Bacterial nucleoid is a riddle wrapped in a mystery inside an enigma.

Bacterial chromosome, the nucleoid, is traditionally modeled as a rosette of DNA mega-loops, organized around proteinaceous central scaffold by nucleoid-associated proteins (NAPs), and mixed with the cytoplasm by transcription and translation. Electron microscopy of fixed cells confirms dispersal of the cloud-like nucleoid within the ribosome-filled cytoplasm. Here, I discuss evidence that the nucleoid in live cells forms DNA phase separate from riboprotein phase, the "riboid." I argue that the nucleoid-riboid interphase, where DNA interacts with NAPs, transcribing RNA polymerases, nascent transcripts, and ssRNA chaperones, forms the transcription zone. An active part of phase separation, transcription zone enforces segregation of the centrally positioned information phase (the nucleoid) from the surrounding action phase (the riboid), where translation happens, protein accumulates, and metabolism occurs. I speculate that HU NAP mostly tiles up the nucleoid periphery-facilitating DNA mobility but also supporting transcription in the interphase. Besides extruding plectonemically supercoiled DNA mega-loops, condensins could compact them into solenoids of uniform rings, while HU could support rigidity and rotation of these DNA rings. The two-phase cytoplasm arrangement allows the bacterial cell to organize the central dogma activities, where (from the cell center to its periphery) DNA replicates and segregates, DNA is transcribed, nascent mRNA is handed over to ribosomes, mRNA is translated into proteins, and finally, the used mRNA is recycled into nucleotides at the inner membrane. The resulting information-action conveyor, with one activity naturally leading to the next one, explains the efficiency of prokaryotic cell design-even though its main intracellular transportation mode is free diffusion.

Relationship of Salmonella Typhimurium 14028 strain and its dam and seqA mutants with gut microbiota dysbiosis in rats.

Introduction. Disruptions in gut microbiota, known as dysbiosis, have been increasingly linked to pathogenic infections, with Salmonella Typhimurium being a notable contributor to these disturbances.Hypothesis. We hypothesize that the S. Typhimurium 14028 WT strain induces significant dysbiosis in the rat gut microbiota and that the dam and seqA genes play crucial roles in this process.Aim. In this study, it was aimed at investigating the dysbiotic activity of the S. Typhimurium 14028 WT strain on the rat gut microbiota and the roles of dam and seqA genes on this activity.Method. Changes in the rat gut microbiota were determined by examining the anal swap samples taken from the experimental groups of these animals using 16S rRNA high-throughput sequencing technology.Results. In the experimental groups, the dominant phyla were determined to be Firmicutes and Bacteroidetes (P<0.05). However, while the rate of Bacteroidetes was significantly reduced in those treated with the WT and seqA mutants, no significant difference was observed in the dam mutant compared to the control group (P<0.05). In all experimental animals, the dominant species was determined to be Prevotella copri, regardless of the experiment time and application. The analysis results of the samples taken on the third day from the rat groups infected with the S. Typhimurium 14028 WT strain (W2) presented the most striking data of this study.Conclusion. Through distance analysis, we demonstrated that a successful Salmonella infection completely changes the composition of the microbiota, dramatically reduces species diversity and richness in the microbiota and encourages the growth of opportunistic pathogens.

Comparative global gene expression analysis of biofilm forms of Salmonella Typhimurium ATCC 14028 and its seqA mutant.

In this study, comparative transcriptomic analyzes (mRNA and miRNA) were performed on the biofilm forms of S. Typhimurium ATCC 14028 wild-type strain and its seqA gene mutant in order to determine the regulation characteristics of the seqA gene in detail. The results of global gene expression analyses showed an increase in the expression level of 54 genes and a decrease in the expression level of 155 genes (p < 0.05) in the seqA mutant compared to the wild-type strain. 10 of the 48 miRNAs identified on behalf of sequence analysis are new miRNA records for Salmonella. Transcripts of 14 miRNAs differed between wild-type strain and seqA mutant (p < 0.05), of which eight were up-regulated and six were down-regulated. Bioinformatic analyzes showed that differentially expressed genes in the wild-type strain and its seqA gene mutant play a role in different metabolic processes as well as biofilm formation, pathogenicity and virulence. When the transcriptomic data were interpreted together with the findings obtained from phenotypic tests such as motility, attachment to host cells and biofilm morphotyping, it was determined that the seqA gene has a critical function especially for the adhesion and colonization stages of biofilm formation, as well as for biofilm stability. Transcriptomic data pointing out that the seqA gene is also a general positive regulator of T3SS effector proteins active in cell invasion in S. Typhimurium wild-type biofilm, proves that this gene is involved in Salmonella host cell invasion.

Plesiomonas shigelloides, an Atypical Enterobacterales with a Vibrio-Related Secondary Chromosome.

About 10% of bacteria have a multichromosome genome with a primary replicon of bacterial origin, called the chromosome, and other replicons of plasmid origin, the chromids. Studies on multichromosome bacteria revealed potential points of coordination between the replication/segregation of chromids and the progression of the cell cycle. For example, replication of the chromid of Vibrionales (called Chr2) is initiated upon duplication of a sequence carried by the primary chromosome (called Chr1), in such a way that replication of both replicons is completed synchronously. Also, Chr2 uses the Chr1 as a scaffold for its partition in the daughter cells. How many of the features detected so far are required for the proper integration of a secondary chromosome in the cell cycle? How many more features remain to be discovered? We hypothesized that critical features for the integration of the replication/segregation of a given chromid within the cell cycle program would be conserved independently of the species in which the chromid has settled. Hence, we searched for a chromid related to that found in Vibrionales outside of this order. We identified one in Plesiomonas shigelloides, an aquatic and pathogenic enterobacterium that diverged early within the clade of Enterobacterales. Our results suggest that the chromids present in P. shigelloides and Vibrionales derive from a common ancestor. We initiated in silico genomic and proteomic comparative analyses of P. shigelloides, Vibrionales, and Enterobacterales that enabled us to establish a list of features likely involved in the maintenance of the chromid within the host cell cycle.

Blocking, Bending, and Binding: Regulation of Initiation of Chromosome Replication During the Escherichia coli Cell Cycle by Transcriptional Modulators That Interact With Origin DNA.

Genome duplication is a critical event in the reproduction cycle of every cell. Because all daughter cells must inherit a complete genome, chromosome replication is tightly regulated, with multiple mechanisms focused on controlling when chromosome replication begins during the cell cycle. In bacteria, chromosome duplication starts when nucleoprotein complexes, termed orisomes, unwind replication origin (oriC) DNA and recruit proteins needed to build new replication forks. Functional orisomes comprise the conserved initiator protein, DnaA, bound to a set of high and low affinity recognition sites in oriC. Orisomes must be assembled each cell cycle. In Escherichia coli, the organism in which orisome assembly has been most thoroughly examined, the process starts with DnaA binding to high affinity sites after chromosome duplication is initiated, and orisome assembly is completed immediately before the next initiation event, when DnaA interacts with oriC's lower affinity sites, coincident with origin unwinding. A host of regulators, including several transcriptional modulators, targets low affinity DnaA-oriC interactions, exerting their effects by DNA bending, blocking access to recognition sites, and/or facilitating binding of DnaA to both DNA and itself. In this review, we focus on orisome assembly in E. coli. We identify three known transcriptional modulators, SeqA, Fis (factor for inversion stimulation), and IHF (integration host factor), that are not essential for initiation, but which interact directly with E. coli oriC to regulate orisome assembly and replication initiation timing. These regulators function by blocking sites (SeqA) and bending oriC DNA (Fis and IHF) to inhibit or facilitate cooperative low affinity DnaA binding. We also examine how the growth rate regulation of Fis levels might modulate IHF and DnaA binding to oriC under a variety of nutritional conditions. Combined, the regulatory mechanisms mediated by transcriptional modulators help ensure that at all growth rates, bacterial chromosome replication begins once, and only once, per cell cycle.

Measuring Plasmid Conjugation Using Fluorescent Reporters.

Fluorescence-based methods are increasingly popular because they (1) offer a faster alternative to labor-intensive traditional methods, (2) enable the development of automated high-throughput screening procedures, and (3) allow direct visualization of biological processes. Here we describe three fluorescence-based methods applicable for the detection and quantitation of plasmid conjugation. The first method uses flow cytometry as a fast and reliable alternative to traditional plating methods. A second one employs fluorescence expression for high-throughput analysis of plasmid conjugation. Finally we review a third method that enables direct visualization of plasmid transfer under the microscope.

Effects of dam and seqA genes on biofilm and pellicle formation in Salmonella.

In this study, the effects of dam and seqA genes on the formation of pellicle and biofilm was determined using five different Salmonella serovars S. Group C1 (DMC2 encoded), S. Typhimurium (DMC4 encoded), S. Virchow (DMC11 encoded), S. Enteritidis (DMC22 encoded), and S. Montevideo (DMC89 encoded). dam and seqA mutants in Salmonella serovars were performed by the single step lambda red recombination method. The mutants obtained were examined according to the properties of biofilm on the polystyrene surfaces and the pellicle formation on the liquid medium. As a result of these investigations, it was determined that the biofilm formation properties on polystyrene surfaces decreased significantly (p < 0.05) in all tested dam and seqA mutants, while the pellicle formation properties were lost in the liquid medium. When pBAD24 vector, containing the dam and seqA genes cloned behind the inducible arabinose promoter, transduced into dam and seqA mutant strains, it was determined that the biofilm formation properties on the polystyrene surfaces reached to the natural strains' level in all mutant strains. Also, the pellicle formation ability was regained in the liquid media. All these data demonstrate that dam and seqA genes play an important role in the formation of biofilm and pellicle structures in Salmonella serovars.

HipA-Mediated Phosphorylation of SeqA Does not Affect Replication Initiation in Escherichia coli.

The SeqA protein of Escherichia coli is required to prevent immediate re-initiation of chromosome replication from oriC. The SeqA protein is phosphorylated at the serine-36 (Ser36) residue by the HipA kinase. The role of phosphorylation was addressed by mutating the Ser36 residue to alanine, which cannot be phosphorylated and to aspartic acid, which mimics a phosphorylated serine residue. Both mutant strains were similar to wild-type with respect to origin concentration and initiation synchrony. The minimal time between successive initiations was also unchanged. We therefore suggest that SeqA phosphorylation at the Ser36 residue is silent, at least with respect to SeqA's role in replication initiation.

Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli, Avoiding Dangers of Runaway Overreplication.

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the "natural" CRC limit of ∼8 (cells divide more slowly); the "functional" CRC limit of ∼22 (cells divide extremely slowly); and the "tolerance" CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.