations and temporally different occasions. Sequence analysis revealed 57 conserved ORFs, three transcriptional terminators and four putative transcriptional promoters. The co-occurrence of an ORF encoding a putative DnaA-like protein and a putative oriC-like origin of replication was found in both brucellaphages genomes, a feature not described in any other phage genome. These elements suggest that DNA replication in brucellaphages differs from other phages, and might resemble that of bacterial chromosomes.

Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons.

During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (ß-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.

Evolution and some functions of the NprR-NprRB quorum-sensing system in the Bacillus cereus group.

Quorum-sensing (QS) is a bacterial mechanism for regulation of gene expression in response to cell density. In Gram-positive bacteria, oligopeptides are the signaling molecules to elicit QS. The RNPP protein family (Rap, NprR, PlcR, and PrgX) are intracellular QS receptors that bind directly to their specific signaling peptide for regulating the transcription of several genes. NprR is the activator of a neutral protease in Bacillus subtilis, and it has been recently related to sporulation, cry genes transcription and extracellular protease activity in strains from the B. cereus group. In the B. thuringiensis genome, downstream nprR, a gene encoding a putative QS signaling propeptide (nprRB) was found. We hypothesized that the nprR and nprRB co-evolved because of their coordinated function in the B. cereus group. A phylogenetic tree of nucleotide sequences of nprR revealed six pherotypes, each corresponding to one putative mature NprRB sequence. The nprR tree does not match the current taxonomic grouping of the B. cereus group or the phylogenetic arrangement obtained when using MLST markers from the same strains. SKPDI and other synthetic peptides encoded in the nprRB gene from B. thuringiensis serovar thuringiensis strain 8741 had effect on temporal regulation of sporulation and expression of a cry1Aa'Z transcriptional fusion, but those peptides that stimulated earlier detection of spores decreased cry1Aa expression suggesting that NprR may either activate or repress the transcription of different genes.

Analysis of some phenotypic traits of feces-borne temperate lambdoid bacteriophages from different immunity groups: a high incidence of cor+, FhuA-dependent phages.

A group of previously isolated heterogeneous mEp lambdoid phages (43) from 19 different immunity groups for phage infection was further characterized to gain insight into some phenotypic traits and to assess their relationship with phage lambda. Interestingly, the FhuA host receptor was required by the majority of mEp phages (37 out of 43; approximately 85%). The cor gene, which has been reported to be involved in FhuA-dependent exclusion of lambdoid phages, was also found in most of the FhuA-dependent phages. Accordingly, no cor amplification by PCR was obtained among the six FhuA-independent mEp lambdoid phages. In contrast, it was found that around 25% of the population (10 out of 43 phages) required the specific and essential lambda N antitermination function, and the lambda site-specific DNA recombination function was observed only in two members (4.6%). Thus, a larger proportion of phages require the FhuA receptor for infection, and this is frequently correlated with the cor gene.

Codon-specific and general inhibition of protein synthesis by the tRNA-sequestering minigenes.

The expression of minigenes in bacteria inhibits protein synthesis and cell growth. Presumably, the translating ribosomes, harboring the peptides as peptidyl-tRNAs, pause at the last sense codon of the minigene directed mRNAs. Eventually, the peptidyl-tRNAs drop off and, under limiting activity of peptidyl-tRNA hydrolase, accumulate in the cells reducing the concentration of specific aminoacylable tRNA. Therefore, the extent of inhibition is associated with the rate of starvation for a specific tRNA. Here, we used minigenes harboring various last sense codons that sequester specific tRNAs with different efficiency, to inhibit the translation of reporter genes containing, or not, these codons. A prompt inhibition of the protein synthesis directed by genes containing the codons starved for their cognate tRNA (hungry codons) was observed. However, a non-specific in vitro inhibition of protein synthesis, irrespective of the codon composition of the gene, was also evident. The degree of inhibition correlated directly with the number of hungry codons in the gene. Furthermore, a tRNA(Arg4)-sequestering minigene promoted the production of an incomplete beta-galactosidase polypeptide interrupted, during bacterial polypeptide chain elongation at sites where AGA codons were inserted in the lacZ gene suggesting ribosome pausing at the hungry codons.

Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development.

Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth). Single-base-pair mutations (bar(-)) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells). When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering tRNA(2)(Ile) as pep-tRNA(2)(Ile). Either tRNA(2)(Ile) or Pth may reverse these effects. In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester tRNA(2)(Ile) as pep-tRNA(2)(Ile). In addition, overexpression of the barI minigene impairs the development even of bar(-) phages in rap cells. Interestingly, tRNA or Pth may reestablish lambda phage development. These results suggest that lambda bar minigenes are expressed and tRNA(2)(Ile) is sequestered as pep-tRNA(2)(Ile) during lambda phage development.

Distribution of minigenes in the bacteriophage lambda chromosome.

The bar loci in the chromosome of bacteriophage lambda inhibit phage vegetative growth in bacteria defective for peptidyl-tRNA hydrolase (Pth). Expression of the bar regions results in accumulation of peptidyl-tRNA, inhibition of protein synthesis, and arrest of mutant cell growth. These effects have been ascribed to the expression of two-codon ORFs present in translatable sequences named 'minigenes' in the lambda bar regions. To investigate the nature, frequency, and distribution of minigenes in the phage genome, we conducted a survey of their location in lambda DNA. A short-fragment random genomic DNA library was constructed for the identification of clones inhibitory of Pth-defective cells (bar-like phenotype). Three new bar-like minigenes were identified in the library but only one was on the sense strand and it had a rare initiation codon. This result contrasted with the in silico identification of over a hundred putative minigenes using an ad hoc computer program on both strands of lambda DNA. Unlike bar constructs, most of the toxic constructed clones were also toxic to wild-type bacteria, thus suggesting a different inhibition mechanism. Sequence analysis of these cloned inserts showed that they harbored minigenes, mini-ORFs, gene starts, gene ends, or combinations thereof. Our data suggest that minigene-like sequences may, at least partly, account for toxicity in wild-type cells. We propose that clustering of minigenes at gene ends may play a role in gene expression. Other minigenes identified in silico were non-toxic. It is still an open question what the in vivo function of these and toxic minigenes might be.

The pair of arginine codons AGA AGG close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating peptidyl-tRNAArg4.

To analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives. The lambda int gene has a high frequency of rare ATA, AGA and AGG codons; two of them (AGA AGG) located at positions 3 and 4 of the int open reading frame (ORF). Escherichia coli pth (rap) cells, which are defective in peptidyl-tRNA hydrolase (Pth) activity, are more susceptible to the inhibitory effects of int expression as compared with wild-type cells. Cell growth and Int protein synthesis were enhanced by overexpression of Pth and tRNAArg4 cognate to AGG and AGA but not of tRNAIle2a specific for ATA. The increase of Int protein synthesis also takes place when the rare arginine codons AGA and AGG at positions 3 and 4 are changed to common arginine CGT or lysine AAA codons but not to rare isoleucine ATA codons. In addition, overexpression of int in Pth defective cells provokes accumulation of peptidyl-tRNAArg4 in the soluble fraction. Therefore, cell growth and Int synthesis inhibition may be due to ribosome stalling and premature release of peptidyl-tRNAArg4 from the ribosome at the rare arginine codons of the first tandem, which leads to cell starvation for the specific tRNA.