Gene function analysis in environmental isolates: the nif regulon of the strict iron oxidizing bacterium Leptospirillum ferrooxidans

Abstract

A random genomic library from an environmental isolate of the Gram-negative bacterium Leptospirillum ferrooxidans has been printed on a microarray. Gene expression analysis was carried out with total RNA extracted from L. ferrooxidans cultures in the presence or absence of ammonium as nitrogen source under aerobic conditions. Although practically nothing is known about the genome sequence of this bacterium, this approach allowed us the selection and sequencing of only those clones bearing genes that showed an altered expression pattern. By sequence comparison, we have identified most of the genes of nitrogen fixation regulon in L. ferrooxidans, like the nifHDKENX operon, encoding the structural components of Mo-Fe nitrogenase; nifSU-hesB-hscBA-fdx operon, for Fe-S cluster assembly; the amtB gene (ammonium transporter); modA (molybdenum ABC type transporter); some regulatory genes like ntrC, nifA (the specific activator of nif genes); or two glnB-like genes (encoding the PII regulatory protein). Our results show that shotgun DNA microarrays are very powerful tools to accomplish gene expression studies with environmental bacteria whose genome sequence is still unknown, avoiding the time and effort necessary for whole genome sequencing projects.

Fig. 1.

L. ferrooxidans growth curves under ammonium-rich (squares) and ammonium-starved (circles) conditions. Arrows indicate the points at which total RNA was extracted.

Fig. 2.

L. ferrooxidans DNA microarray. (A) Differential gene expression after hybridization with a labeled cDNA sample mix prepared from RNA isolated from ammonium-rich (Cy5) or ammonium-starved (Cy3) culture. Spots correspond to DNA fragments preferentially transcribed in ammonium-rich culture (red) or ammonium-starved culture (green) and those whose transcription levels are similar in both conditions (yellow). Enlarged spots correspond to negative (-) and positive (+) controls (see Materials and Methods) located at four different positions on the chip. Other control spots having rDNA, as well as some examples of differential expression (red and green spots), are also displayed. (B) Scatter plot representing the intensity of all microarray spots quantified after overlapping the two scanned images as indicated in Materials and Methods. Red spots are those corresponding to DNA fragments overexpressed more than two times in ammonium-rich conditions, whereas the green ones are those overexpressed more than two times under ammonium-starved conditions.

Fig. 3.

Identification of several operons involved in nitrogen fixation in a cross-labeled experiment. Green (Cy3-labeled) and red (Cy5-labeled) spots correspond to two different hybridized cDNAs from the same total RNA sample extracted from ammonium-starved culture. Clones with the indicated averaged ratio between the two hybridization experiments were selected. Ratio from nifNXlfe188g2nifB-bearing clones was >3.8 when RNA was extracted in early stationary growth phase (not shown). Sequencing both ends of each clone allowed us to overlap them in contigs that contain genes involved in nitrogen fixation and other unknown genes. A thin bar represents each cloned DNA fragment. Green arrows indicate the identified ORFs, whereas dotted lines represent unknown sequences.

Fig. 4.

Regulatory region analysis of some genes and operons whose transcription is induced under nitrogen-fixing conditions. These regions contain regulatory signals characteristic of nitrogen fixation (nif) genes: NifA upstream activating sequences (UAS); consensus -24/-12 sequences for σ⁵⁴-dependent promoters; putative Rho independent transcription terminators (black spots) indicating the free energy; dyad repeats (DyR); direct repeats (DR and contiguous arrows); or inverted repeats (opposing arrows) like that containing the nifH translation start codon (ATG). Underlined and bold-faced sequences are those containing well conserved nucleotides. Numbers indicate the distance (nt) with respect to the translation start codon (ATG).

Fig. 5.

L. ferrooxidans nitrogen fixation regulon. Green arrows and green figures represent genes and proteins, respectively, up-regulated under nitrogen fixation conditions. Yellow arrows indicate genes with the same level of expression in ammonium-rich and nitrogen fixation conditions. We assume, by comparison with other bacterial systems, the presence of a uridylyl-transferase/uridylyl removing (Utase/UR, orange rectangle) enzyme (30) that senses cellular nitrogen status through Gln concentration. The blue oval represents the central role that PII protein may play in regulating different cell functions (black arrows). Electron chain transfer represents all those genes we found known to be involved in such a function, like NADH oxidoreductases, ubiquinones, cytochromes, etc. (Table 1 and data not shown). S (sensor) and E (effector) indicate the two-component sensor systems. Encircled P, phosphate; OM, outer membrane; P, periplasm; IM, inner membrane. See text for explanation.