DNA supercoiling is differentially regulated by environmental factors and FIS in Escherichia coli and Salmonella enterica

Although Escherichia coli and Salmonella enterica inhabit similar niches and employ similar genetic regulatory programmes, we find that they differ significantly in their DNA supercoiling responses to environmental and antibiotic challenges. Whereas E. coli demonstrates large dynamic transitions in supercoiling in response to growth phase, osmotic pressure and novobiocin treatment, supercoiling levels are much less variable in S. enterica. The FIS protein is a global regulator of supercoiling in E. coli, but it was found to have less influence over supercoiling control in S. enterica. These inter-species differences fine-tune gene promoters to endogenous supercoiling and FIS levels. Transferring a Salmonella virulence gene promoter (P(ssrA) ) into a new enteric host (E. coli) caused aberrant expression in response to stimulatory signals. Reciprocal horizontal transfer of topA promoters, which control expression of topoisomerase I, between E. coli and S. enterica revealed how these orthologous promoters have evolved to respond differentially to FIS and supercoiling levels in their cognate species. This also identified a previously unrecognized osmoregulation of topA expression that is independent of FIS and supercoiling in both E. coli and S. enterica. These findings suggest that E. coli and S. enterica may be unexpectedly divergent in their global regulation of cellular physiology.     

利用大肠杆菌质粒检测与分离痘苗病毒的启动子

本文发现,痘苗病毒DNA一些巳知的启动子序列和一些功能尚不清楚的DNA片段,可以在大肠杆菌中起始氯霉素乙酰基转移酶(Chloramphenicol Acetyltrsnsferase,简称CAT)基因的转录和表达,使转化细菌呈现氯霉素抗性表型,这一结果证明,痘苗病毒的启动子可以被大肠杆菌的RNA多聚酶所识别并有效工作。同时发现不同启动子具有不同的强度,利用大肠杆菌质粒分离和检测痘苗病毒的启动子序列,不仅可以研究痘苗病毒基因组的表达调节特点,而且也为组建痘苗病毒表达载体提供了一个快速、简便可靠的方法。     

The phosphotransferase system of Streptomyces coelicolor.

We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion. As in E. coli, the crr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIACrr is expressed in vivo. The functionality of IIACrr was demonstrated by phosphoenolpyruvate-dependent phosphorylation via enzyme I and the histidine-containing phosphoryl carrier protein HPr. Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E. coli IIAGlucose, was mutated. The capacity of IIACrr to operate in sugar transport was shown by complementation of the E. coli glucose-PTS. The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIAGlucose-like protein may be involved in carbon metabolism in S. coelicolor.     

RNase ES of Streptomyces coelicolor A3(2) can complement the rne and rng mutations in Escherichia coli

The Streptomyces coelicolor gene SCC88.10c encodes a protein (RNase ES) which is homologous to endoribonucleases in the RNase E/G family. We expressed S. coelicolor RNase ES as a 6 x His-tagged protein in an Escherichia coli mutant carrying a rng (which encodes RNase G) or a rne (which encodes RNase E) mutation to study whether S. coelicolor RNase ES is able to complement these mutations in host E. coli cells. The results clearly indicated that the S. coelicolor RNase ES can partially abrogate either the rng::cat or rne-1 mutation, as measured by the ability to suppress the several aberrant phenotypes resulting from the rng or rne mutation. Thus, S. coelicolor RNase ES appears to have the dual ability to supplant the functions of both RNase G and RNase E in E. coli.     

Bacillus subtilis rRNA promoters are growth rate regulated in Escherichia coli.

rRNA promoters from the rrnB locus of Bacillus subtilis and from the rrnB locus of Escherichia coli were fused to the gene for chloramphenicol acetyltransferase (CAT). The level of expression of CAT in E. coli showed growth rate dependence when the CAT gene was linked to either E. coli or B. subtilis tandem promoters. The downstream promoter of the tandem Bacillus pair showed growth rate regulation, while the upstream promoter did not, whereas for the E. coli tandem promoters, only the upstream promoter was growth rate regulated.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli.

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.