Bacterial inactivation processes in water disinfection - mechanistic aspects of primary and secondary oxidants - A critical review.

Water disinfection during drinking water production is one of the most important processes to ensure safe drinking water, which is gaining even more importance due to the increasing impact of climate change. With specific reaction partners, chemical oxidants can form secondary oxidants, which can cause additional damage to bacteria. Cases in point are chlorine dioxide which forms free available chlorine (e.g., in the reaction with phenol) and ozone which can form hydroxyl radicals (e.g., during the reaction with natural organic matter). The present work reviews the complex interplay of all these reactive species which can occur in disinfection processes and their potential to affect disinfection processes. A quantitative overview of their disinfection strength based on inactivation kinetics and typical exposures is provided. By unifying the current data for different oxidants it was observable that cultivated wild strains (e.g., from wastewater treatment plants) are in general more resistant towards chemical oxidants compared to lab-cultivated strains from the same bacterium. Furthermore, it could be shown that for selective strains chlorine dioxide is the strongest disinfectant (highest maximum inactivation), however as a broadband disinfectant ozone showed the highest strength (highest average inactivation). Details in inactivation mechanisms regarding possible target structures and reaction mechanisms are provided. Thereby the formation of secondary oxidants and their role in inactivation of pathogens is decently discussed. Eventually, possible defense responses of bacteria and additional effects which can occur in vivo are discussed.

Vibrio natriegens: an ultrafast-growing marine bacterium as emerging synthetic biology chassis.

The marine bacterium Vibrio natriegens is the fastest-growing non-pathogenic bacterium known to date and is gaining more and more attention as an alternative chassis organism to Escherichia coli. A recent wave of synthetic biology efforts has focused on the establishment of molecular biology tools in this fascinating organism, now enabling exciting applications - from speeding up our everyday laboratory routines to increasing the pace of biotechnological production cycles. In this review, we seek to give a broad overview on the literature on V. natriegens, spanning all the way from its initial isolation to its latest applications. We discuss its natural ecological niche and interactions with other organisms, unveil some of its extraordinary traits, review its genomic organization and give insight into its diverse metabolism - key physiological insights required to further develop this organism into a synthetic biology chassis. By providing a comprehensive overview on the established genetic tools, methods and applications we highlight the current possibilities of this organism, but also identify some of the gaps that could drive future lines of research, hopefully stimulating the growth of the V. natriegens research community.

gammaBOriS: Identification and Taxonomic Classification of Origins of Replication in Gammaproteobacteria using Motif-based Machine Learning.

The biology of bacterial cells is, in general, based on information encoded on circular chromosomes. Regulation of chromosome replication is an essential process that mostly takes place at the origin of replication (oriC), a locus unique per chromosome. Identification of high numbers of oriC is a prerequisite for systematic studies that could lead to insights into oriC functioning as well as the identification of novel drug targets for antibiotic development. Current methods for identifying oriC sequences rely on chromosome-wide nucleotide disparities and are therefore limited to fully sequenced genomes, leaving a large number of genomic fragments unstudied. Here, we present gammaBOriS (Gammaproteobacterial oriC Searcher), which identifies oriC sequences on gammaproteobacterial chromosomal fragments. It does so by employing motif-based machine learning methods. Using gammaBOriS, we created BOriS DB, which currently contains 25,827 gammaproteobacterial oriC sequences from 1,217 species, thus making it the largest available database for oriC sequences to date. Furthermore, we present gammaBOriTax, a machine-learning based approach for taxonomic classification of oriC sequences, which was trained on the sequences in BOriS DB. Finally, we extracted the motifs relevant for identification and classification decisions of the models. Our results suggest that machine learning sequence classification approaches can offer great support in functional motif identification.

Exception to the exception rule: synthetic and naturally occurring single chromosome Vibrio cholerae.

The genome of Vibrio cholerae, the etiological agent of cholera, is an exception to the single chromosome rule found in the vast majority of bacteria and has its genome partitioned between two unequally sized chromosomes. This unusual two-chromosome arrangement in V. cholerae has sparked considerable research interest since its discovery. It was demonstrated that the two chromosomes could be fused by deliberate genome engineering or forced to fuse spontaneously by blocking the replication of Chr2, the secondary chromosome. Recently, natural isolates of V. cholerae with chromosomal fusion have been found. Here, we summarize the pertinent findings on this exception to the exception rule and discuss the potential utility of single-chromosome V. cholerae to address fundamental questions on chromosome biology in general and DNA replication in particular.

Stringent response leads to continued cell division and a temporal restart of DNA replication after initial shutdown in Vibrio cholerae.

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi-chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division-replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements.

Functionality of Two Origins of Replication in Vibrio cholerae Strains With a Single Chromosome.

Chromosomal inheritance in bacteria usually entails bidirectional replication of a single chromosome from a single origin into two copies and subsequent partitioning of one copy each into daughter cells upon cell division. However, the human pathogen Vibrio cholerae and other Vibrionaceae harbor two chromosomes, a large Chr1 and a small Chr2. Chr1 and Chr2 have different origins, an oriC-type origin and a P1 plasmid-type origin, respectively, driving the replication of respective chromosomes. Recently, we described naturally occurring exceptions to the two-chromosome rule of Vibrionaceae: i.e., Chr1 and Chr2 fused single chromosome V. cholerae strains, NSCV1 and NSCV2, in which both origins of replication are present. Using NSCV1 and NSCV2, here we tested whether two types of origins of replication can function simultaneously on the same chromosome or one or the other origin is silenced. We found that in NSCV1, both origins are active whereas in NSCV2 ori2 is silenced despite the fact that it is functional in an isolated context. The ori2 activity appears to be primarily determined by the copy number of the triggering site, crtS, which in turn is determined by its location with respect to ori1 and ori2 on the fused chromosome.

Modular Assembly of Synthetic Secondary Chromosomes.

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. Questions of how chromosomes need to be constructed to maintain the genetic information can now be answered by a learning-by-building approach. Here, we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular Modular Cloning system (MoClo).

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae.

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.

Phenotypes of dnaXE145A Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks.

The Escherichia colidnaXE145A mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation parE10 The gene for the clamp loader subunits τ and γ, dnaX, but not the mutant dnaXE145A , was found to suppress parE10(Ts) when overexpressed. Purified mutant protein was found to be functional in vitro, and few phenotypes were found in vivo apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at oriC never reach the terminus in dnaXE145A mutant cells. The SOS response was found to be induced, and a combination of the dnaXE145A mutation with recBC and recA mutations led to reduced viability. The mutant cells exhibited extensive chromosome fragmentation and degradation upon inactivation of recBC and recA, respectively. The results indicate that the dnaXE145A mutant cells suffer from broken replication forks and that these need to be repaired by homologous recombination. We suggest that the dnaX-encoded τ and γ subunits of the clamp loader, or the clamp loader complex itself, has a role in the restart of stalled replication forks without extensive homologous recombination.IMPORTANCE The E. coli clamp loader complex has a role in coordinating the activity of the replisome at the replication fork and loading ß-clamps for lagging-strand synthesis. Replication forks frequently encounter obstacles, such as template lesions, secondary structures, and tightly bound protein complexes, which will lead to fork stalling. Some pathways of fork restart have been characterized, but much is still unknown about the actors and mechanisms involved. We have in this work characterized the dnaXE145A clamp loader mutant. We find that the naturally occurring obstacles encountered by a replication fork are not tackled in a proper way by the mutant clamp loader and suggest a role for the clamp loader in the restart of stalled replication forks.

Exception to the Rule: Genomic Characterization of Naturally Occurring Unusual Vibrio cholerae Strains with a Single Chromosome.

The genetic make-up of most bacteria is encoded in a single chromosome while about 10% have more than one chromosome. Among these, Vibrio cholerae, with two chromosomes, has served as a model system to study various aspects of chromosome maintenance, mainly replication, and faithful partitioning of multipartite genomes. Here, we describe the genomic characterization of strains that are an exception to the two chromosome rules: naturally occurring single-chromosome V. cholerae. Whole genome sequence analyses of NSCV1 and NSCV2 (natural single-chromosome vibrio) revealed that the Chr1 and Chr2 fusion junctions contain prophages, IS elements, and direct repeats, in addition to large-scale chromosomal rearrangements such as inversions, insertions, and long tandem repeats elsewhere in the chromosome compared to prototypical two chromosome V. cholerae genomes. Many of the known cholera virulence factors are absent. The two origins of replication and associated genes are generally intact with synonymous mutations in some genes, as are recA and mismatch repair (MMR) genes dam, mutH, and mutL; MutS function is probably impaired in NSCV2. These strains are ideal tools for studying mechanistic aspects of maintenance of chromosomes with multiple origins and other rearrangements and the biological, functional, and evolutionary significance of multipartite genome architecture in general.

BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci.

Fluorescence-based methods are widely used to analyze elementary cell processes such as DNA replication or chromosomal folding and segregation. Labeling DNA with a fluorescent protein allows the visualization of its temporal and spatial organization. One popular approach is FROS (fluorescence repressor operator system). This method specifically labels DNA in vivo through binding of a fusion of a fluorescent protein and a repressor protein to an operator array, which contains numerous copies of the repressor binding site integrated into the genomic site of interest. Bound fluorescent proteins are then visible as foci in microscopic analyses and can be distinguished from the background fluorescence caused by unbound fusion proteins. Even though this method is widely used, no attempt has been made so far to decrease the background fluorescence to facilitate analysis of the actual signal of interest. Here, we present a new method that greatly reduces the background signal of FROS. BiFCROS (Bimolecular Fluorescence Complementation and Repressor Operator System) is based on fusions of repressor proteins to halves of a split fluorescent protein. Binding to a hybrid FROS array results in fluorescence signals due to bimolecular fluorescence complementation. Only proteins bound to the hybrid FROS array fluoresce, greatly improving the signal to noise ratio compared to conventional FROS. We present the development of BiFCROS and discuss its potential to be used as a fast and single-cell readout for copy numbers of genetic loci.

Establishing a System for Testing Replication Inhibition of the Vibrio cholerae Secondary Chromosome in Escherichia coli.

Regulators of DNA replication in bacteria are an attractive target for new antibiotics, as not only is replication essential for cell viability, but its underlying mechanisms also differ from those operating in eukaryotes. The genetic information of most bacteria is encoded on a single chromosome, but about 10% of species carry a split genome spanning multiple chromosomes. The best studied bacterium in this context is the human pathogen Vibrio cholerae, with a primary chromosome (Chr1) of 3 M bps, and a secondary one (Chr2) of about 1 M bps. Replication of Chr2 is under control of a unique mechanism, presenting a potential target in the development of V. cholerae-specific antibiotics. A common challenge in such endeavors is whether the effects of candidate chemicals can be focused on specific mechanisms, such as DNA replication. To test the specificity of antimicrobial substances independent of other features of the V. cholerae cell for the replication mechanism of the V. cholerae secondary chromosome, we establish the replication machinery in the heterologous E. coli system. We characterize an E. coli strain in which chromosomal replication is driven by the replication origin of V. cholerae Chr2. Surprisingly, the E. coli ori2 strain was not inhibited by vibrepin, previously found to inhibit ori2-based replication.

Design and Assembly of DNA Sequence Libraries for Chromosomal Insertion in Bacteria Based on a Set of Modified MoClo Vectors.

Efficient assembly of large DNA constructs is a key technology in synthetic biology. One of the most popular assembly systems is the MoClo standard in which restriction and ligation of multiple fragments occurs in a one-pot reaction. The system is based on a smart vector design and type IIs restriction enzymes, which cut outside their recognition site. While the initial MoClo vectors had been developed for the assembly of multiple transcription units of plants, some derivatives of the vectors have been developed over the last years. Here we present a new set of MoClo vectors for the assembly of fragment libraries and insertion of constructs into bacterial chromosomes. The vectors are accompanied by a computer program that generates a degenerate synthetic DNA sequence that excludes "forbidden" DNA motifs. We demonstrate the usability of the new approach by construction of a stable fluorescence repressor operator system (FROS).

DNA Replication in Engineered Escherichia coli Genomes with Extra Replication Origins.

The standard outline of bacterial genomes is a single circular chromosome with a single replication origin. From the bioengineering perspective, it appears attractive to extend this basic setup. Bacteria with split chromosomes or multiple replication origins have been successfully constructed in the last few years. The characteristics of these engineered strains will largely depend on the respective DNA replication patterns. However, the DNA replication has not been investigated systematically in engineered bacteria with multiple origins or split replicons. Here we fill this gap by studying a set of strains consisting of (i) E. coli strains with an extra copy of the native replication origin (oriC), (ii) E. coli strains with an extra copy of the replication origin from the secondary chromosome of Vibrio cholerae (oriII), and (iii) a strain in which the E. coli chromosome is split into two linear replicons. A combination of flow cytometry, microarray-based comparative genomic hybridization (CGH), and modeling revealed silencing of extra oriC copies and differential timing of ectopic oriII copies compared to the native oriC. The results were used to derive construction rules for future multiorigin and multireplicon projects.

DistAMo: A Web-Based Tool to Characterize DNA-Motif Distribution on Bacterial Chromosomes.

Short DNA motifs are involved in a multitude of functions such as for example chromosome segregation, DNA replication or mismatch repair. Distribution of such motifs is often not random and the specific chromosomal pattern relates to the respective motif function. Computational approaches which quantitatively assess such chromosomal motif patterns are necessary. Here we present a new computer tool DistAMo (Distribution Analysis of DNA Motifs). The algorithm uses codon redundancy to calculate the relative abundance of short DNA motifs from single genes to entire chromosomes. Comparative genomics analyses of the GATC-motif distribution in γ-proteobacterial genomes using DistAMo revealed that (i) genes beside the replication origin are enriched in GATCs, (ii) genome-wide GATC distribution follows a distinct pattern, and (iii) genes involved in DNA replication and repair are enriched in GATCs. These features are specific for bacterial chromosomes encoding a Dam methyltransferase. The new software is available as a stand-alone or as an easy-to-use web-based server version at http://www.computational.bio.uni-giessen.de/distamo.

Optimization and Characterization of the Synthetic Secondary Chromosome synVicII in Escherichia coli.

Learning by building is one of the core ideas of synthetic biology research. Consequently, building synthetic chromosomes is the way to fully understand chromosome characteristics. The last years have seen exciting synthetic chromosome studies. We had previously introduced the synthetic secondary chromosome synVicII in Escherichia coli. It is based on the replication mechanism of the secondary chromosome in Vibrio cholerae. Here, we present a detailed analysis of its genetic characteristics and a selection approach to optimize replicon stability. We probe the origin diversity of secondary chromosomes from Vibrionaceae by construction of several new respective replicons. Finally, we present a synVicII version 2.0 with several innovations including its full compatibility with the popular modular cloning (MoClo) assembly system.

Synthetic chromosomes.

What a living organism looks like and how it works and what are its components-all this is encoded on DNA, the genetic blueprint. Consequently, the way to change an organism is to change its genetic information. Since the first pieces of recombinant DNA have been used to transform cells in the 1970s, this approach has been enormously extended. Bigger and bigger parts of the genetic information have been exchanged or added over the years. Now we are at a point where the construction of entire chromosomes becomes a reachable goal and first examples appear. This development leads to fundamental new questions, for example, about what is possible and desirable to build or what construction rules one needs to follow when building synthetic chromosomes. Here we review the recent progress in the field, discuss current challenges and speculate on the appearance of future synthetic chromosomes.

Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae.

Recent developments in DNA-assembly methods make the synthesis of synthetic chromosomes a reachable goal. However, the redesign of primary chromosomes bears high risks and still requires enormous resources. An alternative approach is the addition of synthetic chromosomes to the cell. The natural secondary chromosome of Vibrio cholerae could potentially serve as template for a synthetic secondary chromosome in Escherichia coli. To test this assumption we constructed a replicon named synVicII based on the replication module of V. cholerae chromosome II (oriII). A new assay for the assessment of replicon stability was developed based on flow-cytometric analysis of unstable GFP variants. Application of this assay to cells carrying synVicII revealed an improved stability compared to a secondary replicon based on E. coli oriC. Cell cycle analysis and determination of cellular copy numbers of synVicII indicate that replication timing of the synthetic replicon in E. coli is comparable to the natural chromosome II (ChrII) in V. cholerae. The presented synthetic biology work provides the basis to use secondary chromosomes in E. coli to answer basic research questions as well as for several biotechnological applications.