Modularity of Escherichia coli sRNA regulation revealed by sRNA-target and protein network analysis

<p>Abstract</p> <p>Background</p> <p>sRNAs, which belong to the non-coding RNA family and range from approximately 50 to 400 nucleotides, serve various important gene regulatory roles. Most are believed to be <it>trans</it>-regulating and function by being complementary to their target mRNAs in order to inhibiting translation by ribosome occlusion. Despite this understanding of their functionality, the global properties associated with regulation by sRNAs are not yet understood. Here we use topological analysis of sRNA targets in terms of protein-protein interaction and transcription-regulatory networks in <it>Escherichia coli </it>to shed light on the global correlation between sRNA regulation and cellular control networks.</p> <p>Results</p> <p>The analysis of sRNA targets in terms of their networks showed that some specific network properties could be identified. In protein-protein interaction network, sRNA targets tend to occupy more central positions (higher closeness centrality, <it>p-val </it>= 0.022) and more cliquish (larger clustering coefficient, <it>p-val </it>= 0.037). The targets of the same sRNA tend to form a network module (shorter characteristic path length, <it>p-val </it>= 0.015; larger density, <it>p-val </it>= 0.019; higher in-degree ratio, <it>p-val </it>= 0.009). Using the transcription-regulatory network, sRNA targets tend to be under multiple regulation (higher indegree, <it>p-val </it>= 0.013) and the targets usually are important to the transfer of regulatory signals (higher betweenness, <it>p-val </it>= 0.012). As was found for the protein-protein interaction network, the targets that are regulated by the same sRNA also tend to be closely knit within the transcription-regulatory network (larger density, <it>p-val </it>= 0.036), and inward interactions between them are greater than the outward interactions (higher in-degree ratio, <it>p-val </it>= 0.023). However, after incorporating information on predicted sRNAs and down-stream targets, the results are not as clear-cut, but the overall network modularity is still evident.</p> <p>Conclusions</p> <p>Our results indicate that sRNA targeting tends to show a clustering pattern that is similar to the human microRNA regulation associated with protein-protein interaction network that was observed in a previous study. Namely, the sRNA targets show close interaction and forms a closely knit network module for both the protein-protein interaction and the transcription-regulatory networks. Thus, targets of the same sRNA work in a concerted way toward a specific goal. In addition, in the transcription-regulatory network, sRNA targets act as "multiplexor", accepting regulatory control from multiple sources and acting accordingly. Our results indicate that sRNA targeting shows different properties when compared to the proteins that form cellular networks.</p

Low-pass sequencing for microbial comparative genomics

BACKGROUND: We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed Halobacterium sp. NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe. RESULTS: As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits. All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins. Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in H. lacusprofundi and H. marismortui. The core biological functions that govern cellular and genetic mechanisms of H. sp. NRC-1 appear to be conserved in these four other halophiles. Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles. The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between H. sp. NRC-1 and H. lacusprofundi. CONCLUSION: Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively. Identification of multiple IS elements in the genome of H. lacusprofundi and H. marismortui suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich genome of H. sp. NRC-1. Identification of multiple TBP and TFB homologs in these four halophiles are consistent with the hypothesis that different types of complex transcriptional regulation may occur through multiple TBP-TFB combinations in response to rapidly changing environmental conditions. Low-pass shotgun sequence analyses of genomes permit extensive and diverse analyses, and should be generally useful for comparative microbial genomics

Complexity of the Mycoplasma fermentans M64 Genome and Metabolic Essentiality and Diversity among Mycoplasmas

Recently, the genomes of two Mycoplasma fermentans strains, namely M64 and JER, have been completely sequenced. Gross comparison indicated that the genome of M64 is significantly bigger than the other strain and the difference is mainly contributed by the repetitive sequences including seven families of simple and complex transposable elements ranging from 973 to 23,778 bps. Analysis of these repeats resulted in the identification of a new distinct family of Integrative Conjugal Elements of M. fermentans, designated as ICEF-III. Using the concept of “reaction connectivity”, the metabolic capabilities in M. fermentans manifested by the complete and partial connected biomodules were revealed. A comparison of the reported M. pulmonis, M. arthritidis, M. genitalium, B. subtilis, and E. coli essential genes and the genes predicted from the M64 genome indicated that more than 73% of the Mycoplasmas essential genes are preserved in M. fermentans. Further examination of the highly and partly connected reactions by a novel combinatorial phylogenetic tree, metabolic network, and essential gene analysis indicated that some of the pathways (e.g. purine and pyrimidine metabolisms) with partial connected reactions may be important for the conversions of intermediate metabolites. Taken together, in light of systems and network analyses, the diversity among the Mycoplasma species was manifested on the variations of their limited metabolic abilities during evolution

Proteomics Characterization of Cytoplasmic and Lipid-Associated Membrane Proteins of Human Pathogen Mycoplasma fermentans M64

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen

Genome sequence of \u3cem\u3eHalobacterium\u3c/em\u3e species NRC-1

We report the complete sequence of an extreme halophile,Halobacterium sp.NRC-1, harboring a dynamic 2,571,010-bp ge-nome containing 91 insertion sequences representing 12 familiesand organized into a large chromosome and 2 related minichro-mosomes. TheHalobacteriumNRC-1 genome codes for 2,630 pre-dicted proteins, 36% of which are unrelated to any previouslyreported. Analysis of the genome sequence shows the presence ofpathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticatedphotosensory and signal transduction pathways, and DNA repli-cation, transcription, and translation systems resembling morecomplex eukaryotic organisms. Whole proteome comparisonsshow the definite archaeal nature of this halophile with additionalsimilarities to the Gram-positiveBacillus subtilisand other bacteria.The ease of culturingHalobacteriumand the availability of meth-ods for its genetic manipulation in the laboratory, including con-struction of gene knockouts and replacements, indicate this halo-phile can serve as an excellent model system among the archaea

Understanding the Adaptation of Halobacterium Species NRC-1 to Its Extreme Environment through Computational Analysis of Its Genome Sequence

The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity. (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome. (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer. (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution. (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character

Genomic and genetic dissection of an archaeal regulon

The extremely halophilic archaeon Halobacterium sp. NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane. Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen. Analysis of the complete Halobacterium sp. NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes. The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix-turn-helix DNA binding motif. We also provide evidence for involvement of the PAS͞PAC domain of Bat in redoxsensing activity by genetic analysis of a purple membrane overproducer. Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen. Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a ␥-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life. Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine-pyrimidine sequences (RY boxes) near the regulated promoters. H alobacterium species produce a specialized region in the cell membrane, named purple membrane, which consists of a two-dimensional crystalline lattice of a single chromoprotein, bacteriorhodopsin (BR). BR contains a 1:1 complex between a protein component bacterio-opsin and a chromophore retinal, and carries out light-driven proton pumping across the membrane (1, 2). BR is highly induced in late exponential to stationary phase of growth in response to high light intensity and low oxygen tension (3, 4). Proton pumping by BR is used to drive ATP synthesis and can sustain a period of phototrophic growth (1, 2). The mechanism of proton translocation across the membrane by BR has been the subject of extensive study by biophysical and mutational approaches (5, 6). Recently, retinal proteins similar to BR have been discovered in some fungi and uncultivated marine planktonic bacteria, indicating a much wider distribution in nature than originally appreciated In Halobacterium sp. NRC-1, the proteins for biogenesis of the purple membrane are coded by the bop gene, specifying bacterioopsin, and several nearby genes, e.g., brp, bat, blp, and crtB1, thought to be involved in synthesis of the chromophore, genetic regulation, or biogenesis of the membrane In this report, we have used a combination of genomic and genetic approaches to more fully characterize the purple membrane regulon. Evidence is provided for coordinate regulation of bacterio-opsin and retinal chromophore synthesis, and we also discuss mechanisms for sensing oxygen and light, by the pleiotropic regulator, Bat. Materials and Methods Halobacterium Strains and Culturing. Halobacterium NRC-1 is the wild-type strain. Other strains used include Halobacterium S9, a purple membrane (Pum) constitutive strain, and SD20, a Pum Ϫ strain derivative of S9 with an ISH1 insertion in the bat gene Identification of UAS. The UAS consensus sequence (5Ј-ACCcnactagTTnGG-3Ј) (13, 14) was used in FINDPATTERNS analysis (16) (with 1-4 mismatches) of the whole Halobacterium NRC-1 genome sequence. The criteria used for subsequent screening of hits were the following: (i) the UAS should be upstream to a TATA box consensus sequence (5Ј-rtyTT͞aTa-3Ј

Low-pass sequencing for microbial comparative genomics

<p>Abstract</p> <p>Background</p> <p>We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile <it>Haloarcula marismortui</it>; (2) the non-pigmented <it>Natrialba asiatica</it>; (3) the psychrophile <it>Halorubrum lacusprofundi </it>and (4) the Dead Sea isolate <it>Halobaculum gomorrense</it>. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed <it>Halobacterium sp. </it>NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe.</p> <p>Results</p> <p>As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits. All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins. Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in <it>H. lacusprofundi </it>and <it>H. marismortui. </it>The core biological functions that govern cellular and genetic mechanisms of <it>H. sp. </it>NRC-1 appear to be conserved in these four other halophiles. Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles. The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between <it>H. sp. </it>NRC-1 and <it>H. lacusprofundi.</it></p> <p>Conclusion</p> <p>Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively. Identification of multiple IS elements in the genome of <it>H. lacusprofundi </it>and <it>H. marismortui </it>suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich genome of <it>H. sp. </it>NRC-1. Identification of multiple TBP and TFB homologs in these four halophiles are consistent with the hypothesis that different types of complex transcriptional regulation may occur through multiple TBP-TFB combinations in response to rapidly changing environmental conditions. Low-pass shotgun sequence analyses of genomes permit extensive and diverse analyses, and should be generally useful for comparative microbial genomics.</p

Distribution and Genomic Characterization of Third-Generation Cephalosporin-Resistant <i>Escherichia coli</i> Isolated from a Single Family and Home Environment: A 2-Year Longitudinal Study

Third-generation cephalosporin-resistant Escherichia coli (CREC), particularly strains producing extended-spectrum β-lactamases (ESBLs), are a global concern. Our study aims to longitudinally assemble the genomic characteristics of CREC isolates from fecal samples from an index patient with recurrent CREC-related urinary tract infections and his family and swabs from his home environment 12 times between 2019 and 2021 to investigate the distribution of antibiotic resistance genes. CREC identified using the VITEK 2 were subjected to nanopore whole-genome sequencing (WGS). The WGS of 27 CREC isolates discovered in 137 specimens (1 urine, 123 feces, and 13 environmental) revealed the predominance of ST101 and ST131. Among these sequence types, blaCTX-M (44.4%, n = 12) was the predominant ESBL gene family, with blaCTX-M-14 (n = 6) being the most common. The remaining 15 (55.6%) isolates harbored blaCMY-2 genes and were clonally diverse. All E. coli isolated from the index patient’s initial urine and fecal samples belonged to O25b:H4-B2-ST131 and carried blaCTX-M-14. The results of sequence analysis indicate plasmid-mediated household transmission of blaCMY-2 or blaCTX-M-55. A strong genomic similarity was discovered between fecal ESBL-producing E. coli and uropathogenic strains. Furthermore, blaCMY-2 genes were widely distributed among the CREC isolated from family members and their home environment