Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetaterepressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.

The gene responsible for the transport of lactose into Streptococcus thermophilus (lacS) was cloned in Escherichia coli as a 4.2-kilobase fragment from an EcoRI library of chromosomal DNA by using the vector pKK223-3. From deletion analysis, the gene for lactose transport mapped to two Hindlll fragments with a total size of 2.8 kilobases. The gene was transcribed in E. coli from its own promoter. Functional expression of lactose transport activity was shown by assaying for the uptake and exchange of lactose both in intact cells and in membrane vesicles. The nucleotide sequence of lacS and 200 to 300 bases of 3' and 5' flanking regions were determined. The gene was 1,902 base pairs long, encoding a 69,454-dalton protein with an NH2-terminal hydrophobic region and a COOH-terminal hydrophilic region. The NH2-terminal end was homologous with the melibiose carrier of E. coli (23% similarity overall; >50% similarity for regions with at least 16 amino acids), whereas the COOH-terminal end showed 34 to 41% similarity with the enzyme III (domain) of three different phosphoenolpyruvate-dependent phosphotransferase systems. Among the conserved amino acids were two histidyl residues, of which one has been postulated to be phosphorylated by HPr. Since sugars are not phosphorylated during translocation by the lactose transport system, it is suggested that the enzyme III-like region serves a regulatory function in this protein. The lacS gene also appears similar to the partially sequenced lactose transport gene of LactobaciUlus bulgaricus (lacL; >60% similarity). Furthermore, the 3' flanking sequence of the S. thermophilus lactose transport gene showed approximately 50% similarity with the N-terminal portion of the ,-galactosidase gene of L. bulgaricus. In both organisms, the lactose transport gene and the Il-galactosidase appear to be separated by a 3-base-pair intercistronic region.Lactose transport in bacteria generally proceeds via either the phosphoenolpyruvate-dependent sugar transferase system (PEP-PTS) or a proton motive force (pmf)-linked transport system (W. N. Konings, B. Poolman, and A. J. M. Driessen, Crit. Rev. Microbiol., in press). In the former system, lactose enters the cell as lactose 6-phosphate, which is subsequently cleaved into glucose and galactose 6-phosphate by a ,-D-phosphogalactoside galactohydrolase. Lactose accumulated by the pmf-driven transport system enters the cell as free sugar and is hydrolyzed into galactose and glucose by a 3-D-galactoside galactohydrolase (,B-galactosidase).Dairy lactic acid bacteria, including species of both the lactic streptococci and lactobacilli, utilize lactose as their primary energy source. In a number of species which transport lactose by a PEP-PTS, the lac genes involved, their organization on the chromosome or plasmid, and their regulation are well documented (1, 4, 33). In contrast, very little is known about the lac genes in lactic acid bacteria that encode a pmf-linked lactose transport system in conjunction with a P-galactosidase. Representatives of this gr...

The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-gahM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and Il-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose-and glucose-grown S. thermophilus cells matched the mRNA levels.Streptococcus thermophilus transports lactose by means of a proton motive force-linked mechanism (33). Lactose enters the cell as a free sugar, and the disaccharide is hydrolyzed into glucose and galactose by P-galactosidase (20,33). Glucose enters the glycolytic pathway, whereas in the presence of excess lactose, the galactose moiety of lactose is excreted into the medium (39).The lac genes of S. thermophilus have recently been cloned, sequenced, and partially characterized (20, 33; C. J. Schroeder, C. Robert, G. Lenzen, L. L. McKay, and A.Mercenier, submitted for publication). The lactose transport gene (lacS) encodes a 69,454-dalton (Da) protein consisting of an amino-terminal domain with homology to the melibiose carrier of Escherichia coli and a carboxy-terminal domain with homology to enzyme III or enzyme III domains of various phosphoenolpyruvate-dependent phosphotransferase systemns from gram-positive and gram-negative organisms. A similar transport protein has been found in Lactobacillus bulgaricus (33,38), and the function(s) of the different domains of the transport...

The Lactobacillus bulgaricus Il-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'-and 5'-flanking sequences was determined. The amino acid sequence of the 6-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) 0-galactosidase. The cloned P-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus , -galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these PI-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ ,I-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two ,I-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these PI-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic 0-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.Little is known about the specific amino acids involved in the substrate binding and catalysis of the Escherichia coli lacZ P-galactosidase, even though this enzyme has been studied for many years (35). From iodination (8, 15), fluorotyrosine substitution (27), and active-site-directed inhibitor (11,12,23) experiments, Tyr-503 is thought to be the proton-donating species needed for catalysis. Primarily by the use of active-site-directed reagents (11,13)

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