Christine A. Miller

Non-chromosomal antibiotic resistance in bacteria: II. Molecular nature of R-factors isolated from Proteus mirabilis and Escherichia coli☆☆☆

Covalently closed circular species of R-factor† DNA have been isolated by Cs2SO4Hg2+ or CsCl-ethidium bromide centrifugation from both Proteus mirabilis and Escherichia coli strains which have been subjected to detergent lysis. In Proteus, the fi+ R-factors R1 and R6 consist of at least three independently replicating closed circular species of DNA banding at buoyant densities of 1.709, 1.711 and 1.718 g/cm3; in E. coli, a single molecular species having a buoyant density of 1.711 g/cm3 predominates. The respective buoyant densities and calculated molecular weights of isolated R1 and R6 DNA species suggest that the dominant molecular species present in E. coli is the largest of the three forms found in Proteus, and that this species may represent a composite of the two smaller units. Segregants of the R-factor R6 which have lost drug resistance markers but have retained the capacity for transfer have been isolated, and the circular DNA associated with these segregants in both Proteus and E. coli has been characterized by ultracentrifugation and electron microscopy. The results of these experiments suggest that the R6 DNA species banding at ϱ = 1.709 g/cm3 in Proteus is the transfer unit while the species at ϱ = 1.718 g/cm3 contains the drug resistance markers. These findings are consistent with a model depicting certain R-factors as being composed of separately replicating, genetically independent units of DNA.

SIRT1 activators suppress inflammatory responses through promotion of p65 deacetylation and inhibition of NF-κB activity.

Chronic inflammation is a major contributing factor in the pathogenesis of many age-associated diseases. One central protein that regulates inflammation is NF-κB, the activity of which is modulated by post-translational modifications as well as by association with co-activator and co-repressor proteins. SIRT1, an NAD+-dependent protein deacetylase, has been shown to suppress NF-κB signaling through deacetylation of the p65 subunit of NF-κB resulting in the reduction of the inflammatory responses mediated by this transcription factor. The role of SIRT1 in the regulation of NF-κB provides the necessary validation for the development of pharmacological strategies for activating SIRT1 as an approach for the development of a new class of anti-inflammatory therapeutics. We report herein the development of a quantitative assay to assess compound effects on acetylated p65 protein in the cell. We demonstrate that small molecule activators of SIRT1 (STACs) enhance deacetylation of cellular p65 protein, which results in the suppression of TNFα-induced NF-κB transcriptional activation and reduction of LPS-stimulated TNFα secretion in a SIRT1-dependent manner. In an acute mouse model of LPS-induced inflammation, the STAC SRTCX1003 decreased the production of the proinflammatory cytokines TNFα and IL-12. Our studies indicate that increasing SIRT1-mediated NF-κB deacetylation using small molecule activating compounds is a novel approach to the development of a new class of therapeutic anti-inflammatory agents.

Structural and functional analysis of the par region of the pSC101 plasmid

Abstract Three distinct segments (the partition-related, or PR segments) within the 370 bp par region of pSC101 have been shown by deletion analysis to be involved in partitioning of the plasmid to daughter cells. The two lateral segments are direct repeats, each of which potentially can pair with an inverted repeat located between them to form a hairpin-loop structure. Deletion of either lateral segment, together with the middle segment, results in plasmid instability (the Par − phenotype). Deletion of one PR segment yields a stable plasmid that nevertheless shows reduced ability to compete with a coexisting wild-type derivative of the same replicon (the Cmp − phenotype). Deletion of all three segments results in a rate of plasmid loss far in excess of that predicted from the observed copy number of the plasmid. Analysis of the segregation properties of these mutants and of temperature-sensitive and high copy number derivatives of the pSC101 replicon suggests a model in which the par function allows the nonreplicating plasmids of the intracellular pool to be counted as individual molecules, and to be distributed evenly to daughter cells. In the absence of par , the multicopy pool of plasmids behaves as a single segregation unit.

Nucleotide sequence of the partition locus of Escherichia coli plasmid pSC101

We report here the sequence of a 375-bp EcoRI-AvaI DNA fragment that previously has been shown to contain a locus (termed partition or par) responsible for stable maintenance of the pSC101 plasmid in growing cell populations. The DNA sequence of the par region encodes no obvious proteins and contains no segments having the structural characteristics of transcriptional or translational start signals. However, segments of the par locus appear capable of forming regions of intra-strand secondary structure, one of which resembles a rho-independent transcription terminator. Computer analysis shows regions within par that have homology with sequences found near the origin of replication of the Escherichia coli chromosome and of the pBR322 and ColE1 plasmids. The par sequence homology in the pBR322 and ColE1 plasmids maps in the vicinity of sites that interact with the E. coli replication factor Y and accomplishes initiation of DNA synthesis on single-strand templates.

Role of DNA superhelicity in partitioning of the pSC101 plasmid

Previous work has shown that a cis-acting locus (termed par for partitioning) on the pSC101 plasmid accomplishes its stable inheritance in dividing cell populations. We report here that the DNA of pSC101 derivatives lacking the par region shows a decrease in overall superhelical density as compared with DNA of wild-type pSC101. Chemicals and bacterial mutations that reduce negative DNA supercoiling increase the rate of loss of par plasmids and convert normally stable plasmids that have minimal par region deletions into unstable replicons. topA gene mutations, which increase negative DNA supercoiling, reverse the instability of partition-defective plasmids that utilize the pSC101, p15A, F, or oriC replication systems. Our observations show that the extent of negative supercoiling of plasmid DNA has major effects on the plasmids inheritance and suggest a mechanism by which the pSC101 par region may exert its stabilizing effects.

RNase E Maintenance of Proper FtsZ/FtsA Ratio Required for Nonfilamentous Growth of Escherichia coli Cells but Not for Colony-Forming Ability

Inactivation or deletion of the RNase E-encoding rne gene of Escherichia coli results in the growth of bacterial cells as filamentous chains in liquid culture (K. Goldblum and D. Apirion, J. Bacteriol. 146:128-132, 1981) and the loss of colony-forming ability (CFA) on solid media. RNase E dysfunction is also associated with abnormal processing of ftsQAZ transcripts (K. Cam, G. Rome, H. M. Krisch, and J.-P. Bouché, Nucleic Acids Res. 24:3065-3070, 1996), which encode proteins having a central role in septum formation during cell division. We show here that RNase E regulates the relative abundances of FtsZ and FtsA proteins and that RNase E depletion results in decreased FtsZ, increased FtsA, and consequently an altered FtsZ/FtsA ratio. However, while restoration of the level of FtsZ to normal in rne null mutant bacteria reverses the filamentation phenotype, it does not restore CFA. Conversely, overexpression of a related RNase, RNase G, in rne-deleted bacteria restores CFA, as previously reported, without affecting FtsZ abundance. Our results demonstrate that RNase E activity is required to maintain a proper cellular ratio of the FtsZ and FtsA proteins in E. coli but that FtsZ deficiency does not account for the nonviability of cells lacking RNase E.

Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two-component system regulator

We identified a gene (dpiAdestabilizer of plasmid inheritance) which, when overexpressed in Escherichia coli, destabilizes the inheritance of pSC101 and other iteron‐containing plasmids as disparate as mini‐F and RK6 but not the inheritance of P1, RSF1010 and ColD. These effects of DpiA, which functions like an effector protein for a previously undescribed two‐component signal transduction system, were reduced by mutations known to promote pSC101 replication and partitioning. dpiB, a gene encoding the putative histidine kinase of this two‐component system, is located immediately 5′ to dpiA and adjacent to a DpiA‐induced target promoter that transcribes genes having homology to citrate lyase operon genes, citC, citD and citE, of Klebsiella pneumoniae. Disruption of dpiB reversed or reduced the effect of DpiA overproduction on pSC101 inheritance. A second DpiA target, the promoter for a gene (appY ) implicated in E. colis response to anaerobiosis, is repressed by DpiA. A mutation in dpiA at a site commonly conserved and phosphorylated in two‐component system effector proteins abolished the effects of DpiA overproduction on pSC101 inheritance and negative regulation of appY expression. Our findings suggest a possible mechanism by which environmental and/or cellular stimuli may influence plasmid inheritance.

DpiA binding to the replication origin of Escherichia coli plasmids and chromosomes destabilizes plasmid inheritance and induces the bacterial SOS response.

The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size-both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB-both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids-reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.

Gyrase-dependent stabilization of pSC101 plasmid inheritance by transcriptionally active promoters.

The pSC101 plasmid encodes a cis‐acting genetic locus termed par that ensures the stable inheritance of plasmids in a population of dividing cells. In the absence of selection, par‐defective plasmids are lost rapidly from the bacterial population. We report here that the stability of par‐deleted pSC101 derivatives is restored by introducing certain adventitious bacterial promoters onto the plasmid. Stabilization requires active transcription from the inserted promoter and is affected by the site and orientation of the insertion, the length of the nascent transcript and DNA gyrase activity. While a promotor‐associated overall increase in negative superhelicity of plasmid DNA was observed, stabilized inheritance appeared to be dependent on localized rather than generalized supercoiling. Our demonstration that promoter‐induced DNA supercoiling can mimic the effects of the pSC101 par locus provides evidence that the previously reported superhelicity‐generating effects of par are intrinsic to its function.

The partition(par) locus of pSC101 is an enhancer of plasmid incompatibility

The incompatibitity that pSC101‐derived plasmids express toward each other is mediated by directly repeated sequences (iterons) located near the plasmids replication origin. We report here that the pSC101 par locus, which stabilizes plasmid inheritance in dividing cell populations and alters DNA superheliclty, can function as a cis‐acting enhancer of incompatibility, which we show is determined jointly by the copy number of the plasmid and the number of iterons per copy. A single synthetic 32 bp iteron sequence carried by the pUC19 plasmid confers strong pSC101‐specific incompatibility in the absence of any other pSC101 sites but requires the par locus to express strong incompatibility when carried by a lower‐copy‐number plasmid. We propose a model by which the par locus can enchance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.