Atomic Force Microscopy Analysis of the Role of Major DNA-Binding Proteins in Organization of the Nucleoid in Escherichia coli

Bacterial genomic DNA is packed within the nucleoid of the cell along with various proteins and RNAs. We previously showed that the nucleoid in log phase cells consist of fibrous structures with diameters ranging from 30 to 80 nm, and that these structures, upon RNase A treatment, are converted into homogeneous thinner fibers with diameter of 10 nm. In this study, we investigated the role of major DNA-binding proteins in nucleoid organization by analyzing the nucleoid of mutant Escherichia coli strains lacking HU, IHF, H–NS, StpA, Fis, or Hfq using atomic force microscopy. Deletion of particular DNA-binding protein genes altered the nucleoid structure in different ways, but did not release the naked DNA even after the treatment with RNase A. This suggests that major DNA-binding proteins are involved in the formation of higher order structure once 10-nm fiber structure is built up from naked DNA.     

Genomic landscape of single-stranded DNA gapped intermediates in Escherichia coli

Single-stranded (ss) gapped regions in bacterial genomes (gDNA) are formed on W- and C-strands during replication, repair, and recombination. Using non-denaturing bisulfite treatment to convert C to U on ssDNA, combined with deep sequencing, we have mapped gDNA gap locations, sizes, and distributions in Escherichia coli for cells grown in mid-log phase in the presence and absence of UV irradiation, and in stationary phase cells. The fraction of ssDNA on gDNA is similar for W- and C-strands, ∼1.3% for log phase cells, ∼4.8% for irradiated log phase cells, and ∼8.5% for stationary phase cells. After UV irradiation, gaps increased in numbers and average lengths. A monotonic reduction in ssDNA occurred symmetrically between the DNA replication origin of (OriC) and terminus (Ter) for log phase cells with and without UV, a hallmark feature of DNA replication. Stationary phase cells showed no OriC → Ter ssDNA gradient. We have identified a spatially diverse gapped DNA landscape containing thousands of highly enriched ‘hot’ ssDNA regions along with smaller numbers of ‘cold’ regions. This analysis can be used for a wide variety of conditions to map ssDNA gaps generated when DNA metabolic pathways have been altered, and to identify proteins bound in the gaps.     

New genes with old modus operandi. The connection between supercoiling and partitioning of DNA in Escherichia coli

The process of partitioning bacterial sister chromosomes into daughter cells seems to be distinct from chromatid segregation during eukaryotic mitosis. In Escherichia coli, partitioning starts soon after initiation of replication, when the two newly replicated oriCs move from the cell centre to quarter positions within the cell. As replication proceeds, domains of the compact, supercoiled chromosome are locally decondensed ahead of the replication fork. The nascent daughter chromosomes are recondensed and moved apart through the concerted activities of topoisomerases and the SeqA (sequestration) and MukB (chromosome condensation) proteins, all of which modulate nucleoid superhelicity. Thus, genes involved in chromosome topology, once set aside as ‘red herrings’ in the search for ‘true’ partition functions, are again recognized as being important for chromosome partitioning in E. coli.     

Using Mahalanobis distance to compare genomic signatures between bacterial plasmids and chromosomes

Plasmids are ubiquitous mobile elements that serve as a pool of many host beneficial traits such as antibiotic resistance in bacterial communities. To understand the importance of plasmids in horizontal gene transfer, we need to gain insight into the ‘evolutionary history’ of these plasmids, i.e. the range of hosts in which they have evolved. Since extensive data support the proposal that foreign DNA acquires the host's nucleotide composition during long-term residence, comparison of nucleotide composition of plasmids and chromosomes could shed light on a plasmid's evolutionary history. The average absolute dinucleotide relative abundance difference, termed δ-distance, has been commonly used to measure differences in dinucleotide composition, or ‘genomic signature’, between bacterial chromosomes and plasmids. Here, we introduce the Mahalanobis distance, which takes into account the variance–covariance structure of the chromosome signatures. We demonstrate that the Mahalanobis distance is better than the δ-distance at measuring genomic signature differences between plasmids and chromosomes of potential hosts. We illustrate the usefulness of this metric for proposing candidate long-term hosts for plasmids, focusing on the virulence plasmids pXO1 from Bacillus anthracis, and pO157 from Escherichia coli O157:H7, as well as the broad host range multi-drug resistance plasmid pB10 from an unknown host.     

E. coli Fis Protein Insulates the cbpA Gene from Uncontrolled Transcription

The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual “insulation” mechanism. Thus, Fis protects cbpA from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the E. coli chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.     

Insights into the Cellular Function of YhdE,a Nucleotide Pyrophosphatase from Escherichia coli

YhdE, a Maf-like protein in Escherichia coli, exhibits nucleotide pyrophosphatase (PPase) activity, yet its cellular function remains unknown. Here, we characterized the PPase activity of YhdE on dTTP, UTP and TTP and determined two crystal structures of YhdE, revealing ‘closed’ and ‘open’ conformations of an adaptive active site. Our functional studies demonstrated that YhdE retards cell growth by prolonging the lag and log phases, particularly under stress conditions. Morphology studies showed that yhdE-knockout cells transformed the normal rod shape of wild-type cells to a more spherical form, and the cell wall appeared to become more flexible. In contrast, YhdE overexpression resulted in filamentous cells. This study reveals the previously unknown involvement of YhdE in cell growth inhibition under stress conditions, cell-division arrest and cell-shape maintenance, highlighting YhdE’s important role in E. coli cell-cycle checkpoints.     

Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence

The emergence of pathogenic strains of enteric bacteria and their adaptation to unique niches are associated with the acquisition of foreign DNA segments termed ‘genetic islands’. We explored these islands for the occurrence of small RNA (sRNA) encoding genes. Previous systematic screens for enteric bacteria sRNAs were mainly carried out using the laboratory strain Escherichia coli K12, leading to the discovery of ~80 new sRNA genes. These searches were based on conservation within closely related members of enteric bacteria and thus, sRNAs, unique to pathogenic strains were excluded. Here we describe the identification and characterization of 19 novel unique sRNA genes encoded within the ‘genetic islands’ of the virulent strain Salmonella typhimurium. We show that the expression of many of the island-encoded genes is associated with stress conditions and stationary phase. Several of these sRNA genes are induced when Salmonella resides within macrophages. One sRNA, IsrJ, was further examined and found to affect the translocation efficiency of virulence-associated effector proteins into nonphagocytic cells. In addition, we report that unlike the majority of the E. coli sRNAs that are trans regulators, many of the island-encoded sRNAs affect the expression of cis-encoded genes. Our study suggests that the island encoded sRNA genes play an important role within the network that regulates bacterial adaptation to environmental changes and stress conditions and thus controls virulence.     

Ecological fitness, genomic islands and bacterial pathogenicity. A Darwinian view of the evolution of microbes

The compositions of bacterial genomes can be changed rapidly and dramatically through a variety of processes including horizontal gene transfer. This form of change is key to bacterial evolution, as it leads to ‘evolution in quantum leaps’. Horizontal gene transfer entails the incorporation of genetic elements transferred from another organism—perhaps in an earlier generation—directly into the genome, where they form ‘genomic islands’, i.e. blocks of DNA with signatures of mobile genetic elements. Genomic islands whose functions increase bacterial fitness, either directly or indirectly, have most likely been positively selected and can be termed ‘fitness islands’. Fitness islands can be divided into several subtypes: ‘ecological islands’ in environmental bacteria and ‘saprophytic islands’, ‘symbiosis islands’ or ‘pathogenicity islands’ (PAIs) in microorganisms that interact with living hosts. Here we discuss ways in which PAIs contribute to the pathogenic potency of bacteria, and the idea that genetic entities similar to genomic islands may also be present in the genomes of eukaryotes.     

DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study

We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the –10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the –10 and –35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions –15 and –14. Promoter activity is greatest when the ‘non-template’ strand carries T and G at positions –15 and –14, respectively. Promoter activity can be further enhanced by a second T and G at positions –17 and –16, respectively, immediately upstream of the first ‘TG motif’. Our results show that the base sequence of the DNA segment upstream of the –10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of ‘TG motifs’ upstream of the promoters’ –10 elements. We conclude that correctly placed ‘TG motifs’ are found at over 20% of E.coli promoters.     

Integration Host Factor of Mycobacterium tuberculosis,mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.     

APOBEC-1 dependent cytidine to uridine editing of apolipoprotein B RNA in yeast

Cytidine to uridine editing of apolipoprotein B (apoB) mRNA requires the cytidine deaminase APOBEC-1 as well as a tripartite sequence motif flanking a target cytidine in apoB mRNA and an undefined number of auxiliary proteins that mediate RNA recognition and determine site-specific editing. Yeast engineered to express APOBEC-1 and apoB mRNA supported editing under conditions of late log phase growth and stationary phase. The cis-acting sequence requirements and the intracellular distribution of APOBEC-1 in yeast were similar to those described in mammalian cells. These findings suggest that auxiliary protein functions necessary for the assembly of editing complexes, or ‘editosomes’, are expressed in yeast and that the distribution of editing activity is to the cell nucleus.     

The translation of recombinant proteins in E. coli can be improved by in silico generating and screening random libraries of a −70/+96 mRNA region with respect to the translation initiation codon

Recombinant protein translation in Escherichia coli may be limited by stable (i.e. low free energy) secondary structures in the mRNA translation initiation region. To circumvent this issue, we have set-up a computer tool called ‘ExEnSo’ (Expression Enhancer Software) that generates a random library of 8192 sequences, calculates the free energy of secondary structures of each sequence in the −70/+96 region (base 1 is the translation initiation codon), and then selects the sequence having the highest free energy. The software uses this ‘optimized’ sequence to create a 5′ primer that can be used in PCR experiments to amplify the coding sequence of interest prior to sub-cloning into a prokaryotic expression vector. In this article, we report how ExEnSo was set-up and the results obtained with nine coding sequences with low expression levels in E. coli. The free energy of the −70/+96 region of all these coding sequences was increased compared to the non-optimized sequences. Moreover, the protein expression of eight out of nine of these coding sequences was increased in E. coli, indicating a good correlation between in silico and in vivo results. ExEnSo is available as a free online tool.     

Changes in bacterial cells induced by high pressure at subzero temperature

The aim of this study was to examine the effect of pressure treatment at 193 MPa and −20 °C on membrane damage, changes in activity of membrane-bound ATPases and degradation of nucleic acids. The experiments were carried out with three Escherichia coli strains, in the exponential and stationary phases of growth, and differing in sensitivity to pressure. All E. coli strains subjected to pressure in the exponential phase of growth were inactivated by 6 log cycles, independently of the strain, which was accompanied by a total loss of ability to plasmolyse, an increase in irreversible membrane permeability to PI, and a reduction of cellular ATP by more than 80%. After pressure treatment of stationary phase cells, the relationship between the inactivation level and the ability to plasmolyse was not as evident as in the case of exponential phase cells. Pressure treatment of two strains of E. coli K-12 and Ec160/59 in the stationary phase that decreased viability by no more than one log cycle led only to reversible permeabilization of bacterial membranes, while irreversible permeabilization was observed in the pressure sensitive E. coli IBA72 strain phase that was inactivated by 4.6 log cycles. The reduction of ATP and changes in ATPase activity after pressure treatment of tested E. coli strains in the stationary phase of growth depended on the stage of inactivation of the particular strain. Electrophoretic analysis showed degradation of RNA isolated after pressure treatment from cells of all E. coli strains tested in the exponential phase of growth. The changes of RNA induced by pressure were not visible in the case of cells in the stationary phase. The degradation of DNA isolated from pressure treated E. coli strains from the exponential as well as from the stationary phase of growth was not observed.     

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3′-phosphate and 5′-hydroxyl termini. A crystallographic analysis showed that Ba²⁺, Mn²⁺, Co²⁺ and Zn²⁺ bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an ‘in-line’ geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a ‘Trojan horse’ mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a ‘harmless’ species is further transformed in situ into an ‘aggressive’ species carrying a hydroxide anion.     

Neurocognition,Insight and Medication Nonadherence in Schizophrenia: A Structural Equation Modeling Approach

Objective

The aim of this study was to examine the complex relationships among neurocognition, insight and nonadherence in patients with schizophrenia.

Methods

Design: Cross-sectional study. Inclusion criteria: Diagnosis of schizophrenia according to the DSM-IV-TR criteria. Data collection: Neurocognition was assessed using a global approach that addressed memory, attention, and executive functions; insight was analyzed using the multidimensional ‘Scale to assess Unawareness of Mental Disorder;’ and nonadherence was measured using the multidimensional ‘Medication Adherence Rating Scale.’ Analysis: Structural equation modeling (SEM) was applied to examine the non-straightforward relationships among the following latent variables: neurocognition, ‘awareness of positive symptoms’ and ‘negative symptoms’, ‘awareness of mental disorder’ and nonadherence.

Results

One hundred and sixty-nine patients were enrolled. The final testing model showed good fit, with normed χ² = 1.67, RMSEA = 0.063, CFI = 0.94, and SRMR = 0.092. The SEM revealed significant associations between (1) neurocognition and ‘awareness of symptoms,’ (2) ‘awareness of symptoms’ and ‘awareness of mental disorder’ and (3) ‘awareness of mental disorder’ and nonadherence, mainly in the ‘attitude toward taking medication’ dimension. In contrast, there were no significant links between neurocognition and nonadherence, neurocognition and ‘awareness of mental disorder,’ and ‘awareness of symptoms’ and nonadherence.

Conclusions

Our findings support the hypothesis that neurocognition influences ‘awareness of symptoms,’ which must be integrated into a higher level of insight (i.e., the ‘awareness of mental disorder’) to have an impact on nonadherence. These findings have important implications for the development of effective strategies to enhance medication adherence.     

Bridging the membrane lipid divide: bacteria of the FCB group superphylum have the potential to synthesize archaeal ether lipids

Archaea synthesize membranes of isoprenoid lipids that are ether-linked to glycerol-1-phosphate (G1P), while Bacteria/Eukarya produce membranes consisting of fatty acids ester-bound to glycerol-3-phosphate (G3P). This dichotomy in membrane lipid composition (i.e., the ‘lipid divide’) is believed to have arisen after the Last Universal Common Ancestor (LUCA). A leading hypothesis is that LUCA possessed a heterochiral ‘mixed archaeal/bacterial membrane’. However, no natural microbial representatives supporting this scenario have been shown to exist today. Here, we demonstrate that bacteria of the Fibrobacteres–Chlorobi–Bacteroidetes (FCB) group superphylum encode a putative archaeal pathway for ether-bound isoprenoid membrane lipids in addition to the bacterial fatty acid membrane pathway. Key genes were expressed in the environment and their recombinant expression in Escherichia coli resulted in the formation of a ‘mixed archaeal/bacterial membrane’. Genomic evidence and biochemical assays suggest that the archaeal-like lipids of members of the FCB group could possess either a G1P or G3P stereochemistry. Our results support the existence of ‘mixed membranes’ in natural environments and their stability over a long period in evolutionary history, thereby bridging a once-thought fundamental divide in biology.Subject terms: Water microbiology, Cellular microbiology, Molecular evolution, Metagenomics     

The Genome Sequence of ‘Mycobacterium massiliense’ Strain CIP 108297 Suggests the Independent Taxonomic Status of the Mycobacterium abscessus Complex at the Subspecies Level

Members of the Mycobacterium abscessus complex are rapidly growing mycobacteria that are emerging as human pathogens. The M. abscessus complex was previously composed of three species, namely M. abscessus sensu stricto, ‘M. massiliense’, and ‘M. bolletii’. In 2011, ‘M. massiliense’ and ‘M. bolletii’ were united and reclassified as a single subspecies within M. abscessus: M. abscessus subsp. bolletii. However, the placement of ‘M. massiliense’ within the boundary of M. abscessus subsp. bolletii remains highly controversial with regard to clinical aspects. In this study, we revisited the taxonomic status of members of the M. abscessus complex based on comparative analysis of the whole-genome sequences of 53 strains. The genome sequence of the previous type strain of ‘Mycobacterium massiliense’ (CIP 108297) was determined using next-generation sequencing. The genome tree based on average nucleotide identity (ANI) values supported the differentiation of ‘M. bolletii’ and ‘M. massiliense’ at the subspecies level. The genome tree also clearly illustrated that ‘M. bolletii’ and ‘M. massiliense’ form a distinct phylogenetic clade within the radiation of the M. abscessus complex. The genomic distances observed in this study suggest that the current M. abscessus subsp. bolletii taxon should be divided into two subspecies, M. abscessus subsp. massiliense subsp. nov. and M. abscessus subsp. bolletii, to correspondingly accommodate the previously known ‘M. massiliense’ and ‘M. bolletii’ strains.