Jacqueline Plumbridge

Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it has a number of growth defects. Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a large deletion around the fnr (fumarate-nitrate respiration) regulatory gene. Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose. The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar. By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant. Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine. Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects. We confirmed that use of other E. coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest. The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655.

Signal transduction between a membrane‐bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli

The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator. No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICBGlc (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICBGlc. We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICBGlc phosphorylation by the general PTS proteins and also Enzyme IICBGlc‐mediated phosphorylation of α‐methylglucoside. Binding of Mlc to Enzyme IICBGlc in vitro required the IIB domain and the IIC–B junction region. Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross‐dephosphorylation of IIBGlc during transport of other PTS sugars. The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation.

CONTROL OF THE EXPRESSION OF THE MANXYZ OPERON IN ESCHERICHIA COLI : MLC IS A NEGATIVE REGULATOR OF THE MANNOSE PTS

The manXYZ operon of Escherichia coli encodes a sugar transporter of the phosphoenol pyruvate (PEP)‐dependent phosphotransferase system, which is capable of transporting many sugars, including glucose, mannose and the aminosugars, glucosamine and N‐acetylglucosamine. Transcription of manX is strongly dependent on cyclic AMP (cAMP)/cAMP receptor protein (CAP). A cAMP/CAP binding site is located at −40.5, and activation by cAMP/CAP is shown to be typical of a class II promoter. The 5′ end of a transcript, potentially encoding two proteins, is expressed divergently from the manXYZ operon. Previously, two binding sites for the NagC repressor were detected upstream of manX, but a mutation in nagC has very little effect on manX expression. However, a mutation in the mlc gene, encoding a homologue of nagC, results in a threefold derepression of manX expression, suggesting that this protein is a more important regulator of manX expression than NagC. The Mlc protein binds to the NagC operators, binding preferentially to the promoter‐proximal operator. Plasmids overproducing either the NagC protein or the Mlc protein repress the expression of manX, but the effect of the Mlc protein is stronger. The mlc gene is shown to be allelic with the previously characterized dgsA mutation affecting the mannose phosphoenolpyruvate‐dependent phosphotransferase system (PTS).

Expression of ptsG, the gene for the major glucose PTS transporter in Escherichia coli, is repressed by Mlc and induced by growth on glucose

The gene for the glucose‐specific transporter of the phosphotransferase system, ptsG, is expressed from two promoters separated by 141 bp. The expression of the major, shorter transcript is very strongly dependent upon cAMP/CAP. However, unlike other CAP‐activated genes, the expression of ptsG is higher in glucose media than in glycerol, implying that ptsG is controlled by a glucose‐inducible regulator. A mutation in the mlc gene greatly enhances ptsG expression in a glycerol‐grown culture but has no effect on ptsG expression during growth on glucose. The mlc gene encodes a transcriptional regulator that has been shown to affect the expression of manXYZ and malT. ptsG mRNA levels are lower in the mlc strain grown on glucose than in the same strain grown on glycerol. This is presumably because of the greater catabolite repression in the glucose culture than in glycerol. The final level of expression of ptsG in a mlc+ strain in glucose is a compromise between specific induction by glucose and generalized catabolite repression. The result is that ptsG expression is very similar in glucose‐grown cultures of wild‐type and mlc strains. The Mlc protein binds to two sites centred at −6 and −175 upstream of the major ptsG transcript. CAP binds at −40.5 compared with this site, typical of class II CAP‐regulated promoters, and the binding of CAP and Mlc is co‐operative.

Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc

The maltose regulon consists of 10 genes encoding a multicomponent and binding protein‐dependent ABC transporter for maltose and maltodextrins as well as enzymes necessary for the degradation of these sugars. MalT, the transcriptional activator of the system, is necessary for the transcription of all mal genes. MalK, the energy‐transducing subunit of the transport system, acts phenotypically as repressor, particularly when overproduced. We isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK. The affected gene was sequenced and identified as mlc, a known gene encoding a protein of unknown function with homology to the Escherichia coli NagC protein. The loss of Mlc function led to a threefold increase in malT expression, and the presence of mlc on a multicopy plasmid reduced malT expression. By DNaseI protection assay, we found that Mlc protected a DNA region comprising positions + 1 to + 23 of the malT transcriptional start point. Using a mlc–lacZ fusion in a mlc and mlc+ background, we found that Mlc represses its own expression. As Mlc also regulates another operon (manXYZ, see pages 369–379 of this issue), it may very well constitute a new global regulator of carbohydrate utilization.

Sequence of a 1.26-kb DNA fragment containing the structural gene for E.coli initiation factor IF3: presence of an AUU initiator codon.

The nucleotide sequence of a 1.26‐kb pair DNA fragment containing the structural gene for Escherichia coli initiation factor IF3 has been determined. An open reading frame of 540 nucleotides is found at the position predicted by genetic studies. The amino‐acid sequence deduced from the DNA sequence accounts for a molecular weight 20 530. The important feature of the coding DNA sequence is the presence of AUU as the translational initiator codon. It is 11 bases downstream of the center of a GGAGG sequence, which can strongly pair with the sequence CCUCC near the 3′ terminus of 16S rRNA. The primary DNA sequence in the region of the AUU initiator codon and its role in compensating a reduced codon‐anticodon interaction in initiation complex formation are discussed.

Expression of the phosphotransferase system both mediates and is mediated by Mlc regulation in Escherichia coli

The ptsHIcrr operon encodes the cytoplasmic components of the phosphotransferase system (PTS). It is expressed from two major promoters, of which the upstream promoter has previously been shown to be induced by glucose and to be dependent upon cAMP/CAP. This promoter is now shown to be repressed by Mlc. Mlc is a transcriptional regulator controlling, among others, the gene ptsG, encoding EIICBGlc, the glucose‐specific transporter of the PTS. Transcription of ptsH p0 and ptsG are subject to the same regulatory pattern. In addition to induction by glucose and repression by Mlc, mutations in ptsHIcrr, which interrupt the PEP‐dependent phosphate transfer through the soluble components of the PTS, lead to high expression of both ptsH and ptsG, while mutations inactivating EIIBCGlc are non‐inducible. Mutations in mlc lead to high constitutive expression and are dominant, implying that Mlc is the ultimate regulator of ptsHI and ptsG expression. Growth on other PTS sugars, besides glucose, also induces ptsH and ptsG expression, suggesting that the target of Mlc regulation is the PTS. However, induction by these other sugars is only observed in the presence of ptsG+, thus confirming the importance of glucose and EIICBGlc in the regulation of the PTS. The ptsG22 mutation, although negative for glucose transport, shows a weak positive regulatory phenotype. The mutation has been sequenced and its effect on regulation investigated.

Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in Vivo

The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.

Regulation of Sialic Acid Catabolism by the DNA Binding Protein NanR in Escherichia coli

All Escherichia coli strains so far examined possess a chromosomally encoded nanATEK-yhcH operon for the catabolism of sialic acids. These unique nine-carbon sugars are synthesized primarily by higher eukaryotes and can be used as carbon, nitrogen, and energy sources by a variety of microbial pathogens or commensals. The gene nanR, located immediately upstream of the operon, encodes a protein of the FadR/GntR family that represses nan expression in trans. S1 analysis identified the nan transcriptional start, and DNA footprint analysis showed that NanR binds to a region of approximately 30 bp covering the promoter region. Native (nondenaturing) polyacrylamide gel electrophoresis, mass spectrometry, and chemical cross-linking indicated that NanR forms homodimers in solution. The region protected by NanR contains three tandem repeats of the hexameric sequence GGTATA. Gel shift analysis with purified hexahistidine-tagged or native NanR detected three retarded complexes, suggesting that NanR binds sequentially to the three repeats. Artificial operators carrying different numbers of repeats formed the corresponding number of complexes. Among the sugars tested that were predicted to be products of the nan-encoded system, only the exogenous addition of sialic acid resulted in the dramatic induction of a chromosomal nanA-lacZ fusion or displaced NanR from its operator in vitro. Titration of NanR by the nan promoter region or artificial operators carrying different numbers of the GGTATA repeat on plasmids in this fusion strain supported the binding of the regulator to target DNA in vivo. Together, the results indicate that GGTATA is important for NanR binding, but the precise mechanism remains to be determined.

Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP

The chitobiose operon, chbBCARFG, encodes genes for the transport and degradation of the N‐acetylglucosamine disaccharide, chitobiose. Chitobiose is transported by the phosphotransferase system (PTS) producing chitobiose‐6P which is hydrolysed to GlcNAc‐6P by the chbF gene product and then further degraded by the nagBA gene products. Expression of the chb operon is repressed by NagC, which regulates genes involved in amino sugar metabolism. The inducer for NagC is GlcNAc‐6P. NagC binds to two sites separated by 115 bp and the transcription start point of the chb operon lies within the downstream NagC operator. In addition the chb operon encodes its own specific regulator, ChbR, an AraC‐type dual repressor–activator, which binds to two direct repeats of 19 bp located between the two NagC sites. ChbR is necessary for transcription activation in the presence of chitobiose in vivo. Induction of the operon also requires CAP, which binds to a site upstream of the ChbR repeats. In the absence of chitobiose both NagC and ChbR act as repressors. Together these three factors cooperate in switching chb expression from the repressed to the activated state. The need for two specific inducing signals, one for ChbR to activate the expression of the operon and a second for NagC to relieve its repression, ensure that the chb operon is only induced when there is sufficient flux through the combined chb‐nag metabolic pathway to activate expression of both the chb and nag operons.