Non-specific and specific DNA binding modes of bacterial histone, HU, separately regulate distinct physiological processes through different mechanisms.

The histone-like protein HU plays a diverse role in bacterial physiology from the maintenance of chromosome structure to the regulation of gene transcription. HU binds DNA in a sequence-non-specific manner via two distinct binding modes: (i) random binding to any DNA through ionic bonds between surface-exposed lysine residues (K3, K18, and K83) and phosphate backbone (non-specific); (ii) preferential binding to contorted DNA of given structures containing a pair of kinks (structure-specific) through conserved proline residues (P63) that induce and/or stabilize the kinks. First, we show here that the P63-mediated structure-specific binding also requires the three lysine residues, which are needed for a non-specific binding. Second, we demonstrate that substituting P63 to alanine in HU had no impact on non-specific binding but caused differential transcription of diverse genes previously shown to be regulated by HU, such as those associated with the organonitrogen compound biosynthetic process, galactose metabolism, ribosome biogenesis, and cell adhesion. The structure-specific binding also helps create DNA supercoiling, which, in turn, may influence directly or indirectly the transcription of other genes. Our previous and current studies show that non-specific and structure-specific HU binding appear to have separate functions- nucleoid architecture and transcription regulation- which may be true in other DNA-binding proteins.

Hybrid Vigor: Importance of Hybrid λ Phages in Early Insights in Molecular Biology.

Laboratory-generated hybrids between phage λ and related phages played a seminal role in establishment of the λ model system, which, in turn, served to develop many of the foundational concepts of molecular biology, including gene structure and control. Important λ hybrids with phages 21 and 434 were the earliest of such phages. To understand the biology of these hybrids in full detail, we determined the complete genome sequences of phages 21 and 434. Although both genomes are canonical members of the λ-like phage family, they both carry unsuspected bacterial virulence gene types not previously described in this group of phages. In addition, we determined the sequences of the hybrid phages λ imm21, λ imm434, and λ h434 imm21. These sequences show that the replacements of λ DNA by nonhomologous segments of 21 or 434 DNA occurred through homologous recombination in adjacent sequences that are nearly identical in the parental phages. These five genome sequences correct a number of errors in published sequence fragments of the 21 and 434 genomes, and they point out nine nucleotide differences from Sanger's original λ sequence that are likely present in most extant λ strains in laboratory use today. We discuss the historical importance of these hybrid phages in the development of fundamental tenets of molecular biology and in some of the earliest gene cloning vectors. The 434 and 21 genomes reinforce the conclusion that the genomes of essentially all natural λ-like phages are mosaics of sequence modules from a pool of exchangeable segments.

Complete Genome Sequences of Lambdoid Phages 21, 434, and 434B and Several Lambda Hybrids.

Recombinational hybrids between phage λ and its relatives were instrumental in the beginnings of molecular biology. Here, we report the complete genome sequences of lambdoid phages 21 and 434 and three of their λ hybrids. In addition, we describe 434B, where the entire lysis gene region was replaced by cryptic prophage sequences.

Bacteriophage Treatment Rescues Mice Infected with Multidrug-Resistant Klebsiella pneumoniae ST258.

Severe infections caused by multidrug-resistant Klebsiella pneumoniae sequence type 258 (ST258) highlight the need for new therapeutics with activity against this pathogen. Bacteriophage (phage) therapy is an alternative treatment approach for multidrug-resistant bacterial infections that has shown efficacy in experimental animal models and promise in clinical case reports. In this study, we assessed microbiologic, histopathologic, and survival outcomes following systemic administration of phage in ST258-infected mice. We found that prompt treatment with two phages, either individually or in combination, rescued mice with K. pneumoniae ST258 bacteremia. Among the three treatment groups, mice that received combination phage therapy demonstrated the greatest increase in survival and the lowest frequency of phage resistance among bacteria recovered from mouse blood and tissue. Our findings support the utility of phage therapy as an approach for refractory ST258 infections and underscore the potential of this treatment modality to be enhanced through strategic phage selection.IMPORTANCE Infections caused by multidrug-resistant K. pneumoniae pose a serious threat to at-risk patients and present a therapeutic challenge for clinicians. Bacteriophage (phage) therapy is an alternative treatment approach that has been associated with positive clinical outcomes when administered experimentally to patients with refractory bacterial infections. Inasmuch as these experimental treatments are prepared for individual patients and authorized for compassionate use only, they lack the rigor of a clinical trial and therefore cannot provide proof of efficacy. Here, we demonstrate that administration of viable phage provides effective treatment for multidrug-resistant K. pneumoniae (sequence type 258 [ST258]) bacteremia in a murine infection model. Moreover, we compare outcomes among three distinct phage treatment groups and identify potential correlates of therapeutic phage efficacy. These findings constitute an important first step toward optimizing and assessing phage therapy's potential for the treatment of severe ST258 infection in humans.

Single-molecule tracking reveals that the nucleoid-associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA.

HU (Histone-like protein from Escherichia coli strain U93) is the most conserved nucleoid-associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single-molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface-exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over-condensed and mis-segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUß subunit, or deleting nucleoid-associated naRNAs, each previously implicated in HU's high-affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure-binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.

λ Recombineering Used to Engineer the Genome of Phage T7.

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of λ Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.

Correction: Architecture of the Escherichia coli nucleoid.

[This corrects the article DOI: 10.1371/journal.pgen.1008456.].

Nucleoid remodeling during environmental adaptation is regulated by HU-dependent DNA bundling.

Bacterial nucleoid remodeling dependent on conserved histone-like protein, HU is one of the determining factors in global gene regulation. By imaging of near-native, unlabeled E. coli cells by soft X-ray tomography, we show that HU remodels nucleoids by promoting the formation of a dense condensed core surrounded by less condensed isolated domains. Nucleoid remodeling during cell growth and environmental adaptation correlate with pH and ionic strength controlled molecular switch that regulated HUαα dependent intermolecular DNA bundling. Through crystallographic and solution-based studies we show that these effects mechanistically rely on HUαα promiscuity in forming multiple electrostatically driven multimerization interfaces. Changes in DNA bundling consequently affects gene expression globally, likely by constrained DNA supercoiling. Taken together our findings unveil a critical function of HU-DNA interaction in nucleoid remodeling that may serve as a general microbial mechanism for transcriptional regulation to synchronize genetic responses during the cell cycle and adapt to changing environments.

Complete Genome Sequence of Myophage Ec_Makalu_002, Which Infects Uropathogenic Escherichia coli.

We isolated phage Ec_Makalu_002, which infects uropathogenic strains of Escherichia coli Here, we report its complete genome sequence, annotated features, and relatedness to other phages.

Phage Resistance in Multidrug-Resistant Klebsiella pneumoniae ST258 Evolves via Diverse Mutations That Culminate in Impaired Adsorption.

The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. The strategic selection of phage combinations that impose high genetic barriers to resistance and/or high compensatory fitness costs may mitigate this threat. However, for such a strategy to be effective, the evolution of phage resistance must be sufficiently constrained to be consistent. In this study, we isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged coculture in vitro Single- and double-phage-resistant mutants were isolated from independently evolved replicate cocultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures. All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors. Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart). Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design.IMPORTANCE The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.

Architecture of the Escherichia coli nucleoid.

How genomes are organized within cells and how the 3D architecture of a genome influences cellular functions are significant questions in biology. A bacterial genomic DNA resides inside cells in a highly condensed and functionally organized form called nucleoid (nucleus-like structure without a nuclear membrane). The Escherichia coli chromosome or nucleoid is composed of the genomic DNA, RNA, and protein. The nucleoid forms by condensation and functional arrangement of a single chromosomal DNA with the help of chromosomal architectural proteins and RNA molecules as well as DNA supercoiling. Although a high-resolution structure of a bacterial nucleoid is yet to come, five decades of research has established the following salient features of the E. coli nucleoid elaborated below: 1) The chromosomal DNA is on the average a negatively supercoiled molecule that is folded as plectonemic loops, which are confined into many independent topological domains due to supercoiling diffusion barriers; 2) The loops spatially organize into megabase size regions called macrodomains, which are defined by more frequent physical interactions among DNA sites within the same macrodomain than between different macrodomains; 3) The condensed and spatially organized DNA takes the form of a helical ellipsoid radially confined in the cell; and 4) The DNA in the chromosome appears to have a condition-dependent 3-D structure that is linked to gene expression so that the nucleoid architecture and gene transcription are tightly interdependent, influencing each other reciprocally. Current advents of high-resolution microscopy, single-molecule analysis and molecular structure determination of the components are expected to reveal the total structure and function of the bacterial nucleoid.

Phage Therapy in the Twenty-First Century: Facing the Decline of the Antibiotic Era; Is It Finally Time for the Age of the Phage?

Burgeoning problems of antimicrobial resistance dictate that new solutions be developed to combat old foes. Use of lytic bacteriophages (phages) for the treatment of drug-resistant bacterial infections is one approach that has gained significant traction in recent years. Fueled by reports of experimental phage therapy cases with very positive patient outcomes, several early-stage clinical trials of therapeutic phage products have been launched in the United States. Eventual licensure enabling widespread access to phages is the goal; however, new paths to regulatory approval and mass-market distribution, distinct from those of small-molecule antibiotics, must be forged first. This review highlights unique aspects related to the clinical use of phages, including advantages to be reaped as well as challenges to be overcome.

Molecular Basis of Phage Communication.

Recently members of Bacillus phages were found to utilize a small peptide (6 aa long) to communicate with their descendants, aiding in a complex decision-making process. Proteins involved in this remarkable viral communication phenomenon were further investigated at the structural level for better understanding in this issue of Molecular Cell (Gallego del Sol et al., 2019).

Processing generates 3' ends of RNA masking transcription termination events in prokaryotes.

Two kinds of signal-dependent transcription termination and RNA release mechanisms have been established in prokaryotes in vitro by: (i) binding of Rho to cytidine-rich nascent RNA [Rho-dependent termination (RDT)], and (ii) the formation of a hairpin structure in the nascent RNA, ending predominantly with uridine residues [Rho-independent termination (RIT)]. As shown here, the two signals act independently of each other and can be regulated (suppressed) by translation-transcription coupling in vivo. When not suppressed, both RIT- and RDT-mediated transcription termination do occur, but ribonucleolytic processing generates defined new 3' ends in the terminated RNA molecules. The actual termination events at the end of transcription units are masked by generation of new processed 3' RNA ends; thus the in vivo 3' ends do not define termination sites. We predict generation of 3' ends of mRNA by processing is a common phenomenon in prokaryotes as is the case in eukaryotes.

The Developmental Switch in Bacteriophage λ: A Critical Role of the Cro Protein.

Bacteriophage λ of Escherichia coli has two alternative life cycles after infection-host survival with lysogen formation, or host lysis and phage production. In a lysogen, CI represses the two lytic promoters, pR and pL, and activates its own transcription from the auto-regulated pRM promoter. During induction from the lysogenic to lytic state, CI is inactivated, and the two lytic promoters are de-repressed allowing for expression of Cro from pR. Cro is known to repress transcription of CI from pRM to prevent lysogeny. We show here that when Cro and CI are both present but at low levels, the low level of Cro initially stimulates the lytic promoters while CI repressor is still present, stimulating the level of Cro to a concentration required for pRM repression. Cro has no stimulatory effect without the presence of CI. We propose that this early auto-activating role of Cro at lower concentrations is essential in the developmental switch to lytic growth, whereas pRM repression by Cro at relatively higher concentrations avoids restoring lysogeny.

DNA-RNA interactions are critical for chromosome condensation in Escherichia coli.

Bacterial chromosome (nucleoid) conformation dictates faithful regulation of gene transcription. The conformation is condition-dependent and is guided by several nucleoid-associated proteins (NAPs) and at least one nucleoid-associated noncoding RNA, naRNA4. Here we investigated the molecular mechanism of how naRNA4 and the major NAP, HU, acting together organize the chromosome structure by establishing multiple DNA-DNA contacts (DNA condensation). We demonstrate that naRNA4 uniquely acts by forming complexes that may not involve long stretches of DNA-RNA hybrid. Also, uncommonly, HU, a chromosome-associated protein that is essential in the DNA-RNA interactions, is not present in the final complex. Thus, HU plays a catalytic (chaperone) role in the naRNA4-mediated DNA condensation process.

Draft Genome Sequence of the Naturally Competent Bacillus simplex Strain WY10.

We sequenced a naturally competent bacterial isolate, WY10, cultured from a Wyoming soil sample. Sequence analysis revealed that WY10 is a novel strain of Bacillus simplex To our knowledge, WY10 is the first B. simplex strain to be characterized as naturally competent for DNA uptake by transformation.

Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation.

Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub "superspreaders," releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications. IMPORTANCE: Bacteriophages (phages), viruses that infect bacteria, are the planet's most numerous biological entities and kill vast numbers of bacteria in natural environments. Many of these bacteria carry plasmids, extrachromosomal DNA elements that frequently encode antibiotic resistance. However, it is largely unknown whether plasmids are destroyed during phage infection or released intact upon phage lysis, whereupon their encoded resistance could be acquired and manifested by other bacteria (transformation). Because phages are being developed to combat antibiotic-resistant bacteria and because transformation is a principal form of horizontal gene transfer, this question has important implications for biomedicine and microbial evolution alike. Here we report the isolation and characterization of two novel Escherichia coli phages, dubbed "superspreaders," that promote extensive plasmid transformation and efficiently disperse antibiotic resistance genes. Our work suggests that phage superspreaders are not suitable for use in medicine but may help drive bacterial evolution in natural environments.

DNA repeat sequences: diversity and versatility of functions.

Although discovered decades ago, the molecular identification, the diversity and versatility of functions, and the evolutionary origin of repeat DNA sequences (REPs) containing palindromic units in prokaryotes are now bringing attention to a wide range of biological scientists. A brief account of the current state of the repeat DNA sequences is presented here.

Genome-Wide Transcriptional Regulation and Chromosome Structural Arrangement by GalR in E. coli.

The regulatory protein, GalR, is known for controlling transcription of genes related to D-galactose metabolism in Escherichia coli. Here, using a combination of experimental and bioinformatic approaches, we identify novel GalR binding sites upstream of several genes whose function is not directly related to D-galactose metabolism. Moreover, we do not observe regulation of these genes by GalR under standard growth conditions. Thus, our data indicate a broader regulatory role for GalR, and suggest that regulation by GalR is modulated by other factors. Surprisingly, we detect regulation of 158 transcripts by GalR, with few regulated genes being associated with a nearby GalR binding site. Based on our earlier observation of long-range interactions between distally bound GalR dimers, we propose that GalR indirectly regulates the transcription of many genes by inducing large-scale restructuring of the chromosome.