Sigma54 enhancer binding proteins and Myxococcus xanthus fruiting body development

Abstract

A search of the M1genome sequence, which includes 97% of the Myxococcus xanthus genes, identified 53 sequence homologs of sigma54-dependent enhancer binding proteins (EBPs). A DNA microarray was constructed from the M1genome that includes those homologs and 318 other M. xanthus genes for comparison. To screen the developmental program with this array, an RNA extract from growing cells was compared with one prepared from developing cells at 12 h. Previous reporter studies had shown that M. xanthus has initiated development and has begun to express many developmentally regulated genes by 12 h. The comparison revealed substantial increases in the expression levels of 11 transcription factors that may respond to environmental stimuli. Six of the 53 EBP homologs were expressed at significantly higher levels at 12 h of development than during growth. Three were previously unknown genes, and they were inactivated to look for effects on fruiting body development. One knockout mutant produced fruiting bodies of abnormal shape that depended on the composition of the medium.

FIG. 1.

A typical sector of an array after hybridization.

FIG. 2.

Aggregation phenotypes of DK1622 (wild type) and DK1622::pLOJ3320 (Mx_3320 gene disruption mutant) on CF agar. (A) Top view of culture developing for the indicated time. Bar, 0.5 mm. (B) Side view of culture after 72 h. Bar, 0.1 mm. (C) Top view of culture after 72 h. Bar, 0.1 mm.

FIG. 3.

Structure of the putative σ54 EBPs Mx_3320, Mx_3098, and Mx_4756. (A) Amino acid sequence alignment of the central portion of Mx_3320, Mx_3098, and Mx_4756 with the four documented σ54-EBPs E. coli NtrC (GenBank accession no. P06713), E. coli AtoC (GenBank accession no. Q06065), M. xanthus ActB (GenBank accession no. AAK38650), and M. xanthus PilR (GenBank accession no. S70529). Light gray bands indicate 60 to 79% similarity, dark gray bands indicate 80 to 99% similarity, and black indicates identity. The sequences were aligned by Clustal W (7) by using default settings; the alignment is presented by Genedoc (http://www.psc.edu/biomed/genedoc). (B) Domain architecture of Mx_3320, Mx_3098, and Mx_4756. The domains represented as boxes shown to scale were predicted by Pfam. Matches to known domains are indicated. σ54 activation, σ54 interaction domain (PF00158); Receiver, response regulator receiver domain (PF00072); HTH, helix-turn-helix domain (PF02954).