Mutant Ribosomal Protein with Defective RNA Binding Site

THE 30S ribosomal subunits of Escherichia coli contain twenty-one different proteins^1–4, which together with 16S RNA can reassemble in vitro to form functional 30S particles⁵. Five proteins can individually bind to specific sites on the 16S RNA^6–8 and these are S4, S7, S8, S15 and S20 (in the nomenclature recently adopted by several laboratories to report results with the E. coli system⁹). We report here the first identification of a mutation that affects a ribosomal protein-nucleic acid interaction.     

Linear Sequence of Proteins of <Emphasis Type="Italic">Escherichia coli</Emphasis> 50S Ribosomal Particles

THE ribosomal proteins of Escherichia coli have been extensively purified and characterized^1,2; but since the structure of ribosomes is not well understood, we havs studied the linear sequence of the proteins on a ribosomal RNA of E. coli Q13.     

Mechanism of Kasugamycin Resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Strains of Escherichia coli are resistant to the antibiotic kasugamycin due to the partial non-methylation of 16S ribosomal RNA. An RNA methylase activity, absent from resistant strains, is shown here to methylate in vitro the 16S RNA of resistant as well as sensitive strains.     

Gene <Emphasis Type="Italic">yddG</Emphasis> of <Emphasis Type="Italic">Escherichia coli</Emphasis> encoding the putative exporter of aromatic amino acids: Constitutive transcription and dependence of the expression on the cell growth rate

Gene yddG of Escherichia coli encodes a protein of the inner membrane. Data obtained earlier demonstrated that under conditions of aromatic amino acids overproduction YddG promotes their export from E. coli cells. In this work, a method of primer extension was used to localize the P _yddG promoter, which corresponds to E. coli promoters recognized by RNA polymerase in complex with σ⁷⁰ or σ^S subunits. By constructing a gene of the hybrid protein YddG’-LacZ at the intrinsic site of gene yddG location in the E. coli chromosome and analyzing the activity of β-galactosidase in cells growing on laboratory media LB and M9, the constitutive type of yddG expression at a low level was demonstrated (the activity was about 3 to 4% of the LacZ level under induction of the lac operon in E. coli wild-type cells). The expression of yddG had a twofold increase under conditions of retarded cell growth upon the stress caused by the high NaCl content (0.6 M) or by the presence of phenylalanine excess quantities (>1 mM) in the culture medium.     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

Structural relationships between genetically closely related O-antigens of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> spp.

Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using ¹H and ¹³C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.     

<Emphasis Type="Italic">Candida krusei</Emphasis> isolated from fruit juices ultrafiltration membranes promotes colonization of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> on stainless steel surfaces

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm², respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm² and 0.55 log CFU cm² when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm², respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.     

Characterization of the lipopolysaccharide of <Emphasis Type="Italic">Escherichia coli</Emphasis> 126

The lipopolysaccharide (LPS) of Escherichia coli 126 was isolated and studied. The lipid A fatty acid composition of the investigated LPS was similar to that of other members of the family Enterobacteriaceae. The E. coli 126 LPS was more toxic than the LPSs of previously studied E. coli strains and of other members of the Enterobacteriaceae (Budvicia aquatica and Pragia fontium), and was less pyrogenic than pyrogenal. SDS-PAG electrophoresis showed a bimodal distribution typical of S-form LPSs. The LPS of E. coli 126 decreased the adhesive index indicating a possible competition between LPS molecules of E. coli 126 and adhesins of E. coli F-50 on rabbit erythrocytes. The LPS of E. coli 126 in a homologous system showed antigenic activity in the reactions of double immunodiffusion in agar by Ouchterlony. No serological cross-reaction of the LPS of other E. coli strains, as well as of that of the B. aquatica type strain, with the antiserum to E. coli 126 was observed. The structural components of the lipopolysaccharide obtained by mild acid hydrolysis were lipid A, the core oligosaccharide, and the O-specific polysaccharide. Based on the data of monosaccharide analysis and ¹H and ¹³C NMR spectroscopy it was found that the O-specific polysaccharide had the structure characteristic of the representatives of E. coli serogroup O15.     

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g^?1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.     

Dual-species biofilm of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> on stainless steel surface

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ^B, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.     

Functional characterization of a geraniol synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its application in production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geraniol synthase (GES) catalyzes the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used in many industries. Here we report the functional characterization of CaGES from Camptotheca acuminata, a camptothecin-producing plant, and its application in production of geraniol in Escherichia coli. The full-length cDNA of CaGES was obtained from overlap extension PCR amplification. The intact and N-terminus-truncated CaGESs were overexpressed in E. coli and purified to homogeneity. Recombinant CaGES showed the conversion activity from GPP to geraniol. To produce geraniol in E. coli using tCaGES, the biosynthetic precursor GPP should be supplied and transferred to the catalytic pocket of tCaGES. Thus, ispA(S80F), a mutant of farnesyl diphosphate (FPP) synthase, was prepared to produce GPP via the head-to-tail condensation of isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A slight increase of geraniol production was observed in the fermentation broth of the recombinant E. coli harboring tCaGES and ispA(S80F). To enhance the supply of IPP and DMAPP, the encoding genes involved in the whole mevalonic acid biosynthetic pathway were introduced to the E. coli harboring tCaGES and the ispA(S80F) and a significant increase of geraniol yield was observed. The geraniol production was enhanced to 5.85 ± 0.46 mg L^?1 when another copy of ispA(S80F) was introduced to the above recombinant strain. The following optimization of medium composition, fermentation time, and addition of metal ions led to the geraniol production of 48.5 ± 0.9 mg L^?1. The present study will be helpful to uncover the biosynthetic enigma of camptothecin and tCaGES will be an alternative to selectively produce geraniol in E. coli with other metabolic engineering approaches.     

Effect of R Factors on Rifampicm Resistance in <Emphasis Type="Italic">E. coli</Emphasis>

THE bactericidal effect of rifampicin, a semi-synthetic rifamycin, is due to its action on DNA-dependent RNA polymerase¹ and all rifampicin-resistant mutants of Escherichia coli contain an altered RNA polymerase with an increased resistance to rifampicin in vitro^2–4. While studying a possible curing effect of rifampicin on E. coli R factors, we observed that R⁺ recombinants of some rif-r mutants are more sensitive to rifampicin (Table 1). Of the cells harbouring certain R factors, less than 1% are able to form colonies on rifampicin-supplemented agar, while with certain others there is no detectable effect.     

Absence of a 5S RNA Component in the Mitochondrial Ribosomes of <Emphasis Type="Italic">Neurospora crassa</Emphasis>

RIBOSOME-BOUND, low molecular weight RNA, distinct from tRNA, was first observed in E. coli by Rosset and Monier¹. This RNA, which has a sedimentation coefficient of about 5S, is now considered to be a universal component of ribosomes. We report here our attempts to find low molecular weight RNAs other than tRNA in mitochondria of Neurospora. Our evidence suggests that the mitochondrial ribosomes of this organism lack a 5S RNA component.     

Effects of Colistin and Bacteriocins Combinations on the In Vitro Growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains from Swine Origin

Escherichia coli strains from swine origin, either susceptible or resistant to colistin, were grown under planktonic and biofilm cultures. After which, they were treated with antibacterial agents including nisin and enterocin DD14 bacteriocins, colistin and their combinations. Importantly, the combination of colistin, enterocin DD14 and nisin eradicated the planktonic and biofilm cultures of E. coli CIP54127 and the E. coli strains with colistin-resistance phenotype such as E. coli 184 (mcr-1 ⁺) and E. coli 289 (mcr-1 ^?), suggesting therefore that bacteriocins from lactic acid bacteria could be used as agents with antibiotic augmentation capability.     

A homomeric geranyl diphosphate synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its combinatorial optimization for production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5′- and 3′-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L^?1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS^E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L^?1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.