Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae

Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA–ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA–ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA–ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA–ramA genes, strongly suggesting that a secondary regulator may control ramA expression.

Protein kinase B/Akt regulation in diabetic kidney disease

Many reviews have been written on protein kinase B/Akt focusing on its history dating back from the isolation of the Akt8 transforming murine leukemia virus by Staal in 1977, to the co-discovery of the Akt1 gene by the three groups in 1991 (reviewed in 7). There are currently over 22,000 publications in the PubMed database with "Akt" as a keyword - these publications describe a wealth of diverse data on the physiological functions of Akt isoforms. Many of these publications describe roles of Akt ranging from its requirement for cellular processes such as glucose uptake, cell survival and angiogenesis to roles in diseases such as cancer and ischaemia (22). This review will focus on the evidence for Akt signaling in different kidney cells during diabetes, or diabetic nephropathy (DN).

Activated oncogenic pathways and therapeutic targets in extranodal nasal-type NK/T cell lymphoma revealed by gene expression profiling

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-kappa B), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-kappa B p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-kappa B, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

p63 maintains keratinocyte proliferative capacity through regulation of Skp2-p130 levels

p63 is a master regulator of proliferation and differentiation in stratifying epithelia, and its expression is frequently altered in carcinogenesis. However, its role in maintaining proliferative capacity remains unclear. Here, we demonstrate that hypoproliferation and loss of differentiation in organotypic raft cultures of primary neonatal human foreskin keratinocytes (HFKs) depleted of the a and ß isoforms of p63 result from p53-p21-mediated accumulation of retinoblastoma (Rb) family member p130. Hypoproliferation in p63-depleted HFKs can be rescued by depletion of p53, p21(CIP1) or p130. Furthermore, we identified the gene encoding S-phase kinase-associated protein 2 (Skp2), the recognition component of the SCF(Skp2) E3 ubiquitin ligase, as a novel target of p63, potentially influencing p130 levels. Expression of Skp2 is maintained by p63 binding to a site in intron 2 and mRNA levels are downregulated in p63-depleted cells. Hypoproliferation in p63-depleted cells can be restored by re-expression of Skp2. Taken together, these results indicate that p63 plays a multifaceted role in maintaining proliferation in the mature regenerating epidermis, in addition to being required for differentiation.

Comprehensive genomic screens identify a role for PLZF-RAR alpha as a positive regulator of cell proliferation via direct regulation of c-MYC

The t(11; 17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RAR alpha) and RAR alpha-PLZF. Using a combination of chromatin immuno-precipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RAR alpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RAR alpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RAR alpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RAR alpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RAR alpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RAR alpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RAR alpha. (Blood. 2009; 114: 5499-5511)

Bagging statistical network inference from large-scale gene expression data.

Modern biology and medicine aim at hunting molecular and cellular causes of biological functions and diseases. Gene regulatory networks (GRN) inferred from gene expression data are considered an important aid for this research by providing a map of molecular interactions. Hence, GRNs have the potential enabling and enhancing basic as well as applied research in the life sciences. In this paper, we introduce a new method called BC3NET for inferring causal gene regulatory networks from large-scale gene expression data. BC3NET is an ensemble method that is based on bagging the C3NET algorithm, which means it corresponds to a Bayesian approach with noninformative priors. In this study we demonstrate for a variety of simulated and biological gene expression data from S. cerevisiae that BC3NET is an important enhancement over other inference methods that is capable of capturing biochemical interactions from transcription regulation and protein-protein interaction sensibly. An implementation of BC3NET is freely available as an R package from the CRAN repository. © 2012 de Matos Simoes, Emmert-Streib.

A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded Wzz(pHS-2) proteins, respectively. In this study, genes wzzB, wzz(pHS-2) and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz(pHS-2) revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and Wzz(pHS-2) proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.

Growth-phase regulation of lipopolysaccharide O-antigen chain length influences serum resistance in serovars of Salmonella

The amount of lipopolysaccharide (LPS) O antigen (OAg) and its chain length distribution are important factors that protect bacteria from serum complement. Salmonella enterica serovar Typhi produces LPS with long chain length distribution (L-OAg) controlled by the wzz gene, whereas serovar Typhimurium produces LPS with two OAg chain lengths: an L-OAg controlled by Wzz(ST) and a very long (VL) OAg determined by Wzz(fepE). This study shows that serovar Enteritidis also has a bimodal OAg distribution with two preferred OAg chain lengths similar to serovar Typhimurium. It was reported previously that OAg production by S. Typhi increases at the late exponential and stationary phases of growth. The results of this study demonstrate that increased amounts of L-OAg produced by S. Typhi grown to stationary phase confer higher levels of bacterial resistance to human serum. Production of OAg by serovars Typhimurium and Enteritidis was also under growth-phase-dependent regulation; however, while the total amount of OAg increased during growth, the VL-OAg distribution remained constant. The VL-OAg distribution was primarily responsible for complement resistance, protecting the non-typhoidal serovars from the lytic action of serum irrespective of the growth phase. As a result, the non-typhoidal species were significantly more resistant than S. Typhi to human serum. When S. Typhi was transformed with a multicopy plasmid containing the S. Typhimurium wzz(fepE) gene, resistance to serum increased to levels comparable to the non-typhoidal serovars. In contrast to the relevant role for high-molecular-mass OAg molecules, the presence of Vi antigen did not contribute to serum resistance of clinical isolates of serovar Typhi.

O-antigen modal chain length in Shigella flexneri 2a is growth-regulated through RfaH-mediated transcriptional control of the wzy gene

Shigella flexneri 2a 2457T produces lipopolysaccharide (LPS) with two O-antigen (OAg) chain lengths: a short (S-OAg) controlled by WzzB and a very long (VL-OAg) determined by Wzz(pHS-2). This study demonstrates that the synthesis and length distribution of the S. flexneri OAg are under growth-phase-dependent regulation. Quantitative electrophoretic analysis showed that the VL-OAg increased during growth while the S-OAg distribution remained constant. Increased production of VL-OAg correlated with the growth-phase-regulated expression of the transcription elongation factor RfaH, and was severely impaired in a DeltarfaH mutant, which synthesized only low-molecular-mass OAg molecules and a small amount of S-OAg. Real-time RT-PCR revealed a drastic reduction of wzy polymerase gene expression in the DeltarfaH mutant. Complementation of this mutant with the wzy gene cloned into a high-copy-number plasmid restored the bimodal OAg distribution, suggesting that cellular levels of Wzy influence not only OAg polymerization but also chain-length distribution. Accordingly, overexpression of wzy in the wild-type strain resulted in production of a large amount of high-molecular-mass OAg molecules. An increased dosage of either wzzB or wzz(pHS-2) also altered OAg chain-length distribution. Transcription of wzzB and wzz(pHS-2) genes was regulated during bacterial growth but in an RfaH-independent manner. Overall, these findings indicate that expression of the wzy, wzzB and wzz(pHS-2) genes is finely regulated to determine an appropriate balance between the proteins responsible for polymerization and chain-length distribution of S. flexneri OAg.

An expression vector containing a rhamnose-inducible promoter provides tightly regulated gene expression in Burkholderia cenocepacia

Infection of the respiratory tract caused by Burkholderia cepacia complex poses a serious risk for cystic fibrosis (CF) patients due to the high morbidity and mortality associated with the chronic infection and the lack of efficacious antimicrobial treatments. A detailed understanding of the pathogenicity of B. cepacia complex infections is hampered in part by the limited availability of genetic tools and the inherent resistance of these isolates to the most common antibiotics used for genetic selection. In this study, we report the construction of an expression vector which uses the rhamnose-regulated P(rhaB) promoter of Escherichia coli. The functionality of the vector was assessed by expressing the enhanced green fluorescent protein (eGFP) gene (e-gfp) and determining the levels of fluorescence emission. These experiments demonstrated that P(rhaB) is responsive to low concentrations of rhamnose and it can be effectively repressed with 0.2% glucose. We also demonstrate that the tight regulation of gene expression by P(rhaB) promoter allows us to extend the capabilities of this vector to the identification of essential genes.

RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi

We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

O-antigen expression in Salmonella enterica serovar Typhi is regulated by nitrogen availability through RpoN-mediated transcriptional control of the rfaH gene

The authors previously reported increased expression of the Salmonella enterica serovar Typhi (S. typhi) rfaH gene when the bacterial cells reach stationary phase. In this study, using a lacZ fusion to the rfaH promoter region, they demonstrate that growth-dependent regulation of rfaH expression occurs at the level of transcription initiation. It was also observed that production of the lipopolysaccharide (LPS) O-antigen by S. typhi Ty2 correlated with the differential expression of rfaH during bacterial growth. This was probably due to the increased cellular levels of RfaH, since expression of the distal gene in the O-antigen gene cluster of S. typhi Ty2, wbaP, was also increased during stationary growth, as demonstrated by RT-PCR analysis. Examination of the sequences upstream of the rfaH coding region revealed homologies to potential binding sites for the RcsB/RcsA dimer of the RcsC/YopJ/RcsB phosphorelay regulatory system and for the RpoN alternative sigma factor. The expression of the rfaH gene in rpoN and rcsB mutants of S. typhi Ty2 was measured. The results indicate that inactivation of rpoN, but not of rcsB, suppresses the growth-phase-dependent induction of rfaH expression. Furthermore, production of beta-galactosidase mediated by the rfaH-lacZ fusion increased approximately fourfold when bacteria were grown in a nitrogen-limited medium. Nitrogen limitation was also shown to increase the expression of the O-antigen by the wild-type S. typhi Ty2, as demonstrated by a similar electrophoretic profile to that observed during the stationary phase of growth in rich media. It is therefore concluded that the relationship between LPS production and nitrogen limitation parallels the pattern of rfaH regulation under the control of RpoN and is consistent with the idea that RpoN modulates LPS formation via its effect on rfaH gene expression during bacterial growth.

Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster:structural and functional characterization of an upstream untranslated mRNA sequence

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E. coli K1

We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187. Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30. However, in the E. coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used. RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E. coli K-12. In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30. Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems. DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.