RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

ABSTRACT In the nosocomial opportunistic pathogen Acinetobacter baumannii, RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii. Here, we identify a structured 5′ untranslated region (UTR) in the recA transcript which serves as a cis-regulatory element. We show that a predicted stem-loop structure in this 5′ UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5′ UTR influences intracellular RecA protein levels and, in vivo, impairing the formation of the stem-loop structure of the recA 5′ UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5′ UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen. IMPORTANCE Acinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis-acting 5′ UTR regulates recA transcript stability, RecA protein levels, and DNA damage-induced phenotypic variation. Though 5′ UTRs are known to provide stability to transcripts in bacteria, this is the first example in which it regulates a bimodal DDR response through recA transcript stabilization, potentially enabling cells to have enough RecA for survival and genetic variability.

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Role of a Stem-Loop Structure inHelicobacter pylori cagATranscript Stability

Infection and Immunity ◽

10.1128/iai.00692-18 ◽

2018 ◽

Vol 87 (2) ◽

Cited By ~ 3

Author(s):

John T. Loh ◽

Aung Soe Lin ◽

Amber C. Beckett ◽

Mark S. McClain ◽

Timothy L. Cover

Keyword(s):

Steady State ◽

Untranslated Region ◽

Loop Structure ◽

Stem Loop ◽

Content Type ◽

Transcript Stability ◽

Protein Levels ◽

Caga Protein ◽

Stem Loop Structure ◽

Steady State Levels

ABSTRACTHelicobacter pyloriCagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation amongH. pyloristrains in the steady-state levels of CagA and that a strain-specific motif downstream of thecagAtranscriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. ThecagA5′ untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop oncagAtranscript levels andcagAmRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both thecagAtranscript and the CagA protein. Additionally, these mutations resulted in a decreasedcagAmRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-statecagAtranscript and CagA protein levels but did not affectcagAtranscript stability.cagAtranscript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augmentcagAtranscript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in thecagA5′ untranslated region influence the levels ofcagAexpression.

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Role of RNA Secondary Structure and Processing in Stability of thenifH1Transcript in the Cyanobacterium Anabaena variabilis

Journal of Bacteriology ◽

10.1128/jb.02609-14 ◽

2015 ◽

Vol 197 (8) ◽

pp. 1408-1422 ◽

Cited By ~ 6

Author(s):

Brenda S. Pratte ◽

Justin Ungerer ◽

Teresa Thiel

Keyword(s):

Nitrogen Fixation ◽

Secondary Structure ◽

Anabaena Variabilis ◽

Loop Structure ◽

Fixed Nitrogen ◽

Stem Loop ◽

Content Type ◽

Transcript Processing ◽

Stem Loop Structure ◽

In Cells

ABSTRACTIn the cyanobacteriumAnabaena variabilisATCC 29413, aerobic nitrogen fixation occurs in micro-oxic cells called heterocysts. Synthesis of nitrogenase in heterocysts requires expression of the largenif1gene cluster, which is primarily under the control of the promoter for the first gene,nifB1. Strong expression ofnifH1requires thenifB1promoter but is also controlled by RNA processing, which leads to increasednifH1transcript stability. The processing of the primarynifH1transcript occurs at the base of a predicted stem-loop structure that is conserved in many heterocystous cyanobacteria. Mutations that changed the predicted secondary structure or changed the sequence of the stem-loop had detrimental effects on the amount ofnifH1transcript, with mutations that altered or destabilized the structure having the strongest effect. Just upstream from the transcriptional processing site fornifH1was the promoter for a small antisense RNA,sava4870.1. This RNA was more strongly expressed in cells grown in the presence of fixed nitrogen and was downregulated in cells 24 h after nitrogen step down. A mutant strain lacking the promoter forsava4870.1showed delayed nitrogen fixation; however, that phenotype might have resulted from an effect of the mutation on the processing of thenifH1transcript. ThenifH1transcript was the most abundant and most stablenif1transcript, whilenifD1andnifK1, just downstream ofnifH1, were present in much smaller amounts and were less stable. ThenifD1andnifK1transcripts were also processed at sites just upstream ofnifD1andnifK1.IMPORTANCEIn the filamentous cyanobacteriumAnabaena variabilis, thenif1cluster, encoding the primary Mo nitrogenase, functions under aerobic growth conditions in specialized cells called heterocysts that develop in response to starvation for fixed nitrogen. The large cluster comprising more than a dozennif1genes is transcribed primarily from the promoter for the first gene,nifB1; however, this does not explain the large amount of transcript for the structural genesnifH1,nifD1, andnifK1, which are also under the control of the distantnifB1promoter. Here, we demonstrate the importance of a predicted stem-loop structure upstream ofnifH1that controls the abundance ofnifH1transcript through transcript processing and stabilization and show thatnifD1andnifK1transcripts are also controlled by transcript processing.

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Functional Genetic Elements for Controlling Gene Expression inCupriavidus necatorH16

Applied and Environmental Microbiology ◽

10.1128/aem.00878-18 ◽

2018 ◽

Vol 84 (19) ◽

Cited By ~ 8

Author(s):

Swathi Alagesan ◽

Erik K. R. Hanko ◽

Naglis Malys ◽

Muhammad Ehsaan ◽

Klaus Winzer ◽

...

Keyword(s):

Gene Expression ◽

Dynamic Range ◽

Loop Structure ◽

Stem Loop ◽

Control Of Gene Expression ◽

Content Type ◽

Genetic Elements ◽

Stem Loop Structure ◽

Inducible Systems ◽

The Impact

ABSTRACTA robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotrophCupriavidus necatorH16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within a >700-fold dynamic range was compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase geneispSled to isoprene yields that exhibited a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox ofC. necator.IMPORTANCEThis report provides tools for robust and predictable control of gene expression in the model lithoautotrophC. necatorH16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression inC. necatorH16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and two negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.

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Translation of the Flagellar Gene fliO ofSalmonella typhimurium from Putative Tandem Starts

Journal of Bacteriology ◽

10.1128/jb.180.11.2936-2942.1998 ◽

1998 ◽

Vol 180 (11) ◽

pp. 2936-2942 ◽

Cited By ~ 6

Author(s):

Gary J. Schoenhals ◽

May Kihara ◽

Robert M. Macnab

Keyword(s):

Start Codon ◽

Loop Structure ◽

Flagellar Gene ◽

Transmembrane Segment ◽

Stem Loop ◽

E Coli ◽

Protein Levels ◽

Stem Loop Structure ◽

Loop Sequence ◽

Entire Sequence

ABSTRACT The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K. Ohnishi et al., J. Bacteriol. 179:6092–6099, 1997). However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189–197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis genecheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found withcheA. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S. typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between S. typhimurium and E. coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.

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A Stem-Loop Structure inPotato Leafroll VirusOpen Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream

Journal of Virology ◽

10.1128/jvi.01544-17 ◽

2018 ◽

Vol 92 (11) ◽

Cited By ~ 10

Author(s):

Yi Xu ◽

Ho-Jong Ju ◽

Stacy DeBlasio ◽

Elizabeth J. Carino ◽

Richard Johnson ◽

...

Keyword(s):

Capsid Protein ◽

Rna Structure ◽

Stop Codon ◽

Loop Structure ◽

Long Distance ◽

Stem Loop ◽

Content Type ◽

Stem Loop Structure ◽

Rna Domains ◽

Stop Codon Readthrough

ABSTRACTTranslational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of theLuteoviridaeto produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon ofPotato leafroll virus(PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE ofCereal yellow dwarf viruswas not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2′-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.IMPORTANCEProgrammed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such “leaky” stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in theLuteoviridaeby identifying the amino acids inserted at the stop codon and RNA structures that facilitate this “leakiness” of the stop codon. Members of theLuteoviridaeencode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains inPotato leafroll virus(PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.

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Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus

Journal of Virology ◽

10.1128/jvi.01043-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8580-8589 ◽

Cited By ~ 13

Author(s):

Leanne K. Finch ◽

Roger Ling ◽

Sawsan Napthine ◽

Allan Olspert ◽

Thomas Michiels ◽

...

Keyword(s):

Virus Infection ◽

Loop Structure ◽

Theiler's Murine Encephalomyelitis Virus ◽

Stem Loop ◽

Ribosomal Frameshifting ◽

Content Type ◽

Reading Frame ◽

Theiler’S Murine Encephalomyelitis Virus ◽

Stem Loop Structure ◽

Encephalomyelitis Virus

ABSTRACTTheiler's murine encephalomyelitis virus(TMEV) is a member of the genusCardiovirusin thePicornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus,Encephalomyocarditis virus, a programmed −1 ribosomal frameshift (−1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that −1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3′ RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient −1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.IMPORTANCEMany viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, −1 PRF occurs on specific “slippery” heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of −1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of −1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter constructs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved frameshifting as a novel mechanism for removing ribosomes from the message (a “ribosome sink”) to downregulate synthesis of the 3′-encoded replication proteins.

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T-hairpin structure found in the RNA element involved in piRNA biogenesis

RNA ◽

10.1261/rna.078967.121 ◽

2022 ◽

pp. rna.078967.121

Author(s):

Naomi Takase ◽

Maina Otsu ◽

Shigeki Hirakata ◽

Hirotsugu Ishizu ◽

Mikiko C. Siomi ◽

...

Keyword(s):

Dna Damage ◽

Tertiary Structure ◽

Protein A ◽

Hairpin Structure ◽

Loop Structure ◽

Stem Loop ◽

Traffic Jam ◽

Stem Loop Structure ◽

Tudor Domain

PIWI-interacting RNAs (piRNAs) repress transposons to protect the germline genome from DNA damage caused by transposon transposition. In Drosophila, the Traffic jam (Tj) mRNA is consumed to produce piRNA in its 3′ UTR. A cis element located within the 3′-UTR, Tj-cis, is necessary for piRNA biogenesis. In this study, we analyzed the structure of the Tj-cis RNA, a 100 nt RNA corresponding to the Tj-cis element, by the SHAPE and NMR analyses and found that a stable hairpin structure formed in the 5′ half of the Tj-cis RNA. The tertiary structure of the 16 nt stable hairpin was analyzed by NMR, and a novel stem-loop structure, the T-hairpin, was found. In the T-hairpin, four uridine residues are exposed to the solvent, suggesting that this stem loop is the target of Yb protein, a Tudor domain-containing piRNA biogenesis factor. The piRNA biogenesis assay showed that both the T-hairpin and the 3′ half are required for the function of the Tj-cis element, suggesting that both the T-hairpin and the 3′ half are recognized by Yb protein.

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A Conserved Histophilus somni 23S Intervening Sequence Yields Functional, Fragmented 23S rRNA

Microbiology Spectrum ◽

10.1128/spectrum.01431-21 ◽

2021 ◽

Author(s):

Gregory P. Harhay ◽

Dayna M. Harhay ◽

Kerry D. Brader ◽

Timothy P. L. Smith

Keyword(s):

Unknown Function ◽

Central Portion ◽

23S Rrna ◽

Tissue Tropism ◽

Loop Structure ◽

Stem Loop ◽

Content Type ◽

Stem Loop Structure ◽

Histophilus Somni ◽

Environmental Adaptability

The genome biology underlying H. somni virulence, pathogenicity, environmental adaptability, and broad tissue tropism is understood poorly. We identified a novel H. somni 109-nt IVS stem-loop structure, of which the central portion is excised from the 23S rRNA transcript, resulting in the fragmentation of this rRNA in the H. somni isolate USDA-ARS-USMARC-63250 and the release of a 94-nt structured RNA of unknown function.

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