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Research Overview of the group of
Molecular Genetics of Extremely Halophilic Archaea
Prof. Dr. Hua Xiang
Part I. Introduction
The research group for Molecular Genetics of Extremely Halophilic Archaea
(haloarchaea), led by Dr. Hua Xiang, focuses on the genetics of haloarchaea, with
several plasmids and halocins as models for the research of a variety of fundamental
processes in archaea, such as DNA replication, transcriptional regulation, protein
processing/translocation, and signal transduction. This group is also interested in the
biotechnology in thermophilic bacteria, food-grade lactic acid bacteria, as well as
halophilic archaea. A few genetic tools and biotechnological application were
achieved.
Dr. Xiang received his B.S. and M.S. from Beijing Normal University, and
earned his Ph.D. in Biochemistry & Molecular biology in 1997 from the Chinese
Academy of Medical Sciences & Peking Union Medical College (CAMS&PUMC).
He was a postdoctoral fellow in Institute of Microbiology, Chinese Academy of
Sciences from 1997 to 1999, and held another postdoctoral position from 1999 to
2001 in the Department of Molecular Biology, University of Medicine & Dentistry of
New Jersey (UMDNJ), in USA. He established this research group in 2001 after he
came back to this Institute.
Currently, this research group has grown to become a creative team with 12 young
people, including 1 senior technician, 2 assistant professors, 1 postdoc and 7 graduate
students.
Part II. Background and Significance
Archaea have been recognized as the third life domain in this planet since the late
1970s. They are biochemically and genetically different from the other two domains,
Bacteria and Eukaryota. Many archaea thrive in extremely harsh conditions，such as
boiling water or super-salty pools, etc. These types of archaea are often labeled
"extremophiles," meaning creatures that love extreme conditions. The extremely
halophilic archaea require external concentrations of 2 to 4.5 M NaCl for optimal
growth, which indeed represent the salinity limit that an earth life can endure.
Haloarchaea are easy to culture and are genetically tractable, hence they are an
excellent model for archaeal genetics. Furthermore, these extremophiles also have
considerable biotechnological potentials. They possess unique bacteriorhodopsin,
extreme enzymes, liposomes, biopolymers and many other substances with
application potentials. The achievements in genetics and biotechnology would help us
to comprehensively understand these halophiles and effectively utilize these special
microbial resources.
Part III.
Major Achievements
1. Molecular genetics and biotechnology of haloarchaeal plasmids
A novel rolling-circle (RC) replicating plasmid pNB101 (2,538 bp) isolated from
Natronobacterium sp. strain AS7091 has been completely sequenced. It is the first
plasmid isolated from a haloalkaliphilic archaeon. The putative double-stranded origin
(DSO), single-stranded origin (SSO), as well as the rep gene were identified, and the
single-stranded DNA intermediate that corresponds to the leading strand in RC
replication was experimentally demonstrated. A high-level transcription of DSO was
observed and supposed to be involved in the initiation of this RC replication
(Extremophiles, 2004, 8: 81-89). With insertion of the Escherichia coli ColE1
replicon and two antibiotic resistance genes into this plasmid, a novel shuttle vector
(pNB102) between haloarchaea and E.coli was developed. pNB102 was successfully
transformed into two non-haloalkaliphilic archaea, Halobacterium salinarum and
Haloarcula hispanica. It is the first report that the replicon of pNB101 has such a
wide host range, and has taken the first step for construction of the first vector/host
system in haloalkaliphilic archaea (Biotechnol Lett 2004, 26: 1107-1113). This
haloarchaeal vector was patented in P. R. China, and was successfully used to
optimize the bacteriorhodopsin in H. salinarum (Patent No. ZL02100714.4 and No.
200510087721.1).
Recently, we have completely sequenced another novel plasmid pSCM201 (3463
bp), which was isolated from Haloarcula sp. AS7094. This plasmid seems to be
unique as no homology was found with all other reported plasmids. By means of 2D
agarose gel electrophoresis and electron microscopy analysis, the pSCM201 was
demonstrated to be a unidirectional theta-replicating plasmid. To our knowledge, it is
the smallest theta plasmid observed so far in archaea. With this replicon, several
highly stable vectors were developed and successfully transformed into Haloarcula
hispanica, which was also applied for a Chinese patent. Moreover, the minimal
replicon (1.8kb) containing only the putative origin and the replication initiation gene
was determined by gradual deletion of the plasmid sequence. Currently, we are
mapping the precise point of the replication initiation within the putative origin, and
determining the elements that might be involved in the replication initiation. Since the
in vivo analysis system has been established, it would be a good model for study
haloarchaeal DNA replication, and the paper will be prepared for publication in the
journal Nucleic Acids Research in near future.
In the meantime, we have also completely sequenced two other novel haloarchaeal
plasmids pZMX101 (3918 bp) and pZMX201 (1668 bp). Sequence analysis indicates
that pZMX101 is very possible to be a theta-replicating plasmid, while pZMX201 will
be the smallest RC-replicating plasmid so far isolated in halophilic archaea.
2. Molecular biology of the haloarchaeal peptide antibiotics (halocins)
Halocins are bacteriocin-like proteins or peptides produced by many species of
the family Halobacteriaceae. HalC8, a novel halocin isolated from Halobacterium
strain AS7092, is an extremely stable and hydrophobic microhalocin with a wide
inhibitory spectrum against the haloarchaea. Its primary target was found to be
located in the cell wall of the sensitive cells. HalC8 can be desalted, frozen, subjected
to many organic solvents, and boiled for about one hour without losing activity. It was
purified by combination of tangential flow filtration (TFF), Sephadex G50 and
DEAE-sepharose chromatography, and the N-terminal amino acid sequence was
determined by Edman degradation (Extremophiles, 2003, 7: 401-7).
According to the N-terminal amino acid sequence of HalC8, the encoding gene
for this halocin was cloned recently, which strongly supports that HalC8 is derived
from the C-terminus of a 283-amino-acid prepro-protein (ProC8). This was further
verified by MALDI-TOF-MS analysis of the purified HalC8. The production of
HalC8 was controlled through both transcriptional regulation and protein processing:
the halC8 transcripts and HalC8 activity rapidly increased to maximal levels upon
transition from exponential to stationary phase. However, while halC8 transcripts
remained abundant, the HalC8 processing was inhibited during stationary phase.
Remarkably,
agar-diffusion
test
revealed the unprocessed ProC8
and its
207-amino-acid N-terminal peptide (HalI), with or without its putative Tat signal
sequence, were capable to block the halocin activity of HalC8 in vitro. In addition,
heterologous expression of HalI in Haloarcula hispanica rendered this sensitive strain
remarkable resistance to HalC8, indicating that HalI encodes the immunity property
of the producer. In accordance with this immunity function, HalI and ProC8 were both
found localized on the cellular membrane. Protein interaction assay revealed that HalI
likely sequestrated the HalC8 activity by specific binding. To our knowledge, this is
the first report on halocin immunity, and our results that a single gene encodes both
peptide antibiotic and immunity protein also provide a novel immune mechanism for
peptide antibiotics (Mol Microbiol, 2005, 57: 537-549).
Currently, a 9.3-kb gene cluster containing the halocin C8 gene and six other
genes was cloned and sequenced. The novel genes encode a putative regulator and
several transporters, which were speculated to be involved in the halocin expression
regulation and transportation. It would provide a novel and great opportunity for
halocin research in the future.
3. Thermoanaerobacter tengcongensis: genetics and bioengineering
T. tengcongensis is a thermophilic bacterium isolated in Tengcong hot springs in
the south of China, and is the first bacterium whose genome was completely
sequenced in this country. Our group has taken part in partial works to understand its
fundamental genetics and several biotechnological potential genes. As the genetic
tools were still not established for this bacterium, we mainly investigated the
DnaA-based replication initiation and ECF-sigma factor-involved/stress-induced gene
expression regulation by protein-protein interaction assay and other biochemical
techniques, hoping to reveal novel factors or novel mechanisms in these fundamental
processes in this thermophile, especially to understand parts of those uncharacterized
genes in this genome.
In respect of the bioengineering of genes in this thermophile, a thermostable
esterase from this bacterium has been cloned, over-expressed, and biochemically
characterized (Biotechnol Lett, 2003, 25: 1463-1467). We have also identified a
novel trehalose synthesis pathway in this bacterium, where the dominant function of
the T. tengcongensis trehalose phosphorylase (TreP) was proved to catalyze trehalose
synthesis but not degradation. A new approach for production of trehalose from starch
with the thermostable T. tengcongensis enzymes was also proposed (Science in
China-C , 2005, 48: 221-227).
4. Biotechnology in Lactococcus lactis
L. lactis has been safely used in the food industry for centuries and is considered
as potential mucosal delivery vehicles of vaccine antigens. In cooperation with Prof.
Huarong Tan’s group, we have established a novel food-grade gene expression system
in Lactococcus lactis. The food-grade expression of human glutathione S-transferase
(hGST) and Cu/Zn superoxide dismutase (hSOD) in L. lactis and their high-level
expression in E.coli were achieved (Biotechnol Lett, 2002, 24:1669-1672; Biomol
Eng, 2003, 20: 107-112), and the hSOD biotechniques have been transfer to a
company with 3.2 million RMB Yuan. Recently, we have also expressed the
SARS-coronavirus nucleocapsid (N) protein in E. coli for serodiagnosis and in L.
lactis
for
mucosal
vaccination.
Results
indicated
that
orally
delivered
SARS-N-producing L. lactis could induce significant N-specific IgG in the sera (Appl
Microbiol Biotechnol 2005, 68: 220-227).
Publication (SCI journal only) list:
1. Sun C，Li Y, Mei S, Lu Q, Zhou L, Xiang H*. 2005. A single gene directs both production
and immunity of halocin C8 in a haloarchaeal strain AS7092. Molecular Microbiology 57
(2)：537–549.
2. Pei H, Liu J, Cheng Y, Sun C, Wang C, Lu Y, Ding J, Zhou J, Xiang H* . 2005, Expression
of SARS-coronavirus nucleocapsid protein in Escherichia coli and Lactococcus lactis for
serodiagnosis and mucosal vaccination. Appl Microbiol Biotechnol 68: 220–227.
3. Ren Y, Dai X, Zhou J, Liu J, Pei H, Xiang H*. 2005. Gene Expression and molecular
characterization
of
thermostable
trehalose
phosphorylase
in
Thermoanaerobacter
tengcongensis . Sci China (Ser C) 48(3): 221-227.
4. Zhou M, Xiang H*, Sun C, Tan H. 2004. Construction of a novel shuttle vector based on an
RCR-plasmid from a haloalkaliphilic archaeon and transformation into other haloarchaea.
Biotechnol Lett 26: 1107-1113.
5. Zhou M, Xiang H*, Sun C, Li Y, Liu J, Tan H. 2004. Complete sequence and molecular
characterization of pNB101, a rolling-circle replicating plasmid from the haloalkaliphilic
archaeon Natronobacterium sp. strain AS7091. Extremophiles 8(2): 81-89.
6. Li Y, Xiang H*, Liu J, Zhou M, Tan H. 2003. Purification and biological characterization of
halocin C8, a novel peptide antibiotic from Halobacterium strain AS7092. Extremophiles
7(5): 401-7.
7. Xiang H , Wei W, Tan H*. 2003. Food-grade expression of human glutathione S-transferase
and Cu/Zn superoxide dismutase in Lactococcus lactis. Biomol Eng 20(3): 107-112.
8. Zhang J, Liu J, Zhou J, Ren Y, Dai X, Xiang H*. 2003. Thermostable esterase from
Thermoanaerobacter tengcongensis: high-level expression, purification and characterization.
Biotechnol Lett 25: 1463-1467.
9. Wei W, Xiang H*, Tan H*. 2002.Two tandem promoters to increase gene expression in
Lactococcus lactis. Biotechnol Lett, 24:1669-1672.
* Corresponding author
Patent list:
1. Hua Xiang, Meixian Zhou and Huarong Tan. 2002, One haloalkaliphilic archaeal strain and
its plasmid, and their application (granted in 2004 with granted patent No. ZL02100714.4).
2. Hua Xiang, Jian Zhang, Jian Zhou, Yuqing Tian and Huarong Tan. 2003. The application of
a thermostable lipase and its bioengineering bacteria (Application No.03136565.5).
3. Hua Xiang, Chaoming Sun. 2004. A haloarchaeal plasmid and its derived plasmid vectors
(Application No. 2004100497519).
4. Hua Xiang, Yuanyuan Ren, Jingfang Liu, Jian Zhou, Huarong Tan. 2004. The gene encoding
a
novel
thermostable
trehalose
synthase
and
its
application
(Application
No.
200410073883.5).
5. Hua Xiang, Yun Li, Chaoming Sun. 2005. The genes encoding a halocin and its immunity
protein and their application (Application No. 200510055640.3)
6. Hua Xiang, Ligang Zhou, Meixian Zhou, Chaoming Sun, Jian Zhou. 2005. A plasmid for
expression of bacteriorhodopsin mutants in haloarchaea and their bioengineering archaea.
(Application No. 200510087721.1)
Part IV.
Future Research Plan
The future research of this group will focus on the genetics and bioengineering of
halophilic archaea. We will use the halocins and the haloarchaeal plasmids as models
to further explore the fundamental processes of halophilic archaea in replication,
transcriptional regulation, protein processing, protein translocation, and signal
transduction, etc. We will also establish novel haloarchaeal gene expression systems
for bioengineering of bacteriorhodopsin, biopolymers, halophilic enzymes as well as
halocins in halophilic archaea. Moreover, to better understand the differences between
archaea and bacteria, we will also investigate the mechanisms of DNA replication and
gene expression regulation in T. tengcongensis. Our work will provide the
fundamental information and biotechnological potential of these extremophiles.