Additional Nucleotide Sequences in Precursor 16S Ribosomal RNA from <Emphasis Type="Italic">Escherichia coli</Emphasis>

MATURE 5S, 16S and 23S ribosomal RNA species present in E. coli ribosomes are the end products of complex biosyn-thetic pathways. They are formed by reduction in length, and methylation of longer RNA chains transcribed on the ribosomal RNA cistrons of E. coli DNA. While these modifications take place the ribosome structure is formed by progressive addition of ribosomal proteins and conformational changes in the resulting ribonucleoprotein precursor particles¹.     

NMR assignments of the N-terminal domain of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> hibernation promoting factor (SaHPF)

Staphylococcus aureus: hibernation-promoting factor (SaHPF) is a 22.2 kDa stationary-phase protein that binds to the ribosome and turns it to the inactive form favoring survival under stress. Sequence analysis has shown that this protein is combination of two homolog proteins obtained in Escherichia coli—ribosome hibernation promoting factor (HPF) (11,000 Da) and ribosome modulation factor RMF (6500 Da). Binding site of E. coli HPF on the ribosome have been shown by X-ray study of Thermus thermophilus ribosome complex. Hence, recent studies reported that the interface is markedly different between 100S from S. aureus and E. coli. Cryo-electron microscopy structure of 100S S. aureus ribosomes reveal that the SaHPF-NTD binds to the 30S subunit as observed for shorter variants of HPF in other species and the C-terminal domain (CTD) protrudes out of each ribosome in order to mediate dimerization. SaHPF-NTD binds to the small subunit similarly to its homologs EcHPF, EcYfiA, and a plastid-specific YfiA. Furthermore, upon binding to the small subunit, the SaHPF-NTD occludes several antibiotic binding sites at the A site (hygromycin B, tetracycline), P site (edeine) and E site (pactamycin, kasugamycin). In order to elucidate the structure, dynamics and function of SaHPF-NTD from S. aureus, here we report the backbone and side chain resonance assignments for SaHPF-NTD. Analysis of the backbone chemical shifts by TALOS+ suggests that SaHPF-NTD contains two α-helices and four β-strands (β1-α1-β2-β3-β4-α2 topology). Investigating the long-term survival of S. aureus and other bacteria under antibiotic pressure could lead to advances in antibiotherapy.     

Identification of Ribosomal Protein L1-Binding Sites in <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Thermotoga maritima</Emphasis> mRNAs

The conserved two-domain ribosomal protein (r-protein) L1 is a structural part of the L1 stalk of the large ribosomal subunit and regulates the translation of the operon that comprises its own gene. The regulatory properties of the bacterial r-protein L1 have only been studied in detail for Escherichia coli; however, there were no such studies for other bacteria, in particular, Thermus thermophilus and Thermotoga maritima, which are more evolutionarily ancient. It is known that domain I of the r-protein L1 might have regulatory properties of the whole protein. The aim of this study was to identify regulatory sites on the mRNA of T. thermophilus and T. maritima that interact with r-proteins L1, as well as with their domains I from the same organisms. An analysis of the mRNA of the L11 operon T. thermophilus showed the presence of one potential binding site of the L1 r-protein, two such regions were found also in the mRNA sequence of the L11 operon of T. maritima. The dissociation constants for the L1 proteins from T. thermophilus and T. maritima and their domains I with mRNA fragments from the same organisms that contain the supposed L1-binding sites were determined by surface plasmon resonance. It has been shown that the ribosomal proteins L1 as their domains I bind specific fragments of mRNA from the same organisms that may suggest regulatory activity of the L1 protein in the T. thermophilus and T. maritima and conservatism of the principles of L1-RNA interactions.     

<Emphasis Type="Italic">Trueperella pyogenes</Emphasis> isolated from a brain abscess of an adult roebuck (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>)

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S–23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.     

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.     

Influence of Nonconserved Regions of L1 Protuberance of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> Ribosome on the Affinity of L1 Protein to 23s rRNA

The L1 protuberance of the ribosome includes two domain ribosomal protein L1 and three helices of 23S rRNA (H76, H77, and H78) with interconnecting loops A and B. Helix 78 consists of two parts, i.e., H78a and H78b. A comparison of the available structural data of L1-RNA complexes with the obtained kinetic data made it possible to determine the influence of the nonconserved regions of Thermus thermophilus L1-protuberance on the mutual affinity of the L1 protein and 23S rRNA. It has been shown that the N-terminal helix of the protein and 78b helix of 23S rRNA are essential for the formation of an additional intermolecular contact, which is separated in the protein from the main site of L1-rRNA interaction by a flexible connection. This results in a rise in the TthL1-rRNA affinity. At the same time, the elongation of the 76 helix has no effect on rRNA-protein binding.     

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.     

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.     

Identification of <Emphasis Type="Italic">Bacillus</Emphasis> Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, <Emphasis Type="Italic">recA</Emphasis>, <Emphasis Type="Italic">rpoB</Emphasis> Gene Sequencing and RAPD-PCR

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.     

Distinct diversity patterns of <Emphasis Type="Italic">Planctomycetes</Emphasis> associated with the freshwater macrophyte <Emphasis Type="Italic">Nuphar lutea</Emphasis> (L.) Smith

Members of the phylum Planctomycetes were originally described as freshwater bacteria. Most recent studies, however, address planctomycete diversity in other environments colonized by these microorganisms, including marine and terrestrial ecosystems. This study was initiated in order to revisit the specific patterns of planctomycete diversity in freshwater habitats using cultivation-independent approaches. The specific focus was made on planctomycetes associated with Nuphar lutea (L.) Smith, an emergent macrophyte with floating leaves, which is widespread in the Holarctic. As revealed by Illumina pair-end sequencing of 16S rRNA gene fragments, the bacterial assemblages colonizing floating leaf blades of waterlilies sampled from two different boreal lakes displayed similar composition but were distinct from the planktonic bacterial communities. 16S rRNA gene fragments from the Planctomycetes comprised 0.1–1 and 1–2.2% of total 16S rRNA gene reads retrieved from water samples and plant leaves, respectively. Planktonic planctomycetes were mostly affiliated with the class Planctomycetaceae (77–97%), while members of the Phycisphaerae were less abundant (3–22%). The relative proportion of the latter group, however, increased by 13–45% on leaves of N. lutea. The Phycisphaera-related group WD2101, Pirellula-like planctomycetes, as well as Gemmata, Zavarzinella and Planctopirus species were the most abundant groups of planctomycetes associated with plant leaves, which may suggest their involvement in the degradation of plant-derived organic matter.     

The <Emphasis Type="Italic">yajC</Emphasis> gene from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and its role in ethanol tolerance

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-d-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.     

<Emphasis Type="Italic">Thermus thermophilus</Emphasis> L4 ribosomal protein: purification and sensitivity alteration against erythromycin of <Emphasis Type="Italic">E. coli</Emphasis> cells harboring a single amino acid mutant of TthL4 within its extended loop

Summary. Protein L4 from the thermophilic bacterium Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells and purified under native conditions by using ion exchange chromatography. Although it’s known strong binding to RNA (23S rRNA as well as mRNA) the yield of the purified protein was 6 mg per 10 g of cells and it is similar to that referred for Thermotoga maritima L4 ribosomal protein. In addition, E. coli cells harboring the wild type Thermus thermophilus L4 (wtTthL4) ribosomal protein as well as its mutant having changed the highly conserved glutamic acid 56 by alanine (TthL4-Ala 56) were incorporated into E. coli ribosomes after transformation of the host cells with the recombined vector. The cells having incorporated the mutant TthL4-Ala56 are more sensitive against erythromycin related to that containing the wtTthL4 protein. The resistance to the drug indicates that the mutated amino acid Glu56 is probably critical for the local ribosomal conformation and that its mutation induces conformational disturbances that are “transferred” to the entrance of the major exit tunnel, the place where the drug does bind.     

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from <Emphasis Type="Italic">Aspergillus Fumigatus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Chitosanase (CSN) from Aspergillus fumigatus has good thermal stability, wide pH range duration, and effective hydrolysis for chitosan. Inhere, CSN was successfully expressed in Escherichia coli followed by extracellular secretion under the guidance of an N-terminal signal peptide PelB, which effectively prompted its secretion out of E. coli cells. To facilitate its later purification, N-terminal or C-terminal 6xHis epitope tag was added to the PelB-CSN protein complex. Our results indicated that PelB-CSN without 6xHis-tag (PelB-CSN) or with N-terminal 6xHis-tag (PelB-CSN-N) can both be effectively secreted into the medium, while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies. Process optimization strategies were further developed to improve the secretion efficiency of recombinant PelB-CSN-N in E. coli. Under the induction of 10 g/L lactose in shake-flask culture, the extracellular activity of CSN reached 6015 U/mL at 25 °C in TB medium containing 1 % glycine. Moreover, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter, increasing the extracellular CSN activity to 14,000 U/mL in 2-day fermentation with the optimal addition of lactose and glycine.     

Cistron Specific Translation Control Protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

GENE expression may be controlled during translation by ribosomal selection of mRNAs or even individual cistrons. Escherichia coli initiation factors associated with ribosomes affect the binding of ribosomes to mRNA^1,2; initiation factor IF3, for instance, influences the specificity of mRNA-ribosome interaction^3,4. IF3 activity has been separated into several fractions which show various specificities for different mRNA cistrons^4–9. An important problem is the possibility of intracellular changes in IF3 activity^10–12. From uninfected E. coli, we have now isolated a protein which changes the specificity of IF3 toward different mRNAs; we call this interference factor i. Pure factor i binds to IF3 and specifically affects the translation of T4 and MS2 RNA. Whereas the initiation of translation of the MS2 coat protein cistron is inhibited by factor i, the synthetase cistron—when available—is more rapidly initiated in the presence of factor i. The overall translation of T4 mRNA appears unchanged by factor i, but certain cistrons are stimulated at the expense of others. Interfering factors such as factor i could be important in controlling translation in E. coli.     

<Emphasis Type="Italic">Zunongwangia flava</Emphasis> sp. nov., belonging to the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from <Emphasis Type="Italic">Salicornia europaea</Emphasis>

A yellow pigmented bacterium designated strain MBLN094^T within the family Flavobacteriaceae was isolated from a halophyte Salicornia europaea on the coast of the Yellow Sea. This strain was a Gram-stain negative, aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain MBLN094^T was found to be related to the genus Zunongwangia, exhibiting 16S rRNA gene sequence similarity values of 97.0, 96.8, 96.4, and 96.3% to Zunongwangia mangrovi P2E16^T, Z. profunda SM-A87^T, Z. atlantica 22II14-10F7^T, and Z. endophytica CPA58^T, respectively. Strain MBLN094^T grew at 20?37°C (optimum, 25?30°C), at pH 6.0?10.0 (optimum, 7.0?8.0), and with 0.5?15.0% (w/v) NaCl (optimum, 2.0?5.0%). Menaquinone MK-6 was the sole respiratory quinone. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, and four unidentified lipids. Major fatty acids were iso-C_17:0 3-OH, summed feature 3 (C_16:1ω6c and/or C16:1 ω7c), and iso-C_15:0. The genomic DNA G + C content was 37.4 mol%. Based on these polyphasic taxonomic data, strain MBLN094^T is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia flava sp. nov. is proposed. The type strain is MBLN094^T (= KCTC 62279^T = JCM 32262^T).     

<Emphasis Type="Italic">Streptomyces urticae</Emphasis> sp. nov., isolated from rhizosphere soil of <Emphasis Type="Italic">Urtica urens</Emphasis> L.

Two novel Gram-stain positive, spore-forming, aerobic actinomycetes, designated NEAU-PCY-1^T and NEAU-PCY-2, were isolated from rhizosphere soil of Urtica urens L. collected from Anshan, Liaoning Province, northeast China. The 16S rRNA gene sequence analysis showed that strains NEAU-PCY-1^T and NEAU-PCY-2 exhibited 99.8% similarity with each other and are closely related to Streptomyces abietis DSM 42080^T (98.2, 98.3%) and Streptomyces fildesensis DSM 41987^T (98.0, 98.1%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains formed a cluster with these two closely related species. Moreover, DNA–DNA hybridization results and some phenotypic, physiological and biochemical properties differentiated the two strains from their close relatives in the genus Streptomyces. Based on a polyphasic taxonomy study, strains NEAU-PCY-1^T and NEAU-PCY-2 are considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces urticae sp. nov. is proposed, with NEAU-PCY-1^T (=?DSM 105115^T?=?CCTCC AA 2017015^T) as the type strain.     

Functional analysis of <Emphasis Type="Italic">PI-</Emphasis>like gene in relation to flower development from bamboo (<Emphasis Type="Italic">Bambusa oldhamii</Emphasis>)

Bamboo flowering owns many unique characteristics and remains a mystery. To investigate the molecular mechanisms underlying flower development in bamboo, a petal-identity gene was identified as a PISTILLATA homologue named BoPI from Bambusa oldhamii (bamboo family). Expression analysis showed that BoPI was highly expressed in flower organs and gradually increased during flower development stage, suggesting that BoPI played an important role in flower development. Ectopic expression of BoPI in Arabidopsis caused conversion of sepals to petals. 35S::BoPI fully rescued the defective petal formation in the pi-1 mutant. BoPI could interact with BoAP3 protein in vitro. These results suggested that BoPI regulated flower development of bamboo in a similar way with PI. Besides flower organs, BoPI was also expressed in leaf and branch, which revealed that BoPI may involve in leaf and branch development. Similar to other MIKC-type gene, BoPI contained the C-terminal sequence but its function was controversial. Ectopic expression of the C-terminal deletion construct (BoPI- ΔC) in Arabidopsis converted sepals to petals; BoPI- ΔC interacted with BoAP3 on yeast two-hybrid assay, just like the full-length construct. The result implied that the C-terminal sequence may not be absolutely required for organ identity function in the context of BoPI.