(Received for publication, September 25, 1995; and in revised form, October 6, 1995)
From the
The aquaporin family of molecular water channels is widely
expressed throughout the plant and animal kingdoms. No bacterial
aquaporins are known; however, sequence-related bacterial genes have
been identified that encode glycerol facilitators (glpF). By
homology cloning, a novel aquaporin-related DNA (aqpZ) was
identified that contained no surface N-glycosylation
consensus. The aqpZ RNA was not identified in mammalian mRNA
by Northern analysis and exhibited bacterial codon usage preferences.
Southern analysis failed to demonstrate aqpZ in mammalian
genomic DNA, whereas a strongly reactive DNA was present in chromosomal
DNA from Escherichia coli and other bacterial species and did
not correspond to glpF. The aqpZ DNA isolated from E. coli contained a 693-base pair open reading frame encoding
a polypeptide 28-38% identical to known aquaporins. When compared
with other aquaporins, aqpZ encodes a 10-residue insert
preceding exofacial loop C, truncated NH and COOH termini,
and no cysteines at known mercury-sensitive sites. Expression of aqpZ cRNA conferred Xenopus oocytes with a 15-fold
increase in osmotic water permeability, which was maximal after 5 days
of expression, was not inhibited with HgCl
, exhibited a low
activation energy (E
= 3.8
kcal/mol), and failed to transport nonionic solutes such as urea and
glycerol. In contrast, oocytes expressing glpF transported
glycerol but exhibited limited osmotic water permeability. Phylogenetic
comparison of aquaporins and homologs revealed a large separation
between aqpZ and glpF, consistent with an ancient
gene divergence.
The entry and release of water from cells is a fundamental process of life. Although biophysical features of membrane water permeability were recognized, the molecular pathway of transmembrane water movement remained unknown until discovery of the aquaporins, a family of proteins that permits selective, osmotic flow of water across cell membranes(1) . AQP1, the first recognized and characterized homolog, is abundant in mammalian red cells, renal proximal tubules, and other epithelia, where it is constitutively active(2) . cDNAs encoding several other mammalian aquaporins have been isolated by homology cloning(3) . In renal collecting duct AQP2 (4) is regulated by vasopressin(5, 6) , and AQP3 is also slightly permeable to glycerol (7, 8, 9) . Other aquaporins exist in brain (AQP4)(10, 11) , salivary and lacrimal glands, cornea, and lung (AQP5)(12) , and a weak water transporter is in lens (MIP)(13) .
Aquaporins are expressed in diverse species. Plants express numerous different aquaporins in tonoplast or plasma membranes, and these proteins apparently play essential roles in maintenance of turgor and transpiration(14) . A homologous sequence is known to be expressed in Escherichia coli and other bacteria but encodes GlpF, the glycerol facilitator(15) , which exhibits minimal water permeability (16) . Despite phylogenetic analyses suggesting their common prokaryotic origin, no aquaporins have been reported in bacteria.
Changes in the extracellular osmolarity elicit similar physical effects on cells throughout nature, and similarities are observed among the mechanisms of cellular osmoregulation. In bacteria, the cellular turgor is an essential feature based on the high osmotic pressure of the cytoplasm. Although some of the physiological and genetic responses to osmotic shifts among different bacteria are already known, the mechanisms of water transport during bacterial osmoregulation are poorly understood(17, 18) . Here we report the isolation and the molecular characterization of AqpZ, the first prokaryotic water channel. The presence of this aquaporin in Gram-positive and Gram-negative species suggests a widespread existence of this aquaporin among bacteria and suggests structural explanations for the functional differences between the sequence-related proteins AqpZ and GlpF.
Based on codon
usage analysis of aqpZ(19) , amplification of E.
coli chromosomal DNA (DH5 strain) was undertaken by PCR.
Specific oligonucleotides (GCBU3, 5`-TATCTGGTGAATGACTCGCC-3` and GCBD2,
5`-TTGCTTAGCTCATGAAAGGA-3`) were designed corresponding to 5`- and
3`-untranslated sequences of aqpZ and were used as primers (94
°C, 1 min; 51 °C, 1 min; 72 °C, 1 min; 30 cycles). The
resulting 917-bp PCR band was subcloned into pBS-KS(+) and
sequenced.
Bovine genomic DNA as well as equivalent
genomic samples of DNA from E. coli and other bacterial
species were digested with AvaII and BglI and
evaluated by Southern analysis with probes corresponding to the human
water channel protein (AQP1), the novel DNA (aqpZ),
and the glycerol facilitator from E. coli (glpF). As
expected, AQP1 failed to hybridize with E. coli DNA
but reacted with a multiple band in bovine DNA (Fig. 1, left). In contrast, the novel DNA (aqpZ) provided a
striking band at 530 bp with E. coli DNA but failed to
hybridize with bovine DNA (Fig. 1, center); this probe
also hybridized with multiple bands in DNA from Mycobacterium
tuberculosis (atypical Gram-positive), Citrobacter freundii (Gram-negative), and Bacillus subtilis (Gram-positive).
Hybridization of the blot with glpF revealed a weak band in
DNA from B. subtilis (not visible in Fig. 1), one of
the bands in the M. tuberculosis sample, and gave a striking
band of 1100 bp in the E. coli sample (Fig. 1, right).
Figure 1:
Genomic Southern analyses. Mammalian
genomic DNA (18 µg, bovine) and chromosomal DNA samples from
selected bacterial species (50 ng, E. coli DH5, M.
tuberculosis, C. freundii, and B. subtilis) were digested
with AvaII and BglI, electrophoresed into agarose
gels, and transferred to blots. Probes corresponding to coding regions
of human AQP1 (837 bp) and aqpZ (562 bp) and E.
coli glpF (251 bp) were labeled with
P and hybridized
to the blots under conditions of medium stringency. Kb,
kilobases.
The deduced amino acid sequence of aqpZ was compared with sequences of mammalian (AQP1) and plant (TIP) aquaporins and E. coli glpF (Fig. 2A). Alignments revealed the existence of
residues known to be highly conserved in each of the transmembrane
domains as well as the functionally important loops B and E. Hydropathy
analysis of AqpZ was similar to that of other aquaporins (Fig. 2B) and is consistent with the existence of six
transmembrane domains (21) and hydrophobic loops B and E, which
may form the aqueous pore by dipping into the bilayer from opposite
leaflets(22) . The predicted topology of AqpZ contained
distinctive features (Fig. 2C). The NH
- and
COOH-terminal domains of AqpZ were shorter than corresponding domains
in plant and mammalian homologs. A single cassette encoding 10 extra
hydrophobic residues was present at the junction of transmembrane
domain 3 and exofacial loop C in AqpZ, and two cassettes were present
in GlpF (Fig. 2A).
Figure 2:
Comparative alignment of deduced amino
acid sequence, hydrophobicity analysis, and predicted membrane topology
of AqpZ protein. A, deduced amino acid sequence alignments
were performed by PILEUP program analysis of AqpZ, human AQP1, plant
TIP, and E. coli GlpF. Single boldface lines indicate the six predicted transmembrane domains (TM 1 to TM 6). The polypeptide sequences joining transmembrane domains
are doubly underlined (connecting loops A-E). Positions
with fully conserved residues are enclosed. The gaps show the amino
acid cassettes among the sequences in loops A, C, D, and E. B,
Kyte-Doolittle hydrophobicity profile of AqpZ using a 7-residue window. C, predicted topology of AqpZ compared with AQP1. Open
circles represent corresponding residues present in both
structures; closed circles represent extra residues in AqpZ; stippled circles represent residues missing from AqpZ. The
extracellular space corresponds to the E. coli periplasmic
space.
Figure 3:
Comparative transport functional analyses. A, [C]glycerol influx was measured in
oocytes injected with water (control) and 25 ng of cRNA for aqpZ or glpF and measured after 4 days of expression (see
``Materials and Methods''). B, osmotic water
permeability (P
) measured on oocytes
injected similarly. C, osmotic water permeability of oocytes
compared with time of expression. Oocytes were injected with water
(control), 25 ng of cRNA for aqpZ or glpF, or 10 ng
of cRNA for AQP1 and incubated for 2-5 days before
measurement of osmotic water permeability. Each bar or data point represents the mean ± S.E. of 8-12
individual oocytes.
Previous
studies reported an even lower P of oocytes
injected with glpF cRNA and incubated for only 2-3
days(16) , so a time course of expression and water transport
was performed. After 2 days of expression, the AqpZ oocytes exhibited P
values significantly below those expressing AQP1
cRNA, but after additional days of incubation, the water permeability
of AqpZ oocytes rose significantly, whereas the permeability of GlpF
oocytes remained low (Fig. 3C). While it is not known
why the aqpZ cRNA was expressed more slowly, it may reflect
the different preferences in codon usage or it may be a result of
defective membrane targeting of AqpZ protein, which lacks a long
COOH-terminal cytoplasmic domain that is apparently needed for
efficient transit to the plasmalemma(22) . Nevertheless, it is
apparent that despite highly related deduced amino acid sequences, the
two bacterial proteins AqpZ and GlpF perform distinct transport
functions.
Figure 4: Phylogenetic analysis of AqpZ, other aquaporins, and homologs. Deduced amino acid sequences from the indicated aquaporins and homologous proteins were aligned by PILEUP program analysis (version 7.1, Vax computer system). The percent identity compared with AqpZ is indicated.
The relative simplicity of bacterial
genomes is consistent with functional requirements of all genes. While
it is premature to conclude what role AqpZ plays in bacterial
metabolism, it seems highly likely that it may be expressed for
management of osmotic stress(17, 18) , cell growth and
division, or desiccation, which may be involved in chronic dormancy.
The presence of two related genes, aqpZ and glpF,
encoding proteins with distinct transport functions implies the need
for both, and the sequence differences between these genes may provide
structural insight into their distinctive transport functions. For
example, the presence of only one cassette insert in loop C of AqpZ but
two cassette inserts in loop C and another cassette insert in loop E of
GlpF may be of structural significance. Indeed, genomic Southern
analysis (Fig. 1) is consistent with the widespread existence of aqpZ-related genes among bacterial species. In contrast to
plant genomes that contain numerous aquaporin genes, ()when
the entire sequence of the Hemophilus influenzae gene was
recently sequenced(25) , only two genes were identified that
are sequence-related to the aquaporins. Of note, the H. influenzae gene glpF 690 and the E. coli glpF are contained
within operons encoding other known glycerol metabolic enzymes, whereas
the other H. influenzae gene, glpF 1017, and E.
coli aqpZ do not exist within operons. These observations lead us
to speculate that dual existence of the functionally distinct genes aqpZ and glpF may be a general feature of bacterial
species.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38664[GenBank].