scholarly journals Adaptation of Mycobacterium smegmatis to Stationary Phase

1999 ◽  
Vol 181 (1) ◽  
pp. 270-283 ◽  
Author(s):  
Marjan J. Smeulders ◽  
Jacquie Keer ◽  
Richard A. Speight ◽  
Huw D. Williams
Keyword(s):  
Cell Division ◽  
Stationary Phase ◽  
Mycobacterium Smegmatis ◽  
Acid Stress ◽  
Exponential Phase ◽  
Quiescent State ◽  
Dependent Manner ◽  
Wild Type ◽  
Glycerol Uptake ◽  
Dynamic Population

ABSTRACT Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.

scholarly journals Mycothiol Import by Mycobacterium smegmatis and Function as a Resource for Metabolic Precursors and Energy Production

2007 ◽  
Vol 189 (19) ◽  
pp. 6796-6805 ◽  
Author(s):  
Krzysztof P. Bzymek ◽  
Gerald L. Newton ◽  
Philong Ta ◽  
Robert C. Fahey
Keyword(s):  
Stationary Phase ◽  
Mycobacterium Smegmatis ◽  
Type Strain ◽  
Energy Production ◽  
Wild Type Strain ◽  
Krebs Cycle ◽  
Wild Type ◽  
Suitable Target ◽  
Cysteine Desulfhydrase ◽  
And Function

ABSTRACT Mycothiol ([MSH] AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by Mycobacterium smegmatis and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn1) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U-14C]cysteine or with [6-3H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the myo-inositol can serve as a sole carbon source for growth of M. smegmatis, indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be ∼50 h in strains with active MshC and 16 ± 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis.

Formation of ‘non-culturable’ cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1687-1697 ◽  
Author(s):  
Margarita Shleeva ◽  
Galina V. Mukamolova ◽  
Michael Young ◽  
Huw D. Williams ◽  
Arseny S. Kaprelyants
Keyword(s):  
Stationary Phase ◽  
Mycobacterium Smegmatis ◽  
Bacterial Culture ◽  
Simultaneous Action ◽  
Wild Type ◽  
Low Oxygen ◽  
Viable Cells ◽  
Transient Loss ◽  
Isogenic Strains ◽  
Endogenous Synthesis

Conditions were investigated that promote the formation of ‘non-culturable’ (NC) cells of Mycobacterium (Myc.) smegmatis in stationary phase. After cultivation in a rich medium, or under conditions that may be considered optimal for bacterial growth, or starvation for carbon, nitrogen or phosphorus, bacteria failed to enter a NC state. However, when grown under suboptimal conditions, resulting in a reduced growth rate or maximal cell concentration (e.g. in modified Hartman's–de Bont medium), bacteria adopted a stable NC state after 3–4 days incubation in stationary phase. Such conditions are not specific as purF and devR mutants of Myc. smegmatis also showed (transient) loss of culturability following growth to stationary phase in an optimized medium, but under oxygen-limited conditions. The behaviour of the same mutants in oxygen-sufficient but nutrient-inappropriate medium (modified Hartman's–de Bont medium) was similar to that of the wild-type (adoption of a stable NC state). It is hypothesized that adoption of a NC state may represent an adaptive response of the bacteria, grown under conditions when their metabolism is significantly compromised due to the simultaneous action of several factors, such as usage of inappropriate nutrients or low oxygen availability or impairment of a particular metabolic pathway. NC cells of wild-type Myc. smegmatis resume growth when transferred to a suitable resuscitation medium. Significantly, resuscitation was observed when either recombinant Rpf protein or supernatant derived from a growing bacterial culture was incorporated into the resuscitation medium. Moreover, co-culture with Micrococcus (Mcc.) luteus cells (producing and secreting Rpf) also permitted resuscitation. Isogenic strains of Myc. smegmatis harbouring plasmids containing the Mcc. luteus rpf gene also adopt a similar NC state after growth to stationary phase in modified Hartman's–de Bont medium. However, in contrast to the behaviour noted above, these strains resuscitated spontaneously when transferred to the resuscitation medium, presumably because they are able to resume endogenous synthesis of Mcc. luteus Rpf. Resuscitation was not observed in the control strain harbouring a plasmid lacking Mcc. luteus rpf. In contrast to wild-type, the NC cells of purF and devR mutants obtained under oxygen-limited conditions resuscitate spontaneously, presumably because the heterogeneous population contains some residual viable cells that continue to make Rpf-like proteins.

σ B-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3843-3855 ◽  
Author(s):  
David Sue ◽  
Daniel Fink ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Response ◽  
Virulence Genes ◽  
Acid Stress ◽  
Exponential Phase ◽  
Wild Type ◽  
Transcript Accumulation ◽  
Gastrointestinal Infections ◽  
Stress Response Genes ◽  
Foodborne Infections

Listeria monocytogenes must overcome a variety of stress conditions in the host digestive tract to cause foodborne infections. The alternative sigma factor σ B, encoded by sigB, is responsible for regulating transcription of several L. monocytogenes virulence and stress-response genes, including genes that contribute to establishment of gastrointestinal infections. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the virulence genes inlA and bsh, the stress-response genes opuCA and lmo0669 (encoding a carnitine transporter and an oxidoreductase, respectively) and the housekeeping gene rpoB. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting osmotic (0·3 M NaCl) or acid (pH 4·5) conditions typical for the human intestinal lumen. In exponential-phase cells, as well as under osmotic and acid stress, inlA, opuCA and bsh showed significantly lower absolute expression levels in a L. monocytogenes ΔsigB null mutant compared to wild-type. A statistical model that normalized target gene expression relative to rpoB showed that accumulation of inlA, opuCA and bsh transcripts was significantly increased in the wild-type strain within 5 min of acid and osmotic stress exposure; lmo0669 transcript accumulation increased significantly only after acid exposure. It was concluded that σ B is essential for rapid induction of the tested stress-response and virulence genes under conditions typically encountered during gastrointestinal passage. As inlA, bsh and opuCA are critical for gastrointestinal infections in animal models, the data also suggest that σ B contributes to the ability of L. monocytogenes to cause foodborne infections.

scholarly journals Physiological Effects of Crl in Salmonella Are Modulated by σS Level and Promoter Specificity

2007 ◽  
Vol 189 (8) ◽  
pp. 2976-2987 ◽  
Author(s):  
Véronique Robbe-Saule ◽  
Miguel Dias Lopes ◽  
Annie Kolb ◽  
Françoise Norel
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Regulatory Protein ◽  
Acid Stress ◽  
Physiological Effects ◽  
Wild Type ◽  
Serovar Typhimurium

ABSTRACT The small regulatory protein Crl activates σS (RpoS), the stationary-phase and general stress response sigma factor. Crl has been reported to bind σS in vitro and to facilitate the formation of RNA polymerase holoenzyme. In Salmonella enterica serovar Typhimurium, Crl is required for the development of the rdar morphotype and transcription initiation of the σS-dependent genes csgD and adrA, involved in curli and cellulose production. Here, we examined the expression of other σS-dependent phenotypes and genes in a Δcrl mutant of Salmonella. Gene fusion analyses and in vitro transcription assays indicate that the magnitude of Crl activation differs between promoters and is highly dependent on σS levels. We replaced the wild-type rpoS allele in S. enterica serovar Typhimurium strain ATCC 14028 with the rpoS LT2 allele that shows reduced expression of σS; the result was an increased Crl activation ratio and larger physiological effects of Crl on oxidative, thermal, and acid stress resistance levels during stationary phase. We also found that crl, rpoS, and crl rpoS strains grew better on succinate than did the wild type and expressed the succinate dehydrogenase sdhCDBA operon more strongly. The crl and rpoS LT2 mutations also increased the competitive fitness of Salmonella in stationary phase. These results show that Crl contributes to negative regulation by σS, a finding consistent with a role for Crl in sigma factor competition via the facilitation of σS binding to core RNA polymerase.

scholarly journals Chromosome loss from par mutants of Pseudomonas putida depends on growth medium and phase of growth

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 537-548 ◽  
Author(s):  
Richard A Lewis ◽  
Colin R Bignell ◽  
Wei Zeng ◽  
Anthony C Jones ◽  
Christopher M Thomas
Keyword(s):  
Cell Division ◽  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Chromosome Segregation ◽  
Copy Number ◽  
Minimal Medium ◽  
Exponential Phase ◽  
Chromosome Loss ◽  
Chromosome Partitioning ◽  
Mutant Phenotypes

The proteins encoded by chromosomal homologues of the parA and parB genes of many bacterial plasmids have been implicated in chromosome partitioning. Unlike their plasmid counterparts, mutant phenotypes produced by deleting these genes have so far been elusive or weakly expressed, except during sporulation. Here the properties of Pseudomonas putida strains with mutations in parA and parB are described. These mutants do not give rise to elevated levels of anucleate bacteria when grown in rich medium under standard conditions. However, in M9-minimal medium different parA and parB mutations gave between 5 and 10% anucleate cells during the transition from exponential phase to stationary phase. Comparison of the DNA content of bacteria at different stages of the growth curve, in batch culture in L-broth and in M9-minimal medium, suggests that the par genes are particularly important for chromosome partitioning when cell division reduces the chromosome copy number per cell from two to one. This transition occurs in P. putida during the entry into stationary phase in M9-minimal medium, but not in L-broth. It is proposed that the partition apparatus is important to ensure proper chromosome segregation primarily when the bacteria are undergoing cell division in the absence of ongoing DNA replication.

scholarly journals Growth-phase dependence of susceptibility to antimicrobial peptides in Staphylococcus aureus

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1786-1797 ◽  
Author(s):  
Miki Matsuo ◽  
Yuichi Oogai ◽  
Fuminori Kato ◽  
Motoyuki Sugai ◽  
Hitoshi Komatsuzawa
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Antimicrobial Peptides ◽  
Stationary Phase ◽  
Surface Charge ◽  
Growth Phase ◽  
Exponential Phase ◽  
Dependent Manner ◽  
Phase Dependence ◽  
Cell Surface Charge

Bacterial cell surface charge is responsible for susceptibility to cationic antimicrobial peptides. Previously, Staphylococcus aureus dlt and mprF were identified as factors conferring a positive charge upon cell surfaces. In this study, we investigated the regulation of cell surface charge during growth. Using a group of S. aureus MW2 mutants, which are gene-inactivated in 15 types of two-component systems (TCSs), we tested dltC and mprF expression and found that two TCSs, aps and agr, were associated with dltC and mprF expression in a growth phase-dependent manner. The first of these, aps, which had already been identified as a sensor of antimicrobial peptides and a positive regulator of dlt and mprF expression, was expressed strongly in the exponential phase, while its expression was significantly suppressed by agr in the stationary phase, resulting in higher expression of dltC and mprF in the exponential phase and lower expression in the stationary phase. Since both types of expression affected the cell surface charge, the susceptibility to antimicrobial peptides and cationic antibiotics was changed during growth. Furthermore, we found that the ability to sense antimicrobial peptides only functioned in the exponential phase. These results suggest that cell surface charge is tightly regulated during growth in S. aureus.

Characterization of Low Salinity Stress in Vibrio parahaemolyticus

2012 ◽  
Vol 75 (2) ◽  
pp. 231-237 ◽  
Author(s):  
WEI SHEN HUANG ◽  
HIN-CHUNG WONG
Keyword(s):  
Acetic Acid ◽  
Lactic Acid ◽  
Stationary Phase ◽  
Salinity Stress ◽  
Vibrio Parahaemolyticus ◽  
Acid Stress ◽  
Low Ph ◽  
Exponential Phase ◽  
Low Salinity ◽  
Acetic Acid Treatment

Vibrio parahaemolyticus is a marine foodborne pathogenic bacterium commonly found in seawater or seafood. This bacterium often encounters low salinity stress when the contaminated seafood is washed with fresh water during food processing. This study was conducted to investigate the response of exponential- and stationary-phase cells of V. parahaemolyticus ST550 to lethal or sublethal low salinity. Tolerance to lethal low salinity (0.25% NaCl) was enhanced in V. parahaemolyticus cells in the exponential phase by previous adaptation in sublethal low salinity (0.6% NaCl). Low salinity–adapted cells in the exponential phase were also cross-protected against the challenge of lethal low pH, indifferent to heat, and sensitized to bile, acetic acid, and lactic acid stress. The adapted cells in the stationary phase were significantly protected against heat treatment at 44°C for 10 and 15 min, sensitized to bile and acetic acid treatment, and indifferent to low pH and lactic acid.

scholarly journals The impairment of superoxide dismutase coordinates the derepression of the PerR regulon in the response of Staphylococcus aureus to HOCl stress

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 855-861 ◽  
Author(s):  
Sami Maalej ◽  
Ines Dammak ◽  
Sam Dukan
Keyword(s):  
Staphylococcus Aureus ◽  
Superoxide Dismutase ◽  
Hypochlorous Acid ◽  
Antioxidant Properties ◽  
Exponential Phase ◽  
Dependent Manner ◽  
Wild Type ◽  
Sod Activity ◽  
Striking Result ◽  
Phosphoglycerate Dehydrogenase

The response of Staphylococcus aureus to hypochlorous acid (HOCl) exposure was investigated. HOCl challenges were performed on cultures interrupted in the exponential phase. Pretreatment with HOCl conferred resistance to hydrogen peroxide in a PerR-dependent manner. Derepression of the PerR regulon was observed at low HOCl concentration (survival >50 %), using several fusions of different stress promoters to lacZ reporter genes. At least four members of the PerR regulon (katA, mrgA, bcp and trxA) encoding proteins with antioxidant properties were strongly induced following exposure to various HOCl concentrations. A striking result was the link between the derepression of the PerR regulon and the decreased superoxide dismutase (SOD) activity following exposure to increased HOCl concentrations. The sodA mutant was more resistant than the wild-type and also had a higher level of 3-phosphoglycerate dehydrogenase (a measure of PerR regulon activity) without exposure to HOCl. Together, these results imply that derepression of PerR by HOCl is dependent on the level of SOD and protects exponentially arrested cells against HOCl stress.

scholarly journals A Two-Component Regulator of Universal Stress Protein Expression and Adaptation to Oxygen Starvation in Mycobacterium smegmatis

2003 ◽  
Vol 185 (5) ◽  
pp. 1543-1554 ◽  
Author(s):  
Ronan O'Toole ◽  
Marjan J. Smeulders ◽  
Marian C. Blokpoel ◽  
Emily J. Kay ◽  
Kathryn Lougheed ◽  
...  
Keyword(s):  
Heat Stress ◽  
Protein Expression ◽  
Stationary Phase ◽  
Mycobacterium Smegmatis ◽  
Stress Proteins ◽  
Response Regulator ◽  
Stress Protein ◽  
Wild Type ◽  
Two Component ◽  
Oxygen Starvation

ABSTRACT We identified a response regulator in Mycobacterium smegmatis which plays an important role in adaptation to oxygen-starved stationary phase. The regulator exhibits strong sequence similarity to DevR/Rv3133c of M. tuberculosis. The structural gene is present on a multigene locus, which also encodes a sensor kinase. A devR mutant of M. smegmatis was adept at surviving growth arrest initiated by either carbon or nitrogen starvation. However, its culturability decreased several orders of magnitude below that of the wild type under oxygen-starved stationary-phase conditions. Two-dimensional gel analysis revealed that a number of oxygen starvation-inducible proteins were not expressed in the devR mutant. Three of these proteins are universal stress proteins, one of which is encoded directly upstream of devR. Another protein closely resembles a proposed nitroreductase, while a fifth protein corresponds to the α-crystallin (HspX) orthologue of M. smegmatis. None of the three universal stress proteins or nitroreductase, and a considerably lower amount of HspX was detected in carbon-starved wild-type cultures. A fusion of the hspX promoter to gfp demonstrated that DevR directs gene expression when M. smegmatis enters stationary phase brought about, in particular, by oxygen starvation. To our knowledge, this is the first time a role for a two-component response regulator in the control of universal stress protein expression has been shown. Notably, the devR mutant was 104-fold more sensitive than wild type to heat stress. We conclude that DevR is a stationary-phase regulator required for adaptation to oxygen starvation and resistance to heat stress in M. smegmatis.

σ B-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3247-3256 ◽  
Author(s):  
David Sue ◽  
Kathryn J. Boor ◽  
Martin Wiedmann
Keyword(s):  
Listeria Monocytogenes ◽  
Stationary Phase ◽  
Bile Salt ◽  
Expression Patterns ◽  
Exponential Phase ◽  
Compatible Solute ◽  
Bile Salt Hydrolase ◽  
Wild Type ◽  
Rt Pcr ◽  
Solute Transporter

Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor σ B contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific σ B-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative σ B-dependent genes based on the presence of a predicted σ B-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm σ B-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes ΔsigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes ΔsigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0·5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of σ B activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA–gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0·5 M NaCl or 0·5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific σ B-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.

Sign in / Sign up

Export Citation Format

Share Document