Use of a green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata

Abstract

We investigated the applicability of the green fluorescent protein of Aequorea victoria as a reporter for gene expression in the strictly fermentative Gram-negative ethanologenic bacterium Zymomonas mobilis and in the moderately halophilic bacterium Halomonas elongata. We have succeeded to express a mutated gene of green fluorescent protein under the control of different promoters in Z. mobilis and H. elongata grown under various glucose or salt concentrations, respectively. Our results demonstrate that gfp can serve as an easily assayable reporter gene in both organisms. Maximum fluorescence was obtained in Z. mobilis grown aerobically and in H. elongata grown under elevated salt concentration in solid medium. For both bacteria the fluorescence obtained was higher when the gfp gene was placed under the control of a native promoter.

Transcriptional Analysis of a Gene Cluster Involved in Glucose Tolerance in Zymomonas mobilis : Evidence for an Osmoregulated Promoter

“…Plasmid pAEG2 was transferred to Z. mobilis CP4Rif r from E. coli donors by bacterial conjugation. CP4Rif r , a rifampin-resistant derivative of prototype Z. mobilis strain CP4 with the wild-type glucose tolerance phenotype (33), was used as a suitable recipient host of plasmid pAEG1 (13). The amount of fluorescence emitted by CP4Rif r /pAEG2 cells was measured using strain CP4Rif r / pAE1 as a negative control (13).…”

“…CP4Rif r , a rifampin-resistant derivative of prototype Z. mobilis strain CP4 with the wild-type glucose tolerance phenotype (33), was used as a suitable recipient host of plasmid pAEG1 (13). The amount of fluorescence emitted by CP4Rif r /pAEG2 cells was measured using strain CP4Rif r / pAE1 as a negative control (13). The CP4Rif r /pAEG2 isolate showed significantly greater amounts of fluorescence than strain CP4Rif r /pAE1 but smaller amounts of fluorescence than strain CP4Rif r /pAEG1 (Table 3).…”

“…In addition, the promoter activity and expression pattern of this gene cluster were investigated under various growth conditions with different glucose concentrations and osmotic pressures. In this effort the reporter gene gfp was used (4,9), since it was recently shown to be suitable for transcriptional analysis in Z. mobilis (13). Furthermore, the nature of the CU1 mutation was characterized on the molecular level, which provided additional evidence for the autoregulation of the glc operon by the product of ORF4.…”

“…2. The P pdc promoter (36) was excised from plasmid pAEG1 (13) by KpnI digestion and replaced with the region of the putative promoter described above comprising a 0.23-kb fragment. This fragment was obtained by PCR amplification using the following primers carrying a KpnI extension at their 5Ј ends: forward primer 5Ј-GACGAAAGGGTACCTCCGT AACG-3Ј corresponding to bases 101 to 123 and reverse primer 5Ј-GAGTTAT CGGTACCTTGAATTGCCG-3Ј corresponding to bases 311 to 335.…”

Diverse Microhabitats Experienced by Halomonas variabilis on Salt-Secreting Leaves

“…Members of the genus Halomonas are commonly isolated from salt lakes and brines, saline soils, and salted food products and display some of the most extensive salt tolerance observed in prokaryotes (6). These moderate halophiles are gaining considerable attention for their potential use in biotechnological applications because of their ease of culture and amenability to genetic manipulation (7,8). Although there have been many culture-based investigations into the physiology of these organisms (9), detailed studies of in situ colonization by Halomonas species are lacking (6).…”