Transcriptional Organization of Genes for Protocatechuate and Quinate Degradation from Acinetobacter sp. Strain ADP1

손서연

Introduction

dca-pca-qui-pob-hca chromosomal cluster.

All 13 genes of the pca and qui gene cluster are transcribed in the same direction, with the exception of the regulator gene pcaU. The regulation of protocatechuate degradation as well as quinate utilization was studied thoroughly decades ago, and the induction of both enzyme sets by protocatechuate has been shown.

The regulatory pattern of qui gene expression resembles that of pca gene expression-->a function of PcaU for both gene clusters.

The adjacent location of both clusters, with the same direction of transcription for all genes-->to originate from the promoter pcaIp.

Here we describe an investigation of the pca and qui transcripts in the wild type as well as in a strain missing the promoter pcaIp. In addition, we describe a second promoter upstream of quiA, quiAp, that drives the low and constitutive expression of quiA.

Results

Transcriptional readthrough between the pca and the qui genes and between quiX and quiA.

-RT- PCR

-RNAs were isolated from Acinetobacter sp. strain ADP1 after growth on pyruvate, 2 mM protocatechuate.

-pcaG-quiB , quiX-quiA

-Under either condition, we detected a PCR product of the expected size (data not shown)-->there is transcriptional readthrough in both areas.

Promoter search upstream of quiB and of quiA.

The two intergenic regions, pcaG-quiB and quiX-quiA.

5' end of quiA transcript.

Based on the finding of promoter activity in the quiX-quiA region, we expected the presence of a transcriptional start site upstream of quiA.

The primer extension signal was present in total RNAs from cells grown on quinate as well as in RNAs from cells grown on succinate, reflecting the independence of this transcript from the carbon source, as observed earlier for promoter activity.

Transcripts within the pca-qui gene cluster.

-Northern blot analyses

pcaI, quiX , quiA as probes

-Faint signals were obtained before the addition of protocatechuate, whereas strong signals were present 15 min after the addition of protocatechuate, reflecting the induction of expression by protocatechuate.

Transcription of the pca and qui genes in the absence of the promoter pcaIp.

-Created strain ADPU1, which has a 220-bp deletion that eliminates pcaIp.

-Northern blot experiments with probes for pcaC, pcaH, quiX, and quiA.

-Signal intensities between the wild type and the pcaIp deletion strain.

quiX probe-->the mutant had 6% of the wild-type signal.

quiA probe-->the mutant expressed 25% of the wild-type level.

Transcription of pca and qui genes in the absence of the regulator PcaU.

-Strain ADP331, containing a deletion of two-thirds of the pcaU gene.

-Northern blot experiments with the pcaC and pcaH probe, the transcript was present at a level of 8% that of the induced wild type.

-Using the quiX probe, we obtained slightly increased RNA levels in strain ADP331 (14% of the induced wild-type level).

-The use of the quiA probe for strain ADP331 resulted in a transcript level of 25% of the induced wild-type level, which was comparable to the data obtained with the pcaIp-negative strain. This finding supports the presence of a constitutive promoter upstream of quiA.

Discussion-Northern blot analysis with a probe for the pcaI (~ quiX).

-ADP1, ADPU1- 4-kb, 2.4-kb

(1)The promoter activity found upstream of quiA

(2)The observation of a 5’ end of the transcript

(3)The fact that it was not detected with the quiX probe in ADPU1.

2.4-kb: quiA

-quiA promoter, pcaIp

-The basal level of pca-qui operon expression

-Qui genes are active in periplasmthe transport of protocatechuate into the cell is required.(PcaK, VanK)

-Dysfunctional protocatechuate 3,4 dioxygenase

maximal induction of the pca and qui genes

-Dehydroshikimate