Anaerobic Growth,a Property Horizontally Transferred by an Hfr-Like Mechanism among Extreme Thermophiles

Despite the fact that the extreme thermophilic bacteria belonging to the genus Thermus are classified as strict aerobes, we have shown that Thermus thermophilus HB8 (ATCC 27634) can grow anaerobically when nitrate is present in the growth medium. This strain-specific property is encoded by a respiratory nitrate reductase gene cluster (nar) whose expression is induced by anoxia and nitrate (S. Ramírez-Arcos, L. A. Fernández-Herrero, and J. Berenguer, Biochim. Biophys. Acta, 1396:215–1997). We show here that this nar operon can be transferred by conjugation to an aerobic Thermus strain, enabling it to grow under anaerobic conditions. We show that this transfer takes place through a DNase-insensitive mechanism which, as for the Hfr (high frequency of recombination) derivatives of Escherichia coli, can also mobilize other chromosomal markers in a time-dependent way. Three lines of evidence are presented to support a genetic linkage between nar and a conjugative plasmid integrated into the chromosome. First, the nar operon is absent from a plasmid-free derivative and from a closely related strain. Second, we have identified an origin for autonomous replication (oriV) overlapping the last gene of the nar cluster. Finally, the mating time required for the transfer of the nar operon is in good agreement with the time expected if the transfer origin (oriT) were located nearby and downstream of nar.

Most extreme thermophiles that live in geothermal environments are strict anaerobes (3, 11) as a consequence of the adaptation to the low solubility of oxygen at these temperatures. However, members of the genus Thermus constitute an exception to this general rule, being described taxonomically as strictly aerobic chemorganotrophs (2).However, we recently showed that one of the most thermophilic isolates of this genus, Thermus thermophilus HB8, was able to grow anaerobically when nitrate was present in the medium. Biochemical and genetic evidence demonstrated that this ability was related to the synthesis of a membrane-bound respiratory nitrate reductase complex whose protein components, the α (NarG; 136 kDa), β (NarH; 57 kDa), and γ (NarI; 28 kDa) subunits, were homologous (about 48 to 50% sequence identity) to those from mesophilic facultative anaerobes (e.g., Escherichia coli). The genes encoding these subunits were located within a single operon (nar) that was induced under low oxygen concentrations when nitrate was present (21). In contrast to those described for most nitrate reducers, the product of nitrate respiration was secreted to the growth medium through an unknown transporter.We also observed that even a closely related strain, such as T. thermophilus HB27, was unable to grow under such anaerobic conditions (21). Since the main difference between strains HB8 and HB27 of T. thermophilus is the absence of plasmids from the latter, the possibility that the nar operon could be encoded by a transferable genetic element, such as a plasmid, was considered.In this article, we analyze this possibility and demonstrate that the ability to grow by nitrate respiration can be transferred to the aerobic strain T. thermophilus HB27 by conjugation. We also relate this ability to the integration of a nar-carrying conjugative plasmid into the chromosome of T. thermophilus HB8. Moreover, we show that, as for the Hfr strains of E. coli, this integrated plasmid can also mobilize other chromosomal genes in a time-dependent way.