Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicum.

BackgroundAs the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents.Methodology/Principal FindingsFollowing the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays.Conclusions/SignificanceBoth LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.

This study evaluates a novel assay for detecting rifampin resistance in clinical Mycobacterium ulcerans isolates. Although highly susceptible for PCR inhibitors in 50% of the samples tested, the assay was 100% M. ulcerans specific and yielded >98% analyzable sequences with a lower limit of detection of 100 to 200 copies of the target sequence.

20Bacteria regulate chromosome replication and segregation tightly with cell division to 21 ensure faithful segregation of DNA to daughter generations. The underlying 22 mechanisms have been addressed in several model species. It became apparent 23 that bacteria have evolved quite different strategies to regulate DNA segregation and 24 chromosomal organization. We have investigated here how the actinobacterium 25 Corynebacterium glutamicum organizes chromosome segregation and DNA 26 replication. Unexpectedly, we find that C. glutamicum cells are at least diploid under 27 all conditions tested and that these organisms have overlapping C-periods during 28 replication with both origins initiating replication simultaneously. Based on 29 experimentally obtained data we propose growth rate dependent cell cycle models for 30 C. glutamicum. 31 32 65 proteobacteria, Deinococcales, cyanobacteria and also archaea (20-27).66 Besides the distinct cell cycle modes chromosome localization patterns differ 67 between model organisms. In a non-replicating, slow-growing E. coli cell the single 68 4chromosome is placed symmetrically with oriC and terC regions located at midcell 69 and the replichores spatially separated to the two cell halves (28). Upon replication 70 initiation the two sister chromosomes segregate bidirectionally to opposite cell halves 71 with replisomes positioned at midcell (29,30). Finally, oriC and terC are confined to 72 cell quarter regions. Contrary to this, the model organisms C. crescentus, Vibrio 73 cholerae and Pseudomonas aeruginosa localize their nucleoids about the 74 longitudinal axis with chromosome arms adjacent to each other (31-34). Sister 75 replichores move to the opposite cell half with the segregated oriC facing towards the 76 pole, mirroring the second chromosome at the transverse axis. The oriC region of C. 77 crescentus and V. cholera is positioned by polar landmark proteins (35,36), where 78 replisomes assemble and simultaneously move towards midcell in the course of 79 replication (12,17). For the most part, P. aeruginosa places its replication machinery 80 centrally (34). Finally, B. subtilis switches from the longitudinal chromosome 81 organization to the E. coli "left-oriC-right" configuration during replication initiation 82 (37). 83The mitotic-like ParABS system has been identified as a driving force behind 84 coordinated nucleoid partitioning for more than two third of bacterial species 85 analyzed, with exceptions specifically within the phylum of γ-proteobacteria such as 86 E. coli (38). This segregation mechanism involves components similar to the plasmid 87 encoded par genes responsible for active segregation of low-copy-number plasmids 88 (39). Thereby, the ParB protein binds a variable number of centromere-like DNA 89 sequences called parS sites in oriC-proximity (40) and spreads along the DNA 90 forming large protein-DNA complexes (41-43). Interaction of ParB with the Walker-91 type ATPase ParA mediates ATP-hydrolysis and thereby ParA detachment from DNA 92 (44), driving...