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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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales . Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0-4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27-59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis , respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis , R. baikonurensis , R. opacus and R. rhodochrous , would be reclassified either as R. erythropolis or R. qingshengii .

Lactobacillus casei group bacteria improve cheese ripening and may interact with host intestinal cells as probiotics, where surface proteins play a key role. Three complementary methods [trypsin shaving (TS), LiCl-sucrose (LS) extraction, and extracellular culture fluid precipitation] were used to analyze cell surface proteins of Lactobacillus paracasei GCRL163 by label-free quantitative proteomics after culture to the mid-exponential phase in bioreactors at pH 6.5 and temperatures of 30-45 °C. A total of 416 proteins, including 300 with transmembrane, cell wall anchoring, and secretory motifs and 116 cytoplasmic proteins, were quantified as surface proteins. Although LS caused significantly greater cell lysis as growth temperature increased, higher numbers of extracytoplasmic proteins were exclusively obtained by LS treatment. Together with the increased positive surface charge of cells cultured at supra-optimal temperatures, proteins including cell wall hydrolases Msp1/p75 and Msp2/p40, α-fucosidase AlfB, SecA, and a PspC-domain putative adhesin were upregulated in surface or secreted protein fractions, suggesting that cell adhesion may be altered. Prolonged heat stress (PHS) increased binding of L. paracasei GCRL163 to human colorectal adenocarcinoma HT-29 cells, relative to acid-stressed cells. This study demonstrates that PHS influences cell adhesion and relative abundance of proteins located on the surface, which may impact probiotic functionality, and the detected novel surface proteins likely linked to the cell cycle and envelope stress.

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., Competing Interests: The authors declare no conflict of interest.

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 40⁻45 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., Competing Interests: The authors declare no conflict of interest.

Prolonged heat stress is one of the harsh conditions Lactobacillus casei strains encounter as non-starter lactic acid bacteria in dairy product manufacture. To understand the physiological and molecular mechanisms through which Lb. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (log2-fold change of 7) at 45°C. We identified a novel SecB homolog as a protein export chaperone putatively involved in posttranslational translocation systems, which was down-regulated as growth temperature increased and where the modelled 3D-structure shared architectural similarities with the Escherichia coli SecB protein. Membrane lipid analyses revealed temporal changes in fatty acid composition, cyclization of oleic acid to cyclopropane and novel cyclopentenyl moieties, and reduced synthesis of vaccenic acid, at higher temperatures. An 18kDa α-crystallin domain, Hsp20 family heat shock protein was more highly up-regulated in response to heat stress compared to other molecular chaperones, suggesting this protein could be a useful biomarker of prolonged heat stress in Lb. casei GCRL163., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2017 Nahar et al.)

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., (Copyright © 2017 Nahar et al.)

The draft genome sequences of three sub-Antarctic Rhodococcus sp. strains-1159, 1163, and 1168-are reported here. The estimated genome sizes were 7.09 Mb with a 62.3% GC content for strain 1159, 4.45 Mb with a 62.3% GC content for strain 1163, and 5.06 Mb with a 62.10% GC content for strain 1168., (Copyright © 2017 Nahar et al.)

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., (Copyright © 2017 Nahar et al.)

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 of >4 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., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 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.

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 log(10) reductions incurred after pasteurisation (63°C/30 min) and ranked from highest to lowest log(10) 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/15s), 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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background and Objectives: Although members of the Lactobacillus casei group are known to survive under acidic conditions, the underlying mechanisms of growth at acidic condition and the impact of low pH on the relative level of protein expression at the cell surface remain poorly studied., Material and Methods: After confirming the taxonomy of L. casei strain GCRL 12 which was originally isolated from cheese and confirmed by 16S rRNA sequence analysis, the impact of acidic pH on growth rate was determined., Results: Late log-phase cells cultured at pH 4.0 showed obvious changes in Gram staining properties while transmission electron microscopy analysis revealed evidence of structural distortions of the cell surface relative to the controls cultured at pH 6.5. When comparing cytosolic or whole cell preparations on SDS-PAGE, few changes in protein profiles were observed under the two growth conditions. However, analysis of surface protein extracted by 5M LiCl demonstrated changes in the proportions of proteins present in the molecular weight range of 10 to 80 kDa, with some proteins more dominant at pH 6.5 and other at pH 4., Conclusion: These data suggest that surface proteins of this strain are associated with growth and survival at low pH. The function of these proteins is subject to further investigation.

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., (Copyright 2009 Elsevier B.V. All rights reserved.)

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.

Aims: Starvation stress is a condition that nonstarter lactic acid bacteria (NSLAB) normally encounter. This study was aimed to investigate starvation-induced proteins in Lactobacillus casei during stationary growth phase., Methods and Results: The impact of carbohydrate starvation on L. casei GCRL163 was investigated using two different media (a modified de Man, Rogosa and Sharpe broth and a semi-defined medium). Cells were grown in the presence of excess lactose (1%) or starvation (0%) and differences in the patterns of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of the cytosolic protein fractions were investigated. Differentially regulated proteins were identified by MALDI-TOF/TOF mass spectrometry. Many differentially regulated proteins were enzymes of various metabolic pathways involved in carbohydrate metabolism to yield energy. Differences in protein expression were also observed in the two culture conditions tested in this experiment., Conclusion: Numerous glycolytic enzymes were differentially regulated under lactose starvation. The differential expression of these glycolytic enzymes suggests a potential survival strategy under harsh growth conditions (i.e. lactose starvation)., Significance and Impact of the Study: This paper reports improved understanding of stress responses and survival mechanism of NSLAB under lactose-depleted cheese-ripening condition. This knowledge of how NSLAB bacteria adapt to lactose starvation could be applied to predict the performances of bacteria in other industrial applications.

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.

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.

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. Copyright 1998 John Wiley & Sons, Inc.

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 (C 18:0 ), normally a minor component, late in the growth cycle and oleic acid proportionately decreased. All strains had five major types of MAs (C 32:0 , C 34:0 , C 34:1 , C 36:1 , C 36:2 ) 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 C 32:0 relative to increases in the proportion of C 34:1 and C 36:2 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. C 32:0 ) 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.

Resistance to a range of heavy metal ions was determined for lead-resistant and other bacteria which had been isolated from a battery-manufacturing site contaminated with high concentration of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) and Gram-negative (Alcaligenes species) isolates were resistant to lead, mercury, cadmium, cobalt, zinc and copper, although the levels of resistance to the different metal ions were specific for each isolate. Polymerase chain reaction, DNA-DNA hybridization and DNA sequencing were used to explore the nature of genetic systems responsible for the metal resistance in eight of the isolates. Specific DNA sequences could be amplified from the genomic DNA of all the isolates using primers for sections of the mer (mercury resistance determinant on the transposon Tn501) and pco (copper resistance determinant on the plasmid pRJ1004) genetic systems. Positive hybridizations with mer and pco probes indicated that the amplified segments were highly homologous to these genes. Some of the PCR products were cloned and partially sequenced, and the regions sequenced were highly homologous to the appropriate regions of the mer and pco determinants. These results demonstrate the wide distribution of mercury and copper resistance genes in both Gram-positive and Gram-negative isolates obtained from this lead-contaminated soil. In contrast, the czc (cobalt, zinc and cadmium resistance) and chr (chromate resistance) genes could not be amplified from DNAs of some isolates, indicating the limited contribution, if any, of these genetic systems to the metal ion resistance of these isolates.

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.

Cultured tissue slices from normal immature rabbit articular cartilage released latent neutral metalloproteinases into serum-free medium. On activation with 4-aminophenylmercuric acetate, these metalloproteinases could degrade collagen, proteoglycan, and gelatin. Also produced were an acid proteinase with the properties of cathepsin D and an inhibitor of the neutral metalloproteinases. The appearance of both the proteinases and the inhibitor in the culture medium could be prevented by incubation of cultures with cycloheximide. The active and latent forms of the proteinases were characterized using Ultrogel AcA 54 chromatography.

Conversion of leucine to isovaleric (iV) and isocaproic (iC) acids by cell-free extracts of Clostridium bifermentans was demonstrated using two lysis procedures. Sonication resulted in an extract which had the enzymes to convert leucine to alpha-ketoisocaproic acid (alpha-kiC) and thence iV, but failed to produce iC. Extracts prepared by osmotic lysis, which contained intact membranes, could convert leucine to both iV and iC. The enzyme which converts leucine to alpha-kiC was solubilized during osmotic lysis, whereas the decarboxylase and leucine reductase system remained membrane bound. Osmotic lysis also released at least two small molecular weight, heat-stable, anionic components (3500 greater than molecular weight greater than or equal to 1000), which stimulated decarboxylase activity.

Metronidazole-resistant mutants of Bacteroides fragilis, isolated after mutagenesis, had diminished ability to take up and metabolize the drug. All the metronidazole-resistant strains had depressed levels of pyruvate dehydrogenase compared with parent cultures. Their end products of glucose metabolism also differed from normal B. fragilis products and were consistent with deficiencies in pyruvate dehydrogenase activity.

Clostridium perfringens strains resistant to metronidazole and tinidazole were isolated from the sensitive parent strain CM288 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Strain CM288 was already resistant to rifampicin and nalidixic acid; these genetic markers helped to confirm the identity of mutants. All mutants showed similar characteristics: they grew more slowly than the parent strain and failed to reach the same maximum turbidity; uptake of metronidazole and tinidazole from culture fluids was slow and end products of glucose metabolism were different from those of the parent. Pyruvate dehydrogenase activity was not detected in broken cell preparations of the mutant strains although this enzyme was readily detected in the parent strain. Changes in end products of glucose metabolism were consistent with the absence of pyruvate dehydrogenase activity because pyruvate was accumulated during growth and lactate levels were higher whereas acetate, CO2 and ethanol levels were diminished.

Conversion of leucine to isovaleric acid by Clostridium bifermentans is achieved by the action of at least two enzymes. One is a transaminase producing alpha-ketoisocaproic acid, which was purified 30-fold from osmotic lysates of late-exponential phase cells by repeated chromatography on DEAE-Sepharose C16B and Sephacryl S300: this represented a 147-fold purification of activity found in sonically disrupted cells. This enzyme had an apparent molecular weight of approximately 190000 and was composed of six identically sized sub-units (molecular weight 31000 +/- 1000). Transamination required pyridoxal phosphate and pyruvate and was optimal at pH 8.6; the apparent Km for leucine was 7.0 mM. Activity was totally inhibited by 1 mM-p-chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated alpha-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography on DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the Km for alpha-ketoisocaproic acid was 4.2 mM and activity was optimal around pH 8.0. This enzyme was a flavoprotein with absorption maxima at 280, 320 and 400 nm, and a fluorescent maximum at 500 nm. The prosthetic group, FAD, dissociated from the protein during purification resulting in an inactive apoenzyme which was only partially re-activated by FAD. Activity was completely inhibited by several thiol reagents tested at 1 mM.

Freshly compared cell suspensions of clostridia (Clostridium bifermentans, C. botulinum proteolytic type A, C. difficile, C. sordellii, and C. sporogenes) and Peptostreptococcus anaerobius converted leucine to isovaleric (iV) and isocaproic (iC) acids in the absence of other amino acids. The optimal pH for conversion was between 8 and 9 at 37 degrees C. The stoichiometry of reaction was compatible with that expected for the Stickland reaction, as the ratio of iV to iC was 1:2, the amount of CO2 produced was equivalent to that of iV, and ammonium ion concentrations were equal to the total C5 and C6 acids formed. The presence of alanine and valine (proton donors in the Stickland reaction) in incubations effectively increased the concentration of iC at the expense of iV production, implying that leucine acted there primarily as a proton acceptor. Glycine and proline (proton acceptors) stimulated both iV and iC production from leucine, but increases in iV concentrations were proportionately greater than for iC so that leucine was primarily a proton donor in the presence of proton acceptors. Glucose stimulated the conversion of leucine to volatile fatty acids but favoured iC production. Production of iC from leucine was inhibited by surface active compounds (cetyltrimethylammonium bromide and desoxycholate) as well as arsenite and iodoacetate. The redox dyes methyl viologen and phenosafranine inhibited iC production more severely than iV production, as did the nitroimidazole antimicrobial agent, metronidazole.

Chloramphenicol-resistant strains of Bacteroides fragilis (minimum inhibitory concentration, 12.5 mug/ml) were isolated from a stool specimen which contained multiply resistant Escherichia coli. The enzyme responsible for resistance, chloramphenicol acetyltransferase, was produced constitutively by these strains; the specific activity was 10-fold lower than that of the E. coli enzymes. Similar activity was not detected in susceptible B. fragilis strains, nor could it be induced by growth in the presence of chloramphenicol or by mutagenesis. The enzyme had a pH optimum of 7.8 and a molecular weight of approximately 89,000. The K(m) for chloramphenicol was 5.2 muM, and the enzyme was sensitive to inhibition by 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme produced by an E. coli strain isolated from the same specimen had a similar K(m) and sensitivity to 5,5'-dithiobis-2-nitrobenzoic acid.

Beta-Lactamase activity was detected either biologically or using the chromogenic cephalosporin 87/312 in 20 clinical isolates of Bacteroides fragilis with penicillin G minimal inhibitory concentrations of 10 to 100 micrograms/ml. Strain AM78 (minimal inhibitory concentration, greater than 1,000 micrograms/ml) was used to optimize the conditions for production, assay, and storage of the enzyme. The enzymes are cell associated, with less than 1% of activity being found in culture fluids during growth, and can be released from the cell surface by modified osmotic shock procedure. This procedure causes concomitant release of cyclic phosphodiesterase activity. Substrate profiles and the effects of inhibitors were determined for enzymes partially purified by osmotic shock release and gel filtration. The enzymes are cephalosporinases with some penicillinase activity and are inhibited by p-chloromercuribenzoate, cloxacillin, and carbenicillin. The molecular weight, as determined by gel filtration, is 29,000 to 31,000. A method for the purification of the beta-lactamase from strain AM78 is described: the specific activity of the purified enzyme was 3,424 U/mg, about 3,000-fold that of the crude, cell-associated enzyme.