Fluorescence staining of live cyanobacterial cells suggest non-stringent chromosome segregation and absence of a connection between cytoplasmic and thylakoid membranes

<p>Abstract</p> <p>Background</p> <p>In spite of their abundance and importance, little is known about cyanobacterial cell biology and their cell cycle. During each cell cycle, chromosomes must be separated into future daughter cells, i.e. into both cell halves, which in many bacteria is achieved by an active machinery that operates during DNA replication. Many cyanobacteria contain multiple identical copies of the chromosome, but it is unknown how chromosomes are segregated into future daughter cells, and if an active or passive mechanism is operative. In addition to an outer and an inner cell membrane, cyanobacteria contain internal thylakoid membranes that carry the active photosynthetic machinery. It is unclear whether thylakoid membranes are invaginations of the inner cell membrane, or an independent membrane system.</p> <p>Results</p> <p>We have used different fluorescent dyes to study the organization of chromosomes and of cell and thylakoid membranes in live cyanobacterial cells. FM1-43 stained the outer and inner cytoplasmic membranes but did not enter the interior of the cell. In contrast, thylakoid membranes in unicellular <it>Synechocystis </it>cells became visible through a membrane-permeable stain only. Furthermore, continuous supply of the fluorescent dye FM1-43 resulted in the formation of one to four intracellular fluorescent structures in <it>Synechocystis </it>cells, within occurred within 30 to 60 minutes, and may represent membrane vesicles. Using fluorescent DNA stains, we found that <it>Synechocystis </it>genomic DNA is compacted in the cell centre that is devoid of thylakoid membranes. Nucleoids segregated very late in the cell cycle, just before complete closing of the division septum. In striking contrast to <it>Bacillus subtilis</it>, which possesses an active chromosome segregation machinery, fluorescence intensity of stained nucleoids differed considerably between the two <it>Synechocystis </it>daughter cells soon after cell division.</p> <p>Conclusion</p> <p>Our experiments strongly support the idea that the cytoplasmic and thylakoid membranes are not directly connected, but separate entities, in unicellular cyanobacteria. Our findings suggest that a transport system may exist between the cytoplasmic membrane and thylakoids, which could mediate the extension of thylakoid membranes and possibly also protein transport from the cytoplasmic membrane to thylakoid membranes. The cell cycle studies in <it>Synechocystis </it>sp. PCC 6803 show that the multiple chromosome copies per cell segregate very late in the cell cycle and in a much less stringent manner than in <it>B. subtilis </it>cells, indicating that chromosomes may become segregated randomly and in a passive fashion, possibly through constriction of the division septum.</p

Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon-limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light-dependent manner and may be required for processing the L11 r-operon and the SyR7 noncoding RNA, which is antisense to the murF 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ∼10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks

Characteristic of the syr-syp genomic island of Pseudomonas syringae pv.syringae strain B301D

Pseudomonas syringae pv. syringae produces two classes of pore-forming lipodepsipeptide phytotoxins that target host plasma membranes. Both syringomycin and syringopeptin are synthesised by modular nonribosomal peptide synthetases. The syringomycin (syr) and syringopeptin (syp) gene clusters are located adjacent to one another on the chromosome and are estimated to be 55 kb and 90 kb in size, respectively. Here we describe the functional organisation of the clusters, along with flanking DNA regions, which altogether cover an approximately 155-kb region of the genome. The predominant feature of the toxin clusters is the occurrence of peptide synthetase genes for syringomycin (i.e., syrB1 and syrE) and syringopeptin (i.e., sypA, sypB, and sypC). Associated with the toxin clusters are genes that are predicted to function in toxin secretion and regulation of biosynthesis genes. Progress in characterising functions of specific genes of the syr-syp cluster dedicated to syringomycin, syringopeptin, or both toxins is summarised

The contribution of syringopeptin and syringomycin to virulence of Pseudomonas syringae pv. syringae strain B301D on the basis of sypA and syrB1 biosynthesis mutant analysis

Sequencing of an approximately 3.9-kb fragment downstream of the syrD gene of Pseudomonas syringae pv. syringae strain B301D revealed that this region, designated sypA, codes for a peptide synthetase, a multifunctional enzyme involved in the thiotemplate mechanism of peptide biosynthesis. The translated protein sequence encompasses a complete amino acid activation module containing the conserved domains characteristic of peptide synthetases. Analysis of the substrate specificity region of this module indicates that it incorporates 2,3-dehydroaminobutyric acid into the syringopeptin peptide structure. Bioassay and high performance liquid chromatography data confirmed that disruption of the sypA gene in strain B301D resulted in the loss of syringopeptin production. The contribution of syringopeptin and syringomycin to the virulence of P. syringae pv. syringae strain B301D was examined in immature sweet cherry with sypA and syrB1 synthetase mutants defective in the production of the two toxins, respectively. Syringopeptin (sypA) and syringomycin (syrB1) mutants were reduced in virulence 59 and 26%, respectively, compared with the parental strain in cherry, whereas the syringopeptin–syringomycin double mutant was reduced 76% in virulence. These data demonstrate that syringopeptin and syringomycin are major virulence determinants of P. syringae pv. syringae

A physical map of the syringomycin and syringopeptin gene clusters localized to an approximately 145-kb DNA region of Pseudomonas syringae pv. syringae strain B301D

Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv. syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment. The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively. Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases. Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment. In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment. In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P. syringae pv. syringae strain B728a, was localized to the syr gene cluster. A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins. Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters. Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P. syringae pv. syringae that contributes to virulence in plant hosts

Hoyer, Arend: Was Musik andächtig macht : Drei Leipziger Kirchenkantaten Johann Sebastian Bachs, liturgiewissenschaftlich unter die Lupe genommen. Zürich, 2018

<div><p>The CRISPR-Cas (Clustered Regularly Interspaced Short Palindrome Repeats – CRISPR associated proteins) system provides adaptive immunity in archaea and bacteria. A hallmark of CRISPR-Cas is the involvement of short crRNAs that guide associated proteins in the destruction of invading DNA or RNA. We present three fundamentally distinct processing pathways in the cyanobacterium <i>Synechocystis</i> sp. PCC6803 for a subtype I-D (CRISPR1), and two type III systems (CRISPR2 and CRISPR3), which are located together on the plasmid pSYSA. Using high-throughput transcriptome analyses and assays of transcript accumulation we found all CRISPR loci to be highly expressed, but the individual crRNAs had profoundly varying abundances despite single transcription start sites for each array. In a computational analysis, CRISPR3 spacers with stable secondary structures displayed a greater ratio of degradation products. These structures might interfere with the loading of the crRNAs into RNP complexes, explaining the varying abundancies. The maturation of CRISPR1 and CRISPR2 transcripts depends on at least two different Cas6 proteins. Mutation of gene <i>sll7090</i>, encoding a Cmr2 protein led to the disappearance of all CRISPR3-derived crRNAs, providing <i>in vivo</i> evidence for a function of Cmr2 in the maturation, regulation of expression, Cmr complex formation or stabilization of CRISPR3 transcripts. Finally, we optimized CRISPR repeat structure prediction and the results indicate that the spacer context can influence individual repeat structures.</p> </div