Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB.

Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5 degrees C but not at lower temperatures. In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB. The msbB gene has been mapped to 40.5 min on the E. coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB. The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature. The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles. Mutational analysis of msbB shows that the gene is not essential for E. coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30 degrees C not seen with either of the single mutants. Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances. The addition of quaternary ammonium compounds rescues the temperature-sensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2+ or Ca2+. These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function.     

Sequencing, mutational analysis, and transcriptional regulation of the Escherichia coli htrB gene

The Escherichia coli htrB gene was originally discovered because its insertional inactivation led to an exquisitely temperature-sensitive phenotype in rich media, i.e. the ability to form colonies at temperatures below 32 degrees C, but not above 33 degrees C. The htrB gene has been sequenced. It can potentially code for two proteins, with Mr values of 35,407 Da and 8669 Da, that are encoded by overlapping, divergent open reading frames. Our data are consistent with the 35,407 Da protein being HtrB. Northern blot analysis clearly shows that the monocistronic htrB message is not under heat-shock regulation. We have also sequenced the flanking DNA and have discovered a new gene, designated orf39.9, located immediately adjacent to htrB, but divergently transcribed.     

Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization.

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.     

The fumarase genes of Escherichia coli: location of the fumB gene and discovery of a new gene (fumC)

The fumB gene of Escherichia coli, which complements the fumarase deficiency of a fumA mutant when present in multiple copies, has been located at 93.5 min in the E. coli linkage map and its product has been identified as a polypeptide of 61 kDal. Four overlapping ColE1-fumB+ plasmids representing a continuous segment of 23.3 kb of bacterial DNA have been isolated from the Clarke-Carbon E. coli gene bank and the location of the fumB gene relative to the restriction map and the adjacent mel operon has been defined. Hybridization studies have shown that the fumB gene is homologous to the fumA gene, which complements the fumA1 mutation in single and multi-copy situations, and encodes an analogous 61 kDal product formerly regarded as the E. coli fumarase. The hybridization studies also showed that the Bacillus subtilis fumarase gene (citG) is homologous to an independent gene, fumC (formerly g48), which lies adjacent to the fumA gene at 35.5 min in the E. coli linkage map. The N-terminal sequences of the citG and fumC products exhibit a 51% identity over 88 residues. It is possible that the fumC and citG genes are fumarase structural genes of E. coli and B. subtilis, and that the fumA gene may encode a differentially-regulated fumarase or be a positive regulator gene which is essential for the expression of fumC (but not citG). If so, the fumB gene may encode a related enzyme or activator that can replace the fumA function when amplified.     

Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretion

In Escherichia coli the gene htrB codes for an acyltransferase that catalyses the incorporation of laurate into lipopolysaccharide (LPS) as a lipid A substituent. We describe the cloning, expression and characterization of a Porphyromonas gingivalis htrB homologue. When the htrB homologue was expressed in wild-type E. coli or a mutant strain deficient in htrB, a chimeric LPS with altered lipid A structure was produced. Compared with wild-type E. coli lipid A, the new lipid A species contained a palmitate (C16) in the position normally occupied by laurate (C12) suggesting that the cloned gene performs the same function as E. coli htrB but preferentially transfers the longer-chain palmitic acid that is known to be present in P. gingivalis LPS. LPS was purified from wild-type E. coli, the E. coli htrB mutant strain and the htrB mutant strain expressing the P. gingivalis acyltransferase. LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2. LPS from the htrB mutant also had a greatly reduced ability to stimulate interleukin-8 (IL-8) secretion in both endothelial cells and monocytes. In contrast, the activity of LPS from the htrB mutant bacteria expressing the P. gingivalis gene displayed wild-type activity to stimulate IL-8 production from endothelial cells but a reduced ability to stimulate IL-8 secretion from monocytes. The intermediate activation observed in monocytes for the chimeric LPS was similar to the pattern seen in HEK293 cells expressing TLR4/MD2 and CD14. Thus, the presence of a longer-chain fatty acid on E. coli lipid A altered the activity of the LPS in monocytes but not endothelial cell assays and the difference in recognition does not appear to be related to differences in Toll-like receptor utilization.     

Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures.

We identified and cloned an Escherichia coli gene called htrA (high temperature requirement). The htrA gene was originally discovered because mini-Tn10 transposon insertions in it allowed E. coli growth at 30 degrees C but prevented growth at elevated temperatures (above 42 degrees C). The htrA insertion mutants underwent a block in macromolecular synthesis and eventually lysed at the nonpermissive temperature. The htrA gene was located at approximately 3.7 min (between the fhuA and dapD loci) on the genetic map of E. coli and between 180 and 187.5 kilobases on the physical map. It coded for an unstable, 51-kilodalton protein which was processed by removal of an amino-terminal fragment, resulting in a stable, 48-kilodalton protein.     

Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli.

The nature of the Escherichia coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force has been controversial. One E. coli NADH:ubiquinone oxidoreductase has previously been purified to homogeneity, and its corresponding gene (ndh) has been isolated. However, two biochemically distinct E. coli NADH:ubiquinone oxidoreductase activities have been identified by others (K. Matsushita, T. Ohnishi, and H. R. Kaback, Biochemistry 26:7732-7737, 1987). An insertional mutation in the ndh gene has been introduced into the E. coli chromosome, and the resulting strain maintains membrane-bound NADH dehydrogenase activity, demonstrating that a second genetically distinct NADH dehydrogenase must be present. By standard genetic mapping techniques, the map position of a second locus (nuo) involved in the oxidation of NADH has been determined. The enzyme encoded by this locus probably translocates protons across the inner membrane, contributing to the proton motive force.     

Spatial arrangement and macrodomain organization of bacterial chromosomes

Recent developments in fluorescence microscopy have shown that bacterial chromosomes have a defined spatial arrangement that preserves the linear order of genes on the genetic map. These approaches also revealed that large portions of the chromosome in Escherichia coli or Bacillus subtilis are concentrated in the same cellular space, suggesting an organization as large regions defined as macrodomains. In E. coli, two macrodomains of 1 Mb containing the replication origin (Ori) and the replication terminus (Ter) have been shown to relocalize at specific steps of the cell cycle. A genetic analysis of the collision probability between distant DNA sites in E. coli has confirmed the presence of macrodomains by revealing the existence of large regions that do not collide with each other. Two macrodomains defined by the genetic approach coincide with the Ori and Ter macrodomains, and two new macrodomains flanking the Ter macrodomain have been identified. Altogether, these results indicate that the E. coli chromosome has a ring organization with four structured and two less-structured regions. Implications for chromosome dynamics during the cell cycle and future prospects for the characterization and understanding of macrodomain organization are discussed.     

Isolation and characterization of the yeast 3-phosphoglycerokinase gene (PGK) by an immunological screening technique

An immunological screening technique has been used for the detection of a specific antigen-producing clone in a bank of bacterial colonies containing hybrid plasmids. This technique involves covalent attachment of antiserum to cyanogen bromide-activated paper discs, contact of this paper with lysed colonies on agar plates, and finally detection of the bound antigen with 125I-labeled antibody. Using this method, we have identified an Escherichia coli colony, containing a yeast DNA insert in plasmid ColE1, that produces antigen which combines with antibody directed against purified yeast 3-phosphoglycerate kinase. The hybrid plasmid (pYe57E2) obtained by this procedure has been shown by both biochemical and genetic methods to contain the structural gene PGK for yeast 3-phosphoglycerate kinase. The location of the PGK structural gene on pYe56E2 was determined by immunological screening of E. coli colonies bearing plasmids containing various reconstructions of the original yeast DNA insert. Examination of the expression of the cloned yeast PGK gene in both E. coli and yeast has shown that functional enzyme is synthesized from the cloned gene in yeast, but not in E. coli.     

nov: a new genetic locus that affects the response of Escherichia coli K-12 to novobiocin

We have identified a new gene locus (nov) affecting the resistance of Escherichia coli K-12 to novobiocin. The gene also affects, although to a lesser extent, tolerance to another gyrase inhibitor coumermycin. Transductional and complementation analysis show that nov is located between att phi 80 and the osmZ (hns) genes at minute 27 of the E. coli K-12 genetic map. In standard laboratory strains of E. coli K-12 nov exists at least in two allelic forms.     

Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor.

An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition. The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures. A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria. Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene. The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction. The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its six terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues. The E. coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication. Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C. Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C. One of these extragenic suppressors was mapped at 54 min on the E. coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.     

Genetic control of the resistance to phage C1 of Escherichia coli K-12.

Escherichia coli K-12 lytic phage C1 was earlier isolated in our laboratory. Its adsorption is controlled by at least three bacterial genes: dcrA, dcrB, and btuB. Our results provide evidence that the dcrA gene located at 60 min on the E. coli genetic map is identical to the sdaC gene. This gene product is an inner membrane protein recently identified as a putative specific serine transporter. The dcrB gene, located at 76.5 min, encodes a 20-kDa processed periplasmic protein, as determined by maxicell analysis, and corresponds to a recently determined open reading frame with a previously unknown function. The btuB gene product is known to be an outer membrane receptor protein responsible for adsorption of BF23 phage and vitamin B12 uptake. According to our data the DcrA and DcrB proteins are not involved in these processes. However, the DcrA protein probably participates in some cell division steps.     

Tandem duplication and multiple functions of a receptor gene in bacterial chemotaxis

The aspartate receptor in bacterial sensing, previously identified with the tar gene, has been shown to be duplicated in tandem in Escherichia coli. Each gene, which we refer to as tar and tap, respectively, codes for a 60,000-dalton protein. By genetic engineering experiments in which each gene is introduced separately into E. coli strains, it is shown that each transmembrane receptor can respond to the small molecule aspartate, to the maltose-protein-chemoeffector complex, and to repellents.     

Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene

The complete nucleotide sequence of the phoS gene, the structural gene for the phosphate-repressible, periplasmic phosphate-binding protein Escherichia coli K-12, was determined. The phosphate-binding protein is synthesized in a precursor form which includes an additional N-terminal segment containing 25 amino acid residues, with the general characteristics of a signal sequence. The amino acid sequence derived from the nucleotide sequence shows the mature protein to be composed of 321 amino acids with a calculated molecular weight of 34,427. The phoS gene is not part of an operon and is transcribed counterclockwise with respect to the E. coli genetic map. A promoter region has been identified on the basis of homology with the consensus sequence of other E. coli promoter regions. However, an alternative promoter region has been identified on the basis of homology with the promoter regions of the phoA and phoE genes, the structural genes for alkaline phosphatase and outer-membrane pore protein e, respectively.     

Evidence that the two Escherichia coli groE morphogenetic gene products interact in vivo.

The Escherichia coli groEL and groES gene products are essential for both phage morphogenesis and bacterial growth. Although the gene products have been identified, their exact roles in these processes are not known. We have isolated mutations in the groEL gene that suppress defects in the groES gene. These intergenic suppressors were shown to map in the groEL gene by a variety of genetic and biochemical analyses. These results suggest that the two morphogenetic gene products interact in vivo and help to explain why mutations in either gene exhibit the same phenotype with respect to lambda head assembly and bacterial growth.