How Rhizobia Survive in the Absence of a Legume Host, a Stressful World Indeed
In the preceding decade, numerous advances have been made in understanding the genes and proteins involved in rhizobial responses to stress, particularly those caused by desiccation, pH, and nutrient deprivation. Often, the genes and proteins that are expressed under one stress condition overlap with those induced under other stress conditions. Although most studies on rhizobial stress responses employ planktonic cells, one way that rhizobia, as nonspore formers, could maximize their survival under stress conditions is to establish biofilms, surface-attached communities. However, few studies of this life style for rhizobia have been undertaken especially with regard to stress. The knowledge gained from learning about how planktonic cells respond to stress must now be applied to rhizobial biofilms because they serve as reservoirs of bacteria in the rhizosphere when a legume host is absent. By so doing, we will gain a much better understanding of how this fascinating and important group of soil bacteria lives in the “real world.”
J. Seckbach and M. Grube (eds.), Symbioses and Stress: Joint Ventures in Biology,
Cellular Origin, Life in Extreme Habitats and Astrobiology 17, 375–391
DOI 10.1007/978-90-481-9449-0_18, © Springer Science+Business Media B.V. 2010
Biodata of Ann M. Hirsch, author of “How Rhizobia Survive in the Absence of a
Legume Host, A Stressful World Indeed”
Ann M. Hirsch is a Professor in the Department of Molecular, Cell and
Developmental Biology at UCLA (Los Angeles, California). Her major area of
research is in plant–microbe interactions, an area of study in which she combines
her prior training at the University of California-Berkeley (Ph.D.) and Harvard
University (postdoctoral) in plant development, molecular biology, and microbiol-
ogy. Dr. Hirsch was the ﬁrst person to show that early nodulin gene expression was
induced independently of rhizobia by altering the endogenous hormonal levels of
legume roots. Also, in collaboration with Dr. Y. Kapulnik, her group demonstrated
that signal transduction pathways based on common gene expression patterns were
conserved in the nitrogen-ﬁxing symbiosis and in mycorrhizae. Dr. Hirsch’s labora-
tory also unambiguously demonstrated that introducing a lectin gene into a nonhost
altered that legume’s rhizobial host range. With Dr. M. Valdés, Dr. Hirsch identiﬁed
a new group of nitrogen-ﬁxing bacteria, non-Frankia actinomycetes that also have
cellulolytic activity. Her group also developed novel DNA-based systems to authen-
ticate botanical identity and to analyze herbal supplements for contaminants and
adulterants. Recently, Dr. Hirsch and coworkers discovered that the core nodulation
(nod ) genes of Sinorhizobium meliloti, the nitrogen-ﬁxing endosymbiont of alfalfa,
are required for the establishment of mature bioﬁlms. Her expertise in biological
nitrogen ﬁxation has led to valuable and ongoing collaborations with scientists in
Israel, Mexico, Thailand, Brazil, and Pakistan. She was awarded an NSF Faculty
Award for Women and a Research Prize from the Instituto Politecnico Nacional
of México in the Programma Institucional de Medio Ambiente y Desarrollo
Sustentable. She was named a Fellow of the American Society of Plant Biologists
of the Inaugural Class of 2007 and was elected as a corresponding member of the
Mexican Academy of Sciences in 2007.
HOW RHIZOBIA SURVIVE IN THE ABSENCE OF A LEGUME
HOST, A STRESSFUL WORLD INDEED
ANN M. HIRSCH
Department of Molecular, Cell and Developmental Biology
and The Molecular Biology Institute, University of California-
Los Angeles, Los Angeles, CA 90095-1606, USA
Numerous studies have been made of the severe conditions under which Rhizobium–
legume symbioses persist, including exposure to salt, desiccation, acidic or alkaline
pH, temperature extremes, nutrient deﬁciency, and soil toxicity brought about by
heavy metals or hazardous chemicals (Zahran, 1999; Sadowsky, 2005). In addi-
tion, the host plants also inﬂuence rhizobial survival (Hirsch, 1996). Selection of
hosts and their nitrogen-ﬁxing endosymbionts that are tolerant to a broad range
of environmental stresses is important for agriculture in marginal lands as well
as for the inoculant industry, and has been described in numerous reports. This
review asks the question: how do rhizobia survive under stressful conditions in
the absence of their symbiotic partner? The issue of rhizobia as a soil saprophyte
is rarely discussed, but it is extremely relevant because these bacteria are nonspore
formers and do not have a resting stage or form. The most likely bacteria to survive
severe environmental stress such as dehydration are those that are dormant or in
stationery phase, whereas actively growing bacteria usually die (Davet, 2004).
Our hypothesis is that nonspore formers such as rhizobia survive in the soil
environment because they establish bioﬁlms on either biotic or abiotic surfaces
(Fujishige et al., 2006, 2008). We wrote: “It was tempting to speculate that bioﬁlm
formation is important for the overall ﬁtness of rhizobia in the soil and in rhizo-
sphere microenvironments, thereby contributing to an efﬁcient symbiosis” (Rinaudi
et al., 2006). Bioﬁlms are surface-attached communities of bacteria, consisting of
either a single or multiple species, and contained within a self-produced extracellular
matrix (Costerton et al., 1995; Stanley and Lazazzera, 2004). For reasons of carbon
sufﬁciency and in some cases protection from predation, attachment to biotic surfaces
This review is dedicated to memory of the late John G. Streeter, a pioneer in studying the stress
responses of the Bradyrhizobium japonicum–soybean symbiosis.
ANN M. HIRSCH
is more likely to enhance survival. In addition, adherence to abiotic surfaces can be
advantageous because rhizobia in a bioﬁlm are protected from environmental insults
as a consequence of their exopolymeric matrix and lowered metabolic rate. A recent
study of the gram-negative, nonspore-producing Rhizobium NGR234 attached to
dry sand showed a decline in viability after 9 weeks of desiccation stress. Nevertheless,
viable cells per gram of dry sand were detected months later. In addition, the cells
remained symbiotically competent (Gorbushina et al., 2007). In contrast, under the
same conditions, all the vegetative cells of a gram-positive, spore-forming species,
Bacillus megaterium, differentiated into spores within a week (Gorbushina et al.,
2007). Thus, in spite of the fact that rhizobia are nonspore formers, they retain via-
bility following severe stress. Estimates have been made indicating that some rhizo-
bial species survive in soil at least 4–5 years without their host, but a few cases
demonstrated that rhizobia might survive up to 15 years (Fred et al., 1932)!
Nonetheless, the numbers of rhizobia that are present in bulk soil are orders of
magnitude less numerous than those found in the rhizosphere (Hirsch, 1996).
Bioﬁlms comprised of synergistic or syntropic consortia of bacteria would
provide an additional survival advantage to rhizobia. Because the bioﬁlm state is
a means of survival for many bacteria subjected to environmental stress or antimi-
crobial assaults (Costerton et al., 1999; Hogan and Kolter, 2002), it seems logical
to assume that bioﬁlms could be a means that rhizobia use to survive stress.
However, surprisingly little is known about rhizobial bioﬁlms and whether genes
that are expressed in response to stress are also expressed in bioﬁlms. This review
is an attempt to bring some of these literatures together, focusing mainly on desicca-
tion, pH, and nutrient availability. Details about other stresses affecting rhizobia
are found in Zahran (1999), Sadowsky (2005), and Vriezen et al. (2007).
2. Rhizobia and Stress
Rhizobia are found in bulk soil, attached to soil particles, but more frequently
Rhizobium species establish mutualistic or commensal relationships with the roots
of both legume hosts and nonlegumes (Foster et al., 1983; Schwieger and Tebbe,
2000). Attaching to roots or living in the rhizosphere offers a competitive advan-
tage for rhizobial survival in part because of root exudation; bulk soil is a desert
by comparison. One gram of root is estimated to release 50–100 mg of exudate,
enough to support 2 × 10
bacteria (Morgan et al., 2005). Even nonlegumes
can support a substantial number of rhizobia. For example, Rhizobium legumi-
nosarum bv. trifolii cells were recovered from the internal tissues of rice roots at
cells per gram of root fresh weight (Yanni et al., 1997). These rhizobia were
capable of effectively nodulating berseem clover (Trifolium alexandrinum L.)
demonstrating that they were symbiotically competent. However, many rhizo-
bial strains isolated from soil are often classiﬁed as symbiotically incompetent
(Laguerre et al., 1993), leading some to argue that loss of the symbiotic plasmid
enhances survival (Squartini, 2001). Nonsymbiotic bradyrhizobia have also been
HOW RHIZOBIA SURVIVE IN THE ABSENCE OF A LEGUME HOST
isolated (Pongsilp et al. 2002); these bacteria do not have plasmid-borne but rather
have chromosomally located symbiotic genes. However, nonsymbiotic strains can
readily become symbiotic by the acquisition of a symbiotic plasmid or island.
For example, the transfer of a “symbiotic island,” as shown for a soil-inhabiting
Mesorhizobium loti, allowed the new Nod
strain to nodulate Lotus
sp. (Sullivan et al., 1995; Sullivan and Ronson, 1998). In some cases, however, the
strains may become poorly effective or even ineffective after lateral transfer of
a symbiotic island (Nandasena et al., 2006, 2007). Because most studies evalu-
ate symbiotic competence on the basis of whether rhizobia nodulate a particular
legume host, strains defective in a single gene critical for nodulation or for nod-
ule effectiveness, or if tested on the wrong host could be judged as symbiotically
incompetent. Thus, quantiﬁcation of the numbers of symbiotically incompetent
strains in soil could be skewed because of the aforementioned limits on nodula-
tion. Also, many earlier studies evaluating the numbers of rhizobia in the soil have
been culture dependent rather than culture independent, so the actual numbers of
rhizobia and their symbiotic status in bulk soil and the rhizosphere may be under-
estimated. Indeed, several lines of recent evidence suggest that symbiotic genes are
important for rhizobial survival in response to stress (Domínguez-Fererras et al.,
2006) and also for bioﬁlm formation (Fujishige et al., 2008).
With some exceptions, the studies on rhizobial responses to stress have been
performed in vitro on planktonic cells and not in situ on bioﬁlm cells. Nevertheless,
such an approach has facilitated the identiﬁcation of genes that are upregulated/
downregulated or induced in response to stress. Moreover, studies on other bacte-
ria have shown that genes characteristic of various stress responses, e.g., nutrient
deprivation, desiccation, etc. are expressed in bioﬁlms (Whiteley et al., 2001).
Many rhizobia are salt tolerant (NaCl) or osmotically tolerant and thus, capable
of living under severe moisture deﬁciency (Zahran, 1999; Sadowsky, 2005). Several
of the responses of rhizobia to other stresses, such as high temperature toler-
ance and oxygen radical defense mechanisms, overlap with desiccation resistance.
Nonetheless, understanding the details of global gene regulation in response to
various stress parameters in rhizobia is fragmentary. So far not much information
about the signaling pathways/networks that mediate desiccation tolerance, speciﬁ-
cally, or stress resistance, generally, is available for this group.
As free-living rhizobial cells encounter dry conditions, a number of pro-
found changes occur: (i) water activity is reduced; (ii) the osmotic potential
rises as salts, which can be toxic to rhizobia, accumulate; (iii) transcription and
translation slow down and DNA may become damaged; and (iv) membranes
become leaky as they shrink away from the cell wall. To circumvent cellular
damage, many bacteria accumulate various carbohydrates or osmoprotectants.
For example, trehalose, a disaccharide made up of two glucose molecules
ANN M. HIRSCH
joined together by an D,D-1,1 linkage, is employed by many organisms to protect
membranes and proteins from desiccation stress (Streeter and Gomez, 2006).
Rhizobia also accumulate trehalose, among other carbohydrates, and also
betaine and proline, in response to desiccation. Trehalose and sucrose are the
only carbohydrates that are synthesized de novo in response to stress. To syn-
thesize trehalose, bacteria utilize either a single pathway or up to three differ-
ent pathways, the OtsAB, TreYZ, and TreS pathways. For example, the TreYZ
pathway is common to many rhizobia (Streeter and Bhagwat, 1999), whereas
the OtsAB and TreYZ pathways are found in R. leguminosarum bv. trifolii
strain NZP561 (McIntyre et al., 2007). All three pathways have been detected
in Bradyrhizobium japonicum strain USDA110 and B. elkanii (Streeter and
Gomez, 2006). Trehalose at relatively high concentrations is also present in
B. japonicum bacteroids residing within nodules, suggesting that these differen-
tiated nitrogen-ﬁxing cells are under stress. Even adding trehalose to B. japoni-
cum cells enhances their survival in response to dryness (Streeter, 2003).
Saccharides such as trehalose may protect desiccated cells by their ability to
form glasses under dry conditions, in this way maintaining the native confor-
mation of proteins and other macromolecules (Ramos et al., 2001). Trehalose
levels also increase in R. leguminosarum bv. trifolii TA1 cells as they encounter
osmotic stress (Streeter, 1985; Breedveld et al., 1993). R. leguminosarum bv.
trifolii strain NZP561 accumulates trehalose upon entry into stationary phase
(McIntyre et al., 2007), but in this rhizobial strain, trehalose synthesis is con-
stitutive and modiﬁed posttranscriptionally rather than induced as in other
rhizobia. Mutations in otsA or treY individually in strain NZP561 did not
dramatically affect trehalose accumulation, but double otsA treY mutants did
not accumulate trehalose and were more sensitive to desiccation. They were
also less competitive with regard to occupying nodules than were wild-type
strains (McIntyre et al., 2007).
A global approach to study rhizobial stress responses is to do a genome-
wide transcriptional analysis. Domínguez-Fererras et al. (2006) employed a DNA
microarray to examine gene expression in planktonic, exponentially growing
Sinorhizobium meliloti cells following increased NaCl or sucrose stress to monitor
high salinity and hyperosmotic stress, respectively. Overlapping effects on gene
transcription in response to NaCl or sucrose were observed, and a large number
of genes were differentially expressed. As expected, genes for trehalose synthesis
were upregulated. Although many of the genes whose expression levels changed
were unknown, interestingly, a large number of genes on pSymB, one of the two
S. meliloti megaplasmids, was differentially expressed following osmotic stress.
In some case, as for ribosomal proteins and ancillary functions, cognate genes
were repressed by both stresses. Genes encoding proteins important for the
production of the low molecular weight form of succinoglycan, e.g., exoHK, were
induced, and many genes known to be involved in stress-responsiveness were also
induced. As expected, genes encoding proteins for central metabolism and carbon
uptake were downregulated, whereas genes involved in glycogen metabolism were
HOW RHIZOBIA SURVIVE IN THE ABSENCE OF A LEGUME HOST
upregulated. Numerous genes related to chemotaxis and cell motility were
downregulated in response to stress. Similarly, many genes known to be induced
by oxygen, nitrogen, or carbon starvation, as well as other stresses, were upregu-
lated in S. meliloti following increased salt or osmotic stress (Domínguez-Fererras
et al., 2006). Furthermore, rhizobial growth in response to salt stress was found
to require pSymB, and thus, these authors concluded that this large plasmid is
essential for S. meliloti’s saprophytic competence.
Cytryn et al. (2007) also undertook a genome-wide transcriptional analysis,
but this time to analyze B. japonicum’s response to drought. In addition to a large
category of hypothetical proteins, genes encoding proteins involved in trehalose
synthesis were highly upregulated. Other genes were also upregulated, including
many important genes for transcriptional regulation such as rpoN2, DNA repair
and cell cycle regulation, cation uptake and heat shock, pili assembly proteins and
ﬂagellin, transport of sucrose and other molecules, succinylation of osmoregu-
lated periplasmic glucans, energy transfer, and various aspects of metabolism
(Cytryn et al., 2007). The upregulation of genes encoding ﬂagellin differs from the
results of Domínguez-Fererras et al. (2006) where S. meliloti ﬂagellar biosynthesis
genes were downregulated after osmotic upshift. However, like S. meliloti, a
number of genes for succinoglycan (EPSI) biosynthesis, including exoP (suc-
cinoglycan biosynthesis), exoM (UDP-hexose transferase), and exoN (UTP-G1P
uridylyltransferase) were also strongly upregulated upon the induction of drought
stress. A putative LPS synthesis transferase was upregulated in drought-stressed
B. japonicum (Cytryn et al., 2007).
Interestingly, the transcriptional analysis of the genome of B. japonicum
subjected to desiccation stress indicated that genes critical for pilus assembly, e.g.,
pilA, pilA2, and ctpA, are upregulated (Cytryn et al., 2007). Pili, especially type
IV pili, are often important for bioﬁlm formation (Shime-Hattori et al., 2006;
Jurcisek and Bakaletz, 2007). It is extremely likely that desiccation-stressed B.
japonicum cells show some of the same patterns of gene expression, as do cells in
bioﬁlms. Similarly, S. meliloti cells grown under salt and osmotic stress
(Domínguez-Fererras et al., 2006) upregulate some of the same genes uncovered
in transcriptome arrays of bioﬁlm cells of other bacteria (An and Parsek, 2007).
Bioﬁlms are one of the many ways that bacteria use to protect themselves
from desiccation and it is well known that EPS, an important component of
the bioﬁlm matrix, protects bacteria from drought stress. Loss-of-function EPS
mutants of many bacteria show impaired bioﬁlm formation (Yildiz and
Schoolnik, 1999; Danese et al., 2000; Whiteley et al., 2001; Matsukawa and
Greenberg, 2004), as do S. meliloti exoY loss-of-function mutant cells
(Fujishige et al., 2006) and EPS mutants of M. tianshanense (Wang et al.,
2008). Nevertheless, it is not known whether EPS-deﬁcient mutants that are
incapable of bioﬁlm formation are less capable of surviving desiccation stress
under ﬁeld conditions.
Several studies have measured oxygen levels in bioﬁlms using microelec-
trodes (as well as other gases) (see Stewart and Franklin, 2008). Bioﬁlms often
ANN M. HIRSCH
exhibit an oxygen gradient with the lowest levels at the center of the bioﬁlm
(Fig. 1). The gradient, however, may not necessarily be established by the pres-
ence of a polysaccharide matrix. Instead, the physiological heterogeneity of the
bioﬁlm, such that actively respiring cells on the outside reduce the concentration of
oxygen on the inside, may be key. For rhizobia, these types of studies have not yet
been performed. Our preliminary results on a single species bioﬁlm of S. meliloti
carrying gusA fusions to promoters of genes expressed in reduced oxygen levels
demonstrated that the expression of several of these promoters was induced in
the bioﬁlm (V. Tovar, P.L. De Hoff, and A.M. Hirsch, 2008, unpublished results).
Moreover, bioﬁlms are often metbolically diverse because they are com-
posed of multiple species. Thus, rhizobia under natural conditions, as members
of mixed bioﬁlms, may tolerate or survive stress through the efforts of their
Nutrient availability, pH, and concentration of solutes show similar
gradients in bioﬁlms as do oxygen and other gases (Stewart and Franklin, 2008).
Rhizobial bioﬁlms are likely to show the same gradients, but so far this has not
been investigated. S. meliloti is a salt-tolerant species, tolerant to salt
concentrations ranging from 0.3 to 0.7 M (Zahran, 1999). However, bioﬁlm for-
mation in strain Rm1021 was signiﬁcantly decreased at 0.15 M NaCl, a concen-
tration that had no effect on growth (Rinaudi et al., 2006). This suggests that the
higher concentrations of NaCl may be toxic due to the accumulation of charged
ions (Ejlsheikh and Wood, 1989), eliciting either DNA or membrane damage
that precludes bioﬁlm formation. S. meliloti Rm1021 growth in the presence of
0.3 and 0.6 M sorbitol was adversely affected, but bioﬁlm formation was again
more sensitive, showing a decrease at 0.06 M. Although the explanation for this
sensitivity is unknown, it may be that older bioﬁlms, which establish profound
physiological gradients, become more osmotically tolerant.
Figure 1. Diagram of a mature bioﬁlm showing water channels and towers composed of cells of
different developmental states: yellow, cells with intact membranes; speckled, nutrient-deprived cells;
pink, oxygen-deﬁcient cells.
HOW RHIZOBIA SURVIVE IN THE ABSENCE OF A LEGUME HOST
2.2. SOIL pH
Many agricultural soils are either so alkaline or acidic as to hinder rhizobial
growth and subsequent establishment of a viable nitrogen-ﬁxing symbiosis with a
legume host. Even if a legume–rhizobia association is established, the rhizobia may
not persist in alkaline or acidic soils once the legume crop is harvested. Soils may
exhibit different pHs because of the accumulation of ions, particularly Ca
it difﬁcult to distinguish negative growth effects brought about by Ca
(Davet, 2004). Indeed, soil pH is often adjusted by the addition of limestone.
Fungi are thought to be more common in acid soils, whereas bacteria pro-
liferate in neutral or alkaline soils (Davet, 2004). However, some rhizobia are
quite tolerant to acidity. B. japonicum can survive in acid soils down to a pH of
4.0, whereas R. leguminosarum bv. trifolii and bv. viciae cannot grow in soils below
pH 4.7; the lower limit for most S. meliloti strains is pH 5.0 (Hirsch, 1996). Low
soil pH can also result in increased solubility of certain metal ions, e.g., alumi-
num, copper, and zinc, to the point where toxic levels are reached. In contrast,
alkaline soil conditions can lead to deﬁciencies as the metals become increasingly
unavailable. A number of studies have focused on the effects of heavy metals on
rhizobia and the mechanisms of excluding or taking up of metal ions, but these
will not be described here.
Rhizobia employ various mechanisms for maintaining intracellular pH
including (i) decreased membrane permeability, (ii) internal buffering, (iii) amelioration
of external pH, (iv) proton extrusion/uptake, and (v) prevention of metal ion
toxicity (Dilworth and Glenn, 1999). But ﬁrst, a change in pH must be sensed.
One likely pH sensor in S. meliloti and S. medicae is the two component regula-
tory system, ActS/ActR (Tiwari et al., 1996). These genes, which are homologs of
RegBA and PrrBA, are essential for growth in acidic pH. They regulate a number
of genes representing a broad range of metabolic process under low pH condi-
tions including carbon ﬁxation and nitrogen assimilation (Fenner et al., 2004).
Approximately 20–50 genes are predicted to be involved in acid tolerance (act),
and some 15–20 of them may be essential (Glenn et al., 1999). Included among
these are actA, actP, actR/S, exoH, and exoR. However, the mild acid-sensitive
phenotype of an exoR mutant may be indirect because the extra EPS produced
by the mutant may impose a growth defect that is particularly noticeable in cells
grown under acidic conditions. The mutants grow after prolonged incubation on
acidic medium (W.G. Reeve, 2008, personal communication).
Not as many genes have been found that are induced under alkaline condi-
tions in part because fewer studies have been made of alkaline tolerance. An
S. meliloti mutant originally described as Fix
was found to be sensitive to K
able to survive at alkaline pH in the presence of this cation (Putnoky et al., 1998).
The mutation was determined to be in the pha operon, which includes genes that
encode proteins resembling subunits of a Na
transporting system. A recent
investigation showed that tfxG, one of the genes that encodes trifolitoxin, is critical
for S. meliloti to tolerate alkaline growth conditions (Tang et al., 2007). This gene
ANN M. HIRSCH
is upregulated as the pH is changed from 7.0 to 10.0, and mutants show growth
impairment at pH values greater than 9.0.
A strategy to select for acid-tolerant rhizobia among natural genetic
populations has led to the identiﬁcation of a number of strains that grow under
these difﬁcult soil conditions. R. tropici CIAT899, which nodulates common bean
(Phaseolus vulgaris L.), is well adapted to nodulate its host, which grows in highly
acidic soils. Under laboratory conditions, R. tropici CIAT899 grows at a pH as
low as 4.0 (Martinez-Romero et al., 1991; Graham et al., 1994), but under natural
conditions, the cells survive better at pH values over 4.5 (Graham et al., 1982).
Tn5 mutagenesis identiﬁed loci that are involved in acid tolerance in this species.
The mutants were acid sensitive and also incapable of forming nitrogen-ﬁxing
nodules (Vinuesa et al., 2003). One of the mutations was in a gene orthologous to
the avcB gene of Agrobacterium tumefaciens and was renamed atvA by the
authors for acid tolerance and virulence. This gene is induced by acid shock
(Vinuesa et al., 2003). The ﬁrst gene in a putative operon, lpiA, which encodes an
integral membrane protein with 13 transmembrane helices, is also reported to be
Strains of S. meliloti are among the most acid-sensitive rhizobia. They
generally do not grow below pH 5.5 in laboratory media. In contrast, S. medicae
(formerly S. meliloti) WSM419 is one of the more acid-tolerant sinorhizobia
(Goss et al., 1990; Glenn and Dilworth, 1994). Several genes required for the
growth of this strain at low pH have been identiﬁed. Both transcriptional and
proteomic analyses were performed to deduce expression pattern differences
between S. medicae WSM419 grown under neutral and low pH (pH 5.7) condi-
tions (Tiwari et al., 2004; Reeve et al., 2004). Short-term (30 min) exposure to low
pH had only a minor effect on protein expression, whereas long-term exposure
(5 days) resulted in more than 50 proteins showing changes in expression levels
(Reeve et al., 2004). Among these were GroES and DegP, which were upregulated,
and an ATP-binding cassette (ABC) transporter as well as several hypothetical
proteins that were downregulated. Transcriptional analysis identiﬁed phrR, which
encodes a putative repressor; lpiA, a putative membrane protein; kdpBC, a potas-
sium importing ATPase; putative ABC transporters, and several hypothetical
proteins, among others (Tiwari et al., 2004). In S. medicae WSM419, lpiA was
found to be induced 20-fold in low pH conditions relative to that seen at pH 7.0
(Reeve et al., 2006). Although no effect of a mutation in lpiA was observed in cells
grown at pH 5.7, S. medicae lpiA mutant viability was strongly reduced at pH 4.5.
In contrast, a signiﬁcant number of wild-type S. medicae WSM419 cells remained
viable at this low pH. Recent evidence suggests that lpiA expression results in the
synthesis of a lipid, lysyl-phosphatidyglycerol (LPG), in the membranes of R. tropici
CIAT899 when grown at pH 4.5 in minimal medium (Sohlenkamp et al., 2007).
The latter researchers found that mutants with defective lpiA are less likely to
survive at low pH than wild-type rhizobia after challenge with polymyxin B. The
increased LPG in the membrane may alter its surface charge, thereby enhancing
HOW RHIZOBIA SURVIVE IN THE ABSENCE OF A LEGUME HOST
Little connection between what is known regarding genes important for acid
or alkali tolerance and rhizobial bioﬁlm formation has been made so far. Bioﬁlm
formation in response to acidic conditions has been pursued only for S. meliloti
strain Rm1021, the sequenced strain, which is not as acid tolerant as S. medicae
WSM419 and certain other S. meliloti strains. When Rm1021 cells were examined
after 24 h of growth at pH 4.0–8.0, optimal growth and bioﬁlm establishment were
found to occur at pH 7.0 (Rinaudi et al., 2006). Interestingly, cells grown at pH
7.0 on glass cover slips for 6 days established towers and ridges that were typical
of mature S. meliloti Rm1021 bioﬁlms (Fujishige et al., 2006). They also
ﬂuoresced green after staining with the LIVE/DEAD ﬂuorescent stain, indicating
that the cells were viable (Rinaudi et al., 2006). In contrast, Rm1021 cells grown
at pH 4.0 and subsequently treated with the LIVE/DEAD ﬂuorescent stain were
red, suggesting that the cells were either dead or had leaky membranes. The
presence of actively swimming red-staining cells, however, suggested the latter
(Rinaudi et al., 2006).
pH has been shown to inﬂuence the proﬁle of Nod factors secreted by R. tropici
CIAT899, which is tolerant of acidic conditions (Morón et al., 2005). At least
seven different classes of Nod factor structures were identiﬁed, differing in either
the reducing or nonreducing end substitutions on the core N-acetylglucosamine
oligosaccharide. More than 50 different Nod factors were detected at pH 4.5, and
greater induction of the nod genes also occurred at this pH. This diversity of Nod
factor structure may facilitate nodulation of bean at an acidic pH. An earlier
study by McKay and Djordjevic, 1993) demonstrated that R. leguminosarum bv.
trifolii Nod factor production is inﬂuenced by environmental parameters such as
pH, temperature, and nutrient availability.
2.3. NUTRIENT AVAILABILITY
An early study (Wei and Bauer, 1998) demonstrated that C, N, or P starvation
resulted in a loss of motility and a transient increase in chemotaxis in S. meliloti.
Only a subset of cells lost ﬂagella in response to starvation, however. As discussed
in the previous section, genes involved in chemotaxis and motility were down-
regulated in S. meliloti after osmotically induced stress (Domínguez-Fererras
et al., 2006). They were also downregulated in response to phosphate limita-
tion in S. meliloti (Krol and Becker, 2004). Phosphate limitation also resulted
in the upregulation of a number of genes, including the pta-ackA genes coding
for phosphotransacetylase and acetate kinase activity, respectively, heat shock
gene dnaK, and several others (Summers et al., 1999) including those involved
in exopolysaccharide synthesis, both succinoglycan (EPS I) and galactoglycan
(EPS II) (Mendrygal and Gonzalez, 2000; Krol and Becker, 2004). Recently, a
gene for an inducible catalase, katA, found to be upregulated in S. meliloti follow-
ing phosphate stress (Krol and Becker, 2004), is transcribed from a PhoB pro-
moter rather than from an OxyR-dependent promoter as are other catalase genes
ANN M. HIRSCH
(Yuan et al., 2005). A global expression proﬁle of phosphate-limited S. meliloti
cells earlier showed that a number of genes involved in protection against oxida-
tive stress are also induced (Krol and Becker, 2004).
Some recent data implicate an oligopeptide ABC transporter (Opt) in both
stress resistance and symbiosis in R. etli (Vos et al., 2007). Rhizobia mutated in
various genes of the opt operon establish nodules that ﬁxed only about 50% of
the nitrogen levels (monitored by acetylene reduction assays) of controls (Vos
et al., 2007). When challenged with antibiotics or when grown under hyperos-
motic stress, the opt mutant cells grew more slowly than the wild-type controls
did, and were also more susceptible to certain antibiotics such as ampicillin, but
were resistant to others, namely bacitracin. These effects may be a consequence
of the lack of uptake of required peptides, for example, glycine- and proline-
containing peptides, which may protect the cells from osmotic stress. The symbi-
otic defect could potentially be a result of a nutritional defect. The nodules are
infected normally and the bacteroids are surrounded by peribacteroid membrane
(Vos et al., 2007), suggesting that these stages of nodule development are intact.
More studies are needed.
Nutrient availability modulates the depth and structure of many bacterial
bioﬁlms (Stanley and Lazazzera, 2004). Bioﬁlms of gram-positive bacteria such
as Bacillus subtilis, which form spores in response to various stress factors, exhibit
upregulation of sigma factors that are indicative of sporulation and nutrient star-
vation (Stanley et al., 2003). Gram-negative bacterial bioﬁlms also exhibit numer-
ous changes in response to nutrient starvation. The center part of a large bioﬁlm
may be nutrient starved and the bioﬁlm may be undergoing maximal dispersal
of cells to seek new nutrient sources (Fig. 1). Several studies have shown that
rhizobia establish bioﬁlms more quickly when grown in minimal media than when
cultivated in rich media (Fujishige et al., 2006; Rinaudi et al., 2006; Russo et al.,
2006), indicating that changes in physiology brought about by nutrient limitation
positively impact bioﬁlm formation. Both N and P limitation resulted in greater
bioﬁlm formation in S. meliloti (Rinaudi et al., 2006).
3. Symbiosis and Stress
Many of the genes described above inﬂuence symbiosis in some way, most likely
indirectly by causing nutrient deprivation or membrane damage. Little is known
about the global regulation networks that modulate stress responses in rhizo-
bia. A few alternative sigma factors, which are responsible for controlling gene
expression in response to stress in a number of other gram-negative bacteria
(Ramos et al., 2001), namely rpoN2 and rpoE2, have been identiﬁed in rhizobia
(Domínguez-Fererras et al., 2006; Sauviac et al., 2007). RpoE2 is described as a
major global regulator of S. meliloti’s general stress responses. At least 44 genes
identiﬁed by a transcriptome analysis, many of which encode proteins known to
be involved in stress responses such as katC and rpoH2, are controlled by RpoE2
HOW RHIZOBIA SURVIVE IN THE ABSENCE OF A LEGUME HOST
(Sauviac et al., 2007). Transcriptome and proteome analyses have provided
information about many of the downstream players in rhizobial stress responses,
but the details of how these genes are regulated is still unknown. For example,
rpoE2 mutants do not differ from wild-type cells in their resistance to various
stresses in either exponential or stationary phases of growth (Sauviac et al., 2007).
A gene of unknown function that appears to be a master switch for both
symbiosis and environmental stress has been described for S. meliloti (Davies and
Walker, 2008). It was uncovered using a two-part screening strategy to ﬁnd
mutants that were sensitive to H
and at the same time, symbiotically defective
with alfalfa. The white, ineffective nodules were found to be completely devoid of
bacteroids, suggesting that the symbiosis is blocked early in development, before
the release of bacteria from infection threads (Davies and Walker, 2008). Based
on sequence analysis, the gene codes for a putative metal-dependent hydrolase,
with homologs present in a wide range of alpha-proteobacteria, many of which
do not nodulate legumes. Interestingly, the mutant is sensitive to a broad range of
environmental pressures, including oxidative stress, agents of DNA damage, and
inhibitors of cell wall synthesis, among others, suggesting that the wild-type
protein plays a central role in S. meliloti’s stress response (Davies and Walker,
2008). Some genes neighboring the opt operon include two that overlap with pH
effects, lnt and phrR. However, the S. meliloti mutant does not show increased
sensitivity to acid pH, arguing that the stress effect is independent of pH.
4. Future Directions
One would predict that many of the genes described above as being upregulated
by stress are likely to be expressed in bioﬁlms. Based on studies in other bacteria,
genome-wide transcriptional analyses show that this is the case (Whiteley et al.,
2001). However, transcriptome and proteome analyses are fraught with difﬁculties
due to the nonstandard conditions used to study bioﬁlms and because the cells of
the bioﬁlm are highly heterogeneous (An and Parsek, 2007; Stewart and Franklin,
2008). Nevertheless, the various genes identiﬁed from the studies already accom-
plished could provide a tremendous opportunity to learn more about their expres-
sion in rhizobial bioﬁlms. For example, ﬂuorescent gene reporters that respond
to pH or other stress-elicited cues could be followed in ﬂow cells or by epiﬂuores-
cence or confocal microscopy. For this, better reporter genes need to be designed;
for example, green ﬂuorescent protein (GFP) and its derivatives require oxygen
for expression, and as described earlier, many centrally located bioﬁlm cells are
oxygen starved. Perhaps reporter genes that are expressed under anaerobic condi-
tions could be developed to circumvent this difﬁculty. Viability stains such as the
LIVE/DEAD stain are also useful, but can give false-negative results. Details of
methods that could be used are described fully in Stewart and Franklin (2008).
Another possibility is to develop in situ RNA or protein localization methods,
similar to those used in eukaryotic tissue systems. This would require fast freezing of
ANN M. HIRSCH
the bioﬁlm, cryosectioning, and then detecting transcripts with ﬂuorescently labeled
probes. The disadvantage of this method is that it is an end-point protocol, resulting
in the termination of the bioﬁlm. Technological advances are needed to integrate the
knowledge gained from the investigations of planktonic cells to stress and rhizobial
bioﬁlms with the hopes of extrapolating this knowledge to the ﬁeld situation.
The author is grateful to Michael J. Sadowsky (University of Minnesota, USA) and
Wayne G. Reeve (University of Murdoch, Australia) for their helpful comments
on the manuscript. The research in the P.I.’s laboratory is funded by grants from
the National Science Foundation (EF-0626896 and IOS-0747516).
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