Abstract Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) produce free fatty acids (FFAs) because the FFAs generated by deacylation of membrane lipids cannot be recycled. An engineered Aas-deficient mutant of Synechocystis sp. PCC 6803 grew normally under low-light (LL) conditions (50 µmol photons m−2 s−1) but was unable to sustain growth under high-light (HL) conditions (400 µmol photons m−2 s−1), revealing a crucial role of Aas in survival under the HL conditions. Several-times larger amounts of FFAs were produced by HL-exposed cultures than LL-grown cultures. Palmitic acid accounted for ∼85% of total FFAs in HL-exposed cultures, while C18 fatty acids (FAs) constituted ∼80% of the FFAs in LL-grown cultures. Since C16 FAs are esterified to the sn-2 position of lipids in the Synechocystis species, it was deduced that HL irradiation activated deacylation of lipids at the sn-2 position. Heterologous expression of FarB, the FFA exporter protein of Neisseria lactamica, prevented intracellular FFA accumulation and rescued the growth defect of the mutant under HL, indicating that intracellular FFA was the cause of growth inhibition. FarB expression also decreased the ‘per-cell’ yield of FFA under HL by 90% and decreased the proportion of palmitic acid to ∼15% of total FFA. These results indicated that the HL-induced lipid deacylation is triggered not by strong light per se but by HL-induced damage to the cells. It was deduced that there is a positive feedback loop between HL-induced damage and lipid deacylation, which is lethal unless FFA accumulation is prevented by Aas.
Porphyromonas gingivalis , a bacterium implicated in periodontal pathogenesis, has a growth requirement for iron protoporphyrin IX. By complementation with a P. gingivalis 381 chromosomal DNA library, we were able to isolate a clone that enhanced the poor growth of a hemG mutant of Escherichia coli . The DNA sequence analysis of this clone revealed three open reading frames (ORFs). ORF3 encoded a protein of 466 amino acids with a calculated molecular weight of 51 695 Da. The deduced amino acid sequence of the ORF3 gene had significant similarity to sequences of protoporphyrinogen oxidase (PPO) from Myxococcus xanthus (30% identical residues). When the ORF3 gene was overexpressed in E. coli , the extract had much higher PPO activity than a control extract, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Thus, ORF3 was named PgHemG. Furthermore, several porphyrin‐related genes, including hemD , hemN and hemH , were identified in the data bases on the websites available on‐line. We postulated that a porphyrin biosynthetic pathway to heme from preuroporphyrin may be conserved in P. gingivalis .
We investigated the viability of Escherichia coli cells during long-term cultivation in Brain Heart Infusion (BHI) medium and observed that the number of viable cells increased, then decreased, and increased again, in this medium, and finally the cells died out within about 10 days. This cell death may result from an increase in the pH of the medium. After repeated cultivation in BHI, bacterial cells that did not die out even under conditions of further cultivation were obtainable from cultures showing a stabilized viable count. We propose that long-term cultivation in BHI medium is a good system for studying growth phase-specific events in E. coli cells, because the total life-cycle of a population of E. coli, including exponential growth, stationary phase, and extinction, can be seen during a period of only about 10 days. Also, this system clearly allows detection of a phenotype that may not be detectable in orther commonly used media. Moreover, in this report, we show that mutants displaying the GASP (growth advantage in stationary phase) phenotype appear at high frequency under long-term cultivation conditions.
ABSTRACT The heat shock response in alpha proteobacteria is unique in that a combination of two regulators is involved: a positive regulator, RpoH (ς 32 homolog), found in the alpha, beta, and gamma proteobacteria, and a negative regulator, HrcA, widely distributed in eubacteria but not in the gamma proteobacteria. To assess the differential roles of the two regulators in these bacteria, we cloned the hrcA-grpE operon of Agrobacterium tumefaciens , analyzed its transcription, and constructed deletion mutants lacking RpoH and/or HrcA. The ΔrpoH mutant and ΔrpoH ΔhrcA double mutant were unable to grow above 30°C. Whereas the synthesis of heat shock proteins (e.g., DnaK, GroEL, and ClpB) was transiently induced upon temperature upshift from 25 to 37°C in the wild type, such induction was not observed in the ΔrpoH mutant, except that GroEL synthesis was still partially induced. By contrast, the ΔhrcA mutant grew normally and exhibited essentially normal heat induction except for a higher level of GroEL expression, especially before heat shock. The ΔrpoH ΔhrcA double mutant showed the combined phenotypes of each of the single mutants. The amounts of dnaK and groE transcripts before and after heat shock, as determined by primer extension, were consistent with those of the proteins synthesized. The cellular level of RpoH but not HrcA increased significantly upon heat shock. We conclude that RpoH plays a major and global role in the induction of most heat shock proteins, whereas HrcA plays a restricted role in repressing groE expression under nonstress conditions.
The anticodon of the tRNA Lys gene (trnK) in the liverwort, Marchantia polymorpha , was anificially converted to an amber anticodon. This mutant tRNA Lys (CTA) gene carrying either the intron or the C 27 ‐C 43 mismatch at the anticodon‐stem is not functional in Escherichia coli , but without both of them, it does work as a tRNA Lys amber suppressor.
Using Escherichia coli strain VS101, whose hemH gene encoding the ferrochelatase is partially defective, we isolated and analyzed a clone (designated XWH-1) from a X phage library of soybean (Glycine max) cDNA, which exhibited weak complementation activity against the light sensitivity of VS101. In VS101 bacteria lysogenized with lambdaWH-1, a significant decrease in accumulation of protoporphyrin IX (PROTO IX) was detected as compared with that in non-lysogenic bacteria. On the other hand, in the wild-type E. coli strains lysogenized with lambdaWH-1, significant accumulation of delta-aminolevulinic acid (ALA) was observed, although accumulation of other intermediates such as uroporphyrinogen III (UROGEN III) and coproporphyrinogen III (COPROGEN III), was not observed. The growth of the wild-type bacteria in which the insert cDNA from deltaWH-1 had been introduced via a plasmid vector was markedly inhibited. By constructing, testing and sequencing a series of deletion clones of the insert, it was found that the insert encodes two proteins, a trancated LepA and a hypothetical protein ORF296, and that only ORF296 possesses the ability to block the heme biosynthetic pathway. ORF296 showed about 30% identity with the E. coli hypothetical protein YicL. By cloning and examining the gene for YicL in E. coli, we found that YicL shows the same effect as that of the soybean cDNA. From these findings, we concluded that the clone from soybean and yicL from E. coli block a step in an early stage of the heme biosynthetic pathway (probably the step catalyzed by HemB). Consequently, we postulate that the VS101 bacteria harboring these genes became light resistant as a result of a decrease in accumulated PROTO IX, and that the growth of the bacteria harboring these genes was inhibited because of the inhibition of heme biosynthesis at the step catalyzed by HemB.
