FlhD/FlhC Is a Regulator of Anaerobic Respiration and the Entner-Doudoroff Pathway through Induction of the Methyl-Accepting Chemotaxis Protein Aer

Abstract

The regulation by two transcriptional activators of flagellar expression (FlhD and FlhC) and the chemotaxis methyl-accepting protein Aer was studied with glass slide DNA microarrays. An flhD::Kan insertion and an aer deletion were independently introduced into two Escherichia coli K-12 strains, and the effects upon gene regulation were investigated. Altogether, the flhD::Kan insertion altered the expression of 29 operons of known function. Among them was Aer, which in turn regulated a subset of these operons, namely, the ones involved in anaerobic respiration and the Entner-Doudoroff pathway. In addition, FlhD/FlhC repressed enzymes involved in aerobic respiration and regulated many other metabolic enzymes and transporters in an Aerindependent manner. Expression of 12 genes of uncharacterized function was also affected. FlhD increased gltBD, gcvTHP, and ompT expression. The regulation of half of these genes was subsequently confirmed with reporter gene fusions, enzyme assays, and real-time PCR. Growth phenotypes of flhD and flhC mutants were determined with Phenotype MicroArrays and correlated with gene expression.The Escherichia coli flagellar regulon consists of 14 operons expressed in a transcriptional hierarchy (for a review see reference 35). The flagellar master operon flhDC consists of flhD and flhC (4). The active complex is a heterotetramer (C 2 D 2 ) that binds to the upstream sequences of class II promoters (24). FlhD/FlhC also has nonflagellar targets. Previous work with Panorama E. coli gene arrays (Sigma GenoSys, The Woodlands, Tex.) and lacZ fusions determined that FlhD/FlhC regulates the transporter for galactose (mglBAC), the rodshape determination proteins (mreBCD), the tricarboxylic acid (TCA) cycle enzyme malate dehydrogenase (mdh), and five enzymes involved in anaerobic respiration (41).Although FlhD alone does not bind to class II flagellar promoters, FlhD without the involvement of FlhC is involved in the regulation of the cell division rate (36). Mutants with mutations in flhD, but not those with mutations in flhC, continued to divide rapidly as they entered stationary phase. This was mediated by cadA (39), the second gene in the cadBA operon, encoding lysine decarboxylase (28,29,58).In this study, glass slide genomic microarrays were used to study transcriptional regulation by FlhD and FlhC. Altogether, 29 operons that have a characterized function and 12 genes of unknown function were affected by FlhD or FlhD/FlhC. While the regulation of anaerobic respiration involved the oxygen sensor Aer, aerobic regulation appeared to be unaffected by this protein. MATERIALS AND METHODSBacterial strains and plasmids. Strains and plasmids are presented in Table 1. MC1000 was derived by crossing the araD139 ⌬(araA-leu)7697 allele of D7091 Hfr into M182 (M. Casadaban). MC1000 flhD::Kan contains a kanamycin resistance gene inserted into flhD which exhibits a polar effect upon the expression of flhC (26). YK410 was used as a second parental strain (23). YK4131 (flhD A32D) and YK4136 (flhC N70T) contain p...

Differential Activation of Escherichia coli Chemoreceptors by Blue-Light Stimuli

“…The Aer-only UU1250 strains partially restored swarming in semisolid agar. In addition, they were resistant to oxygen depletion in liquid media, consistent with the induction by Aer of anaerobic respiration pathway components at low oxygen tension (39). In brief, the strains remained vigorously motile in the bridged coverslip chambers for tens of minutes after oxygen depletion as monitored by resazurin (51), whereas UU1250 strains expressing Tar or Tsr became immediately immotile.…”

Members of native coral microbiota inhibit glycosidases and thwart colonization of coral mucus by an opportunistic pathogen

“…The GacS/GacA-Csr system is not likely to be affected by the activities of these commensals: first, the phenotype of the gacA mutant (Krediet et al, in review) is distinct from what is observed when the wild type is exposed to the commensals tested here, and, second, the commensals did not affect the expression of the gacA::lacZ reporter (Krediet, unpublished data). It is also unlikely that FlhDC is affected by the commensals: even though FlhDC in Enterobacteriacea is known to contribute to the regulation of both motility and metabolism (Pruss et al, 2003), the fact that none of the commensals inhibited swimming motility suggests that FlhDC is also an unlikely target of these activities. Therefore, these observations suggest that either the compounds target another regulatory cascade, or that different compounds disrupt swarming and the induction of enzymatic activities separately.…”

“…We do not yet know whether the same compound is involved in the inhibition of both swarming and catabolism of mucus components. However, if this were the case, it is possible to hypothesize that a higher-level regulatory system (GacS/GacA-Csr or FlhDC) (Romeo, 1998;Pruss et al, 2003;Park and Forst, 2006;Timmermans and Van Melderen, 2010) may be the target. The GacS/GacA-Csr system is not likely to be affected by the activities of these commensals: first, the phenotype of the gacA mutant (Krediet et al, in review) is distinct from what is observed when the wild type is exposed to the commensals tested here, and, second, the commensals did not affect the expression of the gacA::lacZ reporter (Krediet, unpublished data).…”

Refining the Binding of the Escherichia coli Flagellar Master Regulator, FlhD 4 C 2 , on a Base-Specific Level

“…The activity and stability of FlhDC are regulated by FliZ, FliT, and FliD, the downstream gene products in the flagellar regulon, by YdiV, an EAL-like protein acting as an anti-FlhD 4 C 2 factor, by ClpXP, an ATP-dependent protease, and by DnaK, a protein chaperone (1,45,46,58,59,62,67). Microarray studies suggest that FlhDC also functions as a global regulator involved in carbon metabolism and anaerobic respiration (38,39).…”