Identification of Two myo- Inositol Transporter Genes of Bacillus subtilis

Abstract

Among hundreds of mutants constructed systematically by the Japanese groups participating in the functional analysis of the Bacillus subtilis genome project, we found that a mutant with inactivation of iolT (ydjK) exhibited a growth defect on myo-inositol as the sole carbon source. The putative product of iolT exhibits significant similarity with many bacterial sugar transporters in the databases. In B. subtilis, the iolABCDEFGHIJ and iolRS operons are known to be involved in inositol utilization, and its transcription is regulated by the IolR repressor and induced by inositol. Among the iol genes, iolF was predicted to encode an inositol transporter. Inactivation of iolF alone did not cause such an obvious growth defect on inositol as the iolT inactivation, while simultaneous inactivation of the two genes led to a more severe defect than the single iolT inactivation. Determination of inositol uptake by the mutants revealed that iolT inactivation almost completely abolished uptake, but uptake by IolF itself was slightly detectable. These results, as well as the K m and V max values for the IolT and IolF inositol transporters, indicated that iolT and iolF encode major and minor inositol transporters, respectively. Northern and primer extension analyses of iolT transcription revealed that the gene is monocistronically transcribed from a promoter likely recognized by A RNA polymerase and negatively regulated by IolR as well. The interaction between IolR and the iolT promoter region was analyzed by means of gel retardation and DNase I footprinting experiments, it being suggested that the mode of interaction is quite similar to that found for the promoter regions of the iol divergon.

myo-Inositol transport by Salmonella enterica serovar Typhimurium

“…Overexpression of iolT1 and iolT2 of the same organism leads to a twofold increase of the D-fructose uptake rate, but both transporters show a lower specificity towards this sugar than towards MI (Bäumchen et al, 2009). However, an apparent growth defect of iolT mutants of B. subtilis or S. enterica serovar Typhimurium in the presence of a variety of sugars has not been observed (Yoshida et al, 2002). A major difference between MI utilization by Gram-positive bacteria and that of S. enterica serovar Typhimurium is the extended lag phase of this Gram-negative pathogen in medium containing MI as sole carbon and energy source (Fig.…”

“…1a). Two MI transporters with major and minor transport activity have also been identified in B. subtilis and C. glutamicum (Krings et al, 2006;Yoshida et al, 2002). In B. subtilis, the minor transporter IolF has been shown to have a lower substrate affinity than that of IolT, and IolF can support growth on MI only partially.…”

“…and Paenibacillus sp., are also grouped, indicating that these proteins transport other sugars or sugar alcohols. Interestingly, the Bacillus cereus IolT1 homologue of this group is not identical to the major MI transporter of B. subtilis (Yoshida et al, 2002), but is more closely related to homologues from Gram-negative species. On the other hand, IolT1 homologues of Yersinia intermedia, Yersinia enterocolitica and Klebsiella pneumoniae are more closely related to transporter proteins of Gram-positive bacteria, making it difficult to trace the evolutionary origin of iolT1 and the gene clusters it belongs to.…”

“…An intermediate of MI degradation, 2-deoxy-5-keto-D-gluconic acid 6-phosphate, has been shown to antagonize IolR binding, thus inducing the expression of iol genes (Yoshida et al, 2008(Yoshida et al, , 1999. Two proteins belonging to the major facilitator superfamily (MFS), IolT and IolF, have been identified as the major and minor inositol transporters of B. subtilis, and IolR has been revealed to inhibit the transcription of iolT (Yoshida et al, 2002). MI degradation has also been studied in Corynebacterium glutamicum (Krings et al, 2006), Clostridium perfringens (Kawsar et al, 2004) and Lactobacillus casei (Yebra et al, 2007).…”

Identification of a Functional 2-keto-myo-Inositol Dehydratase Gene ofSinorhizobium frediiUSDA191 Required formyo-Inositol Utilization

“…3,4,8,9) In B. subtilis, the iol divergon, consisting of the operons iolABCDEFGHIJ and iolRS, 1) and iolT 10) are involved in myo-inositol catabolism and transport. Not all of the B. subtilis enzymes encoded by these genes have been fully elucidated.…”