Distribution and molecular evolution of the anti-CRISPR family AcrIF7.

Anti-clustered regularly interspaced short palindromic repeats (CRISPRs) are proteins capable of blocking CRISPR-Cas systems and typically their genes are located on mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPR's function and evolution. Here, we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered 5 subclusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological, and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families.

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.

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.

A Novel Group of Promiscuous Podophages Infecting Diverse Gammaproteobacteria from River Communities Exhibits Dynamic Intergenus Host Adaptation.

Phages are generally described as species specific or even strain specific, implying an inherent limitation for some to be maintained and spread in diverse bacterial communities. Moreover, phage isolation and host range determination rarely consider the phage ecological context, likely biasing our notion on phage specificity. Here we isolated and characterized a novel group of six promiscuous phages, named Atoyac, existing in rivers and sewage by using a diverse collection of over 600 bacteria retrieved from the same environments as potential hosts. These podophages isolated from different regions in Mexico display a remarkably broad host range, infecting bacteria from six genera: Aeromonas, Pseudomonas, Yersinia, Hafnia, Escherichia, and Serratia Atoyac phage genomes are ∼42 kb long and highly similar to each other, but not to those currently available in genome and metagenome public databases. Detailed comparison of the phages' efficiency of plating (EOP) revealed variation among bacterial genera, implying a cost associated with infection of distant hosts, and between phages, despite their sequence similarity. We show, through experimental evolution in single or alternate hosts of different genera, that efficiency of plaque production is highly dynamic and tends toward optimization in hosts rendering low plaque formation. However, adaptation to distinct hosts differed between similar phages; whereas one phage optimized its EOP in all tested hosts, the other reduced plaque production in one host, suggesting that propagation in multiple bacteria may be key to maintain promiscuity in some viruses. Our study expands our knowledge of the virosphere and uncovers bacterium-phage interactions overlooked in natural systems.IMPORTANCE In natural environments, phages coexist and interact with a broad variety of bacteria, posing a conundrum for narrow-host-range phage maintenance in diverse communities. This context is rarely considered in the study of host-phage interactions, typically focused on narrow-host-range viruses and their infectivity in target bacteria isolated from sources distinct to where the phages were retrieved from. By studying phage-host interactions in bacteria and viruses isolated from river microbial communities, we show that novel phages with promiscuous host range encompassing multiple bacterial genera can be found in the environment. Assessment of hundreds of interactions in diverse hosts revealed that similar phages exhibit different infection efficiency and adaptation patterns. Understanding host range is fundamental in our knowledge of bacterium-phage interactions and their impact on microbial communities. The dynamic nature of phage promiscuity revealed in our study has implications in different aspects of phage research such as horizontal gene transfer or phage therapy.

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.

Domains two and three of Escherichia coli ribosomal S1 protein confers 30S subunits a high affinity for downstream A/U-rich mRNAs.

S1, a multi-domain ribosomal protein associated with the 30S subunit, is essential for translation initiation. S1 binds with high affinity to single-stranded mRNA containing A/U-rich patches upstream of the start codon. It was previously reported that domains 1-3 of S1 protein play a role in the docking and unfolding of structured mRNAs to the ribosome. Moreover, S1-deficient 30S subunits are still able to bind to low structured mRNAs. However, mRNAs containing A/U-rich patches in the early base positions after start codon enhance protein synthesis and mRNA binding to the ribosome, which suggests that S1 is also able to interact with these A/U-rich regions. To evaluate the essentiality of S1 domains in the binding to low structured mRNAs containing A/U/G nucleotides after the start codon as well as their role in translation and cell viability, S1 protein deletion variants were generated. We show that S1 domain 3 is necessary to discriminate these mRNAs according to the nucleotide nature since its absence abrogated S1 binding to A/U-rich mRNAs and allowed binding to G-rich mRNAs. Interestingly, domains 2 and 3 were required for the binding of mRNAs containing A/U-rich sequences after the start codon to 30S, in vitro translation and cell viability.

Cor interacts with outer membrane proteins to exclude FhuA-dependent phages.

Superinfection exclusion (Sie) of FhuA-dependent phages is carried out by Cor in the Escherichia coli mEp167 prophage lysogenic strain. In this work, we present evidence that Cor is an outer membrane (OM) lipoprotein that requires the participation of additional outer membrane proteins (OMPs) to exclude FhuA-dependent phages. Two Cor species of ~13 and ~8.5 kDa, corresponding to the preprolipoprotein/prolipoprotein and lipoprotein, were observed by Western blot. Cell mutants for CorC17F, CorA18D and CorA57E lost the Sie phenotype for FhuA-dependent phages. A copurification affinity binding assay combined with LC_ESI_MS/MS showed that Cor bound to OMPs: OmpA, OmpC, OmpF, OmpW, LamB, and Slp. Interestingly, Sie for FhuA-dependent phages was reduced on Cor overexpressing FhuA+ mutant strains, where ompA, ompC, ompF, ompW, lamB, fhuE, genes were knocked out. The exclusion was restored when these strains were supplemented with plasmids expressing these genes. Sie was not lost in other Cor overexpressing FhuA+ null mutant strains JW3938(btuB-), JW5100(tolB-), JW3474(slp-). These results indicate that Cor interacts and requires some OMPs to exclude FhuA-dependent phages.

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.

Core and accessory genome architecture in a group of Pseudomonas aeruginosa Mu-like phages.

BACKGROUND: Bacteriophages that infect the opportunistic pathogen Pseudomonas aeruginosa have been classified into several groups. One of them, which includes temperate phage particles with icosahedral heads and long flexible tails, bears genomes whose architecture and replication mechanism, but not their nucleotide sequences, are like those of coliphage Mu. By comparing the genomic sequences of this group of P. aeruginosa phages one could draw conclusions about their ontogeny and evolution. RESULTS: Two newly isolated Mu-like phages of P. aeruginosa are described and their genomes sequenced and compared with those available in the public data banks. The genome sequences of the two phages are similar to each other and to those of a group of P. aeruginosa transposable phages. Comparing twelve of these genomes revealed a common genomic architecture in the group. Each phage genome had numerous genes with homologues in all the other genomes and a set of variable genes specific for each genome. The first group, which comprised most of the genes with assigned functions, was named "core genome", and the second group, containing mostly short ORFs without assigned functions was called "accessory genome". Like in other phage groups, variable genes are confined to specific regions in the genome. CONCLUSION: Based on the known and inferred functions for some of the variable genes of the phages analyzed here, they appear to confer selective advantages for the phage survival under particular host conditions. We speculate that phages have developed a mechanism for horizontally acquiring genes to incorporate them at specific loci in the genome that help phage adaptation to the selective pressures imposed by the host.

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.