Myxococcus xanthus DK1622 Coordinates Expressions of the Duplicate groEL and Single groES Genes for Synergistic Functions of GroELs and GroES

Abstract

Chaperonin GroEL (Cpn60) requires cofactor GroES (Cpn10) for protein refolding in bacteria that possess single groEL and groES genes in a bicistronic groESL operon. Among 4,861 completely-sequenced prokaryotic genomes, 884 possess duplicate groEL genes and 770 possess groEL genes with no neighboring groES. It is unclear whether stand-alone groEL requires groES in order to function and, if required, how duplicate groEL genes and unequal groES genes balance their expressions. In Myxococcus xanthus DK1622, we determined that, while duplicate groELs were alternatively deletable, the single groES that clusters with groEL1 was essential for cell survival. Either GroEL1 or GroEL2 required interactions with GroES for in vitro and in vivo functions. Deletion of groEL1 or groEL2 resulted in decreased expressions of both groEL and groES; and ectopic complementation of groEL recovered not only the groEL but also groES expressions. The addition of an extra groES gene upstream groEL2 to form a bicistronic operon had almost no influence on groES expression and the cell survival rate, whereas over-expression of groES using a self-replicating plasmid simultaneously increased the groEL expressions. The results indicated that M. xanthus DK1622 cells coordinate expressions of the duplicate groEL and single groES genes for synergistic functions of GroELs and GroES. We proposed a potential regulation mechanism for the expression coordination.

Effects of transcriptional mode on promoter substitution and tandem engineering for the production of epothilones in Myxococcus xanthus

“…The groEL1 gene (MXAN_4895) encodes a type I chaperonin involving in many cellular processes (Kerner et al 2005 ). Similarly, the groEL1 is expressed at a high level in conventional cultivation (Wang et al 2013 ; Zhuo et al 2017 ). The brief information of these three promoters is listed in Table 1 .…”

“…In the previous studies, Wu and Kaiser found that the P pilA activity was increased to the vegetative level during the first 12 h under the developmental conditions and then decreased rapidly (Wu and Kaiser 1997 ). Similarly, the P groEL1 promoter also functioned majorly in the early stage on the TPM development medium (Zhuo et al 2017 ). To investigate the involved mechanisms for the epothilone production decrease driven by the P pilA and the P groEL1 promoters, the above constructed pSWU19-based gfp reporter vectors, which are able to integrate into the chromosome at the attB site in M. xanthus (Wu and Kaiser 1995 ), were introduced into DZ2, respectively, producing the strains of DZ2- gfp , DZ2-P epo - gfp , DZ2-P groEL1 - gfp , and DZ2-P pilA - gfp .…”

Suppressor mutations reveal an NtrC-like response regulator, NmpR, for modulation of Type-IV Pili-dependent motility in Myxococcus xanthus

“…(B) Single copy complementation vectors were integrated at the Mx9 phage attB site and resulting strains assayed for motility. The p Nat = 585 bp upstream of mxan_4236 (See Fig 2A ) and p High = 623 bp upstream of mxan_4894 , groES [ 43 , 44 ] (See S1 Fig). D54A is an unphosphorylatable form of NmpR and D54E is a phosphomimetic.…”

“…Complementation of mutations was performed using expression constructs that are capable of integration in single copy at the Mx9 phage attachment site (see Materials and Methods ). Expression of a wild-type allele of nmpR from its putative native promoter (p Nat ; 585 bp upstream of mxan_4236 ; Fig 2A ) or from a high expression promoter (p High ; 623 bp upstream of mxan_4894 , groES [ 43 , 44 ]; S1 Fig ) was not able to restore motility to the Δ pilR Δ nmpR strain. In contrast, expression of nmpR V87E from its native promoter was sufficient to restore motility to Δ pilR Δ nmpR cells ( Fig 3B ).…”

“…All final deletion mutants were verified by PCR using primers spanning the deletion junction and/or internal to the gene deletion. Complementation of each gene was achieved by cloning the entire opening reading frame down-stream of the heterologous high-expression groES promoter (623 bp upstream of mxan_4894 [ 43 , 44 ]; S1 Fig ) or, the putative native promoter of nmpR (585 bp upstream of mxan_4236 ; Fig 2A ). Mutagenesis of vector constructs was done with QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) or Q5 Site-Directed Mutagenesis Kit (New England Biolabs) per the manufacturer’s instructions and constructed mutations sequenced verified.…”