A single-nucleotide mutation in the −10 promoter region inactivates thenarK2Xpromoter inMycobacterium bovisandMycobacterium bovisBCG and has an application in diagnosis

Chauhan, Santosh; Singh, Alka; Tyagi, Jaya Sivaswami

doi:10.1111/j.1574-6968.2009.01876.x

Cited by 17 publications

(13 citation statements)

References 23 publications

(49 reference statements)

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“…To our knowledge, this is the first analysis of the complete mutagenesis of the Ϫ10 hexamer of a mycobacterial promoter, and it is of interest that the nucleotides at the Ϫ9 and Ϫ10 positions have considerable variations in activity. Other reported mutagenesis studies of equivalent positions in mycobacterial promoters indicate that these positions are important for promoter activity, but the effects of particular bases are not consistent with the results for P R , demonstrating the effect of sequence context (42,43). These positions may thus play a prominent role is modulating the activity of individual promoters in M. smegmatis and M. tuberculosis.…”

Section: Discussioncontrasting

confidence: 40%

Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

Oldfield

Hatfull

2014

Journal of Bacteriology

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The P R promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that P R has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of P R showed that it uses a canonical ؊10 hexamer recognized by SigA, and mutants with mutations to the sequence 5=-TATAMT had the greatest activities. It does not contain a 5=-TGN-extended ؊10 sequence, although mutants with mutations creating an extended ؊10 sequence had substantially increased promoter activity. Mutations in the ؊35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the ؊35 hexamer differentially affected promoter activity, depending on the ؊10 and extended ؊10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout P R generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter.

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Section: Discussioncontrasting

confidence: 40%

Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

Oldfield

Hatfull

2014

Journal of Bacteriology

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“…This study demonstrated that the polymorphism of a single nucleotide in the promoter region is sufficient to significantly change the gene expression. A single-nucleotide mutation in the Ϫ10 promoter region was shown to inactivate the narK2X promoter in Mycobacterium bovis and Mycobacterium bovis BCG (32). Similarly, here, we have demonstrated, using the gene for GFP as a reporter, that alk gene promoter polymorphisms greatly affect the expression of alk genes.…”

Section: Figmentioning

confidence: 58%

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel

Gunasekera

Bowen

Zhou

et al. 2017

Appl Environ Microbiol

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Pseudomonas aeruginosa can utilize hydrocarbons, but different strains have various degrees of adaptation despite their highly conserved genome. P. aeruginosa ATCC 33988 is highly adapted to hydrocarbons, while P. aeruginosa strain PAO1, a human pathogen, is less adapted and degrades jet fuel at a lower rate than does ATCC 33988. We investigated fuel-specific transcriptomic differences between these strains in order to ascertain the underlying mechanisms utilized by the adapted strain to proliferate in fuel. During growth in fuel, the genes related to alkane degradation, heat shock response, membrane proteins, efflux pumps, and several novel genes were upregulated in ATCC 33988. Overexpression of alk genes in PAO1 provided some improvement in growth, but it was not as robust as that of ATCC 33988, suggesting the role of other genes in adaptation. Expression of the function unknown gene PA5359 from ATCC 33988 in PAO1 increased the growth in fuel. Bioinformatic analysis revealed that PA5359 is a predicted lipoprotein with a conserved Yx(FWY)xxD motif, which is shared among bacterial adhesins. Overexpression of the putative resistance-nodulation-division (RND) efflux pump PA3521 to PA3523 increased the growth of the ATCC 33988 strain, suggesting a possible role in fuel tolerance. Interestingly, the PAO1 strain cannot utilize n-C 8 and n-C 10 . The expression of green fluorescent protein (GFP) under the control of alkB promoters confirmed that alk gene promoter polymorphism affects the expression of alk genes. Promoter fusion assays further confirmed that the regulation of alk genes was different in the two strains. Protein sequence analysis showed low amino acid differences for many of the upregulated genes, further supporting transcriptional control as the main mechanism for enhanced adaptation.IMPORTANCE These results support that specific signal transduction, gene regulation, and coordination of multiple biological responses are required to improve the survival, growth, and metabolism of fuel in adapted strains. This study provides new insight into the mechanistic differences between strains and helpful information that may be applied in the improvement of bacterial strains for resistance to biotic and abiotic factors encountered during bioremediation and industrial biotechnological processes.

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“…This result is likely due to polymorphisms in the promoter region of narG that have been shown to prevent the up-regulation of nar genes by NO donor in M. bovis BCG (Stermann et al, 2004; Huang et al, 2015). In addition, narK2 gene is poorly expressed in M. bovis and M. bovis BCG (Honaker et al, 2008) due to a single-nucleotide polymorphism in the narK2X promoter region leading to a reduced promoter activity (Chauhan et al, 2010). In contrast to the nar genes, the rv2617c gene, was highly induced by SNAP in M. bovis BCG, to an extent even greater than the one reported in M. tuberculosis (Ohno et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Endogenous and Exogenous KdpF Peptide Increases Susceptibility of Mycobacterium bovis BCG to Nitrosative Stress and Reduces Intramacrophage Replication

Olvera

Vivès

Molle

et al. 2017

Front. Cell. Infect. Microbiol.

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Emerging antibiotic resistance in pathogenic bacteria like Mycobacterium sp., poses a threat to human health and therefore calls for the development of novel antibacterial strategies. We have recently discovered that bacterial membrane peptides, such as KdpF, possess anti-virulence properties when overproduced in pathogenic bacterial species. Overproduction of the KdpF peptide in Mycobacterium bovis BCG decreased bacterial replication within macrophages, without presenting antibacterial activity. We propose that KdpF functions as a regulatory molecule and interferes with bacterial virulence, potentially through interaction with the PDIM transporter MmpL7. We demonstrate here that KdpF overproduction in M. bovis BCG, increased bacterial susceptibility to nitrosative stress and thereby was responsible for lower replication rate within macrophages. Moreover, in a bacterial two-hybrid system, KdpF was able to interact not only with MmpL7 but also with two membrane proteins involved in nitrosative stress detoxification (NarI and NarK2), and a membrane protein of unknown function that is highly induced upon nitrosative stress (Rv2617c). Interestingly, we showed that the exogenous addition of KdpF synthetic peptide could affect the stability of proteins that interact with this peptide. Finally, the exogenous KdpF peptide presented similar biological effects as the endogenously expressed peptide including nitrosative stress susceptibility and reduced intramacrophage replication rate for M. bovis BCG. Taken together, our results establish a link between high levels of KdpF and nitrosative stress susceptibility to further highlight KdpF as a potent molecule with anti-virulence properties.

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A single-nucleotide mutation in the −10 promoter region inactivates thenarK2Xpromoter inMycobacterium bovisandMycobacterium bovisBCG and has an application in diagnosis

Cited by 17 publications

References 23 publications

Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel

Endogenous and Exogenous KdpF Peptide Increases Susceptibility of Mycobacterium bovis BCG to Nitrosative Stress and Reduces Intramacrophage Replication

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