Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli

 Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB. Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996     

Properties of plasmids from Methylomonas clara

Three independently isolated clones of the obligate methylotrophic bacterium Methylomonas clara (ATCC 31226) were tested for their plasmid content. Strains B45-BE-2 and B45-BE-3 were shown to harbor one class of plasmid molecules each, with molecular sizes of 28 MDa (46 kb) and 10 MDa (16 kb), respectively. Strain B45-BE-1 does not contain extrachromosomal DNA. Restriction maps of plasmid pBE-2 from strain B45-BE-2 and of plasmid pBE-3 from strain B45-BE-3 were established and the smaller plasmid pBE-3 shown to be a linear deletion derivative of plasmid pBE-2. Two other deletions were characterized. When subcloned into the Escherichia coli vector pBR322, plasmid pBE-3 was unable to complement for the polymerase A dependence of the pBR322 replicon. Additional experiments indicate a general failure of the M. clara specific replicon to function in E. coli.     

High level production of supra molecular weight poly (3-hydroxybutyrate) by metabolically engineered<Emphasis Type="Italic">Escherichia coli</Emphasis>

The supra molecular weight poly ([R]-3-hydroxybutyrate) (PHB), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineeredEscherichia coli strain and its fermentation for high level production of supra molecular weight PHB. RecombinantE. coli strains, harboring plasmids of different copy numbers containing theAlcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinantE. coli XL 1-Blue, harboring a medium-copy-number pJC2 containing theA. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced inRalstonia eutropha or recombinantE. coli     

Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits

Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracelular erythrocruorins (Ers), ∼ 3.5 MDa, comprized of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions forLumbricus andRiftia assemblies and their globin and linker subassemblies, based on theLumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses ofLumbricus andRiftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of ∼ 9% due to the breadth of the mass distributions. TheLumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 ± 0.481 MDa.     

Isolation of a conjugative plasmid in Escherichia coli determining formaldehyde resistance

A clinical isolate of Escherichia coli which was resistant to the disinfectant formaldehyde was investigated. The strain harboured a plasmid of 62 MDa size. It was shown by conjugation, transformation and plasmid-curing experiments that the formaldehyde resistance is plasmid-mediated and transferable to other strains.     

Complete nucleotide sequences of Bacillus plasmids pUB110dB, pRBH1 and its copy mutants

Summary The deletion plasmids, pRBH1 (1.5 MDa, kanamycin resistance, Km^r) and pUB110dB (1.5 MDa, Km^r), were obtained from pTB913 (2.9 MDa, Km^r, isolated from a thermophilic bacillus) and pUB110 (3.0 MDa, Km^r, from Staphylococcus aureus), respectively. All the nucleotide sequences of these deletion plasmids were determined. Replication origin regions of pRBH1 and pUB110dB contained, respectively, 63 base-pair inverted repeat and a large open reading frame, encoding RepB protein (235 amino acid residues). The nucleotide sequences were identical to each other except for one base in the center of the inverted repeat. Two copy number mutant plasmids, pRBHC3 and pRBHC7, were obtained from pRBH1. The mutation points were located at different positions in the RepB protein coding region (Gly to Asp for pRBHC3 and Gly to Glu for pRBHC7). RepB protein was shown to be involved in the copy number control of these plasmids.     

Antimicrobial Resistance,Virulence Factors and Genetic Diversity of Escherichia coli Isolates from Household Water Supply in Dhaka,Bangladesh

Background

Unsafe water supplies continue to raise public health concerns, especially in urban areas in low resource countries. To understand the extent of public health risk attributed to supply water in Dhaka city, Bangladesh, Escherichia coli isolated from tap water samples collected from different locations of the city were characterized for their antibiotic resistance, pathogenic properties and genetic diversity.

Methodology/Principal Findings

A total of 233 E. coli isolates obtained from 175 tap water samples were analysed for susceptibility to 16 different antibiotics and for the presence of genes associated with virulence and antibiotic resistance. Nearly 36% (n = 84) of the isolates were multi-drug(≥3 classes of antibiotics) resistant (MDR) and 26% (n = 22) of these were positive for extended spectrum β-lactamase (ESBL). Of the 22 ESBL-producers, 20 were positive for bla _CTX-M-15, 7 for bla _OXA-1-group (all had bla _OXA-47) and 2 for bla _CMY-2. Quinolone resistance genes, qnrS and qnrB were detected in 6 and 2 isolates, respectively. Around 7% (n = 16) of the isolates carried virulence gene(s) characteristic of pathogenic E. coli; 11 of these contained lt and/or st and thus belonged to enterotoxigenic E. coli and 5 contained bfp and eae and thus belonged to enteropathogenic E. coli. All MDR isolates carried multiple plasmids (2 to 8) of varying sizes ranging from 1.2 to >120 MDa. Ampicillin and ceftriaxone resistance were co-transferred in conjugative plasmids of 70 to 100 MDa in size, while ampicillin, trimethoprim-sulfamethoxazole and tetracycline resistance were co-transferred in conjugative plasmids of 50 to 90 MDa. Pulsed-field gel electrophoresis analysis revealed diverse genetic fingerprints of pathogenic isolates.

Significance

Multi-drug resistant E. coli are wide spread in public water supply in Dhaka city, Bangladesh. Transmission of resistant bacteria and plasmids through supply water pose serious threats to public health in urban areas.     

Genetic structure of natural populations of Nitrobacter in an aquatic environment

Nitrification has been essentially studied as a chemical process and the studies of the microbial population have been slightly worked out. This work constitutes a first attempt to study the diversity of Nitrobacter strains with the aim of working out the genetic structure of natural populations. The Nitrobacter population structure was compared between freshwater and sediments of the same lake. Nitrobacter isolates were identified and characterized by DNA/DNA hybridization, restriction pattern of rRNA genes, PCR/RFLP analysis of the ribosomal intergenic spacer and plasmid patterns. It was shown that this lake population was split into 3 subpopulations: a specific freshwater subpopulation, a specific sediment subpopulation and a nonspecific one. A 60 MDa plasmid was detected in 3,3% of freshwater isolates. A 37 MDa plasmid was detected in 59% of sediments isolates. This 37 MDa plasmid was not associated with a genomic group, but associated with a localization: the sediments. These results suggest that this 37 MDa plasmid could be implicated in the adaptation to sediment environment.     

Electron Microscopy of Bacillus subtilis GroESL Chaperonin and Interaction with the Bacteriophage φ29 Head-Tail Connector

The Bacillus subtilis GroESL chaperonin was isolated by sucrose density gradient centrifugation and the constituent GroES and GroEL moieties were purified by electrophoresis in agarose. Electron microscopic images of negatively stained GroEL and GroES oligomers and GroESL complexes were averaged using a reference-free alignment method. The GroEL and GroES particles had the sevenfold symmetry characteristic of their Escherichia coli counterparts. GroESL complexes, reconstituted efficiently in vitro from GroEL and GroES in the absence of added ADP or ATP, had the characteristic bullet- and football-like shapes in side view. Purified bacteriophage φ29 head-tail connectors having a mass in excess of 0.4 MDa were shown to bind to GroESL at the end opposite to the GroES. The same GroESL-connector complexes were isolated from phage-infected cells in which capsid assembly was blocked, and thus the complex may have functional significance in φ29 morphogenesis.     

A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus

Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50–70MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasm id-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. cob strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The 8FP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.     

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene

Summary A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato bectle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73115 dalton protein and a flat, diamond-shaped crystal composed of a protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.     

Stability and transmissibility of the cryptic plasmids of Rhizobium meliloti GR4

Rhizobium meliloti strain GR4 harbours two cryptic plasmids sharing extensive regions of homology between them and with other non-symbiotic plasmids of different strains of R. meliloti. They both are very stable showing a segregation rate of less than 0.1% loss per generation. pRmeGR4a (115 MDa) is a self-transmissible plasmid at a variable frequency to other species, and it is also responsible for promoting, at low frequency, the contransfer of pRmeGR4b (140 MDa), the other cryptic plasmid of the strain. A 4.8 kb PstI fragment of pRmeGR4a, responsible for the high stability in cis of this plasmid, has been isolated and several recombinant plasmids have been constructed showing different segregation rates in the strains used in this study. Their stabilities can be considerably improved by insertion of the stabilization mrs/par region of RK2.     

Survey of plasmids and resistance factors among veterinary isolates of Salmonella enteritidis in Malaysia

Thirty-five veterinary isolates of Salmonella enteritidis were characterized by their susceptibility to 10 antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All were susceptible to carbenicillin, chloramphenicol and nalidixic acid but 89% were resistant to tetracycline. When examined, 91% of the isolates harboured plasmids, with sizes ranging from 9.8 to 60 MDa. However, it was only possible to associate the presence of plasmids with tetracycline resistance; plasmids occurring in 90% of the tetracycline-resistant isolates. In conjugation experiments, with Escherichia coli K12 Nal^r as recipient, the tetracycline resistance in three selected S. enteritidis isolates was observed to transfer at frequencies of 3.0×10^-3 to 1.0×10^-2/donor cell. The concomitant transfer of a 56-MDa or 60-MDa plasmid in these three S. enteritidis isolates was also detected.R. Son. A. Ansary and I. Salmah are with the Department of Genetics and Cellular Biology. University of Malaya, 59100 Kuala Lumpur, Malaysia     

Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system

The type VI secretion system (T6SS) is an anti‐bacterial weapon comprising a contractile tail anchored to the cell envelope by a membrane complex. The TssJ, TssL, and TssM proteins assemble a 1.7‐MDa channel complex that spans the cell envelope, including the peptidoglycan layer. The electron microscopy structure of the TssJLM complex revealed that it has a diameter of ~18 nm in the periplasm, which is larger than the size of peptidoglycan pores (~2 nm), hence questioning how the T6SS membrane complex crosses the peptidoglycan layer. Here, we report that the MltE housekeeping lytic transglycosylase (LTG) is required for T6SS assembly in enteroaggregative Escherichia coli. Protein–protein interaction studies further demonstrated that MltE is recruited to the periplasmic domain of TssM. In addition, we show that TssM significantly stimulates MltE activity in vitro and that MltE is required for the late stages of T6SS membrane complex assembly. Collectively, our data provide the first example of domestication and activation of a LTG encoded within the core genome for the assembly of a secretion system.     

Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC₄ strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac⁺), lactococcin-producing (Lap⁺), lactococcin-resistant (Lap^r), sucrosefermenting (Suc⁺) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Ery^r) at the frequencies of 10⁴ transformants/g of DNA. PC₄ protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac^–Lap^–Ery^r, Lac⁺Suc⁺Lap⁺Lap^rEry^r, and Lac^–Suc⁺Lap⁺ Lap^rLap^r, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc⁺Lap⁺Lap^r and Lac⁺ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC₄ transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.     

Isolation and characterization of exocellular polysaccharides produced by Bifidobacterium longum

When grown anaerobically at pH values above 5.0, on ultrafiltered complex media containing excess lactose, Bifidobacterium longum formed up to 140 mg 1^–1 (glucose equiv.) exopolysaccharides. The highest yield was obtained when the cells were cultivated in a peptone/yeast extract medium with pH controlled by additions of NH₄OH. Whatever the conditions under study, exopolysaccharides represented about 30% of the polysaccharides produced by B. longum after 48 h of culture. Crude pronase-treated exopolysaccharide preparations were adsorbed on ion-exchange chromatographic resin to yield an anionic heteropolysaccharide fraction. Two subfractions with apparent molecular masses of 1.2 MDa and 0.36 MDa respectively were subsequently recovered after gel filtration on Sepharose 4B. In both subfractions, glucose, galactose and small amounts of uronic acids and hexosamines were present in similar molar proportions, suggesting that the excreted polymers may be synthesized from the same base unit and may have a structure resulting from repeating subunits.     

Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: Isolation of putative respiratory strings or patches

Here, we expand the application of blue native electrophoresis to the separation of mega protein complexes larger than 10 MDa by introducing novel large pore acrylamide gels. We tailored the bis‐acrylamide cross‐linker amounts relative to the acrylamide monomer to enlarge the pore size of acrylamide gels and to obtain elastic and sufficiently stable gels. The novel gel types were then used to search for suprastructures of mitochondrial respiratory supercomplexes, the hypothetical respiratory strings, or patches. We identified 4–8 MDa assemblies that contain respiratory complexes I, III, and IV and most likely represent dimers, trimers, and tetramers of respiratory supercomplexes. We also isolated multimeric respiratory supercomplexes with apparent masses of 35–45 MDa, the presumed core pieces of respiratory strings or patches. Electron microscopic investigations will be required to clarify whether the isolated assemblies of complexes are ordered and specific, as predicted for respiratory strings and patches in the mitochondrial membrane.     

Assessment of virulence determinants in Yersinia enterocolitica 1A,O:5 strains isolated from chicken carcasses

The virulence potential associated with 42–45 MDa plasmid pYV, of three Yersinia enterocolitica, 1A, O:5, strains isolated previously from chicken carcasses was tested by plasmid extraction, and by four in vitro tests: (1) Auto-agglutination; (2) Calcium dependence for growth at 37 °C; (3) Congo Red and Crystal Violet binding; and (4) Resistance to the bactericidal effect of serum. The in vitro tests were negative in all strains tested, but from strain PP131 it was possible to extract a plasmid which was quite similar in size to the wild type pYV, but heavier and not observed in any other isolated strains. These contradictions between the presence of a plasmid of approximately 42–45 MDa and the in vitro tests negative results may suggest that there is no homology between the PP131 plasmid and pYV. From these results we conclude that the plasmid found in PP131 is not pYV and that the three Y. enterocolitica 1A, O:5, strains are non-pathogenic on pYV classical mechanism basis, which do not exclude the possibility of them becoming infectious by a different mechanism, especially in children or immune-depressed individuals.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Summary Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.