Papers by Gintaras Deikus
Genome announcements, Jan 28, 2016
Carbapenem resistance in Klebsiella pneumoniae, frequently conferred by the blaKPC gene, is a maj... more Carbapenem resistance in Klebsiella pneumoniae, frequently conferred by the blaKPC gene, is a major public health threat. We sequenced a blaKPC-containing strain of K. pneumoniae belonging to the emergent lineage ST941, in order to better understand the evolution of blaKPC within this species.
The Journal of Biological Chemistry, May 16, 2007
Transcription termination in the leader region of the Bacillus subtilis trp operon is regulated b... more Transcription termination in the leader region of the Bacillus subtilis trp operon is regulated by binding of the 11-mer TRAP complex to nascent trp RNA, which results in formation of a terminator structure. Rapid decay of trp leader RNA, which is required to release the TRAP complex and maintain a sufficient supply of free TRAP, is mediated by polynucleotide phosphorylase (PNPase). Using purified B. subtilis PNPase, we showed that, when TRAP was present, PNPase binding to the 3 end of trp leader RNA and PNPase digestion of trp leader RNA from the 3 end were inefficient. These results suggested that initiation of trp leader RNA may begin with an endonuclease cleavage upstream of the transcription terminator structure. Such cleavage was observed in vivo. Mutagenesis of nucleotides at the cleavage site abolished processing and resulted in a 4-fold increase in trp leader RNA half-life. This is the first mapping of a decay-initiating endonuclease cleavage site on a native B. subtilis RNA.
Proteins, 2003
Prolonged exposure of Ca 2؉ -loaded or Ca 2؉ -depleted human ␣-lactalbumin to ultraviolet light (... more Prolonged exposure of Ca 2؉ -loaded or Ca 2؉ -depleted human ␣-lactalbumin to ultraviolet light (270 -290 nm, 1 mW/cm 2 , for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms:
Analysis of mRNA decay in Bacillus subtilis
Methods in Enzymology, Feb 1, 2008
Studies of mRNA turnover in B. subtilis are less well known than in E. coli. Here we provide rese... more Studies of mRNA turnover in B. subtilis are less well known than in E. coli. Here we provide researchers who have an interest in gram-positive RNA processing with several protocols for RNA isolation, for 5'- and 3'-mapping of mRNAs and mRNA decay fragments, and we also include a comprehensive listing of B. subtilis mutants that are deficient in ribonucleases thought to be involved in mRNA decay.
Interactions of α-lactalbumin with lipid vesicles studied by tryptophan fluorescence
Biochemistry and molecular biology international
ABSTRACT
Whole-genome sequencing identifies emergence of a quinolone resistance mutation in a case of Stenotrophomonas maltophilia bacteremia
Antimicrobial agents and chemotherapy, Jan 31, 2015
Whole genome sequences for Stenotrophomonas maltophilia serial isolates from a bacteremic patient... more Whole genome sequences for Stenotrophomonas maltophilia serial isolates from a bacteremic patient before and after development of levofloxacin resistance were assembled de novo and differed by one single-nucleotide variant in smeT, a repressor for multidrug efflux operon smeDEF. Along with sequenced isolates from five contemporaneous cases, they displayed considerable diversity compared against all published complete genomes. Whole genome sequencing and complete assembly can conclusively identify resistance mechanisms emerging in S. maltophilia strains during clinical therapy.
Extensive sequencing of seven human genomes to characterize benchmark reference materials
Bacillus subtilis trp Leader RNA
Journal of Biological Chemistry
In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter... more In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter terminates to give a 140-nucleotide trp leader RNA. Turnover of trp leader RNA has been shown to depend on RNase J1 cleavage at a single-stranded, AU-rich region just upstream of the 3′ transcription terminator. The small size of trp leader RNA and its strong dependence on RNase J1 cleavage for decay make it a suitable substrate for analyzing the requirements for RNase J1 target site specificity. trp leader RNAs with nucleotide changes around the RNase J1 target site were more stable than wild-type trp leader RNA, showing that sequences on either side of the cleavage site contribute to RNase J1 recognition. An analysis of decay intermediates from these mutants suggested limited 3′-to-5′ exonuclease processing from the native 3′ end. trp leader RNAs were designed that contained wild-type or mutant RNase J1 targets elsewhere on the molecule. The presence of an additional RNase J1 cleavage...
Methicillin-Susceptible, Vancomycin-Resistant Staphylococcus aureus , Brazil
Emerging Infectious Diseases, 2015
We report characterization of a methicillin-susceptible, vancomycin-resistant bloodstream isolate... more We report characterization of a methicillin-susceptible, vancomycin-resistant bloodstream isolate of Staphylococcus aureus recovered from a patient in Brazil. Emergence of vancomycin resistance in methicillin-susceptible S. aureus would indicate that this resistance trait might be poised to disseminate more rapidly among S. aureus and represents a major public health threat.
Assembly and diploid architecture of an individual human genome via single-molecule technologies
Nature Methods, 2015
We present the first comprehensive analysis of a diploid human genome that combines single-molecu... more We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.
Nature communications, 2015
Beyond its role in host defense, bacterial DNA methylation also plays important roles in the regu... more Beyond its role in host defense, bacterial DNA methylation also plays important roles in the regulation of gene expression, virulence and antibiotic resistance. Bacterial cells in a clonal population can generate epigenetic heterogeneity to increase population-level phenotypic plasticity. Single molecule, real-time (SMRT) sequencing enables the detection of N6-methyladenine and N4-methylcytosine, two major types of DNA modifications comprising the bacterial methylome. However, existing SMRT sequencing-based methods for studying bacterial methylomes rely on a population-level consensus that lacks the single-cell resolution required to observe epigenetic heterogeneity. Here, we present SMALR (single-molecule modification analysis of long reads), a novel framework for single molecule-level detection and phasing of DNA methylation. Using seven bacterial strains, we show that SMALR yields significantly improved resolution and reveals distinct types of epigenetic heterogeneity. SMALR is a...
Parallel Epidemics of Community-Associated Methicillin-Resistant Staphylococcus aureus USA300 in North and South America
Journal of Infectious Diseases, 2015
The community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) epidemic in the U... more The community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) epidemic in the United States is attributed to the spread of the USA300 clone. An epidemic of CA-MRSA closely related to USA300 has occurred in northern South America (USA300 Latin-American variant, USA300-LV). Using phylogenomic analysis we aimed to understand the relationships between these two epidemics. We sequenced the genomes of 51 MRSA clinical isolates collected between 1999 and 2012 from United States, Colombia, Venezuela and Ecuador. Phylogenetic analysis was used to infer the relationships and times since the divergence of the major clades. Phylogenetic analyses revealed two dominant clades that segregated by geographical region with a putative common ancestor in 1975, individually originating in 1989 and 1985 for North American and South American clades, respectively. Emergence of these parallel epidemics coincides with the independent acquisition of the arginine catabolic mobile element (ACME) in North American isolates and a novel copper and mercury resistance (COMER) mobile element in South American isolates. Our results reveal the existence of two parallel USA300 epidemics that shared a recent common ancestor. The simultaneous rapid dissemination of these two epidemic clades suggests the presence of shared, potentially convergent, adaptations that enhance fitness and ability to spread.
Analysis of mRNA decay in Bacillus subtilis
Methods in enzymology, 2008
Studies of mRNA turnover in B. subtilis are less well known than in E. coli. Here we provide rese... more Studies of mRNA turnover in B. subtilis are less well known than in E. coli. Here we provide researchers who have an interest in gram-positive RNA processing with several protocols for RNA isolation, for 5'- and 3'-mapping of mRNAs and mRNA decay fragments, and we also include a comprehensive listing of B. subtilis mutants that are deficient in ribonucleases thought to be involved in mRNA decay.
Global analysis of mRNA decay intermediates in Bacillus subtilis wild-type and polynucleotide phosphorylase-deletion strains
Molecular microbiology, 2014
Messenger RNA decay in Bacillus subtilis is accomplished by a combination of exoribonucleases and... more Messenger RNA decay in Bacillus subtilis is accomplished by a combination of exoribonucleases and endoribonucleases. Intermediates in the decay process have not been readily detectable, and previous studies on mRNA decay have used a handful of highly expressed transcripts as models. Here, we use RNA-Seq analysis to probe mRNA turnover globally. A significant fraction of messages showed differential accumulation of RNA fragments that mapped near the 5' or 3' end of the coding sequence, consistent with initiation of decay from either the 5' end or from an internal cleavage site. Patterns of mRNA decay in the wild type were compared with patterns in a mutant strain lacking polynucleotide phosphorylase (PNPase), which is considered the major 3' exonuclease activity in mRNA decay and which is one of four known 3' exonucleases in B. subtilis. The results showed a striking dependence on PNPase for mRNA turnover in many cases, suggesting specificity in the ability of 3&#...
Methods in Enzymology, 2008
Development of such assays is also necessary for detailed biochemical analysis of enzyme properti... more Development of such assays is also necessary for detailed biochemical analysis of enzyme properties. In this chapter, we describe the purification and assay of 12 RNases of B. subtilis thought to be involved in stable RNA maturation or RNA degradation.
Genome announcements, 2014
Staphylococcus aureus 502A was a strain used in bacterial interference programs during the 1960s ... more Staphylococcus aureus 502A was a strain used in bacterial interference programs during the 1960s and early 1970s. Infants were deliberately colonized with 502A with the goal of preventing colonization with more invasive strains. We present the completed genome sequence of this organism.
Molecular microbiology, 2014
Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There ... more Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole-genome analysis of a methicillin-resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives of this strain. Of the multiple copies of IS256 found in the USA500 genome, one was inserted in the promoter sequence of repressor of toxins (Rot), a master transcriptional regulator responsible for the expression of virulence factors in S. aureus. We show that insertion into the rot promoter by IS256 results in the derepres...
Proteins: Structure, Function, and Bioinformatics, 2003
Prolonged exposure of Ca 2؉ -loaded or Ca 2؉ -depleted human ␣-lactalbumin to ultraviolet light (... more Prolonged exposure of Ca 2؉ -loaded or Ca 2؉ -depleted human ␣-lactalbumin to ultraviolet light (270 -290 nm, 1 mW/cm 2 , for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms:
Proceedings of the National Academy of Sciences, 2004
When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp op... more When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3-to-5 exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase ؊ strain showed accumulation of a stable, TRAPprotected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase ؊ strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription.
Nucleic Acids Research, 2009
In the presence of Mn 2Y , an activity in a preparation of purified Bacillus subtilis RecN degrad... more In the presence of Mn 2Y , an activity in a preparation of purified Bacillus subtilis RecN degrades singlestranded (ss) DNA with a 3' ! 5' polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn 2Y and low-level inorganic phosphate (P i ), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg 2Y or high-level P i . In contrast, the RNase activity of PNPase requires Mg 2Y and P i , suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation ("pnpA) is not epistatic with "recA, but is epistatic with "recN and "ku, which by themselves are non-epistatic. The addA5, "recO, "recQ ("recJ), "recU and "recG mutations (representative of different epistatic groups), in the context of "pnpA, demonstrate gain-or loss-offunction by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways.
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Papers by Gintaras Deikus