Coexpression of ω subunit in E. coli is required for the maintenance of enzymatic activity of Xanthomonas campestris pv. campestris RNA polymerase

Cheng, Ching-Yuan; Yu, Yu-Jen; Yang, Ming-Te

doi:10.1016/j.pep.2009.07.002

Cited by 6 publications

(5 citation statements)

References 35 publications

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“…Only mild phenotypes can be observed when v is knocked out, suggesting a level of redundancy in the role(s) of v in vivo. Recently, purified RNAP from Xanthomonas campestris was characterized, revealing a strong correlation between the presence of v and both the expression level and activity of the resultant enzyme when overproduced from a plasmid in E. coli (Cheng et al, 2010). Mukherjee et al (1999) found that small amounts of the chaperone protein GroEL co-purified with RNAP from wild-type E. coli cells.…”

Section: Discussionmentioning

confidence: 99%

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition

et al. 2010

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Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core a, b and b9 subunits. The v subunit is conserved between Gram-positive and Gramnegative bacteria, while the d subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the d and v subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed d to be present at equimolar levels with RNAP and v to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP b9 and v subunits in Escherichia coli was also investigated. Similar to B. subtilis, b9 and v closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike v in B. subtilis, v levels in E. coli were close to parity with those of b9. These results indicate that d is likely to be an integral RNAP subunit in Gram-positives, whereas v levels differ substantially between Grampositives and -negatives. The v subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

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Section: Discussionmentioning

confidence: 99%

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition

et al. 2010

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“…The ω subunit was originally described as a molecular latch stabilizing the structure of the RNAP core in T. thermophilus (Vassylyev et al ., 2002). Although interactions between ω and β′ are less extensive in the RNAP of E. coli than in T. thermophilus (Figure 4 and 5), the ω subunit increases the stability of RNAP in E. coli and also in Xanthomonas campestris (Cheng et al ., 2010). Furthermore, the β′ subunit tends to fragment in isolated RNAPs of ω‐less strains of Mycobacterium smegmatis and Stafylococcus aureus (Mathew et al ., 2005; Mao et al ., 2018), which also points to a structure stabilizing role of ω.…”

Section: Introductionmentioning

confidence: 99%

Revealing secrets of the enigmatic omega subunit of bacterial RNA polymerase

Kurkela

Fredman

Salminen

et al. 2020

Molecular Microbiology

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The conserved omega (ω) subunit of RNA polymerase (RNAP) is the only nonessential subunit of bacterial RNAP core. The small ω subunit (7 kDa–11.5 kDa) contains three conserved α helices, and helices α2 and α3 contain five fully conserved amino acids of ω. Four conserved amino acids stabilize the correct folding of the ω subunit and one is located in the vicinity of the β′ subunit of RNAP. Otherwise ω shows high variation between bacterial taxa, and although the main interaction partner of ω is always β′, many interactions are taxon‐specific. ω‐less strains show pleiotropic phenotypes, and based on in vivo and in vitro results, a few roles for the ω subunits have been described. Interactions of the ω subunit with the β′ subunit are important for the RNAP core assembly and integrity. In addition, the ω subunit plays a role in promoter selection, as ω‐less RNAP cores recruit fewer primary σ factors and more alternative σ factors than intact RNAP cores in many species. Furthermore, the promoter selection of an ω‐less RNAP holoenzyme bearing the primary σ factor seems to differ from that of an intact RNAP holoenzyme.

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“…Crude extracts were prepared for the RNAP assay as described by Liao and Kuo (26), except that the buffer described in Cheng et al (10) was used (designated as RPA [RNase protection assay] buffer herein). Two cultures (100 ml) of X. campestris pv.…”

Section: Sds-page and Lc-ms/ms Analysismentioning

confidence: 99%

Genomic Characterization of the Intron-Containing T7-Like Phage phiL7 ofXanthomonas campestris

Lee

Lin

Weng

et al. 2009

Appl Environ Microbiol

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The lytic phage phiL7, which morphologically belongs to the Siphoviridae family, infects Xanthomonas campestris pv. campestris. Nucleotide sequence analysis has revealed that phiL7 contains a linear doublestranded DNA genome (44,080 bp, 56% G؉C) with a 3-protruding cos site (5-TTACCGGAC-3) and 59 possible genes. Among the deduced proteins, 32 have homologs with known functions and 18 show no database similarities; moreover, the genes encoding these 18 proteins mostly have varying G؉C contents and form clusters dispersed along the genome. Only 39 genes have sequences related (27% to 78%) to those of sequenced genes of X. oryzae pv. oryzae phages, although the genome size and architecture of these Xanthomonas phages are similar. These findings suggest that phiL7 acquired genes by horizontal transfer, followed by evolution via various types of mutations. Major differences were found between phiL7 and the X. oryzae pv. oryzae phages: (i) phiL7 has a group I intron inserted in the DNA polymerase gene, the first such intron observed in Xanthomonas phages; (ii) although infection of phiL7 exerted inhibition to the host RNA polymerase, similar to the situations in X. oryzae pv. oryzae phages Xp10 and Xop411, sequence analysis did not identify a homologue of the Xp10 p7 that controls the shift from host RNA polymerase (RNAP) to viral RNAP during transcription; and (iii) phiL7 lacks the tail fiber protein gene that exhibits domain duplications thought to be important for host range determination in OP1, and sequence analysis suggested that p20 (tail protein III) instead has the potential to play this role.The genus Xanthomonas in the gamma subdivision of the Proteobacteria is a diverse and economically important group of bacterial plant pathogens consisting of 27 species (35), causing tremendous loss in agriculture worldwide. Individual species of this genus comprise multiple pathogenic variants (pathovars) and collectively cause diseases on at least 124 monocot species and 268 dicot species: for example, Xanthomonas campestris pv. campestris causes black rot in crucifers, X. campestris pv. vesicatoria causes spot disease in peppers and tomatoes, X. axonopodis pv. citri causes citrus canker, and X. oryzae pv. oryzae causes bacterial leaf blight on rice plants (17). In addition, members of the genus Xanthomonas commonly produce great amounts of an exopolysaccharide (xanthan gum) and X. campestris pv. campestris has been the organism of choice in industry for production of xanthan, which is useful in food, agriculture, industry and cosmetics (55).For controlling bacterial plant diseases, chemical control with antibiotics and copper compounds has been the standard (31, 46). However, many agrochemicals are toxic and may pose significant environmental and health risks (14). Therefore, there has been a resurgence of interest recently in using bacteriophages to replace the currently prevalent chemical measures, in which phages can be used effectively as part of integrated disease management strategies (12,15,16,32). Although pha...

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Coexpression of ω subunit in E. coli is required for the maintenance of enzymatic activity of Xanthomonas campestris pv. campestris RNA polymerase

Cited by 6 publications

References 35 publications

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition

Revealing secrets of the enigmatic omega subunit of bacterial RNA polymerase

Genomic Characterization of the Intron-Containing T7-Like Phage phiL7 ofXanthomonas campestris

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