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The distinctive flavours in hard cheeses are attributed largely to the activity of nonstarter lactic acid bacteria (NSLAB) which dominate the cheese matrix during maturation after lactose is consumed. Understanding how different strains of NSLAB survive, compete, and scavenge available nutrients is fundamental to selecting strains as potential adjunct starters which may influence product traits. Three Lacticaseibacillus paracasei isolates which dominated at different stages over 63-week maturation periods of Australian Cheddar cheeses had the same molecular biotype. They shared many phenotypic traits, including salt tolerance, optimum growth temperature, growth on N-acetylglucosamine and N-acetylgalactosamine plus delayed growth on D-ribose, carbon sources likely present in cheese due to bacterial autolysis. However, strains 124 and 163 (later named GCRL163) survived longer at low pH and grew on D-tagatose and D-mannitol, differentiating this phenotype from strain 122. When cultured on growth-limiting lactose (0.2%, wt/vol) in the presence of high concentrations of L-leucine and other amino acids, GCRL163 produced, and subsequently consumed lactate, forming acetic and formic acids, and demonstrated temporal accumulation of intermediates in pyruvate metabolism in long-term cultures. Strain GCRL163 grew in Tween 80-tryptone broths, a trait not shared by all L. casei-group dairy isolates screened in this study. Including citrate in this medium stimulated growth of GCRL163 above citrate alone, suggesting cometabolism of citrate and Tween 80. Proteomic analysis of cytosolic proteins indicated that growth in Tween 80 produced a higher stress state and increased relative abundance of three cell envelope proteinases (CEPs) (including PrtP and Dumpy), amongst over 230 differentially expressed proteins.

Auxotrophic mutants of

In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis.

In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis.

Bacterial cell wall hydrolases, including amidases and peptidases, play a critical role in peptidoglycan turnover during growth, impacting daughter cell separation, and cell death, through autolysis. When exploring the regulation of protein expression across the growth cycle of an acid-resistant strain of Lactobacillus paracasei, GCRL 46, we observed temporal up-regulation of proteins in the 40&ndash, 45 kDa molecular weight range for whole-cell extracts when culturing in fermenters at a controlled pH of 4.0 versus optimum growth pH of 6.3. Up-regulation of proteins in this size range was not detected in SDS-PAGE gels of the cytosolic fraction, but was routinely detected following growth at low pH in whole cells and cell debris obtained after bead beating and centrifugation, indicating a cell surface location. N-terminal sequencing and in silico analyses showed sequence similarity with proteins in the L. casei group (L. casei, L. paracasei and L. rhamnosus) which were variously annotated as uncharacterized proteins, surface antigens, possible TrsG proteins, CHAP (cysteine, histidine-dependent amidohydrolases/peptidases)-domain proteins or putative peptidoglycan d,l-endopeptidase due to the presence of a CwlO domain. This protein is a homologue of the p40 (Msp2) secreted protein of L. rhamnosus LGG, which is linked to probiotic functionality in this species, and is phylogenetically related to structurally-similar proteins found in Enterococcus, Streptococcus and Bifidobacterium species, including the glucan-binding (GbpB), surface antigen (SagA) proteins detected in pathogenic group A streptococci species as secreted, immunoglobulin-binding (SibA) proteins (also named PcsB). Three-dimensional (3D) modelling predicted structural similarities in the CHAP proteins from the L. casei group and streptococcal species, indicating retention of overall architecture despite sequence divergence, and an implied retention of function during evolution. A phylogenetically-related hydrolase also contained the CwlO domain with a NLPC_P60 domain, and showed similar overall but distinct architecture to the CHAP proteins. We concluded that the surface-located, CHAP protein in L. casei is up-regulated during long-term exposure to acidic conditions during growth but not during acid shock.

This study investigated the prevalence, seasonality, and species variety of enterococci present in raw milk factory silos and pasteurized milk in 3 dairying regions in Victoria, Australia, over a 1-yr period. Additionally, the growth ability of thermoduric enterococci isolated in this study (Enterococcus faecalis, E. faecium, E. hirae, and E. durans) was determined in milk at temperatures likely to occur during storage, transport, and distribution, and before domestic consumption (4 and 7°C). Enterococci were detected in 96% of 211 raw milk samples, with an average count of 2.48 log10 cfu/mL. Counts were significantly lower in winter than summer (average 1.84 log10 cfu/mL) and were different between factories but not regions. Enterococcus faecalis was the most prevalent species isolated from raw milk in every factory, comprising between 61.5 and 83.5% of enterococcal species across each season. Enterococci were detected in lower numbers in pasteurized milk than in raw milk and were below the limit of detection on spread plates (10 cfu/mL) after factory pasteurization. Residual viable cells were only detected following enrichment using 100-mL samples of milk, with 20.8% of the samples testing positive; this equated to a decrease in the average raw milk enterococci count of4 log10 cfu/mL following pasteurization. Although E. faecalis predominated in raw milk and E. durans was found in only 2.9% of raw milk samples, E. durans was the most prevalent species detected in pasteurized milk. The detection of enterococci in the pasteurized milk did not correlate with higher enterococci counts in the raw milk. This suggested that the main enterococci populations in raw milk were heat-sensitive and that thermoduric enterococci survived pasteurization in a small numbers of instances. All of the thermoduric enterococci that were assessed for growth at likely refrigeration temperatures were able to grow at both 4 and 7°C in sterile milk, with generation times of 35 to 41h and 16 to 22h, respectively. Thermoduric enterococci were detected in pasteurized milk stored at 4°C for 2 wk (typically 1 to 9 cells/100mL, up to 2.82 log10 cfu/mL), demonstrating the potential of enterococci to survive pasteurization and contribute to milk spoilage at refrigeration temperatures. This is particularly relevant for milk that is aseptically packaged to exclude gram-negative psychrotrophic bacteria and kept above the recommended storage temperature of ≤5°C.

Zinc (Zn) occurs in high concentrations in heavy metal polluted ecosystems which are colonized by bacterial populations. Some members of these communities are attractive candidates for phytoremediation of Zn-contaminated soils as they are able to convert insoluble forms of Zn into soluble forms, making Zn available to plants as a micronutrient. However, mechanisms of bacterial Zn solubilization and homeostasis are often not well understood. In the present study, two Gram positive rhizosphere strains, Bacillus sp. ZM20 and Bacillus subtilis ZM63, which were shown previously to solubilize phosphate and produce indole acetic acid plus siderophores, and two laboratory strains selected as controls (Bacillus cereus and Paenibacillus polymyxa), were tested for their ability to solubilize zinc oxide (ZnO). All of the strains tested could solubilize Zn to some degree, as they produced acids that lowered the medium pH. Two strains, Bacillus sp. ZM20 and B. cereus, were more Zn and acid resistant. When cultured aerobically in a minimum salt medium with glucose as carbon source plus 0.1% (w/v) Zn as ZnO, Bacillus sp. ZM20 and B. cereus both produced significantly higher concentrations of lactic and acetic acids than detected in control broths lacking Zn, with formic, citric, succinic, isobutyric and isovaleric acids detected as minor end-products. In in vitro tests, mixtures and individual acids at concentrations seen in broth cultures were shown to solubilize ZnO. However, the less acid- and Zn-resistant strains showed lower Zn solubility efficiency at higher concentrations of ZnO, as their growth was impaired, and higher concentrations of acids were not induced to the same extent by culture in ZnO. Induction of lactic, acetic, succinic, formic, isobutyric and isovaleric acids during culture correlated with ZnO solubilization, which has not been shown before for Gram positive species.

MiSeq Illumina shotgun sequencing technology was used to sequence two Lactobacillus casei strains, designated strains GCRL 163 and MJA 12. The estimated genome sizes for GCRL 163 and MJA 12 were 2.9 Mb and 3.1 Mb, with 46.35% and 46.31% GC contents, respectively.

MiSeq Illumina shotgun sequencing technology was used to sequence two Lactobacillus casei strains, designated strains GCRL 163 and MJA 12. The estimated genome sizes for GCRL 163 and MJA 12 were 2.9 Mb and 3.1 Mb, with 46.35% and 46.31% GC contents, respectively.

Illumina MiSeq shotgun sequencing technology was used to sequence the genomes of two novel sub-Antarctic Williamsia species, designated strains 1135 and 1138. The estimated genome sizes for strains 1135 and 1138 are 5.99 Mb and 6.08 Mb, respectively. This genome sequence information will aid in understanding the lipid metabolic pathways of cold-tolerant Williamsia species.

Survival in harsh environments is critical to both the industrial performance of lactic acid bacteria (LAB) and their competitiveness in complex microbial ecologies. Among the LAB, members of the Lactobacillus casei group have industrial applications as acid-producing starter cultures for milk fermentations and as specialty cultures for the intensification and acceleration of flavor development in certain bacterial-ripened cheese varieties. They are amongst the most common organisms in the gastrointestinal (GI) tract of humans and other animals, and have the potential to function as probiotics. Whether used in industrial or probiotic applications, environmental stresses will affect the physiological status and properties of cells, including altering their functionality and biochemistry. Understanding the mechanisms of how LAB cope with different environments is of great biotechnological importance, from both a fundamental and applied perspective: hence, interaction between these strains and their environment has gained increased interest in recent years. This paper presents an overview of the important features of stress responses in Lb. casei, and related proteomic or gene expression patterns that may improve their use as starter cultures and probiotics.

The draft genome sequence of subantarctic Rhodococcus sp. strain 1139 is reported here. The genome size is 7.04 Mb with high G+C content (62.3%) and it contains a large number of genes involved in lipid synthesis. This lipid synthesis system is characteristic of oleaginous Actinobacteria, which are of interest for biofuel production.

The draft genome sequence of subantarctic Rhodococcus sp. strain 1139 is reported here. The genome size is 7.04 Mb with high G+C content (62.3%) and it contains a large number of genes involved in lipid synthesis. This lipid synthesis system is characteristic of oleaginous Actinobacteria , which are of interest for biofuel production.

In this research, changes were observed in cell surface proteins of a typical strain of the Lactobacillus casei was investigated in response to acidic growth conditions. Two dimensional electrophoresis and Western blot analyses were carried out to detect changes in relative abundance of proteins at the cell surface. The identity of the differentially expressed proteins extracted by LiCl, a chemical routinely used to extract surface proteins of lactic acid bacteria, was determined by MALDI-TOF/TOF MS. Many enzymes involved in glycolysis were up-regulated in the cell surface fraction following growth at low pH, including enolase, lactate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase. Several of these proteins were also related to adhesion and generalized stress responses. It is demonstrated that growth of L. casei under acidic conditions caused molecular changes at the cell surface to develop an adaptive strategy corresponding to slower growth at low pH.

This study describes the characterisation of G8 rotavirus strains isolated from humans with acute gastroenteritis. Six G8 strains were detected in Australia between 2002 and 2008. Four were G8P[14] strains, one was G8P[8] + [14] and one was G8 P non-typeable. By polyacrylamide gel electrophoresis and enzyme immunoassay analysis, four G8 strains with visible RNA exhibited a long electropherotype and five G8 strains displayed subgroup I specificity. Sequence analysis of the VP7 gene indicated that the G8 strains exhibited the highest nucleotide and amino acid identity with a G8P[11] bovine rotavirus strain detected in Japan. VP4 sequence data of one G8P[14] strain revealed that the closest identity was to another human-bovine-like strain detected in Australia, MG6, a G6P[14] strain. The identification of G8 strains causing disease further extends the number of G8P[14] strains detected in Australian children, and indicates that there is a rare but ongoing presence of uncommon human strains within the community in Australia.

Bovine rotavirus (BRV) has been detected in both dairy and beef cattle herds worldwide. Stool samples collected from calves in the Gippsland region of Victoria, Australia were screened to determine the presence of BRV. A total of 100 faecal samples were collected from calves with and without diarrhoea across three farms during 2004 and 2005. Group A BRV was detected in 26% of faecal samples (22 from diarrheic calves and four from asymptomatic calves). Genotyping analysis of rotavirus positive samples indicated that G6P[5] was the most prevalent genotype (38.5%) followed by G6P[5+11] (15.4%). G10P[11] and G6+G10P[5] were each detected at a rate of 7.7%, and G6+G10P[11] was found in a single sample (3.8%). Seven samples (26.9%) could not be G and/or P typed. Thirty percent of the BRV positive samples were mixed infections, indicating that individual calves were co-infected with more than one strain of rotavirus. The G6P[5] strains exhibited high VP7 identity (>97% amino acid identity) with B-60, a G6 strain identified in Victorian calves during 1988. A G10P[11] isolate was closely related (>97% amino acid identity in VP7 and VP4 proteins) to a Victorian G10P[11] strain (B-11) also identified during 1988. This study demonstrates that BRV is a contributing pathogen to diarrhoeal disease in Victorian calves, with sequence analysis suggesting long-term conservation of the VP7 protein over a 16-year period.

Physiological and metabolic traits of Lactobacillus casei strain GCRL163 were studied in a buffered semi-defined medium supplemented with different concentrations of lactose. The extent of growth (maximum OD 600 reached) was limited by concentrations of ≤1% lactose in this medium. Survival of the strain during extended incubation periods (up to 30 days) in cultures without lactose was found to be better than in cultures supplemented with lactose. Important metabolites detected in lactose-starved (0% lactose) cultures were acetate, as a major end product, as well as ethanol, diacetyl, 3-methyl-1-butanol, benzyl alcohol, 1-butanol, isopropanol, 3-(methylthio)-propanoic acid and other compounds. Viability of recovered cells declined but metabolic products continued accumulating, when the pH was kept constant at pH 5.2. It was proposed that cells entered a non-culturable state but remained metabolically active. Results indicated the potential of this strain, as a possible non-starter lactic acid bacteria adjunct, to survive and utilize alternative available substrates as energy source in cheese curd.

As the protein composition of Cheddar cheese varies substantially from that of milk, it is important to investigate whether the standard dairy nitrogen conversion factor (NCF), 6.38, accurately reflects the protein content of Cheddar cheese. Forty-eight Cheddar cheeses of a wide range of composition were manufactured. Amino acid compositions of measured casein subtypes, peptides and amino acids in the cheeses were used to calculate a nitrogen conversion factor for each cheese. Average NCF values were calculated as 6.394±0.004, for the full dataset, which is slightly higher than the standard dairy nitrogen conversion factor of 6.38. Crown Copyright © 2007.

To investigate impediments to plasmid transformation inBrevibacterium flavum BF4 andB. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties.B. lactofermentum was more sensitive to growth inhibition by glycine thanB. flavum. Release of cellular proteins during sonication was more rapid forB. lactofermentum than forB. flavum. Plasmid DNA (pCSL17) isolated fromB. flavum transformed recipient McrBC+ strains ofEscherichia coli with lower efficiency than McrBC−.McrBC digestion of this DNA confirmed thatB. flavum contain methylated cytidines in the target sequence ofMcrBC sequences butB. lactofermentum contained a different methylation pattern. DNA derived from theB. lactofermentum transformed recipient EcoKR+ strains ofE. coli with lower efficiency than EcoKR−, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.

Bioaugmentation of polycyclic aromatic hydrocarbon (PAH)-contaminated soil was investigated using a mixed bacterial culture (community five) isolated from an abandoned industrial site. Community fi...

The ability of Stenotrophomonas maltophilia strain VUN 10,003 to degrade and detoxify high molecular weight polycyclic aromatic hydrocarbons (PAHs) was evaluated in a basal liquid medium. Using high cell density inocula of strain VUN 10,003, the concentration of pyrene, fluoranthene, benz[a]anthracene, benzo[a]pyrene, dibenz[a, h]anthracene and coronene decreased by 98, 45, 26, 22, 22 and 55% over periods ranging from 5 to 42 d. When a PAH mixture containing three- to seven-ring compounds was used, degradation of both low and high molecular weight compounds occurred concurrently. Mutagenicity assays (Ames Test) demonstrated a decrease in the mutagenic potential of dichloromethane culture extracts from all cultures containing single PAH over the incubation period, corresponding to the decrease in the concentration of the PAH. These observations indicate that strain VUN 10,003 could be used for the detoxification of PAH-contaminated wastes.

Creosote was evaluated as an inexpensive carbon source for growing inocula of a polycyclic aromatic hydrocarbon (PAH)-degrading bacterial community (community five). Creosote was a poor growth substrate when provided as sole carbon source in a basal salts solution (BSM). Alternatively, peptone, yeast extract or glucose in BSM supported high growth rates, but community five could not subsequently degrade pyrene. A combination of creosote and yeast extract in BSM (CYEM) supported growth and maintained the pyrene-degrading capacity of community five. Optimum pyrene-degrading activity occurred when the inocula were grown in creosote and yeast extract concentrations of 2 ml L−1 and 1 g L−1 respectively: concentrations outside these values resulted in either low biomass yields or loss of PAH-degrading activity. CYEM-grown community five inocula degraded 250 mg L−1 of pyrene in BSM at a rate comparable to cultures inoculated with community five grown in BSM-pyrene. However, the CYEM-grown community showed a 40% lower rate of PAH degradation in a synthetic PAH mixture compared with pyrene-grown cells and there was an increase in the lag period before the onset of PAH degradation. This appears to reflect a weaker induction of PAH catabolism by CYEM compared to BSM-pyrene. Journal of Industrial Microbiology & Biotechnology (2000) 24, 277–284.

The objectives of this study were to isolate and evaluate microorganisms with the ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) in the presence of synthetic surfactants. Stenotrophomonas maltophilia VUN 10,010, isolated from PAH-contaminated soil, utilized pyrene as a sole carbon and energy source and also degraded other high molecular weight PAHs containing up to seven benzene rings. Various synthetic surfactants were tested for their ability to improve the PAH degradation rate of strain VUN 10,010. Anionic and cationic surfactants were highly toxic to this strain, and the Tween series was used as a growth substrate. Five nonionic surfactants (Brij 35, Igepal CA-630, Triton X-100, Tergitol NP-10, and Tyloxapol) were not utilized by, and were less toxic to, strain VUN 10,010. MSR and log Km values were determined for fluoranthene, pyrene, and benzo[a]pyrene in the presence of these nonionic surfactants and their apparent solubility was increased by a minimum of 250-fold in the presence of 10 g L−1 of all surfactants. The rate of pyrene degradation by strain VUN 10,010 was enhanced by the addition of four of the nonionic surfactants (5–10 g L−1); however, 5 g L−1 Igepal CA-630 inhibited pyrene degradation and microbial growth. The specific growth rate of VUN 10,010 on pyrene was increased by 67% in the presence of 10 g L−1 Brij 35 or Tergitol NP-10. The addition of Brij 35 and Tergitol NP-10 to media containing a single high molecular weight PAH (four and five benzene rings) as the sole carbon source increased the maximum specific PAH degradation rate and decreased the lag period normally seen for PAH degradation. The addition of Tergitol NP-10 to VUN 10,010 cultures which contained a PAH mixture (three to seven benzene rings) substantially improved the overall degradation rate of each PAH and increased the specific growth rate of VUN 10,010 by 30%. Evaluation of the use of VUN 10,010 for degrading high molecular weight PAHs in leachates from surfactant-flushed, weathered, PAH-contaminated sites is warranted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:482–494, 1998.

A Burkholderia cepacia strain (VUN 10,001) isolated from manufacturing gas plant soil was selected for its ability to grow on and degrade pyrene as a sole source of carbon and energy. VUN 10,001 was able to grow on fluorene, phenanthrene benz[a]anthracene, fluoranthene and pyrene, but not benzo[a]pyrene and dibenz[a,h]anthracene when tested using inocula with low cell numbers. However, a small decrease in the concentrations of these latter compounds was observed. When media were inoculated at a high cell density, VUN 10,001 degraded the high molecular weight PAHs benzo[a]pyrene, dibenz[a,h]anthracene and coronene when these were present as individual substrates or as components of a complex mixture. Degradation of the high molecular weight PAHs was found to increase in the presence of the lower molecular weight compounds.

Auxotrophic mutants of Corynebacterium glutamicum strain ATCC 13059 (parent of AS019, a rifampicin-resistant variant), which were morphologically distinct from the parent and formed protoplasts more readily, had been isolated previously. Mutants MLB130-133 and MLB194 were more sensitive to growth inhibition by isonicotinic acid hydrazide (INH) and glycine, which caused branching and budding. Fatty acid and mycolic acid (MA) profiles were determined after growth in LBG (Luria broth plus glucose), LBG-glycine (LBG- and LBG-INH (LBG-I). The fatty acid profiles of all strains were similar, except that mutant MLB133 showed some increase in stearic acid (C18:0), normally a minor component, late in the growth cycle and oleic acid proportionately decreased. All strains had five major types of MAs (C32:0, C34:0, C34:1, C36:1, C36:2) but the relative proportion of each varied with the strain, age of culture and medium composition. Mutants MLB133 and MLB194 showed slightly higher levels of non-covalentiy bound MAs than the parent and normally showed a higher proportion of longer-chained, unsaturated MAs. The proportion of extracellular MAs increased with culture age for these mutants. Typically, by late stationary phase, mycolic acids in culture fluids increased to 6.5% of the total MAs for MLB194 and 7.9% for MLB133 compared with 3.5% for the parent strain grown in LBG. The main effect of glycine (2%, w/v) addition was to increase the proportion of mycolic acids found in extracellular fluids (16.1 % for AS019 and 31% for MLB133). The most significant effects of INH were seen when strains were cultured in LBG with 8 mg INH ml−1. When harvested at late stationary phase, strains MLB133 and MLB194 had 18.8% and 21.2% extracellular mycolic acids respectively, with a significant increase in the relative proportion of unsaturated mycolic acids. This effect was not as marked for AS019, which also showed a similar decrease in C32:0 relative to increases in the proportion of C34:1 and C36:2 plus a corresponding increase in the overall proportion of unsaturated mycolic acids and increased extracellular mycolates (8.5%). These results suggest that the mutations in strains MLB133 and MLB194 are associated with synthesis of specific mycolic acids (e.g. C32:0) and attachment of mycolic acids to the cell surface, both of which are likely target sites for glycine and INH action for cell-surface modifications. In addition to previously reported targeting of the peptidoglycan cross-linking, these results show that glycine affects mycolic acid attachment to the cell surface of C. glutamicum.

Microbiological analysis of soils from a polycyclic aromatic hydrocarbon (PAH)-contaminated site resulted in the enrichment of five microbial communities capable of utilizing pyrene as a sole carbon and energy source. Communities 4 and 5 rapidly degraded a number of different PAH compounds. Three pure cultures were isolated from community 5 using a spray plate method with pyrene as the sole carbon source. The cultures were identified as strains of Burkholderia (Pseudomonas) cepacia on the basis of biochemical and growth tests. The pure cultures (VUN 10 001, VUN 10 002 and VUN 10 003) were capable of degrading fluorene, phenanthrene and pyrene (100 mg l−1) to undetectable levels within 7–10 d in standard serum bottle cultures. Pyrene degradation was observed at concentrations up to 1000 mg l−1. The three isolates were also able to degrade other PAHs including fluoranthene, benz[a]anthracene and dibenz[a,h]anthracene as sole carbon and energy sources. Stimulation of dibenz[a,h]anthracene and benzo[a]pyrene degradation was achieved by the addition of small quantities of phenanthrene to cultures containing these compounds. Substrate utilization tests revealed that these micro-organisms could also grow on n-alkanes, chlorinated- and nitro-aromatic compounds.

Resistance to a range of heavy metal ions wasdetermined for lead-resistant and other bacteria whichhad been isolated from a battery-manufacturing sitecontaminated with high concentrations of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) andGram-negative (Alcaligenes species) isolateswere resistant to lead, mercury, cadmium, cobalt,zinc and copper, although the levels of resistance tothe different metal ions were specific for eachisolate. Polymerase chain reaction, DNA-DNAhybridization and DNA sequencing were used to explorethe nature of genetic systems responsible for themetal resistance in eight of the isolates. SpecificDNA sequences could be amplified from the genomic DNAof all the isolates using primers for sections of themer (mercury resistance determinant on thetransposon Tn501) and pco (copperresistance determinant on the plasmid pRJ1004) geneticsystems. Positive hybridizations with mer andpco probes indicated that the amplified segmentswere highly homologous to these genes. Some of thePCR products were cloned and partially sequenced, andthe regions sequenced were highly homologous to theappropriate regions of the mer and pcodeterminants. These results demonstrate the widedistribution of mercury and copper resistance genes inboth Gram-positive and Gram-negative isolates obtainedfrom this lead-contaminated soil. In contrast, theczc (cobalt, zinc and cadmium resistance) andchr (chromate resistance) genes could not beamplified from DNAs of some isolates, indicating thelimited contribution, if any, of these genetic systemsto the metal ion resistance of these isolates.

The global proteomic response of the nonstarter lactic acid bacteria Lactobacillus casei strain GCRL163 under carbohydrate depletion was investigated to understand aspects of its survival following cessation of fermentation. The proteome of L. casei GCRL163 was analyzed quantitatively after growth in modified MRS (with and without Tween 80) with different levels of lactose (0% lactose, starvation; 0.2% lactose, growth limiting; 1% lactose, non-growth-limited control) using gel-free proteomics. Results revealed that carbohydrate starvation lead to suppression of lactose and galactose catabolic pathways as well as pathways for nucleotide and protein synthesis. Enzymes of the glycolysis/gluconeogenesis pathway, amino acid synthesis, and pyruvate and citrate metabolism become more abundant as well as other carbohydrate catabolic pathways, suggesting increased optimization of intermediary metabolism and scavenging. Tween 80 did not affect growth yield; however, proteins related to fatty acid biosynthesis were repressed in the presence of Tween 80. The data suggest that L. casei adeptly switches to a scavenging mode, using both citrate and Tween 80, and efficiently adjusts energetic requirements when carbohydrate starved and thus can sustain survival for weeks to months. Explaining the adaptation of L. casei during lactose starvation will assist efforts to maintain viability of L. casei and extend its utility as a beneficial dietary adjunct and fermentation processing aid.

Listeria monocytogenes is a foodborne human pathogen capable of causing life-threatening disease in susceptible populations. Previous proteomic analysis we performed demonstrated that different strains of L. monocytogenes initiate a stringent response when subjected to alkaline growth conditions. Here, using multidimensional protein identification technology (MudPIT), we show that in L. monocytogenes EGD-e this response involves an energy shift to anaerobic pathways in response to the extracellular pH environment. Importantly we show that this supports a reduction in relative lag time following an abrupt transition to low oxygen tension culture conditions. This has important implications for the packaging of fresh and ready-to-eat foods under reduced oxygen conditions in environments where potential exists for alkaline adaptation.

Photobacterium phosphoreum is a marine bacterium which is used extensively as a bioluminescent indicator of pollutants, where the presence of toxicants diminishes light output. To evaluate the utility of cell immobilisation in continuous toxicity testing, the sensitivity of P. phosphoreum to five gelling agents was evaluated relative to the retention of bioluminescence in 3% NaCl-glycerol suspensions. Following storage at 4°C, the control cultures retained light output for up to 2 weeks before significant decline; alginate-glycerol suspensions were stable for up to 4 weeks and bioluminescence was detectable for up to 6 weeks. Cells stored in agar were no more stable than the control, whereas cells gelled in agarose and low-melting point agarose showed a significant decline in bioluminescence within 2 weeks of storage. Bioluminescence was totally retained in alginate-glycerol suspensions stored at −80°C for up to 12 weeks. P. phosphoreum was successfully immobilised in strontium alginate and showed a dose-related response to four of the five heavy metal ions, SDS and pentachlorophenol tested when responses were followed over a time-course. A flow-through system for Sr-alginate immobilised cells was developed and conditions for operation were optimised. When cells were exposed to a pulse of 4-nitrophenol or salicylate then the nutrient feed continued, bioluminescence declined in response (pulse of 4–6 min) to these pollutants then recovered to a new stable rate of decline which was faster than the pre-exposure rate. These results demonstrate the potential of using immobilised P. phosphoreum in a continuous flow-through system for real-time environmental monitoring of water quality.

Plasmid DNA (pCSL17) isolated from Corynebacterium glutamicum transformed recipient McrBC+ strains of Escherichia coli with lower efficiency than McrBC- strains, confirming a previous report by Tauch et al. (FEMS Microbiol. Lett. 123 (1994) 343-348) which inferred that C. glutamicum DNA contains methylcytidine. Analysis of nucleotides in C. glutamicum-derived chromosomal and plasmid DNA failed to detect significant levels of methylated adenosine, but methylated cytidine was readily detected. Restriction enzymes which are inhibited by the presence of methylcytidine in their recognition sequence failed to cut pCSL17 from C. glutamicum, whereas enzymes which require methylation at adenosine in GATC sequences failed to cut. Failure of HaeIII to cut two specific sites of C. glutamicum-derived pCSL17 identified the first cytidine in the sequence GGCCGC as one target of methylation in this species, which contains the methyltransferase recognition sequence. Although Brevibacterium lactofermentum-derived DNA showed a similar methylation pattern by HPLC analysis, HaeIII cleaved these GGCCGC sites, suggesting differences in the specificity of methylation between these two species. Results for all analyses of B. flavum DNA were identical to those for C. glutamicum.

Samples of five brands of commercial yoghurt were obtained directly from the processors and enumeration of viable Lactobacillus acidophilus and Bifidobacterium bifidum and determination of pH was carried out at three-day intervals over a five-week period. Three of the five products contained 10 7 –10 8 g −1 viable cells of L. acidophilus , whereas the other two products contained ⩽ 10 5 of this organism initially. Similarly, the initial count of B. bifidum was 10 6 –10 7 g −1 in two of five products, while the viable numbers of this organism were ⩽ 10 3 in the other three products. All the products showed a constant decline in the viable count of L. acidophilus and B. bifidum during storage. Initial pH values ranged from 4.07 to 4.36 and these declined after five weeks of storage to pH 3.8–4.26.

Enterococci are reported to survive pasteurisation but the extent of their survival is unclear. Sixty-one thermoduric enterococci isolates were selected from laboratory pasteurised milk obtained from silos in six dairy factories. The isolates were screened to determine log10 reductions incurred after pasteurisation (63 °C/30 min) and ranked from highest to lowest log10 reduction. Two isolates each of Enterococcus faecalis, Enterococcus faecium, Enterococcus durans and Enterococcus hirae, exhibiting the median and the greatest heat resistance, as well as E. faecalis ATCC 19433, were selected for further heat resistance determinations using an immersed coil apparatus. D values were calculated from survival curves plotted from viable counts obtained after heating isolates in Brain Heart Infusion Broth at 63, 69, 72, 75 and 78 °C followed by rapid cooling. At 72 °C, the temperature employed for High Temperature Short Time (HTST) pasteurisation (72 °C/15 s), the D values extended from 0.3 min to 5.1 min, depending on the isolate and species. These data were used to calculate z values, which ranged from 5.0 to 9.8 °C. The most heat sensitive isolates were E. faecalis (z values 5.0, 5.7 and 7.5 °C), while the most heat resistant isolates were E. durans (z values 8.7 and 8.8 °C), E. faecium (z value 9.0 °C) and E. hirae (z values 8.5 and 9.8 °C). The data show that heat resistance in enterococci is highly variable.

SUMMARY: A rapid and efficient method of transforming Corynebacterium glutamicum by electroporation is described. A number of factors are important in determining the level of transformation obtained. Cells grown in the presence of glycine and isonicotinic acid hydrazide and harvested in early exponential growth phase were much easier to transform. The recovery medium on which transformants were isolated also had a significant effect on the number of transformants obtained because cells were osmotically or electrochemically sensitive following electroporation. Transformation efficiencies of up to 5 x 105 transformants per μg plasmid DNA with homologously derived DNA and 2 x 103 transformants per μg of DNA derived from Escherichia coli were obtained.

Large inocula of Stenotrophomonas maltophilia VUN 10,003 were used to investigate bacterial degradation of benzo[a]pyrene and dibenz[a,h]anthracene. Although strain VUN 10,003 was capable of degrading 10-15 mg (-1) of the five-ring compounds in the presence of pyrene after 63 days, further addition of pyrene after degradation of the five-ring polycyclic aromatic hydrocarbons (PAHs) ceased did not stimulate significant decreases in the concentration of benzo[a]pyrene or dibenz[a,h]anthracene. However, pyrene was degraded to undetectable levels 21 days after its addition. The amount of benzo[a]pyrene and dibenz[a,h]anthracene degraded by strain VUN 10,003 was not affected by the initial concentration of the compounds when tested at 25-100 mg l(-1), by the accumulation of by-products from pyrene catabolism or a loss of ability by the cells to catabolise benzo[a]pyrene or dibenz[a,h]anthracene. Metabolite or by-product repression was suspected to be responsible for the inhibition: By-products from the degradation of the five-ring compounds inhibited their further degradation.

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria ( Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus ( Penicillium janthinellum VUO 10,201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[ a ]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[ a ]anthracene, benzo[ a ]pyrene, and dibenz[ a , h ]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[ a ]anthracene, benzo[ a ]pyrene, and dibenz[ a , h ]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[ a ]pyrene was mineralized to CO 2 by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[ a ]pyrene mineralization (53% of added [ 14 C]benzo[ a ]pyrene was recovered as 14 CO 2 in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.

Many agricultural and industrial practices have led to environmental pollution by heavy metal ions. Metals, such as zinc, cobalt, cadmium, nickel, copper and chromium, are used for several industrial applications, such as production of steel and other alloys, galvanisation of iron, electroplating, manufacture of batteries, TV tubes and pigments. Copper has been used for a number of years as an active ingredient of bactericides and fungicides and as a growth enhancer of pigs. Mercury is a by-product of burning coal and petroleum products, and it is also used in household and hospital disinfectant. Contamination of soil by lead occurs following the use of lead arsenate insecticides. High levels of lead in wastewaters may originate from combustion of leaded gasoline, coal burning or metal smelting. Mining and leaching from natural deposits also contribute to environmental contamination. A number of sites contaminated by heavy metals around the world are associated with human activities, such as discharge of wastes into natural waterways, various metallurgical industries, accidental spills or mining [1–8].

Twenty-five lead-tolerant strains of Paecilomyces lilacinus which had been isolated from a lead-contaminated site in Melbourne, Australia, were screened for lead biosorption capacity using biomass collected following culture in a basal salts medium supplemented with 0.5 gN l−1 as either ammonium or nitrate. All of these strains showed similar patterns of biosorption and lead uptake by dried biomass was always superior to freshly-harvested biomass and nitrate-grown biomass was superior to ammonium-grown biomass. Six strains were selected for detailed analysis of lead biosorption following growth in media with different C:N ratios and in lead solutions of pH between 3 and 7, comparing lead uptake for the lead-tolerant strains with the type strain of P. lilacinus (FRR 895), Aspergillus niger and Penicillium fumigatus. In comparison to the latter reference strains, all strains of P. lilacinus showed better biosorptive capacity following growth in media supplemented with high levels of nitrate and lead sorption was poor for all strains if biomass was exposed to lead in low pH solutions. Growth in high levels of ammonium (0.5 gNl−1) was accompanied by decreases in medium pH and relatively poor biosorption by dried biomass. Supplying low levels of ammonium (0.1 gN l−1) improved lead biosorption but controlling pH at 6–7 for media containing higher levels of ammonium improved the normally poor biosorption capacity. Biosorption isotherms for ammonium-grown biomass fitted the Freundlich model, but nitrate-grown biomass did not. Maximum biosorption values were 9–12% w/w and most of the lead-tolerant strains had higher Qmax values than the type strain, for nitrate-grown biomass. The lead-resistant strains could initiate growth in media containing 5,500 μg Pb ml−1, taking up lead into or onto cells (insensitive to EDTA treatment) early in growth, after which cell-associated lead declined as growth progressed. If lead was added later in growth, uptake was lower, suggesting that lead was excluded. Under the same growth conditions, the type strain FRR 895 was completely inhibited. Cells grown in lower concentrations of lead bound less lead but growth was still impaired.

A Burkholderia cepacia strain (VUN 10,001) isolated from manufacturing gas plant soil was selected for its ability to grow on and degrade pyrene as a sole source of carbon and energy. VUN 10,001 was able to grow on fluorene, phenanthrene benz[ a ]anthracene, fluoranthene and pyrene, but not benzo[ a ]pyrene and dibenz[ a , h ]anthracene when tested using inocula with low cell numbers. However, a small decrease in the concentrations of these latter compounds was observed. When media were inoculated at a high cell density, VUN 10,001 degraded the high molecular weight PAHs benzo[ a ]pyrene, dibenz[ a , h ]anthracene and coronene when these were present as individual substrates or as components of a complex mixture. Degradation of the high molecular weight PAHs was found to increase in the presence of the lower molecular weight compounds.

Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous environmental pollutants which have been detected in numerous aquatic and terrestrial ecosystems (Cemiglia, 1992). There is concern about the presence of these compounds in the environment as they have been shown to exhibit toxic, mutagenic and carcinogenic effects (Mersch-Sundermann et al., 1992; Nylund et al., 1992). PAHs may enter the environment via many mechanisms; however, anthropogenic processes such as the combustion of fossil fuels, the accidental spilling of hydrocarbons and oils, coal gasification and liquefaction, wood treatment processes, open burning and the incineration of wastes are the major sources of PAHs in the environment (Guerin and Jones, 1988; Freeman and Cattell, 1990).

Microbial species respond to changes in their environmental conditions by producing a range of new proteins which assist their survival and adaptation to new conditions. Normally, sudden changes in temperature (heat shock), exposure to potentially toxic substances (ethanol, hydrogen peroxide, heavy metal ions) or the onset of starvation cause the production of stress-related proteins. The most thoroughly studied stress proteins include the heat shock proteins (HSP), where the mostly highly conserved responses across genera and shock conditions is the production of the HSP60 (GroEL) and HSP70 (DnaK) group of proteins [1]. The major HSPs synthesised by Escherichia coli (HtpG, GroEL and DnaK) were designated as molecular chaperones because of their role in protein folding for both newly synthesised proteins and proteins partially denatured by stressing conditions [1], where elevated activity of these proteins is essential for cell recovery [2]. Although the heat-shock response of E. coli occurs even for mild, non-lethal temperature upshifts, the intensity of the response depends on the severity of the temperature shift: a 2-fold increase in GroEL and DnaK may occur with a shift from 30°C to 42°C, whereas a 10-fold increase is caused by a shift to 46°C [3]. Other environmental hazards also cause similar stress responses in cells, including cold shock [4], nutrient starvation [5] and exposure to pollutants such as heavy metal ions and organic compounds [6–8]. Blom et al. [6] reported that E. coli synthesises up to 39 new proteins after exposure to a range of different pollutants, where there was some commonality in the types of proteins produced in response to the different chemicals.

When inoculated at high cell densities, three strains of Pseudomonas cepacia degraded the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene, dibenz[a,h]anthracene and coronene as sole carbon and energy sources. After 63 days incubation, there was a 20 to 30% decrease in the concentration of benzo[a]pyrene and dibenz[a,h]anthracene and a 65 to 70% decrease in coronene concentration. The three strains were also able to degrade all the PAHs simultaneously in a PAH substrate mixture containing three-, four-, five- and seven-benzene ring compounds. Furthermore, improved degradation of the five- and seven-ring PAHs was observed when low molecular weight PAHs were present.

Mankind has used microbes from the dawn of history to perform services and produce useful chemicals and bioactives. Mixed complex communities, which are resilient over time, preserved food, made alcoholic beverages and treated wastes, all in the absence of an understanding of the underlying biological processes. Moving to single microbial transformation systems led to high-level production of primary (amino acids, nucleotides, vitamins ? used as flavour-enhancing agents, nutritional supplements and pharmaceuticals ? solvents and organic acids, including biofuels) and secondary (pharmaceuticals, enzyme inhibitors, bio-herbicides and pesticides, plant growth regulators) metabolites and bioactives (including bacteriocins and enzymes). Several hallmark discoveries in microbiology and other sciences over the last 60 years transformed our ability to discover, manipulate, enhance and derive commercial benefit from industrial applications of microorganisms. This article attempts to capture some of the key discoveries that revolutionised industrial microbiology and speculates about where the ?omics? revolution will take us next.

Professor Arnold (Arny) Lester Demain is one of the few scientists who have witnessed the progress of biotechnology in a career that has spanned almost 60 years. He is one of the world?s leading industrial microbiologists who has pioneered discovery in genetic and nutritional regulation of biosynthetic pathways leading to overproduction of a suite of primary and secondary metabolites, and their subsequent scale-up in manufacturing processes. These metabolites have huge economic value due to their application in the food, pharmaceutical and agricultural sectors. In this article, Arny?s story is summarised and put in context of the changing face of biotechnology in the various ?golden ages? of biotechnology. A former Rubbo Orator in 1979, Arny will be visiting Australia again in 2010 to present the closing plenary address at the Genetics of Industrial Microorganisms Symposium (GIMS) in Melbourne, a role he played before at the first GIMS in 1970.

This paper describes two quite different mechanisms of resistance to clinically important antimicrobial agents in anaerobic bacteria, which also represent two distinct genetic processes. The first is resistance to chloramphenicol (Cm) in Bacteroides species (in particular, Bacteroides fragilis) and Clostridium perfringens, which arises due to the enzymic inactivation of the drug by acetylation. When Cm resistance occurs in other genera, it is typically plasmid determined; it is also plasmid associated in Cm-resistant strains of Cl. perfringens (Sebald and Brfort, 1975) and B. ochraceus (Guiney and Davis, 1978). The second is resistance to a group of antimicrobial agents, the 5-nitroimidazoles, which includes metronidazole (Mz) and tinidazole (Tz). In this case, resistance in strains of B. fragilis and Cl. perfringens is associated with a change in the biochemical pathway normally involved in activation of the drugs. This is a result of a chromosomal mutation which can be induced by treatment with mutagens such as N-methyl-N'-nitro N-nitrosoguanidine (MNNG) (Britz & Wilkinson, 1979). © 1981 The British Society for Antimicrobial Chemotherapy. All Rights Reserved.