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1.
Nucleic Acids Res ; 48(19): 11054-11067, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045733

RESUMO

The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Shewanella/metabolismo , Sistemas Toxina-Antitoxina
2.
Microb Biotechnol ; 13(4): 1132-1144, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32246813

RESUMO

Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf4 inhibition toxin and Rorf0727 be named PfiA for PfiT antitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5'-AATTCN5 GTTAA-3') overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.

3.
J Antimicrob Chemother ; 74(9): 2559-2565, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31203365

RESUMO

OBJECTIVES: To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates. METHODS: Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing. RESULTS: In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing. CONCLUSIONS: CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.


Assuntos
Antibacterianos/farmacologia , Sistemas CRISPR-Cas , Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Plasmídeos/genética , Proteínas de Bactérias/genética , Conjugação Genética , Elementos de DNA Transponíveis/genética , Escherichia coli/efeitos dos fármacos , Humanos , RNA Guia/genética , Recombinação Genética
4.
Cell Rep ; 27(3): 737-749.e4, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995473

RESUMO

Cells are social, and self-recognition is a conserved aspect of group behavior where cells assist kin and antagonize non-kin. However, the role of phage in self-recognition is unexplored. Here we find that a demarcation line is formed between different swimming Escherichia coli strains but not between identical clones; hence, motile cells discriminate between self and non-self. The basis for this self-recognition is a 49 kb, T1-type, lytic phage of the family Siphoviridae (named here SW1) that controls formation of the demarcation line by utilizing one of the host's cryptic prophage proteins, YfdM of CPS-53, to propagate. Critically, SW1 provides a conditional benefit to E. coli K-12 compared with the identical strain that lacks the phage. A demarcation line is also formed when strains harbor either the lysogenic phage ϕ80 or lambda and encounter siblings that lack the lysogen. In summary, bacteria can use phage to distinguish siblings that lack phage.


Assuntos
Escherichia coli/fisiologia , Siphoviridae/fisiologia , Bacteriófago lambda/fisiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lisogenia , Prófagos/fisiologia , Proteínas Virais/metabolismo , Replicação Viral
5.
Mar Drugs ; 17(4)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987346

RESUMO

Toxin-antitoxin (TA) systems are ubiquitous and abundant genetic elements in bacteria and archaea. Most previous TA studies have focused on commensal and pathogenic bacteria, but have rarely focused on marine bacteria, especially those isolated from the deep sea. Here, we identified and characterized three putative TA pairs in the deep-sea-derived Streptomyces sp. strain SCSIO 02999. Our results showed that Orf5461/Orf5462 and Orf2769/Orf2770 are bona fide TA pairs. We provide several lines of evidence to demonstrate that Orf5461 and Orf5462 constitute a type-II TA pair that are homologous to the YoeB/YefM TA pair from Escherichia coli. Although YoeB from SCSIO 02999 was toxic to an E. coli host, the homologous YefM antitoxin from SCSIO 02999 did not neutralize the toxic effect of YoeB from E. coli. For the Orf2769/Orf2770 TA pair, Orf2769 overexpression caused significant cell elongation and could lead to cell death in E. coli, and the neighboring Orf2770 could neutralize the toxic effect of Orf2769. However, no homologous toxin or antitoxin was found for this pair, and no direct interaction was found between Orf2769 and Orf2770. These results suggest that Orf2769 and Orf2770 may constitute a novel TA pair. Thus, deep-sea bacteria harbor typical and novel TA pairs. The biochemical and physiological functions of different TAs in deep-sea bacteria warrant further investigation.


Assuntos
Organismos Aquáticos/fisiologia , Proteínas de Bactérias/genética , Streptomyces/fisiologia , Sistemas Toxina-Antitoxina/genética , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas , Escherichia coli/fisiologia , Proteínas de Escherichia coli/fisiologia , Loci Gênicos/fisiologia , Sedimentos Geológicos/microbiologia , Interações Microbianas/fisiologia , Oceanos e Mares , Homologia de Sequência do Ácido Nucleico
6.
Environ Microbiol ; 21(8): 2707-2723, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30882983

RESUMO

Toxin/antitoxin (TA) systems are ubiquitous in bacteria and archaea and participate in biofilm formation and stress responses. The higBA locus of the opportunistic pathogen Pseudomonas aeruginosa encodes a type II TA system. Previous work found that the higBA operon is cotranscribed and that HigB toxin regulates biofilm formation and virulence expression. In this study, we demonstrate that HigA antitoxin is produced at a higher level than HigB and that higA mRNA is expressed separately from a promoter inside higB during the late stationary phase. Critically, HigA represses the expression of mvfR, which is an important virulence-related regulator, by binding to a conserved HigA palindrome (5'-TTAAC GTTAA-3') in the mvfR promoter, and the binding of HigB to HigA derepresses this process. During the late stationary phase, excess HigA represses the expression of mvfR and higBA. However, in the presence of aminoglycoside antibiotics where Lon protease is activated, the degradation of HigA by Lon increases P. aeruginosa virulence by simultaneously derepressing mvfR and higB transcription. Therefore, this study reveals that the antitoxin of the P. aeruginosa TA system is integrated into the key virulence regulatory network of the host and functions as a transcriptional repressor to control the production of virulence factors.


Assuntos
Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genética , Sistemas Toxina-Antitoxina , Proteínas de Bactérias/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Sistemas Toxina-Antitoxina/genética , Virulência/genética , Fatores de Virulência
7.
Microb Biotechnol ; 12(2): 392-404, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30656833

RESUMO

C-tail anchored inner membrane proteins are a family of proteins that contain a C-terminal transmembrane domain but lack an N-terminal signal sequence for membrane targeting. They are widespread in eukaryotes and prokaryotes and play critical roles in membrane traffic, apoptosis and protein translocation in eukaryotes. Recently, we identified and characterized in Escherichia coli a new C-tail anchored inner membrane, ElaB, which is regulated by the stationary phase sigma factor RpoS. ElaB is important for resistance to oxidative stress but the exact mechanism is unclear. Here, we show that ElaB functions as part of the adaptive oxidative stress response by maintaining membrane integrity. Production of ElaB is induced by oxidative stress at the transcriptional level. Moreover, elaB expression is also regulated by the key regulator OxyR via an OxyR binding site in the promoter of elaB. OxyR induces the expression of elaB in the exponential growth phase, while excess OxyR reduces elaB expression in an RpoS-dependent way in the stationary phase. In addition, deletion of elaB reduced fitness compared to wild-type cells after prolonged incubation. Therefore, we determined how ElaB is regulated under oxidative stress: RpoS and OxyR coordinately control the expression of inner membrane protein ElaB.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Estresse Fisiológico , Proteínas de Bactérias , Escherichia coli/genética , Transcrição Genética
8.
Front Microbiol ; 10: 3015, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31998280

RESUMO

Colistin is considered the last-resort antibiotic used to treat multidrug resistant bacteria-related infections. However, the discovery of the plasmid-mediated colistin resistance gene, mcr-1, threatens the clinical utility of colistin antibiotics. In this study, the physiological function of MCR-1, which encodes an LPS-modifying enzyme, was investigated in E. coli K-12. Specifically, the impact of mcr-1 on membrane permeability and antibiotic resistance of E. coli was assessed by constructing an mcr-1 deletion mutant and by a complementation study. The removal of the mcr-1 gene from plasmid pHNSHP45 not only led to reduced resistance to colistin but also resulted in a significant change in the membrane permeability of E. coli. Unexpectedly, the removal of the mcr-1 gene increased cell viability under high osmotic stress conditions (e.g., 7.0% NaCl) and led to increased resistance to hydrophobic antibiotics. Increased expression of mcr-1 also resulted in decreased growth rate and changed the cellular morphology of E. coli. Collectively, our results revealed that the spread of mcr-1-carrying plasmids alters other physiological functions in addition to conferring colistin resistance.

9.
Mol Microbiol ; 111(2): 495-513, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30475408

RESUMO

Pf filamentous prophages are prevalent among clinical and environmental Pseudomonas aeruginosa isolates. Pf4 and Pf5 prophages are integrated into the host genomes of PAO1 and PA14, respectively, and play an important role in biofilm development. However, the genetic factors that directly control the lysis-lysogeny switch in Pf prophages remain unclear. Here, we identified and characterized the excisionase genes in Pf4 and Pf5 (named xisF4 and xisF5, respectively). XisF4 and XisF5 represent two major subfamilies of functional excisionases and are commonly found in Pf prophages. While both of them can significantly promote prophage excision, only XisF5 is essential for Pf5 excision. XisF4 activates Pf4 phage replication by upregulating the phage initiator gene (PA0727). In addition, xisF4 and the neighboring phage repressor c gene pf4r are transcribed divergently and their 5'-untranslated regions overlap. XisF4 and Pf4r not only auto-activate their own expression but also repress each other. Furthermore, two H-NS family proteins, MvaT and MvaU, coordinately repress Pf4 production by directly repressing xisF4. Collectively, we reveal that Pf prophage excisionases cooperate in controlling lysogeny and phage production.


Assuntos
DNA Nucleotidiltransferases/metabolismo , Lisogenia , Prófagos/enzimologia , Prófagos/crescimento & desenvolvimento , Fagos de Pseudomonas/enzimologia , Pseudomonas aeruginosa/virologia , Proteínas Virais/metabolismo , Replicação Viral , Regulação Viral da Expressão Gênica , Prófagos/genética , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/crescimento & desenvolvimento
10.
Front Microbiol ; 9: 2514, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405572

RESUMO

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.

11.
Environ Microbiol ; 20(3): 1224-1239, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29411516

RESUMO

Toxin/antitoxin (TA) loci are commonly found in mobile genetic elements such as plasmids and prophages. However, the physiological functions of these TA loci in prophages and cross-regulation among these TA loci remain largely unexplored. Here, we characterized a newly discovered type II TA pair, ParESO /CopASO , in the CP4So prophage in Shewanella oneidensis. We demonstrated that ParESO /CopASO plays a critical role in the maintenance of CP4So in host cells after its excision. The toxin ParESO inhibited cell growth, resulting in filamentous growth and eventually cell death. The antitoxin CopASO neutralized the toxicity of ParESO through direct protein-protein interactions and repressed transcription of the TA operon by binding to a DNA motif in the promoter region containing two inverted repeats [5'-GTANTAC (N)3 GTANTAC-3']. CopASO also repressed transcription of another TA system PemKSO /PemISO in megaplasmid pMR-1 of S. oneidensis through binding to a highly similar DNA motif in its promoter region. CopASO homologs are widely spread in Shewanella and other Proteobacteria, either as a component of a TA pair or as orphan antitoxins. Our study thus illustrated the cross-regulation of the TA systems in different mobile genetic elements and expanded our understanding of the physiological function of TA systems.


Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , Sequências Repetitivas Dispersas/genética , Prófagos/genética , Shewanella/genética , Sistemas Toxina-Antitoxina/genética , Proteínas de Bactérias/metabolismo , Sequências Repetidas Invertidas/genética , Óperon/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Shewanella/fisiologia
12.
Front Microbiol ; 8: 1822, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28983293

RESUMO

Pseudoalteromonas is an important bacterial genus present in various marine habitats. Many strains of this genus are found to be surface colonizers on marine eukaryotes and produce a wide range of pigments. However, the exact physiological role and mechanism of pigmentation were less studied. Pseudoalteromonas sp. SM9913 (SM9913), an non-pigmented strain isolated from the deep-sea sediment, formed attached biofilm at the solid-liquid interface and pellicles at the liquid-air interface at a wide range of temperatures. Lower temperatures and lower nutrient levels promoted the formation of attached biofilm, while higher nutrient levels promoted pellicle formation of SM9913. Notably, after prolonged incubation at higher temperatures growing planktonically or at the later stage of the biofilm formation, we found that SM9913 released a brownish pigment. By comparing the protein profile at different temperatures followed by qRT-PCR, we found that the production of pigment at higher temperatures was due to the induction of melA gene which is responsible for the synthesis of homogentisic acid (HGA). The auto-oxidation of HGA can lead to the formation of pyomelanin, which has been shown in other bacteria. Fourier Transform Infrared Spectrometer analysis confirmed that the pigment produced in SM9913 was pyomelanin-like compound. Furthermore, we demonstrated that, during heat stress and during biofilm formation, the induction level of melA gene was significantly higher than that of the hmgA gene which is responsible for the degradation of HGA in the L-tyrosine catabolism pathway. Collectively, our results suggest that the production of pyomelanin of SM9913 at elevated temperatures or during biofilm formation might be one of the adaptive responses of marine bacteria to environmental cues.

13.
Front Microbiol ; 8: 840, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28536573

RESUMO

Bacterial toxin/antitoxin (TA) systems have received increasing attention due to their prevalence, diverse structures, and important physiological functions. In this study, we identified and characterized a type II TA system in a soil bacterium Pseudomonas putida KT2440. This TA system belongs to the MqsR/MqsA family. We found that PP_4205 (MqsR) greatly inhibits cell growth in P. putida KT2440 and Escherichia coli, the antitoxin PP_4204 (MqsA) neutralizes the toxicity of the toxin MqsR, and the two genes encoding them are co-transcribed. MqsR and MqsA interact with each other directly in vivo and MqsA is a negative regulator of the TA operon through binding to the promoter. Consistent with the MqsR/MqsA pair in E. coli, the binding of the toxin MqsR to MqsA inhibits the DNA binding ability of MqsA in P. putida KT2440. Disruption of the mqsA gene which induces mqsR expression increases persister cell formation 53-fold, while overexpressing mqsA which represses mqsR expression reduces persister cell formation 220-fold, suggesting an important role of MqsR in persistence in P. putida KT2440. Furthermore, both MqsR and MqsA promote biofilm formation. As a DNA binding protein, MqsA can also negatively regulate an ECF sigma factor AlgU and a universal stress protein PP_3288. Thus, we revealed an important regulatory role of MqsR/MqsA in persistence and biofilm formation in P. putida KT2440.

14.
J Bacteriol ; 199(9)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28242719

RESUMO

Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coliIMPORTANCEEscherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Membrana/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Proteínas de Membrana/genética , Estresse Oxidativo , Regiões Promotoras Genéticas , Fator sigma/genética , Fatores de Transcrição/metabolismo
15.
Toxins (Basel) ; 9(3)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28257056

RESUMO

Toxin/antitoxin (TA) systems are widespread in prokaryotic chromosomes and in mobile genetic elements including plasmids and prophages. The first characterized Type IV TA system CbtA/CbeA was found in cryptic prophage CP4-44 in Escherichia coli K-12. Two homologous TA loci of CbtA/CbeA also reside in cryptic prophages of E. coli K-12, YkfI/YafW in CP4-6 and YpjF/YfjZ in CP4-57. In this study, we demonstrated that YkfI and YpjF inhibited cell growth and led to the formation of "lemon-shaped" cells. Prolonged overproduction of YkfI led to the formation of "gourd-shaped" cells and immediate cell lysis. YafW and YfjZ can neutralize the toxicity of YkfI or YpjF. Furthermore, we found that YkfI and YpjF interacted with cell division protein FtsZ in E. coli, but ectopic expression in Pseudomonas and Shewanella did not cause the formation of "lemon-shaped" cells. Moreover, deletion of all of the three toxin genes together decreased resistance to oxidative stress and deletion of the antitoxin genes increased early biofilm formation. Collectively, these results demonstrated that the homologous Type IV TA systems in E. coli may target cell division protein FtsZ in E. coli and may have different physiological functions in E. coli.


Assuntos
Antitoxinas , Toxinas Bacterianas , Escherichia coli K12 , Prófagos , Antitoxinas/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Proteínas de Transporte/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Prófagos/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas putida/crescimento & desenvolvimento , Shewanella/crescimento & desenvolvimento
16.
ISME J ; 10(12): 2787-2800, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27482926

RESUMO

Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation.


Assuntos
Prófagos/fisiologia , Shewanella/fisiologia , Shewanella/virologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Temperatura Baixa , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Prófagos/genética , Shewanella/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação Viral
17.
Toxins (Basel) ; 8(7)2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27376329

RESUMO

Toxin-antitoxin (TA) systems are small genetic elements that are ubiquitous in prokaryotes. Most studies on TA systems have focused on commensal and pathogenic bacteria; yet very few studies have focused on TAs in marine bacteria, especially those isolated from a deep sea environment. Here, we characterized a type II VapC/VapB TA system from the deep-sea derived Streptomyces sp. SCSIO 02999. The VapC (virulence-associated protein) protein belongs to the PIN (PilT N-terminal) superfamily. Overproduction of VapC strongly inhibited cell growth and resulted in a bleb-containing morphology in E. coli. The toxicity of VapC was neutralized through direct protein-protein interaction by a small protein antitoxin VapB encoded by a neighboring gene. Antitoxin VapB alone or the VapB/VapC complex negatively regulated the vapBC promoter activity. We further revealed that three conserved Asp residues in the PIN domain were essential for the toxic effect of VapC. Additionally, the VapC/VapB TA system stabilized plasmid in E. coli. Furthermore, VapC cross-activated transcription of several TA operons via a partially Lon-dependent mechanism in E. coli, and the activated toxins accumulated more preferentially than their antitoxin partners. Collectively, we identified and characterized a new deep sea TA system in the deep sea Streptomyces sp. and demonstrated that the VapC toxin in this system can cross-activate TA operons in E. coli.


Assuntos
Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Glicoproteínas de Membrana/metabolismo , Streptomyces/metabolismo , Microbiologia da Água , Antitoxinas/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicoproteínas de Membrana/genética , Viabilidade Microbiana , Oceanos e Mares , Óperon , Regiões Promotoras Genéticas , Protease La/metabolismo , Domínios Proteicos , Streptomyces/genética , Relação Estrutura-Atividade , Fatores de Tempo , Transcrição Genética , Ativação Transcricional
18.
Sci Rep ; 5: 16074, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26530864

RESUMO

Rac or rac-like prophage harbors many genes with important physiological functions, while it remains excision-proficient in several bacterial strains including Escherichia coli, Salmonella spp. and Shigella spp. Here, we found that rac excision is induced during biofilm formation, and the isogenic stain without rac is more motile and forms more biofilms in nutrient-rich medium at early stages in E. coli K-12. Additionally, the presence of rac genes increases cell lysis during biofilm development. In most E. coli strains, rac is integrated into the ttcA gene which encodes a tRNA-thioltransferase. Rac excision in E. coli K-12 leads to a functional change of TtcA, which results in reduced fitness in the presence of carbenicillin. Additionally, we demonstrate that YdaQ (renamed as XisR) is the excisionase of rac in E. coli K-12, and that rac excision is induced by the stationary sigma factor RpoS through inducing xisR expression. Taken together, our results reveal that upon rac integration, not only are new genes introduced into the host, but also there is a functional change in a host enzyme. Hence, rac excision is tightly regulated by host factors to control its stability in the host genome under different stress conditions.


Assuntos
Biofilmes/crescimento & desenvolvimento , DNA Nucleotidiltransferases/metabolismo , Escherichia coli K12/crescimento & desenvolvimento , Prófagos/metabolismo , Proteínas Virais/metabolismo , Ativação Viral/genética , Liberação de Vírus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Carbenicilina/farmacologia , DNA Nucleotidiltransferases/genética , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/virologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Dados de Sequência Molecular , Prófagos/genética , Alinhamento de Sequência , Fator sigma/genética , Sulfurtransferases/genética , Proteínas Virais/genética , Ativação Viral/fisiologia
19.
Microb Biotechnol ; 8(6): 961-73, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26112399

RESUMO

Toxin-antitoxin (TA) systems are prevalent in bacteria and archaea. However, related studies in the ecologically and bioelectrochemically important strain Shewanella oneidensis are limited. Here, we show that SO_3166, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibited cell growth in S. oneidensis and Escherichia coli. SO_3165, a putative minimal nucleotidyltransferase (MNT), neutralized the toxicity of SO_3166. Gene SO_3165 lies upstream of SO_3166, and they are co-transcribed. Moreover, the SO_3165 and SO_3166 proteins interact with each other directly in vivo, and antitoxin SO_3165 bound to the promoter of the TA operon and repressed its activity. Finally, the conserved Rx4-6H domain in HEPN family was identified in SO_3166. Mutating either the R or H abolished SO_3166 toxicity, confirming that Rx4-6H domain is critical for SO_3166 activity. Taken together, these results demonstrate that SO_3166 and SO_3165 in S. oneidensis form a typical type II TA pair. This TA pair plays a critical role in regulating bacterial functions because its disruption led to impaired cell motility in S. oneidensis. Thus, we demonstrated for the first time that HEPN-MNT can function as a TA system, thereby providing important insights into the understanding of the function and regulation of HEPNs and MNTs in prokaryotes.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Shewanella/genética , Shewanella/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Mapeamento de Interação de Proteínas , Shewanella/efeitos dos fármacos , Shewanella/crescimento & desenvolvimento , Transcrição Genética
20.
Mol Biol Rep ; 41(9): 5863-75, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24962047

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant viral diseases in swine, which causes large economic losses to the swine industry worldwide. There is considerable strain variation in PRRSV and two examples of this are the highly virulent Chinese-type PRRSV (H-PRRSV) and the classical North American type PRRSV (N-PRRSV), both with different pathogenesis. These differences may be due in part to genetic and phenotypic differences in virus replication, but also interaction with the host cell. MicroRNAs (miRNAs) are crucial regulators of gene expression and play vital roles in virus and host interactions. However, the regulation role of miRNAs during PRRSV infection has not been systematically investigated. In order to better understand the differential regulation roles of cellular miRNAs in the host response to PRRSV, miRNA expression and a global mRNA transcriptome profile was determined in primary cells infected with either H-PRRSV or N-PRRSV as multiple time points during the viral lifecycle. miRNA-mRNA interactome networks were constructed by integrating the differentially expressed miRNAs and inversely correlated target mRNAs. Using gene ontology and pathway enrichment analyses, cellular pathways associated with deregulated miRNAs were identified, including immune response, phagosome, autophagy, lysosome, autolysis, apoptosis and cell cycle regulation. To our knowledge, this is the first global analysis of strain-specific host miRNA molecular signatures associated with H- and N-PRRSV infection by integrating miRNA and mRNA transcriptomes and provides a new perspective on the contribution of miRNAs to the pathogenesis of PRRSV infection.


Assuntos
Macrófagos Alveolares/virologia , MicroRNAs/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , RNA Mensageiro/genética , Transcriptoma , Animais , Macrófagos Alveolares/citologia , MicroRNAs/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Suínos , Replicação Viral
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