A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.     

H-NS Facilitates Sequence Diversification of Horizontally Transferred DNAs during Their Integration in Host Chromosomes

Bacteria can acquire new traits through horizontal gene transfer. Inappropriate expression of transferred genes, however, can disrupt the physiology of the host bacteria. To reduce this risk, Escherichia coli expresses the nucleoid-associated protein, H-NS, which preferentially binds to horizontally transferred genes to control their expression. Once expression is optimized, the horizontally transferred genes may actually contribute to E. coli survival in new habitats. Therefore, we investigated whether and how H-NS contributes to this optimization process. A comparison of H-NS binding profiles on common chromosomal segments of three E. coli strains belonging to different phylogenetic groups indicated that the positions of H-NS-bound regions have been conserved in E. coli strains. The sequences of the H-NS-bound regions appear to have diverged more so than H-NS-unbound regions only when H-NS-bound regions are located upstream or in coding regions of genes. Because these regions generally contain regulatory elements for gene expression, sequence divergence in these regions may be associated with alteration of gene expression. Indeed, nucleotide substitutions in H-NS-bound regions of the ybdO promoter and coding regions have diversified the potential for H-NS-independent negative regulation among E. coli strains. The ybdO expression in these strains was still negatively regulated by H-NS, which reduced the effect of H-NS-independent regulation under normal growth conditions. Hence, we propose that, during E. coli evolution, the conservation of H-NS binding sites resulted in the diversification of the regulation of horizontally transferred genes, which may have facilitated E. coli adaptation to new ecological niches.     

Disruption of a ∼23–24 nucleotide small RNA pathway elevates DNA damage responses in Tetrahymena thermophila

Endogenous RNA interference (RNAi) pathways regulate a wide range of cellular processes in diverse eukaryotes, yet in the ciliated eukaryote, Tetrahymena thermophila, the cellular purpose of RNAi pathways that generate ∼23–24 nucleotide (nt) small (s)RNAs has remained unknown. Here, we investigated the phenotypic and gene expression impacts on vegetatively growing cells when genes involved in ∼23–24 nt sRNA biogenesis are disrupted. We observed slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1Δ, RDN2Δ, and RDF2Δ. In addition, RSP1Δ and RDN2Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are nonnuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1Δ and RDN2Δ strains, with Rad51 and double-stranded DNA break marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci was also found in knockouts of TWI8, a macronucleus-localized PIWI protein. Together, our findings suggest that an evolutionarily conserved role for RNAi pathways in maintaining genome integrity may be extended even to the early branching eukaryotic lineage that gave rise to Tetrahymena thermophila.     

The yhhP Gene Encoding a Small Ubiquitous Protein Is Fundamental for Normal Cell Growth of Escherichia coli

H-NS is a major constituent of the Escherichia coli nucleoid, whereas ς^S is a stress-induced sigma factor. An hns null mutation affects the cellular content of ς^S in such a way that a remarkable accumulation of ς^S is observed in the logarithmic growth phase, which results in enhanced expression of a number of ς^S-dependent genes, including the katE gene. We isolated an extragenic mutation that affects the expression of the katE-lacZ fusion gene in the Δhns background. The relevant gene was identified as yhhP, which encodes a small polypeptide of 81 amino acids. Lesion of this gene seemed to affect the stability of ς^S. A deletion analysis of yhhP revealed that this small protein plays a fundamental role in the general physiology of E. coli. The yhhP-deficient cell is not capable of growing in standard laboratory rich medium (i.e., Luria broth), resulting in the formation of filamentous cells. Homologs of this intriguing protein occur in a wide variety of bacterial species, including archaeal species.     

Fate of the H-NS–Repressed bgl Operon in Evolution of Escherichia coli

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS–repressed locus is the bgl (aryl-β,D-glucoside) operon of E. coli. This locus is “cryptic,” as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli.     

Natural variation of the Y chromosome suppresses sex ratio distortion and modulates testis-specific gene expression in Drosophila simulans

X-linked sex-ratio distorters that disrupt spermatogenesis can cause a deficiency in functional Y-bearing sperm and a female-biased sex ratio. Y-linked modifiers that restore a normal sex ratio might be abundant and favored when a X-linked distorter is present. Here we investigated natural variation of Y-linked suppressors of sex-ratio in the Winters systems and the ability of these chromosomes to modulate gene expression in Drosophila simulans. Seventy-eight Y chromosomes of worldwide origin were assayed for their resistance to the X-linked sex-ratio distorter gene Dox. Y chromosome diversity caused males to sire ∼63% to ∼98% female progeny. Genome-wide gene expression analysis revealed hundreds of genes differentially expressed between isogenic males with sensitive (high sex ratio) and resistant (low sex ratio) Y chromosomes from the same population. Although the expression of about 75% of all testis-specific genes remained unchanged across Y chromosomes, a subset of post-meiotic genes was upregulated by resistant Y chromosomes. Conversely, a set of accessory gland-specific genes and mitochondrial genes were downregulated in males with resistant Y chromosomes. The D. simulans Y chromosome also modulated gene expression in XXY females in which the Y-linked protein-coding genes are not transcribed. The data suggest that the Y chromosome might exert its regulatory functions through epigenetic mechanisms that do not require the expression of protein-coding genes. The gene network that modulates sex ratio distortion by the Y chromosome is poorly understood, other than that it might include interactions with mitochondria and enriched for genes expressed in post-meiotic stages of spermatogenesis.     

Genetic Architecture and Fitness of Bacterial Interspecies Hybrids

Integration of a conjugative plasmid into a bacterial chromosome can promote the transfer of chromosomal DNA to other bacteria. Intraspecies chromosomal conjugation is believed responsible for creating the global pathogens Klebsiella pneumoniae ST258 and Escherichia coli ST1193. Interspecies conjugation is also possible but little is known about the genetic architecture or fitness of such hybrids. To study this, we generated by conjugation 14 hybrids of E. coli and Salmonella enterica. These species belong to different genera, diverged from a common ancestor >100 Ma, and share a conserved order of orthologous genes with ∼15% nucleotide divergence. Genomic analysis revealed that all but one hybrid had acquired a contiguous segment of donor E. coli DNA, replacing a homologous region of recipient Salmonella chromosome, and ranging in size from ∼100 to >4,000 kb. Recombination joints occurred in sequences with higher-than-average nucleotide identity. Most hybrid strains suffered a large reduction in growth rate, but the magnitude of this cost did not correlate with the length of foreign DNA. Compensatory evolution to ameliorate the cost of low-fitness hybrids pointed towards disruption of complex genetic networks as a cause. Most interestingly, 4 of the 14 hybrids, in which from 45% to 90% of the Salmonella chromosome was replaced with E. coli DNA, showed no significant reduction in growth fitness. These data suggest that the barriers to creating high-fitness interspecies hybrids may be significantly lower than generally appreciated with implications for the creation of novel species.     

Phenotypic Analysis of Random hns Mutations Differentiate DNA-Binding Activity from Properties of fimA Promoter Inversion Modulation and Bacterial Motility

H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of the proU and fimB genes, increased fimA promoter inversion rates, and repression of the flhCD master operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proU expression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimB occurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hns mutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.     

Hir Proteins Are Required for Position-Dependent Gene Silencing in Saccharomyces cerevisiae in the Absence of Chromatin Assembly Factor I

Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacΔ mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacΔ mutants, although hirΔ mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacΔ mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families.     

Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli

Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused ∼5-fold increase of the lysis protein. Gel mobility shift assays established that both the single-stranded loop of the T1 stem–loop distal to cei, and the putative CsrA binding site overlapping the Shine–Dalgarno sequence (SD) of the cel gene are important for CsrA binding. Substitution mutations at SD relieved CsrA-dependent repression of the cel gene in vivo. Steady-state levels and half-life of the cel mRNA were not affected by CsrA, implying that regulation is mediated at the translational level. Levels of CsrB and CsrC sRNAs, which bind to and antagonize CsrA, were drastically reduced upon induction of the SOS response, while the CsrA protein itself remained unaffected. Thus, CsrA is a trans-acting modulator that downregulates the expression of lysis protein, which may confer a survival advantage on colicinogenic E. coli under environment stress conditions.     

Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H,a Bacteriocin Produced by Bacillus thuringiensis SF361

Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared.     

Systematic Production of Inactivating and Non-Inactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli

In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into relA⁺ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase.     

Temperature-Sensitive Lethal Mutations on Yeast Chromosome I Appear to Define Only a Small Number of Genes

A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N -nitrosoguanidine and screened for temperature-sensitive (Ts^- ) mutants capable of growth on rich, glucose-containing medium at 25° but not at 37°. Recessive mutations induced on chromosome I are expressed, whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely. — Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss. — The available information about the size of chromosome I suggests that it should contain approximately 60–100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts^--lethal phenotype. — During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.     

Xenogeneic nucleoid-associated EnrR thwarts H-NS silencing of bacterial virulence with unique DNA binding

Type III and type VI secretion systems (T3/T6SS) are encoded in horizontally acquired genomic islands (GIs) that play crucial roles in evolution and virulence in bacterial pathogens. T3/T6SS expression is subjected to tight control by the host xenogeneic silencer H-NS, but how this mechanism is counteracted remains to be illuminated. Here, we report that xenogeneic nucleoid-associated protein EnrR encoded in a GI is essential for virulence in pathogenic bacteria Edwardsiella and Salmonella. We showed that EnrR plays critical roles in T3/T6SS expression in these bacteria. Various biochemical and genetic analyses demonstrated that EnrR binds and derepresses the promoter of esrB, the critical regulator of T3/T6SS, to promote their expression by competing with H-NS. Additionally, EnrR targets AT-rich regions, globally modulates the expression of ∼363 genes and is involved in various cellular processes. Crystal structures of EnrR in complex with a specific AT-rich palindromic DNA revealed a new DNA-binding mode that involves conserved HTH-mediated interactions with the major groove and contacts of its N-terminal extension to the minor groove in the symmetry-related duplex. Collectively, these data demonstrate that EnrR is a virulence activator that can antagonize H-NS, highlighting a unique mechanism by which bacterial xenogeneic regulators recognize and regulate foreign DNA.