ABSTRACT - Shodhganga

ABSTRACT
The pro U locus has been identified
in both Escherichia coli and Salmonella
typhimurium as an osmoresponsive locus which is induced several hundred-fold, at the level
of transcription, in media of elevated osmolarity. ProU is involved in the transport of Lproline and glycine betaine: both substrates, when provided exogenously, are known to exert an
osmoprotective effect (consequent to their accumulation inside the cell) on the growth of the
bacteria in media of low water activity. The structural organisation of proU in E.coli, both at
genetic and molecular levels, and the regulation of proU expression were investigated in this
study.
Using the bacteriophage Mu dll(lac Amp), of Casada ban and Chou, several
osmoresponsive proU::lac gene fusions were isolated. All of them were induced about 100-fold
in media of high osmolarity. When these fusion strains were tested for
complementa~ion
using
the plasmids carrying different functional regions of the proU locus, all insertions mapped to
only one of the two complementation groups that had been identified ealier by Gowrishankar
et a[ (1986).
In order to characterise the other complementation group, additional lac fusions were
isolated using A. placMu55, which creates operon fusions. Characterisation of about 80
osmoresponsive lac fusions in proU unexpectedly revealed the presence of/ not one but two
complementation groups in this region, that is,
a total of three complementation groups at the
proU locus. The three cistrons representing the three complementation groups were designated
as pro V, proW, and pro X, and the designation proU was retained to represent the entire locus.
Each of the Mu dii (lac Amp) insertions was replaced by the mini-Mu lac fusion
phage, Mu dll1734 (also called Mu dK) that encodes Kanr, by a process of gene conversion.
Detailed complementation analysis of the Mu dK-converted strains indicated that the gene
fusions characterised earlier in this study were in· fact distributed into two of the three
complementation groups above (i.e., proW and proX).
The lac expression pattern of the fusion strains belonging to the different
complementation groups indicated that they were all induced approximately to the same
extent (about 100-fold) by increase in the medium osmolarity. Chromosome-mobilisation
experiments indicated that the direction of transcription of each of the three classes of fusions
was the same i.e., clockwise on the E. coli genetic map. These observations, considered in
conjunction with the complementation results, suggested that the proU locus forms an operon
comprising three genes.
Physical characterisation of the extent of the three genes of proU were carried out
using the technique of Tn1000 insertion mutagenesis and also by direct mapping of the lac
fusions. Tn1000 insertions that abolished prou+ function on a multicopy plasmid could be
classified into three categories based on their ability to grow in rich medium without and
with NaCl supplementation. The three classes were designated as non-osmosensitive,
osmosensitive and extremely osmosensitive. Restriction mapping of the three classes of
insertions revealed that the insertions belonging to each of the three classes were discretely
clustered on the proU DNA.
All the Mu dK-converted gene fusions (in proW and proX) were transferred onto the
plasmid pHYD58 (a pBR322 derivative carrying the entire region of proU locus) by
homologous recombination and the positions of the gene fusions were determined by restriction
enzyme digestion analysis. The operon fusions (in proV and proW) were directly mapped on
the chromosome by the technique of Southern blot hybridization. The results indicated that
there was one-to-one correspondence between the three classes of TnJOOO insertions and the
three classes of lac fusions, and the two taken together defined the extent of the three genes at
the proU locus.
Plasmids carrying Tn1000 insertions were also used to identify the protein products
encoded by the proU locus. Maxicell analysis of the plasmid-encoded proteins (of a prou+
plasmid and of the plasmids representing the three classes of Tn1000 insertions) identified
three proteins of molecular weight 44 kDa, 37 kDa and 33 kDa as the products of proV, proW,
and proX genes respectively. The polar effect observed in the case of Tn1000 insertion in proV
on the expression of the downstream genes (proW and proX), and of Tn1000 in proW on proX
expression provided unequivocal evidence that the three genes of the proU locus constitute an
operon.
May et al (1986) and Barron et al (1986) had identified a 32-kDa protein present in
the peri plasmic compartment to be the product of the pro U locus. Purification and
characterisation of this protein by these workers had shown that the protein binds glycine
betaine with very high affinity (Ko of 0.7 !J.M). The pcriplasmic protein profiles of the three
classes of lac fusion strains were also analysed in this study, and the data permitted
identification of the 33-kDa glycine betaine-binding protein as the product of the last gene
(proX) of the proU operon.
The sizes of the hybrid proteins encoded by the gene fusions were also determined.
Based on the sizes of the hybrid proteins and the position of the corresponding lac fusions, the
translation start-sites of pro V, proW and pro X were deduced to be at 0.7, 2.1 and 3.0 kb
respectively, from a unique EcoRV site present in the vicinity of proU.
Information from sequencing of the proU operon obtained by Gowrishankar (1989)
provided confirmation for all the observations made from the genetic analysis described
above, and in turn the genetic studies corroborated the predictions of the sequencing data that
IV
the three open-reading frames (ORFs) of proU code for three proteins. Since the sequence
information of proU was available, the position of each of the lac insertions was accurately
mapped (to the basepair level) by sequencing across the fusion joint. In all the cases, the lac
fusion had occurred in the correct reading frame of proU as was predicted from the sequencing
studies, and thereby validated the prediction that the three ORFs code for the three proteins
ProV, ProW and ProX respectively.
Studies on lac expression of the gene fusions on plasmids carrying different lengths of
proU upstream sequences indicated that all the information necessary in cis for osmotic
regulation of proU expression resides in the sequences that are present downstream of the
EcoRV site (0.69 kb upstream of the translation start-site of pro V).
Regulation of proU expression was very interesting from two diffe_rent angles:
(i)
proU
transcription is induced several hundred-fold by the change in osmolarity, which represents a
physical (or mechanical) signal. (ii) Attempts by many workers to identify trans-acting
regulatory mutants that would completely abolish proU regulation have so far been
unsuccessful.
In the course of creating proV::lac gene fusions, two fusions which were mapped to
positions 751 bp and 883 bp respectively from the EcoRV site (i.e., within the first structural
gene) were found to express very high levels of !5-galactosidase activity (2000 U) even in lowosmolarity conditions, while a fusion at 1528 bp (near the C-terminal end of the same gene)
showed normal osmotic induction (in particular, 30 U of 15-galactosidase activity after growth
at low osmolarity). Another fusion at a position 1000 bp from EcoRV exhibited a slightly
elevated level of expression (about 100 U). These observations demonstrated for the first time
that proU is negatively regulated, and that this negative regulatory element (NRE) resides
well within the first structural gene. Each of the four gene fusions identified in proV produced
an appropriate-sized hybrid protein as was expected from its physical map position; the
results, therefore, indicated that all the fusions had occurred in the correct reading .frame and
that the constitutive expression observed in three of these fusion strains was not due to any
deletion or mutation in that region.
Two alternative hypotheses were considered in order to explain the mechanism of
action of the NRE in proU regulation:
(i) In the same region of
proU where the two constitutive fusions map, there exists an ORF on
the complementary strand (that is, on the strand opposite that encoding ProV) which can code
for a presumptive polypeptide of 70 amino acid residues. The putative protein was postulated
to act as a repressor of proU expression. However, the various experiments carried out to test
the action of the putative repressor, failed to convincingly demonstrate the presence of such a
molecule.
v
.
(ii) The NRE was postulated to exert its effect entirely in a cis manner on proU regulation. One
such model put forth by ljiggins et al (1988) has postulated that regulation of the proU locus
was entirely governed by the extent of DNA superhelicity in the region of the proU promoter.
However, it has been very difficult to directly te_st such a hypothesis because of the known
inherent complication of transcription-induced supercoiling changes. In this study, the NRE
region alone (without the proU promoter) was cloned into two d.ifferent plasmid vectors pBluescript SK and pUC18 - and the effect of this region ml. plasmid supercoiling was
analysed. The results clearly demonstrated that plasmids carrying the NRE showed a
significant reduction in linking number (i.e., increase in negative DNA supercoiling) in an
osmolarity-dependent fashion as compared with the resptive parental plasmid vectors
themselves.
Based on these observations, a model to explain proU regulation has been proposed in
this study. Under low osmolarity conditions, it is suggested that the NRE is in the form of BONA, and that the transcription of proU is repressed by the binding of one or more proteins in
this region. One protein which can fit into such a role would be osmZ-cncoded H-NS. Under
high-osmolarity conditions, the DNA in the NRE region is postulated to undergo a structural
transition (probably as a result of the bound proteins falling off from the DNA) and thus to
extrude a cruciform structure, a process which is suggested to be associated with derepression
of proU expression. Such cruciform extrusions are known to absorb DNA supercoils and
therefore can satisfactorily explain the observed changes in DNA supercoiling of plasmids
carrying the NRE region. Moreover, the hypothesis is also supported by the facts that (i) an
RNA secondary-structure prediction algorithm identified the region of NRE (between
nucleotide positions 831 and 919) capable of exhibiting alternative secondary structure. (ii)
the region of NRE which is postulated to undergo cruciform extrusion is absolutely conserved
between E. coli and S. typhimurium. However, the occurrence of cruciform structure at the NRE
was not directly demonstrated in this study.
The above studies on proU regulation, when considered in conjunction with other
information available on proU regulation, illustrated that the regulation of proU is quite
complex and that the NRE forms the major component of its osmotic regulation. Nevertheless,
the signal responsible for induction of proU expression remains to be identified.

Download Report