Functional and regulatory analysis of the OmpF-like porin, OpnP, of the symbiotic bacterium Xenorhabdus nematophilus.
The function and novel regulation of OpnP of the symbiotic/pathogenic bacterium, Xenorhabdus nematophilus was studied. In vitro pore-function analysis of purified OpnP indicated that the single-channel-conductance values were similar to that measured for the porin protein, OmpF, of Esherichia coli. Nucleotide sequence analysis revealed that the mature OpnP protein contained 348 amino acid residues and shared 55% amino acid sequence identity with OmpF. Similar to ompF, opnP mapped between asnS and aspC. The 16 transmembrane beta-sheet structures and the internal loop 3 were highly conserved, while the remaining external loop domains were more divergent. Primer extension analysis identified the start site of transcription of opnP. A sigma 70-type promoter, a perfect 20 bp OmpR-binding site, and a binding site for the antisense molecule, micF RNA, were found in the upstream region of opnP. While the overall sequence identity of the asn-opnP-aspC region was high, the intergenic region between asnS and opnP had diverged markedly. The asnS-opnP region was 313 bp shorter than the intergenic region between asnS and ompF and lacked the OmpR-binding site that is required for ompF repression by high osmolarity in E. coli. Results from osmolarity-shift experiments indicated that OpnP was not repressed by high osmolarity. It was also found that OpnP was thermally regulated.
Forst
S
S
Department of Biological Sciences, University of Wisconsin, Milwaukee 53201, USA. sforst@alpha 2.csd uwm.edu
Waukau
J
J
Leisman
G
G
Exner
M
M
Hancock
R
R
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004755:Enterobacteriaceae / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D018272:Porins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C102238:OpnP protein, Xenorhabdus nematophilus; D018272:Porins; D016372:RNA, Antisense