R. Jayaraman

Bacterial persistence: some new insights into an old phenomenon

Bigger discovered more than 60 years ago, at the very beginning of the antibiotic era, that populations of antibiotic-sensitive bacteria contained a very small fraction (approximately 10−6) of antibiotic-tolerant cells (persisters). Persisters are different from antibiotic-resistant mutants in that their antibiotic tolerance is non-heritable and reversible. In spite of its importance as an interesting biological phenomenon and in the treatment of infectious diseases, persistence did not attract the attention of the scientific community for more than four decades since its discovery. The main reason for this lack of interest was the difficulty in isolating sufficient numbers of persister cells for experimentation, since the proportion of persisters in a population of wild-type cells is extremely small. However, with the discovery of high-persister (hip) mutants of Escherichia coli by Moyed and his group in the early 1980s, the phenomenon attracted the attention of many groups and significant progress has occurred since then. It is now believed that persistence is the end result of a stochastic switch in the expression of some toxin-antitoxin (TA) modules (of which the hipA and hipB genes could be examples), creating an imbalance in their intracellular levels. There are also models invoking the involvement of the alarmone (p) ppGpp in the generation of persisters. However, the precise mechanisms are still unknown. Bacterial persistence is part of a wider gamut of phenomena variously called as bistability, multistability, phenotypic heterogeneity, stochastic switching processes, etc. It has attracted the attention of not only microbiologists but also a diverse band of researchers such as biofilm researchers, evolutionary biologists, sociobiologists, etc. In this article, I attempt to present a broad overview of bacterial persistence to illustrate its significance and the need for further exploration.

Nitrofuratoin-resistant mutants of Escherichia coli: Isolation and mapping

SummaryMutants of Escherichia coli resistant to nitrofurantoin have been isolated. The mutations, designated nfnA and nfnB were introduced individually into a multiply auxotrophic E. coli F− strain and mapped by conjugation and transduction. nfnA is located at 79.8 min and nfnB at 13.0 min on the E. coli chromosome.

Intragenic suppression of the temperature-sensitivity caused by a mutation in a gene controlling transcription (fit) in Escherichia coli.

SummaryStarting from a transcription-defective strain harbouring a temperature-sensitive mutation in the fit gene, a rifampicin-resistant, temperature-insensitive derivative has been isolated. Genetic analysis of this derivative demonstrated the presence of a second temperature-sensitive mutation in the same gene. The two mutations mutually suppress each others phenotype. From cotransduction experiments, the fit gene has been mapped 0.32 min and 0.16 min clockwise from the aroD and pps loci, respectively, at 37.5 min on the linkage map. The mutants harbouring either or both of the fit mutations are defective in RNA synthesis at the non-permissive temperature. The fit gene product is suggested to function as an accessory transcription factor.

Mapping of two transcription mutations (tlnI and tlnII) conferring thiolutin resistance, adjacent to dnaZ and rho in Escherichia coli

SummaryTwo mutations in Escherichia coli conferring resistance to the transcription initiation inhibitor, thiolutin, have been mapped. One of these mutations (tln-I) maps at 10.2 min on the genetic map and is cotransducible with dnaZ at a frequency of approximately 50%. The other mutation (tln-II) maps between metE and ilvD, probablyclose to rho, and is cotransducible with ilvD at a frequency of approximately 65%. The presence of both the mutations in the same cell confers resistance to thiolutin in minimal medium. Either one of them alone renders the cell ‘conditionally auxotrophic’ in the presence of the drug. The implications of these findings are discussed in relation to the mode of action of the thiolutin sensitive factors in transcription.

Molecular cloning, characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.

Modulation of gene expression by the product offitA gene inEscherichia coli

Physiological parameters such as viability, gross RNA synthesis,β-galactosidase induction, development of phages T4, T7 andλ have been studied in temperature-sensitiveEscherichia coli strains harbouring fit A76,fit A24 andfit A76fit A24 mutations in rpoB+ andrpoB240 genetic backgrounds. The efficiently of expression of these functions is influenced by thefit A alleles depending upon the medium of growth and/or temperature. Strains harbouring therpoB240 mutation and thefit A76 mutation, either alone or together with thefit A24 mutation, are rifampicin-sensitive even at the perfssive temperature. The results suggest possible interaction between thefit A gene product and RNA polymerase invivo.

Aberrant transcriptionin fit mutants ofEscherichia coli and its alleviation by suppressor mutations

Earlier work from this laboratory had identified, mapped and characterised an intragenic suppressor(fitA24) as well as an extragenic suppressor(fitB) for the temperature-sensitive transcription defective mutationfitA76 inEscherichia coli In this communication we report the results of experiments on RNA synthesis and decay of pulse labelled RNA in strains harbouringfit A76,fitB, fitA24, fitA76-fitA24, fitA76-fitB mutation(s) as well as in the isogenicfitA+ fitB+ strain. Taken together with earlier results, this indicates that thefitA andfitB gene products could be involved in the expression of some classes of genes including genes coding for ribosomal proteins. The implications of these results for thein vivo control of transcription inEscherichia coli are discussed.

Inhibitors of nitrofuran reduction in Escherichia coli: Evidence for their existence, partial purification, binding of nitrofurantoin in vitro, and implications for nitrofuran resistance

Nitrofurantoin (NF)-resistant mutants of Escherichia coli were isolated as described previously (18). One of the mutants (SSJ-2) was found to possess NF reductase activity equal to that of its parent (E. coli KL16). Two NF-resistant transductional derivatives, SSJ-2A and SSJ-2B, were isolated using SSJ-2 as the donor. SSJ-2 was found to be a double mutant carrying two mutations, nfnA and nfnB, while SSJ-2A (nfnA) and SSJ-2B (nfnB) carried these mutations individually. Heated extracts from SSJ-2A and SSJ-2B were found to inhibit the reduction of NF by unheated extracts of the NF-sensitive strain E. coli KL16 in vitro. Unheated extracts of these mutants reduced NF poorly relative to E. coli KL16. The poor reduction of NF by unheated extracts of SSJ-2A and SSJ-2B was greatly stimulated by heated extracts of SSJ-2B and SSJ-2A, respectively, and also by heated extracts of E. coli KL16. When heated extracts of SSJ-2A and SSJ-2B were mixed in a particular ratio and added to unheated extracts of E. coli KL16 they lost their inhibitory activity. Two proteins, designated inhibitor A and inhibitor B, have been partially purified from heated extracts of SSJ-2B and SSJ-2A, respectively. Their respective molecular weights, as determined by gel chromatography, were 37,000 and 20,500. The two inhibitors bound nitrofurantoin in vitro, and the NF-binding ability was lost when mixed in the molar ration of 3/1 (B/A). These observations were rationalized in terms of a hypothesis which explains (i) maximal NF reduction in wild-type cells, (ii) maximal NF reduction of nfnA-nfnB- double mutant, and (iii) poor NF reduction in nfnA- or nfnB- single mutants. The possible role of these inhibitors in nitrofurantoin resistance is also discussed.

Elucidation of the lesions present in the transcription defectivefitA76 mutant ofEscherichia coli: Implication of phenylalanyl tRNA synthetase subunits as transcription factors

Earlier reports from our laboratory dealt with the identification, mapping and characterization of a temperature sensitive mutant (fitA76) with a primary transcription defect at 42‡C and two of its suppressors (fitA24 andfitB). We report here the cloning and molecular characterization of a 2-1 kb DNA fragment which complemented the Ts phenotype of thefitA76 andfitA24 mutants but not that due to thefitB mutant. Cloning of this fragment in the T7 expression vector pT7.5 revealed the synthesis of a 33 kDa protein. The fragment hybridized with the Kohara phages 322 and 323 whose overlapping regions includepheS,pheT andrplT genes. Nucleotide sequencing showed that the fragment contains the entirepheS gene and the N-terminal portion ofpheT. Although these results implied that thefitA andpheS genes could be one and the same, earlier data had ruled out such a possibility. In order to know whether thefitA76 mutation defines a novel allele ofpheS, thepheS region of thefitA76 mutant was also sequenced, revealing a G → A nucleotide transition at position 293 of the coding region. This lesion is the same as that reported for thepheS5 mutant. However, it is shown that thefitA76 mutant is primarily transcription-defective while thepheS5 mutant is primarily translation-defective. These results suggested that thefitA76 mutant might harbour another mutation, in addition topheS5. In this report, we present genetic evidence for a second mutation (namedfit95) in thefitA76 mutant. Thefit95 by itself confers a Ts phenotype on rich media devoid of sodium chloride. It is proposed that the subunits of phenylalanyl tRNA synthetase could act as transcription factors (Fit) also.

Transient suppression of F-plasmid incompatibility in a strain of Escherichia coli

SummaryThe incompatibility between F plasmids is transiently suppressed in Escherichia coli strain CSH54. As a result this strain is able to maintain two F′ factors or an F′ factor and a mini-F plasmid for considerably longer periods than normal strains. When selective pressure for two markers carried by two separate F′s (or an F′ and mini-F) is imposed on normal strains, the two plasmids tend to form a cointegrate structure which can be detected genetically by the joint transfer of both the markers upon mating. This does not happen in CSH54; instead, the two plasmids are maintained and transferred independently. Physical evidence for the maintenance of an F′ and a mini-F plasmid is provided by agarose gel electrophoresis.