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PubMed1981

RNA polymerase binding sites on the broad host range plasmid RP4.

Binding sites of Escherichia coli RNA polymerase on RP4 plasmid DNA were determined electron microscopically. Comparison of the RNA polymerase binding map and the genetic map of RP4 revealed several strong binding sites outside the well-known RP4 genes. RNA polymerase binding sites for the three antibiotic resistance genes were also detected. Two binding sites were observed for the tra-1 region, whereas the tra-2 and tra-3 regions showed no prominent affinity for RNA polymerase. The genomic regions for the replication origins, oriV (for vegetative replication) and oriT (for transfer replication, equivalent to rlx), both exhibited strong binding to RNA polymerase, as did genomic regions which code for trans-acting replication functions (trfA and trfB).

Binding Sites↗
PubMed1981

Phage X: a plasmid-dependent, broad host range, filamentous bacterial virus.

Phage X was isolated from sewage as plating on Escherichia coli or Salmonella typhimurium strains harbouring the incompatibility group X plasmid R6K. It also plated on a strain of Serratia marcescens carrying this plasmid. It failed to form plaques on Proteus mirabilis, P. morganii or Providencia alcalifaciens harbouring R6K, but did multiply on them. No phage increase occurred with homologous R- strains. Phage X also plated or registered an increase in titre on E. coli or S. typhimurium strains carrying various plasmids of incompatibility groups M, N, P-1, U or W as well as the unassigned plasmid R775. It adsorbed to pili determined by a group P-10 plasmid in a Pseudomonas aeruginosa strain but did not multiply on this organism. The phage was filamentous and curly, resistant to ribonuclease and diethyl ether and sensitive to chloroform. It adsorbed to the tips of pili.

Adsorption↗
PubMed1985

Isolation and preliminary characterization of lytic and lysogenic phages with wide host range within the streptomycetes.

Examination of approximately 700 soil samples yielded about 100 actinophages. Restriction analysis of phage DNA indicated that 57 are unique, and of these, 20 produce turbid plaques on one or more of the streptomycetes tested. Five phages are shown to insert into the genome of Streptomyces avermitilis. None of the phages was able to perform generalized transduction of S. avermitilis.

Anthelmintics↗
PubMed1981

An R plasmid of broad host-range, coding for resistance to nine antimicrobial agents endemic in Gram-negative nosocomial isolates.

Of 3952 clinical isolates of Enterobacteriaceae, 246 exhibited resistance to at least carbenicillin, gentamicin and tobramycin. All these isolates, representing eight genera, were resistant to at least nine antimicrobial agents in common, including the three key antibiotics and streptomycin, kanamycin, sisomycin, ampicillin, cephalothin and sulphonamide. The strains could be subdivided into seven groups depending upon additional resistance traits and some were resistant to as many as 15 antibiotics. When mated with a standard strain of Escherichia coli, 85% of 123 randomly selected donors transferred resistance to at least the nine core antibiotics. Some donors occasionally transferred resistance to two additional antibiotics, neomycin and tetracycline, while one Citrobacter freundi donor always transferred linked resistance to all 11 drugs. Although many donors were found to harbour more than one species of plasmid DNA, all except a strain of C. freundi contained at least a plasmid of mol. wt 89 x 10(6). Analysis of E. coli transconjugants showed this plasmid to be responsible for transferable resistance to the nine core antibiotics. Restriction-endonuclease analysis indicates that the 89 x 10(6) plasmids originating from different isolates were essentially identical with each other. These results show that a particular R plasmid has established itself among the Enterobacteriaceae at Hines VA Hospital. This R plasmid appears to be the predominant genetic element responsible for linked resistance to carbenicillin, gentamicin and tobramycin among these hospital-associated bacteria.

Anti-Bacterial Agents↗
PubMed1994

Nucleotide sequence and structural organization of the small, broad-host-range plasmid pCI411 from Leuconostoc lactis 533.

The nucleotide sequence of the Leuconostoc lactis 533 cryptic plasmid pCI411 (2926 bp) was determined. Analysis revealed the presence of three open reading frames (ORFs). ORF 1 was capable of encoding a 24.9 kDa peptide which shared homology with the replication initiation protein (RepB) from a number of Gram-positive rolling circle plasmids. ORF 2 could encode a peptide of 6.6 kDa which was homologous to the RepC protein of the lactococcal plasmid pWV01. A function could not be assigned to ORF 3, which was capable of encoding a 12.1 kDa peptide. Transcription-translation analysis indicated the presence of three peptides of the predicted molecular masses. A putative double strand origin of replication (DSO) was identified which showed strong similarity with the DSO of a number of Gram-positive plasmids including pE194 from Staphylococcus. Structural analysis identified a number of direct and indirect repeats in addition to putative recombination-specific sites (RSA and RSB) in the non-coding region of pCI411. The observed characteristics suggest that this plasmid replicates using the rolling circle mechanism. pCI411, which could be introduced into Leuconostoc, Lactococcus, Streptococcus, Lactobacillus and Bacillus is the first plasmid from the genus Leuconostoc to be characterized in such detail.

Amino Acid Sequence↗
PubMed2006

The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501.

The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular Streptomyces lividans, as well as in Gram-negative Escherichia coli. The 15 putative pIP501 transfer (tra) genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 tra genes in exponentially growing Enterococcus faecalis JH2-2 cells, as shown by RT-PCR. The tra genes are expressed throughout the life cycle of Ent. faecalis, and the expression level is independent of the growth phase. Electrophoretic mobility shift assays indicated that the TraA relaxase, the first gene of the tra operon, binds to the tra promoter P(tra), which partially overlaps with the origin of transfer (oriT). DNase I footprinting experiments further delimited the TraA binding region and defined the nucleotides bound by TraA. Beta-Galactosidase assays with P(tra)-lacZ fusions proved P(tra) promoter activity, which was strongly repressed when TraA was supplied in trans. Thus, it is concluded that the pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA.

Bacterial Proteins↗
PubMed2004

Relationships and host range of human, canine, simian and porcine isolates of simian virus 5 (parainfluenza virus 5).

Sequence comparison of the V/P and F genes of 13 human, canine, porcine and simian isolates of simian virus 5 (SV5) revealed a surprising lack of sequence variation at both the nucleotide and amino acid levels (0-3%), even though the viruses were isolated over 30 years and originated from countries around the world. Furthermore, there were no clear distinguishing amino acid or nucleotide differences among the isolates that correlated completely with the species from which they were isolated. In addition, there was no evidence that the ability of the viruses to block interferon signalling by targeting STAT1 for degradation was confined to the species from which they were isolated. All isolates had an extended cytoplasmic tail in the F protein, compared with the original W3A and WR monkey isolates. Sequence analysis of viruses that were derived from human bone-marrow cells isolated in London in the 1980s revealed that, whilst they were related more closely to one another than to the other isolates, they all had identifying differences, suggesting that they were independent isolates. These results therefore support previous data suggesting that SV5 can infect humans persistently, although the relationship of SV5 to any human disease remains highly contentious. Given that SV5 has been isolated on multiple occasions from different species, it is proposed that the term simian virus 5 is inappropriate and suggested that the virus should be renamed parainfluenza virus 5.

Amino Acid Sequence↗
PubMed1976

Alteration of the in vitro host range of rabies virus after serial chick embryo cell passage using alkaline maintenance medium.

HEP Flury strain of rabies virus maintained by 7-day chicken egg passage (parent line) and the same strain serially passaged in primary chick embryo (CE) cells using alkaline maintenance medium (AM line) were inoculated to cells of various species. Growth was negative in primary mouse embryo, L and HeLa cells, and positive in primary hamster kidney and BHK21 cells with both lines. An all-or-none difference between the two lines was observed in primary monkey kidney and Vero cells. The parent line did not multiply in these monkey cells, whereas the AM line grew to high titers. In the case of Vero cells a unique cytopathic effect (CPE) was induced by the AM line. After five consecutive passages in Vero cells, the CPE-inducing agent was identified as rabies virus by a neutralization test. It was infective to intracerebrally inoculated suckling mice but not to adult mice, and its Vero cell-infective titer determined by CPE induction was about 1 log lower than the baby mouse-infective and CE plaque-forming titers. In contrast to the AM line, HEP Flury strain receiving 150 CE cell passages under neutral maintenance medium and three other strains receiving similar CE cell passages all failed to grow in Vero cells.

Animals↗
PubMed1991

Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range gram-negative cosmid vector pJRD215.

The transformation of Mycobacterium aurum and Mycobacterium smegmatis with the Gram-negative RSF1010-derived cosmid pJRD215 is described. The plasmid is stably maintained in both species and the antibiotic resistance determinants for kanamycin and streptomycin are expressed. Southern blot analysis shows that rearrangements take place both in M. aurum and in M. smegmatis. The use of pJRD215 in mycobacterial cloning systems is discussed.

Blotting, Southern↗
PubMed1979

The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1.

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.

Amino Acid Sequence↗
PubMed1999

A small derivative of the broad-host-range plasmid RK2 which can be switched from a replicating to a non-replicating state as a response to an externally added inducer.

TrfA is the only plasmid-encoded protein required for RK2 replication. We report here the construction and characterization of an RK2-based vector in which trfA is expressed from the inducible promoter Pm. The resulting construct, pJBSD1, was found to replicate in Escherichia coli DH5a (recA(-)) only in the presence of a Pm inducer. In two tested E. coli recA(+) strains pJBSD1 could replicate in the absence of inducer, but a replication inducer-dependent phenotype was obtained in these strains by introducing a mutation known to reduce the trfA expression level. The plasmid construct could be used as a conditional suicide vector system for targeted chromosomal integration via homologous recombination. This feature may potentially be used for many types of studies in microbial molecular biology.

Bacterial Proteins↗
PubMed1999

The broad host range plasmid pLF1311 from Lactobacillus fermentum VKM1311.

The complete nucleotide sequence (2389 bp) of the cryptic plasmid pLF1311 from Lactobacillus fermentum VKM1311 was determined. DNA sequence analysis revealed the putative coding regions for a replicative protein (RepB), its repressor (RepA) and double-stranded (dso) and single-stranded (sso) origins. pLF1311 belongs to the pE194 family of rolling circle-replicating plasmids. A derivative of pLF1311 that contains the cat gene of plasmid pC194 of Staphylococcus aureus and the oriT of RP4 was constructed and transferred by conjugative mobilization from Escherichia coli to various Gram-positive bacteria. The stable maintenance of this derivative was shown in some strains of Lactobacillus, Lactococcus, Enterococcus and Bacillus under non-selective conditions.

Amino Acid Sequence↗