Return of Streptococcus faecalis DNA cloned in Escherichia coli to its original host via transformation of Streptococcus sanguis followed by conjugative mobilization.

It is possible to select transmembrane potential (delta psi)-altered mutants in Streptococcus pneumoniae on the basis of their resistance to the antifolate methotrexate. Comparison of such a mutant strain ( amiA9 ) with its parent was used to evaluate the role of delta psi in the uptake of certain amino acids. The delta psi-dependent uptake of isoleucine, leucine, valine, and asparagine showed a reduced maximum velocity of uptake, and decrease in the transport constant of the energy-dependent, delta psi-independent uptake of lysine, methionine, and glutamine was observed. No reduction of the intracellular pool of ATP or of lactate excretion could be detected in the mutant strain. Moreover, studies on membrane preparations suggest that the phenotype expressed by the amiA mutation is not a consequence of alteration of its ATPase activity or susceptibility to N,N'-dicyclohexylcarbodiimide. Therefore, it is unlikely that the amiA mutation affects the H+ F1F0 ATPase which is involved in the establishment of the proton motive force in anaerobic bacteria. We propose that another function contributes to delta psi in S. pneumoniae. The amiA gene may be the structural gene of that function.     

Role of the Escherichia coli Tat pathway in outer membrane integrity

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.     

Characterization of an amber suppressor in Pneumococcus.

Summary Partial revertant has been isolated, with resistance to aminopretin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su⁺) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su⁺ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidilate synthetase gene are suppressible. Thymidilate synthetase activity is partially restored by su⁺. Optochin mutants can also be suppressed. Thus su⁺ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycine is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor.The t-RNA extracted from the suppressor strain su⁺ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su⁺ gene is amber specific.Thus su⁺ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su⁺; this is additional evidence that both categories contain point mutations.     

Integration efficiencies of spontaneous mutant alleles of amiA locus in pneumococcal transformation

The distribution of integration efficiencies of independent mutations spontaneously occurring in the amiA locus was determined in two strains of pneumococcus. Strain Cl(3) integrates genetic markers with different efficiencies during transformation, whereas strain 401, isogenic with strain Cl(3), does not discriminate between markers and integrates all markers with the same high efficiency. The discriminating strain Cl(3) gives rise to spontaneous mutations in the locus amiA, which fall into four classes with respect to their individual integration efficiencies. Mutations with a low efficiency of integration are equal in number to mutations with a high efficiency. Mutations from the two other classes corresponding to intermediate and very high efficiencies are about five times less frequent. The same four classes were also found among amiA mutants spontaneously occurring in strain 401. However, the two distributions of integration efficiencies of amiA mutants arisen either in strain Cl(3) or strain 401 are significantly different. The number of spontaneous amiA mutants, estimated by two methods, was found to be higher in strain 401 than in strain Cl(3). The increase of the mutation rate in strain 401 could be accounted for by the excess of mutations falling in the two less-efficient classes observed in this strain. The discriminating process which acts during transformation presumably also intervenes in the appearance of spontaneous mutations.     

Prospecting for novel biocatalysts in a soil metagenome

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.     

Possible promoter regions within the proteolytic system in Streptococcus thermophilus and their interaction with the CodY homolog

Possible promoter regions preceding 14 genes belonging to the proteolytic system of Streptococcus thermophilus KLDS 3.0503 were predicted by a promoter analysis software nnpp . The 14 genes included an extracellular protease gene prtS , an oligopeptide ABC transport system gene amiA1 , and 12 genes, respectively, encoding peptidases pepA, pepS , pepN, pepC , pepB, pepQ , pepV, pepT , pepM, pepXP , pepP , and pepO . These predicted promoter sequences were cloned and inserted into the upstream of a promoterless Escherichia coli gusA (β-glucuronidase) gene in a promoter probe vector pNZ273. The resulting vectors were, respectively, introduced into S. thermophilus KLDS 3.0503 and all 14 predicted promoter sequences were able to drive gusA expression, which indicated that these sequences were functional promoters. These promoters were able to interact with the S. thermophilus CodY homolog in an in vitro DNA binding assay but they did not contain a conserved CodY-box sequence identified in Lactococcus lactis . These results were useful for further studies on the regulation of protein metabolism in S. thermophilus .     

Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus

The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.     

Expression in Escherichia coli of the Saccharomyces cerevisiae CCT gene encoding cholinephosphate cytidylyltransferase.

The coding region of the CCT gene from the yeast Saccharomyces cerevisiae was cloned into the pUC18 expression vector. The plasmid directed the synthesis of an active cholinephosphate cytidylyltransferase in Escherichia coli, confirming that CCT is the structural gene for this enzyme. The enzyme produced in E. coli efficiently utilized cholinephosphate and N,N-dimethylethanolaminephosphate, but N-methylethanolamine-phosphate and ethanolaminephosphate were poor substrates. Consistently, disruption of the CCT locus in the wild-type yeast cells resulted in a drastic decrease in activities with respect to the former two substrates. When activity was expressed in E. coli, over 90% was recovered in the cytosol, whereas most of the activity of yeast cells was associated with membranes, suggesting that yeast cells possess a mechanism that promotes membrane association of cytidylyltransferase.     

Alteration of Streptococcus pneumoniae membrane properties by the folate analog methotrexate

The antifolate compound methotrexate (MTX) is toxic to the gram-positive bacterium Streptococcus pneumoniae. Interaction of MTX with this bacterium resulted in an increase in the electric transmembrane potential (delta psi) and enhanced the delta psi-dependent uptake of isoleucine and MTX. In contrast, delta psi-independent uptake of glutamine was not changed. Folate, a nontoxic analog of MTX, did not exhibit these membrane effects, nor did it prevent the effect of MTX, suggesting that the NH2 in position 4 of the pteridine ring of the MTX molecule is involved in the MTX response. A strain bearing the nonsense mutation amiA9, selected for MTX resistance, did not exhibit increased membrane potential after MTX pretreatment. This suggests that MTX interacts with a specific membrane component in S. pneumoniae. A resulting change in ion permeability could lead to changes in the magnitude of the delta psi. The MTX-sensitive component is altered or absent in mutant amiA9.     

Prevalence of ompT among Escherichia coli isolates of human origin

OmpT is a protease associated with the outer membrane of Escherichia coli and possesses a high degree of homology to the plasminogen activator, Pla, of Yersinia pestis. We show here that OmpT from intact cells can indeed activate plasminogen. Clinical specimens of E. coli were examined for protease activity and for the ompT gene. Few isolates (12%) were found to be positive for OmpT activity, whereas most (77%) carried the ompT gene and expressed the cloned protease gene. In this report we present evidence suggesting that the surface architecture of E. coli influences the activity of OmpT and that OmpT may be indicative of the pathogenic potential of the organism.     

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.     

“Host Shutoff” Function of Bacteriophage T7: Involvement of T7 Gene 2 and Gene 0.7 in the Inactivation of Escherichia coli RNA Polymerase

The "host shutoff" function of bacteriophage T7 involves an inactivation of the host Escherichia coli RNA polymerase by an inhibitor protein bound to the enzyme. When this inhibitor protein, termed I protein, was removed from the inactive RNA polymerase complex prepared from T7-infected cells by glycerol gradient centrifugation in the presence of 1 M KCl, the enzyme recovered its activity equivalent to about 70 to 80% of the activity of the enzyme from uninfected cells. Analysis of the activity of E. coli RNA polymerase from E. coli cells infected with various T7 mutant phages indicated that the T7 gene 2 codes for the inhibitor I protein. The activity of E. coli RNA polymerase from gene 2 mutant phage-infected cells, which was about 70% of that from uninfected cells, did not increase after glycerol gradient centrifugation in the presence of 1 M KCl, indicating that the salt-removable inhibitor was not present with the enzyme. It was found that the reduction in E. coli RNA polymerase activity in cells infected with T7(+) or gene 2 mutant phage, i.e., about 70% of the activity of the enzyme compared to that from uninfected cells after glycerol gradient centrifugation in the presence of 1 M KCl, results from the function of T7 gene 0.7. E. coli RNA polymerase from gene 0.7 mutant phage-infected cells was inactive but recovered a full activity equivalent to that from uninfected cells after removal of the inhibitor I protein with 1 M KCl. E. coli RNA polymerase from the cells infected with newly constructed mutant phages having mutations in both gene 2 and gene 0.7 retained the full activity equivalent to that from uninfected cells with or without treatment of the enzyme with 1 M KCl. From these results, we conclude that both gene 2 and gene 0.7 of T7 are involved in accomplishing complete shutoff of the host E. coli RNA polymerase activity in T7 infection.     

Role of AmiA in the morphological transition of Helicobacter pylori and in immune escape

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-beta(1,4)-N-acetylmuramyl-L-Ala-D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-kappaB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection.     

The isolation and characterization of glutamine-requiring strains of Escherichia coli K12.

Mutants of Escherichia coli K12 requiring glutamine as a nitrogen source were isolated, and characterized as lacking glutamine synthetase activity. Temperature sensitive revertants of one of the mutants had a heat labile glutamine synthetase, while temperature insensitive revertants had a glutamine synthetase which was thermostable in vitro, indicating that the mutation was in the structural gene for the enzyme. All of the mutations mapped in the same region of the chromosome suggesting that they might all be in the same gene. The glutamine synthetase gene (gln) was located on the E. coli chromosome by conjugation and P1-mediated transduction at minute 77. The gln gene cotransduced with the genes for oleate degradation (old), and the genes for L-rhamnose utilization (rha). The most probable gene order is old-gln-rha.     

大肠杆菌副产物合成途径删除提高外源蛋白表达水平

大肠杆菌是应用最广泛的外源基因表达宿主。为探索阻断副产物产生途径对提高大肠杆菌表达外源蛋白的能力,本实验以野生型大肠杆菌菌株为基础,删除其乳酸脱氢酶基因(ldhA),磷酸烯醇式丙酮酸合成酶基因(pps)和丙酮酸甲酸裂解酶基因(pflB)。在此基础上,以甘露聚糖酶基因man为报告基因,考察阻断以上代谢途径对大肠杆菌产酶能力的影响。结果显示,以上述三个基因叠加删除的三重突变株为宿主时,重组茵产酶水平最高,比酶活达到158.3 U/mg,相比野生出发菌株提高82.3%。     

Differential expression of protein S genes during Myxococcus xanthus development

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.