Essential role of aspartokinase in L-threonine production by Escherichia coli W mutants.

The heptose-less mutant of Escherichia coli, D31m4, bound complement subcomponent C1q and its collagen-like fragments (C1qCLF) with Ka values of 1.4 x 10(8) and 2.0 x 10(8) M-1 respectively. This binding was suppressed by chemical modification of C1q and C1qCLF using diethyl pyrocarbonate (DEPC). To investigate the role of lipopolysaccharides (LPS) in this binding, biosynthetically labelled [14C]LPS were purified from E. coli D31m4 and incorporated into liposomes prepared from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) [PC/PE/LPS, 2:2:1, by wt.]. Binding of C1q or its collagen-like fragments to the liposomes was estimated via a flotation test. These liposomes bound C1q and C1qCLF with Ka values of 8.0 x 10(7) and 2.0 x 10(7) M-1; this binding was totally inhibited after chemical modification of C1q and C1qCLF by DEPC. Liposomes containing LPS purified from the wild-strain E. coli K-12 S also bound C1q and C1qCLF, whereas direct binding of C1q or C1qCLF to the bacteria was negligible. Diamines at concentrations which dissociate C1 into C1q and (C1r, C1s)2, strongly inhibited the interaction of C1q or C1qCLF with LPS. Removal of 3-deoxy-D-manno-octulosonic acid (2-keto-3-deoxyoctonic acid; KDO) from E. coli D31m4 LPS decreases the binding of C1qCLF to the bacteria by 65%. When this purified and modified LPS was incorporated into liposomes, the C1qCLF binding was completely abolished. These results show: (i) the essential role of the collagen-like moiety and probably its histidine residues in the interaction between C1q and the mutant D31m4; (ii) the contribution of LPS, particularly the anionic charges of KDO, to this interaction.

Download Full-text

Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.00159-18 ◽

2018 ◽

Vol 200 (12) ◽

Author(s):

Chunyou Mao ◽

Yan Zhu ◽

Pei Lu ◽

Lipeng Feng ◽

Shiyun Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Rna Polymerase ◽

Essential Role ◽

Β Subunit ◽

Content Type ◽

E Coli ◽

Core Enzyme ◽

Loop Region

ABSTRACT The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis . Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly. IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α 2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

Download Full-text