Quorum‐sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA)

The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteriophage that packages almost random genomic DNA segments that may be transferred to other R. capsulatus cells. Gene transfer agents are produced by a number of prokaryotes; however, no receptors have been identified. We investigated the RcGTA recipient capability of wild‐type R. capsulatus cells at different culture growth phases, and found that the frequency of RcGTA‐dependent acquisition of an allele increases as cultures enter the stationary phase. We also found that RcGTA adsorption to cells follows a similar trend. RcGTA recipient capability and adsorption were found to be dependent on the GtaR/I quorum‐sensing (QS) system. Production of an extracellular polysaccharide was found to be regulated by GtaR/I QS, as was production of the cell capsule. A number of QS‐regulated putative polysaccharide biosynthesis genes were identified, and mutagenesis of two of these genes, rcc01081 and rcc01932, yielded strains that lack a capsule. Furthermore, these mutants were impaired in RcGTA recipient capability and adsorption, as was a non‐encapsulated wild‐type isolate of R. capsulatus. Overall, our results indicate that capsular polysaccharide is a receptor for the gene transfer agent of R. capsulatus, RcGTA.     

Importance of widespread gene transfer agent genes in alpha-proteobacteria

The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) is a model for several virus-like elements that seem to function solely for mediating gene exchange. Several genes that encode RcGTA are clearly related to bacteriophage genes but the cellular regulatory mechanisms that control RcGTA production indicate that RcGTA is more than just a defective prophage. Genome sequencing projects show that seemingly functional RcGTA-like structural gene clusters are present in many other species of alpha-proteobacteria, which might also produce RcGTA-like particles. Here, we use the genomic sequence data that are currently available to identify candidate GTA-producing species and propose an evolutionary scheme for RcGTA-like elements in the alpha-proteobacteria.     

One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus

The gene transfer agent (RcGTA) of Rhodobacter capsulatus is the model for a family of novel bacteriophage-related genetic elements that carry out lateral transfer of essentially random host DNA. Genuine and putative gene transfer agents have been discovered in diverse genera and are becoming recognized as potentially an important source of genetic exchange and microbial evolution in the oceans. Despite being discovered over 30 years ago, little is known about many essential aspects of RcGTA biology. Here, we validate the use of direct fluorescence reporter constructs, which express the red fluorescent protein mCherry in R. capsulatus. A construct containing the RcGTA promoter fused to mCherry was used to examine the single-cell expression profiles of wild type and RcGTA overproducer R. capsulatus populations, under different growth conditions and growth phases. The majority of RcGTA production clearly arises from a small, distinct sub-set of the population in the wild type strain and a larger sub-set in the overproducer. The most likely RcGTA release mechanism concomitant with this expression pattern is host cell lysis and we present direct evidence for the release of an intracellular enzyme accompanying RcGTA release. RcGTA ORF s is annotated as a 'cell wall peptidase' but we rule out a role in host lysis and propose an alternative function as a key contributor to RcGTA invasion of a target cell during infection.     

Rhodobacter capsulatus DprA is essential for RecA‐mediated gene transfer agent (RcGTA) recipient capability regulated by quorum‐sensing and the CtrA response regulator

Gene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best‐studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA‐mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA‐protecting protein A) homologue is essential for RcGTA‐mediated gene acquisition, and that dprA expression is induced by gtaI‐dependent quorum‐sensing and non‐phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C‐terminal region that resembles a dsDNA‐binding protein domain. Purified His‐tagged R. capsulatus DprA protein bound to both single‐stranded (ss)DNA and double‐stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single‐cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.     

Regulation of genes involved in nitrogen utilization on different C/N ratios and nitrogen sources in the model ectomycorrhizal fungus Hebeloma cylindrosporum

Nitrogen (N) utilization by ectomycorrhizal fungi is an essential aspect of their ecosystem function. N deposition changes both the N pools and the carbon/nitrogen (C/N) ratio of the substrates where ectomycorrhizal fungi are found, and it is important to understand how these changes affect the N forms used by ectomycorrhizal fungi. To overcome the difficulties of studying ectomycorrhizal fungi in situ, we investigated all known N genes in the model fungus, Hebeloma cylindrosporum in a culture study. In addition to studying the regulation of all known N utilization genes, we aimed to understand whether there are gene clusters that undergo similar regulation. Lastly we studied how C/N ratio, N transporter type, and N source affected relative gene expression levels. We grew the D2 strain of H. cylindrosporum on a range of inorganic and organic N sources under low, medium, and high C/N ratios. We found three gene clusters that were regulated in a similar pattern. Lastly, we found C/N ratio, N source and N transporter type all affected gene expression levels. Relative expression levels were highest on the high C/N ratio, BSA and diLeucine N sources, and inorganic N transporters were always expressed at higher levels than organic N transporters. These results suggest that inorganic N sources may always the default preference for H. cylindrosporum, regardless of both the N sources and the C/N ratio of the substrate.     

Molecular evolution and expression of the CRAL_TRIO protein family in insects

CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in Lepidoptera, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.     

Repurposing Hsp90 inhibitors as antibiotics targeting histidine kinases

To address the growing need for new antimicrobial agents, we explored whether inhibition of bacterial signaling machinery could inhibit bacterial growth. Because bacteria rely on two-component signaling systems to respond to environmental changes, and because these systems are both highly conserved and mediated by histidine kinases, inhibiting histidine kinases may provide broad spectrum antimicrobial activity. The histidine kinase ATP binding domain is conserved with the ATPase domain of eukaryotic Hsp90 molecular chaperones. To find a chemical scaffold for compounds that target histidine kinases, we leveraged this conservation. We screened ATP competitive Hsp90 inhibitors against CckA, an essential histidine kinase in Caulobacter crescentus that controls cell growth, and showed that the diaryl pyrazole is a promising scaffold for histidine kinase inhibition. We synthesized a panel of derivatives and found that they inhibit the histidine kinases C. crescentus CckA and Salmonella PhoQ but not C. crescentus DivJ; and they inhibit bacterial growth in both Gram-negative and Gram-positive bacterial strains.     

Valinomycin Biosynthetic Gene Cluster in Streptomyces: Conservation,Ecology and Evolution

Many Streptomyces strains are known to produce valinomycin (VLM) antibiotic and the VLM biosynthetic gene cluster (vlm) has been characterized in two independent isolates. Here we report the phylogenetic relationships of these strains using both parsimony and likelihood methods, and discuss whether the vlm gene cluster shows evidence of horizontal transmission common in natural product biosynthetic genes. Eight Streptomyces strains from around the world were obtained and sequenced for three regions of the two large nonribosomal peptide synthetase genes (vlm1 and vlm2) involved in VLM biosynthesis. The DNA sequences representing the vlm gene cluster are highly conserved among all eight environmental strains. The geographic distribution pattern of these strains and the strict congruence between the trees of the two vlm genes and the housekeeping genes, 16S rDNA and trpB, suggest vertical transmission of the vlm gene cluster in Streptomyces with no evidence of horizontal gene transfer. We also explored the relationship of the sequence of vlm genes to that of the cereulide biosynthetic genes (ces) found in Bacillus cereus and found them highly divergent from each other at DNA level (genetic distance values≥95.6%). It is possible that the vlm gene cluster and the ces gene cluster may share a relatively distant common ancestor but these two gene clusters have since evolved independently.