Characterization of Linear Chromosome Segregation and Condensation of Streptomyces coelicolor

DNA segregation and condensation must occur accurately during cell division to ensure the survival of daughter cells. Most prokaryotes have a single, circular genome, which is simultaneously replicated, segregated and condensed during cell division. Streptomyces coelicolor is a sporulating, filamentous bacterium with a large, linear genome. Syncytial aerial hyphae contain numerous copies of the genome, which must be synchronously segregated into prespore compartments while up to 100 septa form. The proteins that control and accomplish this complicated process are of interest. Genetic analysis revealed that S. coelicolor can survive without three DNA segregation proteins, SMC, FtsK and ParB, which normally result in synthetic lethal phenotypes in unicellular bacteria. A ∆smc ∆ftsK ∆parB triple mutant was still able to segregate genetic material to 90% of its spores, but exhibited a fourfold decrease in viability when compared to wild type. For S. coelicolor, there must be considerable redundancy in genome segregation to overcome the loss of these genes. Furthermore, the large genome of S. coelicolor has to be properly condensed in order for it to fit inside a spore which is 1 μm in length. Genetic analysis of scpA and scpB, the gene products are thought to be involved in DNA condensation by interaction with SMC, revealed that neither were required for viability, but produced spores with a bilobed DNA architecture, unlike wild type or ∆smc mutant spores. It was concluded that this morphological phenotype was not the result of an interaction of the scpAB with smc, as the smc mutant did not present this phenotype. This bilobed nature of the scp mutants prompted the investigation of spore ploidy. Using several different but complementary methods, evidence was obtained suggesting that S. coelicolor spores and that of other species are diploid. A specific and dynamic movement of the origin of replication was discovered very late in spore development and a novel gene, parA2, was found to directly or indirectly play a role in this process. Together, these data reveal new information in order to better understand how linear chromosomal DNA is segregated, condensed and localized in this sporulating bacterium

Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease

Mycobacteriophage; Nontuberculous mycobacteria; Phage therapyMicobacteriófago; Micobacterias no tuberculosas; Terapia de fagosMicobacteriòfag; Micobacteris no tuberculosos; Teràpia de fagsBackground Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. Methods Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. Results No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. Conclusions Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections

Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes

Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous freestanding HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution

Comparative genomics of Cluster O mycobacteriophages

Mycobacteriophages - viruses of mycobacterial hosts - are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages - Corndog, Catdawg, Dylan, Firecracker, and YungJamal - designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange

Comparative Genomics of Cluster O Mycobacteriophages

Mycobacteriophages – viruses of mycobacterial hosts – are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages – Corndog, Catdawg, Dylan, Firecracker, and YungJamal – designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8–9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange

Phage Therapy of Mycobacterium Infections : Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease

Background Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. Methods Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. Results No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. Conclusions Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.We describe 20 consecutive cases of bacteriophage treatment of Mycobacterium infections. We observed no adverse reactions, favorable outcomes in at least 50% of patients, no evidence of phage resistance, and neutralizing immune reactions that do not correlate with treatment success.Peer reviewe

Genetic Interactions of smc, ftsK, and parB Genes in Streptomyces coelicolor and Their Developmental Genome Segregation Phenotypes▿ †

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK′ truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid

Expression and evolutionary patterns of mycobacteriophage D29 and its temperate close relatives

Abstract Background Mycobacteriophages are viruses that infect Mycobacterium hosts. A large collection of phages known to infect the same bacterial host strain – Mycobacterium smegmatis mc2155 – exhibit substantial diversity and characteristically mosaic architectures. The well-studied lytic mycobacteriophage D29 appears to be a deletion derivative of a putative temperate parent, although its parent has yet to be identified. Results Here we describe three newly-isolated temperate phages – Kerberos, Pomar16 and StarStuff – that are related to D29, and are predicted to be very close relatives of its putative temperate parent, revealing the repressor and additional genes that are lost in D29. Transcriptional profiles show the patterns of both lysogenic and lytic gene expression and identify highly-expressed, abundant, stable, small non-coding transcripts made from the Pleft early lytic promoter, and which are toxic to M. smegmatis. Conclusions Comparative genomics of phages D29, Kerberos, Pomar16 and StarStuff provide insights into bacteriophage evolution, and comparative transcriptomics identifies the pattern of lysogenic and lytic expression with unusual features including highly expressed, small, non-coding RNAs

Additional file 1: Table S1. of Expression and evolutionary patterns of mycobacteriophage D29 and its temperate close relatives

Insertions and deletions identified by Mauve whole genome alignment of four D29 relatives, indicating the evolutionary history and nucleotide size of the event. Table S2. Total number of single nucleotide polymorphisms (SNPs) for each pairwise comparison identified by Mauve whole genome alignment of four D29 relatives. Table S3. A comparison of the stoperators and their coordinates in each genome. Table S4. The percentage of RNAseq mapped reads of host vs. phage for the samples indicated on the left. Table S5. Oligonucleotides used in this paper to generate PCR fragments, plasmids, as well as Northern blot probes. Coordinates are identified for all Northern blot probes on the right. (DOCX 28 kb

Mycobacteriophage–antibiotic therapy promotes enhanced clearance of drug-resistant Mycobacterium abscessus

International audienceABSTRACT Infection by multidrug-resistant Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF) patients, leaving clinicians with few therapeutic options. A compassionate study showed the clinical improvement of a CF patient with a disseminated M. abscessus (GD01) infection, following injection of a phage cocktail, including phage Muddy. Broadening the use of phage therapy in patients as a potential antibacterial alternative necessitates the development of biological models to improve the reliability and successful prediction of phage therapy in the clinic. Herein, we demonstrate that Muddy very efficiently lyses GD01 in vitro, an effect substantially increased with standard drugs. Remarkably, this cooperative activity was retained in an M. abscessus model of infection in CFTR-depleted zebrafish, associated with a striking increase in larval survival and reduction in pathological signs. The activity of Muddy was lost in macrophage-ablated larvae, suggesting that successful phage therapy relies on functional innate immunity. CFTR-depleted zebrafish represent a practical model to rapidly assess phage treatment efficacy against M. abscessus isolates, allowing the identification of drug combinations accompanying phage therapy and treatment prediction in patients. This article has an associated First Person interview with the first author of the paper