scholarly journals Role of the kdpDE Regulatory Operon of Mycobacterium tuberculosis in Modulating Bacterial Growth in vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Moloko C. Cholo ◽  
Maborwa T. Matjokotja ◽  
Ayman G. Osman ◽  
Ronald Anderson
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Growth ◽  
Deletion Mutant ◽  
Regulatory System ◽  
Growth Conditions ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Uptake Efficiency ◽  
Rates Of Growth

Bacteria use K+-uptake transporters differentially for adaptation in varying growth conditions. In Mycobacterium tuberculosis, two K+-uptake systems, the Trk comprising the CeoB and CeoC proteins and the Kdp consisting of the two-component system (TCS), KdpDE and KdpFABC, have been characterized, but their selective utilization during bacterial growth has not been completely explored. In the current study, the roles of the M. tuberculosis KdpDE regulatory system alone and in association with the Trk transporters in bacterial growth were investigated by evaluating the growth of M. tuberculosis KdpDE-deletion and KdpDE/Trk (KT)-double knockout mutant strains in planktonic culture under standard growth conditions. The KT-double knockout mutant strain was first constructed using homologous recombination procedures and was evaluated together with the KdpDE-deletion mutant and the wild-type (WT) strains with respect to their rates of growth, K+-uptake efficiencies, and K+-transporter gene expression during planktonic growth. During growth at optimal K+ concentrations and pH levels, selective deletion of the TCS KdpDE (KdpDE-deletion mutant) led to attenuation of bacterial growth and an increase in bacterial K+-uptake efficiency, as well as dysregulated expression of the kdpFABC and trk genes. Deletion of both the KdpDE and the Trk systems (KT-double knockout) also led to severely attenuated bacterial growth, as well as an increase in bacterial K+-uptake efficiency. These results demonstrate that the KdpDE regulatory system plays a key role during bacterial growth by regulating K+ uptake via modulation of the expression and activities of both the KdpFABC and Trk systems and is important for bacterial growth possibly by preventing cytoplasmic K+ overload.

scholarly journals Identification and Characterization of a Regulatory Sequence Recognized by Mycobacterium tuberculosis Persistence Regulator MprA

2005 ◽  
Vol 187 (1) ◽  
pp. 202-212 ◽  
Author(s):  
Hongjun He ◽  
Thomas C. Zahrt
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Response Regulator ◽  
Regulatory System ◽  
Direct Repeat ◽  
Regulatory Sequence ◽  
Spacer Length ◽  
Signaling Systems ◽  
Downstream Effector

ABSTRACT Establishment and maintenance of persistent, latent infection by Mycobacterium tuberculosis are dependent on expression of the mprA-mprB regulatory system. Previously, MprA and MprB were shown to participate in phosphotransfer reactions characteristic of two-component signaling systems. To begin identifying downstream effector genes regulated by mprA-mprB during persistent stages of infection, a search for the regulatory sequence(s) recognized by response regulator MprA was carried out. Here, evidence is presented demonstrating that MprA recognizes a 19-bp sequence comprising two loosely conserved 8-bp direct repeat subunits separated by 3 nucleotides. This motif, termed the MprA box, is found upstream of the mprA coding sequence and that of downstream gene pepD (Rv0983). Protein phosphorylation was not required for binding to this DNA sequence by MprA in vitro; however, phosphorylation enhanced DNA binding by MprA and was required for the regulation of mprA and pepD by MprA in vivo. Binding of MprA to the MprA box was dependent on conserved nucleotides contained within repeat subunits and on the spacer length separating these repeats. In addition, recognition of this sequence proceeded via tandem binding of two monomers of MprA. Identification of the genetic determinants regulated by MprA will ultimately enhance our understanding of the mechanisms utilized by M. tuberculosis to undergo latency.

scholarly journals Inactivation of Rv2525c, a Substrate of the Twin Arginine Translocation (Tat) System of Mycobacterium tuberculosis, Increases β-Lactam Susceptibility and Virulence

2006 ◽  
Vol 188 (18) ◽  
pp. 6669-6679 ◽  
Author(s):  
Brigitte Saint-Joanis ◽  
Caroline Demangel ◽  
Mary Jackson ◽  
Priscille Brodin ◽  
Laurent Marsollier ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biochemical Analysis ◽  
Species Conservation ◽  
Knockout Mutant ◽  
Orthologous Genes ◽  
Twin Arginine Translocation ◽  
Cell Wall Biogenesis ◽  
Tat System ◽  
Folded Proteins

ABSTRACT The twin arginine translocation (Tat) system is used by many bacteria to export fully folded proteins containing cofactors. Here, we show genetically that this system is essential for Mycobacterium tuberculosis, as the tatAC operon and tatB genes could be inactivated only in partially diploid strains. Using comparative genomics, the rv2525c gene of M. tuberculosis was identified as encoding a histidine-rich protein, with a twin arginine signal peptide, and orthologous genes were shown to be present in several but not all actinobacterial species. Conservation of this gene by Mycobacterium leprae, which has undergone reductive evolution, suggested an important role for rv2525c. An rv2525c knockout mutant was constructed, and biochemical analysis indicated that the mature Rv2525c protein is secreted. Upon exposure to antituberculous drugs, rv2525c expression is significantly up-regulated together with those of other genes involved in cell wall biogenesis. Phenotypic comparison of the mutant with the parental strain revealed an increase in susceptibility to some β-lactam antibiotics and, despite slower growth in vitro, enhanced virulence in both cellular and murine models of tuberculosis. The Tat system thus contributes in multiple ways to survival of the tubercle bacillus.

scholarly journals Ribonucleotide Reduction in Mycobacterium tuberculosis: Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

2003 ◽  
Vol 71 (11) ◽  
pp. 6124-6131 ◽  
Author(s):  
Stephanie S. Dawes ◽  
Digby F. Warner ◽  
Liana Tsenova ◽  
Juliano Timm ◽  
John D. McKinney ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Class Ii ◽  
Growth Conditions ◽  
Small Subunit ◽  
Reverse Transcription Pcr ◽  
Hypoxic Conditions ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Class Ib

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a ΔnrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O2-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O2 restriction. However, survival of the ΔnrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F1 mice infected with the ΔnrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.

A C-terminal deletion mutant of Mycobacterium tuberculosis FtsZ shows fast polymerization in vitro

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1119-1121 ◽  
Author(s):  
Syam Prasad Anand ◽  
Haryadi Rajeswari ◽  
Prabuddha Gupta ◽  
Ramanujam Srinivasan ◽  
Shantinath Indi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Deletion Mutant ◽  
Terminal Deletion

scholarly journals Role of the dosR-dosS Two-Component Regulatory System in Mycobacterium tuberculosis Virulence in Three Animal Models

2008 ◽  
Vol 77 (3) ◽  
pp. 1230-1237 ◽  
Author(s):  
Paul J. Converse ◽  
Petros C. Karakousis ◽  
Lee G. Klinkenberg ◽  
Anup K. Kesavan ◽  
Lan H. Ly ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Animal Models ◽  
Hollow Fiber ◽  
Regulatory System ◽  
Growth Defect ◽  
Bacterial Survival ◽  
Wild Type ◽  
Fiber Model

ABSTRACT The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (ΔdosR-dosS [ΔdosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the ΔdosR-S mutant exhibited a growth defect. In the rabbit, the ΔdosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.

scholarly journals Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

1994 ◽  
Vol 36 (4) ◽  
pp. 301-310 ◽  
Author(s):  
Maria Cristina de Cunto Brandileone ◽  
Rosemeire Cobo Zanella ◽  
Vera Simonsen Dias Vieira ◽  
Claudio Tavares Sacciii ◽  
Lucimar Gonçalves Milagres ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Culture Supernatant ◽  
Culture Media ◽  
Membrane Vesicles ◽  
Normal Growth ◽  
Growth Conditions ◽  
Defined Medium ◽  
Outer Membrane Vesicles ◽  
Iron Deficient

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

scholarly journals Identification of a Novel Essential Two-Component Signal Transduction System, YhcSR, in Staphylococcus aureus

2005 ◽  
Vol 187 (22) ◽  
pp. 7876-7880 ◽  
Author(s):  
Junsong Sun ◽  
Li Zheng ◽  
Christina Landwehr ◽  
Junshu Yang ◽  
Yinduo Ji
Keyword(s):  
Signal Transduction ◽  
Staphylococcus Aureus ◽  
Bacterial Growth ◽  
Antisense Rna ◽  
Regulatory System ◽  
Gene Replacement ◽  
Bacterial Survival ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT Two-component signal transduction systems play an important role in the ability of bacteria to adapt to various environments by sensing changes in their habitat and by altering gene expression. In this study, we report a novel two-component system, YhcSR, in Staphylococcus aureus which is required for bacterial growth in vitro. We found that the down-regulation of yhcSR expression by induced yhcS antisense RNA can inhibit and terminate bacterial growth. Moreover, without complementary yhcS or yhcR, no viable yhcS or yhcR gene replacement mutant was recoverable. Collectively, these results demonstrated that the YhcSR regulatory system is indispensable for S. aureus growth in culture. Moreover, induced yhcS antisense RNA selectively increased bacterial susceptibility to phosphomycin. These data suggest that YhcSR probably modulates the expression of genes critical for bacterial survival and may be a potential target for the development of novel antibacterial agents.

scholarly journals Strains of the East Asian (W/Beijing) Lineage of Mycobacterium tuberculosis Are DosS/DosT-DosR Two-Component Regulatory System Natural Mutants

2010 ◽  
Vol 192 (8) ◽  
pp. 2228-2238 ◽  
Author(s):  
Ashley Fallow ◽  
Pilar Domenech ◽  
Michael B. Reed
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Frameshift Mutation ◽  
East Asian ◽  
Regulatory System ◽  
Bacterial Respiration ◽  
Low Oxygen ◽  
Gene Encoding ◽  
Two Component ◽  
Dosr Regulon

ABSTRACT As part of our ongoing efforts to uncover the phenotypic consequences of genetic variability among clinical Mycobacterium tuberculosis isolates, we previously reported that isolates of the “East Asian” or “W/Beijing” lineage constitutively overexpress the coordinately regulated transcriptional program known as the DosR regulon under standard in vitro conditions. This phenotype distinguishes the W/Beijing lineage from all other M. tuberculosis lineages, which normally induce expression of this regulon only once exposed to low oxygen or nitric oxide, both of which result in inhibition of bacterial respiration and replication. Transcription of the DosR regulon is controlled through a two-component regulatory system comprising the transcription factor DosR and two possible cognate histidine sensor kinases, DosS and DosT. Through sequence analysis of a carefully selected set of isolates representing each of the major M. tuberculosis lineages, we describe herein a naturally occurring frameshift mutation in the gene encoding the DosT sensor kinase for isolates of the most recently evolved W/Beijing sublineages. Intriguingly, the occurrence of the frameshift mutation correlates precisely with the appearance of the constitutive DosR regulon phenotype displayed by the same “modern” W/Beijing strains. However, complementation studies have revealed that the mutation in dosT alone is not directly responsible for the constitutive DosR regulon phenotype. Our data serve to highlight the evolutionary pressure that exists among distinct M. tuberculosis lineages to maintain tight control over DosR regulon expression.

scholarly journals TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis

2005 ◽  
Vol 202 (12) ◽  
pp. 1715-1724 ◽  
Author(s):  
Andre Bafica ◽  
Charles A. Scanga ◽  
Carl G. Feng ◽  
Cynthia Leifer ◽  
Allen Cheever ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Double Knockout ◽  
Susceptibility To Infection ◽  
Pathogen Challenge ◽  
In Vitro Response ◽  
Ifn Γ ◽  
Aerosol Infection

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9−/− as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9−/− but not TLR2−/− mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-γ responses in vivo, but in common with TLR2−/− animals, the TLR9−/− mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9−/− mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-γ recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9−/− mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.

scholarly journals PafR, a Novel Transcription Regulator, Is Important for Pathogenesis in Uropathogenic Escherichia coli

2014 ◽  
Vol 82 (10) ◽  
pp. 4241-4252 ◽  
Author(s):  
Mordechai Baum ◽  
Mobarak Watad ◽  
Sara N. Smith ◽  
Christopher J. Alteri ◽  
Noa Gordon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Gene Knockout ◽  
Sensory System ◽  
Phosphotransferase System ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Content Type

ABSTRACTThemetVgenomic island in the chromosome of uropathogenicEscherichia coli(UPEC) encodes a putative transcription factor and a sugar permease of the phosphotransferase system (PTS), which are predicted to compose a Bgl-like sensory system. The presence of these two genes, hereby termedpafRandpafP, respectively, has been previously shown to correlate with isolates causing clinical syndromes. We show here that deletion of both genes impairs the ability of the resulting mutant to infect the CBA/J mouse model of ascending urinary tract infection compared to that of the parent strain, CFT073. Expressing the two genes intransin the two-gene knockout mutant complemented full virulence. Deletion of either gene individually generated the same phenotype as the double knockout, indicating that bothpafRandpafPare important to pathogenesis. We screened numerous environmental conditions but failed to detect expression from the promoter that precedes thepafgenesin vitro, suggesting that they arein vivoinduced (ivi). Although PafR is shown here to be capable of functioning as a transcriptional antiterminator, its targets in the UPEC genome are not known. Using microarray analysis, we have shown that expression of PafR from a heterologous promoter in CFT073 affects expression of genes related to bacterial virulence, biofilm formation, and metabolism. Expression of PafR also inhibits biofilm formation and motility. Taken together, our results suggest that thepafgenes are implicated in pathogenesis and that PafR controls virulence genes, in particular biofilm formation genes.

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