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Cellulolytic bacteria employ a variety of different strategies for the degradation of cellulose. Our laboratory is interested in biofilm-mediated breakdown of cellulose and possible roles of exopolysaccharides in this process. As part of an effort to isolate new strains of biofilm-forming, cellulolytic bacteria, soil samples were collected from sites rich in decaying vegetation and used to inoculate a minimal growth medium containing a MOPS/Tris buffer (pH 7.4), a combination of inorganic salts, 0.05% yeast extract, and 1% microcrystalline cellulose (MCC) as the primary source of carbon and energy (MSYE-MCC media). These cellulose-enrichment cultures were grown aerobically at 30 C for 2 weeks in a shaking incubator, and culture samples were then spread over the surface of tryptic soy agar (TSA) plates. Colonies on TSA were streaked onto agar plates containing the MSYE basal medium plus either 1% carboxymethyl-cellulose (MSYE-CMC media) or 1% filter paper cellulose (MSYE-FPC). Over one dozen new, cellulose-degrading strains were isolated and initially characterized according to colony morphologies, relative cell size, cellular arrangements, Gram stain reaction, and other outstanding features. Approximately one quarter of the new strains were Gram-negative, and large inclusion bodies were noted in some. The remainder of the strains were either Gram-positive or Gram-variable. Many Gram-positive strains were bacilli arranged in chains (streptobacilli) and one strain exhibited filamentous growth. Phase-contrast microscopy was used to observe cultures grown in MSYE-MCC media, and strains which attached to MCC particles, aggregated on and around the particles, and grew as biofilms on the surfaces of the particles were selected for further study. These strains were also screened for the production of extracellular material as detected by phase-contrast microscopy. A few strains produced possible glycocalyx layers during biofilm formation and utilization of cellulose. These extracellular layers, and any exopolysaccharides associated with them, will be the primary focus of future research., Acknowledgements University of West Georgia Recommended Citation Authors John D. Butler (*), University of West Georgia Follow Emma S. Young (*), University of West Georgia Follow John P. Escate (*), [...]

Carbon-energy source starvation is a commonly encountered stress that can influence the epidemiology and virulence of Salmonella enterica serovars. Salmonella responds to C-starvation by eliciting the starvation-stress response (SSR), which allows for long-term C-starvation survival and cross-resistance to other stresses. The stiC locus was identified as a C-starvation-inducible, [[sigma].sup.s]-dependent locus required for a maximal SSR. We report here that the stiC locus is an operon composed of the yohC (putative transport protein) and pbpG (penicillin-binding protein-7/8) genes, yohC pbpG transcription is initiated from a [[sigma].sup.s]-dependent C-starvation-inducible promoter upstream of yohC. Another ([[sigma].sup.s]-independent) promoter, upstream of pbpG, drives lower constitutive pbpG transcription, primarily during exponential phase. C-starvation-inducible pbpG expression was required for development of the SSR in 5 h, but not 24 h, C-starved cells; yohC was dispensable for the SSR. Furthermore, the yohC pbpG operon is induced within MDCK epithelial cells, but was not essential for oral virulence in BALB/c mice. Thus, PBP 7 is required for physiological changes, occurring within the first few hours of C-starvation, essential for the development of the SSR. Lack of PBP 7, however, can be compensated for by further physiological changes developed in 24 h C-starved cells. This supports the dynamic overlapping and distinct nature of resistance pathways within the Salmonella SSR.

Salmonella enterica serovar Typhimurium (S. Typhimurium) elicits the starvation-stress response (SSR) due to starvation for an essential nutrient, e.g. a carbon/energy source (C-source). As part of the SSR, the alternative sigma factor [[sigma].sup.E] is activated and induced. The authors suspect that this activation is, in part, triggered by changes in the S. Typhimurium cell envelope occurring during the adaptation from growth to carbon/energy starvation (C-starvation), and resulting in an increased need for [[sigma].sup.E]-regulated factors involved in the proper folding and assembly of newly synthesized proteins destined for this extracytoplasmic compartment. This led to the hypothesis that a [[sigma].sup.E] activation signal might arise during C-source shifts that cause the induction of proteins localized to the extracytoplasmic compartment, i.e. the outer membrane or periplasm, of the cell. To test this hypothesis, cultures were grown in minimal medium containing enough glucose to reach mid-exponential-phase, plus a non-limiting amount of a secondary 'less-preferred' but utilizable carbon/energy source. The [[sigma].sup.E] activity was then monitored using plasmids carrying rpoEP1- and rpoEP2-lacZ transcriptional fusions, which exhibit [[sigma].sup.E]-independent and -dependent lacZ expression, respectively. The secondary C-sources maltose, succinate and citrate, which have extracytoplasmic components involved in their utilization (e.g. LamB), resulted in a discernible diauxic lag period and a sustained increase in 0-E activity. Growth transition from glucose to other utilizable phosphotransferase (PTS) and non-PTS C-sources, such as trehalose, mannose, mannitol, fructose, glycerol, D-galactose or L-arabinose, did not cause a discernible diauxic lag period or a sustained increase in [[sigma].sup.E] activity. Interestingly, a shift from glucose to melibiose, which does not use an extracytoplasmic-localized protein for uptake, did cause an observable diauxic lag period but did not result in a sustained increase in [[sigma].sup.E] activity. In addition, overexpression of LamB from an arabinose-inducible promoter leads to a significant increase in GE activity in the absence of a glucose to maltose shift or C-starvation. Furthermore, a [DELTA]lamb::[OMEGA]-[Km.sup.r] mutant, lacking the LamB maltoporin, exhibited an approximately twofold reduction in the sustained [[sigma].sup.E] activity observed during a glucose to maltose shift, again supporting the hypothesis. Interestingly, the LamB protein lacks the typical Y-X-F terminal tripeptide of the OmpC-like peptides that activate DegS protease activity leading to [[sigma].sup.E] activation. It does, however, possess a terminal pentapeptide (Q-M-E-I-W-W) that may function as a ligand for a putative class II PDZ-binding site. The authors therefore propose that the [[sigma].sup.E] regulon of S. Typhimurium not only is induced in response to deleterious environmental conditions, but also plays a role in the adaptation of cells to new growth conditions that necessitate changes in the extracytoplasmic compartment of the cell, which may involve alternative signal recognition and activation pathways that are independent of DegS.

Research indicates that carbon and energy starvation in salmonella Typhimurium triggers induction of rpoE regulon, which produces one or more sigma(sup)E regulon to restore maximal starvation-stress response. Data further suggest that carbon-starvation does not induce Cpx regulon and it is not involved in the long-term carbon-starvation survival.

The article highlights that the stiC locus, identified as a C-starvation-inducible, σlocus required for maximal starvation-stress response (SSR), is an operon composed of the putative transport protein and penicillin-binding protein-7/8 genes. Penicillin-binding protein-7 (PBP 7) is required for physiological changes, occurring within the first few hours of C-starvation, essential for the development of the SSR. PBP 7 is required for the SSR in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) elicits the starvation-stress response (SSR) due to starvation for an essential nutrient, e.g. a carbon/energy source (C-source). As part of the SSR, the alternative sigma factor σE is activated and induced. The authors suspect that this activation is, in part, triggered by changes in the S. Typhimurium cell envelope occurring during the adaptation from growth to carbon/energy starvation (C-starvation), and resulting in an increased need for σE-regulated factors involved in the proper folding and assembly of newly synthesized proteins destined for this extracytoplasmic compartment. This led to the hypothesis that a σE activation signal might arise during C-source shifts that cause the induction of proteins localized to the extracytoplasmic compartment, i.e. the outer membrane or periplasm, of the cell. To test this hypothesis, cultures were grown in minimal medium containing enough glucose to reach mid-exponential-phase, plus a non-limiting amount of a secondary ‘less-preferred’ but utilizable carbon/energy source. The σE activity was then monitored using plasmids carrying rpoEP1- and rpoEP2-tacZ transcriptional fusions, which exhibit σE-independent and -dependent lacZ expression, respectively. The secondary C-sources maltose, succinate and citrate, which have extracytoplasmic components involved in their utilization (e.g. LamB), resulted in a discernible diauxic lag period and a sustained increase in σE activity. Growth transition from glucose to other utilizable phosphotransferase (PTS) and non-PTS C-sources, such as trehalose, mannose, mannitol, fructose, glycerol, D-galactose or L-arabinose, did not cause a discernible diauxic lag period or a sustained increase in σE activity. Interestingly, a shift from glucose to melibiose, which does not use an extracytoplasmic-localized protein for uptake, did cause an observable diauxic lag period but did not result in a sustained increase in σE activity. In addition, overexpression of LamB from an arabinose-inducible promoter leads to a significant increase in σE activity in the absence of a glucose to maltose shift or C-starvation. Furthermore, a Δlamb::Ω-Kmr mutant, lacking the LamB maltoporin, exhibited an approximately twofold reduction in the sustained σE activity observed during a glucose to maltose shift, again supporting the hypothesis. Interestingly, the LamB protein lacks the typical Y-X-F terminal tripeptide of the OmpC-like peptides that activate DegS protease activity leading to σE activation. It does, however, possess a terminal pentapeptide (Q-M-E-I-W-W) that may function as a ligand for a putative class II PDZ-binding site. The authors therefore propose that the σE regulon of S. Typhimurium not only is induced in response to deleterious environmental conditions, but also plays a role in the adaptation of cells to new growth conditions that necessitate changes in the extracytoplasmic compartment of the cell, which may involve alternative signal recognition and activation pathways that are independent of DegS. [ABSTRACT FROM AUTHOR]

The broad host-range pathogen Serratia marcescens survives in diverse host and non-host environments, often enduring conditions in which the concentration of essential nutrients is growth-limiting. In such environments, carbon and energy source starvation (carbon-starvation) is one of the most common forms of stress encountered by S. marcescens. Related members of the family Enterobacteriaceae are known to undergo substantial changes in gene expression and physiology in response to the specific stress of carbon-starvation, enabling non-spore-forming cells to survive periods of prolonged starvation and exposure to other forms of stress (i.e., starvation-induced cross-resistance). To determine if carbon-starvation also results in elevated levels of cross-resistance in S. marcescens, both log-phase and carbon-starved cultures, depleted of glucose before the onset of high cell-density stationary-phase, were grown in minimal media at either 30 °C or 37 °C and were then challenged for resistance to high temperature (50 °C), low pH (pH 2.8), and oxidative stress (15 mM H₂O₂). In general, carbon-starved cells exhibited a higher level of resistance to thermal stress, acid stress, and oxidative stress compared to log-phase cells. The extent of carbon-starvation-induced cross-resistance was dependent on incubation temperature and on the particular strain of S. marcescens. In addition, strain- and temperature-dependent variations in long-term starvation survival were also observed. The enhanced stress-resistance of starved S. marcescens cells could be an important factor in their survival and persistence in many non-host environments and within certain host microenvironments where the availability of carbon sources is suboptimal for growth.