Abstract
Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can ...divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype–genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.
We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (Reizer, ...J., et al., 1992, J. Biol. Chem. 267, 9158–9169). We now report the sequencing of the ptsl gene of B. subtilis encoding Enzyme I (570 amino acids and 63, 076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source‐dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate:phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequenced HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross‐reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N‐terminal and the C‐terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms. Quantitative analysis of this bias indicated that the ptsH genes have higher bias toward codons favored in highly expressed genes than the ptsI genes, in agreement with their relative levels of expression.
In spite of many efforts and achievements to optimize the prokaryotic expression systems, there are still general and specific problems in obtaining sufficient yields of the functionally active gene ...products. The main problems concern the formation of inclusion bodies, incorrect folding, toxicity for the producer cells and degradation by proteases. One way to overcome these problems is with expression systems alternative to those of
Escherichia coli. For the first time, cell wall-less L-form bacteria were used to establish such an alternative expression system and test its practicability. The results showed that various recombinant proteins can be synthesized in considerable amounts as soluble, functionally active products with these cell wall-less strains.
The structural gene of the carboxypeptidase T (
cpt) was successfully expressed in cell wall-less L-form cells of
Proteus mirabilis. The DNA sequence encoding the PhoA leader peptide was fused with a ...truncated
cpt gene encoding the mature enzyme. The modified gene in a pUC-based kanamycin resistance vector under the control of the
lac promoter was transformed into L-form cells of
P. mirabilis. They were able to produce the recombinant CpT both as a secretory and as a cell-bound insoluble form. The co-secretory processing of the PhoA leader peptide was quite efficient. The yield of the secreted CpT was not less than 20 mg l
−1 and should be improvable.
Previous studies have demonstrated the involvement of a carrier system in glutamate secretion by
Corynebacterium glutamicum under biotin limitation (Hoischen, C. and Krämer, R. (1989) Arch. ...Microbiol. 151, 342–347). In a detailed analysis of the export process we found secretion to be independent of secondary forces: (i) glutamate was secreted at high rate even when external glutamate exceeded the internal concentration, (ii) movement of neither protons nor potassium or chloride ions was found to be coupled to glutamate secretion, and (iii) secretion continued unaffected after breakdown of the membrane potential. Instead, under conditions leading to variation of glutamate secretion activity, a correlation of secretion rate and the intracellular ATP-pool was observed. Thus, ATP or a related high-energy metabolite is thought to be involved in the activity of the glutamate secretion system.
Observations of astrophysical transients have brought many novel discoveries and provided new insights into physical processes at work under extreme conditions in the Universe. Multi-wavelength and ...multi-messenger observations of variable objects require dedicated procedures and follow-up systems capable of digesting and reacting to external alerts to execute coordinated follow-up campaigns. The main functions of such follow-up systems are the processing, filtering, and ranking of the incoming alerts, the fully automated rapid execution of the observations according to an observation strategy tailored to the instrument, and real-time data analysis with feedback to the operators and other instruments. H.E.S.S. has been searching for transient phenomena since its inauguration in 2003. In this paper, we describe the transients follow-up system of H.E.S.S. which became operational in 2016. The system allows H.E.S.S. to conduct a more versatile, optimised, and largely autonomous transient follow-up program, combining all major functionalities in one systematic approach. We describe the design, central functionalities, and interfaces of the follow-up system in general and its three main components in detail: the Target of Opportunity (ToO) alert system, the data acquisition and central control system, and the real-time analysis. We highlight architectural decisions and features that enable fully automatic ToO follow-up and indicate key performance metrics of the sub-systems. We discuss the system's capabilities and highlight the need for a fine-tuned interplay of the different sub-systems in order to react quickly and reliably. Lessons learned from the development, integration, and operation of the follow-up system are reviewed in light of new and large science infrastructures and associated challenges in this exciting new era of inter-operable astronomy.
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