Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

Abstract

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacteriumLeptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillumchemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. ThelcrI gene showed both ς28-like and ς70-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP fromEscherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed inE. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.

Helicobacter pyloristrain ATCC700392 encodes a methyl-accepting chemotaxis receptor protein (MCP) for arginine and sodium bicarbonate

“…To express HP0099, electroporants were grown to exponential density in LB Amp Kan medium at 30°C, shifted to 42°C for 30 min to induce the expression of T7 RNA polymerase, and incubated at 37°C for 30 min. Cells were collected by centrifugation, resuspended in chemotaxis buffer and sonicated, and intracellular proteins were analyzed by polyacrylamide gel electrophoresis [21]. Cells were also assayed for their chemotactic responses to a variety of substrates before and after heat induction, using the method of Mazunder et al [15].…”

Metabolic diversity and adaptive mechanisms of iron‐ and/or sulfur‐oxidizing autotrophic acidophiles in extremely acidic environments

“…Acidithiobacillus spp., except for A. ferrooxidans and A. ferrivorans , express genes involved in flagellar biosynthesis and chemotaxis (Valdés et al ., ; Valdés et al ., ), while all Leptospirillum spp. express the flagellar operon and chemotaxis genes (Delgado et al ., ; Tyson et al ., ; Mi et al ., ; Goltsman et al ., ; Cárdenas et al ., ). Interestingly, a strain of A. ferrooxidans (ATCC 23270) is reported to lack the expression of genes involved in both flagellar biosynthesis and Che signaling (chemotaxis) (Valdés et al ., ), but this finding is inconsistent with the results from previous studies (DISpirito et al ., ; Chakraborty and Roy, ).…”

Integrative approaches for assessing the ecological sustainability ofin situbioremediation

“…With the advent of whole‐genome sequencing and transcriptome analysis, it is being realized that the distribution of chemotaxis‐related genetic elements is much wider than was initially expected (Joseph & Beier, 2007; Lange et al , 2007; Li et al , 2007a, b; Porter et al , 2007). Earlier, some studies had characterized DNA fragments harboring genes associated with bacterial chemotaxis (Froyen et al , 1997; Delgado et al , 1998; Ditty et al , 1998; Hauwaerts et al , 2002). Based on the limited information available on the chemotactic response, it could be stated that regulation of bacterial chemotaxis is largely based on ‘phosphorylation’ and ‘dephosphorylation’ of transducer and effector proteins arranged in a cascade manner.…”