Stephen J. Poole

A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria

Bacteria have developed mechanisms to communicate and compete with one another in diverse environments. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA ‘exoprotein’ onto the cell surface. An additional small immunity protein (CdiI) protects CDI+ cells from autoinhibition. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI+ cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.

Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI+ cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These “orphan” cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems.

Isolation of a family of Drosophila POU domain genes expressed in early development.

The POU domain is a approximately 140 amino acid domain shared by several mammalian transcriptional activators. We have isolated genomic and cDNA clones from three Drosophila POU domain genes, designated pdm-1, pdm-2, and Cf1a. All three genes encode a conserved POU-specific domain and a POU homeo domain but are otherwise divergent. By Northern analysis, all three of the genes are expressed in early embryos; the pdm-1 and Cf1a genes are also expressed at lower levels throughout development. The spatial expression patterns of the pdm-2 and Cf1a genes show that they probably play multiple roles during development: an early role in specific ectodermal cells, and a subsequent role in the embryonic nervous system.

The toxin/immunity network of Burkholderia pseudomallei contact‐dependent growth inhibition (CDI) systems

Burkholderia pseudomallei is a category B pathogen and the causative agent of melioidosis – a serious infectious disease that is typically acquired directly from environmental reservoirs. Nearly all B. pseudomallei strains sequenced to date (> 85 isolates) contain gene clusters that are related to the contact‐dependent growth inhibition (CDI) systems of γ‐proteobacteria. CDI systems from Escherichia coli and Dickeya dadantii play significant roles in bacterial competition, suggesting these systems may also contribute to the competitive fitness of B. pseudomallei. Here, we identify 10 distinct CDI systems in B. pseudomallei based on polymorphisms within the cdiA‐CT/cdiI coding regions, which are predicted to encode CdiA‐CT/CdiI toxin/immunity protein pairs. Biochemical analysis of three B. pseudomallei CdiA‐CTs revealed that each protein possesses a distinct tRNase activity capable of inhibiting cell growth. These toxin activities are blocked by cognate CdiI immunity proteins, which specifically bind the CdiA‐CT and protect cells from growth inhibition. Using Burkholderia thailandensis E264 as a model, we show that a CDI system from B. pseudomallei 1026b mediates CDI and is capable of delivering CdiA‐CT toxins derived from other B. pseudomallei strains. These results demonstrate that Burkholderia species contain functional CDI systems, which may confer a competitive advantage to these bacteria.

Genetic modifiers of abnormal organelle biogenesis in a Drosophila model of BLOC-1 deficiency

Biogenesis of lysosome-related organelles complex 1 (BLOC-1) is a protein complex formed by the products of eight distinct genes. Loss-of-function mutations in two of these genes, DTNBP1 and BLOC1S3, cause Hermansky-Pudlak syndrome, a human disorder characterized by defective biogenesis of lysosome-related organelles. In addition, haplotype variants within the same two genes have been postulated to increase the risk of developing schizophrenia. However, the molecular function of BLOC-1 remains unknown. Here, we have generated a fly model of BLOC-1 deficiency. Mutant flies lacking the conserved Blos1 subunit displayed eye pigmentation defects due to abnormal pigment granules, which are lysosome-related organelles, as well as abnormal glutamatergic transmission and behavior. Epistatic analyses revealed that BLOC-1 function in pigment granule biogenesis requires the activities of BLOC-2 and a putative Rab guanine-nucleotide-exchange factor named Claret. The eye pigmentation phenotype was modified by misexpression of proteins involved in intracellular protein trafficking; in particular, the phenotype was partially ameliorated by Rab11 and strongly enhanced by the clathrin-disassembly factor, Auxilin. These observations validate Drosophila melanogaster as a powerful model for the study of BLOC-1 function and its interactions with modifier genes.

Mechanisms and Biological Roles of Contact-Dependent Growth Inhibition Systems

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. CDI(+) cells bind to susceptible target bacteria and deliver a toxic effector domain derived from the carboxyl terminus of CdiA (CdiA-CT). More than 60 distinct CdiA-CT sequence types have been identified, and all CDI toxins characterized thus far display RNase, DNase, or pore-forming activities. CDI systems also encode CdiI immunity proteins, which specifically bind and inactivate cognate CdiA-CT toxins to prevent autoinhibition. CDI activity appears to be limited to target cells of the same species, suggesting that these systems play a role in competition between closely related bacteria. Recent work on the CDI system from uropathogenic Escherichia coli (UPEC 536) has revealed that its CdiA-CT toxin binds tightly to a cysteine biosynthetic enzyme (CysK) in the cytoplasm of target cells. The unanticipated complexity in the UPEC CDI pathway raises the possibility that these systems perform other functions in addition to growth inhibition. Finally, we propose that the phenomenon of CDI is more widespread than previously appreciated. Rhs (rearrangement hotspot) systems encode toxin-immunity pairs, some of which share significant sequence identity with CdiA-CT/CdiI proteins. A number of recent observations suggest that Rhs proteins mediate a distinct form of CDI.

Selection of Orphan Rhs Toxin Expression in Evolved Salmonella enterica Serovar Typhimurium

Clonally derived bacterial populations exhibit significant genotypic and phenotypic diversity that contribute to fitness in rapidly changing environments. Here, we show that serial passage of Salmonella enterica serovar Typhimurium LT2 (StLT2) in broth, or within a mouse host, results in selection of an evolved population that inhibits the growth of ancestral cells by direct contact. Cells within each evolved population gain the ability to express and deploy a cryptic “orphan” toxin encoded within the rearrangement hotspot (rhs) locus. The Rhs orphan toxin is encoded by a gene fragment located downstream of the “main” rhs gene in the ancestral strain StLT2. The Rhs orphan coding sequence is linked to an immunity gene, which encodes an immunity protein that specifically blocks Rhs orphan toxin activity. Expression of the Rhs orphan immunity protein protects ancestral cells from the evolved lineages, indicating that orphan toxin activity is responsible for the observed growth inhibition. Because the Rhs orphan toxin is encoded by a fragmented reading frame, it lacks translation initiation and protein export signals. We provide evidence that evolved cells undergo recombination between the main rhs gene and the rhs orphan toxin gene fragment, yielding a fusion that enables expression and delivery of the orphan toxin. In this manner, rhs locus rearrangement provides a selective advantage to a subpopulation of cells. These observations suggest that rhs genes play important roles in intra-species competition and bacterial evolution.

Proteobacterial ArfA peptides are synthesized from non-stop messenger RNAs

Background: The Escherichia coli ArfA peptide mediates alternative ribosome rescue. Results: Proteobacterial arfA transcripts lack in-frame stop codons, leading to ssrA tagging and degradation of ArfA peptides. Conclusion: ArfA synthesis from non-stop mRNA is conserved, ensuring that alternative rescue is regulated by tmRNA. Significance: The alternative ribosome rescue pathway acts as a failsafe to the tmRNA system in bacteria that contain arfA homologues. The translation of non-stop mRNA (which lack in-frame stop codons) represents a significant quality control problem for all organisms. In eubacteria, the transfer-messenger RNA (tmRNA) system facilitates recycling of stalled ribosomes from non-stop mRNA in a process termed trans-translation or ribosome rescue. During rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which promotes polypeptide degradation after release from the stalled ribosome. Escherichia coli possesses an additional ribosome rescue pathway mediated by the ArfA peptide. The E. coli arfA message contains a hairpin structure that is cleaved by RNase III to produce a non-stop transcript. Therefore, ArfA levels are controlled by tmRNA through ssrA-peptide tagging and proteolysis. Here, we examine whether ArfA homologues from other bacteria are also regulated by RNase III and tmRNA. We searched 431 arfA coding sequences for mRNA secondary structures and found that 82.8% of the transcripts contain predicted hairpins in their 3′-coding regions. The arfA hairpins from Haemophilus influenzae, Proteus mirabilis, Vibrio fischeri, and Pasteurella multocida are all cleaved by RNase III as predicted, whereas the hairpin from Neisseria gonorrhoeae functions as an intrinsic transcription terminator to generate non-stop mRNA. Each ArfA homologue is ssrA-tagged and degraded when expressed in wild-type E. coli cells, but accumulates in mutants lacking tmRNA. Together, these findings show that ArfA synthesis from non-stop mRNA is a conserved mechanism to regulate the alternative ribosome rescue pathway. This strategy ensures that ArfA homologues are only deployed when the tmRNA system is incapacitated or overwhelmed by stalled ribosomes.

Mechanistic Model of Rothia mucilaginosa Adaptation toward Persistence in the CF Lung, Based on a Genome Reconstructed from Metagenomic Data

The impaired mucociliary clearance in individuals with Cystic Fibrosis (CF) enables opportunistic pathogens to colonize CF lungs. Here we show that Rothia mucilaginosa is a common CF opportunist that was present in 83% of our patient cohort, almost as prevalent as Pseudomonas aeruginosa (89%). Sequencing of lung microbial metagenomes identified unique R. mucilaginosa strains in each patient, presumably due to evolution within the lung. The de novo assembly of a near-complete R. mucilaginosa (CF1E) genome illuminated a number of potential physiological adaptations to the CF lung, including antibiotic resistance, utilization of extracellular lactate, and modification of the type I restriction-modification system. Metabolic characteristics predicted from the metagenomes suggested R. mucilaginosa have adapted to live within the microaerophilic surface of the mucus layer in CF lungs. The results also highlight the remarkable evolutionary and ecological similarities of many CF pathogens; further examination of these similarities has the potential to guide patient care and treatment.

Regulation of expression domains and effects of ectopic expression reveal gap gene-like properties of the linked pdm genes of Drosophila

The closely linked POU domain genes pdm-1 and pdm-2 are first expressed early during cellularization in the presumptive abdomen in a broad domain that soon resolves into two stripes. This expression pattern is regulated by the same mechanisms that define gap gene expression domains. The borders of pdm-1 expression are set by the terminal system genes torso and tailless, and the gradient morphogen encoded by hunchback. The resolution into two stripes is controlled by the gap gene knirps. Ectopic expression of pdm-1 at the cellular blastoderm stage leads to disruptions in pair rule gene expression and in anterior segmentation. The broad abdominal domain of pdm-1 protein is lacking in nanos- mutant embryos, and ectopic pdm-1 expression in nanos- embryos leads to a partial restoration of abdominal segmentation. These data suggest that the pdm genes may act in segmentation near the level of the zygotic gap genes.